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 casein hydrolysate and at least one plant-derived proteose peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate. When Clostridium botulinum is cultured using the culture medium according to the present invention containing plant-derived proteose peptone, casein hydrolysate, and minerals, the growth rate of the bacteria in the culture medium is higher than that in each of the currently used culture medium and the culture medium containing only plant-derived proteose peptone. In addition, when the culture medium of the present invention is used, it is possible to produce a high concentration of botulinum toxin by culturing the bacteria in a safe manner.

Description

Field of the Invention

[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 strain of Clostridium capable of producing botulinum toxin. The culture medium composition of the present invention comprises casein hydrolysate and at least one plant-derived peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate, and wheat gluten hydrolysate. Background of the Invention

[0003] Since the 1890s, various Clostridium sp. that secrete neurotoxic toxins have been discovered, and characterization of the toxins secreted from these bacteria has been ongoing for the past 70 years (Schant, EJ et al., Microbiol. Rev., 56:80, 1992).

[0004] The neurotoxin derived from Clostridium species, i.e. botulinum toxin, is divided into seven serotypes (A to G) according to its serological properties. Each toxin has a toxin protein of about 150 kDa in size and naturally contains a complex of several non-toxic proteins bound thereto. The medium complex (300 kDa) is composed of toxin protein and non-toxic non-hemagglutinin protein, while the large complex (450 kDa) and very large complex (900 kDa) are composed of medium-sized complexes bound to hemagglutinin (Sugiyama, H., Microbiol. Rev., 44: 419, 1980). It is known that the function of such non-toxic hemagglutinin proteins is to protect the toxin from the low pH and various proteases in the intestine.

[0005] The toxin is synthesized in cells as a single polypeptide with a molecular weight of approximately 150 kDa. It is then cleaved into two units at a position 1 / 3 from the N-terminal end by the action of intracellular proteases or by treatment with artificial enzymes such as trypsin: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). Compared to a single polypeptide, the cleaved toxin has greatly increased toxicity. The two units are linked to each other by a disulfide bond and have different functions. The heavy chain binds to a receptor on a target cell (Park. MK 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 made cell-permeable by using a detergent or introduced by electroporation or the like (Poulain, B. et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).

[0006] The toxin inhibits acetylcholine exocytosis at the cholinergic presynapse of the neuromuscular junction to cause weakness. It has been found that even treatment with very small amounts of the toxin exhibits toxicity, suggesting that the toxin has any enzymatic activity (Simpson, LL et al., Ann. Rev. Pharmacol. Toxicol., 26:427, 1986).

[0007] According to recent reports, the toxin has metallopeptidase activity, and its substrates include synaptobrevin, syntaxin, 25 kDa synaptosome-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 type B, D, F, and G toxins cleave synaptobrevin at a specific site, type A and E toxins cleave SNAP25 at a specific site, and type C cleaves 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 at pH 4.0-6.8. It is known that the stable, non-toxic protein separates from the neurotoxin at a pH above approximately 7, and thus gradually loses its toxicity. In particular, it is known that toxicity decreases with increasing pH and temperature.

[0009] Botulinum toxin is lethal to humans even in small quantities and is easily produced in large quantities. Therefore, it constitutes one of the four major bioterrorist weapons, along with Bacillus anthracis, Yersinia pestis, and smallpox virus. However, it has been found that when botulinum toxin type A is injected at a dose that does not systematically affect the human body, it can paralyze local muscles at the injection site. Based on this characteristic, botulinum toxin type A can be used for a wide range of applications, including wrinkle removing agents, medicaments for treating spastic hemiplegia and cerebral palsy, and the like. Therefore, the demand for botulinum toxin type A has increased, and research on methods for producing botulinum toxin has been actively carried out to meet the demand.

[0010] Typical commercial products currently available are those from Allergan, Inc., USA (Purified neurotoxin complex of botulinum toxin type A). 100 units The vial consists of approximately 5 ng of the 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 with preservative-free sterile saline (0.9% Sodium Chloride Injection). Other commercial products include those available from Ipsen Ltd., UK. (a complex of Clostridium botulinum type A toxin and hemagglutinin with lactose and human serum albumin in a pharmaceutical composition comprising botulinum toxin and reconstituted with 0.9% sodium chloride prior to use), available from Solstice Neurosciences, Inc. (an injectable solution (pH of approximately 5.6) containing type B toxin, human serum albumin, sodium succinate, and sodium chloride).

[0011] Culture media for culturing Clostridium botulinum, which are commonly used in methods for producing botulinum toxin, as disclosed in Korean Patent No. 10-1339349, contain animal components. Therefore, if animal abnormal prions, known as agents that cause transmissible spongiform encephalopathies, are contained in the animal components due to contamination, it can cause problems in the process used to produce botulinum toxin.

[0012] Transmissible spongiform encephalopathy (TSE) is called neurodegenerative disease that causes severe degeneration of neurons, and its example includes bovine spongiform encephalopathy (BSE), sheep scabies (Scrapie), Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, kuru (Kuru), transmissible mink encephalopathy (transmissible mink encephalopathy), chronic wasting disease (chronic wasting disease), feline spongiform encephalopathy (feline spongiform encephalopathy) etc., which affects humans and animals. BSE has been reported to cross species barriers and even infect humans.

