Method for producing spores of bacteria belonging to genus bacillus

Abstract

Disclosed is a method for producing spores of a bacterium belonging to the genus Bacillus, which comprises a step for culturing a bacterium belonging to the genus Bacillus using a liquid culture medium having a C / N ratio (the weight ratio of carbon content to nitrogen content) of more than 4 and less than 9.5.

Description

[0001] This application is a divisional application of the patent application with international application number PCT / JP2016 / 061604, international filing date of April 8, 2016, Chinese application number 201680020658.6, and invention title of "Method for producing spores of bacteria of the genus Bacillus". TECHNICAL FIELD

[0002] The present application relates to a method for efficiently producing spores of bacteria of the genus Bacillus. BACKGROUND

[0003] Bacteria of the genus Bacillus are used in various fields such as production of enzymes or useful substances, production of fermented foods, decomposition of organic matter, microbial pesticides or microbial fertilizers, and the like. In the case of using such microbial pesticides, microbial fertilizers, and the like, spores of bacteria of the genus Bacillus are generally used. However, even if a strain that exhibits excellent performance in such use is available, it is difficult to commercialize if spores cannot be efficiently formed.

[0004] As a medium that is frequently used in liquid culture of bacteria of the genus Bacillus, Nutrient Broth (DIFCO), Luria Bertani broth, Trypticase Soy Broth (Becton Dickinson), and the like can be given, however, these media do not provide sufficient proliferative ability, and there are cases where spore formation is not observed at all.

[0005] In Patent Literature 1, a method for forming spores by performing culture including a process of reducing the dissolved oxygen concentration after proliferation is given, however, in some bacteria of the genus Bacillus, it is difficult to efficiently form spores even if the same method is used. In addition, since the stirring and aeration conditions need to be adjusted in the culture process in this method, the manufacturing process can become complicated.

[0006] In Patent Literature 2, a method for forming spores by continuing culture for a long time after the carbon source is consumed is given, however, since the culture time is long, the cost of culture increases, and it is not suitable for practical production. In addition, in some bacteria of the genus Bacillus, it is difficult to form spores even if the same method is used.

[0007] In Patent Literature 3, a method for producing spores by regulating the oxygen supply amount, the range of stirring speed, and the like by the range of the phosphate concentration of the culture solution and the culture conditions is given, however, in some bacteria of the genus Bacillus, it is difficult to efficiently form spores even if the same method is used. In addition, in the implementation, it is necessary to use a culture device that can achieve the prescribed culture conditions.

[0008] Prior Art Documents

[0009] Patent Literature

[0010] Patent Literature 1: Japanese Patent Application Laid-Open (JP-A) No. 2007-236286

[0011] Patent Literature 2: Japanese Patent Application Laid-Open (JP-A) No. 2000-217567

[0012] Patent Literature 3: Japanese Patent Application Laid-Open (JP-A) No. 2007-195542 SUMMARY

[0013] PROBLEMS TO BE SOLVED BY THE INVENTION

[0014] An object of the present application is to provide a culture method which can effectively produce spores for Bacillus bacteria for which it is difficult to form spores with a general liquid medium for bacteria.

[0015] METHOD FOR SOLVING THE PROBLEM

[0016] The present inventors have conducted intensive studies in order to solve the above problem, and as a result, have found a liquid medium composition which, for Bacillus bacteria for which the production efficiency of spores is not sufficient in culture using a general liquid medium for bacteria, also effectively proliferates the bacterial cells and is suitable for the formation of spores, thereby completing the present application.

[0017] The present application is as follows.

[0018] [1] A method for producing spores of Bacillus bacteria, the method comprising a step of culturing Bacillus bacteria using a liquid medium having a C / N ratio (weight ratio of carbon content to nitrogen content) of greater than 4.0 and less than 9.5.

[0019] [2] The method for producing spores of Bacillus bacteria according to [1], wherein the C / N ratio of the liquid medium used in the culture is 4.5 or greater and less than 9.5.

