MEDIUM COMPOSITION FOR PREPARING BOTULINUM TOXIN

Abstract

The present invention relates to a medium composition for production of botulinum toxin and, more particularly, to a medium composition for culture of Clostridium sp. capable of producing botulinum toxin. The medium composition of the present invention comprises at least one plant-derived peptone selected from the group consisting of a garden pea hydrolysate, a cotton seed hydrolysate and a wheat gluten hydrolysate. When the medium according to the present invention, which contains plant-derived peptones and minerals, is used for culture of Clostridium botulinum, the growth rate of the bacterium in the medium is about 1.5-2 times higher than that in the medium that is in current use. In addition, when botulinum toxin is produced by culturing the bacterium in the medium, infection with transmissible spongiform encephalopathy (TSE) or the like can be prevented by blocking introduction of animal-derived components.

Claims

1. A medium composition for culture of Clostridium botulinum, the medium composition comprising: at least one plant-derived peptone selected from the group consisting of a garden pea hydrolysate, a cotton seed hydrolysate and a wheat gluten hydrolysate.

2. The medium composition of claim 1, wherein the plant-derived peptone is comprised with content of 0.1-10 w/v %.

3. The medium composition of claim 1, wherein the medium composition comprising the garden pea hydrolysate, the cotton seed hydrolysate and the wheat gluten hydrolysate in the medium composition with a ratio of 1:0.24-43.62:0.01-50.57 by weight, provided if the medium composition comprising the garden pea hydrolysate, the cotton seed hydrolysate and the wheat gluten hydrolysate.

4. The medium composition of claim 1, wherein the plant-derived peptone is subjected to an enzyme treatment.

5. The medium composition of claim 1, further comprising a carbon source, and at least one mineral selected from the group consisting of K.sub.2HPO.sub.4 (dipotassium phosphate), Na.sub.2HPO.sub.4 (disodium phosphate) and KH.sub.2PO.sub.4 (monopotassium phosphate).

6. The medium composition of claim 5, wherein the mineral is comprised in the medium composition with content of 0.05-3.5 w/v %.

7. A method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the medium composition of claim 1 to produce botulinum toxin; and (b) recovering the produced botulinum toxin.

8. The method of claim 7, wherein the culture is performed under anaerobic conditions.

9. The method of claim 7, wherein the botulinum toxin is selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F and G.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows the growth of Clostridium botulinum in a medium (APF medium) containing plant-derived peptones.

[0020] FIG. 2 shows the growth of Clostridium botulinum in a medium containing plant-derived peptones, minerals, amino acids and vitamins.

[0021] FIG. 3 shows the results of examining whether a precipitate is formed after sterilization of a medium containing plant-derived peptones, minerals, amino acids and vitamins.

[0022] FIG. 4 shows the results of examining whether a precipitate is formed after sterilization of a medium containing plant-derived peptones and minerals.

[0023] FIG. 5 shows the growth of Clostridium botulinum in media obtained by additionally adding vitamins, amino acids and “BD Recharge™ without Glucose and L-Glutamine” to media for culture of the bacterium, which contain plant-derived peptones and minerals.

[0024] FIG. 6 shows the growth of Clostridium botulinum in media for culture of the bacterium, which contain various kinds of plant-derived peptones.

[0025] FIG. 7 shows contour plots of FFD for mineral screening, and response optimization. FIG. 7a contour plot for high setting; FIG. 7b contour plot for middle setting; FIG. 7c contour plot for low setting; and FIG. 7d response optimization for maximum OD.

[0026] FIG. 8 shows contour plots of FFD for mineral screening, and response optimization. FIG. 8a contour plot for high setting; FIG. 8b contour plot for middle setting; FIG. 8c contour plot for low setting; and FIG. 8d response optimization for maximum OD.

[0027] FIG. 9 shows contour plots for plant peptone screening, and response optimization. FIG. 9a contour plot for middle setting; FIG. 9b contour plot for low setting; and FIG. 9c response optimization for maximum OD.

[0028] FIG. 10 shows the growth curve of Clostridium botulinum in the finally selected APF medium, and a change in toxin concentration.

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] In the present invention, it was attempted to prepare a medium that further increases the growth rate of Clostridium botulinum compared to a medium that is in current use (original medium) and have no risk of infection with TSE or the like. Thus, an animal protein-free (APF) medium containing plant-derived peptones were used, and the growth of a bacterium in the APF medium was examined. As a result, the APF medium showed an increased growth rate of the bacterium compared to a medium that is in current use. Thus, if the APF medium is used, a high concentration of botulinum toxin can be produced by culturing a bacterium in a safe manner under TSE-free conditions.

[0030] As used herein, the term “medium that is in current use or original medium” means a medium comprising casein hydrolysate, yeast extract and thioglycollate medium, which are animal-derived medium components. The term “APF medium (animal protein-free medium)” means a medium that contains no animal-derived protein and that contains plant-derived peptones, minerals and glucose.

[0031] In an example of the present invention, in order to produce botulinum toxin by culturing Clostridium botulinum under transmissible spongiform encephalopathy (TSE)-free conditions, an APF medium comprising TSE-free plant-derived peptone was prepared and compared with a medium that is in current use (containing an animal component). As a result, it could be seen that an optimal medium composition for culturing Clostridium botulinum is one comprising a plant-derived peptone, at least one mineral selected from the group consisting of KH.sub.2PO.sub.4, K.sub.2HPO.sub.4 and Na.sub.2HPO.sub.4, and a carbon source (e.g., glucose), and the optimal growth of the bacterium in this medium was found. As a result, as shown in Table 13, it was determined that the optimal contents of plant-derived peptones in the finally selected medium composition for culture of Clostridium botulinum are 5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cotton and 5 g/L HyPep™ 4601N, and the optimal contents of minerals in the medium composition are 5.5 g/L K.sub.2HPO.sub.4 and 3 g/L Na.sub.2HPO.sub.4.

