Sporulation method of <i>Bacillus </i>bacterium

Abstract

A method of producing Bacillus spores comprising a step of culturing the Bacillus bacterium using a liquid medium having a C/N ratio (weight ratio of carbon content to nitrogen content) of greater than 4 and less than 9.5.

Claims

1. A method for increasing a spore production in Bacillus simplex, the method comprising culturing Bacillus simplex with a liquid medium having a C/N ratio (weight ratio of carbon content to nitrogen content) of from 4.5 to less than 9.5, wherein a potassium content in the liquid medium is 1.9 g/L or less.

2. The method of claim 1, wherein the C/N ratio in the liquid medium is from 4.5 to 7.5.

3. The method of claim 1, wherein the C/N ratio in the liquid medium is from 6.0 to 7.5.

4. The method of claim 1, wherein the carbon content in the liquid medium is 50 g/L or less.

5. The method of claim 1, wherein the carbon content in the liquid medium is 25 g/L or less.

6. The method of claim 1, wherein the carbon and nitrogen sources contained in the liquid medium are carbon and nitrogen sources which can be utilized by Bacillus simplex.

7. The method of claim 6, wherein the carbon source which can be utilized by Bacillus simplex is one or more carbon sources selected from the group consisting of starch, glucose, lactose, glycerol, arabinose, ribose, xylose, galactose, fructose, mannose, inositol, mannitol, sorbitol, glucosamine, N-acetylglucosamine, cellobiose, maltose, sucrose, trehalose, xylitol, alcohols, organic acids, organic salts, and alkanes; and the nitrogen source which can be utilized by Bacillus simplex is one or more nitrogen sources selected from the group consisting of soybean-derived components, yeast-derived components, corn-derived components, animal and plant proteins and hydrolysates thereof, and ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium acetate, ammonia, sodium nitrate, potassium nitrate, sodium glutamate, urea and the like.

8. The method of claim 1, wherein the liquid medium contains KH.sub.2PO.sub.4.

9. The method of claim 1, wherein the liquid medium contains KH.sub.2PO.sub.4 in an amount of from 0.5 to 5.5 g/L.

10. The method of claim 1, wherein the liquid medium contains KH.sub.2PO.sub.4 in an amount of from 0.5 to 2.0 g/L.

11. The method according to claim 1, wherein the culturing is performed for 40 hours to 72 hours.

Description

EXAMPLES

(1) The present invention will be described in detail below with reference to Examples, but is not limited to the following Examples.

Example 1

(2) Evaluation of Bacillus simplex NBRC15720 Strain Using a 500 ml Erlenmeyer flask, each 100 ml of media containing glucose (Wako Pure Chemicals), defatted soy flour (Ajinomoto Healthy Supply), yeast extract (Difco), CSL (Corn Steep Liquor: ROQUETTE), peptone (Difco), and KH.sub.2PO.sub.4 (Wako Pure Chemicals) so that the final concentrations listed in Table 1 were achieved and further containing 100 ppm of MnCl.sub.2 (Wako Pure Chemicals), 400 ppm of NaCl (Wako Pure Chemicals), 250 ppm of MgCl.sub.2 (Wako Pure Chemicals), 75 ppm of CaCl.sub.2(Wako Pure Chemicals), and 0.3 ppm of FeSO.sub.4 (Wako Pure Chemicals) were prepared, and autoclave sterilization was carried out with a SILICOSEN (glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction).

(3) First, one loopful of Bacillus simplex NBRC15720 strain was taken from a colony grown on a nutrient agar plate, aseptically inoculated into the medium described in the medium condition 1 in Table 1 and cultured overnight with shaking at 37° C. and 150 rpm to obtain a preculture medium.

(4) Three milliliters from the obtained preculture medium was aseptically inoculated into various media described in Table 1 and cultured overnight with shaking at 37° C. and 150 rpm for 40 hours to 72 hours to obtain a culture medium.

