CN107022597A - Microbial limit tests in anti-inflammatory capsule - Google Patents
Microbial limit tests in anti-inflammatory capsule Download PDFInfo
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- CN107022597A CN107022597A CN201710211991.1A CN201710211991A CN107022597A CN 107022597 A CN107022597 A CN 107022597A CN 201710211991 A CN201710211991 A CN 201710211991A CN 107022597 A CN107022597 A CN 107022597A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/10—Enterobacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
Abstract
The invention discloses microbial limit tests in a kind of anti-inflammatory capsule, it is desirable to provide a kind of simple to operate, microbial limit tests in the feasible anti-inflammatory capsule of method, technical points comprise the steps successively：1) prepared by bacterium solution；2) prepared by test liquid；3) measure of aerobic bacteria sum is carried out using dilution method, the measure of yeast and mold sum is carried out using conventional method；4) foundation and checking of the bacterium method of inspection are controlled, belongs to technical field of chemical detection.
Description
Technical field
The present invention relates to a kind of microbial limit tests, limit test of microbe side in especially a kind of anti-inflammatory capsule
Method, belongs to technical field of chemical detection.
Background technology
JINJI JIAONANG is the anti-inflammatory drugs such as treatment adnexitis, endometritis, pelvic infecton, is removed with clearing heat and detoxicating, invigorating the spleen
Wet, dredging collateral and promoting blood circulation effect, medicine contains the compositions such as cherokee rose root, reticulate millettia, waras, leatherleaf mahonia, Radix zanthoxyli, Herba Andrographitis, its
Middle Radix zanthoxyli, Herba Andrographitis have certain bacteriostasis.According to its route of administration and preparation method, should carry out aerobic bacteria sum, mould and
The measure of saccharomycete sum, and control bacterium --- EHEC, bile tolerance gram-negative bacteria, the inspection of salmonella.
The content of the invention
For above-mentioned technical problem, it is an object of the invention to provide one kind is simple to operate, in the feasible anti-inflammatory capsule of method
Microbial limit tests.
In order to solve the above technical problems, the technical scheme is that such：
A kind of microbial limit tests in anti-inflammatory capsule, the capsule is JINJI JIAONANG, is comprised the steps successively：
1) prepared by bacterium solution
Learn from else's experience 33 DEG C of staphylococcus aureuses for cultivating 24h, bacillus subtilis, pseudomonas aeruginosa pancreas junket soya peptone liquid
The Candida albicans Sabouraud dextrose liquid medium of body nutrient solution and 25 DEG C of culture 24h, with 0.9% aseptic sodium chloride solution system
It is standby into containing the bacteria suspension that bacterium number is 1000~10000cfu/ml；
Learn from else's experience 25 DEG C the aspergillus niger Sabouraud's dextrose agar inclined-plane cultivated 7 days, add 5ml containing 0.05% polyoxyethylene sorbitan monoleate
0.9% aseptic sodium chloride solution, spore is eluted, and spore suspension is suctioned out with the pipette with cotton, is gathered with containing 0.05%
0.9% aseptic sodium chloride solution of sorb ester 80 is made containing the spore suspension that spore count is 1000~10000cfu/ml, standby；
2) prepared by test liquid
This product 10g is taken, the pancreas junket soya peptone fluid nutrient medium for being preheated to 45 DEG C is added to 100ml, 45 DEG C of gas baths shake
30min, mixes, is used as 1:10 test liquids；
3) method is set up and recovery test
The measure of aerobic bacteria sum is carried out using dilution method, the measure of yeast and mold sum is carried out using conventional method.
4) foundation and checking of the bacterium method of inspection are controlled
EHEC, bile tolerance gram-negative bacteria and salmonella are carried out using conventional method to check.