[0032] In another example of the present invention, the growth pattern of Clostridium botulinum in the finally selected APF medium comprising plant-derived peptones and minerals, and the toxin concentration were measured. As a result, as shown in Table 12 and FIG. 10, the OD value started to increase after 12 hours of culture of Clostridium botulinum, and at 24 hours of culture, the culture medium showed an OD.sub.540 nm of 3.5465 and an OD.sub.600 nm of 3.0695. Then, the OD value decreased gradually, and at 48 hours of culture, the culture medium showed an OD.sub.540 nm of 0.792 and an OD.sub.600 nm of 0.7224. The toxin concentration in the culture supernatant of Clostridium botulinum started to increase after 5 hours of culture and showed a final value of 31.41 μg/ml. When the toxin concentration was measured after rupturing the bacterium, the toxin started to be produced after 5 hours of culture, and the toxin concentration continued to increase, was maintained at a uniform level after 28 hours of culture, and showed a final value of 38.39 μg/ml.

[0033] Based on this, in one aspect, the present invention is directed to a medium composition for culture of Clostridium botulinum, the medium composition comprising: at least one plant-derived peptone selected from the group consisting of a garden pea hydrolysate, a cotton seed hydrolysate and a wheat gluten hydrolysate.

[0034] As used herein, the term “plant-derived peptone” means a peptone extracted from garden pea, cotton seed or wheat gluten. Preferably, the plant-derived peptone may be commercially available Hy-Pea™ 7404, UltraPep™ Cotton, HyPep™ 7504 or HyPep™ 4601N, but is not limited thereto.

[0035] As used herein, the term “plant-derived peptone” or “plant-derived hydrolysate” means a product obtained by degrading a protein isolated from a plant. For example, the garden pea peptone (garden pea hydrolysate) means a product obtained by degrading a total protein isolated from garden pea.

[0036] Degradation of the plant-derived protein is preferably performed by partial digestion. Degradation of the protein is preferably performed by acid treatment, base treatment, enzyme treatment, high-pressure treatment, heat treatment or physical treatment. More preferably, the plant-derived peptone may be one obtained by enzyme treatment. The physical treatment is, for example, grinding.

[0037] The plant-derived peptone that is used in the present invention is a partial degradation product of plant-derived protein, is a mixture comprising not only amino acids that are single molecules, but also peptides composed of several to several tens of amino acids, and intact protein molecules.

[0038] In the present invention, the content of the plant-derived peptones in the 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).

[0039] In the present invention, the medium composition contains all the garden pea hydrolysate, the cotton seed hydrolysate and the wheat gluten hydrolysate, and the content ratio of the garden pea hydrolysate, the cotton seed hydrolysate and the wheat gluten hydrolysate in the 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, more preferably 1:1.6-2.4:0.6-1.4 by weight.

[0040] In the present invention, the medium composition for culture of Clostridium botulinum may further contains a carbon source and at least one mineral selected from the group consisting of K.sub.2HPO.sub.4 (dipotassium phosphate), Na.sub.2HPO.sub.4 (disodium phosphate) and KH.sub.2PO.sub.4 (monopotassium phosphate).

[0041] Herein, examples of the carbon source include, but are not limited to, monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, starch, etc.), sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.).

[0042] In the present invention, the content of the mineral in the 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).

[0043] In another aspect, the present invention is directed to a method for producing botulinum toxin, comprising the steps of: (a) culturing Clostridium botulinum using the above-described medium composition to produce botulinum toxin; and (b) recovering the produced botulinum toxin.

[0044] In the present invention, the culturing may be performed under anaerobic conditions, and the botulinum toxin may be selected from the group consisting of botulinum toxin types A, B, C, D, E, F and G.

Examples

[0045] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Example 1: Culture of Clostridium botulinum in Plant-Derived Peptone Medium

[0046] 1-1: Composition of a Medium Currently Used in Culture

[0047] The reagents and 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).

[0048] A medium that is in current use having a composition comprising 2% casein hydrolysate (20 g/L), 1% yeast extract (10 g/L), 1% glucose (10 g/L) and 0.5% thioglycollate medium (5 g/L) was used for the seed culture and main culture of Clostridium botulinum to produce botulinum toxin. 5 g of the thioglycollate medium per liter of the medium that is in current use is composed of 2.52 g of an enzymatic digest of casein, 0.84 g of yeast extract, 0.925 g of dextrose, 0.085 g of sodium thioglycollate, 0.42 g of NaCl, 0.085 g of L-cysteine, 0.00014 g of Resazurin and 0.125 g of bacteriological agar.

[0049] 1-2: Composition of APF Medium Used in Culture

[0050] A negative control medium was prepared by removing casein hydrolysate, yeast extract and thioglycollate medium from the medium that is in current use (original medium) for culture of Clostridium botulinum, and an animal protein-free (APF) medium was prepared by adding four plant-derived peptone candidates (Hy-Pea™ 7404, UltraPep™ Cotton, HyPep™ 7504, and HyPep™ 4601N) to the negative control medium (Table 1).

[0051] Table 1 shows the components of the plant-derived peptone-containing APF medium for culture of Clostridium botulinum in comparison with the medium that is in current use.

