CN109797108A - Aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof - Google Patents
Aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof Download PDFInfo
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- CN109797108A CN109797108A CN201910149171.3A CN201910149171A CN109797108A CN 109797108 A CN109797108 A CN 109797108A CN 201910149171 A CN201910149171 A CN 201910149171A CN 109797108 A CN109797108 A CN 109797108A
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- sample
- bacterial strain
- employment
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Abstract
The invention belongs to the field of quality control in terms of drug microbiologic inhibition tests, in particular to a kind of aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof.Aspergillus niger employment and suitability test (E & ST) bacterial strain only includes a certain amount of aspergillus niger.Uniformity of the present invention, stability meet employment and suitability test (E & ST) bacterial strain requirement, bacterium number contained by bacterial strain utmostly meets the needs such as employment and suitability test (E & ST) during drug microorganism detection, growth promotion test, the preparation method of sample within the scope of employment and suitability test (E & ST) standard requirements, simple process, success rate are high.
Description
Technical field
The invention belongs to the field of quality control in terms of microbiologic inhibition tests, in particular to a kind of aspergillus niger employment and suitability test (E & ST)
Bacterial strain and preparation method thereof.
Background technique
Between the particularity of drug, drug microbiological Test field it is very special, it is desirable that every time detection before cultivate
The test samples such as base are both needed to carry out applicability inspection, have defined concentration range requirement to test bacterial strain.Current existing pure bacterium
Strain is most of not to be quantified, only for qualitative use, only a small number of quantitative bacterial strains.Bacterial strain, uniformity are quantified for such
It is particularly important with stability, the problems such as that there are stability is poor for existing quantitative bacterial strain, and traffic condition is more demanding.
Aspergillus niger is a Common Species in aspergillus fungi, is distributed widely on soil, air and cereal, can cause to eat
The Biodeterioration of object, cereal and fruits and vegetables, some can produce the aflatoxin of carcinogenicity.If in the aspergillus niger sample of preparation,
Aspergillus niger easily changes (breeding and death etc.), and the storage life that will lead to sample is short, and the uniformity and stability of sample are poor.
Therefore, comprehensively consider the biochemical characteristic of bacterium, choose the metastable aspergillus niger of property to the uniformity and stabilization for guaranteeing sample
Property is particularly significant.The process specifications of aspergillus niger sample preparation are relatively high, and the uniformity of sample and stability and freeze-drying
Strain, the process conditions of freeze-drying, the use of freeze drying protectant, the medium of rehydration etc. have close relationship.
Aspergillus niger index in drug directly affects the hygienic quality and safety of drug.Therefore, uniformity and stability are prepared
All good aspergillus niger employment and suitability test (E & ST) bacterial strain, can be to avoid pathogenic risk.If there are aspergillus nigers in drug, easily patient is made
At infection etc., the life of patient is directly threatened.The randomness and uncertainty that aspergillus niger is examined, are truly reflected testing laboratory
Ability, this to improve Good Laboratory controlled level, ensure drug safety, or even break International trade practices, improve China
Drug international competitiveness all has special important meaning.
Summary of the invention
The object of the present invention is to provide it is a kind of meet employment and suitability test (E & ST) during drug microorganism detection require it is quantitative black
Aspergillus employment and suitability test (E & ST) bacterial strain；Meanwhile overcoming the number of bacteria of work total in aspergillus niger employment and suitability test (E & ST) bacterial strain in transport, storage
With test etc. during clump count can all change the problem of, it is a further object to provide the employment and suitability test (E & ST) bacterium
The preparation method of strain, simple process, success rate are high.
Present invention technical solution used for the above purpose is: aspergillus niger employment and suitability test (E & ST) bacterial strain, it is characterized in that:
It only include a kind of object bacteria: aspergillus niger, the aimed concn of object bacteria is 500~2000CFU/ branch, and every is equipped with the freeze-drying of 0.2g
Powder.