As present invention further optimization, microbial limit tests in a kind of above-mentioned anti-inflammatory capsule are described
Conventional method comprises the steps successively：
Test group：Draw 1:10 test liquid 9.9ml, add 0.1ml experiment bacteria suspension, shake up by totally 5 parts in every part, point
1ml is not drawn from test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Separately inhaled respectively from fungi test tube
Take 1ml into plate, pour into Sabouraud glucose agar；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, 0.1ml experiment is added in every part
Bacteria suspension, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；Separately from true
1ml is drawn in bacterium test tube respectively into plate, Sabouraud glucose agar is poured into；The parallel preparation of every plant of test organisms 2 is put down
Ware；
Test sample group：Draw 1:10 test liquid 1ml are to plate, parallel 4 plates of preparation, wherein 2 are poured into pancreas junket soybean
Peptone agar medium, 2 are poured into Sabouraud glucose agar in addition；
Diluent group：Pancreas junket soya peptone fluid nutrient medium 1ml is drawn to plate, parallel 4 plates of preparation, wherein 2 are inclined
Pancreas junket soya peptone agar medium is noted, 2 are poured into Sabouraud glucose agar in addition；
Each plate pours into defined agar medium, it is to be solidified after, set point of temperature culture 3-5d is put respectively, is determined aerobic
Bacterium sum, yeast and mold number, calculate its rate of recovery, calculation formula is as follows；
Further, microbial limit tests in a kind of anti-inflammatory capsule on, under described dilution method includes successively
State step：
It is prepared by test liquid
This product 10g is taken, addition is preheated in 45 DEG C of pancreas junket soya peptone fluid nutrient medium to 100ml, 45 DEG C of gas bath shakings
30min, mixes, is used as 1:10 test liquids；Draw 1:10 test liquid 20ml are added in 80ml pancreas junket soya peptone fluid nutrient mediums, are shaken
It is even to be made 1:50 test liquids；
Dilution method
Test group：Draw 1:50 test liquid 9.9ml, add 0.1ml experiment bacteria suspension, shake up by totally 5 parts in every part, point
1ml is not drawn from test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Draw 1:50 test liquid 9.9ml,
Totally 5 parts, 0.1ml experiment bacteria suspension is added in every part, is shaken up, drawn 1ml from test organisms test tube respectively into plate, pour into
Pancreas junket soya peptone agar medium；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, 0.1ml experiment is added in every part
Bacteria suspension, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；Every plant of examination
Test bacterium 2 plates of parallel preparation；
Test sample group：Draw 1:50 test liquid 1ml are to plate, parallel 2 plates of preparation, pour into the training of pancreas junket soya peptone agar
Support base；
Each plate pours into defined agar medium, it is to be solidified after, set point of temperature culture 3d is put respectively, aerobic bacteria is determined total
Number, calculates its rate of recovery, calculation formula is as follows；
As present invention further optimization, microbial limit tests in a kind of above-mentioned anti-inflammatory capsule are described
The control bacterium method of inspection comprises the steps successively：
1) bacteria suspension is prepared
The P. aeruginosa bacteria suspension that bacteria containing amount is 1000~10000cfu/ml is taken, it is dilute with 0.9% aseptic sodium chloride solution
Release and the bacteria suspension containing bacterium number is 10~100cfu/ml is made, it is standby；
Learn from else's experience 33 DEG C and cultivate 24h EHEC, pseudomonas aeruginosa pancreas junket soya peptone liquid medium, with 0.9%
Aseptic sodium chloride solution is made containing the bacteria suspension that bacterium number is 10~100cfu/ml, standby；
3) test liquid is prepared
This product 10g is taken, the pancreas junket soya peptone fluid nutrient medium for being preheated to 45 DEG C is added to 100ml, 45 DEG C of gas baths shake
30min, mixes, is used as 1:10 test liquids；
3) test method
Take test liquid sample to carry out EHEC, bile tolerance gram-negative bacteria, salmonella to check；
Take test solution sample to add corresponding positive bacteria bacterium solution 1ml to be cultivated, carry out EHEC, bile tolerance leather blue cloudy
Property bacterium, salmonella check；
Negative control experiments take dilution 10ml to carry out EHEC, bile tolerance gram-negative bacteria, salmonella and checked,
It is used as negative control.
As present invention further optimization, microbial limit tests in a kind of above-mentioned anti-inflammatory capsule are described
EHEC inspection method comprises the steps successively：
Take 1：10 test liquid 10ml are seeded in 100ml pancreas junket soya peptone fluid nutrient mediums, 33 DEG C of culture 24h；Take culture
Thing 1.0ml, is seeded in 100ml Mai Kangkai fluid nutrient mediums, 44 DEG C of culture 24h, takes Mai Kangkai liquid culture streak inoculations
In maconkey agar flat board, 33 DEG C of culture 24h observe its colonial morphology.
As present invention further optimization, microbial limit tests in a kind of above-mentioned anti-inflammatory capsule are described
Bile tolerance gram-negative bacteria inspection method comprises the steps successively：
10ml enterobacteriaceae enrichment liquid body cultures base tube 1 is taken, 1 is added:10 test liquid 1mL, 33 DEG C of culture 24h；Take training
Thing streak inoculation is supported in purplish red cholate dextrose agar plate, 33 DEG C of culture 24h observe its colonial morphology.
As present invention further optimization, microbial limit tests in a kind of above-mentioned anti-inflammatory capsule are described
Salmonella inspection method comprises the steps successively：This product 10g is taken, is added in 100ml pancreas junket soya peptone fluid nutrient mediums, 33
DEG C culture 24h；Culture 0.1ml is taken, is seeded in 10mlRV detection of Salmonella enrichment liquid body culture mediums, 33 DEG C of culture 24h take RV husky
Door bacterium enrichment liquid body culture streak inoculation observes its bacterium in xylose-lysine-desoxycholate agar plate, 33 DEG C of culture 24h
Fall form.
