CN107727863A - The detection method of endotoxin content in yeast extract - Google Patents
The detection method of endotoxin content in yeast extract Download PDFInfo
- Publication number
- CN107727863A CN107727863A CN201710828600.0A CN201710828600A CN107727863A CN 107727863 A CN107727863 A CN 107727863A CN 201710828600 A CN201710828600 A CN 201710828600A CN 107727863 A CN107727863 A CN 107727863A
- Authority
- CN
- China
- Prior art keywords
- endotoxin
- solution
- yeast extract
- test
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/579—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving limulus lysate
Abstract
The invention discloses a kind of detection method of endotoxin content in yeast extract, it is tested by xeothermic program effectiveness, Sensilivily Check is tested, interference test and gel sxemiquantitative success of the test establish endotoxin content detection method in yeast extract, its is simple and easy to operate, it is reproducible, endotoxic appraisal procedure in yeast extract can be used as, is worthy of popularization.Test specimen and detection method meet《Pharmacopoeia of People's Republic of China》Issued with state food pharmaceuticals administration general bureau《Bacterial endotoxin test method routinely with jumping to criticize examine by monitoring》The relevant regulatory requirements of professional standard.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to the detection side of endotoxin content in a kind of yeast extract
Method.
Background technology
Yeast extract is using the food yeast of rich in protein as raw material, using biotechnology, by yeast cells
Interior protein, nucleic acid etc. refine the natural flavouring formed after being degraded, and it contains 20 kinds of amino acid and polypeptide, additionally
Contain the plurality of active ingredients such as nucleotides, vitamin, organic acid and mineral matter.Amino acid balance is good in yeast extract, taste
Road is delicious strong, has meat-like flavor, thus yeast extract is the excellent food for having nutritious, seasoning and health care three zones concurrently
Flavoring, it is widely used in biology laboratory, pharmacy industry, fermentation industry, health-care medical and Animal nutrition etc..Due to ferment
Female extract can provide amino acid and vitamins and other nutritious components for the growth of cell, can promote cell growth.In recent years, it is domestic
Have started to carry out application study using it as the adding ingredient of Zooblast culture medium outside.
At present, the existing mature yeast breaking-wall cell in China obtains yeast extract technology, possesses and have developed culture early stage
Base yeast extract, Bacteria Culture, fermentation industry start to apply at home, to carry out the research of Zooblast culture medium accumulation
Experience and technology, the said firm develop culture ground level yeast extract, can as the addition factor of serum free medium, and
The composition of non-animal source, completely can be as the compositing formula of serum free medium.As serum free medium except ensureing to cultivate
Composition meets outside Hemapoiesis and the needs of development in base, and important requirement is that the middle endotoxin content of culture medium must be tight
Lattice control within the limits prescribed.Therefore, yeast extract wants the important set for successfully substituting serum as serum free medium
Into composition, it is necessary to have a clear and definite sign to its endotoxin content, to prepare culture medium selection application, but currently not
Seeing has yeast extract endotoxin detection method research.
The content of the invention
It is an object of the invention to provide a kind of detection method of endotoxin content in yeast extract, can fast and accurately examine
Endotoxic content in yeast extract is found, guidance is provided for its application.
In order to solve the above technical problems, the technical solution adopted in the present invention is：Endotoxin contains in a kind of yeast extract
The detection method of amount, it is characterised in that comprise the following steps：
1) xeothermic program effectiveness experiment
Not xeothermic endotoxin serial solution and the endotoxin serial solution after dry heat treatment are prepared respectively, using TAL
Tested, calculate endotoxin and decay and determine specific effectively dry heat treatment condition.
2) Sensilivily Check is tested
Prepare the endotoxin working standard serial solution of TAL to be checked and various concentrations, then by TAL with
Endotoxin working standard serial solution is mixed respectively, while does negative control with water using inspection, calculates TAL spirit
The measured value λ of sensitivityc, when its result is between the λ of 0.5 λ~2.0, then sensitivity is qualified；If unqualified, TAL need to be changed
Inspection test is carried out until qualified.In addition, the TAL of the different production batch of purchase must also carry out review experiment.
3) interference test
Yeast extract test sample serial solution, endotoxin standard work product and yeast extract test sample is set to mix respectively
Close serial solution, endotoxin standard work product serial solution and check and use water negative control solution, then utilize and answered through sensitivity
The qualified TAL of core is tested, it is ensured that test sample not interference test under what concentration；
4) gel sxemiquantitative is tested
Yeast extract test sample serial solution is set respectively, and endotoxin standard work product mix with yeast extract test sample
Close solution, endotoxin standard work product serial solution and check and use water negative control solution, it is qualified through Sensilivily Check to recycle
TAL tested, finally at calculating need testing solution endotoxin concns；Wherein yeast extract test sample passes through step
It is rapid 3) in interference test.