[0020] [3] The method for producing spores of Bacillus bacteria according to [1], wherein the C / N ratio of the liquid medium used in the culture is 4.5 or greater and 7.5 or less.

[0021] [4] The method for producing spores of Bacillus bacteria according to [1], wherein the C / N ratio of the liquid medium used in the culture is 6.0 or greater and 7.5 or less.

[0022] [5] The method for producing spores of Bacillus bacteria according to any one of [1] to [4], wherein the carbon content in the liquid medium is 50 g / L or less.

[0023] [6] The method for producing spores of Bacillus bacteria according to any one of [1] to [4], wherein the carbon content in the liquid medium is 25 g / L or less.

[0024] [7] The method for producing spores of Bacillus bacteria according to any one of [1] to [6], wherein the potassium content in the liquid medium is less than 2.0 g / L.

[0025] [8] The method for producing spores of Bacillus bacteria according to any one of [1] to [6], wherein the potassium content in the liquid medium is 1.9 g / L or less.

[0026] [9] The method for producing spores of Bacillus bacteria according to any one of [1] to [8], wherein the carbon source and the nitrogen source contained in the liquid medium are carbon and nitrogen sources that can be assimilated by the Bacillus bacteria.

[0027]

[10] The method for producing spores of Bacillus bacteria according to [9], wherein the carbon source that can be assimilated by the Bacillus bacteria 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, an alcohol, an organic acid, a salt of an organic acid, and an alkane, and the nitrogen source that can be assimilated by the Bacillus bacteria is one or more nitrogen sources selected from the group consisting of components derived from soybeans, components derived from yeast, components derived from corn, animal and plant proteins and their decomposed products, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium acetate, and other ammonium salts, ammonia, sodium nitrate, potassium nitrate, sodium glutamate, urea, and the like.

[0028]

[11] The method for producing spores of a Bacillus bacterium according to any one of [1] to

[10] , wherein the Bacillus bacterium is Bacillus simplex, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus megaterium, Bacillus thuringiensis, Bacillus popilliae, Bacillus cereus, Bacillus licheniformis, Bacillus firmus, Bacillus velezensis, Bacillus stearothermophilus, Bacillus pichinotyi, Bacillus acidocaldarius, Bacillus alcalophilus, Bacillus alkalicola, Bacillus coagulans, Bacillus azotoformans, Bacillus anthracis, Bacillus siamensis, Bacillus badius, Bacillus bataviensis, Bacillus brevis, Bacillus cycloheptanicus, Bacillus circulans, Bacillus aneurinilyticus, Bacillus migulanus, Bacillus abyssalis, Bacillus a estuarii, Bacillus polymyxa, or Bacillus sp.

[0029]

[12] The method for producing spores of the Bacillus bacteria according to any one of [1] to

[10] , wherein the Bacillus bacteria is Bacillus siamensis, Bacillus simplex, or Bacillus megaterium.

[0030] Effects of the Invention

[0031] According to the present application, stable proliferation of the Bacillus bacteria, spore formation, and proliferation at a higher concentration can be observed, and spores can be formed at a high ratio. DETAILED DESCRIPTION

[0032] In the present application, as the Bacillus bacteria, there is no particular limitation as long as it is a bacterium classified as Bacillus, and, for example, Bacillus simplex, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus megaterium, Bacillus thuringiensis, Bacillus popilliae, Bacillus cereus, Bacillus licheniformis, Bacillus firmus, Bacillus velezensis, Bacillus stearothermophilus, Bacillus pichinotyi, Bacillus acidocaldarius, Bacillus alcalophilus, Bacillus alkalicola, Bacillus coagulans, Bacillus azotoformans, Bacillus anthracis, Bacillus siamensis, Bacillus badius, Bacillus bataviensis, Bacillus brevis, Bacillus cycloheptanicus, Bacillus circulans, Bacillus aneurinilyticus, Bacillus migulanus, Bacillus abyssalis, Bacillus aestuarii, Bacillus polymyxa, or Bacillus sp. can be cited.