The present invention compared with prior art, has the advantage that：
The method that the present invention is provided, it is determined that using dilution method (1:50) measure of aerobic bacteria sum is carried out, using conventional method
The measure of yeast and mold sum is carried out, the inspection of bacterium is controlled using conventional method.Through being fitted to the method used
Tested with property, meet version in 2015《Chinese Pharmacopoeia》Four 1105 non-sterile product limit test of microbe：Microorganism count method,
1106 non-sterile product limit test of microbe：Control bacteria examination method, 1107 non-sterile medicine limitation standard in microbe it is relevant
Regulation, method is feasible.
Embodiment
Claims of the present invention is described further with reference to specific embodiment, but not constituted to the present invention's
Any limitation, any available technical scheme of limited number of time modification of being made to the present invention still falls within invention which is intended to be protected.
Embodiment 1
1st, experimental article
1.l instrument and equipments
Biohazard Safety Equipment, biochemical cultivation case, full-automatic pressure sterilizer, Constant Temp. Oven, refrigerator, micro-wave oven,
Constant water bath box, electronic balance (sensibility reciprocal 0.1g), Thermo Scientific Maxi Mix II turbula shakers, gas bath are permanent
Warm oscillator, Syrup-homogenizing instrument.
1.2 glassware
Conical flask, plate, beaker, indexing suction pipe, test tube and silica gel plug, in 180 DEG C of hot air sterilization 2h or high steam
121 DEG C of sterilizing 15min.
1.3 apparatus
Aseptic clothing, cap, mouth mask, gloves, skin nipple, oese, alcolhol burner, cotton ball soaked in alcohol and Iodophor cotton balls, sterilizing scissors,
Sterilize tweezers, sterilising filtration cylinder, stainless steel spoon, rack for test tube, lighter, marking pen, experimental record paper etc..
1.4 test solutions and culture medium
L.4.1 thimerosal
0.1% benzalkonium bromide solution (for washing one's hands, wiping operation table top), 5% phenol solution (loads glass to disappear after preparing
Carried disease germs suction pipe for sterilization in malicious cylinder), 75% ethanol solution, iodophor solution.
1.4.2 diluent
It is 0.9% aseptic sodium chloride solution (dilution bacterium solution use), 0.9% containing 0.05% (ml/ml) polyoxyethylene sorbitan monoleate sterile
Sodium chloride solution, pancreas junket soya peptone fluid nutrient medium (dilution test sample use).
1.4.3 culture medium
Pancreas junket soya peptone agar medium, Sabouraud glucose agar, pancreas junket soya peptone fluid nutrient medium, Mai Kangkai
Fluid nutrient medium, maconkey agar culture medium, enterobacteriaceae enrichment liquid body culture medium, purplish red cholate glucose agar medium, RV
Detection of Salmonella enrichment liquid body culture medium, xylose-lysine-desoxycholate agar medium.
1.5 sample
JINJI JIAONANG, lot number：7516016th, 7516017,7516018, manufacturer：Lingfeng Pharmaceutical Co., Ltd., Guangxi
2nd, the foundation and checking of aerobic bacteria sum, mould and yeast total bacteria count method of counting employment and suitability test (E ＆ ST)
2.1 strain
Pseudomonas aeruginosa (Pseudomonas aeruginosa) [CMCC (B) 10104]
Staphylococcus aureus (Staphylococcus aureus) [CMCC (B) 26003]
Bacillus subtilis (.Bacillus Sulticis) [CMCC (B) 63501]
Candida albicans (Candida albicans) [CMCC (F) 98001]
Aspergillus niger (Aspeigillus niger) [CMCC (F) 98003]
National Institute for Food and Drugs Control provides
It is prepared by 2.2 bacterium solutions
Learn from else's experience 33 DEG C of staphylococcus aureuses for cultivating 24h, bacillus subtilis, pseudomonas aeruginosa pancreas junket soya peptone liquid
The Candida albicans Sabouraud dextrose liquid medium of body nutrient solution and 25 DEG C of culture 24h, with 0.9% aseptic sodium chloride solution system
It is standby into containing the bacteria suspension that bacterium number is 1000~10000cfu/ml；Learn from else's experience 25 DEG C the aspergillus niger Sabouraud dextrose fine jade cultivated 7 days
Fat inclined-plane, adds 0.9% aseptic sodium chloride solution that 5ml contains 0.05% (ml/ml) polyoxyethylene sorbitan monoleate, spore is eluted, with band
The pipette for having cotton suctions out spore suspension, with 0.9% aseptic sodium chloride solution containing 0.05% (ml/ml) polyoxyethylene sorbitan monoleate
It is made containing the spore suspension that spore count is 1000~10000cfu/ml, it is standby.