TABLE-US-00001 TABLE 1 Conc. medium that is Negative APF Components of Medium (g/L) in current use Control Medium Glucose 10 10 10 10 Sodium Chloride (NaCl) 0.42 0.42 0.42 0.42 Casein hydrolysate 20 20 — — Yeast extract 10 10 — — Thioglycollate medium 5 5 — — Hy-Pea ™ 7404 20 — — 20 UltraPep ™ Cotton 10 — — 10 HyPep ™ 7504 10 — — 10 HyPep ™ 4601N 10 — — 10

[0052] 1-3: Seed Culture of Clostridium botulinum

[0053] 20 μl of Clostridium botulinum (the Korean Centers for Disease Control and Prevention Accession No.: 4-029-CBB-IS-001) was inoculated into a culture tube containing 10 ml of a sterile medium having each of the compositions described in Examples 1-1 and 1-2 and was subjected to primary seed culture (stationary culture) at 35° C. for 22-30 hours under anaerobic conditions. When the growth of the bacterium in the primary seed culture was confirmed, 8 ml of the primary seed culture was inoculated into a 1-liter culture bottle containing 800 ml of a sterile medium having the same medium composition and was subjected to secondary seed culture (stationary culture) at 35° C. for 8-15 hours under anaerobic conditions.

[0054] 1-4: Main Culture of Clostridium botulinum

[0055] In order to produce a botulinum toxin by culturing Clostridium botulinum, the main culture of the bacterium was performed. Specifically, 9.3 L of a 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 of the medium. Nitrogen was supplied to make anaerobic conditions, and the growth of the bacterium was performed at a temperature of 35° C. and an agitation speed of 50 rpm.

[0056] The secondary seed culture in the 1-liter culture bottle in Example 1-3 was inoculated into a 10-liter incubator through an inoculation line connected to the inoculation port of the 10-liter incubator. Clostridium botulinum in the 10-liter incubator was cultured under the conditions of 35° C. and 50 rpm and the set culture conditions were monitored and recorded. When the bacterium was cultured for 100 hours or more, the main culture was terminated.

[0057] The growth of Clostridium botulinum in the animal protein-free (APF) medium prepared by adding four plant-derived peptone candidates (Hy-Pea™ 7404, UltraPep™ Cotton, HyPep™ 7504, and HyPep™ 4601N) to the negative control medium was compared with that of the bacterium in the negative control medium prepared by removing casein hydrolysate, yeast extract and thioglycollate medium from the medium that is in current use (original medium) (Table 1).

[0058] As a result, as shown in Table 1 and FIG. 1, Clostridium botulinum did not grow in the negative control medium, but started to grow in the original medium (medium that is in current use) at 24 hours after inoculation of the bacterium and started to grow in the plant-derived peptone-containing medium at 30 hours after inoculation of the bacterium.

Example 2: Culture of Clostridium botulinum in Medium Containing Plant-Derived Peptones, Minerals, Amino Acids and Vitamins

[0059] Because the growth of Clostridium botulinum in the medium prepared by adding four plant-derived peptones in Example 1 was slower than that in the original medium, solutions thereto were provided as follows.

[0060] 1) To examine the effect of thioglycollate functioning to make anaerobic conditions, thioglycollate was removed from the original medium (medium that is in current use), and a change in the growth rate of the bacterium in the thioglycollate-free medium was analyzed.

[0061] 2) Because the slower growth rate could be because of the lack of the nitrogen source, the peptone concentration in the medium used for culture of the bacterium was increased two times.

[0062] 3) The growth of Clostridium botulinum in a medium obtained by adding minerals, amino acids and vitamins to the plant-derived peptone-containing medium was compared with the growth of Clostridium botulinum in an APF medium disclosed in U.S. Pat. No. 8,012,716 (Allergan) (Table 2).

[0063] Table 2 shows the components of the medium for culture of Clostridium botulinum, which contains plant-derived peptones, minerals, amino acids and vitamins.

TABLE-US-00002 TABLE 2 medium 1 2 3 4 APF that is in (APF (APF (APF (APF Medium of current Medium Medium Medium Medium Allergan Components of Medium g/L use Candidate) Candidate) Candidate) Candidate) Company Glucose 10 10 10 10 10 10 15 Sodium Chloride (NaCl) 0.42 0.42 0.42 0.42 0.42 0.42 — Casein hydrolysate 20 20 20 — — — — Yeast extract 10 10 10 — — — 12 Thioglycollate medium 5 5 — — — — — Hy-Pea ™ 7404 20 — — 20 40 20 — UltraPep ™ Cotton 10 — — 10 20 10 — HyPep ™ 7504 10 — — 10 20 10 — HyPep ™ 4601N 10 — — 10 20 10 — KH.sub.2PO.sub.4 7 — — — — 7 — K.sub.2HPO.sub.4 5.5 — — — — 5.5 — Na.sub.2HPO.sub.4 5 — — — — 5 — MgSO.sub.47H.sub.2O 10 — — — — 10 — Vitamin Kit 100X — — — — 1X — Amino acid mixture 50X — — — — 1X — Soy peptone 32.5 — — — — — 32.5

[0064] As a result, as shown in Table 2 and FIG. 2, when the bacterium was cultured in the medium that is in current use without thioglycollate, the growth rate of the bacterium in the medium was slower than that in the thioglycollate-containing medium, indicating that thioglycollate influences the growth rate of the bacterium. When the peptone concentration in the medium was increased two times, the bacterium did not grow in the medium. When in the case in which mineral components, amino acids and vitamins were added to the peptone-containing medium, the growth rate of the bacaterium was similar to that in the medium that is in current use, but a precipitate was formed after sterilization of the medium. In addition, it was seen that the growth rate of the bacterium in the Allergan's APF medium was similar to that in the medium that is in current use.