[0033] Among them, Bacillus simplex, Bacillus siamensis or Bacillus megaterium is preferred.

[0034] In the present application, a liquid medium having a C / N ratio (weight ratio of carbon content to nitrogen content) of more than 4.0 and less than 9.5 is used in the culture. Here, the C / N ratio is preferably 4.5 or more and less than 9.5, more preferably 4.5 or more and 7.5 or less, and further preferably 6.0 or more and 7.5 or less.

[0035] The C / N ratio is calculated as shown below.

[0036] C / N ratio = total of carbon content contained in each medium component ÷ total of nitrogen content contained in each medium component.

[0037] In the liquid medium used in the present application, the carbon content is preferably 50 g / L or less, and more preferably 25 g / L or less. On the other hand, the carbon content is preferably 3 g / L or more.

[0038] The carbon source and nitrogen source of the liquid medium used in the culture can use a carbon source and nitrogen source that can be dissimilated by Bacillus bacteria. As a carbon source that can be dissimilated, a sugar (starch, glucose, lactose, glycerol, arabinose, ribose, xylose, galactose, fructose, mannose, inositol, mannitol, sorbitol, glucosamine, N-acetylglucosamine, cellobiose, maltose, sucrose, trehalose, xylitol, etc.) that can be dissimilated by Bacillus bacteria, an alcohol, an organic acid, an organic acid salt, an alkane, or other general carbon sources can be exemplified, and as a nitrogen source that can be dissimilated, a component from soybean, a component from yeast, a component from corn, an animal and plant protein and its decomposition product, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium acetate, etc., ammonia, sodium nitrate, potassium nitrate, sodium glutamate, urea, etc. can be exemplified.

[0039] In addition, in the liquid medium used in the present application, in order to achieve a higher sporulation rate, the potassium content is preferably less than 2 g / L, and more preferably 1.9 g / L or less. The potassium content is preferably 0.2 g / L or more. As a source of potassium, at least one or more selected from a component from soybean, a component from yeast, a component from corn, an animal and plant protein and its decomposition product, KH2PO4, K2HPO4, KCl, etc. is used for the culture.

[0040] As other medium components, a trace metal salt or the like that is generally used in the culture of Bacillus bacteria can be added without adversely affecting sporulation, and if necessary, an amino acid or a vitamin or the like can be added.

[0041] As the culture conditions, those used in general liquid culture of Bacillus bacteria can be used, for example, conditions of 20 to 40°C, under aerobic conditions (e.g., oxygen concentration of 15 to 50%), with stirring, for 10 to 100 hours. The pH of the culture medium is preferably 6.5 to 8.5, more preferably 7.0 to 8.0.

[0042] Note that, prior to the culture with the liquid culture medium having the above C / N ratio, a preculture can be performed.

[0043] By so doing, a bacterial cell of Bacillus bacteria having a high sporulation rate (e.g., 50% or more, preferably 80% or more) can be obtained. The bacterial cell of Bacillus bacteria having a high sporulation rate can be used for the intended purpose after appropriate concentration or removal of the culture medium, drying, and the like.

[0044] [Example]

[0045] Hereinafter, the present application will be specifically described by way of examples, but the present application is not limited to the following examples.

[0046] [Example 1]

[0047] Evaluation of Bacillus simplex NBRC 15720 strain

[0048] Using 500 ml Erlenmeyer flasks, 100 ml each of a culture medium containing glucose (Wako Pure Chemical), defatted soybean powder (Ajinomoto HEALTHY SUPPLY), yeast extract (Difco), CSL (Corn Steep Liquor: ROQUETTE), peptone (Difco), KH2PO4 (Wako Pure Chemical), further containing 100 ppm of MnCl2 (Wako Pure Chemical), 400 ppm of NaCl (Wako Pure Chemical), 250 ppm of MgCl2 (Wako Pure Chemical), 75 ppm of CaCl2 (Wako Pure Chemical), and 0.3 ppm of FeSO4 (Wako Pure Chemical) were prepared so as to achieve the final concentrations described in Table 1, and autoclave sterilization was performed with a silicone plug (in order to avoid the Maillard reaction, the glucose was mixed aseptically after separate sterilization).