(5) After cultivation, the bacterial cell concentration in the culture medium and the sporulation rate of the bacterial cells were measured using an optical microscope and a bacterial cell counter.

(6) Methods for measuring bacterial cell concentration, spore concentration and sporulation rate are as follows. The Bacillus simpler strain grown in the culture medium was diluted with, for example, sterile water containing 0.01% Tween 20, and then the bacterial cell concentration (vegetative cells and spores) and the spore concentration were counted with a bacterial cell counter. The sporulation rate was calculated by: spore concentration/bacterial cell concentration.

(7) The C/N ratio was calculated from the weight ratio of the carbon content to the nitrogen content in each medium component. C/N ratio=total carbon content in each media component/total nitrogen content in each media component.

(8) The carbon content in each medium component was calculated by determining the reducing sugar concentration by Somogyi method after hydrolysis in acid and subsequently multiplying the total sugar amount by 0.4.

(9) The nitrogen content in each medium component was determined by the Kjeldahl method.

(10) The potassium content in each medium component was determined by atomic absorption spectrophotometry (measurement wavelength: 766.5 nm).

(11) TABLE-US-00001 TABLE 1 Medium composition Medium g/L Defatted Yeast conditions Glucose soy flour extract CSL Peptone KH.sub.2PO.sub.4 1 4 4.0 1.6 0.8 1.6 2.0 2 10.0 4.0 1.6 0.8 1.6 2.0 3 9.0 4.0 1.6 0.8 1.6 2.0 4 8.0 4.0 1.6 0.8 1.6 2.0 5 6.0 4.0 1.6 0.8 1.6 2.0 6 5.5 4.0 1.6 0.8 1.6 2.0 7 1.5 4.0 1.6 0.8 1.6 2.0 8 0.0 4.0 1.6 0.8 1.6 2.0 9 4.0 4.0 1.6 0.8 1.6 5.5 10 4.0 4.0 1.6 0.8 1.6 1 11 4.0 4.0 1.6 0.8 1.6 0 12 10.0 10.0 4.0 2.0 4.0 5.0 13 10.0 10.0 4.0 2.0 4.0 1.0 14 0.5 4.0 1.6 0.8 1.6 6.4 15 10 4.0 1.6 0.8 1.6 6.4 16 10 4.0 1.6 0.8 1.6 0 17 4 2.5 3.0 2.0 0 1.0 18 4 6.0 0 0 0 1.0

(12) The results are shown in Table 2. At the C/N ratio of 9.5 or more, although growth of the bacterial cells was observed, a decrease in the sporulation rate was detected. On the other hand, at the C/N ratio of 4.0 or less, although growth of the bacterial cells was observed, a decrease in the sporulation rate was detected. The preferred potassium content was found to be in the range of 0.2 to 1.9 g/L.

(13) TABLE-US-00002 TABLE 2 Culture results for NBRC15720 strain Content of each component (g/L) and Culture result C/N ratio in medium Bacterial cell Sporulation Spore Medium Potassium C/N Carbon Nitrogen concentration rate concentration condition content ratio content content cell/ml % spore/ml 1 0.8 6.0 4.1 0.7 2.4E+09 75% 1.8E+09 2 0.8 9.5 6.5 0.7 1.6E+09  8% 1.3E+08 3 0.8 9.0 6.1 0.7 1.3E+09 89% 1.2E+09 4 0.8 8.4 5.7 0.7 1.6E+09 89% 1.5E+09 5 0.8 7.2 4.9 0.7 1.6E+09 67% 1.1E+09 6 0.8 6.9 4.7 0.7 1.8E+09 76% 1.4E+09 7 0.8 4.5 3.1 0.7 1.8E+09 70% 1.3E+09 8 0.8 3.7 2.5 0.7 1.1E+09 50% 5.4E+08 9 1.8 6.0 4.1 0.7 1.4E+09 97% 1.4E+09 10 0.5 6.0 4.1 0.7 2.0E+09 90% 1.8E+09 11 0.2 6.0 4.1 0.7 1.2E+09 94% 1.2E+09 12 1.9 6.0 10.2 1.7 2.2E+09 86% 1.9E+09 13 0.8 6.0 10.2 1.7 2.7E+09 82% 2.2E+09 14 2.0 4.0 2.7 0.7 1.3E+07  7% 9.4E+05 15 2.0 9.5 6.5 0.7 1.3E+07  0% 0.0E+00 16 0.2 9.5 6.5 0.7 1.2E+09 25% 3.1E+08 17 0.5 6.5 3.5 0.4 4.0E+09 68% 2.8E+09 18 0.4 7.5 3.5 0.5 1.5E+09 77% 1.2E+09