Employment and suitability test (E & ST) bacterial strain in the sample category Medicine inspection is applied to applicability during drug microorganism detection
Test.
The employment and suitability test (E & ST) bacterial strain is using trehalose, gelatin and sterile water as matrix, and wherein trehalose volume fraction is
10%, gelatin volume fraction is 1%.
The preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain, it is characterized in that: including the preparation of sample addition bacterial strain, freeze-drying guarantor
Preparation, sample freeze-drying, the uniformity of sample and five stability test, sample definite value steps of agent are protected, wherein sample was lyophilized
Journey are as follows: bacterial strain recovery passage-increasing bacterium-bacterium solution packing-bacteria suspension preparation-freeze-drying and packaging-storage, detailed process are as follows:
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that it is recovered and is grown at 20~25 DEG C, and right
The bacterial strain of recovery is identified；
(2) increase bacterium
Object bacteria culture scrapes bacterium colony from inclined-plane, is added in the freeze drying protectant of 10mL and bacterium is made to the logarithmic growth end of term
Liquid；
(3) bacterium solution dispenses
The bacterium solution that a upper process obtains is mixed with freeze drying protectant, 100mL bacterium solution is prepared, this bacterium solution is placed in magnetic force and is stirred
Mix be stirred continuously on device it is lower be averagely dispensed into bacterium solution storage bottle, every bottle of 5mL, is placed in liquid nitrogen and saves backup by totally 20 bottles.
(4) bacteria suspension is prepared
According to target aimed concn 500~2000CFU/ branch of bacterium takes appropriate above-mentioned 5mL bacterium solution and 1500mL freeze drying protectant
Mixing, is placed on magnetic stirring apparatus to be stirred continuously down and is dispensed into sample bottle (cillin bottle), and 0.3mL/ bottles, in addition bottle stopper, but want
There are gaps, not cover reality；
(5) it is lyophilized and packs
Sample bottle equipped with bacteria suspension is uncapped and is put into freeze dryer, 40~50h is freeze-dried, is tied when sample is freeze-dried
Shu Hou directly carries out jumping a queue in case, closing machine, and sample is vacuum state in sample bottle；
(6) it stores
It is kept in dark place under the conditions of sample is placed on -18 DEG C, random selected sample is issued to laboratory from whole samples
Or carry out uniformity and stability test.
The freeze drying protectant is the sterile water containing trehalose and gelatin, and wherein volume fraction is respectively as follows: trehalose
10%, gelatin 1%；The disposable bacterium solution for preparing same concentrations, the repeatable applicability examination for preparing multiple batches of same magnitude range
Test bacterial strain.
The uniformity of the sample and stability test are according to uniformity and stability test reference GB/T 15000.3-
2008 " rules and statistical method of standard sample work directive/guide (3) standard sample definite value " carry out.
Strain of the present invention is selected using aspergillus niger as object bacteria.
Aspergillus niger employment and suitability test (E & ST) bacterial strain of the invention has the advantage that characteristic: being a kind of with certain satisfaction detection side
Method requires the employment and suitability test (E & ST) bacterial strain of levels, and sets about from sample stability, passes through the research of special process, stability
With the research of uniformity, make that microorganism living, quantity do not change, biochemical character does not morph, from meeting special transport
Condition, form a set of perfect employment and suitability test (E & ST) bacterial strain technology of preparing, be suitable for the preparation of aspergillus niger employment and suitability test (E & ST) bacterial strain.
Detailed description of the invention
Fig. 1 is process flow chart of the invention.
Specific embodiment
With reference to the accompanying drawing and specific embodiment present invention is further described in detail, but the invention is not limited to tools
Body embodiment.