[0049] First, 1 platinum ear was taken from a colony of Bacillus simplex NBRC 15720 strain grown on a general agar plate medium, and aseptically inoculated into the culture medium described in Medium Condition 1 of Table 1, and subjected to overnight shaking culture at 37°C at 150 rpm to obtain a preculture.

[0050] From the resulting pre-culture solution, 3 ml was aseptically inoculated into each of the various media described in Table 1, and subjected to shaking culture at 37°C at 150 rpm for 40 to 72 hours to obtain a culture solution.

[0051] After the culture, the concentration of the bacterial cells and the sporulation rate of the bacterial cells in the culture solution were measured using an optical microscope and a bacterial cell counter.

[0052] The measurement method of the concentration of the bacterial cells, the concentration of the spores, and the sporulation rate is shown below.

[0053] The simple Bacillus proliferated in the culture solution was diluted with sterilized water, 0.01% Tween 20 solution, etc., and the concentration of the bacterial cells (vegetative cells and spores) and the concentration of the spores were counted using a bacterial cell counter. The sporulation rate was calculated as the concentration of the spores ÷ the concentration of the bacterial cells.

[0054] The C / N ratio was calculated from the weight ratio of the carbon content to the nitrogen content contained in each medium component.

[0055] C / N ratio = total of the carbon content contained in each medium component ÷ total of the nitrogen content contained in each medium component.

[0056] The measurement of the carbon content of each medium component was performed by quantifying the concentration of reducing sugars after hydrolysis in acid using the Somogy method, and then multiplying the total sugar amount by 0.4.

[0057] The measurement of the nitrogen content of each medium component was performed using the Kjeldahl method.

[0058] The measurement of the potassium content contained in each medium component was performed using atomic absorption spectrophotometry (measurement wavelength 766.5 nm).

[0059]

[0060] The results are shown in Table 2. Under conditions where the C / N ratio was 9.5 or more, proliferation of the bacterial cells was observed, but a decrease in the sporulation rate was confirmed. On the other hand, under conditions where the C / N ratio was 4.0 or less, proliferation of the bacterial cells was observed, but a decrease in the sporulation rate was confirmed. In addition, it was found that the potassium content was preferably in the range of 0.2 to 1.9 g / L.

[0061]

[0062] [Example 2]

[0063] Evaluation of the simple Bacillus NBRC 104473 strain

[0064] Using 500 ml Erlenmeyer flasks, each 100 ml of a medium containing glucose (and pure chemical industries), defatted soybean powder (Ajinomoto HEALTHY SUPPLY), yeast extract (Difco), CSL (ROQUETTE), proteose peptone (Difco), KH2PO4 (and pure chemical industries), and further containing 100 ppm of MnCl2 (and pure chemical industries), 400 ppm of NaCl (and pure chemical industries), 250 ppm of MgCl2 (and pure chemical industries), 75 ppm of CaCl2 (and pure chemical industries), 0.3 ppm of FeSO4 (and pure chemical industries) in the final concentrations described in Medium Condition 1 to 3 described in Table 3 was prepared in such a way that the medium was autoclaved with a silicon plug (in order to avoid Maillard reaction, after separate sterilization, glucose was mixed aseptically).

[0065] From the colony of the B. simplex NBRC 104473 strain grown on a general agar plate medium, 1 platinum ear was taken, and aseptically inoculated into the medium described in Medium Condition 1 described in Table 3, and incubated at 37°C with shaking at 150 rpm for 1 night to obtain a preculture. From the obtained preculture of the B. simplex NBRC 104473 strain, 3 ml was aseptically inoculated into each of the media described in Table 3, and incubated at 37°C with shaking at 150 rpm for 40 hours to 72 hours to obtain a culture. After the incubation, the cell concentration and the sporulation rate of the cells in the culture were measured using an optical microscope and a bacterial cell counter.