[0013] The agent that causes transmissible spongiform encephalopathies (TSEs) is characterized by its lack of immunogenicity and a long incubation period. Histopathological analysis of brain tissue from BSE-affected cattle reveals the formation of distinctive spongiform vacuoles in the brain due to neuronal damage and the deposition of abnormal protein fibers.

[0014] The cause of TSE is a proteinaceous infectious particle called abnormal prions. Unlike general viruses that require nucleic acids, abnormal prions are infectious particles composed of a single protein that does not contain nucleic acids. Regarding TSE, it is known that when abnormal prions (PrPsc), which are infectious particles, bind to normal prions (PrPc), they are converted into pathogenic prions, which are then accumulated in the brain (Prusiner SB, Alzheimer Dis Assoc Disord., 3:52-78, 1989).

[0015] Creutzfeldt-Jakob disease is a rare neurodegenerative disease of the human transmissible spongiform encephalopathy (TSE) in which the infectious agent is apparently an abnormal isoform of the prion protein. Within six months, individuals with Creutzfeldt-Jakob disease can deteriorate from apparently perfect health to akinetic mutism. Therefore, there is a potential risk of acquiring prion-mediated diseases such as Creutzfeldt-Jakob disease from the administration of pharmaceutical compositions containing biologics obtained using products of animal origin, such as botulinum toxin. Therefore, if a pharmaceutical composition is prepared from a drug substance produced using ingredients of animal origin, it can expose the patient to the potential risk of exposure to various pathogens or infectious agents.

[0016] Against this technical background, the present inventors have found that when a culture medium comprising transmissible spongiform encephalopathy (TSE)-free plant-derived peptone, mineral components, and TSE-free casein hydrolysate (e.g., TSE-certified casein hydrolysate) is used for the cultivation of Clostridium botulinum to prevent the risk of the occurrence of the above-mentioned prion-mediated diseases, the risk of the occurrence of prion-mediated diseases that may be present in currently used culture media (initial culture media) can be eliminated, and the growth rate of Clostridium botulinum in this culture medium can be increased as compared to the growth rate of Clostridium botulinum in currently used culture media and culture media containing plant-derived peptone, thereby completing the present invention. SUMMARY OF THE INVENTION

[0018] Technical issues

[0019] The present invention aims to provide a culture medium composition comprising plant-derived peptone and casein hydrolysate without the risk of transmissible spongiform encephalopathy (TSE) infection, and a method for producing botulinum toxin, which improves the production of botulinum toxin by culturing Clostridium botulinum in the culture medium composition.

[0020] Technical Solution

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

[0022] The present invention also provides a method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the culture medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown is the growth of Clostridium botulinum in a medium containing peptone of plant origin (APF medium).

[0025] Figure 2 Shown is the growth of Clostridium botulinum in a medium containing peptone of plant origin, minerals, amino acids and vitamins.

[0026] Figure 3 Shows the results of examining whether a precipitate is formed after sterilization of a culture medium containing plant-derived peptone, minerals, amino acids, and vitamins.

[0027] Figure 4 Shows the results of examining whether a precipitate forms after sterilization of a culture medium containing plant-derived peptone and minerals.

[0028] Figure 5 It was shown that by adding vitamins, amino acids and "BD Recharge without glucose and L-glutamine" to the culture medium for culturing bacteria (which contains plant-derived peptone and minerals), TM ” Growth of Clostridium botulinum in the culture medium obtained.

[0029] Figure 6 Shown is the growth of Clostridium botulinum in culture media containing various types of plant-derived peptones used to cultivate the bacteria.

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

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

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

[0033] Figure 10 The growth curve of Clostridium botulinum in the final selected APF medium and the changes in toxin concentration are shown.

[0034] FIG. 11 shows a contour plot of a culture medium 24 hours after inoculation of Clostridium botulinum into a culture medium containing plant-derived peptone to which casein hydrolysate had been added. Figure 11a Contour plot at low setting; and Figure 11b Contour plot of the middle setting.

[0035] Figure 12 Shown is the optimization of the response of the culture medium 24 hours after inoculation of Clostridium botulinum into a culture medium comprising vegetable-derived peptone to which casein hydrolysate has been added.

[0036] Figure 13 Contour plots of a CCF (central composite surface) design are shown. (A) Graph of the statistical procedure; and (B) Graph of the statistical procedure.

[0037] Figure 14 Shown are the contour plot (A) and optimization plot (B) of the CCF (Central Composite Face) design.

[0038] Figure 15 is a set of graphs showing time-dependent OD values ​​indicating the growth of Clostridium botulinum in the medium currently used, a medium comprising peptone of plant origin, and a medium comprising peptone of plant origin and casein hydrolysate.

[0039] Best Mode for Carrying Out the Invention

[0040] In the present invention, an animal protein-free (APF) medium composition shows an increased growth rate of Clostridium botulinum compared to a currently used medium (initial medium), but does not consider adding a medium component that further increases the growth rate of the bacteria, and there is no risk of infection such as TSE. Therefore, a casein hydrolyzate (e.g., a TSE-certified casein hydrolyzate) that has not been reported to cause TSE infection is added to the APF medium, and the growth of bacteria in the APF medium is examined. As a result, the above-mentioned medium showed an increased bacterial growth rate compared to a currently used medium and a medium containing only plant-derived peptone. Therefore, if the APF medium is used, it is possible to produce a high concentration of botulinum toxin by culturing bacteria in a safe manner under TSE-free conditions.