It is prepared by 2.3 test liquids
This product 10g is taken, the pancreas junket soya peptone fluid nutrient medium for being preheated to 45 DEG C is added to 100ml, 45 DEG C of gas baths shake
30min, mixes, is used as 1:10 test liquids.
2.4 methods are set up and recovery test
2.4.1 conventional method：
Test group：Draw 1:10 test liquid 9.9ml, add 0.1ml experiment bacteria suspension, shake up by totally 5 parts in every part, point
1ml is not drawn from test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Separately inhaled respectively from fungi test tube
Take 1ml into plate, pour into Sabouraud glucose agar；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, 0.1ml experiment is added in every part
Bacteria suspension, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；Separately from true
1ml is drawn in bacterium test tube respectively into plate, Sabouraud glucose agar is poured into；The parallel preparation of every plant of test organisms 2 is put down
Ware；
Test sample group：Draw 1:10 test liquid 1ml are to plate, parallel 4 plates of preparation, wherein 2 are poured into pancreas junket soybean
Peptone agar medium, 2 are poured into Sabouraud glucose agar in addition；
Diluent group：Pancreas junket soya peptone fluid nutrient medium 1ml is drawn to plate, parallel 4 plates of preparation, wherein 2 are inclined
Pancreas junket soya peptone agar medium is noted, 2 are poured into Sabouraud glucose agar in addition.
Each plate pours into defined agar medium, it is to be solidified after, set point of temperature culture 3-5d is put respectively, is determined aerobic
Bacterium sum, yeast and mold number, calculate its rate of recovery, are shown in Table 1.
The conventional method recovery test result of table 1
As a result show, the pseudomonas aeruginosa of aerobic bacteria sum, the staphylococcus aureus test organisms rate of recovery are respectively less than
50%, show that JINJI JIAONANG has inhibitory action to bacterium, carrying out aerobic bacteria sum using conventional method determines infeasible.
Each strain test organisms rate of recovery of yeast and mold shows JINJI JIAONANG to fungi without suppression in 50%-200%
Make and use, carrying out yeast and mold sum using conventional method determines feasible.
2.4.2 the total counting number of aerobic bacteria --- --- dilution method：
It is prepared by test liquid
This product 10g is taken, addition is preheated in 45 DEG C of pancreas junket soya peptone fluid nutrient medium to 100ml, 45 DEG C of gas bath shakings
30min, mixes, is used as 1:10 test liquids.Draw 1:10 test liquid 20ml are added in 80ml pancreas junket soya peptone fluid nutrient mediums, are shaken
It is even to be made 1:50 test liquids；
Dilution method：
Test group：Draw 1:50 test liquid 9.9ml, add 0.1ml experiment bacteria suspension, shake up by totally 5 parts in every part, point
1ml is not drawn from test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Draw 1:50 test liquid 9.9ml,
Totally 5 parts, 0.1ml experiment bacteria suspension is added in every part, is shaken up, drawn 1ml from test organisms test tube respectively into plate, pour into
Pancreas junket soya peptone agar medium；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, 0.1ml experiment is added in every part
Bacteria suspension, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；Every plant of examination
Test bacterium 2 plates of parallel preparation；
Test sample group：Draw 1:50 test liquid 1ml are to plate, parallel 2 plates of preparation, pour into the training of pancreas junket soya peptone agar
Support base；
Each plate pours into defined agar medium, it is to be solidified after, set point of temperature culture 3d is put respectively, aerobic bacteria is determined total
Number, calculates its rate of recovery, is shown in Table 2.
The dilution method recovery test result of table 2
As a result show, each strain test organisms rate of recovery of aerobic bacteria sum shows dilution method (1 in 50%-200%:50)
Inhibitory action of the JINJI JIAONANG to bacterium can be eliminated, using dilution method (1:50) carry out aerobic bacteria sum and determine feasible.
2.5 method validation
1 batch of test sample is taken, lot number is：7516016, by dilution method (1:50) progress aerobic bacteria sum, conventional method carry out mould
Bacterium and the method for counting checking of saccharomycete sum, the results are shown in Table 3 to table 7：
Table 3 first time result of the test
Second of the result of the test of table 4
The third time result of the test of table 5
3 times checking test is summarized：
The JINJI JIAONANG of the table 6 ----aerobic bacteria sum method of counting employment and suitability test (E ＆ ST) rate of recovery
The JINJI JIAONANG of the table 7 ----yeast and mold sum method of counting employment and suitability test (E ＆ ST) rate of recovery
3 times checking test result shows, with dilution method (1:50) carry out JINJI JIAONANG aerobic bacteria sum, carried out with conventional method
Yeast and mold sum is determined, and the rate of recovery of five plants of test organisms meets in 50%-200%《Chinese Pharmacopoeia》Version in 2015
Four 1105 non-sterile product limit test of microbe：The regulation of microorganism count method, method is feasible.
