WO2008043368A2 - Method for obtaining endospores of sporogenous fermentable thermophilic strains of microorganism and use of the obtained endospores to inoculate fermentation - Google Patents
Method for obtaining endospores of sporogenous fermentable thermophilic strains of microorganism and use of the obtained endospores to inoculate fermentation Download PDFInfo
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- WO2008043368A2 WO2008043368A2 PCT/EE2007/000019 EE2007000019W WO2008043368A2 WO 2008043368 A2 WO2008043368 A2 WO 2008043368A2 EE 2007000019 W EE2007000019 W EE 2007000019W WO 2008043368 A2 WO2008043368 A2 WO 2008043368A2
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- endospores
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Definitions
- the invention is related to the field of biotechnology, more specifically to the field of obtaining and using endospores of sporogenous fermentable microorganisms, and is usable in microbiological production of lactic acid, where fermentation of sporogenous thermophilic fermentable microorganisms is initiated by an inoculum consisting of endospores .
- Microbiological production of lactic acid uses microorganisms of various genera ⁇ Lactobacillus sp. ; Lactococcus sp. ; Bacillus sp.) . Homofermentative microorganisms share several common characteristics - for example, high demands as to the composition of the medium
- Another known method is the aerobic cultivation of sporogenous bacteria necessary for obtaining the D- isomer of lactic acid in a medium with a low saccharide content and an subsequent immediate or post- immobilization anaerobic pour plating in a medium with a high saccharide content
- the objective of the present invention is to provide a method for obtaining endospores of thermophilic sporogenous fermentative strains of microorganism and the use of the obtained endospores to inoculate fermentation.
- the object of the present invention is a method for obtaining endospores of thermophilic sporogenous fermentable strains of microorganism and the use of the obtained endospores to inoculate fermentation.
- Mn and Ca compounds are added to the medium and limitation of phosphor and carbon source is generated in the presence of a nitrogen source.
- the obtained unimmobilized endospores are used to inoculate fermentation medium,- the obtained culture is synchronized by the activation of endospores before cultivation.
- Endospores are dormant forms of cells, which are highly resistant to many influences of the external environment (high/low temperature; radiation (UV) ; high/low pH; low molecular weight diols, etc.). Maintaining a microorganism culture in the form of endospores is robust, as such a stock culture does not germinate without special activation. In addition, the preparation of a preservation culture based on endospores is less expensive than the alternative lyophilization of vegetative cells. Suitably activated endospores can be used directly as a fermentation inoculum.
- An advantage compared to generally used vegetative cell inoculum is the short preparation time - the prepared endospores only need activation. What is also important in the case of using endospores is the possibility to separate in time and space the preparation of the cell material needed for the inoculum and using it in the basic fermentation (as the fermentation inoculum) .
- vegetative cells a time consuming and spatially complex solution for preparing a fresh inoculum is essential .
- an inoculum consisting of endospores these processes can easily be separated.
- the synchronization of cells can be applied in the case of an inoculum consisting of endospores. Activation of the endospores of the inoculum causes synchronization of the bacterial culture, since the cells are cultivated simultaneously from the same growth phase.
- the sporulation of B. coagulans is a complicated process. In the case of the described species, sporulation is complicated due to several essentially contradictory- requirements. On the one hand, the high demands of the described species to the complexity of the medium (the nitrogen source is especially important) . On the other hand, stress inducing conditions - lack of nutrients, presence of Mn 2+ and Ca 2+ ions, limitation of phosphor and the carbon source - must be provided in the culture medium in order to initiate sporulation.
- under limitation is meant that the environment contains the necessary compounds, but in an insufficient concentration for the vegetative reproduction of cells.
- low molecular weight diols are used as sporulation inducers.
- the preferred pH range for the production of endospores is 5-6.5. The most preferred pH is 5.5.
- B. coagulans endospores several specific features of this species have to be taken into account. What is important is the fact that the formed endospore of this species may germinate fast. This basically means that in the case of B. coagulans the endospores may germinate (including regermination) in the conditions of a longer sporulation process. Special attention should be paid to this endospore "circulation" process to produce endospores in a liquid medium.
- Low molecular weight diols such as 2 , 3-butanediol (2,3-BD) may be formed by several microorganisms as by-products of fermentation in particular conditions. Addition of a low molecular diol (e.g. 2,3-BD) into the sporulation medium increases the effectiveness of sporulation up to 10 times.