[0041] As used herein, the term "currently used culture medium or initial culture medium" means a culture medium containing casein hydrolysate, yeast extract, and thioglycolate medium (which is an animal-derived culture medium component). The term "APF medium (animal protein-free medium)" means a culture medium that does not contain animal-derived protein and contains plant-derived peptone, minerals, and glucose.

[0042] In the present invention, in order to produce botulinum toxin by culturing Clostridium botulinum under conditions free of transmissible spongiform encephalopathy (TSE), an APF culture medium containing TSE-free plant-derived peptone was prepared and compared with the currently used culture medium (containing animal components). As a result, it can be seen that the optimal culture medium composition for culturing Clostridium botulinum is a culture medium containing plant-derived peptone, at least one mineral selected from KH2PO4, K2HPO4, and Na2HPO4, and a carbon source (e.g., glucose), and the best growth of bacteria in this culture medium was found. As shown in Table 13, the optimal content of plant-derived peptone in the final selected culture medium composition for culturing Clostridium botulinum was determined to be 5 g / L Hy-Pea. TM 7404, 10g / L UltraPep TM Cotton and 5g / L HyPep TM4601N, and the optimal content of minerals in the culture medium composition is 5.5g / L K2HPO4 and 3g / L Na2HPO4.

[0043] In another example of the present invention, the growth pattern and toxin concentration of Clostridium botulinum in the final selected APF medium containing plant-derived peptone and minerals were measured. The results are shown in Table 12 and Figure 10 As shown in FIG, the OD value of Clostridium botulinum began to increase after 12 hours of culture, and at 24 hours of culture, the culture medium showed an OD of 3.5465. 540nm and an 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 an OD of 0.7224 600nm The toxin concentration in the Clostridium botulinum culture supernatant began to increase after 5 hours of culture, reaching a final value of 31.41 μg / ml. When the toxin concentration was measured after disrupting the bacteria, toxin production began after 5 hours of culture and continued to increase, remaining at a consistent level after 28 hours of culture, reaching a final value of 38.39 μg / ml.

[0044] In another embodiment of the present invention, the growth pattern of Clostridium botulinum in a culture medium containing plant-derived peptone and casein hydrolysate was examined. Figure 15 As shown in Figure 2, the currently used culture medium showed an increase in the growth rate of Clostridium botulinum after 24 hours of culture and showed a peak OD at 39 hours of culture. 540nm APF medium containing plant-derived peptone showed an increase in growth rate after 18 hours of culture and a peak OD value of 3.384 at 28 hours of culture. 540nm The value is 3.526.

[0045] In addition, APF medium containing plant-derived peptone + casein hydrolysate showed an increase in growth rate after 18 h of culture and a peak OD at 26 h of culture. 540nm The value is 5.628. Therefore, the OD value at 24 hours of culture is compared. 540nm The culture medium currently used shows OD 540nm APF medium containing plant-derived peptone showed an OD of 1.2382. 540nm value of 2.8595, and the APF medium containing plant-derived peptone + casein hydrolysate showed an OD 540nmThe OD value of the APF medium containing plant-derived peptone and casein hydrolyzate at 24 hours of culture was approximately 4.23 times higher than that of the medium currently used and approximately 1.83 times higher than that of the APF medium containing plant-derived peptone. Furthermore, the peak OD value of the APF medium containing plant-derived peptone and casein hydrolyzate was approximately 1.6 times higher than that of the APF medium containing plant-derived peptone.

[0046] As shown in Table 18, it was determined that the optimal content of plant-derived peptone in the final selected medium composition for culturing Clostridium botulinum (which contains transmissible spongiform encephalopathy (TSE)-free casein hydrolysate) was 5 g / L Hy-Pea TM 7404, 10g / L UltraPep TM Cotton and 5g / L HyPep TM 4601N, and the optimal mineral content in the culture medium composition is 5.5g / L K2HPO4 and 3g / L Na2HPO4, and the content of casein hydrolysate is 20g / L NZ-Amine A and 11g / L NZ-Case TT.

[0047] Based on this, in one aspect, the present invention relates to a culture medium composition for culturing Clostridium botulinum, the culture medium composition comprising: at least one plant-derived peptone selected from the group consisting of pea hydrolysate, cottonseed hydrolysate and wheat gluten hydrolysate; and casein hydrolysate.

[0048] As used herein, the term "plant-derived peptone" means peptone extracted from pea, cottonseed or wheat gluten. Preferably, the plant-derived peptone may be commercially available Hy-Pea TM 7404、UltraPep TM Cotton, HyPep TM 7504 or HyPep TM 4601N, but not limited thereto. The term "casein hydrolysate" means a component extracted from milk. Preferably, the casein hydrolysate may be a casein hydrolysate containing approximately 85.5-94.5 wt% of peptides with a molecular weight of 500 Da or less, or a casein hydrolysate containing approximately 63.56-70.25 wt% of peptides with a molecular weight of 500 Da or less. More preferably, the casein hydrolysate may be commercially available NZ-Amine A or NZ-Case TT, but not limited thereto.