Embodiment 1
Aspergillus niger employment and suitability test (E & ST) bacterial strain only includes a kind of object bacteria: aspergillus niger (Aspergillus niger), target
The aimed concn of bacterium is 500~2000CFU/ branch, and every is equipped with the freeze-dried powder of 0.2g.The employment and suitability test (E & ST) bacterial strain is with seaweed
Sugar, gelatin and sterile water are matrix, and wherein trehalose volume fraction is 10%, gelatin volume fraction is 1%.
Employment and suitability test (E & ST) bacterial strain in the sample category Medicine inspection is applied to applicability during drug microorganism detection
Test.Specific test process steps are as follows:
After sample is opened, 2mL sterile water is added immediately and carries out rehydration, the concussion that is vortexed mixes well, and 100 μ L is taken to be inoculated into
Fluid nutrient medium to be measured is applied on culture medium to be measured, and 20~25 DEG C are cultivated 5~7 days.Observe its whether good, bacterium of growth
Whether consistent fall size, morphological feature.If well-grown, bacterium colony size, morphological feature are consistent, then illustrate tested culture medium and side
Method is suitable for test product inspection, on the contrary then illustrate that tested culture medium or method are not suitable for test sample inspection, should according to method into
The further treatment measures of row.
Embodiment 2
As shown in Figure 1, in a kind of embodiment 1 aspergillus niger employment and suitability test (E & ST) bacterial strain preparation method, including sample add bacterium
The preparation of strain, the preparation of freeze drying protectant, sample freeze-drying, the uniformity of sample and five stability test, sample definite value steps,
Specific step is as follows:
1, the selection of sample addition bacterial strain
It only include a kind of object bacteria: aspergillus niger, the bacterial strain is purchased from specified mechanism, government, and has bacterial strain certificate,
It ensure that the traceability of bacterial strain.
2, the preparation of freeze drying protectant
Sample is prepared as matrix (volume fraction: trehalose 10%, gelatin 1%) using trehalose, gelatin and sterile water and is lyophilized
Protective agent.
3, sample is lyophilized
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that it is recovered and is grown at 20~25 DEG C, and right
The bacterial strain of recovery is identified；
(2) increase bacterium
Object bacteria culture scrapes bacterium colony from inclined-plane, is added in the freeze drying protectant of 10mL and bacterium is made to the logarithmic growth end of term
Liquid；
(3) bacterium solution dispenses
The bacterium solution that a upper process obtains is mixed with freeze drying protectant, 50mL bacterium solution is prepared, this bacterium solution is placed in magnetic force and is stirred
Mix be stirred continuously on device it is lower be averagely dispensed into bacterium solution storage bottle, every bottle of 5mL, totally 10 bottles；
(4) bacteria suspension is prepared
According to target aimed concn 500~2000CFU/ branch of bacterium is prepared, and appropriate above-mentioned 5mL bacterium solution and 1500mL freeze-drying is taken to protect
Agent mixing is protected, is placed on magnetic stirring apparatus to be stirred continuously down and is dispensed into sample bottle (cillin bottle), 0.3mL/ bottles, in addition bottle stopper,
But reality is not covered there are gap；
(5) it is lyophilized and packs
Sample bottle equipped with bacteria suspension is uncapped and is put into freeze dryer, freeze drier parameter is by following setting:
Chilling rate (Cooling Rate) 0.5 DEG C/min
- 1 DEG C of 15min of early stage cold point (Incipient Freezing Point)
- 40 DEG C of cryogenic temperature (Cooling temperature)
- 29 DEG C of eutectic point (Melting Point Eutectic temperature)
20 DEG C of 120min of heating temperature (Heating temperature)
Start freeze drier, machine is directly entered the freezing dry process of sequencing, entire freeze-drying process 40h.
After sample freeze-drying, directly carry out jumping a queue in case；Closing machine.The not tight cillin bottle of plug is rejected,
Sample is vacuum state in cillin bottle；
(6) it stores
It is kept in dark place under the conditions of sample is placed on -18 DEG C, and the sample of preservation is suitably managed and detected, from complete
Random selected sample is issued to laboratory or carries out uniformity and stability test in portion's sample.