It is determined that using dilution method (1:50) aerobic bacteria in the non-sterile product limit test of microbe of JINJI JIAONANG is carried out total
Number is checked, yeast and mold sum in the non-sterile product limit test of microbe of JINJI JIAONANG is carried out using conventional method and is examined
Look into.
3rd, the foundation and checking of the bacterium method of inspection are controlled
3.1 strain
EHEC (Escherichia Coli) [CMCC (B) 44102]
Pseudomonas aeruginosa (Pseudomonas aeruginosa) [CMCC (B) 10104]
Moscow' paratyphi B (Salmonella paratyphi) [CMCC (B) 50094]
National Institute for Food and Drugs Control provides
3.1.1 bacteria suspension is prepared with 2.2
The P. aeruginosa bacteria suspension for taking 2.2 times bacteria containing amounts to be 1000~10000cfu/ml, uses 0.9% sterile NaCl
Solution dilution is made containing the bacteria suspension that bacterium number is 10~100cfu/ml, standby.
Learn from else's experience 33 DEG C and cultivate 24h EHEC, pseudomonas aeruginosa pancreas junket soya peptone liquid medium, with 0.9%
Aseptic sodium chloride solution is made containing the bacteria suspension that bacterium number is 10~100cfu/ml, standby；
The preparation of 3.2 test liquids is with 2.3
3.3 test method：
Test sample group takes the sample of ormal weight to do the inspection of Control bacteria examination method according to " 3.3.1 ", " 3.3.2 ", " 3.3.3 " operation
Look into.
Positive control experiment takes the sample of ormal weight according to " 3.3.1 ", " 3.3.2 ", " 3.3.3 " operation, and adds corresponding
Positive bacteria bacterium solution 1ml is cultivated, and is controlled accordingly bacterium inspection technique inspection.
Negative control experiments take dilution 10ml to replace test liquid to do control bacterium inspection according to " 3.3.1 ", " 3.3.2 ", " 3.3.3 "
Method inspection is looked into, negative control is used as.
3.3.1 EHEC：
Take 1：10 test liquid 10ml are seeded in 100ml pancreas junket soya peptone fluid nutrient mediums, 33 DEG C of culture 24h.Take culture
Thing 1.0ml, is seeded in 100ml Mai Kangkai fluid nutrient mediums, 44 DEG C of culture 24h, takes Mai Kangkai liquid culture streak inoculations
In maconkey agar flat board, 33 DEG C of culture 24h observe its colonial morphology.
3.3.2 bile tolerance gram-negative bacteria：
10ml enterobacteriaceae enrichment liquid body cultures base tube 1 is taken, 1 is added:10 test liquid 1mL, 33 DEG C of culture 24h.Take training
Thing streak inoculation is supported in purplish red cholate dextrose agar plate, 33 DEG C of culture 24h observe its colonial morphology.
3.3.3 salmonella：
This product 10g is taken, is added in 100ml pancreas junket soya peptone fluid nutrient mediums, 33 DEG C of culture 24h.Culture 0.1ml is taken,
It is seeded in 10mlRV detection of Salmonella enrichment liquid body culture mediums, 33 DEG C of culture 24h take the line of RV detection of Salmonella enrichment liquid bodies culture to connect
Plant in xylose-lysine-desoxycholate agar plate, 33 DEG C of culture 24h, observe its colonial morphology.
3.4 result of the test
3.3.1 EHEC：
Test sample group：Without colony growth in maconkey agar flat board；
Positive controls：There is typical EHEC colony growth in maconkey agar flat board；Gram stain microscopy
For G-Bacillus, biochemical identification result is typical ETEC biochemical reaction, is shown in Table 8.
The EHEC testing result of table 8
Negative control group：Without colony growth in maconkey agar flat board；
Show that JINJI JIAONANG can carry out EHEC inspection using conventional method, method is feasible.
3.3.2 bile tolerance gram-negative bacteria：
Test sample group：Without colony growth in purplish red cholate dextrose agar plate；
Positive controls：
EHEC positive controls：There is colony growth in purplish red cholate dextrose agar plate；
Pseudomonas aeruginosa positive controls：There is colony growth in purplish red cholate dextrose agar plate；
Negative control group：Without colony growth in purplish red cholate dextrose agar plate；
Show that JINJI JIAONANG can carry out bile tolerance gram-negative bacteria inspection using conventional method, method is feasible.
