CN106434520B - The preparation method of bacillus spore - Google Patents
The preparation method of bacillus spore Download PDFInfo
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- CN106434520B CN106434520B CN201610856595.XA CN201610856595A CN106434520B CN 106434520 B CN106434520 B CN 106434520B CN 201610856595 A CN201610856595 A CN 201610856595A CN 106434520 B CN106434520 B CN 106434520B
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Abstract
This case is related to the preparation method of bacillus spore, comprising: 1 part of bacillus stearothermophilus is inoculated in 10 parts of eutrophy culture medium, after cultivating 12h at 50-55 DEG C, 150-250rpm/min；90 parts of eutrophy culture medium is then added, continues to cultivate 12h, obtains vegetative cell culture solution；1 part of inorganic ions culture medium is added into 1 part of vegetative cell culture solution, cultivates 12h at 55-60 DEG C, 100-150rpm/min；1 part of inorganic ions culture medium is added every 12h, until the total number of inorganic ions culture medium and the number ratio of eutrophy culture medium that are added reach 7: 1-10: 1；Obtain the culture solution containing bacillus spore.This case only needs two kinds of culture mediums, easy to operate, reproducible；Incubation time is shortened, and time-consuming only needs 60-72h；Spore production has reached 109Every milliliter, gemma rate is 90% or more.
Description
Technical field
The present invention relates to a kind of microbial spores preparation fields, and in particular to a kind of preparation method of bacillus spore.
Background technique
Bacillus stearothermophilus is gram positive bacteria, and aerobic or amphimicrobian, bacillus stearothermophilus can produce gemma,
Optimum growth temp is 55-60 DEG C, and maximum growth temperature is 65-75 DEG C, belongs to Thermophilic Bacteria.Chinese industrial Microbiological Culture Collection
Administrative center (CICC) collection number is CICC:10267；Or ATCC:7953.
It is the produced gemma no pathogenicity of bacillus stearothermophilus, apyrogeneity, nontoxic.The gemma has really certain sterilizing factor
Fixed resistance, therefore can be used as indicator microoraganism and be used to monitoring sterilization effect, including pressuresteam sterilization, hydrogen peroxide
(H2O2) Low-temperature Plasma Sterilization, low-temperature formaldehyde vapor sterilizing etc. sterilization methods.As sterile biological indicator bacteria, according to China
Pharmacopeia code requirement, Number of spores are generally 5 × 105To 5 × 106A, statoblast content need to account for 90% or more.
Currently, in the prior art, it is already possible to it realizes and low dose of preparation is carried out to the gemma of bacillus stearothermophilus,
But in scale of mass production, but there are many technology barriers, is primarily present that spore forming rate is not high, production cost is excessively high asks
Topic, while time-consuming, technique is cumbersome.
Summary of the invention
In view of the deficiencies of the prior art, it is an object of the invention to propose a kind of preparation side of bacillus spore
Method, by liquid cultivating method easy to operate, the principle generated using gemma passes through turn of two kinds of different liquids culture mediums
It changes, to guarantee the productivity and yield of gemma.
To achieve the above object, technical solution of the present invention is summarized as follows:
A kind of preparation method of bacillus spore comprising following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1 parts by weight is inoculated in the eutrophy culture medium of 10 parts by weight, in 50-55 DEG C of environment
In, after revolving speed cultivates 12h under the conditions of being 150-250rpm/min；The eutrophy culture medium of 90 parts by weight is then added, continues to train
12h is supported, vegetative cell culture solution is obtained；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1 parts by weight is added into the vegetative cell culture solution of 1 parts by weight, at 55-60 DEG C
In environment, revolving speed cultivates 12h under the conditions of being 100-150rpm/min；The inorganic ions culture of 1 parts by weight is then added every 12h
Base, until gross mass part of inorganic ions culture medium and the mass parts ratio of eutrophy culture medium for being added reach 7: 1-10: 1；
Obtain the culture solution containing bacillus spore.
Preferably, the preparation method of the bacillus spore, wherein the eutrophy culture medium includes: 5-20
The soy peptone of parts by weight, the beef extract of 5 parts by weight, 3-5 parts by weight yeast extract, 5 parts by weight sodium chloride and
The distilled water of 1000 parts by weight.
Preferably, the preparation method of the bacillus spore, wherein the pH of the eutrophy culture medium is 7.2-
7.5。
Preferably, the preparation method of the bacillus spore, wherein the inorganic ions culture medium includes: 1 weight
Measure the MnSO of NaCl, 0.5-1 parts by weight of part4·H2O, the MgSO of 0.1-0.5 parts by weight4·2H2O, 0.01-1 parts by weight
CaCl2·2H2O, the KH of 0.01-0.1 parts by weight2PO4With the distilled water of 1000 parts by weight.Bacillus spore is that thallus is being disliked
The hypopus generated under bad environment, and the effect of inorganic ions culture medium is exactly to reduce nutriment, environment becomes severe.