4, the uniformity and stability test of sample
Uniformity and stability inspection to object bacteria in sample are the main methods of verification sample preparation process validity.
Check sample homogeneity and stability according to uniformity and stability test according to GB/T 15000.3-2008 " standard sample work
Make the rule and statistical method of directive/guide (3) standard sample definite value " it carries out.Gained sample homogeneity and Detection of Stability method
And result is as follows:
12 samples are randomly selected respectively, using PetrifilmTMQuick mould yeast testing piece (RYM) test method,
Repeat condition tests the aspergillus niger of 2 × 12 parts of samples.Result data carries out statistical disposition, statistic procedure and knot with method of analysis of variance
Fruit is as follows::
1 variance analysis formula of table
In upper table,
2 sample homogeneity testing result of table
3 sample homogeneity of table tests the results of analysis of variance
Conclusion: under 95% fiducial probability, compared with other factors are to the influence of test result, the inhomogeneities of sample is
It is acceptable.
Using two kinds of stability test: one is the stability test under storage temperature (4 DEG C), another kind is
Stability test at high temperature (traffic condition of analog sample), selects three temperature spots, respectively 20 DEG C, 36 DEG C and
42℃.Periodic detection sample tests 3 samples for different temperature points different holding time, by 2 × 3 parts of sample results
Mean value (after logarithmic transformed) carries out statistical disposition, F value < F with method of analysis of varianceCritical value, then illustrate the stabilization of employment and suitability test (E & ST) bacterial strain
Property meet the requirements, while determining and meeting longest holding time of employment and suitability test (E & ST) bacterial strain requirement under condition of different temperatures.Stablize
Property test result is shown in Table 4 and table 5.
4 sample short-term stability testing result of table
5 sample short-term stability of table tests the results of analysis of variance
5, sample definite value
Specific steps are as follows:
Using PetrifilmTMQuick mould yeast testing piece (RYM) method, detects sample, according to GB/T
In the 15000.3-2008 rule and statistical method of " standard sample work directive/guide (3) standard sample definite value " corresponding principle into
The calculating of row characteristic value and uncertain assessment.
Embodiment 3
Each step of the preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain described in the present embodiment with phase in embodiment 2
Together, different technical parameter are as follows: freeze-drying process 45h.
Embodiment 4
Each step of the preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain described in the present embodiment with phase in embodiment 3
Together, different technical parameter are as follows: freeze-drying process 50h.
Claims (6)
1. aspergillus niger employment and suitability test (E & ST) bacterial strain, it is characterized in that: only including a kind of object bacteria: aspergillus niger, the aimed concn of object bacteria
For 500~2000CFU/ branch, every is equipped with the freeze-dried powder of 0.2g.
2. aspergillus niger employment and suitability test (E & ST) bacterial strain according to claim 1, it is characterized in that: being fitted in the sample category Medicine inspection
With property test bacterial strain, it is applied to employment and suitability test (E & ST) during drug microorganism detection.
3. aspergillus niger employment and suitability test (E & ST) bacterial strain according to claim 1, it is characterized in that: the employment and suitability test (E & ST) bacterial strain is with sea
Algae sugar, gelatin and sterile water are matrix, and wherein trehalose volume fraction is 10%, gelatin volume fraction is 1%.
