Medium composition for producing botulinum toxin

Abstract

A culture medium composition for producing botulinum toxin is disclosed, particularly a culture medium composition for culturing Clostridium species capable of producing botulinum toxin. When Clostridium botulinum strains are cultured using a medium containing plant peptone, glucose, and yeast extract and free of animal-derived components, the growth rate of the strains can be increased compared to existing media, and the strains can be safely cultured due to the absence of animal-derived components, thereby increasing the yield of botulinum toxin.

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 spp. strain capable of producing botulinum toxin. The culture medium composition of the present invention comprises plant protein extract, D-(+)-glucose and yeast extract, wherein the plant protein extract comprises wheat protein extract and pea protein extract. Prior Art

[0002] Various strains of Clostridium species that secrete neurotoxins 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).

[0003] Botulinum toxin, a neurotoxin derived from strains of Clostridium species, is classified into seven types from A to G based on its serological characteristics. Each toxin contains a toxin protein of about 150 kDa and is naturally composed of a complex bound to several non-toxic proteins. The medium complex (300 kDa) is composed of the toxin protein and a non-toxic non-hemagglutinin protein, and the large complex (450 kDa) and giant complex (900 kDa) are in such a form that the medium complex is bound to hemagglutinin (Sugiyama, H, Microbiol Rev., 44:419, 1980). This non-toxic hemagglutinin protein is known to function to protect the toxin from the low pH and various proteases in the intestine.

[0004] The toxin is synthesized in cells as a single polypeptide with a molecular weight of about 150 kDa, and then cut into two units at the 1 / 3 position from the N-terminus by the action of intracellular proteases or artificial enzymatic treatment with trypsin: a light chain (L; molecular weight: 50 kDa) and a heavy chain (H; molecular weight: 100 kDa). Compared with a single polypeptide, the toxicity of the toxin cut in this way is greatly increased. The two units are connected to each other by a disulfide bond and have different functions. The heavy chain binds to the receptors of target cells (Park. MK et al., FEMS Microbiol. Lett., 72:243, 1990) and reacts with biological membranes at low pH (pH 4) to form channels (Mantecucco, C. et al., TIBS., 18:324, 1993). The light chain has pharmacological activity and, when introduced into cells through electroporation, can confer cell permeability using detergents or interfere with the secretion of neurotransmitters (Poulain, B. et al., Proc. Natl. Acad. Sci. USA., 85:4090, 1988).

[0005] The toxin inhibits the exocytosis of acetylcholine at the pre-cholinergic synapses of the neuromuscular junction, causing general weakness. It is believed that the toxin is toxic even in very small amounts, indicating that the toxin has any enzymatic activity (Simpson, LL et al., Ann. Rev. Pharmae​​ol. Toxicol., 26:427, 1986).

[0006] According to the latest report, the toxin has metallopeptidase activity, and its substrates are synaptobrevin, syntaxin, and 25 KDa synaptosome-associated protein (SNAP25), which are unit proteins of the exocytosis machinery complex. Each type of toxin uses any one of these three proteins as a substrate, and it is known that toxins B, D, F, and G cleave synaptobrevin at a specific site, toxins A and E cleave SNAP25 at a specific site, and toxin C cleaves syntaxin at a specific site (Binz, T. et al., J. Biol. Chem., 265:9153, 1994).

[0007] In particular, it is known that botulinum toxin type A is soluble in dilute aqueous solutions at a pH of 4.0-6.8. It is known that at a pH of about 7 or higher, a stable non-toxic protein separates from the neurotoxin, whereby the toxicity gradually disappears. In particular, it is known that the toxicity decreases with increasing pH and temperature.

[0008] Botulinum toxin is lethal to humans even in small amounts and is easy to produce on a large scale, so it forms one of the four major biological terrorist 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 equal to or less than the dose that will not affect the human body systemically, it may paralyze local muscles at the injection site. Based on such characteristics, botulinum toxin type A can be used in a wide range of applications, including wrinkle removers, treatments for spastic hemiplegia and cerebral palsy, etc. Therefore, the demand for this botulinum toxin has increased dramatically, and in order to meet this demand, people are conducting in-depth research on methods for producing botulinum toxin.

[0009] A representative example of a currently commercially available product is BOTOX® (a purified neurotoxin complex of botulinum toxin type A) from Allergan in the United States. A bottle of 100 units of BOTOX® consists of approximately 5 ng of purified botulinum toxin type A complex, 0.5 mg of human serum albumin, and 0.9 mg of sodium chloride, and is provided in a vacuum-dried form and reconstituted with preservative-free sterile saline (0.9% sodium chloride for injection). Examples of other commercially available products include Dysport® (a complex of Clostridium botulinum toxin type A and hemagglutinin, which has lactose and human serum albumin in a pharmaceutical composition containing botulinum toxin and is reconstituted with 0.9% sodium chloride before use) from Ipsen in the United Kingdom, MyoBloc™ (an injection solution with a pH of approximately 5.6, containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride) from Solstice Neurosciences, and the like.

[0010] The method for producing botulinum toxin disclosed in Korean Patent No. 10-1339349 and the culture medium generally used for culturing Clostridium botulinum strains contain animal-derived components. Therefore, if the animal-derived components contain animal-derived abnormal prions, which are known as the cause of transmissible spongiform encephalopathy, due to contamination, problems may arise in the production of botulinum toxin.

[0011] Transmissible spongiform encephalopathies (TSEs) are a class of degenerative neurological diseases that cause fatal neurodegenerative disorders in humans and animals, including bovine spongiform encephalopathy (BSE) (mad cow disease), scrapie, Creutzfeldt-Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome, kuru, transmissible mink encephalopathy, chronic wasting disease in deer, feline spongiform encephalopathy, etc. BSE has been reported to cross the species barrier and infect humans.