Example 2

(14) Evaluation of Bacillus simplex NBRC104473 Strain

(15) Using a 500 ml Erlenmeyer flask, each 100 ml of media containing glucose (Wako Pure Chemicals), defatted soy flour (Ajinomoto Healthy Supply), yeast extract (Difco), CSL (ROQUETTE), peptone (Difco), and KH.sub.2PO.sub.4 (Wako Pure Chemicals) so that the final concentrations of medium conditions 1 to 3 listed in Table 3 were achieved and each further containing 100 ppm of MnCl.sub.2 (Wako Pure Chemicals), 400 ppm of NaCl (Wako Pure Chemicals), 250 ppm of MgCl.sub.2 (Wako Pure Chemicals), 75 ppm of CaCl.sub.2 (Wako Pure Chemicals), and 0.3 ppm of FeSO.sub.4 (Wako Pure Chemicals) were prepared, and autoclave sterilization was carried out with a SILICOSEN (glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction).

(16) One loopful of Bacillus simplex NBRC104473 strain was taken from a colony grown on a nutrient agar plate, aseptically inoculated into the medium described in the medium condition 1 listed in Table 3 and cultured overnight with shaking at 37° C. and 150 rpm to obtain a preculture medium. Each 3 ml from the obtained preculture medium used for culturing the Bacillus simplex NBRC104473 strain was aseptically inoculated into each medium described in Table 3 and cultured overnight with shaking at 37° C. and 150 rpm for 40 hours to 72 hours to obtain a culture medium. After cultivation, the bacterial cell concentration in the culture medium and the sporulation rate of the bacterial cells were measured using an optical microscope and a bacterial cell counter.

(17) TABLE-US-00003 TABLE 3 Medium composition Medium g/L Defatted Yeast conditions Glucose soy flour extract CSL Peptone KH.sub.2PO.sub.4 1 4.0 4.0 1.6 0.8 1.6 2.0 2 0.5 4.0 1.6 0.8 1.6 6.4 3 10 4.0 1.6 0.8 1.6 6.4

(18) The results are shown in Table 4. NBRC104473 strain also showed the same tendency as in Example 1.

(19) TABLE-US-00004 TABLE 4 Culture results for NBRC104473 strain Content of each component (g/L) Culture result and C/N ratio in medium Bacterial cell Sporulation Spore Medium Potassium C/N Carbon Nitrogen concentration rate concentration condition content ratio content content cell/ml % spore/ml 1 0.77 6.01 4.09 0.68 1.2E+09 100% 1.2E+09 2 2.03 3.95 2.69 0.68 4.4E+07  0% 0.0E+00 3 2.03 9.54 6.49 0.68 3.2E+07  0% 0.0E+00

Example 3

(20) Evaluation in Jar Fermentation System

(21) Using a 5 L culture tank, each 2,000 ml of media containing glucose (Wako Pure Chemicals), defatted soy flour (Ajinomoto Healthy Supply), yeast extract (Difco), CSL (ROQUETTE), peptone (Difco), and KH.sub.2PO.sub.4 (Wako Pure Chemicals) so that the final concentrations of medium conditions 1 to 3 listed in Table 5 were achieved and each further containing 100 ppm of MnCl.sub.2 (Wako Pure Chemicals), 400 ppm of NaCl (Wako Pure Chemicals), 250 ppm of MgCl.sub.2 (Wako Pure Chemicals), 75 ppm of CaCl.sub.2 (Wako Pure Chemicals), and 0.3 ppm of FeSO.sub.4 (Wako Pure Chemicals) were prepared, and autoclave sterilization was carried out (glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction).