Example 3: Production of Precipitate by Sterilization of Medium Containing Plant-Derived Peptones, Minerals, Amino Acid and Vitamin

[0065] In Example 2, it was observed that the growth rate of Clostridium botulinum in the medium containing plant-derived peptones, minerals, amino acids and vitamins, among the APF medium candidates 2 to 4 shown in Table 2, was similar that in the medium that is in current use. However, formation of a precipitate appeared after sterilization of the medium, and thus the cause thereof was examined (Table 3).

[0066] Table 3 shows the components of a medium for culture of Clostridium botulinum, which was used in sterilization and contain plant-derived peptones, minerals, amino acids and vitamins.

TABLE-US-00003 TABLE 3 medium 1 2 3 4 that is in (APF (APF (APF (APF current Medium Medium Medium Medium Components of Medium g/L use Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 Sodium Chloride (NaCl) 0.5 0.5 0.5 0.5 0.5 0.5 Casein hydrolysate 20 20 — — — — Yeast extract 10 10 — — — — Thioglycollate medium 5 5 — — — — Hy-Pea ™ 7404 20 — 20 20 20 20 UltraPep ™ Cotton 10 — 10 10 10 10 HyPep ™ 7504 10 — 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 KH.sub.2PO.sub.4 7 — 7 7 — — K.sub.2HPO.sub.4 5.5 — 5.5 5.5 — — Na.sub.2HPO.sub.4 5 — 5 5 — — MgSO.sub.47H.sub.2O 10 — 10 10 — — Vitamin Kit 100X — 1X — 1X 1X (Adding after Sterilization) Amino acid mixture 50X — 1X — 1X 1X (Adding after Sterilization)

[0067] As a result, as shown in Table 3 and FIG. 3, only in the case in which minerals were added to the plant-derived peptone-containing medium, a precipitate was formed after sterilization of the medium, indicating that the main cause of formation of the precipitate was the minerals. This is believed to be because the mineral components interacted with one another under the conditions of high temperature and high pressure during sterilization of the medium.

Example 4: Formation of Precipitate by Sterilization of Medium Containing Plant-Derived Peptones and Minerals

[0068] In order to identify the mineral components involved in the formation of precipitate caused by sterilization as confirmed in Example 3, various combinations of different components were added to media, followed by sterilization (Table 4).

[0069] Table 4 shows the components of media for culture of Clostridium botulinum, which contain plant-derived peptones and minerals, and the results of sterilization of the media.

TABLE-US-00004 TABLE 4 medium 1 2 3 4 5 that is in (APF (APF (APF (APF (APF Components of current Medium Medium Medium Medium Medium Medium g/L use Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein hydrolysate 20 20 — — — — — Yeast extract 10 10 — — — — — Thioglycollate 5 5 — — — — — medium Hy-Pea ™ 7404 20 — 20 20 20 20 20 UltraPep ™ Cotton 10 — 10 10 10 10 10 HyPep ™ 7504 10 — 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 10 KH.sub.2PO.sub.4 7 — — 7 — 7 7 K.sub.2HPO.sub.4 5.5 — — 5.5 5.5 — 5.5 Na.sub.2HPO.sub.4 5 — — 5 5 5 — MgSO.sub.47H.sub.2O 10 — — 10 10 10 10 precipitation, x o o o o aggregation 6 7 8 9 10 11 12 (APF (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Medium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 05 0.5 0.5 0.5 0.5 (NaCl) Casein hydrolysate — — — — — — — Yeast extract — — — — — — — Thioglycollate — — — — — — — medium Hy-Pea ™ 7404 20 20 20 20 20 20 20 UltraPep ™ Cotton 10 10 10 10 10 10 10 HyPep ™ 7504 10 10 10 10 10 10 10 HyPep ™ 4601N 10 10 10 10 10 10 10 KH.sub.2PO.sub.4 7 — 7 7 — — 10 K.sub.2HPO.sub.4 5.5 — — 5.5 5.5 5.5 — Na.sub.2HPO.sub.4 5 5 — — 5 — 5 MgSO.sub.47H.sub.2O — 10 10 — — 10 — precipitation, x o x x x o x aggregation

[0070] As a result, as shown in Table 4 and FIG. 4, among the media containing plant-derived peptones and minerals, the medium containing MgSO.sub.4.7H.sub.2O and K.sub.2HPO.sub.4 and the medium containing MgSO.sub.4.7H.sub.2O and Na.sub.2HPO.sub.4 formed a precipitate after sterilization.

Example 5: Culture of Clostridium botulinum Under Conditions in which No Precipitate is Formed in APF Medium

[0071] An experiment was performed to determine whether culture of Clostridium botulinum is possible when vitamins and amino acids are additionally added to the APF medium of Example 4 containing plant-derived peptones and minerals. In addition, an experiment was performed to examine whether culture of the bacterium is possible in a medium which is free of plant-derived peptone and mineral and contains vitamins, amino acids and/or “BD Recharge™ without Glucose and L-Glutamine” (Cat No. 670002, BD Bioscience) (a yeast extract-based medium component free of glucose and L-glutamine) (Table 5).

[0072] Table 5 shows the components of media obtained by additionally adding vitamins, amino acids and “BD Recharge™ without Glucose and L-Glutamine” to the medium for culture of Clostridium botulinum, which contains plant-derived peptones and minerals, and the growth rates of the bacterium in the media.

