CN112941005A - Method for rapidly producing spores by bacillus coagulans - Google Patents
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- 241000193749 Bacillus coagulans Species 0.000 title claims abstract description 53
- 229940054340 bacillus coagulans Drugs 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000010438 heat treatment Methods 0.000 claims abstract description 35
- 230000001332 colony forming effect Effects 0.000 claims abstract description 10
- 230000001580 bacterial effect Effects 0.000 claims description 53
- 239000000725 suspension Substances 0.000 claims description 53
- 238000010790 dilution Methods 0.000 claims description 27
- 239000012895 dilution Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 239000002504 physiological saline solution Substances 0.000 claims description 7
- 230000028070 sporulation Effects 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 125000004122 cyclic group Chemical group 0.000 abstract 1
- 239000011248 coating agent Substances 0.000 description 8
- 238000000576 coating method Methods 0.000 description 8
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 241001112741 Bacillaceae Species 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000193833 Bacillales Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000701 coagulant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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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
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses a method for rapidly producing spores by bacillus coagulans, and belongs to the technical field of biology. The method comprises the following steps: the bacillus coagulans is subjected to heat treatment at 80-100 ℃ and then is coated on a flat plate for culture, a single colony forming spores is selected for repeated heat treatment and coated on the flat plate for culture, and the single colony forming the spores is the bacillus coagulans capable of producing the spores rapidly and can be used for amplification culture. The spore-forming activity of the strain can be maintained by subjecting the strain to a cyclic heat treatment. The invention can lead the bacillus coagulans to form spores within 15-24h, leads the formation time of the spores to be 12-48h ahead of the formation time of the spores, and leads the spore rate to be more than 90 percent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for rapidly producing spores by bacillus coagulans.
Background
Bacillus coagulans bacterium (A), (B) and (C)Bacillus coagulans) Is a group of lactic acid producing bacteria capable of forming spores, gram-positive, belonging to the phylum firmicutes, class Bacillaceae, order Bacillales, family Bacillaceae, genus Bacillus, listed as safe strains for feed in 1989 by the United states Food and Drug Administration (FDA), and re-approved 42 included in 1992Of the 5 species of Bacillus, Bacillus coagulans is ranked first. The bacillus coagulans can promote the balance of microbial flora in human intestinal tracts, improve the immunity of organisms, and can also generate a large amount of bacteriostatic substances such as coagulants, lactic acid and the like for inhibiting the growth of harmful bacteria, and the bacillus coagulans is more and more widely applied to the aspects of food and medicines.
Because the spores have the characteristics of drying resistance, high temperature resistance, strong acid and alkali resistance and the like, most of the products sold in the market are stored in the form of spores. In the prior art, bacillus coagulans has poor spore production capacity, long period and low spore rate, and a method with fast spore production and short spore production period is very important based on the research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for rapidly producing spores of bacillus coagulans, wherein the method can shorten the sporulation period of the bacillus coagulans and increase the sporulation rate.
The purpose of the invention is realized by the following technical scheme:
a method for rapidly producing spores of bacillus coagulans comprises the following steps: the bacillus coagulans is subjected to heat treatment at 80-100 ℃ and then is coated on a flat plate for culture, a single colony forming spores is selected for repeated heat treatment and coated on the flat plate for culture, and the single colony forming the spores is the bacillus coagulans capable of producing the spores rapidly and can be used for amplification culture. The spore-forming activity of the bacillus coagulans can be maintained by performing circulating heat treatment on the strain by the method.
Preferably, the heat treatment is to prepare bacillus coagulans into bacterial suspension for heat treatment; the heat treatment is carried out in hot water; the heat treatment time is 15-30 min.
Preferably, the method for rapidly producing spores of bacillus coagulans comprises the following steps:
(1) preparing bacillus coagulans into bacterial suspension, performing gradient dilution, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 48-72 h.
(2) Selecting single colony forming spore to make into bacterial suspension, and heat treating in 80-100 deg.C hot water for 15-30 min.
(3) And (3) carrying out gradient dilution on the heat-treated bacterial suspension, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 24-72 h.
(4) Selecting single colony forming spore to make into bacterial suspension, and performing secondary heat treatment in 80-100 deg.C hot water for 15-30 min.
(5) And (3) carrying out gradient dilution on the bacterial suspension subjected to heat treatment, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 15-72h to obtain a single colony of spores, namely the bacillus coagulans capable of producing spores rapidly, which can be used for expanded culture.
Preferably, the above steps are performed by preparing a bacterial suspension and performing gradient dilution with sterilized physiological saline. Further, 1mL of sterilized physiological saline is used to prepare a single colony into a bacterial suspension; a suitable dilution gradient of 0.5mL of bacterial suspension was selected to coat the plates.