- a low molecular diol e.g. 2,3-BD
- endospores Use of endospores. The following should be taken into consideration when using endospores to inoculate lactate fermentation: Unimmobilized endospores of the species Bacillus coagulans are used for the inoculum; for activation and sterilization it is heated at up to 100 0 C for up to 2 hours. The total inoculum consisting of endospores is activated simultaneously, obtaining a synchronized microbial culture. After inoculation of endospores to fermentation medium, the pH of the medium is temporarily acidified (pH 5.5). Using of endospores inoculum, the different osmoresistance of vegetative cells and endospores must be taken into account. Endospores themselves are not sensitive to osmotic pressure, but the latter may influence the germination and outgrowth processes of endospores.
- the Bacillus sp. endospores used in the example of embodiment of the current method were obtained from the strain Bacillus coagulans SIM-7 DSM 14043, which has been described in the following patent applications and patents with regard to the invention "Thermophilic microorganism Bacillus coagulans strain SIM-7 DSM 14043 for the production of L (+) -lactate from fermentable sugars and by means of named microorganisms" of the University of Tartu: Estonian patent EE04529, European patent EP1409642, US patent US7183088, Russian patent RU2288263, Chinese patent CN1283781, HongKong patent HK1065069, Indian patent IN196725, Japanese patent application 2002-574,236, Canadian patent application CA2441915, etc.
- the medium was inoculated with vegetative cells and/or activated endospores and incubated at 55 0 C for 24h.
- the bacterial cell mass was precipitated from the suspension by centrifugation (5000 x g) and the obtained sediment was washed 3 times with cooled distilled water.
- the purity and endospore content of the prepared suspension was controlled with a microscope, observing the preparation in the phase contrast system with a 100Ox magnification.
- the effeciency of sporulation in these conditions was over 90% (i.e. over 90% of the cells were endospores) .
- Inoculation of L-lactic acid fermentation using Bacillus coagulans SIM-7 DSM 14043 endospores and vegetative cells Fig.
- the endospores used as the inoculum were synchronized before inoculation by activation at 70 0 C for 30 minutes. Chemical and mechanical activation was also used.
- the medium for producing lactate 25 g/L YE, 2.16 mM phosphate buffer (pH 6.2), 893 ul/1 Mg 2+ mixture (0.35 M MgCl ⁇ and 0.35 M MgSOO , 2.6 ml/1 microelements (Bauchop, T., Elsden, S. R. (1960) The growth of microorganisms in relation to their energy supply. J. Gen. Mirobiol., 23: 469 - 475) ; fermentable sugar (glucose 100 - 500 mM) .
- the medium was made in tap water and mixed in unsterile conditions. Fermentation was carried out at 55 0 C, using 4.1 M ammonium hydroxide as the neutralizer.
- the final concentration of the inoculum in the case of both vegetative cells and endospores was 10 7 vegetative cells or endospores per millilitre.
- the following symbols mark the endospore inoculum influence to lactate production at •-lOO mM; ⁇ -250 mM and A -500 mM glucose,- the symbol of a vegetative cell inoculum at D- 500 mM glucose.
- an inoculum consisting of endospores provides a significant advantage to production rate of lactic acid.
- a culture based on endospores enables an almost 20% decrease in time of the production cycle, as compared to unsynchronized vegetative cells (Fig.).
- the present invention makes it possible to activate cells of a thermophilic microorganism culture that are in the form of endospores due to dormancy characteristic of endospores, and thus to prepare a synchronized culture that provides a considerable economic advantage in the fermentation process.
- Unimmobilized endospores were used in the current invention.
- the most effective pH range for producing endospores according to the present invention is considerably more acidic (pH 5.5) than the neutral pH value (pH 6.8-8.4) well known in the state of the art .
- Using the present invention reduces significantly the duration of the sporulation process, which will be up to 24 hours instead of the minimum of 36 hours known in the state of the art .
- a low molecular weight diol is used in the invention as a novel sporulation-enhancing factor.
Abstract
The present invention relates to the method of producing endospores of thermophilic sporogenic microbial strains and the use thereof for inoculation of fermentation process. Unimmobilized endospores will be proposed for inoculation of fermentation whereas the obtained culture will be synchronized by activation of endospores before outgrowth. The strain, used in the examples of implementation for production of endospores, has been previously described and patented is Bacillus coagulans SIM7 DSM 14043.