TABLE-US-00005 TABLE 5 medium 1 2 3 4 5 that is in (APF (APF (APF (APF (APF Components of current Medium Medium Medium Medium Medium Medium g/L use Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein 20 20 — — — — — hydrolysate Yeast extract 10 10 — — — — — Thioglycollate 5 5 — — — — — medium Sodium 1 — — — — — — thioglycollate Hy-Pea ™ 7404 20 — 20 20 20 20 20 UltraPep ™ 10 — 10 10 10 10 10 Cotton HyPep ™ 7504 10 — 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 10 KH.sub.2PO.sub.4 7 — — 7 7 7 — K.sub.2HPO.sub.4 5.5 — — 5.5 — 5.5 5.5 Na.sub.2HPO.sub.4 5 — — 5 — — 5 MgSO.sub.47H.sub.2O 10 — — — 10 — — Vitamin Kit 100X — — 1X 1X 1X 1X Amino acid — — 1X 1X 1X 1X mixture 50X w/o Glucose and 45.42 — — — — — — L-glutamine Growth x o x o o Details Growing Growing Growing in 24 hrs in 24 hrs in 24 hrs 6 7 8 9 10 11 12 (APF (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Medium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein — — — — — — — hydrolysate Yeast extract — — — — — — — Thioglycollate — — — — — — — medium Sodium — — — 1 — — — thioglycollate Hy-Pea ™ 7404 20 20 20 20 20 — — UltraPep ™ 10 10 10 10 10 — — Cotton HyPep ™ 7504 10 10 10 10 10 — — HyPep ™ 4601N 10 10 10 10 10 — — KH.sub.2PO.sub.4 7 — — — — — — K.sub.2HPO.sub.4 — — — — — — — Na.sub.2HPO.sub.4 5 — — — — — — MgSO.sub.47H.sub.2O — — — — — — — Vitamin Kit 100X 1X 1X — — 1X — 1X Amino acid 1X 1X — — 1X — 1X mixture 50X w/o Glucose and — — 45.42 — 45.42 45.42 45.42 L-glutamine Growth o x x x x x o Details Growing Growing in 24 hrs in 48 hrs

[0073] As a result, as shown in Table 5 and FIG. 5, only in the case in which the medium contained plant-derived peptones and a combination of two or more minerals of KH.sub.2PO.sub.4, K.sub.2HPO.sub.4 and Na.sub.2HPO.sub.4 and further contained vitamins and amino acids, Clostridium botulinum grew within 24 hours after inoculation of the bacterium. In addition, in the case in which the medium was free of plant-derived peptone and mineral and contained vitamins, amino acids and “BD Recharge™ without Glucose and L-Glutamine”, the bacterium grew within 48 hours after inoculation of the bacterium. In conclusion, the most suitable medium composition for culture of Clostridium botulinum comprises plant-derived peptones, KH.sub.2PO.sub.4, K.sub.2HPO.sub.4, Na.sub.2HPO.sub.4, amino acids and vitamins.

Example 6: Culture of Clostridium botulinum in Media Containing Different Plant-Derived Peptones

[0074] An experiment was performed to examine whether culture of Clostridium botulinum is possible when different combinations of plant-derived peptones are added to the APF medium of Example 5.

[0075] Table 6 shows the components of media for culture of Clostridium botulinum, which contain different plant-derived peptones, and the results of examining whether the bacterium grew in the media.

TABLE-US-00006 TABLE 6 medium 1 2 3 4 5 6 that is in (APF (APF (APF (APF (APF (APF Components of current Medium Medium Medium Medium Medium Medium Medium g/L use Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein 20 20 — — — — — — hydrolysate Yeast extract 10 10 — — — — — — Thioglycollate 5 5 — — — — — — medium Sodium 0.1 — — — — — — — thioglycollate Hy-Pea ™ 7404 10 — 10 10 — — — — UltraPep ™ 10 — 10 — 10 — — — Cotton HyPep ™ 7504 10 — 10 — — 10 — 10 HyPep ™ 4601N 10 — 10 — — — 10 10 KH.sub.2PO.sub.4 7 — 7 7 7 7 7 7 K.sub.2HPO.sub.4 5.5 — 5.5 5.5 5.5 5.5 5.5 5.5 Na.sub.2HPO.sub.4 5 — 5 5 5 5 5 5 MgSO.sub.47H.sub.2O 10 — — — — — — — Vitamin Kit 100X — — 1X 1X 1X 1X 1X Amino acid — — 1X 1X 1X 1X 1X mixture 50X w/o Glucose and 45.42 — — — — — — — L-glutamine Growth o o o o o o 7 8 9 10 11 12 13 (APF (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Medium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein — — — — — — — hydrolysate Yeast extract — — — — — — — Thioglycollate — — — — — — — medium Sodium — — — — — 0.1 — thioglycollate Hy-Pea ™ 7404 — — 10 10 10 — — UltraPep ™ 10 10 — — 10 — — Cotton HyPep ™ 7504 — 10 — 10 — — — HyPep ™ 4601N 10 — 10 — — — — KH.sub.2PO.sub.4 7 7 7 7 7 — 7 K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 — 5.5 Na.sub.2HPO.sub.4 5 5 5 5 5 — 5 MgSO.sub.47H.sub.2O — — — — — — — Vitamin Kit 100X 1X 1X 1X 1X 1X 1X 1X Amino acid 1X 1X 1X 1X 1X 1X 1X mixture 50X w/o Glucose and — — — — — 45.42 45.43 L-glutamine Growth o o o o o x x

[0076] As a result, as shown in Table 6 and FIG. 6, even when only or two of the four plant-derived peptones were added to the medium, culture of Clostridium botulinum was possible.

[0077] Taking the results of Examples 5 and 6 into account, it could be seen that at least one plant-derived peptone should be contained in the medium and that the plant-derived peptone cannot be substituted with “BD Recharge™ without Glucose and L-Glutamine” (Cat No. 670002, BD Bioscience) (a yeast extract-based medium component free of glucose and L-glutamine).