[0049] As used herein, the term "plant-derived peptone" or "plant-derived hydrolysate" refers to a product obtained by degrading proteins isolated from plants. For example, pea peptone (pea hydrolysate) refers to a product obtained by degrading total proteins isolated from peas. In addition, the term "casein hydrolysate" refers to a product obtained by degrading casein protein.

[0050] The degradation of plant-derived proteins or casein proteins is preferably carried out by partial digestion. Protein degradation is preferably carried out by acid treatment, alkali treatment, enzyme treatment, high pressure treatment, heat treatment or physical treatment. More preferably, the plant-derived peptone or casein hydrolysate can be obtained by enzyme treatment. Physical treatment is, for example, grinding.

[0051] The plant-derived peptone or casein hydrolyzate used in the present invention is a partially degraded product of protein and is a mixture containing not only amino acids as single molecules but also peptides composed of several to several dozen amino acids and intact protein molecules.

[0052] In the present invention, the content of plant-derived peptone in the culture medium composition may be 0.1-10 w / v% (1-100 g / L), preferably 0.2-5 w / v% (2-50 g / L), more preferably 0.5-2 w / v% (5-20 g / L).

[0053] In the present invention, the culture medium composition contains all of pea hydrolysate, cottonseed hydrolysate and wheat gluten hydrolysate, and the content ratio of pea hydrolysate, cottonseed hydrolysate and wheat gluten hydrolysate in the culture medium composition may 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.

[0054] In the present invention, the content of casein hydrolyzate in the culture medium composition may be 0.22-15.5 w / v% (2.2-155 g / L), preferably 0.44-7.75 w / v% (4.4-77.5 g / L), and more preferably 1.1-3.1 w / v% (11-31 g / L).

[0055] In the present invention, the casein hydrolyzate may be a casein hydrolyzate containing about 85.5-94.5 wt % of peptides having a molecular weight of 500 Da or less and / or a casein hydrolyzate containing about 63.56-70.25 wt % of peptides having a molecular weight of 500 Da or less.

[0056] In the present invention, the casein hydrolysate includes both a casein hydrolysate containing about 85.5-94.5 wt% of peptides having a molecular weight of 500 Da or less and a casein hydrolysate containing about 63.56-70.25 wt% of peptides having a molecular weight of 500 Da or less, and the content ratio of the casein hydrolysate containing about 85.5-94.5 wt% of peptides having a molecular weight of 500 Da or less and the casein hydrolysate containing about 63.56-70.25 wt% of peptides having a molecular weight of 500 Da or less may be 0.01-40 by weight:0.01-22, preferably 10-30 by weight:5.5-16.5, more preferably 16-24 by weight:8.8-13.2.

[0057] In the present invention, the culture medium composition for culturing Clostridium botulinum may further contain a carbon source and at least one mineral selected from K2HPO4 (dipotassium hydrogen phosphate), Na2HPO4 (sodium dihydrogen phosphate), and KH2PO4 (potassium dihydrogen phosphate).

[0058] Herein, examples of carbon sources 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.).

[0059] In the present invention, the content of minerals in the culture medium composition may 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).

[0060] In another aspect, the present invention relates to a method for producing a botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the above-mentioned medium composition to produce a botulinum toxin; and (b) recovering the produced botulinum toxin.

[0061] In the present invention, the culture may be performed under anaerobic conditions, and the botulinum toxin may be selected from botulinum toxin types A, B, C, D, E, F, and G. Example

[0062] Hereinafter, with reference to embodiment, the present invention is described in more detail. It is apparent to those skilled in the art that these embodiments are merely illustrative purposes and should not be construed as limiting the scope of the present invention. Therefore, the essential scope of the present invention will be limited by the appended claims and their equivalents.

[0063] Example 1: Cultivation of Clostridium botulinum in plant-derived peptone medium

[0064] 1-1: Composition of culture media currently used for culture

[0065] The reagents and culture medium components used in the present invention were purchased from Sigma (USA), Kerry Inc. (USA), BD Biosciences (USA), Gibco Life Technologies (USA), and Quest (USA).

[0066] Seed culture and main culture of Clostridium botulinum for the production of botulinum toxin were performed using a medium currently in use, which has a composition comprising 2% casein hydrolysate (20 g / L), 1% yeast extract (10 g / L), 1% glucose (10 g / L), and 0.5% thioglycolate medium (5 g / L). 5 g of thioglycolate medium per liter of the medium currently in use consists of 2.52 g of enzymatic digest of casein, 0.84 g of yeast extract, 0.925 g of dextrose, 0.085 g of sodium thioglycolate, 0.42 g of NaCl, 0.085 g of L-cysteine, 0.00014 g of resazurin, and 0.125 g of bacteriological agar.

[0067] 1-2: Composition of APF medium used in culture

[0068] Negative control media were prepared by removing casein hydrolysate, yeast extract, and thioglycolate medium from the medium currently used for culturing Clostridium botulinum (initial medium), and by adding four plant-derived peptone candidates (Hy-Pea TM 7404、UltraPep TM Cotton, HyPep TM 7504, and HyPep TM 4601N) was added to the negative control medium to prepare animal protein-free (APF) medium (Table 1).

