CN102331405A - Detection method for titer of maduramicin fermentation medium - Google Patents
Detection method for titer of maduramicin fermentation medium Download PDFInfo
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Abstract
The invention discloses a detection method for the titer of a maduramicin fermentation medium. The detection method comprises the following steps: 1, carrying out enlargement processing on the aperture of the head of a pipette gun with the standard of 1mL, pipetting the undefoamed maduramicin fermentation medium with the pipette gun with the processed head, putting the weighed fermentation medium in a volumetric flask, adding waterless methanol to the volumetric flask, shaking, carrying out supersonic extraction, adding waterless methanol to a constant volume, and filtering to obtain a filtrate; and 2, preparing a solution to be detected, a standard solution and a reference solution in order, carrying out water bath heating processing, cooling, and constant volume on the three liquids, respectively detecting absorbances with a spectrophotometer, and carrying out titer calculation according to a titer formula. The detection method of the invention which is a weighing detection method has the advantages of convenient operation, accurate datum, and energy saving and emission reduction.
Description
Technical field
The present invention relates to a kind of detection method of titer of antibodies, particularly relate to a kind of detection method of maduramicin fermentation liquor.
Background technology
At present; The production of antibiotics field is about the detection of tiring of maduramicin fermentation liquor; All adopt AAS based on volume; The method first step adopts the standard pipettor to pipette the sample of certain volume, in testing process, because fermentation liquor has foam, needs to add foam-breaking agent and carries out defoaming treatment in advance; Sometimes the large particulate matter that exists metabolic product crystallization or supplementary material to form in the fermentation liquor simultaneously; And the tire initial step that detects of this AAS is to carry out lixiviate with the fermentation liquor that pipettor or suction pipe are measured certain volume; Not only there is even problem in fermentation liquor behind the adding foam-breaking agent; And large particulate matter that exists in the fermentation liquor or oarse-grained metabolic product crystallization, in the time of can causing moving the liquid instrument and moving liquid with standard, large particulate matter can not suck; Perhaps large particulate matter sucks too much, all can cause bigger detection deviation.
Tiring based on the spectrophotometry of volume, influenced by fermentation liquor physical behavior, fermentation liquor temperature, ambient temperature, personnel operation difference etc. bigger; And fermentation liquor pretreatment needs to carry out froth breaking in advance with the organic solvent normal butyl alcohol in this AAS testing process; Not only increased detection cost and detection time thus, and environment has been caused certain pollution.
Summary of the invention
The technical matters that the present invention will solve is: the AAS based on volume of tiring to above-mentioned maduramicin fermentation liquor detects the defective that exists, the detection method that the present invention provides a kind of maduramicin fermentation liquor to tire.Detection method of the present invention is a kind of AAS based on weight, and is easy to operate, data are accurate, and energy-saving and emission-reduction.
In order to address the above problem, the technical scheme that the present invention adopts is:
The detection method that the present invention provides a kind of maduramicin fermentation liquor to tire, said detection method may further comprise the steps:
A, at first the rifle draught animals with 1mL standard liquid-transfering gun directly enlarges processing; The 1mL liquid-transfering gun rifle draught animals after expansion is handled directly is 3～5 times of 1mL standard liquid-transfering gun rifle draught animals footpath; 1mL liquid-transfering gun rifle head with after handling pipettes the maduramicin fermentation liquor without defoaming treatment; And take by weighing 1.0g with electronic balance, and the maduramicin fermentation liquor that takes by weighing is put into volumetric flask, in volumetric flask, add the absolute methanol of 1～2 mL then; Shaking volumetric flask mixes fermentation liquor and absolute methanol; And then add an amount of absolute methanol and mix, add that total addition of absolute methanol accounts for 85～90% of volumetric flask volume behind an amount of absolute methanol, the mixed liquor after mixing is carried out ultrasound wave lixiviate 8～12min or static placement;
B, lixiviate or static back add the scale mark that absolute methanol is settled to volumetric flask in volumetric flask, the mixed liquor behind the constant volume is filtered, and obtain filtrating after the filtration;
C, pipette the filtrating and the 2mL vanillic aldehyde solution that obtain after 1mL step b filters with the standard transfer pipet and put into the brown volumetric flask of 10 mL, add the absolute methanol of 5～6mL then, obtain liquid to be measured after shaking up;
D, with the standard transfer pipet pipette 1mL maduramicin standard solution, 2mL vanillic aldehyde solution is put into an other brown volumetric flask of 10mL, adds the absolute methanol of 5～6mL then, obtains titer after shaking up;
E, in the brown volumetric flask of another 10mL, add the absolute methanol of 2mL vanillic aldehyde solution and 5～6mL, obtain reference liquid after shaking up, reference liquid is as spectrophotometer school zero solution;
F, liquid to be measured, titer and reference liquid that step c, d, e are made respectively are put in water-bath heating 22～28min in 68～72 ℃ the water-bath; Take out respectively after the water-bath heating and be cooled to room temperature, add the scale mark that absolute methanol is settled to the brown volumetric flask of 10mL after the cooling respectively;
G, liquid to be measured, titer and reference liquid after step f handled utilize spectrophotometer to survey its absorbance respectively, and be last according to the formula of the tiring calculating of tiring.