Further, dry heat treatment condition in step 1)：Baking temperature be 220~280 DEG C, soaking time be 70~
110min。
In addition, in the experiment of xeothermic program effectiveness, endotoxin used is endotoxin indicator, not xeothermic endogenous toxic material prime system
The speed of row solution dilution is between 1000-50000；The extension rate of endotoxin serial solution through dry heat treatment is at 1-20 times
Between.Preferably in scheme, the speed of not xeothermic endotoxin serial solution dilution is respectively 2500,5000,10000,20000 and
40000；The extension rate of endotoxin serial solution through dry heat treatment is respectively 1,2,4,6,8.
Preferably, in step 2), sensitivity of the limulus reagent sign value λ is 0.015~0.500EU/mL；More preferably
0.125EU/mL；Concentration after the work product dilution of endotoxin standard is in 0.1~2.0EU/mL, further preferred diluted concentration point
It is not：0.125、0.250、0.500、1.000EU/mL.
Preferably, sensitivity of the limulus reagent sign value λ is 0.030~1.000EU/mL in step 3), more preferably
0.500EU/mL；Concentration after the work product dilution of endotoxin standard is 0.1~2.0EU/mL, further preferred diluted concentration point
It is not：0.250、0.500、1.000、2.000EU/mL；Concentration after yeast extract dilution is 0.01~0.500mg/mL, is entered
One step is preferably 0.025mg/mL.
Preferably, sensitivity of the limulus reagent sign value λ is 0.015~0.500EU/mL in step 4)；Endotoxin standard works
Concentration after product dilution is 0.1~2.0EU/mL.It is further preferred that sensitivity of the limulus reagent sign value λ is 0.125EU/mL；It is interior
Concentration after the work product dilution of toxin standard is respectively 0.250,0.500,1.000,2.000EU/mL.
Wherein, step 3) is primary dcreening operation scope, and step 4) is the refinement on the basis of primary dcreening operation scope, in sensitivity of the limulus reagent
Selection on can make corresponding adjustment.
The invention further relates to application of the methods described in the detection of yeast extract endotoxin content.
The invention further relates to methods described the various microorganisms containing yeast extract, zooblast culture medium in
The application of content of toxins detection.
Method provided by the invention passes through in actually detected middle application, it was demonstrated that test specimen and detection method meet《China
People's republic's pharmacopeia》Issued with state food pharmaceuticals administration general bureau《Bacterial endotoxin test method routinely monitoring with
Jump batch inspection》The relevant regulatory requirements of professional standard.This detection method is tried by interference program efficiency assay, Sensilivily Check
Test, interference test and gel sxemiquantitative experiment, be successfully established endotoxin content detection method in yeast extract, it is easy easily
Operation, it is reproducible, endotoxic appraisal procedure in yeast extract can be used as, is worthy of popularization.
The present invention also has the advantages that：
Cell and the mankind are very sensitive to induced by endotoxin, when the infection concentration of cell and people respectively in 10.0EU/mL and
During more than 1.0EU/mL, strong adverse reaction can be caused, therefore culture medium to cell and biological products and its for producing
The strict control of endotoxin content requirement of the supplementary material of biological products.This research overcomes complicated component in yeast extract, held
The deficiencies of easy moisture absorption, endotoxin detection method in yeast extract is established first, check that yeast soaks with commercially available TAL
It is feasible to go out endotoxin method in thing.This method is trained for endotoxic detection in yeast extract and the cell containing yeast extract
The research and development for supporting base are respectively provided with important directive significance.
Embodiment
The present invention is further illustrated with reference to embodiment, but it is as described below, only it is to presently preferred embodiments of the present invention
, not limit the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents are changed to the equivalent embodiment changed on an equal basis.It is every without departing from the present invention program content, according to the present invention's
Technical spirit is all fallen within protection scope of the present invention to any simple modification made for any of the above embodiments or equivalent variations.
Embodiment 1：
1st, test specimen
Cultivate ground level yeast extract：FM902, lot number：2017030601B9, Angel Yeast Co., Ltd provide.
2nd, main agents
Endotoxin indicator：Zhanjiang Andusi Biology Co., Ltd.；Bacterial endotoxin working standard：Spend this life in comfort in Zhanjiang
Thing Co., Ltd；TAL：Specification 0.1mL/ branch, sensitivity：0.5、0.25、0.125、0.06、0.03、0.015EU/mL：It is profound
This biological Co., Ltd of Jiang'an degree；Without thermal source sky ampoule bottle (10mL), without heat source check water, without thermal source pipettor gun head：It is profound
This biological Co., Ltd of Jiang'an degree.
3rd, key instrument
BET-40 type instrument for testing endotoxin：Tianjin Pharmacopoeia Standard Instrument Factory；Vortex mixed instrument, U.S. Scientific
Industries companies；Superclean bench, Beijing Dong Lianhaer instrument manufacturings Co., Ltd；Ultra-low temp, Qingdao Haier
Extraordinary Electrical Appliances Co., Ltd；Automatic distilled water distiller, Shanghai Ya Rong biochemical equipments Instrument Ltd..