[0069] Table 1 shows the composition of APF medium containing plant-derived peptone for culturing Clostridium botulinum compared to currently used media.

[0070] Table 1

[0071]

[0072] 1-3: Seed culture of Clostridium botulinum

[0073] 20 μl of Clostridium botulinum (Korea Centers for Disease Control and Prevention registration number: 4-029-CBB-IS-001) was inoculated into a culture tube containing 10 ml of a sterile culture medium having each of the compositions described in Examples 1-1 and 1-2, and primary seed culture (stationary culture) was performed at 35° C. under anaerobic conditions for 22 to 30 hours. When 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 a sterile culture medium having the same medium composition, and secondary seed culture (stationary culture) was performed at 35° C. under anaerobic conditions for 8 to 15 hours.

[0074] 1-4: Primary culture of Clostridium botulinum

[0075] To produce botulinum toxin by culturing Clostridium botulinum, a primary bacterial culture was performed. Specifically, 9.3 L of culture medium having each of the compositions described in Examples 1-1 and 1-2 was prepared and placed in a 10-liter incubator, followed by sterilization. Nitrogen was supplied to create anaerobic conditions, and bacterial growth was performed at a temperature of 35°C and a stirring speed of 50 rpm.

[0076] The secondary seed culture from the 1-L culture flask described in Examples 1-3 was inoculated into a 10-L incubator via an inoculation line connected to the inoculation port of the 10-L incubator. Clostridium botulinum was cultured in the 10-L incubator at 35°C and 50 rpm. The established culture conditions were monitored and recorded. The primary culture was terminated after the bacteria had been cultured for at least 100 hours.

[0077] By combining four plant-derived peptone candidates (Hy-Pea TM 7404、UltraPep TM Cotton, HyPep TM 7504 and HyPep TM The growth of Clostridium botulinum in an animal protein-free (APF) medium prepared by adding 4601N to a negative control was compared with the bacterial growth in a negative control medium prepared by removing casein hydrolysate, yeast extract, and thioglycolate medium from the medium currently used (initial medium) (Table 1).

[0078] Therefore, as shown in Table 1 and Figure 1 As shown in , Clostridium botulinum did not grow in the negative control medium, but began to grow in the initial medium (the medium currently used) 24 hours after inoculation of the bacteria, and began to grow in the medium containing plant-derived peptone 30 hours after inoculation of the bacteria.

[0079] Example 2: Cultivation of Meat in a Medium Containing Plant-Derived Peptone, Minerals, Amino Acids and Vitamins Clostridium difficile

[0080] Since the growth of Clostridium botulinum in the culture medium prepared by adding four plant-derived peptones in Example 1 was slower than the growth of Clostridium botulinum in the initial culture medium, a solution thereof was provided as follows.

[0081] 1) To examine the effect of thioglycolate functioning to produce anaerobic conditions, thioglycolate was removed from the initial culture medium (the culture medium currently used), and changes in bacterial growth rate in the thioglycolate-free culture medium were analyzed.

[0082] 2) Since the slower growth rate may be due to a lack of nitrogen source, the peptone concentration in the culture medium used for bacterial culture was increased 2-fold.

[0083] 3) The growth of Clostridium botulinum in a medium obtained by adding minerals, amino acids, and vitamins to a medium containing plant-derived peptone was compared with the growth of Clostridium botulinum in an APF medium disclosed in US Pat. No. 8,012,716 (Allergan) (Table 2).

[0084] Table 2 shows the composition of the culture medium for culturing Clostridium botulinum, which contains plant-derived peptone, minerals, amino acids, and vitamins.

[0085] Table 2

[0086]

[0087] The results are shown in Table 2 and Figure 2 As shown in , when bacteria were cultured in a medium that does not contain thioglycolate and is currently used, the growth rate of the bacteria in the medium was slower than that in a medium containing thioglycolate, indicating that thioglycolate affects the growth rate of bacteria. When the peptone concentration in the medium was doubled, the bacteria did not grow in the medium. When mineral components, amino acids, and vitamins were added to the medium containing peptone, the growth rate of the bacteria was similar to that of the bacteria in the medium currently used, but a precipitate was formed after the medium was sterilized. In addition, it was seen that the growth rate of bacteria in Allergan's APF medium was similar to that of bacteria in the medium currently used.

[0088] Example 3: Production of a precipitate by sterilizing a culture medium containing plant-derived peptone, minerals, amino acids and vitamins

[0089] In Example 2, the growth rate of Clostridium botulinum in the medium containing plant-derived peptone, minerals, amino acids, and vitamins in APF medium candidates 2 to 4 shown in Table 2 was observed to be similar to the growth rate of Clostridium botulinum in the medium currently used. However, precipitate formation occurred after sterilization of the medium, and the cause was examined (Table 3).

[0090] Table 3 shows the composition of a culture medium for culturing Clostridium botulinum that is used in sterilization and contains plant-derived peptone, minerals, amino acids, and vitamins.

[0091] Table 3

[0092]

[0093] The results are shown in Table 3 and Figure 3 As shown in , only when minerals were added to a medium containing plant-derived peptone did a precipitate form after sterilization of the medium, indicating that the minerals were the primary cause of precipitate formation. This is believed to be due to the interaction of the mineral components under high temperature and high pressure conditions during sterilization of the medium.