[0012] The pathogens of transmissible spongiform encephalopathies are characterized by non-immunogenicity and a long incubation period. Postmortem analysis of the brains of cattle infected with bovine spongiform encephalopathy (BSE) revealed the presence of distinctive sponge-like vacuoles in the brain, which are caused by the destruction of nerve cells and the deposition of abnormal protein fibers.

[0013] The pathogen that causes transmissible spongiform encephalopathies is an infectious protein called abnormal prion. Unlike typical viruses that require nucleic acids, abnormal prions are infectious particles composed entirely of protein and do not contain nucleic acids. It is known that transmissible spongiform encephalopathies occur when normal prions (PrPc) are converted into pathogenic prions due to binding to infectious agents such as abnormal prions (PrPsc), and these pathogenic prions accumulate in the brain (Prusiner SB, Alzheimer Dis. Assoc. Disord., 3:52-78, 1989).

[0014] Creutzfeldt-Jakob disease is a rare human transmissible spongiform encephalopathy (TSE) neurodegenerative disorder in which the infectious agent is apparently an abnormal isoform of the prion protein. Individuals with Creutzfeldt-Jakob disease may deteriorate from seemingly perfect health to akinetic mutism within 6 months. Therefore, there is a risk of contracting prion-mediated diseases such as Creutzfeldt-Jakob disease when administering pharmaceutical compositions containing biologics such as botulinum toxin obtained using animal-derived products. When manufacturing pharmaceuticals using raw materials produced from animal-derived components, patients are at risk of being exposed to various pathogens or infectious agents.

[0015] Therefore, it has been reported that TSE infection can be prevented by adding casein hydrolysate (e.g., TSE-certified casein hydrolysate) to APF (animal protein-free) medium, thereby increasing the growth rate of the strain and the yield of botulinum toxin (Korean Patent No. 17-1729251, etc.). However, since the APF medium to which casein hydrolysate is added still contains casein as an animal-derived component, it is necessary to study how to achieve a high growth rate and high toxin yield of Clostridium botulinum strains in an APF medium that does not contain animal-derived components. Summary of the invention

[0016] Therefore, in order to prevent the risk of infection with the above-mentioned prion-mediated diseases, the present inventors have devoted themselves to developing a culture medium that contains plant-derived protein spores with no TSE infection risk and does not contain animal-derived components when culturing Clostridium botulinum strains, and confirmed that the culture medium can improve the growth rate and toxin yield of Clostridium botulinum strains compared with existing culture media, thereby completing the present invention.

[0017] The object of the present invention is to provide a culture medium composition for culturing Clostridium botulinum strains, which does not contain animal-derived components.

[0018] Another object of the present invention is to provide a method for producing botulinum toxin, which can increase the yield of botulinum toxin by culturing Clostridium botulinum in the above-mentioned culture medium composition.

[0019] In order to achieve the above-mentioned object, the present invention provides a culture medium composition for culturing Clostridium botulinum strains, comprising plant protein extract (including wheat protein extract and pea protein extract), D-(+)-glucose and yeast extract, and does not contain animal-derived components.

[0020] The present invention further provides a method for producing botulinum toxin, comprising (a) producing botulinum toxin by culturing Clostridium botulinum using the above-mentioned culture medium composition, and (b) recovering the produced botulinum toxin. Simple diagram description

[0021] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0022] FIG1 shows the custom design of JMP to confirm the appropriate ratio of plant protein extracts using three types of plant protein extracts, including pea protein extract, wheat protein extract, and protein extract from potato No. 2 as factors with an OD 540nm response of 3 or above;

[0023] FIG2 shows a design evaluation of the power analysis and design space score plots to confirm the custom design;

[0024] Figure 3 shows a culture medium comprising an APF culture medium composition;

[0025] FIG4A is a graph showing the results of measuring the growth rate of a Clostridium botulinum strain over time in an APF medium containing dextrin as a carbon source, and FIG4B is a graph showing the results of measuring the growth rate of a Clostridium botulinum strain over time in an APF medium containing glucose as a carbon source;

[0026] Figure 5 is a graph showing the measurement results of the growth rate of Clostridium botulinum strains over time in 16 APF media;

[0027] Figure 6 shows the distribution analysis of the D-optimal design experiment, confirming that the experimental results are uniformly distributed overall;

[0028] Figure 7 shows a scatter plot matrix analysis of the D-optimal design results, confirming that wheat protein and pea protein were weakly positively correlated with OD 540nm and toxin concentration;

[0029] Figure 8 shows the residual analysis, confirming that there are no outliers in the experimental results;

[0030] Figure 9 shows the process used to build the OD 540nm model, confirming that the model is fine because the VIF value does not exceed 10 and is at the level of 1, and all studentized residuals fall within the 95% confidence interval;

[0031] Figure 10 shows the prediction formula for OD 540nm;

[0032] Figure 11 shows the interaction curve at OD 540nm;

[0033] Figure 12 shows the process of building the OD 540nm model, confirming that there is no problem with the model because the VIF value does not exceed 10 and is at the level of 1, and all studentized residuals fall within the 95% confidence interval;

[0034] Figure 13 shows the prediction formula for toxin concentration;

[0035] Figure 14 shows the interaction curve;

[0036] FIG15 shows the process of finding the media conditions for OD 540nm and toxin concentration using a prediction profiler based on a prediction formula for each response;

[0037] 16A and 16B show the Y distribution in the specification (FIG. 16A) and the edge of failure analysis (FIG. 16B) after 5000 analogies of random variations of the factors and model noise using the simulator function;

[0038] Figure 17 shows the capability analysis of pea, where the range corresponding to 3σ is set as NOR, and the range corresponding to 4.5σ is set as PAR;