Description
METHOD FOR OBTAINING ENDOSPORES OF SPOROGENOUS FERMENTABLE THERMOPHILIC STRAINS OF MICROORGANISM AND USE OF THE OBTAINED ENDOSPORES TO INOCULATE FERMENTATION
TECHNICAL FIELD OF THE INVENTION
The invention is related to the field of biotechnology, more specifically to the field of obtaining and using endospores of sporogenous fermentable microorganisms, and is usable in microbiological production of lactic acid, where fermentation of sporogenous thermophilic fermentable microorganisms is initiated by an inoculum consisting of endospores .
BACKGROUND OF THE INVENTION
Microbiological production of lactic acid uses microorganisms of various genera {Lactobacillus sp. ; Lactococcus sp. ; Bacillus sp.) . Homofermentative microorganisms share several common characteristics - for example, high demands as to the composition of the medium
(especially the nitrogen source) ; often an anaerobic culture medium is required.
An important stage in lactic acid fermentation is the preparation of the starter culture necessary for inoculation. The preparation of the starter culture, in its turn, presupposes an available stock culture that is usually maintained at -70 0C. Manipulations with preservation and starter cultures are time-consuming and labour-intensive and require a special laboratory and fermenters for precultivation steps. The cells of a microbial culture obtained like this are in a different growth phase. Using a synchronized microbial culture (the culture/bacterial cells are in the same growth phase) provides a considerable economic advantage. However, it is known that synchronization of a vegetative cell culture for a successful inoculation of fermentation is a difficult
process (Helmstetter CE. , Thornton M., Grover N. B.. Cell- cycle research with synchronous cultures: an evaluation. Biochimie. 2001 Jan; 83 (1) : 83-9) .
The method of lactic acid production by immobilized Bacillus coagulans is well-known (Rosenberg M, Rebros M, Kristofikova L, Malatova K. High temperature lactic acid production by Bacillus coagulans immobilized in LentiKats. Biotechnol . Lett. 2005 Dec; 27 (23-24) : 1943-7) , where endospores were used to prepare an immobilized culture.
Another known method is the aerobic cultivation of sporogenous bacteria necessary for obtaining the D- isomer of lactic acid in a medium with a low saccharide content and an subsequent immediate or post- immobilization anaerobic pour plating in a medium with a high saccharide content
(JP58040093, C12P 7/56, Nakayama Tomoki, 1983).
The technical solutions well known in the art have the following disadvantages: long duration of the sporulation process; need for immobilizing spores on or in a medium,-
- using an environment with a neutral pH to produce spores; thus the sporulation process of e.g. Bacillus coagulans is slow and ineffective; if spores exist in the starter culture, they are not activated;
- there is no usable technology for synchronizing the culture during fermentation.
DISCLOSURE OF THE INVENTION
The objective of the present invention is to provide a method for obtaining endospores of thermophilic sporogenous fermentative strains of microorganism and the use of the obtained endospores to inoculate fermentation.
The object of the present invention is a method for obtaining endospores of thermophilic sporogenous fermentable strains of microorganism and the use of the obtained endospores to inoculate fermentation. In order to obtain spores , Mn and Ca compounds are added to the medium and limitation of phosphor and carbon source is generated in the presence of a nitrogen source. The obtained unimmobilized endospores are used to inoculate fermentation medium,- the obtained culture is synchronized by the activation of endospores before cultivation.
Some strains of microorganism form endospores in certain conditions. Endospores are dormant forms of cells, which are highly resistant to many influences of the external environment (high/low temperature; radiation (UV) ; high/low pH; low molecular weight diols, etc.). Maintaining a microorganism culture in the form of endospores is robust, as such a stock culture does not germinate without special activation. In addition, the preparation of a preservation culture based on endospores is less expensive than the alternative lyophilization of vegetative cells. Suitably activated endospores can be used directly as a fermentation inoculum. An advantage compared to generally used vegetative cell inoculum is the short preparation time - the prepared endospores only need activation. What is also important in the case of using endospores is the possibility to separate in time and space the preparation of the cell material needed for the inoculum and using it in the basic fermentation (as the fermentation inoculum) . In the case of vegetative cells, a time consuming and spatially complex solution for preparing a fresh inoculum is essential . In the case of an inoculum consisting of endospores these processes can easily be separated. In addition, the synchronization of cells can be applied in the case of an inoculum consisting of endospores. Activation of the endospores of the inoculum causes synchronization of the bacterial culture, since the
cells are cultivated simultaneously from the same growth phase.