3.3.3 bile tolerance gram-negative bacteria：
Test sample group：Without colony growth in xylose-lysine-desoxycholate agar plate；
Positive controls：There is typical salmonella colony growth in xylose-lysine-desoxycholate agar plate；Leather
Blue Albert'stain Albert microscopy is G-Bacillus, preliminary biochemical identification result is typical Salmonella biochemical reaction, is shown in Table 9.
The bile tolerance gram-negative bacteria testing result of table 9
Negative control group：Without colony growth in xylose-lysine-desoxycholate agar plate；
Show that JINJI JIAONANG can carry out bile tolerance gram-negative bacteria inspection using conventional method, method is feasible.
3.5 determine that Control bacteria examination method is conventional method in the non-sterile product limit test of microbe of JINJI JIAONANG.
4 conclusions
By checking：It is determined that using dilution method (1:50) in the non-sterile product limit test of microbe for carrying out JINJI JIAONANG
Aerobic bacteria sum is checked, yeast and mold in the non-sterile product limit test of microbe of JINJI JIAONANG is carried out using conventional method
Sum is checked.
EHEC inspection, resistance to courage in the non-sterile product limit test of microbe of JINJI JIAONANG are carried out using conventional method
Salt gram-negative bacteria, salmonella.
The limit test of microbe of 5 three batches of JINJI JIAONANGs
By the non-sterile product microbial decolorization of the JINJI JIAONANG set up, three batches of samples are tested, as a result
It is shown in Table 10：
Table 10
It should be noted that the technical parameter that does not refer in detail to of the present invention, according to this area conventional technical means or
Parameter as defined in Chinese Pharmacopoeia is carried out.
Claims (7)
1. microbial limit tests in a kind of anti-inflammatory capsule, it is characterised in that comprise the steps successively：
1) prepared by bacterium solution
Learn from else's experience 33 DEG C of staphylococcus aureuses for cultivating 24h, bacillus subtilis, pseudomonas aeruginosa pancreas junket soya peptone liquid training
The Candida albicans Sabouraud dextrose liquid medium of nutrient solution and 25 DEG C of culture 24h, is made of 0.9% aseptic sodium chloride solution and contained
Bacterium number is 1000~10000cfu/ml bacteria suspension, standby；
Learn from else's experience 25 DEG C the aspergillus niger Sabouraud's dextrose agar inclined-plane cultivated 7 days, add 5ml containing 0.05% polyoxyethylene sorbitan monoleate
0.9% aseptic sodium chloride solution, spore is eluted, and spore suspension is suctioned out with the pipette with cotton, is gathered with containing 0.05%
0.9% aseptic sodium chloride solution of sorb ester 80 is made containing the spore suspension that spore count is 1000~10000cfu/ml, standby；
2) prepared by test liquid
This product 10g is taken, the pancreas junket soya peptone fluid nutrient medium for being preheated to 45 DEG C is added to 100ml, 45 DEG C of gas baths shake 30min,
Mix, be used as 1:10 test liquids；
3) method is set up and recovery test
The measure of aerobic bacteria sum is carried out using dilution method, the measure of yeast and mold sum is carried out using conventional method.
4) foundation and checking of the bacterium method of inspection are controlled
EHEC, bile tolerance gram-negative bacteria and salmonella are carried out using conventional method to check.