Example 7: Experiment for Selection of Two of Three Types of Minerals Contained in Medium

[0078] In Examples 1 to 7, it was determined that the APF medium composition used for culture of Clostridium botulinum comprises glucose, sodium chloride (NaCl), four plant-derived peptones, three minerals, amino acids, and vitamins. Among these medium components, medium components having no significant effect on the growth of the bacterium were removed to reduce the number of the medium components. Thus, it was judged that amino acids and vitamins have 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 three types of minerals, the bacterium was cultured using the medium compositions shown in Table 7, and the OD (540 nm and 600 nm) values at 24 hours and 48 hours after inoculation of the bacterium were measured and compared.

[0079] Table 7 shows the compositions of media resulting from the first-stage selection of minerals and the growth of Clostridium botulinum in the media.

TABLE-US-00007 TABLE 7 medium 1 2 3 4 5 that is in (APF (APF (APF (APF (APF Components of current Medium Medium Medium Medium Medium Medium g/L use Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein 20 20 — — — — — hydrolysate Yeast extract 10 10 — — — — — Thioglycollate 5 5 — — — — — medium Hy-Pea ™ 7404 10 — 10 10 10 10 10 UltraPep ™ 10 — 10 10 10 10 10 Cotton HyPep ™ 7504 10 — 10 10 10 10 10 HyPep ™ 4601N 10 — 10 10 10 10 10 KH.sub.2PO.sub.4 7 — — 7 — 7 — K.sub.2HPO.sub.4 5.5 — — — 5.5 5.5 — Na.sub.2HPO.sub.4 5 — — — — — 5 Culture 24 hr OD 540 nm 0.942 −0.017 −0.024 4.396 3.226 4.218 600 nm 0.780 −0.016 −0.020 3.832 2.691 3.593 Culture 48 hr OD 540 nm 2.459 −0.014 −0.019 4.716 5.220 3.502 600 nm 2.057 −0.015 −0.018 3.852 4.288 2.989 6 7 8 9 10 11 (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein — — — — — — hydrolysate Yeast extract — — — — — — Thioglycollate — — — — — — medium Hy-Pea ™ 7404 10 10 10 10 10 10 UltraPep ™ 10 10 10 10 10 10 Cotton HyPep ™ 7504 10 10 10 10 10 10 HyPep ™ 4601N 10 10 10 10 10 10 KH.sub.2PO.sub.4 7 — 7 3.5 3.5 3.5 K.sub.2HPO.sub.4 — 5.5 5.5 2.75 2.75 2.75 Na.sub.2HPO.sub.4 5 5 5 2.5 2.5 2.5 Culture 24 hr OD 3.214 4.964 3.991 3.951 3.938 3.594 2.680 4.304 3.351 3.341 3.335 3.036 Culture 48 hr OD 5.460 2.056 2.603 5.726 5.682 5.434 4.480 1.587 2.020 4.688 4.647 4.459

[0080] As a result, as shown in Table 7, at 24 hours after inoculation of the bacterium, the medium that is in current use showed an OD (540 nm) value of 0.942, and the APF medium containing K.sub.2HPO.sub.4 and Na.sub.2HPO.sub.4 showed the highest OD (540 nm) value of 4.964 among the APF media. In addition, at 48 hours after inoculation of the bacterium, the APF medium containing KH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 showed the highest OD value and active bacterial growth.

[0081] Meanwhile, as shown in FIG. 7a to FIG. 7d, contour plots of K.sub.2HPO.sub.4 and Na.sub.2HPO.sub.4 showing high main effects were drawn. As a result, as the concentrations of K.sub.2HPO.sub.4 and Na.sub.2HPO.sub.4 increased, the OD value increased. And Clostridium botulinum showed the highest growth when minerals were added to the medium at the concentrations of KH.sub.2PO.sub.4=0 g/L, K.sub.2HPO.sub.4=5.5 g/L, and Na.sub.2HPO.sub.4=5 g/L.

[0082] Meanwhile, in order to confirm the results of bacterial culture according to more precise addition of minerals, a second-stage experiment was performed using response surface methodology. Because the medium composition cannot have a negative value, the experiment was planned using a CCF (central composite faced) design and performed by culturing the bacterium in the medium compositions shown in Table 8. Then, the experimental results were combined with the results of the previously performed FFD and subjected to statistical analysis.

[0083] Table 8 shows the compositions of media obtained by the second-stage selection of minerals and the growth of Clostridium botulinum in the media.

TABLE-US-00008 TABLE 8 1 2 3 4 5 6 7 8 9 medium (APF (APF (APF (APF (APF (APF (APF (APF (APF that is in Components of Medium Medium Medium Medium Medium Medium Medium Medium Medium current Medium g/L Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) use Glucose 10 10 10 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 — (NaCl) Casein 20 — — — — — — — — — 20 hydrolysate Yeast extract 10 — — — — — — — — — 10 Thioglycollate 5 — — — — — — — — — 5 medium Hy-Pea ™ 7404 10 10 10 10 10 10 10 10 10 10 — UltraPep ™ 10 10 10 10 10 10 10 10 10 10 — Cotton HyPep ™ 7504 10 10 10 10 10 10 10 10 10 10 — HyPep ™ 4601N 10 10 10 10 10 10 10 10 10 10 — KH.sub.2PO.sub.4 7 — 7 3.5 3.5 3.5 3.5 3.5 3.5 3.5 — K.sub.2HPO.sub.4 5.5 2.75 2.75 — 5.5 2.75 2.75 2.75 2.75 2.75 — Na.sub.2HPO.sub.4 5 2.5 2.5 2.5 2.5 — 5 2.5 2.5 2.5 — OD 24 hr 540 nm 4.408 3.587 2.233 4.639 1.778 4.332 3.904 3.907 4.046 1.556 600 nm 3.836 3.086 1.896 4.068 1.503 3.777 3.366 3:368 3.505 1.307 OD 48 hr 540 nm 5.021 5.760 4.359 3.594 4.529 4.054 6.492 5.621 5.473 3.622 600 nm 4.284 4.925 3.695 3.049 3.832 3.457 5.603 4.830 4.677 3.062