Specifically detection method is：
1) xeothermic program effectiveness experiment
Not xeothermic endotoxin serial solution is prepared and detection
Specific test procedure is as follows：
1. 1 endotoxin indicator (ECV) of not xeothermic mistake is dissolved with inspection water 1mL, on vortex mixer acutely
Shake 15min；
2. taking the solution 100 μ L, add 900 μ L inspection water, dilute 3 times, every time dilution be take last time solution 100 and
Detection is mixed with the μ L of water 900, and to mix 30~60s before dilution every time, obtains the solution that concentration is 1.25EU/ μ L；Then
Dilute successively：Take 800 μ L solution to check with 1200 μ L to be diluted with water, obtain 0.5EU/ μ L solution；1000 μ L solution and 1000 μ are taken again
L is checked and is diluted with water, and obtains 0.25EU/ μ L solution；Take 1000 μ L solution to check with 1000 μ L again to be diluted with water, obtain 0.125EU/ μ L
Solution；Take 1000 μ L solution to check with 1000 μ L again to be diluted with water, obtain 0.06EU/ μ L solution；1000 μ L solution and 1000 μ are taken again
L is checked and is diluted with water, and obtains 0.03EU/ μ L solution.
3. each strength solution takes 0.1mL to add in the TAL original ampoule that 0.1mL/ is propped up (parallel to do 2 pipes) respectively, and pipe is light
After light mixing, with aluminium foil sealed tube mouth, vertically it is put into 37 DEG C ± 1 DEG C of thermostat, is incubated 60 ± 2 minutes.
4. test tube is gently taken out from thermostat, 180 ° are slowly reversed.If pipe in formed gel, and gel it is indeformable,
Do not slipped from tube wall as positive (avoiding shaking).
5. reaction end refers to the concentration of last result that is positive in the endotoxin concns that series is successively decreased.
Specific result of the test is as shown in table 1.
The not xeothermic endotoxin detection result of the test of table 1
+ sign positive findings ,-sign negative findings.
Prepare and detect through dry heat treatment endotoxin serial solution
Specific test procedure is as follows：
1. taking 1 EVC, breaking off ampoule neck, sealed a bottle with aluminium foil, be put into baking oven (250 DEG C, 90 minutes) it is xeothermic after take
Go out.
2. dissolving the ECV of xeothermic mistake with inspection water 1mL, 15min is acutely shaken on vortex mixer.
3. diluting 2 times successively, dilute four times, mixed every time with 500 μ L solution with 500 μ L inspections with water.Every time before dilution
Mix 30~60s.
4. each strength solution takes 0.1mL to add in the TAL original ampoule that 0.1mL/ is propped up (parallel to do 2 pipes) respectively, and pipe is light
After light mixing, with aluminium foil sealed tube mouth, vertically it is put into 37 DEG C ± 1 DEG C of thermostat, is incubated 60 ± 2 minutes.
5. test tube is gently taken out from thermostat, 180 ° are slowly reversed, sees whether gel.Tied to appearance is negative
Fruit.
Specific test result is shown in Table 2.
Table 2 detects result of the test through xeothermic endotoxin
+ sign positive findings ,-sign negative findings.
Calculated according to Tables 1 and 2 testing result：
Recyclable endotoxin concns：Rc=λ D1mL (λ=0.125EU/mL)=0.125 × 20000 × 1=2500,
Remaining endotoxin：Rs=λ D1mL (λ=0.125EU/mL)=0.125 × 1 × 1=0.125.
Endotoxin decay test result：LgRd=lgRc-lgRs=lg2500-lg0.125=4.301>4.
The professional standard in China：Endotoxin is decayed：(States Pharmacopoeia specifications of China 2015 editions work as lgRd to lgRd=lgRc-lgRs>4
Or lgRd>When 3, xeothermic program is effective).
From experimental result：The xeothermic program drafted：250 DEG C, effectively (subsequent experimental is carried out 90min according to this program
Remove exogenous endotoxin operation).
2) Sensilivily Check is tested
Selected sensitivity of the limulus reagent sign value λ=0.5EU/mL, bacterial endotoxin working standard 14EU/ branch.
Specific test procedure is as follows：1. taking 18 TALs to be separately added into 0.1mL inspections to be dissolved with water, obtaining every concentration is
0.5EU/mL is standby.2. bacterial endotoxin working standard gradient dilution is as follows：1 bacterial endotoxin working standard is taken, is added
Enter 1mL inspection water, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration；100 μ L solution and 1300 μ L are taken to check
Mixed with water, it is 1EU/mL (2 λ) to obtain concentration；700 μ L solution are taken to be mixed with 700 μ L inspections with water again, obtaining concentration is
0.5EU/mL(λ)；700 μ L solution are taken to be mixed with 700 μ L inspections with water again, it is 0.25EU/mL (0.5 λ) to obtain concentration；Take again
700 μ L solution are mixed with 700 μ L inspections with water, and it is 0.125EU/mL (0.25 λ) to obtain concentration；Above-mentioned each step dilution all should be
Mixed 30 seconds on eddy mixer.3. 0.1mL is taken to add in 18 test tubes respectively the TAL of 18 dissolvings, wherein 16 pipes
The endotoxin standard liquid of 0.1mL various concentrations is separately added into, each concentration is parallel to do 4 pipes, and 2 pipes add 0.1mL inspections in addition
Look into and do negative control with water, after solution gently mixes in test tube, close the mouth of pipe, be vertically put into 37 DEG C ± 1 DEG C of insulating box,
Gently taken out after 60 ± 2min of insulation, slowly reverse 180 ° of observations.