Preferably, the preparation method of the bacillus spore, wherein the pH of the inorganic ions culture medium is
7.2-7.5。
Preferably, the preparation method of the bacillus spore, wherein the inorganic ions culture medium further includes having
The ZnCl of 0.1-0.2 parts by weight2。
Preferably, the preparation method of the bacillus spore, wherein the inorganic ions culture medium further includes having
The FeCl of 0.1-0.2 parts by weight3。
The beneficial effects of the present invention are: this case is by the improvement to existing bacillus spore preparation process, so that gemma
Preparation method only needs two kinds of culture mediums, and operating procedure is simple, reproducible；Incubation time is further shortened, consumption
When only need 60-72h；Spore production has reached 109Every milliliter, gemma rate is 90% or more.
Specific embodiment
The present invention will be further described in detail below with reference to the embodiments, to enable those skilled in the art referring to specification
Text can be implemented accordingly.
Embodiment 1
A kind of preparation method of bacillus spore comprising following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1g is inoculated in the eutrophy culture medium of 10g, in 50 DEG C of environment, revolving speed is
After cultivating 12h under the conditions of 150rpm/min；The eutrophy culture medium of 90g is then added, continues to cultivate 12h, obtains vegetative cell
Culture solution；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1g is added into the vegetative cell culture solution of 1g, in 55 DEG C of environment, revolving speed is
12h is cultivated under the conditions of 100rpm/min；The inorganic ions culture medium of 1g is then added every 12h, until the inorganic ions being added
Gross mass part of culture medium and the mass parts ratio of eutrophy culture medium reach 7: 1；Obtain the culture solution containing bacillus spore.
Wherein, eutrophy culture medium preferably includes: the soy peptone of 5-20 parts by weight, the beef extract of 5 parts by weight, 3-
The distilled water of 5 parts by weight yeast extracts, the sodium chloride of 5 parts by weight and 1000 parts by weight.The pH control of eutrophy culture medium exists
7.2-7.5。
Wherein, inorganic ions culture medium preferably includes: the MnSO of NaCl, 0.5-1 parts by weight of 1 parts by weight4·H2O、0.1-
The MgSO of 0.5 parts by weight4·2H2O, the CaCl of 0.01-1 parts by weight2·2H2O, the KH of 0.01-0.1 parts by weight2PO4With 1000 weights
Measure the distilled water of part.The pH of inorganic ions culture medium is controlled in 7.2-7.5.
Embodiment 2
A kind of preparation method of bacillus spore comprising following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1g is inoculated in the eutrophy culture medium of 10g, in 55 DEG C of environment, revolving speed is
After cultivating 12h under the conditions of 250rpm/min；The eutrophy culture medium of 90g is then added, continues to cultivate 12h, obtains vegetative cell
Culture solution；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1g is added into the vegetative cell culture solution of 1g, in 60 DEG C of environment, revolving speed is
12h is cultivated under the conditions of 150rpm/min；The inorganic ions culture medium of 1g is then added every 12h, until the inorganic ions being added
Gross mass part of culture medium and the mass parts ratio of eutrophy culture medium reach 10: 1；Obtain the culture containing bacillus spore
Liquid.
Wherein, eutrophy culture medium preferably includes: the soy peptone of 5-20 parts by weight, the beef extract of 5 parts by weight, 3-
The distilled water of 5 parts by weight yeast extracts, the sodium chloride of 5 parts by weight and 1000 parts by weight.The pH control of eutrophy culture medium exists
7.2-7.5。
Wherein, inorganic ions culture medium preferably includes: the MnSO of NaCl, 0.5-1 parts by weight of 1 parts by weight4·H2O、0.1-
The MgSO of 0.5 parts by weight4·2H2O, the CaCl of 0.01-1 parts by weight2·2H2O, the KH of 0.01-0.1 parts by weight2PO4With 1000 weights
Measure the distilled water of part.The pH of inorganic ions culture medium is controlled in 7.2-7.5.