4. the preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain according to claim 1, it is characterized in that: including that sample adds
The preparation of bacterial strain, the preparation of freeze drying protectant, sample freeze-drying, the uniformity of sample and stability test, sample definite value five steps
Suddenly, wherein sample freeze-drying process are as follows: bacterial strain recovery passage-increasing bacterium-bacterium solution packing-bacteria suspension preparation-freeze-drying and packaging-
Storage, detailed process are as follows:
(1) bacterial strain recovery passage
Reference culture is inoculated into nutrient agar slant medium, so that it is recovered and is grown at 20~25 DEG C, and to recovery
Bacterial strain identified；
(2) increase bacterium
Object bacteria culture scrapes bacterium colony from inclined-plane, is added in the freeze drying protectant of 10mL and bacterium solution is made to the logarithmic growth end of term；
(3) bacterium solution dispenses
The bacterium solution that a upper process obtains is mixed with freeze drying protectant, 100mL bacterium solution is prepared, this bacterium solution is placed in magnetic stirring apparatus
On be stirred continuously it is lower be averagely dispensed into bacterium solution storage bottle, every bottle of 5mL, is placed in liquid nitrogen and saves backup by totally 20 bottles.
(4) bacteria suspension is prepared
According to target 500~2000CFU/mL of aimed concn of bacterium takes appropriate above-mentioned 5mL bacterium solution and 1500mL freeze drying protectant mixed
It closes, is placed on magnetic stirring apparatus to be stirred continuously down and is dispensed into sample bottle (cillin bottle), 0.3mL/ bottles, in addition bottle stopper, but to stay
There is gap, not cover reality；
(5) it is lyophilized and packs
Sample bottle equipped with bacteria suspension is uncapped and is put into freeze dryer, 40~50h is freeze-dried, when sample freeze-drying terminates
Afterwards, it directly carries out jumping a queue in case, closing machine, sample is vacuum state in sample bottle；
(6) it stores
Be kept in dark place under the conditions of sample is placed on -18 DEG C, from whole samples random selected sample be issued to laboratory or into
Row uniformity and stability test.
5. the preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain according to claim 4, it is characterized in that: the frozen-dried protective
Agent is the sterile water containing trehalose and gelatin, and wherein volume fraction is respectively as follows: trehalose 10%, gelatin 1%；It is disposable to prepare
The bacterium solution of same concentrations, the repeatable employment and suitability test (E & ST) bacterial strain for preparing multiple batches of same magnitude range.
6. the preparation method of aspergillus niger employment and suitability test (E & ST) bacterial strain according to claim 4, it is characterized in that: the sample is equal
Even property and stability test are according to uniformity and stability test reference GB/T 15000.3-2008 " standard sample work directive/guide
(3) rule and statistical method of standard sample definite value " it carries out.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910149171.3A CN109797108A (en) | 2019-02-28 | 2019-02-28 | Aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof |
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| CN201910149171.3A CN109797108A (en) | 2019-02-28 | 2019-02-28 | Aspergillus niger employment and suitability test (E & ST) bacterial strain and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982712A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
| CN111321084A (en) * | 2020-04-10 | 2020-06-23 | 商城北纳创联生物科技有限公司 | Aspergillus niger quantitative bacterial tablet and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974607A (en) * | 2010-10-26 | 2011-02-16 | 冯广青 | Reagent and kit for detecting sensitivity of culture medium |
| CN106635802A (en) * | 2016-11-22 | 2017-05-10 | 浙江泰林生物技术股份有限公司 | Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof |
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
-
2019
- 2019-02-28 CN CN201910149171.3A patent/CN109797108A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974607A (en) * | 2010-10-26 | 2011-02-16 | 冯广青 | Reagent and kit for detecting sensitivity of culture medium |
| CN106635802A (en) * | 2016-11-22 | 2017-05-10 | 浙江泰林生物技术股份有限公司 | Solid-state freeze-dried product of quantitative strain and preparation method and using method thereof |
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110982712A (en) * | 2020-01-04 | 2020-04-10 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
| CN110982712B (en) * | 2020-01-04 | 2023-04-11 | 广东环凯生物科技有限公司 | Stabilizing agent for aspergillus niger spores and application thereof |
| CN111321084A (en) * | 2020-04-10 | 2020-06-23 | 商城北纳创联生物科技有限公司 | Aspergillus niger quantitative bacterial tablet and preparation method thereof |
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