Preferably, the heat treatment conditions in the above step are preferably treatment in hot water at 90 ℃ for 15 min.
Preferably, the temperature for the culture in the above step is preferably 42 ℃.
Compared with the prior art, the invention has the following advantages and effects: the bacillus coagulans screened by the method can form spores within 15-24 hours, the formation time of the spores is 12-48 hours earlier than that of the common spores, and the spore rate can reach more than 90%.
Drawings
FIG. 1 is a microscopic image of a suspension of cells prepared before 72 hours of culturing, showing no sporulation of the cells.
FIG. 2 is a microscopic image of 48h of the primary heat-treated single colony culture, showing the formation of spores.
FIG. 3 is a microscopic examination image of single colony culture for 24h in secondary heat treatment, the spore ratio is significantly improved, and the spore production time is significantly advanced.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The bacillus coagulans BC99 used in the following examples has a preservation number of CGMCC No.19487, a preservation time of 3 months and 18 days in 2020, a preservation unit of China general microbiological culture Collection center (CGMCC), and a preservation address of No. 3 Hospital No.1 of North Chen West Lu of the Chaoyang district in Beijing. Bacillus coagulans BC99 has been disclosed in application No. 202010469207.9, entitled A Bacillus coagulans BC99 and method of screening the same.
The plate medium used in the following examples was prepared: 0.1g of glucose, 5g of yeast extract powder, 10g of peptone and 5g of sodium chloride, adding water to a constant volume of 1000mL, adjusting the pH value to 7.0, subpackaging 500mL of triangular bottles with 300mL of each bottle, weighing 18g/L of agar powder in each bottle, plugging a cotton plug, externally wrapping kraft paper, and sterilizing at 121 ℃ for 20 min. The sterilized medium is cooled to 45-50 ℃ and the plate medium is poured.
Example 1: primary heat treatment of Bacillus coagulans BC99
(1) Preparing the BC99 bacterial liquid into bacterial suspension with a ratio of 1:10 (V/V) by using sterilized normal saline, and then performing gradient dilution by 10 times by using the sterilized normal saline to a proper gradient.
(2) 0.5mL of bacterial suspension with a proper dilution gradient is uniformly coated on a plate culture medium by using a sterile glass rake, 3 plates are coated in parallel, and the plate culture medium is placed in an incubator at 42 ℃ for culture for 48 to 72 hours.
(3) And observing the growth state of the colonies on the plate culture medium during 48-72h, taking a single colony for microscopic examination, and picking the single colony to 1mL of sterile physiological saline for uniform mixing when spores appear.
(4) And (4) putting the bacterial suspension obtained in the step (3) into a sealed freezing pipe, and immersing the bacterial suspension into hot water at 90 ℃ for treatment for 15 min.
(5) And (3) carrying out gradient dilution on the heat-treated bacterial suspension by 10 times, selecting 0.5mL of bacterial suspension with proper dilution gradient for plate coating, placing the bacterial suspension in an incubator at 42 ℃ for culturing for 24-72h, and observing the appearance condition of single colony, quantity and spore during the culture.
The results are shown in FIG. 1, FIG. 2 and Table 1.
TABLE 1 Observation of 72 h-cultured colonies
| Processing items | Single colony morphology | Number of | Microscopic examination of spore condition |
| Initial bacterial suspension coating plate | Off-white, smooth and moist | Spread over the flat plate | Individual spores |
| Primary heat treatment bacterial suspension coating flat plate | Off-white, smooth and moist | About 30 | 50% of spores |
Example 2: secondary heat treatment of bacillus coagulans BC99
(1) The single colonies with spores after the primary heat treatment of example 1 and the coated plates were picked up to 1mL of sterile physiological saline and mixed well.
(2) And (2) taking 1mL of the bacterial suspension obtained in the step (1), placing the bacterial suspension in a sealed freezing tube, and immersing the bacterial suspension in hot water at 90 ℃ for treatment for 15 min.
(3) And (3) carrying out gradient dilution on the heat-treated bacterial suspension by 10 times, selecting 0.5mL of bacterial suspension with proper dilution gradient for plate coating, placing the bacterial suspension in an incubator at 42 ℃ for culturing for 15-72h, and observing the appearance condition of single colony, quantity and spore during the culture.
The results are shown in FIG. 3 and Table 2.
TABLE 2 Observation of colonies cultured for 48h
| Processing items | Single colony morphology | Number of | Microscopic examination of spore condition |
| Secondary heat treatment bacterial suspension coating flat plate | Off-white, smooth and moist | 20 to 30 | 80-90% of spores |
Example 3: bacillus coagulans BC 993 times of heat treatment
(1) After the plate was coated by the second heat treatment as in example 2, the single colony with spores was picked up to 1mL of sterile physiological saline and mixed well.