[0039] Figure 18 shows the capability analysis for wheat, where the range corresponding to 3σ is set as NOR and the range corresponding to 4.5σ is set as PAR;

[0040] FIG19 shows the contour profiler results for the APF medium, confirming that when NOR (3σ, red line) and PAR (4.5σ, blue line) are represented on the contour plot, they do not fall outside the white range corresponding to the range that satisfies the model;

[0041] Figure 20 shows the growth curve of botulinum strains in APF medium; and

[0042] FIG. 21 shows the extent of toxin expression in APF culture medium. Implementation

[0043] Although APF (animal protein free) medium compositions have also been shown to increase the growth rate of Clostridium botulinum strains compared to existing culture media, there is still a need to add medium components that can further increase the growth rate of the strain without causing infection problems such as TSE. Recently, the strain was allowed to grow and produce toxins by adding casein hydrolysate (e.g., TSE certified casein hydrolysate) to APF medium, and no cases of TSE infection have been reported so far.

[0044] However, casein hydrolysate is a component extracted from milk, and casein accounts for about 80% of the total protein contained in milk. Although the APF medium supplemented with casein hydrolysate does not contain animal-derived components such as fetal bovine serum (FBS), casein hydrolysate itself is an animal-derived component, and therefore a medium for culturing Clostridium botulinum strains that is very safe by containing plant protein without adding animal-derived components such as casein hydrolysate is needed.

[0045] Therefore, one aspect of the present invention relates to a culture medium composition for culturing a Clostridium botulinum strain, comprising plant protein extract, D-(+)-glucose and yeast extract, wherein the plant protein extract comprises wheat protein extract and pea protein extract, and does not contain animal-derived components. Here, the term "plant protein extract" refers to protein extract extracted from wheat or peas, and preferred examples thereof include but are not limited to commercially available Millipore TM96174, Millipore TM93492-500G-F, etc.

[0046] The term "existing medium" used herein refers to a medium containing casein hydrolysate as an animal-derived component, yeast extract, and thioglycolate medium. In addition, APF (animal protein-free) medium means that it does not contain animal-derived protein.

[0047] In one embodiment of the present invention, under TSE (transmissible spongiform encephalopathy)-free conditions, an APF medium containing various plant-based culture medium components and no animal-derived components was prepared, wherein the plant-based culture medium components included pea protein, wheat protein, protein from potato No. 2, corn steep solid, gluten from wheat, potato protein E210, banana powder, tomato juice and malt extract, and the growth rate and toxin expression degree of the strain were compared. Based on the results, when the plant-based culture medium components used to effectively culture Clostridium botulinum strains included plant protein (which included pea protein, wheat protein and protein from potato No. 2), it was confirmed that the strain growth and toxin expression degree were excellent. Therefore, as shown in Table 4, Table 5 and Figure 4, the plant protein contained in the culture medium composition for culturing Clostridium botulinum strains finally selected was determined to be pea protein, wheat protein and potato protein.

[0048] In another embodiment of the present invention, an APF medium containing plant protein (including pea protein, wheat protein and potato protein) without animal-derived components in various composition ratios under TSE-free conditions was prepared, and the strain growth rate and toxin expression degree were compared. Based on the results, when the culture medium composition for effectively culturing Clostridium botulinum strains includes at least one plant protein selected from the group consisting of wheat protein and pea protein and adding a carbon source (e.g., glucose) and a nitrogen source (e.g., yeast extract) thereto, it is confirmed that the strain growth and toxin expression degree are excellent. Therefore, as shown in Tables 8 to 12 and Figures 5 to 19, the plant protein contained in the culture medium composition finally selected for culturing Clostridium botulinum strains consists of 18 g / L pea protein and 7 g / L wheat protein.

[0049] In another embodiment of the present invention, an experiment was conducted to compare the growth pattern and toxin concentration of the Clostridium botulinum strain during growth in the APF medium containing the plant protein extract finally selected according to the present invention and the culture medium disclosed in Korean Patent Application Publication No. 10-2022-0140696 (Comparative Example 7) disclosed before the present invention. As shown in Tables 13 to 15 and Figures 20 and 21, in the culture medium of the present invention, after culturing the Clostridium botulinum strain for 28 hours, the OD 540nm was 7.1021, and then the OD value gradually decreased, and after 48 hours, the OD 540nm was 5.5636, and the toxin concentration in the supernatant of the Clostridium botulinum toxin after 48 hours of culturing was 588.8 μg (10 ml of culture fluid). On the other hand, in the culture medium of Comparative Example 7, as shown in Tables 13 to 15 and Figures 20 and 21, after culturing the Clostridium botulinum strain for 28 hours, the OD 540nm was 6.7739, and after culturing for 48 hours, the toxin concentration in the Clostridium botulinum toxin supernatant was 263.7 μg (10 ml of culture medium), indicating that the toxin expression level was reduced by 55.2% compared with the culture medium of the present invention.

[0050] In another embodiment of the present invention, an experiment was conducted to compare the growth pattern and toxin concentration of Clostridium botulinum strains when the culture medium components disclosed in Korean Patent Application Publication No. 10-2022-0140696, such as L-cysteine ​​hydrochloride monohydrate and / or medical defoamer C emulsion (Dow Corning®) were additionally included in the culture medium of the present invention (Comparative Examples 1 to 3). As shown in Tables 13 to 15 and Figures 20 and 21, Comparative Examples 1 to 3 had low OD 540nm values ​​compared to the present invention, and the toxin expression degree after 48 hours of culture was reduced by 14.9 to 76.6% compared to the present invention.