[0066]

[0067] The results are shown in Table 4. The same tendency as in Example 1 was observed also for the NBRC 104473 strain.

[0068]

[0069] [Example 3]

[0070] <Assessment in Large Scale Culture>

[0071] Using a 5 L capacity culture tank, 2000 ml of each of the media containing glucose (and pure chemicals), defatted soybean powder (Ajinomoto HEALTHY SUPPLY), yeast extract (Difco), CSL (ROQUETTE), proteose peptone (Difco), KH2PO4 (and pure chemicals), and further containing 100 ppm of MnCl2 (and pure chemicals), 400 ppm of NaCl (and pure chemicals), 250 ppm of MgCl2 (and pure chemicals), 75 ppm of CaCl2 (and pure chemicals), and 0.3 ppm of FeSO4 (and pure chemicals) were prepared so as to achieve the final concentrations of the medium conditions 1 to 3 described in Table 5, and autoclave sterilization was performed (in order to avoid the Maillard reaction, the glucose was mixed aseptically after separate sterilization).

[0072] From a colony of the B. simplex NBRC 15720 strain grown on a general agar plate medium, 1 platinum loop was taken, and aseptically inoculated into 2000 ml of the medium of the medium condition 1 described in Example 1 (Table 1) prepared in a 500 ml capacity Erlenmeyer flask, and subjected to 1 night of shaking culture at 37°C at 150 rpm to obtain a preculture. From the obtained preculture of the B. simplex NBRC 15720 strain, 60 ml was aseptically inoculated into each of the media described in Table 5, and subjected to 40 hours of aeration stirring culture at 37°C at 400 rpm to obtain a culture solution. After the culture, the cell concentration and the sporulation rate of the cells in the culture solution were measured using an optical microscope and a bacterial cell counter.

[0073]

[0074] The results are shown in Table 6. If the C / N is within a certain range, even in a large volume culture system, a sporulation rate of 50% or more of the B. simplex NBRC 15720 strain can be obtained.

[0075]

[0076] [Example 4]

[0077] Evaluation of B. siamensis in a large volume culture system

[0078] Using a 5 L capacity culture tank, 2000 ml of each of the media containing glucose (and pure chemical industry), defatted soybean powder (Ajinomoto HEALTHY SUPPLY), yeast extract (Difco), CSL (ROQUETTE), proteose peptone (Difco), KH2PO4 (and pure chemical industry), and further containing 100 ppm of MnCl2 (and pure chemical industry), 400 ppm of NaCl (and pure chemical industry), 250 ppm of MgCl2 (and pure chemical industry), 75 ppm of CaCl2 (and pure chemical industry), and 0.3 ppm of FeSO4 (and pure chemical industry) in the final concentrations of the medium conditions 1 to 3 described in Table 7 were prepared, and autoclave sterilization was performed (in order to avoid Maillard reaction, after separate sterilization, the glucose was mixed aseptically).

[0079] From the colonies of Bacillus siamensis grown on a general agar plate medium, 1 platinum ear was taken, and aseptically inoculated into the medium of the medium condition 1 described in Table 7 prepared in a 500 ml capacity Erlenmeyer flask, and subjected to 1 night shaking culture at 37°C at 150 rpm to obtain a preculture. From the obtained preculture of Bacillus siamensis, 60 ml was aseptically inoculated into each of the various media described in Table 7, and subjected to 40 hours of aeration stirring culture at 37°C at 400 rpm to obtain a culture solution. After the culture, the cell concentration and the sporulation rate of the cells in the culture solution were measured using an optical microscope and a bacterial cell counter.