(2) And (2) taking 1mL of the bacterial suspension obtained in the step (1), placing the bacterial suspension in a sealed freezing tube, and immersing the bacterial suspension in hot water at 90 ℃ for treatment for 15 min.
(3) And (3) carrying out gradient dilution on the heat-treated bacterial suspension by 10 times, selecting 0.5mL of bacterial suspension with proper dilution gradient for plate coating, placing the bacterial suspension in an incubator at 42 ℃ for culturing for 15-72h, and observing the appearance condition of single colony, quantity and spore during the culture.
The results are given in Table 3 below.
TABLE 3 Observation of colonies cultured for 48h
| Processing items | Single colony morphology | Number of | Microscopic examination of spore condition |
| Three-time heat treatment bacterial suspension coating flat plate | Off-white, smooth and moist | 20 to 30 | 80-90% of spores |
Example 4: heat treatment comparison of Bacillus coagulans products on the market
A bacillus coagulans live bacterial tablet (the general name Shubao) sold by Qingdao east-sea pharmaceutical industry Co., Ltd is purchased for dilution, separation and purification to obtain a bacillus coagulans single bacterial colony, and the experiment is carried out according to the method.
The strain was heat-treated twice in the same manner as in example 1 and example 2, and a single colony having spores on the plate after the second heat-treatment was suspended in a sterile physiological saline solution.
And (3) carrying out gradient dilution on the bacterial suspension by 10 times, selecting 0.5mL of bacterial suspension with proper dilution gradient for plate coating, placing the bacterial suspension in an incubator at 42 ℃ for culturing for 15-72h, and observing the appearance condition of single colony, quantity and spore during the culture. Meanwhile, single colonies which are not subjected to heat treatment are taken for parallel culture comparison.
In a comparison mode, lawn cultured in different time periods is prepared into suspension, the suspension is taken to be subjected to water bath at 80 ℃ for 10min, and the ratio of the number of spores to the total number of bacteria is calculated. The following table 4 shows the results of spore ratios with and without heat treatment:
TABLE 4
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for rapidly producing spores of bacillus coagulans is characterized by comprising the following steps: the method comprises the following steps: the bacillus coagulans is subjected to heat treatment at 80-100 ℃ and then is coated on a flat plate for culture, a single colony forming spores is selected for repeated heat treatment and coated on the flat plate for culture, and the single colony forming the spores is the bacillus coagulans capable of producing the spores rapidly.
2. The method for rapid sporulation of bacillus coagulans according to claim 1, wherein: the heat treatment is to prepare bacillus coagulans into bacterial suspension for heat treatment.
3. The method for rapid sporulation of bacillus coagulans according to claim 1, wherein: the heat treatment is carried out in hot water.
4. The method for rapid sporulation of bacillus coagulans according to claim 1, wherein: the time of the heat treatment is 15-30 min.
5. The method for rapid sporulation of bacillus coagulans according to claim 1, wherein: the method comprises the following steps:
(1) preparing bacillus coagulans into bacterial suspension, performing gradient dilution, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 48-72 h;
(2) selecting single colony forming spore to make into bacterial suspension, and heat treating in 80-100 deg.C hot water for 15-30 min;
(3) carrying out gradient dilution on the heat-treated bacterial suspension, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 24-72 h;
(4) selecting single colony forming spore to make into bacterial suspension, and performing secondary heat treatment in 80-100 deg.C hot water for 15-30 min;
(5) and (3) carrying out gradient dilution on the bacterial suspension subjected to heat treatment, selecting the bacterial suspension with proper dilution gradient to coat a flat plate, and culturing for 15-72h to obtain a single colony of spores, namely the bacillus coagulans capable of producing spores rapidly.
6. The method for rapid spore production of bacillus coagulans according to claim 5, wherein the bacillus coagulans is selected from the group consisting of: sterile saline was used to prepare the bacterial suspension and gradient dilution.
7. The method for rapid spore production of bacillus coagulans according to claim 5, wherein the bacillus coagulans is selected from the group consisting of: in steps (2) and (4), 1mL of sterilized physiological saline is used to prepare a single colony into a bacterial suspension.
8. The method for rapid spore production of bacillus coagulans according to claim 5, wherein the bacillus coagulans is selected from the group consisting of: the plate was coated with 0.5mL of bacterial suspension at the appropriate dilution gradient selected in step (3) and step (5).
9. The method for rapid spore production of bacillus coagulans according to claim 5, wherein the bacillus coagulans is selected from the group consisting of: the conditions of the heat treatment in the step (2) and the step (4) are treatment in hot water at 90 ℃ for 15 min.
10. The method for rapid spore production of bacillus coagulans according to claim 5, wherein the bacillus coagulans is selected from the group consisting of: the temperature for the cultivation in the step (1), the step (3) and the step (5) was 42 ℃.
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