Result of the test is shown in Table 3.
The sensitivity of the limulus reagent inspection test result (sensitivity of the limulus reagent of table 3：0.5EU/mL)
+ sign positive findings ,-sign negative findings.
If gel is formed in pipe, and gel is indeformable, is not determined as the positive from tube wall slippage person；Gel or shape are not formed
Into gel it is not solid, deform and be determined as feminine gender from tube wall slippage person.As shown in Table 3, when the λ pipes of Cmax 2 are the positive,
The λ pipes of least concentration 0.25 are feminine gender, and negative control pipe is feminine gender, and experiment is valid.It is dense that reaction end is calculated as follows
The measured value (λ c) of the geometrical mean of degree, as sensitivity of the limulus reagent, wherein λ c=antilg (∑ X/4), X are reaction end
Log concentration value lg, reaction end concentration refer to the concentration of last result that is positive in the endotoxin concns that series is successively decreased.
When λ c are in 0.5 λ~2 λ (including 0.5 λ and 2 λ), baterial endotoxin test can be used for, and to indicate sensitivity λ as this batch of horseshoe crab
The sensitivity of reagent.
Calculated according to the experimental result of table 3：X1=X2=X3=lg0.25, X4=lg0.50.λ c=antilg (∑ X/
4)=antilg (lg0.25 × 3+lg0.50)/4=antilg (- 0.5268)=0.2973.0.25=0.5 λ≤λ c=
0.2973≤2 λ=1.0.From experimental result：This batch sensitivity is that 0.5EU/mL TAL is qualified, available in YE
Toxin Inspection and analysis.Other sensitivity are：0.25th, 0.125,0.06,0.03,0.015EU/mL TALs review method with it is above-mentioned
Method is similar.
3) FM902 gel methods interference test
Selected sensitivity of the limulus reagent sign value λ=0.5EU/mL, bacterial endotoxin working standard 14EU/ branch.Specific examination
It is as follows to test step：The preparation (C groups) of product mother liquor 1. the 4.2mL endotoxins standard that concentration is 2 λ works：Take 1 bacterial endotoxin work
Make standard items, add 1mL inspection water, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration, takes 150 μ L respectively
Solution mixes the mother liquor for producing that two pipe concentration are 2 λ, common 4.2mL with 1.950mL inspections with water.2. concentration is 0.1mg/mL for examination
The preparation (A groups) of product solution：Weigh 0.5mg yeast bacterium powders to be dissolved in having in the test tube of 5mL inspection water, shake up and produce concentration and be
0.1mg/mL need testing solutions.Then 2.5mL solution is taken to be mixed with 2.5mL inspections with water successively, it is 0.05mg/mL to obtain concentration
Need testing solution；2.5mL solution is taken to be mixed with 2.5mL inspections with water again, it is 0.025mg/mL need testing solutions to obtain concentration；Again
2.5mL solution is taken to be mixed with 2.5mL inspections with water, it is 0.0125mg/mL need testing solutions to obtain concentration.3. concentration is 2 λ's
The preparation (B groups solution) of 2mL endotoxins standard work product and test sample mixed liquor：1 bacterial endotoxin working standard is taken, is added
Enter 1mL inspection water, 15min mixed on eddy mixer, it is 14EU/mL standard items to obtain concentration, then take 150 μ l liquid with
1950 μ L need testing solutions mix.4. take 42 sensitivity to be separately added into 0.1mL inspections for 0.5EU/mL TALs to be dissolved with water
It is stand-by.5. often managing the TAL and corresponding solution that each addition 0.1mL sensitivity is 0.5EU/mL, result of the test table is shown in specific dilution
4。
According to《Pharmacopoeia of People's Republic of China》The regulation of (2015 editions) bacterioscopy method：Only when solution A and feminine gender are right
According to solution D all parallel pipes all for feminine gender, and serial solution C result sensitivity of the limulus reagent review in the range of when, examination
Test valid.
Foundation：Es=antilg (∑ Xs/4)=antilg (0.5 × 4 ÷ 4)=0.5 λ,
Et=antilg (∑ Xt/4)=antilg (0.25 × 4 ÷ 4)=0.25 λ
(X：For reaction end when concentration logarithm value, Es be C reaction end concentration geometrical mean, Et be B reaction eventually
The geometrical mean of point concentration), if meet Es the λ (including 0.5 λ and 2 λ) and Et of 0.5 λ~2 0.5Es~2Es (including
0.5Es and 2Es) regulation.So finally think test sample noiseless effect under the concentration.Carried out according to the experimental result of table 4
Calculate：Above-mentioned experiment determines that the experiment is effective when the concentration that batch is FM902 is 0.025mg/mL.
4) sxemiquantitative of FM902 gels is tested
Selected sensitivity of the limulus reagent sign value λ=0.125EU/mL, bacterial endotoxin working standard 14EU/ branch.