Embodiment 3
A kind of preparation method of bacillus spore comprising following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1g is inoculated in the eutrophy culture medium of 10g, in 52 DEG C of environment, revolving speed is
After cultivating 12h under the conditions of 200rpm/min；The eutrophy culture medium of 90g is then added, continues to cultivate 12h, obtains vegetative cell
Culture solution；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1g is added into the vegetative cell culture solution of 1g, in 57 DEG C of environment, revolving speed is
12h is cultivated under the conditions of 125rpm/min；The inorganic ions culture medium of 1g is then added every 12h, until the inorganic ions being added
Gross mass part of culture medium and the mass parts ratio of eutrophy culture medium reach 8.5: 1；Obtain the culture containing bacillus spore
Liquid.
Wherein, eutrophy culture medium preferably includes: the soy peptone of 5-20 parts by weight, the beef extract of 5 parts by weight, 3-
The distilled water of 5 parts by weight yeast extracts, the sodium chloride of 5 parts by weight and 1000 parts by weight.The pH control of eutrophy culture medium exists
7.2-7.5。
Wherein, inorganic ions culture medium preferably includes: the MnSO of NaCl, 0.5-1 parts by weight of 1 parts by weight4·H2O、0.1-
The MgSO of 0.5 parts by weight4·2H2O, the CaCl of 0.01-1 parts by weight2·2H2O, the KH of 0.01-0.1 parts by weight2PO4With 1000 weights
Measure the distilled water of part.The pH of inorganic ions culture medium is controlled in 7.2-7.5.
Embodiment 4
A kind of preparation method of bacillus spore comprising following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1kg is inoculated in the eutrophy culture medium of 10kg, in 50 DEG C of environment, revolving speed is
After cultivating 12h under the conditions of 150rpm/min；The eutrophy culture medium of 90kg is then added, continues to cultivate 12h, obtains vegetative cell
Culture solution；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1kg is added into the vegetative cell culture solution of 1kg, in 55 DEG C of environment, revolving speed is
12h is cultivated under the conditions of 100rpm/min；The inorganic ions culture medium of 1kg is then added every 12h, until the inorganic ions being added
Gross mass part of culture medium and the mass parts ratio of eutrophy culture medium reach 7: 1；Obtain the culture solution containing bacillus spore.
Wherein, eutrophy culture medium preferably includes: the soy peptone of 5-20 parts by weight, the beef extract of 5 parts by weight, 3-
The distilled water of 5 parts by weight yeast extracts, the sodium chloride of 5 parts by weight and 1000 parts by weight.The pH control of eutrophy culture medium exists
7.2-7.5。
Wherein, inorganic ions culture medium preferably includes: the MnSO of NaCl, 0.5-1 parts by weight of 1 parts by weight4·H2O、0.1-
The MgSO of 0.5 parts by weight4·2H2O, the CaCl of 0.01-1 parts by weight2·2H2O, the KH of 0.01-0.1 parts by weight2PO4With 1000 weights
Measure the distilled water of part.The pH of inorganic ions culture medium is controlled in 7.2-7.5.
Embodiment 5
It further include the ZnCl for having 0.1-0.2 parts by weight in inorganic ions culture medium2, remaining is same as Example 1.
Embodiment 6
It further include the FeCl for having 0.1-0.2 parts by weight in inorganic ions culture medium3, remaining is same as Example 5.
Comparative example 1
Rate-determining steps 2) in be added inorganic ions culture medium gross mass part and eutrophy culture medium mass parts ratio
It is 6: 1；Remaining is same as Example 1.
Comparative example 2
Rate-determining steps 2) in be added inorganic ions culture medium gross mass part and eutrophy culture medium mass parts ratio
It is 11: 1；Remaining is same as Example 1.
Table 1 lists the method spore production obtained and gemma rate using embodiment and comparative example:
Table 1
Table 1 statistics indicate that, embodiment 4 be the injected volume of embodiment 1 is exaggerated to 1000 times, but acquired results there is no
Decaying, illustrates the process stabilizing, has volume production basis.Embodiment 5 and embodiment 6 illustrate that the addition of zinc ion is conducive to gemma
The promotion of rate to a certain extent.
Table 2 is the investigation to this process repeatability, lists embodiment 1, embodiment 5 and embodiment 6 and is repeated 10 times respectively
The floating situation of obtained gemma rate:
Table 2
| Embodiment 1 | Embodiment 5 | Embodiment 6 | |
| 1st time | 91% | 96% | 98% |
| 2nd time | 92% | 97% | 98% |
| 3rd time | 95% | 98% | 97% |
| The 4th | 93% | 97% | 98% |
| The 5th | 91% | 98% | 99% |
| 6th time | 96% | 96% | 98% |
| 7th time | 94% | 98% | 98% |
| 8th time | 93% | 96% | 99% |
| 9th time | 94% | 96% | 98% |
| 10th time | 91% | 98% | 97% |
| Average gemma rate | 93% | 97% | 98% |
Table 2 shows that zinc ion and iron ion can reduce the fluctuating range of gemma rate, improves the repeatability of the technique, is conducive to
Guarantee product quality in large-scale production.And being applied in combination for zinc ion and iron ion can be with compared to independent addition zinc ion
Obtain higher data reappearance.In addition, it should be noted that, the influence of zinc ion and iron ion to spore production is little；And
The reproducibility of spore production in embodiment 1-4 is inherently very stable, no longer needs to be improved further.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details.