[0080]

[0081] The results are shown in Table 8. If the C / N is within a certain range, in a large capacity culture system, a sporulation rate of 88% or more of Bacillus siamensis can be obtained.

[0082]

[0083] [Example 5]

[0084] Evaluation of Bacillus megaterium in a large capacity culture system

[0085] Using a 5 L capacity culture tank, 2000 ml of each of the media containing glucose (and pure chemical industry), defatted soybean powder (Ajinomoto HEALTHY SUPPLY), yeast extract (Difco), CSL (ROQUETTE), proteose peptone (Difco), KH2PO4 (and pure chemical industry), and further containing 100 ppm of MnCl2 (and pure chemical industry), 400 ppm of NaCl (and pure chemical industry), 250 ppm of MgCl2 (and pure chemical industry), 75 ppm of CaCl2 (and pure chemical industry), and 0.3 ppm of FeSO4 (and pure chemical industry) in the final concentrations of the medium conditions 1 to 3 described in Table 9 were prepared, and autoclave sterilization was performed (in order to avoid the Maillard reaction, after separate sterilization, the glucose was mixed aseptically).

[0086] From the colony of the Bacillus megaterium grown on the ordinary agar plate medium, 1 platinum ear was taken, and aseptically inoculated into the medium of the medium condition 1 described in Table 9 prepared in a 500 ml capacity Erlenmeyer flask, and subjected to 1 night shaking culture at 37°C at 150 rpm to obtain a preculture. From the obtained Bacillus megaterium preculture, 60 ml was aseptically inoculated into each of the various media described in Table 9, and subjected to 40 hours of aeration stirring culture at 37°C at 400 rpm to obtain a culture solution. After the culture, the cell concentration and the sporulation rate of the cells in the culture solution were measured using an optical microscope and a bacterial cell counter.

[0087]

[0088] The results are shown in Table 10. If the C / N is within a certain range, in a large capacity culture system, a sporulation rate of 78% or more of the Bacillus megaterium can be obtained.

[0089] [Table 10]

[0090] Table 10

[0091] Culture results of Bacillus megaterium (large capacity culture system)

[0092]

Claims

1. A method for manufacturing spores of Bacillus spp., the method comprising a step of culturing Bacillus spp. using a liquid culture medium having a carbon content of 3 g / L or more and 25 g / L or less, a C / N ratio (i.e., the weight ratio of carbon content to nitrogen content) of 4.5 or more and 9.0 or less, and a potassium content of 0.2 g / L or more and 1.9 g / L or less, wherein the Bacillus spp. is Bacillus simplex.

2. The method for manufacturing spores of Bacillus bacteria according to claim 1, wherein, The C / N ratio of the liquid culture medium used in the culture should be above 4.5 and below 7.

5.

3. The method for manufacturing spores of Bacillus bacteria according to claim 1, wherein, The C / N ratio of the liquid culture medium used in the culture should be above 6.0 and below 7.

5.

4. The method for manufacturing spores of Bacillus bacteria according to claim 1, wherein, The cultivation process is carried out at 20-40℃ under aerobic conditions, with stirring for 10-100 hours.

5. The method for manufacturing spores of Bacillus bacteria according to claim 1, wherein, The method achieves a spore formation rate of at least 67%.

6. The method of producing spores of bacteria of the genus Bacillus according to any one of claims 1 to 5, wherein, The carbon and nitrogen sources contained in the liquid culture medium are the carbon and nitrogen sources that Bacillus bacteria can metabolize.

7. The method for manufacturing spores of Bacillus bacteria according to claim 6, wherein, Bacillus bacteria can metabolize carbon sources selected from one or more of the following: 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 acid salts, and alkanes. Bacillus bacteria can metabolize nitrogen sources selected from one or more of the following: components from soybeans, components from yeast, components from corn, animal and plant proteins and their decomposition products, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, and ammonium acetate, ammonia, sodium nitrate, potassium nitrate, monosodium glutamate, and urea.

Images