(22) One loopful of Bacillus simplex NBRC15720 was taken from a colony grown on a nutrient agar plate, aseptically inoculated into the medium of medium condition 1 described in Example 1 (Table 1) prepared in a 500 ml Erlenmeyer flask and cultured overnight with shaking at 37° C. and 150 rpm to obtain a preculture medium. Each 60 ml from the obtained preculture medium used for culturing the Bacillus simplex NBRC15720 strain was aseptically inoculated into each medium described in Table 5 and cultured overnight with aeration and agitation at 37° C. and 400 rpm for 40 hours to obtain a culture medium. After cultivation, the bacterial cell concentration in the culture medium and the sporulation rate of the bacterial cells were measured using an optical microscope and a bacterial cell counter.

(23) TABLE-US-00005 TABLE 5 Medium composition Medium g/L Defatted Yeast conditions Glucose soy flour extract CSL Peptone KH.sub.2PO.sub.4 1 10.0 10.0 12.0 8.0 0 1.0 2 20.0 16.0 12.0 8.0 0 1.0 3 35.0 20.0 12.0 8.0 0 0

(24) The results are shown in Table 6. As long as C/N is within a certain range, 50% or more of sporulation rate of the Bacillus simplex NBRC15720 strain was obtained even in a jar fermentation system.

(25) TABLE-US-00006 TABLE 6 Culture results for NBRC15720 strain (jar fermentation system) Content of each component (g/L) Culture result and C/N ratio in medium Bacterial cell Sporulation Spore Medium Potassium C/N Carbon Nitrogen concentration rate concentration condition content ratio content content cell/ml % spore/ml 1 1.24 5.41 11.67 2.16 9.0E+09 60% 5.4E+09 2 1.36 6.68 17.53 2.62 1.5E+10 53% 7.9E+09 3 1.15 8.45 24.76 2.93 1.8E+10 61% 1.1E+10

Example 4

(26) Evaluation of Bacillus siamensis in Jar Fermentation System

(27) Using a 5 L culture tank, each 2,000 ml of media containing glucose (Wako Pure Chemicals), defatted soy flour (Ajinomoto Healthy Supply), yeast extract (Difco), CSL (ROQUETTE), peptone (Difco), and KH.sub.2PO.sub.4 (Wako Pure Chemicals) so that the final concentrations of medium conditions 1 to 3 listed in Table 7 were achieved and each further containing 100 ppm of MnCl.sub.2 (Wako Pure Chemicals), 400 ppm of NaCl (Wako Pure Chemicals), 250 ppm of MgCl.sub.2 (Wako Pure Chemicals), 75 ppm of CaCl.sub.2 (Wako Pure Chemicals), and 0.3 ppm of FeSO.sub.4 (Wako Pure Chemicals) were prepared, and autoclave sterilization was carried out (glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction).

(28) One loopful of Bacillus siamensis was taken from a colony grown on a nutrient agar plate, aseptically inoculated into the medium of medium condition 1 described in Table 7 prepared in a 500 ml Erlenmeyer flask and cultured overnight with shaking at 37° C. and 150 rpm to obtain a preculture medium. Each 60 ml from the obtained preculture medium used for culturing the Bacillus siamensis was aseptically inoculated into various media described in Table 7 and cultured overnight with aeration and agitation at 37° C. and 400 rpm for 40 hours to obtain a culture medium. After cultivation, the bacterial cell concentration in the culture medium and the sporulation rate of the bacterial cells were measured using an optical microscope and a bacterial cell counter.