Claims (4)
1. a kind of preparation method of bacillus spore, which comprises the following steps:
Step 1) vegetative cell proliferation:
The bacillus stearothermophilus of 1 parts by weight is inoculated in the eutrophy culture medium of 10 parts by weight, in 50-55 DEG C of environment,
After revolving speed cultivates 12h under the conditions of being 150-250rpm/min；The eutrophy culture medium of 90 parts by weight is then added, continues to cultivate
12h obtains vegetative cell culture solution；
The conversion of step 2) gemma:
The inorganic ions culture medium of 1 parts by weight is added into the vegetative cell culture solution of 1 parts by weight, in 55-60 DEG C of environment
In, revolving speed cultivates 12h under the conditions of being 100-150rpm/min；The inorganic ions culture medium of 1 parts by weight is then added every 12h,
Until gross mass part of inorganic ions culture medium and the mass parts ratio of eutrophy culture medium for being added reach 7: 1-10: 1；It obtains
Culture solution containing bacillus spore；
Wherein, the inorganic ions culture medium includes: the MnSO of NaCl, 0.5-1 parts by weight of 1 parts by weight4·H2O、0.1-0.5
The MgSO of parts by weight4·2H2O, the CaCl of 0.01-1 parts by weight2·2H2O, the KH of 0.01-0.1 parts by weight2PO4, 1000 parts by weight
Distilled water, 0.1-0.2 parts by weight ZnCl2With the FeCl of 0.1-0.2 parts by weight3。
2. the preparation method of bacillus spore according to claim 1, which is characterized in that the eutrophy culture medium packet
It includes: the chlorination of the soy peptone of 5-20 parts by weight, the beef extract of 5 parts by weight, 3-5 parts by weight yeast extract, 5 parts by weight
The distilled water of sodium and 1000 parts by weight.
3. the preparation method of bacillus spore according to claim 1, which is characterized in that the eutrophy culture medium
PH is 7.2-7.5.
4. the preparation method of bacillus spore according to claim 1, which is characterized in that the inorganic ions culture medium
PH be 7.2-7.5.
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| CN107475172B (en) * | 2017-08-30 | 2020-05-15 | 武汉工控工业技术研究院有限公司 | Method for obtaining Geobacillus stearothermophilus spores |
| CN108865937B (en) * | 2018-07-11 | 2021-05-25 | 青岛创通生物科技有限公司 | Method for producing spores by using Bacillus stearothermophilus mutant |
| CN110438060A (en) * | 2019-07-22 | 2019-11-12 | 南京巨鲨显示科技有限公司 | A kind of preparation of Geobacillus stearothermophilus gemma, enrichment method |
| CN114410540B (en) * | 2022-02-07 | 2024-01-19 | 山东新华医疗器械股份有限公司 | Bacillus stearothermophilus culture solution for improving spore rate and culture method |
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| CN104593315A (en) * | 2015-01-06 | 2015-05-06 | 中国科学院苏州生物医学工程技术研究所 | Method for preparing spores of bacillus stearothermophilus |
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| DE19844469A1 (en) * | 1998-09-28 | 2000-03-30 | Herzog Mesmer Artur | Production of an antimicrobial agent useful for pharmaceutical or disinfectant purposes comprises culturing a bacterium, washing the culture and heat treating the wash liquid |
| MY162974A (en) * | 2004-02-20 | 2017-07-31 | Univ Putra Malaysia | Production of protease from bacillus stearothermophilus f1 |
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| CN105018409A (en) * | 2014-04-22 | 2015-11-04 | 中国科学院沈阳应用生态研究所 | Methyl nutritional type bacillus spore-production fermentation medium and culture method thereof |
| CN104593315A (en) * | 2015-01-06 | 2015-05-06 | 中国科学院苏州生物医学工程技术研究所 | Method for preparing spores of bacillus stearothermophilus |
| CN105368746A (en) * | 2015-12-01 | 2016-03-02 | 山东新华医疗器械股份有限公司 | Preparation method of Bacillus stearothermophilus spores |
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