Production of endospores . The sporulation of B. coagulans is a complicated process. In the case of the described species, sporulation is complicated due to several essentially contradictory- requirements. On the one hand, the high demands of the described species to the complexity of the medium (the nitrogen source is especially important) . On the other hand, stress inducing conditions - lack of nutrients, presence of Mn2+ and Ca2+ ions, limitation of phosphor and the carbon source - must be provided in the culture medium in order to initiate sporulation. Within the scope of the present invention by the term "under limitation" is meant that the environment contains the necessary compounds, but in an insufficient concentration for the vegetative reproduction of cells. In addition, low molecular weight diols are used as sporulation inducers. However, a sufficient amount of nutrients in the medium is also necessary, since sporulation is a metabolically costly process. The preferred pH range for the production of endospores is 5-6.5. The most preferred pH is 5.5. When producing B. coagulans endospores, several specific features of this species have to be taken into account. What is important is the fact that the formed endospore of this species may germinate fast. This basically means that in the case of B. coagulans the endospores may germinate (including regermination) in the conditions of a longer sporulation process. Special attention should be paid to this endospore "circulation" process to produce endospores in a liquid medium. The process according to the invention has not been described in the case of the genus Bacillus sp. ; however, such behaviour has been observed among of representatives of the sporogenous microorganisms belonging to genus Clostridium (Annelis A., Berkowitz D., Kemper D., Rowley
D. B. Production of types A and B spores of Clostridium botulinum by the biphasic method: effect on spore population, radiation resistence, and toxigenicity. Appl Microbiol. 1972, Apr., 23 (4), 734-9). Therefore, the usual methods (long-term incubation (7-10 days) , exhausting media, high initial density of the culture etc.) might not be effective for the production of endospores of this species. Using the species according to the invention, a relatively rich nitrogen source (10 g/1 YE at the minimum or an equivalent amount of an analogue) is necessary in order to achieve a high level of sporulation, but limitation of the carbon and phosphor source must be generated. A short incubation period (24h) is essential in the production of endospores. In the case of this species, a longer incubation period would cause a significant decrease in the endospore yield.
Low molecular weight diols, such as 2 , 3-butanediol (2,3-BD), may be formed by several microorganisms as by-products of fermentation in particular conditions. Addition of a low molecular diol (e.g. 2,3-BD) into the sporulation medium increases the effectiveness of sporulation up to 10 times.
Use of endospores. The following should be taken into consideration when using endospores to inoculate lactate fermentation: Unimmobilized endospores of the species Bacillus coagulans are used for the inoculum; for activation and sterilization it is heated at up to 100 0C for up to 2 hours. The total inoculum consisting of endospores is activated simultaneously, obtaining a synchronized microbial culture. After inoculation of endospores to fermentation medium, the pH of the medium is temporarily acidified (pH 5.5). Using of endospores inoculum, the different osmoresistance of vegetative cells and endospores must be taken into account. Endospores themselves are not sensitive to osmotic pressure,
but the latter may influence the germination and outgrowth processes of endospores.
DESCRIPTION OF EMBODIMENT The Bacillus sp. endospores used in the example of embodiment of the current method were obtained from the strain Bacillus coagulans SIM-7 DSM 14043, which has been described in the following patent applications and patents with regard to the invention "Thermophilic microorganism Bacillus coagulans strain SIM-7 DSM 14043 for the production of L (+) -lactate from fermentable sugars and by means of named microorganisms" of the University of Tartu: Estonian patent EE04529, European patent EP1409642, US patent US7183088, Russian patent RU2288263, Chinese patent CN1283781, HongKong patent HK1065069, Indian patent IN196725, Japanese patent application 2002-574,236, Canadian patent application CA2441915, etc.