[0051] In another embodiment of the present invention, experiments were conducted to compare the growth pattern and toxin concentration of Clostridium botulinum strains when soy protein was added to the culture medium of the present invention instead of pea protein and / or wheat protein, and further including L-cysteine ​​hydrochloride monohydrate and medical defoamer C emulsion (Dow Corning®) (Comparative Examples 4 to 6). As shown in Tables 13 to 15 and Figures 20 and 21, Comparative Examples 4 to 6 had very low OD 540nm values ​​compared to the present invention, and the toxin expression level after 48 hours of culture was reduced by 87.0 to 94.9% compared to the present invention.

[0052] In the present invention, the culture medium composition for culturing the Clostridium botulinum strain may include at least one plant-derived component selected from the group consisting of pea peptone, wheat peptone, peptone from No. 2 potato, corn steep solids, gluten from wheat, potato peptone E210, banana powder, tomato juice and malt extract, and preferably includes pea peptone, wheat peptone and peptone from No. 2 potato, more preferably pea peptone and wheat peptone, but the present invention is not limited thereto.

[0053] The term "plant protein hydrolysate" or "plant hydrolysate" as used herein refers to a decomposition product of a protein obtained from a plant. For example, wheat protein hydrolysate (wheat hydrolysate) means a product obtained by decomposing a total protein obtained from wheat. In addition, "casein hydrolysate" means a decomposition product of casein.

[0054] The degradation of vegetable protein or casein is preferably carried out by partial digestion. The degradation of protein is preferably carried out by acid treatment, alkali treatment, enzyme treatment, high pressure treatment, heat treatment or physical treatment. More preferably, the vegetable protein or casein hydrolysate is characterized in that it has been subjected to enzyme treatment. Physical treatment is, for example, grinding.

[0055] The plant protein extract or casein hydrolyzate in the present invention is a partial decomposition product of protein, which is in the form of a mixture, and the mixture includes both small molecule amino acids and peptides composed of several to dozens of amino acids and complete protein molecules.

[0056] In the present invention, the amount of the plant protein extract may be 0.1 to 10 w / v% (1 to 100 g / L), such as 0.2 to 5 w / v% (2 to 50 g / L), such as 0.5 to 2 w / v% (5 to 20 g / L).

[0057] In the present invention, the amount of wheat peptone can be 0.1 to 4.0 w / v% (1 to 40 g / L), for example, 0.45 to 0.95 w / v% (4.5 to 9.5 g / L), for example, 0.48 to 0.92 w / v% (4.8 to 9.2 g / L), for example, 0.55 to 0.85 w / v% (5.5 to 8.5 g / L), for example, 0.7 w / v% (7 g / L).

[0058] In the present invention, the amount of pea protein extract can be 0.1 to 3.5 w / v% (1 to 35 g / L), for example, 1.55 to 2.05 w / v% (15.5 to 20.5 g / L), for example, 1.58 to 2.03 w / v% (15.8 to 20.3 g / L), for example, 1.65 to 1.95 w / v% (16.5 to 19.5 g / L), for example, 1.8 w / v% (18 g / L).

[0059] In the present invention, the culture medium composition for culturing Clostridium botulinum strains does not contain L-cysteine ​​hydrochloride monohydrate and / or a defoaming agent.

[0060] The term "defoaming agent" as used herein refers to a material that completely or partially prevents the formation of foam, and can be used interchangeably with "foaming inhibitor". The defoaming agent may include a medical defoaming agent or a defoaming agent from Dow Corning®, but is not limited thereto.

[0061] In the present invention, the culture medium composition for culturing the Clostridium botulinum strain may include a carbon source and a nitrogen source.

[0062] In the present invention, the carbon source can be a monosaccharide (such as glucose, fructose, etc.), a disaccharide (such as maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (such as dextrin, cyclodextrin, starch, etc.) or a sugar alcohol (such as xylitol, sorbitol, erythritol, etc.), preferably D-(+)-glucose, but not limited thereto.

[0063] In the present invention, the amount of the carbon source can be 0.1 to 1.5 w / v% (1 to 15 g / L), for example 0.25 to 1.5 w / v% (2.5 to 15 g / L), for example 1.0 w / v% (10 g / L), but is not limited thereto.

[0064] As used herein, "nitrogen source" refers to an organic or inorganic substance containing nitrogen atoms that is necessary for the synthesis of proteins, purines, pyrimidines and polysaccharide chitosan for the growth of the strain. The nitrogen source is preferably, but not limited to, yeast extract.

[0065] In the present invention, the nitrogen source can be yeast extract, polypeptone, trypsin, malt extract, corn steep liquor or soy protein, preferably yeast extract, but not limited thereto.

[0066] In the present invention, the amount of the nitrogen source can be 0.5 to 2.0 w / v% (5 to 20 g / L), for example 0.5 to 1.0 w / v% (5 to 10 g / L), for example 1.0 w / v% (10 g / L), but is not limited thereto.

[0067] Another aspect of the present invention relates to a method for producing botulinum toxin, comprising (a) producing botulinum toxin by culturing Clostridium botulinum using the above-mentioned culture medium composition, and (b) recovering the produced botulinum toxin.

[0068] In the present invention, the culturing can be carried out under anaerobic conditions, the culturing can be carried out at 33 to 37° C., the culturing can be carried out for 20 to 48 hours, and the toxin can be selected from the group consisting of botulinum toxin types A, B, C, D, E, F and G.

[0069] The present invention can be better understood through the following examples. These examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention, which is obvious to those with ordinary knowledge in the technical field to which the present invention belongs.