Specific test procedure is as follows：The preparation (C groups) of product mother liquor 1. the endotoxin standard that concentration is 2 λ works：Take 1 thin
Bacterium endotoxin working standard, 1mL inspection water is added, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration, then
Take 25 μ l solution and 675 μ L inspections to mix to obtain 0.5EU/mL solution with water, then take 0.5mL solution to be mixed with 0.5ml inspections with water
Produce 0.25EU/ml (2 λ) mother liquor.2. the preparation (A groups) of need testing solution：Pass through the need testing solution of interference test.3. concentration
For the preparation (B groups solution) of 2 λ endotoxin standard work product and test sample mixed liquor：Take 1 bacterial endotoxin working stamndard
Product, 1mL inspection water is added, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration, then takes 25 μ L solution and 675 μ
L need testing solutions mix to obtain 0.5EU/mL solution, then take 0.5mL solution to mix the endogenous toxic material for producing 2 λ with 0.5mL need testing solutions
Plain standard work product and test sample mixed liquor.4. taking 20 TALs to be separately added into 0.1mL inspections to be dissolved with water, every concentration is obtained
It is stand-by for 0.5EU/mL.5. often manage the TAL and corresponding solution that each addition 0.1mL sensitivity is 0.5EU/mL.Specific dilution is shown in
Result of the test table 5.
The FM902 gel method sxemiquantitative result of the test (sensitivity of the limulus reagent of table 5：λ=0.125EU/mL, FM902 concentration：
0.025mg/mL)
+ sign positive findings ,-sign negative findings.
According to the regulation of usp bacteria inspection technique：If negative control solution D parallel pipe is feminine gender, the test sample positive is right
Parallel pipe according to solution B is the positive, and the geometrical mean of serial solution C reaction end concentration is between the λ of 0.5 λ~2, examination
Test effectively.The λ of Es=antilg (∑ Xs/4)=0.5.Thus judge, the experiment is effective.Try to achieve the endotoxin of YE need testing solutions
(the end dilution multiple in serial solution A per series of parallel pipe is multiplied by λ to concentration, for the reaction end concentration of each series, institute
The geometrical mean for having parallel pipe reaction end concentration is the endotoxin concns of need testing solution).
Calculated according to the experimental result of table 5：
CE=antilg (∑ X/2)=antilg [(lg0.125+lg0.125)/2]=antilg (- 0.9031)=
0.125EU/mL.So the endotoxin content that the inside is contained when FM902 concentration is 0.025mg/mL is 0.125EU/mL.I.e.：
Contained endotoxin is 5EU/mg in FM902 extracts.
Conclusion (of pressure testing)
Pass through the experiment of xeothermic program effectiveness, Sensilivily Check experiment, FM902 gel methods interference test and FM902 gels
Sxemiquantitative is tested, and it is 5EU/mg to measure contained endotoxin in FM902 extracts, is met《Pharmacopoeia of People's Republic of China》(2015
Version) limit standard.Result of the test shows that in serum free medium it is feasible to be properly added yeast extract FM902.
Embodiment 2：
1st, test specimen
Cultivate ground level yeast extract：FM905, lot number：2017030611A3, Angel Yeast Co., Ltd provide.
2nd, main agents
With embodiment 1.
3rd, key instrument
With embodiment 1.
4th, test method
1) xeothermic program effectiveness experiment
Not xeothermic endotoxin serial solution is prepared and detected referring to embodiment 1, and result of the test is shown in Table 6.
Prepare and detected referring to embodiment 1 through xeothermic endotoxin serial solution, result of the test is shown in Table 7.
The not xeothermic endotoxin detection result of the test of table 6
+ sign positive findings ,-sign negative findings.
Table 7 detects result of the test through xeothermic endotoxin
+ sign positive findings ,-sign negative findings.
Calculated according to table 6 and the testing result of table 7：
Recyclable endotoxin concns：Rc=λ D1mL (λ=0.125EU/mL)=0.125 × 20000 × 1=2500,
Remaining endotoxin：Rs=λ D1mL (λ=0.125EU/mL)=0.125 × 1 × 1=0.125.
Endotoxin decay test result：LgRd=lgRc-lgRs=lg2500-lg0.125=4.301>4.The row in China
Industry standard：Endotoxin is decayed：(States Pharmacopoeia specifications of China 2015 editions work as lgRd to lgRd=lgRc-lgRs>4 or lgRd>When 3, xeothermic journey
Sequence is effective).From experimental result：The xeothermic program that we draft：250 DEG C, effectively (subsequent experimental is according to this program by 90min
Carry out removing exogenous endotoxin operation).
2) Sensilivily Check is tested
Referring to embodiment 1, result of the test is shown in Table 8.
The sensitivity of the limulus reagent inspection test result (sensitivity of the limulus reagent of table 8：0.5EU/mL)
+ sign positive findings ,-sign negative findings.