1. Preparation of endospores of Bacillus coagulans SIM-7 DSM 14043. In order to obtain endospores, the microorganism was cultivated in the following medium: 10 g/L YE (yeast extract, LABM); 0.7 mM Mg-mixture (0.35 M MgCl2/0.35 M MgSOO ; 4OmM MOPS (3- (N-morpholine) propanesulfonic acid, pH 5.5); 2.6 ml/1 microelements (Bauchop, T., Elsden, S. R. (1960) The growth of microorganisms in relation to their energy supply. J. Gen. Mirobiol . , 23: 469 - 475). The medium was inoculated with vegetative cells and/or activated endospores and incubated at 55 0C for 24h. The bacterial cell mass was precipitated from the suspension by centrifugation (5000 x g) and the obtained sediment was washed 3 times with cooled distilled water. The purity and endospore content of the prepared suspension was controlled with a microscope, observing the preparation in the phase contrast system with a 100Ox magnification. The effeciency of sporulation in these conditions was over 90% (i.e. over 90% of the cells were endospores) .
2. Inoculation of L-lactic acid fermentation using Bacillus coagulans SIM-7 DSM 14043 endospores and vegetative cells (Fig.) .
The endospores used as the inoculum were synchronized before inoculation by activation at 70 0C for 30 minutes. Chemical and mechanical activation was also used.
The medium for producing lactate: 25 g/L YE, 2.16 mM phosphate buffer (pH 6.2), 893 ul/1 Mg2+ mixture (0.35 M MgCl∑ and 0.35 M MgSOO , 2.6 ml/1 microelements (Bauchop, T., Elsden, S. R. (1960) The growth of microorganisms in relation to their energy supply. J. Gen. Mirobiol., 23: 469 - 475) ; fermentable sugar (glucose 100 - 500 mM) . The medium was made in tap water and mixed in unsterile conditions. Fermentation was carried out at 55 0C, using 4.1 M ammonium hydroxide as the neutralizer. The final concentration of the inoculum in the case of both vegetative cells and endospores was 107 vegetative cells or endospores per millilitre. In the Figure, the following symbols mark the endospore inoculum influence to lactate production at •-lOO mM; ^-250 mM and A -500 mM glucose,- the symbol of a vegetative cell inoculum at D- 500 mM glucose.
As a result of the experiment it can be stated that even equal number of inoculated cells, either endospores or vegetative cells, an inoculum consisting of endospores provides a significant advantage to production rate of lactic acid. A culture based on endospores enables an almost 20% decrease in time of the production cycle, as compared to unsynchronized vegetative cells (Fig.).
The present invention makes it possible to activate cells of a thermophilic microorganism culture that are in the form of endospores due to dormancy characteristic of endospores, and thus to prepare a synchronized culture that provides a considerable economic advantage in the fermentation process. Unimmobilized endospores were used in the current invention.
The most effective pH range for producing endospores according to the present invention is considerably more acidic (pH 5.5) than the neutral pH value (pH 6.8-8.4) well known in the state of the art . Using the present invention reduces significantly the duration of the sporulation process, which will be up to 24 hours instead of the minimum of 36 hours known in the state of the art .
A low molecular weight diol is used in the invention as a novel sporulation-enhancing factor.
Claims
1. A method for obtaining endospores of thermophilic sporogenous fermentable strains of microorganism, by the cultivation of microorganisms on a medium, characterized in that in order to provide sporulation- enhancing conditions, a medium with a pH adjusted to 5-6.5, preferably to 5.5, is prepared, wherein Mn and Ca compounds are added and limitation of phosphor and carbon source is caused in the presence of a nitrogen source.
2. The method according to Claim 1, characterized in that low molecular weight diols are used in the medium as sporulation inducers.
3. The method according to Claim 1, characterized in that endospores are obtained from the microorganism species Bacillus coagulans.
4. The method according to Claim 3, characterized in that endospores are obtained from the strain of microorganism Bacillus coagulans SIM-7 DSM 14043.
5. A method of using thermophilic sporogenous fermentable endospores to inoculate fermentation, characterized in that unimmobilized endospores of thermophilic sporogenous fermentable microbes are used for the inoculum, and the named endospores are simultaneously activated before outgrowth, thus obtaining a synchronized culture.
6. The method according to Claim 5, characterized in that endospores of the strain Bacillus coagulans are used for the inoculum.
7. The method according to Claim 6, characterized in that endospores of the strain Bacillus coagulans SIM- 7 DSM 14043 are used for the inoculum.
8. The method according to Claim 5, characterized in that in fermentation inoculated with endospores the pH of the medium is temporarily changed after inoculation with the endospores, adjusting a pH to 4-8, preferably to 5-7, and most preferably 5.4-6.