[0070] [Example] [1] [:The choice of plant protein]

[0071] [1-1] [:used for cultivation] [CYG] [Medium composition (control)]

[0072] To prepare CYG (casein, yeast extract, and glucose) medium for culturing Clostridium botulinum, 2% casein hydrolysate [20 g / L], 1% yeast extract [10 g / L], and 1% glucose [10 g / L] were dissolved in 90 ml of distilled water, the final volume was adjusted to 100 ml, and 10 ml of the resulting medium was dispensed into each 15 ml culture tube. Subsequently, the medium was autoclaved at 121°C for 30 minutes and then maintained under anaerobic conditions in an anaerobic incubator.

[0073] [1-2] [:used for cultivation] [APF] [Medium composition]

[0074] As shown in Tables 1 and 2 below, each of nine 2% plant protein extract candidates (Sigma TMC8160-500G, Millipore TM96174, Millipore TM93492-500G-F, Sigma-Aldrich TMG5004-500G, Millipore TM38143-500G, Organotechnie TM19425, Sigma-Aldrich TMB4032-500G, Sigma-Aldrich TM17218-500G, and Merck TM1.05391.0500) [10 g / L] was added to 1% yeast extract [10 g / L] and 1% dextrin or glucose [10 g / L], and then dissolved in 90 ml of water for injection, after which the final volume was adjusted to 100 ml, the pH of each medium was adjusted to 7.3, and 10 ml of the resulting medium was distributed to each 15 Subsequently, the culture medium was autoclaved at 121 °C for 30 min and then maintained under anaerobic conditions in an anaerobic incubator.

[0075] 〔Table 1〕 [category] [Culture medium (] [g / L] [)] [1] [2] [3] [4] [5] [6] [7] [8] [9] Carbon source dextrin 10 10 10 10 10 10 10 10 10 glucose Plant-derived ingredients Corn Steep Corn Steep Solids 10 pea Pea protein 10 Wheat Gluten Wheat Gluten 10 Gluten from wheat 10 potato Protein from No. 2 potatoes 10 Potato protein E210 10 banana Banana powder 10 tomato Tomato juice 10 malt Malt Extract 10 N Source Yeast Extract 10 10 10 10 10 10 10 10 10

[0076] 〔Table 2〕 [category] [Culture medium (] [g / L] [)]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18] Carbon source dextrin glucose 10 10 10 10 10 10 10 10 10 Plant-derived ingredients Corn Steep Corn Steep Solids 10 pea Pea protein 10 Wheat Gluten Wheat Gluten 10 Gluten from wheat 10 potato Protein from No. 2 potatoes 10 Potato protein E210 10 banana Banana powder 10 tomato Tomato juice 10 malt Malt Extract 10 N Source Yeast Extract 10 10 10 10 10 10 10 10 10

[0077] [1-3] [:Cultivation of Clostridium botulinum strains]

[0078] Each of the culture tubes containing the culture medium having the culture medium components 1 to 18 of Tables 1 and 2 was inoculated with 20 μl of Clostridium botulinum (Korea Center for Disease Control and Prevention Management Number: 4-029-CBB-MF-2020001), and then sampled by fixed culture at 35°C±1°C under anaerobic conditions for 20, 24, 28, and 48 hours. Thereafter, the growth rate was determined by measuring the OD 540nm value of the culture solution sampled over time using a UV spectrophotometer, with the blank being the culture medium. Based on the measured OD 540nm value, the plant protein candidate in the culture medium was selected by comparison with the control.

[0079] [1-4:] [Strain growth rate]

[0080] Among the plant protein extract candidates, corn steep solids, banana powder and gluten from wheat were excluded from the plant protein extract candidates because they formed sediments after the culture medium was sterilized, which made the subsequent filtration process and the measurement of accurate OD 540nm values ​​difficult. The culture medium components including the plant protein extract candidates are shown in Table 3 below.

[0081] 〔Table 3〕 [Culture medium (] [g / L] [)] [1] [2] [3] [4] [5] [6] [7] [8] [9]

[10]

[11]

[12] dextrin 10 10 10 10 10 10 glucose 10 10 10 10 10 10 pea Pea protein 10 10 Wheat Gluten Wheat Gluten 10 10 potato Protein from No. 2 potatoes 10 10 Potato protein E210 10 10 tomato Tomato juice 10 10 malt Malt Extract 10 10 Yeast Extract 10 10 10 10 10 10 10 10 10 10 10 10

[0082] The OD540nm values ​​of the culture broth sampled over time in the medium having the medium components 1 to 12 of Table 3 and two control mediums (CYG medium) were measured (Table 4), and a graph of the OD540nm values ​​over time in each medium containing a carbon source (dextrin or glucose) was compared (Figure 4).

[0083] 〔Table 4〕

[0084] As shown in Table 4 and Figure 4, media 1, 2, 3, 7, 8, and 9 using components derived from pea, wheat, and potato collectively exhibited higher strain growth rates.

[0085] Furthermore, even when the same plant protein extracts derived from pea, wheat, and potato were used, mediums 7, 8, and 9 using glucose as a carbon source showed high strain growth rates compared to mediums 1, 2, and 3 using dextrin. In particular, for the wheat-derived component, a growth trend was still observed 48 hours after the strain was inoculated in medium 2 using dextrin ( FIG. 4 ), which may unnecessarily delay the subsequent raw material production process, confirming that the use of glucose as a carbon source for the medium is appropriate.

[0086] [1-5] [:Botulinum toxin concentration]

[0087] The amount of toxin protein in culture medium 1, 2, 3, 7, 8 and 9 showing high strain growth rate in Examples 1-4 was measured. In conventional studies, the toxin is recovered during the death phase, so the results of quantitative detection of botulinum toxin type A culture fluid at 28 and 48 hours corresponding to the second half of the culture using ELISA are shown in the following Table 5 (ICH Guidelines, Q2 (R1)).