[0084] Contour plots were drawn and used for comparison. As shown in FIGS. 8a to 8d, the OD value increased as the concentration of KH.sub.2PO.sub.4 decreased. When the optimal conditions were compared, the results were different from the results of FFD due to the curvature effect, and the value of K.sub.2HPO.sub.4 was the same, but the value of Na.sub.2HPO.sub.4 changed from 5 g/L to 3.1313 g/L. Thus, it was confirmed that the optimal mineral conditions of the medium by statistical analysis are 5.5 g/L K.sub.2HPO.sub.4 and 3 g/L Na.sub.2HPO.sub.4.

Example 8: Experiment for Selection of Plant-Derived Peptones Contained in Medium

[0085] As shown in Tables 9 and 10, plant-derived peptones were combined according to a mixture design, and the growth of Clostridium botulinum in a medium containing the combined plant-derived peptones was examined.

[0086] Table 9 shows the compositions of media obtained by the first-stage selection of plant-derived peptones and the growth of Clostridium botulinum using the media.

TABLE-US-00009 TABLE 9 1 2 3 4 5 6 (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium g/L Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (NaCl) Casein 20 — — — — — — hydrolysate Yeast extract 10 — — — — — — Thioglycollate 5 — — — — — medium Hy-Pea ™ 7404 10 5 10 5 5 — — UltraPep ™ 10 5 — 5 5 10 — Cotton HyPep ™ 7504 10 5 10 5 5 — 10 HyPep ™ 4601N 10 5 — 5 5 10 10 K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Na.sub.2HPO.sub.4 3 3 3 3 3 3 3 OD 24 hr 540 nm 3.541 2.440 3.345 3.305 3.317 2.852 600 nm 3.058 2.066 2.868 2.831 2.853 2.445 OD 48 hr 540 nm 0.811 0.935 0.731 0.799 1.400 0.777 600 nm 0.714 0.795 0.647 0.694 1.199 0.680 7 8 9 10 11 medium (APF (APF (APF (APF (APF that is in Components of Medium Medium Medium Medium Medium current Medium Candidate) Candidate) Candidate) Candidate) Candidate) use Glucose 10 10 10 10 10 10 Sodium Chloride 0.5 0.5 0.5 0.5 0.5 — (NaCl) Casein — — — — — 20 hydrolysate Yeast extract — — — — — 10 Thioglycollate — — — — — 5 medium Hy-Pea ™ 7404 6.667 6.667 — — 10 — UltraPep ™ 6.667 6.667 20 — 10 — Cotton HyPep ™ 7504 — 6.667 — 20 20 — HyPep ™ 4601N 6.667 — — — 20 — K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 — Na.sub.2HPO.sub.4 3 3 3 3 3 — OD 24 hr 3.695 2.772 2.353 1.688 4.842 2.239 3.183 2.376 2.014 1.419 4.245 1.893 OD 48 hr 1.660 1.090 1.810 1.402 2.093 3.341 1.403 0.929 1.548 1.210 1.764 2.812

[0087] Table 10 shows the compositions of media obtained by the second-stage selection of plant-derived peptones and the growth of Clostridium botulinum using the media.

TABLE-US-00010 TABLE 10 1 2 3 4 5 6 7 (APF (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Medium g/L Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 10 Sodium Chloride 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 (NaCl) Casein 20 — — — — — — — hydrolysate Yeast extract 10 — — — — — — — Thioglycollate 5 — — — — — — — medium Hy-Pea ™ 7404 10 5 5 — — 10 10 5 UltraPep ™ 10 5 5 10 6.667 10 — 5 Cotton HyPep ™ 7504 10 5 5 10 6.667 — — 5 HyPep ™ 4601N 10 5 5 — 6.667 — 10 5 K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Na.sub.2HPO.sub.4 3 3 3 3 3 3 3 3 OD 24 hr 540 nm 3.425 3.640 2.349 2.581 3.272 1.289 3.514 600 nm 2.969 3.159 2.029 2.244 1.096 1.096 3.032 OD 48 hr 540 nm 0.769 0.836 1.633 0.961 1.501 1.148 0.803 600 nm 0.675 0.732 1.420 0.854 1.270 0.982 0.698 8 9 10 11 12 13 medium (APF (APF (APF (APF (APF (APF that is in Components of Medium Medium Medium Medium Medium Medium current Medium Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) use Glucose 10 10 10 10 10 10 10 Sodium Chloride 0.05 0.05 0.05 0.05 0.05 0.05 — (NaCl) Casein — — — — — — 20 hydrolysate Yeast extract — — — — — — 10 Thioglycollate — — — — — — 5 medium Hy-Pea ™ 7404 20 — 6.667 10 10 10 — UltraPep ™ — — — 10 10 10 — Cotton HyPep ™ 7504 — — 6.667 10 10 10 — HyPep ™ 4601N — 20 6.667 10 10 10 — K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 5.5 — Na.sub.2HPO.sub.4 3 3 3 3 3 3 — OD 24 hr 0.776 1.257 3.457 5.376 5.235 4.809 2.208 0.649 1.098 2.950 4.689 4.534 4.246 1.863 OD 48 hr 0.880 1.278 0.962 1.986 1.994 2.010 3.185 0.744 1.124 0.818 1.708 1.710 1.717 2.708

[0088] As a result, as shown in FIGS. 9a to 9c, contour plots were drawn and used for analysis. It was determined that HyPep™ 7504 corresponding to the component C has the lowest effect on the growth of Clostridium botulinum. Based on this determination, HyPep™ 7504 was excluded from medium components. In conclusion, it was determined that the composition of the finally selected plant-derived peptones that are contained in the medium comprises 5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cotton and 5 g/L HyPep™ 4601N.