2. microbial limit tests in a kind of anti-inflammatory capsule according to claim 1, it is characterised in that described is normal
Rule method comprises the steps successively：
Test group：Draw 1:10 test liquid 9.9ml, totally 5 parts, in every part add 0.1ml experiment bacteria suspension, shake up, respectively from
1ml is drawn in test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Separately drawn respectively from fungi test tube
1ml pours into Sabouraud glucose agar into plate；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, the test organisms that 0.1ml is added in every part hangs
Liquid, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；It is another to be tried from fungi
1ml is drawn in pipe respectively into plate, Sabouraud glucose agar is poured into；Every plant of test organisms is parallel to prepare 2 plates；
Test sample group：Draw 1:10 test liquid 1ml are to plate, parallel 4 plates of preparation, wherein 2 are poured into pancreas junket soya peptone fine jade
Fat culture medium, 2 are poured into Sabouraud glucose agar in addition；
Diluent group：Pancreas junket soya peptone fluid nutrient medium 1ml is drawn to plate, parallel 4 plates of preparation, wherein 2 are poured into pancreas
Junket soya peptone agar medium, 2 are poured into Sabouraud glucose agar in addition；
Each plate pours into agar medium, it is to be solidified after, set point of temperature culture 3-5d is put respectively, aerobic bacteria sum, mould is determined
And yeast count, its rate of recovery is calculated, calculation formula is as follows；
3. microbial limit tests in a kind of anti-inflammatory capsule according to claim 1, it is characterised in that described is dilute
Interpretation of the law comprises the steps successively：
It is prepared by test liquid
This product 10g is taken, addition is preheated in 45 DEG C of pancreas junket soya peptone fluid nutrient medium to 100ml, 45 DEG C of gas bath shakings
30min, mixes, is used as 1:10 test liquids；Draw 1:10 test liquid 20ml are added in 80ml pancreas junket soya peptone fluid nutrient mediums, are shaken
It is even to be made 1:50 test liquids；
Dilution method
Test group：Draw 1:50 test liquid 9.9ml, totally 5 parts, in every part add 0.1ml experiment bacteria suspension, shake up, respectively from
1ml is drawn in test organisms test tube into plate, pancreas junket soya peptone agar medium is poured into；Draw 1:50 test liquid 9.9ml, totally 5
Part, 0.1ml experiment bacteria suspension is added in every part, is shaken up, 1ml is drawn from test organisms test tube respectively into plate, pours into pancreas
Junket soya peptone agar medium；Every plant of test organisms is parallel to prepare 2 plates；
Bacterium solution control group：Pancreas junket soya peptone fluid nutrient medium 9.9ml is drawn, totally 5 parts, the test organisms that 0.1ml is added in every part hangs
Liquid, shakes up, and draws 1ml from test organisms test tube respectively into plate, pours into pancreas junket soya peptone agar medium；Every plant of test organisms
2 plates of parallel preparation；
Test sample group：Draw 1:50 test liquid 1ml are to plate, and parallel 2 plates of preparation pour into pancreas junket soya peptone agar culture
Base；
Each plate pours into agar medium, it is to be solidified after, set point of temperature culture 3d is put respectively, aerobic bacteria sum is determined, calculates it
The rate of recovery, calculation formula is as follows；
4. microbial limit tests in a kind of anti-inflammatory capsule according to claim 1, it is characterised in that described control
The bacterium method of inspection processed comprises the steps successively：
1) bacteria suspension is prepared
The P. aeruginosa bacteria suspension that bacteria containing amount is 1000~10000cfu/ml is taken, is diluted and made with 0.9% aseptic sodium chloride solution
It is standby into containing the bacteria suspension that bacterium number is 10~100cfu/ml；
Learn from else's experience 33 DEG C and cultivate 24h EHEC, pseudomonas aeruginosa pancreas junket soya peptone liquid medium, it is sterile with 0.9%
Sodium chloride solution is made containing the bacteria suspension that bacterium number is 10~100cfu/ml, standby；
2) test liquid is prepared
This product 10g is taken, the pancreas junket soya peptone fluid nutrient medium for being preheated to 45 DEG C is added to 100ml, 45 DEG C of gas baths shake 30min,
Mix, be used as 1:10 test liquids；
3) test method
Take test liquid sample to carry out EHEC, bile tolerance gram-negative bacteria, salmonella to check；
Take test solution sample to add corresponding positive bacteria bacterium solution 1ml to be cultivated, carry out EHEC, bile tolerance Grain-negative
Bacterium, salmonella check；
Negative control experiments take dilution 10ml to carry out EHEC, bile tolerance gram-negative bacteria, salmonella and checked, as
Negative control.
5. microbial limit tests in a kind of anti-inflammatory capsule according to claim 3, it is characterised in that described is big
The uncommon bacterium inspection method of intestines angstrom comprises the steps successively：
Take 1：10 test liquid 10ml are seeded in 100ml pancreas junket soya peptone fluid nutrient mediums, 33 DEG C of culture 24h；Take culture
1.0ml, is seeded in 100ml Mai Kangkai fluid nutrient mediums, 44 DEG C culture 24h, take the streak inoculation of Mai Kangkai liquid cultures in
Maconkey agar flat board, 33 DEG C of culture 24h, observes its colonial morphology.
6. microbial limit tests in a kind of anti-inflammatory capsule according to claim 3, it is characterised in that described is resistance to
Cholate gram-negative bacteria inspection method comprises the steps successively：
10ml enterobacteriaceae enrichment liquid body cultures base tube 1 is taken, 1 is added:10 test liquid 1mL, 33 DEG C of culture 24h；Take culture
Streak inoculation observes its colonial morphology in purplish red cholate dextrose agar plate, 33 DEG C of culture 24h.