If gel is formed in pipe, and gel is indeformable, is not determined as the positive from tube wall slippage person；Gel or shape are not formed
Into gel it is not solid, deform and be determined as feminine gender from tube wall slippage person.As shown in Table 8, when the λ pipes of Cmax 2 are the positive,
The λ pipes of least concentration 0.25 are feminine gender, and negative control pipe is feminine gender, and experiment is valid.It is dense that reaction end is calculated as follows
The measured value (λ c) of the geometrical mean of degree, as sensitivity of the limulus reagent, wherein λ c=antilg (∑ X/4), X are reaction end
Log concentration value lg, reaction end concentration refer to the concentration of last result that is positive in the endotoxin concns that series is successively decreased.
When λ c are in 0.5 λ~2 λ (including 0.5 λ and 2 λ), baterial endotoxin test can be used for, and to indicate sensitivity λ as this batch of horseshoe crab
The sensitivity of reagent.
Calculated according to the experimental result of table 8：X1=X2=X3=lg0.25, X4=lg0.50.λ c=antilg (∑ X/
4)=antilg (lg0.25 × 3+lg0.50)/4=antilg (- 0.5268)=0.2973.0.25=0.5 λ≤λ c=
0.2973≤2 λ=1.0.From experimental result：This batch sensitivity is that 0.5EU/mL TAL is qualified, available in YE
Toxin Inspection and analysis.Other sensitivity are：0.25th, 0.125,0.06,0.03,0.015EU/mL TALs review method with it is above-mentioned
Method is similar.
3) FM905 gel methods interference test
Referring to embodiment 1, result of the test is shown in Table 9.
According to《Pharmacopoeia of People's Republic of China》The regulation of (2015 editions) bacterioscopy method：Only when solution A and feminine gender are right
According to solution D all parallel pipes all for feminine gender, and serial solution C result sensitivity of the limulus reagent review in the range of when, examination
Test valid.Foundation：Es=antilg (∑ Xs/4)=antilg (0.5 × 4 ÷ 4)=0.5 λ, Et=antilg (∑ Xt/
4)=antilg (0.25 × 4 ÷ 4)=0.25 λ (X：For reaction end when concentration logarithm value, Es is C reaction end concentration
Geometrical mean, Et are the geometrical mean of B reaction end concentration), if meet Es in the λ (including 0.5 λ and 2 λ) of 0.5 λ~2 and
Regulations of the Et in 0.5Es~2Es (including 0.5Es and 2Es).So finally think test sample noiseless effect under the concentration.
Calculated according to the experimental result of table 9：Above-mentioned experiment determines there there is the experiment when concentration that batch is FM905 is 0.025mg/mL
Effect.
4) sxemiquantitative of FM905 gels is tested
Selected sensitivity of the limulus reagent sign value λ=0.125EU/mL, bacterial endotoxin working standard 14EU/ branch.Specifically
Test procedure is as follows：The preparation (C groups) of product mother liquor 1. the endotoxin standard that concentration is 2 λ works：Take 1 bacterial endotoxin work
Standard items, add 1mL inspections water, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration, then take 25 μ l solution and
675 μ L inspections mix to obtain 0.5EU/mL solution with water, then take 0.5mL solution to be mixed with 0.5ml inspections with water and produce 0.25EU/ml
(2 λ) mother liquor.2. the preparation (A groups) of need testing solution：Pass through the need testing solution of interference test.3. concentration is 2 λ endotoxin
The preparation (B groups solution) of standard work product and test sample mixed liquor：1 bacterial endotoxin working standard is taken, 1mL is added and checks
With water, 15min is mixed on eddy mixer, it is 14EU/mL to obtain concentration, then takes 25 μ L solution to be mixed with 675 μ L need testing solutions
Even 0.5EU/mL solution, then take 0.5mL solution mixed with 0.5mL need testing solutions produce 2 λ endotoxin standard work product with
Test sample mixed liquor.4. taking 20 TALs to be separately added into 0.1mL inspections to be dissolved with water, obtain every concentration and treated for 0.5EU/mL
With.5. often manage the TAL and corresponding solution that each addition 0.1mL sensitivity is 0.5EU/mL.Result of the test table is shown in specific dilution
10。
The FM905 gel method sxemiquantitative result of the test (sensitivity of the limulus reagent of table 10：λ=0.125EU/mL, FM905 concentration：
0.025mg/mL)
+ sign positive findings ,-sign negative findings.
According to the regulation of usp bacteria inspection technique：If negative control solution D parallel pipe is feminine gender, the test sample positive is right
Parallel pipe according to solution B is the positive, and the geometrical mean of serial solution C reaction end concentration is between the λ of 0.5 λ~2, examination
Test effectively.The λ of Es=antilg (∑ Xs/4)=0.5.Thus judge, the experiment is effective.Try to achieve the endotoxin of YE need testing solutions
(the end dilution multiple in serial solution A per series of parallel pipe is multiplied by λ to concentration, for the reaction end concentration of each series, institute
The geometrical mean for having parallel pipe reaction end concentration is the endotoxin concns of need testing solution).
Calculated according to the experimental result of table 10：
CE=antilg (∑ X/2)=antilg [(lg0.125+lg0.125)/2]=antilg (- 0.9031)=
0.125EU/mL.So the endotoxin content that the inside is contained when FM905 concentration is 0.025mg/mL is 0.125EU/mL.I.e.：
Contained endotoxin is 5EU/mg in FM905 extracts.