[0088] Antitoxin was diluted and dispensed into each well of a 96-well plate, and then coated for 16 hours or more. The contents of the plate were removed, each well was blocked by dispensing blocking solution, and the plate was washed three times with washing buffer. Standards and culture medium were diluted and dispensed into each well in triplicate, and then reacted, after which the plate was washed three times with washing buffer. Primary antibody dilution solution was dispensed into each well, and then reacted, and the plate was washed three times with washing buffer. Secondary antibody dilution solution was dispensed into each well, and then reacted, and the plate was washed three times with washing buffer. After the substrate solution was dispensed into each well and allowed to develop color, the reaction was stopped by dispensing stop solution into each well. The absorbance was measured at a wavelength of 450-540 nm.

[0089] 〔Table 5〕 [Culture medium] [plant] [ / ] [Animal-derived ingredients] [Carbon source] [Toxin concentration(] [ng / ml] [)]

[28] [Hour]

[48] [Hour] Control 1 (CYG) Casein glucose 17826.0 20007.7 1 pea dextrin 11716.8 6676.7 2 wheat 4294.4 5703.5 3 potato 14732.2 9945.9 7 pea glucose 20775.2 11772.9 8 wheat 5135.8 17415.1 9 potato 14440.7 16602.9

[0090] As shown in Table 5, the order of high toxin expression was pea, potato, and wheat. In addition, when the same plant protein was used, the toxin expression level in the medium using glucose as the carbon source was higher than that in the medium using dextrin, confirming that the toxin production of Clostridium botulinum strains in the medium using glucose as the carbon source was more appropriate.

[0091] Based on the experimental results of Example 1, three plant protein extracts were finally selected from the candidate plant protein extracts: pea protein extract, wheat protein extract and protein extract from potato No. 2.

[0092] [Example] [2] [:Determination of the ratio of plant protein to protein]

[0093] [2-1] [:Method for determining the ratio of plant protein to protein]

[0094] In order to determine the ratio of the three plant protein extract candidates finally selected in Examples 1-4 and 1-5, the response OD 540nm value was designed to be 3 or more using the custom design of JMP, and the ratio of the three plant protein extracts as factors was designed to be within 2%. In addition, as in Examples 1-1 and 1-2, the amount of carbon source and yeast extract was fixed to 1%, and the culture under each medium condition was repeated (Figure 1; protein extract 1: pea protein extract; protein extract 2: wheat protein extract; protein extract 3: potato protein extract).

[0095] For the design evaluation, the efficacy analysis and design space score plot were confirmed (Figure 2; Protein 1: pea protein; Protein 2: wheat protein; Protein 3: potato protein).

[0096] According to the ratio of plant protein extracts obtained through custom design, the experimental design of OD 540nm value is shown in Table 6 below, and the toxin concentration is measured after the cultivation under individual conditions and added to the analysis items (protein extract 1: pea protein extract; protein extract 2: wheat protein extract; protein extract 3: potato protein extract).

[0097] 〔Table 6〕

[0098] [2-2] [:used for cultivation] [APF] [Medium composition]

[0099] As shown in Table 7 below, each of the three 2% plant protein extract candidates [10 g / L] was added to 1% glucose [10 g / L] and 1% yeast extract [10 g / L] as a carbon source and then dissolved in 90 ml of water for injection, after which the final volume was adjusted to 100 ml, and 10 ml of the resulting medium was dispensed into each 15 ml culture tube. Subsequently, the culture medium was autoclaved at 121°C for 30 minutes and then maintained under anaerobic conditions in an anaerobic incubator.

[0100] 〔Table 7〕

[0101] [2-3] [:Cultivation of Clostridium botulinum strains]

[0102] Each of the culture tubes containing the culture medium with the culture medium components 1 to 16 of Table 7 was inoculated with 20 μl of Clostridium botulinum (Korea Center for Disease Control and Prevention Management Number: 4-029-CBB-MF-2020001) and then sampled by fixed culture for 20, 24, 28 and 48 hours under anaerobic conditions at 35°C±1°C. Thereafter, the growth rate was determined by measuring the OD 540nm value of the culture solution sampled over time using a UV spectrophotometer, with the blank being the culture medium. Based on the measured OD 540nm value, the ratio of the plant protein added to the culture medium was determined.

[0103] [2-4:] [Strain growth rate]

[0104] The OD 540 nm values ​​of the culture broths sampled over time in the culture media comprising the medium components 1 to 16 of Table 7 were measured (Table 8), and a graph of the OD 540 nm values ​​over time in each culture medium was compared ( FIG. 5 ).

[0105] 〔Table 8〕

[0106] [2-5] [:Experimental Design(] [DoE] [)Analysis of results]

[0107] In the culture medium containing the culture medium components 1 to 16 of Table 7, the OD 540nm value at 24 hours was used for analysis, which is the main strain culture time based on the conventional Clostridium botulinum research results, and because the toxin is recovered during the death stage, the ELISA measurement value at 48 hours was used as the toxin protein amount (Table 9), and on this basis, the results were statistically analyzed using JMP.

[0108] 〔Table 9〕

[0109] Through the analysis of the result distribution under the conditions in Table 9, it is determined that the experimental design is D optimal, and the overall distribution of the experimental results is uniform (Figure 6).

[0110] According to the scatter plot matrix analysis, all correlation values ​​were 0.75 or lower, indicating weak correlation. The R values ​​between the X variables (pea, wheat, potato) showed very weak correlation, close to 0, indicating no interaction, while in the Y variables (OD 540nm, toxin concentration), pea and Y variables as well as wheat and OD 540nm values ​​showed weak positive correlation (Figure 7), and outlier analysis confirmed that there were no outliers in the experimental results (Figure 8).