7. microbial limit tests in a kind of anti-inflammatory capsule according to claim 3, it is characterised in that described sand
Door Salmonella inspection method comprises the steps successively：This product 10g is taken, is added in 100ml pancreas junket soya peptone fluid nutrient mediums, 33 DEG C
Cultivate 24h；Culture 0.1ml is taken, is seeded in 10mlRV detection of Salmonella enrichment liquid body culture mediums, 33 DEG C of culture 24h take RV sramana
The body culture streak inoculation of bacterium enrichment liquid observes its bacterium colony in xylose-lysine-desoxycholate agar plate, 33 DEG C of culture 24h
Form.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048524A (en) * | 2017-12-20 | 2018-05-18 | 江苏红豆杉药业有限公司 | The detection method of microbial limit in a kind of injection activated carbon |
| CN108300755A (en) * | 2018-01-26 | 2018-07-20 | 北京朗依制药有限公司 | The microbial limit tests of dispersible tablet of pidotimod |
| CN108300756A (en) * | 2018-01-26 | 2018-07-20 | 北京朗依制药有限公司 | The microbial limit tests of Nifuratel capsule |
| CN108315384A (en) * | 2018-01-26 | 2018-07-24 | 北京朗依制药有限公司 | The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule |
| CN109777856A (en) * | 2018-12-20 | 2019-05-21 | 武汉科诺生物科技股份有限公司 | A kind of detection method of 1,000,000,000,000 live spores per gram Bacillus subtillis original powder brood cell's number living |
| CN113122611A (en) * | 2019-12-30 | 2021-07-16 | 海南先声药业有限公司 | Method for checking microbial limit of montmorillonite powder |
| CN114563369A (en) * | 2022-03-08 | 2022-05-31 | 正大制药（青岛）有限公司 | Quality detection method for cyclobenzaprine hydrochloride sustained-release capsule |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533931A (en) * | 2012-03-13 | 2012-07-04 | 陕西省食品药品检验所 | Inspection method and verification for salmonella in lip care cosmetics |
| CN102586393A (en) * | 2012-02-24 | 2012-07-18 | 陕西省食品药品检验所 | Microbial limit detection method of miconazole nitrate suppository |
| CN103834718A (en) * | 2012-11-23 | 2014-06-04 | 青岛康地恩动物药业有限公司 | Microbe limit inspection method of Qinglu tincture |
| US20160002697A1 (en) * | 2013-08-30 | 2016-01-07 | Tsumura & Co. | Microorganism detection method |
-
2017
- 2017-04-01 CN CN201710211991.1A patent/CN107022597A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586393A (en) * | 2012-02-24 | 2012-07-18 | 陕西省食品药品检验所 | Microbial limit detection method of miconazole nitrate suppository |
| CN102533931A (en) * | 2012-03-13 | 2012-07-04 | 陕西省食品药品检验所 | Inspection method and verification for salmonella in lip care cosmetics |
| CN103834718A (en) * | 2012-11-23 | 2014-06-04 | 青岛康地恩动物药业有限公司 | Microbe limit inspection method of Qinglu tincture |
| US20160002697A1 (en) * | 2013-08-30 | 2016-01-07 | Tsumura & Co. | Microorganism detection method |
Non-Patent Citations (2)
| Title |
|---|
| FDA: "Pharmaceutical Microbiology Manual 2014", 《HTTPS://WWW.FDA.GOV/FILES/ABOUT%20FDA/PUBLISHED/PHARMACEUTICAL-MICROBIOLOGY-MANUAL.PDF》 * |
| 罗达龙 等: "盐酸氨溴索缓释胶囊微生物限度检查的方法学验证", 《临床医药文献杂志》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048524A (en) * | 2017-12-20 | 2018-05-18 | 江苏红豆杉药业有限公司 | The detection method of microbial limit in a kind of injection activated carbon |
| CN108300755A (en) * | 2018-01-26 | 2018-07-20 | 北京朗依制药有限公司 | The microbial limit tests of dispersible tablet of pidotimod |
| CN108300756A (en) * | 2018-01-26 | 2018-07-20 | 北京朗依制药有限公司 | The microbial limit tests of Nifuratel capsule |
| CN108315384A (en) * | 2018-01-26 | 2018-07-24 | 北京朗依制药有限公司 | The microbial limit tests of Nifuratel nystatin expandable vaginal soft capsule |
| CN109777856A (en) * | 2018-12-20 | 2019-05-21 | 武汉科诺生物科技股份有限公司 | A kind of detection method of 1,000,000,000,000 live spores per gram Bacillus subtillis original powder brood cell's number living |
| CN113122611A (en) * | 2019-12-30 | 2021-07-16 | 海南先声药业有限公司 | Method for checking microbial limit of montmorillonite powder |
| CN114563369A (en) * | 2022-03-08 | 2022-05-31 | 正大制药（青岛）有限公司 | Quality detection method for cyclobenzaprine hydrochloride sustained-release capsule |
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