(29) TABLE-US-00007 TABLE 7 Medium composition Medium g/L Defatted Yeast conditions Glucose soy flour extract CSL Peptone KH.sub.2PO.sub.4 1 8.0 5.0 6.0 4.0 0 0.5 2 24.0 15.0 18.0 12.0 0 0.8 3 36.0 15.0 18.0 12.0 0 0.8

(30) The results are shown in Table 8. As long as C/N is within a certain range, 88% or more of sporulation rate of the Bacillus siamensis was obtained even in a jar fermentation system.

(31) TABLE-US-00008 TABLE 8 Culture results for Bacillus siamensis (jar fermentation system) Content of each component (g/L) Culture result and C/N ratio in medium Bacterial cell Sporulation Spore Medium Potassium C/N Carbon Nitrogen concentration rate concentration condition content ratio content content cell/ml % spore/ml 1 0.62 6.52 7.04 1.08 3.1E+09 93% 2.9E+09 2 1.64 6.52 21.11 3.24 6.8E+09 88% 6.0E+09 3 1.64 8.00 25.91 3.24 8.0E+10 95% 7.6E+10

Example 5

(32) Culture Results for Bacillus megaterium (Jar Fermentation System)

(33) Using a 5 L culture tank, each 2,000 ml of media containing glucose (Wako Pure Chemicals), defatted soy flour (Ajinomoto Healthy Supply), yeast extract (Difco), CSL (ROQUETTE), peptone (Difco), and KH.sub.2PO.sub.4 (Wako Pure Chemicals) so that the final concentrations of medium conditions 1 to 3 listed in Table 9 were achieved and each further containing 100 ppm of MnCl.sub.2 (Wako Pure Chemicals), 400 ppm of NaCl (Wako Pure Chemicals), 250 ppm of MgCl.sub.2 (Wako Pure Chemicals), 75 ppm of CaCl.sub.2 (Wako Pure Chemicals), and 0.3 ppm of FeSO.sub.4 (Wako Pure Chemicals) were prepared, and autoclave sterilization was carried out (glucose was separately sterilized and aseptically mixed in order to avoid Maillard reaction).

(34) One loopful of Bacillus megaterium was taken from a colony grown on a nutrient agar plate, aseptically inoculated into the medium of medium condition 1 described in Table 9 prepared in a 500 ml Erlenmeyer flask and cultured overnight with shaking at 37° C. and 150 rpm to obtain a preculture medium. Each 60 ml from the obtained preculture medium used for culturing the Bacillus megaterium was aseptically inoculated into various media described in Table 9 and cultured overnight with aeration and agitation at 37° C. and 400 rpm for 40 hours to obtain a culture medium. After cultivation, the bacterial cell concentration in the culture medium and the sporulation rate of the bacterial cells were measured using an optical microscope and a bacterial cell counter.

(35) TABLE-US-00009 TABLE 9 Medium composition Medium g/L Defatted Yeast conditions Glucose soy flour extract CSL Peptone KH.sub.2PO.sub.4 1 8.0 5.0 6.0 4.0 0 0.5 2 24.0 15.0 18.0 12.0 0 0.8 3 36.0 15.0 18.0 12.0 0 0.8

(36) The results are shown in Table 10. As long as C/N is within a certain range, 78% or more of sporulation rate of the Bacillus megaterium was obtained even in a bulk culture system.

(37) TABLE-US-00010 TABLE 10 Culture results for Bacillus megaterium (jar fermentation system) Content of each component (g/L) Culture result and C/N ratio in medium Bacterial cell Sporulation Spore Medium Potassium C/N Carbon Nitrogen concentration rate concentration condition content ratio content content cell/ml % spore/ml 1 0.62 6.52 7.04 1.08 2.7E+09 78% 2.1E+09 2 1.64 6.52 21.11 3.24 8.4E+09 95% 8.0E+09 3 1.64 8.00 25.91 3.24 9.2E+10 93% 8.6E+10