9. The method according to Claim 5, characterized in that in fermentation inoculated with endospores in order to activate the endospore inoculum and sterilize the medium, the medium is heated at up to 100 °C for up to 2 hours.
10. The method according to Claim 5, characterized in that the inoculum is activated chemically or mechanically.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EEP200600034 | 2006-10-11 | ||
| EEP200600034A EE200600034A (en) | 2006-10-11 | 2006-10-11 | Method for Preparation of Sporogenic Fermented Thermophilic Microorganism T Water Endospores and Use of Endospores Resulting to Inoculate Fermentation |
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| WO2008043368A2 true WO2008043368A2 (en) | 2008-04-17 |
| WO2008043368A3 WO2008043368A3 (en) | 2008-05-29 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018163094A1 (en) * | 2017-03-08 | 2018-09-13 | Symbiosis International University | A method of inducing sporulation in bacillus coagulans |
| CN109207406A (en) * | 2018-10-24 | 2019-01-15 | 广东绿百多生物开发有限公司 | A kind of method and culture medium for improving bacillus coagulans cell and forming gemma |
| US11591563B2 (en) | 2019-07-26 | 2023-02-28 | American Sterilizer Company | Post-sporulation modification of spores and biological indicator |
| JP2023508764A (en) * | 2020-03-24 | 2023-03-03 | トリプルダブリュー リミテッド | Production of lactic acid from organic waste using a composition of Bacillus coagulans spores |
| US11708558B2 (en) | 2019-07-26 | 2023-07-25 | American Sterilizer Company | Liquid sporulation method and sporulation broth |
| US11760585B2 (en) | 2019-08-22 | 2023-09-19 | American Sterilizer Company | Momentum arresting ramp |
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| WO2002074934A1 (en) * | 2001-03-16 | 2002-09-26 | University Of Tartu | Thermophilic microorganism bacillus coagulans strain sim-t dsm 14043 for the production of l(+)-lactate from fermentable sugars and their mixtures |
| US6849256B1 (en) * | 1999-11-08 | 2005-02-01 | Ganeden Biotech Incorporated | Inhibition of pathogens by probiotic bacteria |
| EP1067945B1 (en) * | 1998-04-01 | 2006-01-04 | Ganeden Biotech, Inc. | Methods for reducing cholesterol using bacillus coagulans spores, systems and compositions |
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2006
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| EP1067945B1 (en) * | 1998-04-01 | 2006-01-04 | Ganeden Biotech, Inc. | Methods for reducing cholesterol using bacillus coagulans spores, systems and compositions |
| US6849256B1 (en) * | 1999-11-08 | 2005-02-01 | Ganeden Biotech Incorporated | Inhibition of pathogens by probiotic bacteria |
| WO2002074934A1 (en) * | 2001-03-16 | 2002-09-26 | University Of Tartu | Thermophilic microorganism bacillus coagulans strain sim-t dsm 14043 for the production of l(+)-lactate from fermentable sugars and their mixtures |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018163094A1 (en) * | 2017-03-08 | 2018-09-13 | Symbiosis International University | A method of inducing sporulation in bacillus coagulans |
| CN110382686A (en) * | 2017-03-08 | 2019-10-25 | 共生国际大学 | The method that inducing spore is formed in bacillus coagulans |
| CN109207406A (en) * | 2018-10-24 | 2019-01-15 | 广东绿百多生物开发有限公司 | A kind of method and culture medium for improving bacillus coagulans cell and forming gemma |
| US11591563B2 (en) | 2019-07-26 | 2023-02-28 | American Sterilizer Company | Post-sporulation modification of spores and biological indicator |
| US11708558B2 (en) | 2019-07-26 | 2023-07-25 | American Sterilizer Company | Liquid sporulation method and sporulation broth |
| US11760585B2 (en) | 2019-08-22 | 2023-09-19 | American Sterilizer Company | Momentum arresting ramp |
| JP2023508764A (en) * | 2020-03-24 | 2023-03-03 | トリプルダブリュー リミテッド | Production of lactic acid from organic waste using a composition of Bacillus coagulans spores |
| JP7353689B2 (en) | 2020-03-24 | 2023-10-02 | トリプルダブリュー リミテッド | Production of lactic acid from organic waste using Bacillus coagulans spore composition |
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| Publication number | Publication date |
|---|---|
| WO2008043368A3 (en) | 2008-05-29 |
| EE200600034A (en) | 2008-06-16 |
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