[0094] Example 4: Formation of a precipitate by sterilizing a culture medium containing plant-derived peptone and minerals

[0095] To identify the mineral components involved in the formation of the precipitate induced by sterilization as confirmed in Example 3, various combinations of different components were added to the culture medium and then sterilized (Table 4).

[0096] Table 4 shows the composition of the culture medium containing plant-derived peptone and minerals for culturing Clostridium botulinum, and the results of sterilization of the culture medium.

[0097] Table 4

[0098]

[0099] The results are shown in Table 4 and Figure 4 As shown in , in the culture medium containing plant-derived peptone and minerals, the culture medium containing MgSO4·7H2O and K2HPO4 and the culture medium containing MgSO4·7H2O and Na2HPO4 formed precipitates after sterilization.

[0100] Example 5: Cultivation of Clostridium botulinum in APF medium without precipitation

[0101] When vitamins and amino acids were additionally added to the APF medium of Example 4 containing plant-derived peptone and minerals, an experiment was conducted to determine whether culturing of Clostridium botulinum was possible. In addition, an experiment was conducted to examine whether culturing of bacteria was possible in a medium containing no plant-derived peptone and minerals and containing vitamins, amino acids and / or BD Recharge without glucose and L-glutamine. TM ” (Cat. No. 670002, BD Bioscience) (a yeast extract-based medium component without glucose and L-glutamine) (Table 5).

[0102] Table 5 shows the effect of adding vitamins, amino acids and "BD Recharge without glucose and L-glutamine" to the medium for culturing Clostridium botulinum containing plant-derived peptone and minerals. TM ” The culture medium components obtained, as well as the growth rate of bacteria in the culture medium.

[0103] Table 5

[0104]

[0105] The results are shown in Table 5 and Figure 5 As shown in , Clostridium botulinum grew within 24 hours after bacterial inoculation only in the case of a 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, in the case of a medium containing no plant-derived peptone and minerals and containing vitamins, amino acids, and "BD Recharge without glucose and L-glutamine", TM In summary, the most suitable medium composition for culturing Clostridium botulinum includes plant-derived peptone, KH2PO4, K2HPO4, Na2HPO4, amino acids and vitamins.

[0106] Example 6: Cultivation of Clostridium botulinum in media containing proteins from different plant sources

[0107] An experiment was conducted to examine whether cultivation of Clostridium botulinum was possible when different combinations of plant-derived peptones were added to the APF medium of Example 5.

[0108] Table 6 shows the composition of culture media containing peptones of different plant origins for culturing Clostridium botulinum and the results of examining whether the bacteria grew in the culture media.

[0109] Table 6

[0110]

[0111] The results are shown in Table 6 and Figure 6 , it was shown that cultivation of Clostridium botulinum was possible even when only one or two of four plant-derived peptones were added to the culture medium.

[0112] Considering the results of Examples 5 and 6, it can be seen that the culture medium should contain at least one plant-derived peptone, and plant-derived peptone cannot be used with "BD Recharge without glucose and L-glutamine". TM ” (Cat. No. 670002, BD Bioscience) (a yeast extract-based medium component without glucose and L-glutamine) was substituted.

[0113] Example 7: Experiment for selecting two of the three types of minerals contained in the culture medium

[0114] In Examples 1 to 7, the APF medium composition for culturing Clostridium botulinum was determined to contain glucose, sodium chloride (NaCl), four plant-derived peptones, three minerals, amino acids, and vitamins. Among these medium components, the medium components that did not significantly affect bacterial growth were removed to reduce the number of medium components. Therefore, it was judged that amino acids and vitamins had no significant effect on the growth of Clostridium botulinum, and under this judgment, amino acids and vitamins were removed from the medium components. In addition, in order to select two from the three types of minerals, bacteria were cultured using the medium composition shown in Table 7, and the OD (540nm and 600nm) values ​​24 hours and 48 hours after inoculation of the bacteria were measured and compared.

[0115] Table 7 shows the composition of the mineral derived first stage selection medium and the growth of C. botulinum in the medium.

[0116] Table 7

[0117]

[0118] As shown in Table 7, 24 hours after bacterial inoculation, the currently used medium showed an OD (540 nm) value of 0.942, and the APF medium containing K2HPO4 and Na2HPO4 showed the highest OD (540 nm) value of 4.964 among the APF medium. In addition, 48 hours after bacterial inoculation, the APF medium containing KH2PO4 and Na2HPO4 showed the highest OD value and active bacterial growth.

[0119] As shown in FIG7 , contour plots of K₂HPO₄ and Na₂HPO₄, which have high main effects, were plotted. As a result, the OD value increased as the concentrations of K₂HPO₄ and Na₂HPO₄ increased. Furthermore, Clostridium botulinum exhibited the highest growth when the minerals were added to the culture medium at concentrations of K₂HPO₄ = 0 g / L, K₂HPO₄ = 5.5 g / L, and Na₂HPO₄ = 5 g / L.

[0120] At the same time, in order to confirm the bacterial culture results based on more accurate mineral addition, a second phase of experiments was conducted using response surface methodology. Since the culture medium composition cannot have negative values, the experiment was planned using a CCF (Central Composite Surface) design and was conducted by culturing bacteria in the culture medium composition shown in Table 8. The experimental results were then combined with the results of the previously performed FFD and statistically analyzed.