Example 9: Culture of Clostridium botulinum in Medium Containing or not Containing NaCl

[0089] The medium compositions used in Examples 1 to 8 contained a small amount (0.5 g/L) of NaCl. In order to examine the growth of Clostridium botulinum according to the concentration change of NaCl, the content of NaCl in the medium was adjusted to a range from 0 to 1 g/L, followed by culture of the bacterium in the medium.

[0090] Table 11 shows the components of NaCl-containing media for culture of Clostridium botulinum and the growth of Clostridium botulinum in the media.

TABLE-US-00011 TABLE 11 1 2 3 4 5 6 7 8 9 (APF (APF (APF (APF (APF (APF (APF (APF (APF Components of Medium Medium Medium Medium Medium Medium Medium Medium Medium Medium g/L Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Candidate) Glucose 10 10 10 10 10 10 10 10 10 10 Sodium Chloride 0.5 — — — 0.5 0.5 0.5 1 1 1 (NaCl) Hy-Pea ™ 7404 5 5 5 5 5 5 5 5 5 5 UltraPep ™ 10 10 10 10 10 10 10 10 10 10 Cotton HyPep ™ 4601N 5 5 5 5 5 5 5 5 5 5 K.sub.2HPO.sub.4 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 5.5 Na.sub.2HPO.sub.4 3 3 3 3 3 3 3 3 3 3 OD 24 hr 540 nm 2.166 2.154 2.151 2.148 2.115 2.120 2.145 2.147 2.140 600 nm 1.940 1.923 1.922 1.922 1.892 1.896 1.919 1.922 1.917

[0091] As a result, as shown in FIG. 11, there was no difference in the growth of the bacterium whether the medium contained NaCl or not. Thus, NaCl was excluded from the final APF medium components.

Example 10: Measurement of Growth Pattern of Clostridium botulinum in Finally Selected APF Medium and Toxin Concentration

[0092] Clostridium botulinum was inoculated into the finally selected Clostridium botulinum culture medium (10 g/L glucose, 5 g/L Hy-Pea™ 7404, 10 g/L UltraPep™ Cotton, 5 g/L HyPep™ 4601N, 5.5 g/L K.sub.2HPO.sub.4, and 3 g/L Na.sub.2HPO.sub.4) determined based on the results of Examples 1 to 9, and then the growth pattern of the bacterium and the toxin concentration were measured.

[0093] Table 12 shows the time-dependent OD value and the toxin concentration of Clostridium botulinum grown in the finally selected APF medium.

TABLE-US-00012 TABLE 12 Time of Total Toxin Conc. Culture OD Toxin Conc. in after rupturing (hr) 540 nm 600 nm Supernatant (μg/ml) strain (μg/ml) 0 0 0 0 0 6 0.0953 0.0393 0.00 0.00 9 0.0648 0.0525 0.00 0.00 12 0.5003 0.4411 0.00 0.00 14 1.1328 0.9958 2.18 2.04 16 1.6252 1.4484 4.64 10.22 18 2.3435 2.0215 6.77 18.15 20 2.777 2.4015 8.47 29.26 22 3.3485 2.896 9.46 31.86 24 3.5465 3.0695 — 31.73 28 3.452 2.982 — 37.31 36 2.5955 2.242 21.20 38.00 48 0.792 0.7224 31.41 38.39

[0094] As a result, as shown in Table 12 and FIG. 10, the OD value started to increase after 12 hours of culture of Clostridium botulinum, and at 24 hours of culture, the culture medium showed an OD.sub.540 nm of 3.5465 and an OD.sub.600 nm of 3.0695. Then, the OD value decreased gradually, and at 48 hours of culture, the culture medium showed an OD.sub.540 nm of 0.792 and an OD.sub.600 nm of 0.7224. The toxin concentration in the supernatant of Clostridium botulinum started to increase after 24 hours of culture and showed a final value of 31.41 μg/ml. When the toxin concentration was measured after rupturing the bacterium, the toxin started to be produced after 5 hours of culture, and the toxin concentration continued to increase, was maintained at a uniform level after 28 hours of culture, and showed a final value of 38.39 μg/ml.

[0095] In conclusion, the finally selected APF (animal protein-free medium) composition determined based on the results of Examples 1 to 10 is summerized in Table 13.

TABLE-US-00013 TABLE 13 Components of Medium g/L Carbon Source Glucose 10 Nitrogen Hy-Pea ™ 7404 5 Source UltraPep ™ Cotton 10 (Vegetable HyPep ™ 4601N 5 Peptone) Mineral K.sub.2HPO.sub.4 5.5 Na.sub.2HPO.sub.4 3

INDUSTRIAL APPLICABILITY

[0096] As described above, when the medium according to the present invention, which contains plant-derived peptones and minerals, is used for culture of Clostridium botulinum, the growth rate of the bacterium in the medium is about 1.5-2 times higher than that in the medium that is in current use. In addition, when botulinum toxin is produced by culturing the bacterium in the medium, infection with transmissible spongiform encephalopathy (TSE) or the like can be prevented by blocking introduction of animal-derived components.

[0097] Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.