Conclusion (of pressure testing)
Pass through the experiment of xeothermic program effectiveness, Sensilivily Check experiment, FM905 gel methods interference test and FM905 gels
Sxemiquantitative is tested, and it is 5EU/mg to measure contained endotoxin in FM905 extracts, is met《Pharmacopoeia of People's Republic of China》(2015
Version) limit standard.Result of the test shows that in serum free medium it is feasible to be properly added yeast extract FM905.
For the FM902 that Angel company provides and FM905 by detecting level of endotoxin, it is the same to find its endotoxin content
's.
In summary, endotoxin content detection method in the yeast extract of foundation, this method is simple and easy to operate, repeatability
It is good, endotoxic appraisal procedure in yeast extract can be used as.
Claims (10)
1. the detection method of endotoxin content in a kind of yeast extract, it is characterised in that comprise the following steps：
1）Xeothermic program effectiveness experiment
Not xeothermic endotoxin serial solution and the endotoxin serial solution after dry heat treatment are prepared respectively, are carried out using TAL
Test, calculate endotoxin and decay and determine specific effective dry heat treatment condition, done in follow-up test according to determined above
Heat treatment condition removes exogenous endotoxin；
2）Sensilivily Check is tested
The endotoxin working standard serial solution of TAL to be checked and various concentrations is prepared, then by TAL and endogenous toxic material
Plain working standard serial solution is mixed respectively, while does negative control with water using inspection, calculates sensitivity of the limulus reagent
Measured value λc, when its result is between the λ of 0.5 λ ~ 2.0, then sensitivity is qualified；If unqualified, TAL need to be changed and answered
Nuclear test is until qualified；
3）Interference test
Yeast extract test sample serial solution, endotoxin standard work product and yeast extract test sample mixed stocker are set respectively
Water negative control solution is used in row solution, endotoxin standard work product serial solution and inspection, is then utilized and is closed through Sensilivily Check
The TAL of lattice is tested, it is ensured that test sample not interference test under what concentration；
4）Gel sxemiquantitative is tested
Yeast extract test sample serial solution is set respectively, and endotoxin standard work product mix molten with yeast extract test sample
Water negative control solution is used in liquid, endotoxin standard work product serial solution and inspection, is recycled through the qualified horseshoe crab of Sensilivily Check
Reagent is tested, finally at calculating need testing solution endotoxin concns；Wherein yeast extract test sample passes through step 3）
In interference test.
2. according to the method for claim 1, it is characterised in that：Step 1）Middle dry heat treatment condition：Baking temperature be 220 ~
280 DEG C, soaking time is 70 ~ 110min.
3. according to the method for claim 1, it is characterised in that：In xeothermic program effectiveness experiment, endotoxin used is
Endotoxin indicator, the speed of not xeothermic endotoxin serial solution dilution is between 1000-50000；Endogenous toxic material through dry heat treatment
The extension rate of plain serial solution is between 1-20 times.
4. according to the method for claim 3, it is characterised in that：The speed of xeothermic endotoxin serial solution dilution is not respectively
2500th, 5000,10000,20000 and 40000；The extension rate of endotoxin serial solution through dry heat treatment is respectively 1,2,4,
6、8。
5. according to the method for claim 1, it is characterised in that：Step 2）In, sensitivity of the limulus reagent sign value λ be 0.015 ~
0.500EU/mL；Concentration after the work product dilution of endotoxin standard is in 0.1 ~ 2.0EU/mL.
6. according to the method for claim 1, it is characterised in that：Step 3）Middle sensitivity of the limulus reagent sign value λ be 0.030 ~
1.000EU/mL, the concentration after the work product dilution of endotoxin standard is 0.1 ~ 2.0EU/mL；Concentration after yeast extract dilution
For 0.01 ~ 0.500mg/mL.
7. according to the method for claim 1, it is characterised in that：Step 4）Middle sensitivity of the limulus reagent sign value λ be 0.015 ~
0.500EU/mL；Concentration after the work product dilution of endotoxin standard is 0.1 ~ 2.0EU/mL.
8. according to the method for claim 8, it is characterised in that：Sensitivity of the limulus reagent sign value λ is 0.125EU/mL；Endogenous toxic material
Concentration after the work product dilution of plain standard is respectively 0.250,0.500,1.000,2.000EU/mL.
9. according to application of the claim 1-8 any one methods described in the detection of yeast extract endotoxin content.