[0121] Table 8 shows the composition of the culture medium obtained by the second stage selection of minerals and the growth of Clostridium botulinum in the culture medium.

[0122] Table 8

[0123]

[0124] Draw isovalue map and be used for comparison.As shown in Figure 8, along with KH PO Concentration reduces, and OD value increases.When comparing optimum condition, result is different from the result of FFD due to curvature effect, and K HPO Value is identical, but Na HPO Value changes to 3.1313g / L from 5g / L.Therefore, confirming that the optimum mineral condition of culture medium is 5.5g / LK HPO and 3g / L Na HPO by statistical analysis.

[0125] Example 8: Experiment for selecting plant-derived peptone contained in culture medium

[0126] As shown in Tables 9 and 10, the plant-derived peptones were combined according to the mixture design, and the growth of Clostridium botulinum was examined in the culture media containing the combined plant-derived peptones.

[0127] Table 9 shows the composition of the culture medium obtained by the first-stage selection of plant-derived peptone and the growth of Clostridium botulinum using the culture medium.

[0128] Table 9

[0129]

[0130] Table 10 shows the composition of the culture medium obtained by the second stage selection with plant-derived peptone and the growth of Clostridium botulinum using the culture medium.

[0131] Table 10

[0132]

[0133] As a result, as shown in FIG. 9, contour plots were drawn and compared. It was determined that HyPep TM 7504 had the lowest effect on the growth of C. botulinum. Based on this assay, HyPep TM 7504 was excluded from the medium components. In summary, it was determined that the composition of the final selected plant-derived proteose peptone to be contained in the medium included 5 g / L Hy-Pea TM 7404, 10 g / L UltraPep TM cotton, and 5 g / L HyPep TM 4601N.

[0134] Example 9: Culturing of C. botulinum in Medium Containing NaCl or Not Containing NaCl

[0135] 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 change of NaCl, the content of NaCl in the medium was adjusted to the range of 0 to 1 g / L, and then the bacteria were cultured in the medium.

[0136] Table 11 shows the composition of the medium containing NaCl for culturing C. botulinum and the growth of C. botulinum in the medium.

[0137] Table 11

[0138]

[0139] 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. Accordingly, NaCl was excluded from the final APF medium composition.

[0140] Example 10: Measuring the Growth Pattern and Toxin Concentration of Clostridium botulinum in the Final Selected APF Medium

[0141] C. botulinum was inoculated into the final selected C. botulinum medium (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.

[0142] Table 12 shows the time-dependent OD values ​​and toxin concentrations of C. botulinum grown in the final selected APF medium.

[0143] Table 12

[0144]

[0145] The results are shown in Table 12 and Figure 10 As shown in FIG, the OD value began to increase after culturing Clostridium botulinum for 12 hours, and the culture medium showed an OD of 3.5465 after culturing for 24 hours. 540nm and an OD of 3.0695 600nm Then, the OD value gradually decreased, and after culturing for 48 h, the culture medium showed an OD of 0.792 540nm and an OD of 0.7224 600nm The toxin concentration in the supernatant of Clostridium botulinum began to increase after 24 hours of culture, reaching a final value of 31.41 μg / ml. When the toxin concentration was measured after disrupting the bacteria, toxin production began after 5 hours of culture and continued to increase, remaining at a consistent level after 28 hours of culture, reaching a final value of 38.39 μg / ml.

[0146] In summary, the final selected APF (animal protein-free medium) composition determined based on the results of Examples 1 to 10 is summarized in Table 13.

[0147] Table 13

[0148]

[0149] Example 11: Selection of a culture medium composition containing plant-derived peptone and casein hydrolysate

[0150] The APF medium composition determined based on the results of Examples 1 to 10 showed an increased bacterial growth rate compared to the currently used medium (initial medium), but the addition of medium components further increased the bacterial growth rate without the risk of infection by TSEs, etc. was considered. Therefore, TSE-free casein hydrolyzate (e.g., TSE-certified casein hydrolyzate) was added to the APF medium, and the growth of bacteria in the medium was examined.

[0151] One or two TSE-free casein hydrolysates were added to the APF culture medium determined based on the results of Examples 1 to 10. Specifically, Clostridium botulinum was cultured for 24 and 48 hours in culture medium A (obtained by adding casein hydrolysate NZ-Amine A to a culture medium composition containing plant-derived peptone), culture medium B (obtained by adding NZ-CaseTT to a culture medium composition), culture medium C (obtained by adding casamino acids to a culture medium composition), or culture medium D (obtained by adding tryptone to a culture medium composition), and bacterial growth was examined by measuring OD (540 nm, 600 nm) values ​​during the culture period (Table 14). Furthermore, the measurement results were statistically analyzed using a statistical program to select the culture medium composition that showed the highest growth when the bacteria were cultured for 24 hours.

[0152] Table 14 shows the results of time-dependent growth of Clostridium botulinum in medium compositions containing casein hydrolysate in addition to plant-derived peptone.