[0111] [2-6] [:Analysis based on the type of plant protein extract] [OD, 540nm , ] [value]

[0112] According to the type of plant protein extract, the fitting model was established based on the analysis results of OD 540nm value. The least square method was used to establish the model through the effect lever, and the response surface method was used to analyze the X variable. In order to select the best model, it is necessary to consider both the R square and RMSE values, and delete the insignificant items after measuring the effect summary and the p value of the effect test.

[0113] Based on the measurement results of the VIF value of the optimized model, the value did not exceed 10 and was at the level of 1, thus confirming that there was no problem with the model. In addition, in the residual analysis, the points were evenly distributed above and below, and all studentized residuals fell within the 95% confidence interval (Figure 9).

[0114] Based on the results of the through-effect test, the factors that had a great influence on the model were confirmed. Pea and wheat had an influence on the model (Table 10), and the prediction formula of OD 540nm was determined through the model (Figure 10). Based on the results of the main effect and interaction profiles, it was confirmed that there was an interaction between pea and potato, and between wheat and potato, but there was no interaction between the remaining factors (Figure 11).

[0115] 〔Table 10〕

[0116] [2-7] [:Analysis of toxin expression levels according to different plant protein extract types]

[0117] The fitting model was established based on the results of ELISA analysis. The least square method was used to establish the model through the effect lever, and the response surface method was used to analyze the X variable. In order to select the best model, it is necessary to consider both the R square and RMSE values, and delete the insignificant items after measuring the effect summary and the p value of the effect test.

[0118] Based on the measurement results of the VIF value of the optimized model, the value did not exceed 10 and was at the level of 1, thus confirming that there was no problem with the model. In addition, in the residual analysis, the points were evenly distributed above and below, and all studentized residuals fell within the 95% confidence interval (Figure 12).

[0119] Based on the results of the through-effect test to confirm the factors that have a great influence on the model, pea has a significant effect on the model (Table 11), and the prediction formula of OD 540nm is determined through the model (Figure 13). Based on the results of the main effect and interaction curves, it was confirmed that there was an interaction between pea and potato, and between wheat and potato, but there was no interaction between the remaining factors (Figure 14).

[0120] 〔Table 11〕

[0121] [2-8] [:Robust Optimization]

[0122] In order to determine the culture medium conditions that can obtain appropriate OD 540nm and maximum toxin concentration based on the prediction formula established for each response, the robust optimization conditions were confirmed using the profiler. In the actual culture process, for the OD 540nm value, cells that can maintain continuous growth in the exponential phase after subculture are selected. Therefore, since the minimum cell concentration OD 540nm of subculture in the exponential phase is 3 to 7, the OD 540nm value is set to match the target: 5 based on the established model formula to search for suitable conditions. For toxin concentration, the ELISA value is set to maximize to find the composition that produces the highest toxin concentration. According to the analysis of maximizing the desirability of the two responses, it is confirmed that only peas and wheat are used, and potatoes are excluded from subsequent analysis (Figure 15).

[0123] After simulating 5,000 random variations of the factors and model noise using the simulator function (Figure 16A), the simulation results are presented through failure boundary analysis. It was confirmed that all responses were within the specification limits except for one case out of 5,000 (Figure 16B).

[0124] In order to obtain the results under appropriate conditions, the acceptable range of each component was determined, and the range corresponding to 3σ was set as NOR (representing the range of 99.7% of the actual result distribution), and the range corresponding to 4.5σ was set as PAR (Figures 17 and 18). The allowable range of each component determined based on this is shown in Table 12 below.

[0125] 〔Table 12〕 [Component(] [g / L] [)] [Set point] [NOR] [(] [3σ] [)] [PAR] [(] [4.5σ] [)] Pea protein 18 g / L 16.5-19.5 15.8-20.3 Wheat Gluten 7 g / L 5.5-8.5 4.8-9.2

[0126] In order to facilitate operators during the actual process, the pea protein set point was determined to be 18 g / L and the wheat protein set point was 7 g / L, and the range that met the model was confirmed. When NOR (3σ, red line) and PAR (4.5σ, blue line) were expressed on the contour map, they did not fall out of the white range that met the process conditions. This confirmed that the NOR and PAR set by this test and statistical analysis were appropriate (Figure 19).

[0127] [Example] [3] [:] [APF] [Comparative measurement of culture medium absorbance and toxin expression level]

[0128] [3-1] [: Medium composition used for culture]

[0129] Pea gluten and wheat gluten were added to the APF medium of Clostridium botulinum in the amounts determined 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.

[0130] At the same time, in order to evaluate the effect of L-cysteine ​​hydrochloride monohydrate or antifoam emulsion on APF medium, 0.2 g / L of L-cysteine ​​hydrochloride monohydrate and / or 0.24 g / L of medical antifoam emulsion C (Dow Corning®) were added to APF medium. In addition, in order to evaluate the effect of soy protein on APF medium, 18 g / L of soy protein was added to APF medium instead of pea protein and / or wheat protein, and 0.2 g / L of L-cysteine ​​hydrochloride monohydrate and 0.24 g / L of medical antifoam emulsion C (Dow Corning®) were added.

[0131] 10 ml of the resulting medium was distributed to each 15 ml culture tube. Subsequently, the medium was autoclaved at 121°C for 30 minutes and then maintained under anaerobic conditions in an anaerobic incubator. The APF medium and specific components of the medium used for culturing Clostridium botulinum are shown in Table 13 below.