10. according to claim 1-8 any one methods described in various microorganisms containing yeast extract, zooblast
The application that endotoxin content detects in culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710828600.0A CN107727863A (en) | 2017-09-14 | 2017-09-14 | The detection method of endotoxin content in yeast extract |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710828600.0A CN107727863A (en) | 2017-09-14 | 2017-09-14 | The detection method of endotoxin content in yeast extract |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107727863A true CN107727863A (en) | 2018-02-23 |
Family
ID=61206260
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710828600.0A Pending CN107727863A (en) | 2017-09-14 | 2017-09-14 | The detection method of endotoxin content in yeast extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107727863A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109387637A (en) * | 2018-10-12 | 2019-02-26 | 四川升和药业股份有限公司 | Danshen injections Test for Bacterial Endotoxins |
| CN109613250A (en) * | 2018-12-25 | 2019-04-12 | 中国大冢制药有限公司 | The inspection method of pyrophosphoric acid ironic citrate raw material bacterial endotoxin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1030138A (en) * | 1987-05-20 | 1989-01-04 | 雷夫·贝克 | The method that endotoxin exists in a kind of working sample |
| WO2008128329A1 (en) * | 2007-04-19 | 2008-10-30 | Viventia Biotech Inc. | Endotoxin removal |
| CN104155442A (en) * | 2008-08-18 | 2014-11-19 | 拜奥泰克公司 | Enhancing Endotoxin Detection |
-
2017
- 2017-09-14 CN CN201710828600.0A patent/CN107727863A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1030138A (en) * | 1987-05-20 | 1989-01-04 | 雷夫·贝克 | The method that endotoxin exists in a kind of working sample |
| WO2008128329A1 (en) * | 2007-04-19 | 2008-10-30 | Viventia Biotech Inc. | Endotoxin removal |
| CN104155442A (en) * | 2008-08-18 | 2014-11-19 | 拜奥泰克公司 | Enhancing Endotoxin Detection |
Non-Patent Citations (3)
| Title |
|---|
| 国家药典委员会: "《中华人民共和国药典2010版一部》", 31 January 2010 * |
| 汪穗福: "《微生物检测验证技术》", 31 May 2005 * |
| 洪明章: "FM888：安琪超滤级酵母浸出物", 《豆丁》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109387637A (en) * | 2018-10-12 | 2019-02-26 | 四川升和药业股份有限公司 | Danshen injections Test for Bacterial Endotoxins |
| CN109613250A (en) * | 2018-12-25 | 2019-04-12 | 中国大冢制药有限公司 | The inspection method of pyrophosphoric acid ironic citrate raw material bacterial endotoxin |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107314980B (en) | Detection kit and detection method for multiple antibiotic residues in animal-derived food | |
| CN110453011A (en) | A kind of method and application based on CRISPR/Cas12a fast accurate detection African swine fever virus | |
| CN106290890B (en) | A kind of the human body fluid development process fungi 1,3- calloses detection kit and its application process of improvement | |
| CN107727863A (en) | The detection method of endotoxin content in yeast extract | |
| Kistner | An improved method for viable counts of bacteria of the ovine rumen which ferment carbohydrates | |
| CN106048052A (en) | Kit and method for detecting staphylococcus aureus and integron in food | |
| CN1811436B (en) | Method for detecting Ennoxacin and its special enzyme-linked immune reagent kit | |
| CN101712989B (en) | Method for quickly, qualitatively and quantitatively measuring Lactobacillus casei in probiotic dairy products | |
| CN103224989B (en) | Method for quickly detecting duck-derived components in food | |
| CN107988401A (en) | The dry powdered LAMP quick detection kits of salmonella | |
| CN101718709A (en) | Method for detecting bacterial endotoxin of xylitol injection | |
| CN105510590B (en) | The detection method of cysteine hydrochloride bacterial endotoxin | |
| Sperber | The identification of staphylococci in clinical and food microbiology laboratories | |
| CN103592442B (en) | A kind of method detecting bacteria endotoxin content in Recombinant Swine Interferon α1 freeze drying powder injection | |
| CN206768082U (en) | A kind of magnetic stirring Analysis of Drug Susceptibility instrument and matched reagent box | |
| CN108277289A (en) | Escherichia coli O157:The dry powdered LAMP quick detection kits of H7 | |
| Varela et al. | Prevalence of Campylobacter spp. isolated from grower-finisher pigs in Ontario | |
| CN203965445U (en) | A kind of dynamic color method limulus kit for endotoxin detection | |
| Papagianni et al. | Design of a tubular loop bioreactor for scale‐up and scale‐down of fermentation processes | |
| CN103205493B (en) | LAMP method for detecting Brucella | |
| CN110343766A (en) | A method of based on calf-derived Cyclospora in QPCR quantitative detection livestock meat | |
| CN101936986A (en) | Method for detecting diazepam and chemoluminescence immunoassay kit special for same | |
| CN104931314A (en) | Stool sample handling method and application thereof | |
| CN105445467B (en) | The detection method of sodium pyrosulfite bacterial endotoxin | |
| CN105203724A (en) | Method for inspecting bacterial endotoxin of ibuprofen raw material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180619 Address after: No. 168 Chengdong Avenue, Yichang, Hubei Province, Hubei Applicant after: Anqi Yeast Co., Ltd., Hubei Address before: 443002 No. 8, University Road, Yichang, Hubei Applicant before: China Three Gorges University Applicant before: Anqi Yeast Co., Ltd., Hubei |
|
| TA01 | Transfer of patent application right | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180223 |
|
| RJ01 | Rejection of invention patent application after publication |