[0153] Table 14

[0154]

[0155] At the same time, as shown in FIG11 , in order to find the conditions showing the highest growth at 24 hours of culturing bacteria, a mixed contour map of the lower limit setting and the intermediate setting conditions was drawn. As a result, it can be seen that Clostridium botulinum showed the highest growth in the area where medium A and medium B were present, and medium C and medium D showed a value of 0. In addition, as Figure 12 As shown in , the conditions showing the highest growth of bacteria were investigated, and as a result, it was shown that the bacteria showed the highest growth under the following conditions: A = 0.8725, B = 1.1275, C = 0, and D = 0. Based on these results, medium A (NZ-Amine A) and medium B (NZ-Case TT) were finally selected from the four medium components.

[0156] Example 12: Final selection of the concentration of the culture medium containing plant-derived peptone and casein hydrolysate

[0157] In order to determine the optimal medium composition of the two casein hydrolysate medium components selected in Example 11, a response surface experiment was performed using CCF (central composite surface) design.

[0158] Table 15 shows the results of analyzing the time-dependent growth of Clostridium botulinum in a medium composition containing plant-derived peptone and casein hydrolysate using CCF response surface methodology.

[0159] Table 15

[0160]

[0161] As shown in Table 15, C. botulinum growth was found.

[0162] At the same time, contour plots of the model set using the statistical program were drawn. As shown in Figure 13 As shown in (A), the medium component A was increased outside the experimental range, indicating that there were optimal conditions. To confirm this, the range of the medium component A (N-Z-Amine A) was extended outside the experimental range using the statistical program, and contour plots were drawn. As a result, as shown in Figure 13 As shown in (B), bacterial growth increased as the concentration of the medium component A increased. However, since an increase in the content of casein hydrolysate can cause problems regarding solubility, optimal conditions were selected within the experimental range.

[0163] Therefore, the bacteria showed the highest growth after 24 hours of culture under the following conditions: A = 2.0 and B = 1.1402. Based on these results, the medium composition containing casein hydrolysate added to the animal protein-free (APF) medium, which showed the highest growth of C. botulinum, was determined, as shown in Table 16.

[0164] Table 16

[0165]

[0166] Example 13: Growth model of Clostridium botulinum in medium containing plant-derived peptone and casein hydrolysate Mode

[0167] C. botulinum was cultured using the medium composition containing a plant-derived proteose peptone and / or casein hydrolysate determined based on the results of Examples 1 to 10, and the growth pattern of the bacteria was examined.

[0168] Table 17 shows the time-dependent OD values of C. botulinum growth between the currently used medium, the medium containing a plant-derived protein (APF medium), and the final medium containing a plant-derived protein and casein hydrolysate (APF medium + casein).

[0169] Table 17

[0170]

[0171] Therefore, as shown in Table 17 and Figure 15 the currently used medium showed an increase in the growth rate after 24 hours of culture of C. botulinum, and showed a peak OD 540nmThe APF medium containing plant-derived protein showed an increase in growth rate after 18 hours of culture and a peak OD value of 3.384 at 28 hours of culture. 540nm The value is 3.526.

[0172] In addition, APF medium containing plant-derived protein + casein hydrolysate showed an increase in growth rate after 18 h of culture and a peak OD at 26 h of culture. 540nm The value is 5.628. When comparing the OD of culture for 24 hours 540nm The culture medium currently used shows OD 540nm The APF medium containing plant-derived protein showed an OD of 1.2382. 540nm value of 2.8595, and the APF medium containing plant-derived protein + casein hydrolysate showed an OD 540nm The OD value of the APF medium containing plant-derived protein and casein hydrolyzate at 24 hours of culture was approximately 4.23 times higher than that of the conventional medium and approximately 1.83 times higher than that of the APF medium containing plant-derived protein. Furthermore, the peak OD value of the APF medium containing plant-derived protein and casein hydrolyzate was approximately 1.6 times higher than that of the APF medium containing plant-derived protein.

[0173] Industrial Applicability

[0174] As described above, when the culture medium according to the present invention containing plant-derived peptone, casein hydrolyzate, and minerals is used to culture Clostridium botulinum, the growth rate of the bacteria in the culture medium is higher than the growth rate of the bacteria in each of the currently used culture medium and the culture medium containing only plant-derived protein. In addition, when the culture medium of the present invention is used, high concentrations of botulinum toxin can be produced by culturing the bacteria in a safe manner.

[0175] Although the present invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is only directed to preferred embodiments and does not limit the scope of the invention. Therefore, the true scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A culture medium composition for culturing Clostridium botulinum, comprising: 10 g / L glucose; Plant-derived peptone, comprising 5 g / L of pea hydrolysate, 10 g / L of cottonseed hydrolysate, and 5 g / L of wheat gluten hydrolysate; 5.5 g / L K2HPO4; 3g / L Na2HPO4; 20g / L NZ-AMINE A; and 11g / L NZ-CASE TT. 2 . The medium composition according to claim 1 , wherein the plant-derived peptone or the casein hydrolysate is subjected to enzyme treatment.

3. A method for producing botulinum toxin, comprising the following steps: (a) using the medium composition of claim 1 or 2 to cultivate Clostridium botulinum to produce botulinum toxin; and (b) Recovery of produced botulinum toxin. The method according to claim 3 , wherein the culturing is carried out under anaerobic conditions.

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

Images