[0132] 〔Table 13〕 [Component] [(] [g / L] [)] [APF] [Culture medium] [Comparative Example] [1] [Comparative Example] [2] [Comparative Example] [3] [Comparative Example] [4] [Comparative Example] [5] [Comparative Example] [6] [Comparative Example] [7] [Pea protein]

[18] 18 18 18 - - - - [wheat] [Protein] [7] 7 7 7 - 7 7

[20] [Yeast Extract]

[10] 10 10 10 10 10 10

[20] [D-] [(] [+] [)] [-] [glucose]

[10] 10 10 10 10 10 10

[10] [Soybean protein] - - - - 18 18 18 [L-] [Cysteine] [HCl] - 0.2 - 0.2 - - 0.2 0.2 [Defoaming agent] [C] [Lotion] - - 0.24 0.24 - - 0.24 0.24

[0133] [3-2] [:Cultivation of Clostridium botulinum strains]

[0134] Each of the culture tubes containing the culture medium shown in Table 13 was inoculated with 20 μl of Clostridium botulinum (Korea Center for Disease Control and Prevention Management Number: 4-029-CBB-MF-2020001) and then sampled by stationary culture at 35°C±1°C under anaerobic conditions for 20, 24, 28, and 48 hours. Thereafter, the growth rate was determined by measuring the OD 540nm value of the culture solution sampled over time using a UV spectrophotometer, with the blank being the culture medium.

[0135] [3-3] [:Strain growth rate and toxin expression level]

[0136] The OD 540nm value of the culture broth of the medium in Table 13 was sampled over time, and the toxin expression level was measured after 48 hours of cultivation (Tables 14 and 15 and Figures 20 and 21).

[0137] 〔Table 14〕 [APF] [Culture medium] [Comparative Example] [1] [Comparative Example] [2] [Comparative Example] [3] [Comparative Example] [4] [Comparative Example] [5] [Comparative Example] [6] [Comparative Example] [7] OD at 20 hours 4.0474 3.7144 1.7721 3.9557 2.2205 2.8330 1.7535 3.7871 OD at 24 hours 5.9563 5.3636 2.0319 5.6034 2.9668 3.4903 2.7539 5.4549 OD at 28 hours 7.1021 [6.2576] 1.7567 [7.1667] 3.5252 4.2801 3.2109 6.7739 OD at 48 hours 4.5636 5.3044 1.5032 4.5062 4.4182 5.3889 3.7297 [7.8900] Toxin at 48 hours (μg) 588.8 452.1 137.9 501.1 75.2 76.4 30.1 263.7 Compared with control Toxin expression (%) - -23.2 -76.6 -14.9 -87.2 -87.0 -94.9 -55.2

[0138] 〔Table 15〕

[0139] As shown in Table 14 and Figures 20 and 21, when Clostridium botulinum was cultured in the APF medium of the present invention, it grew best at 20 to 28 hours in the APF medium, and the toxin expression level was the highest 48 hours after the strain grew.

[0140] 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 defoamer C emulsion was additionally added to the APF medium of the present invention, and Comparative Example 3 in which both L-cysteine ​​HCl monohydrate and medical defoamer C emulsion were added to the APF medium of the present invention, the strain growth rate was low and the respective toxin expression levels were reduced by 23.2%, 76.6% and 14.9% compared to the APF medium of the present invention. Therefore, it can be confirmed that even when L-cysteine ​​HCl monohydrate and / or medical defoamer C emulsion are additionally added to the APF medium composition of the present invention, the APF medium composition of the present invention is still excellent in terms of strain growth and toxin expression.

[0141] Meanwhile, in Comparative Example 4 in which soybean protein was added to the APF medium of the present invention instead of pea protein and wheat protein, Comparative Example 5 in which soybean protein was added to the APF medium of the present invention instead of pea protein, Comparative Example 6 in which soybean protein was added to the APF medium of the present invention instead of pea protein and L-cysteine ​​HCl monohydrate and medical defoamer C emulsion were additionally added, and Comparative Example 7 in which the same composition as that in Korean Patent Application Publication No. 10-2022-0140696 was applied, the strain growth rate was low and the respective toxin expression levels were reduced by 87.2%, 87.0%, 94.9% and 55.2% compared with the APF medium of the present invention. Therefore, considering that the time required for the death stage in which the toxin can be restored is expected to be 72 hours or more, it can be confirmed that the APF medium composition of the present invention is very excellent in terms of strain growth and toxin expression.

[0142] From the above, it can be seen that according to the present invention, when a Clostridium botulinum strain is cultured using a culture medium containing plant protein, glucose and yeast extract and free of animal-derived components, the growth rate of the strain is increased compared to the existing culture medium, and since there are no animal-derived components, the strain can be safely cultured, thereby increasing the yield of botulinum toxin.

[0143] none

Claims

1. A culture medium composition for culturing Clostridium botulinum strains, comprising essentially the following: plant protein, wherein the plant protein is 0.48 to 0.92 w / v% wheat protein and 1.58 to 2.03 w / v% pea protein; 1 w / v% D-(+)-glucose; and 1 w / v% yeast extract, wherein the culture medium composition does not contain animal-derived components, L-cysteine ​​salt monohydrate, and medical defoamer C emulsion.

2. The culture medium composition as described in claim 1, wherein, The animal-derived component is a casein hydrolysate.

3. The culture medium composition as described in claim 1, wherein, The amount of wheat protein is 0.55 to 0.85 w / v% and the amount of pea protein is 1.65 to 1.95 w / v.

4. A method for producing botulinum toxin, wherein, include: (a) Producing botulinum toxin by culturing Clostridium botulinum using a culture medium composition as described in any one of claims 1 to 3; (b) The botulinum toxin produced is recovered.

5. The method as described in claim 4, wherein, The culture was carried out under anaerobic conditions.

6. The method as described in claim 4, wherein, The culture was carried out at 33 to 37°C.

7. The method as described in claim 4, wherein, The culture lasts for 20 to 48 hours.

8. The method as described in claim 4, wherein, The toxins are selected from the group consisting of: botulinum toxins of types A, B, C, D, E, F, and G.

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