According to the detection method that above-mentioned maduramicin fermentation liquor is tired, volumetric flask described in the step a is 25mL volumetric flask or 50mL volumetric flask or 100mL volumetric flask.
According to the detection method that above-mentioned maduramicin fermentation liquor is tired, the ultrasound wave model that the lixiviate of ultrasound wave described in the step a is adopted is KQ ﹣ 250B, and frequency of operation is 40KHz, and power is 250W; Be 20～30min the standing time during said static place.
Detection method according to above-mentioned maduramicin fermentation liquor is tired removes by filter the large granular impurity in the solution with the mixed liquor behind the constant volume described in the step b, obtains filtrating.
The detection method of tiring according to above-mentioned maduramicin fermentation liquor; The compound method of vanillic aldehyde solution is described in step c, d and the e: take by weighing 15g vanillic aldehyde, 100mL absolute methanol and mass concentration and be 98% concentrated sulphuric acid 7.5mL and join in the 500 mL volumetric flasks and shake up; Shake up back adding absolute methanol and be settled to the volumetric flask scale mark, obtain vanillic aldehyde solution behind the constant volume.
According to the detection method that above-mentioned maduramicin fermentation liquor is tired, liquid to be measured, titer and the reference liquid of described in the step f step c, d, e being made respectively be put in 70 ℃ water-bath cross in water-bath heating 25min.
According to the detection method that above-mentioned maduramicin fermentation liquor is tired, utilizing spectrophotometer to survey its absorbance respectively described in the step g is to survey its absorbance respectively in the 515nm wavelength.
According to the detection method that above-mentioned maduramicin fermentation liquor is tired, the computing formula of tiring described in the step g is:
Positive beneficial effect of the present invention:
1, the present invention adopts the AAS based on weight to detect tiring of maduramicin fermentation liquor; This method is simple to operate, data accurately, save time, reduce blowdown, can reach the purpose that detection of antibiotics maduramicin fermentation liquor is accurately and fast tired through technical scheme of the present invention.Detection method of the present invention is simple to operate, practice thrift detection time, all can use in antibiotic fermentation enterprise.
2, adopt technical scheme of the present invention to detect tiring of maduramicin fermentation liquor; Need not adopt the organic solvent normal butyl alcohol to carry out defoaming treatment to the maduramicin fermentation liquor; Not only avoided foreign matter to cause the inhomogenous defective of fermentation liquor, and can be because of adopting the organic solvent normal butyl alcohol that environment is polluted; Detection cost and detection time that fermentation liquor is tired have been reduced thus.Therefore, the present invention has certain economic benefits and social benefit.
3, the rifle draught animals to the standard liquid-transfering gun has directly carried out enlarging processing in the technical scheme of the present invention; Can evenly move into volumetric flask with containing oarse-grained maduramicin fermentation liquor thus, thereby overcome because the osculum liquid-transfering gun that large particulate matter causes can not move into uniform fermentation liquor the defective of volumetric flask.
4, detection method of the present invention can effectively shorten working time, improve accuracy of detection, energy-saving and emission-reduction, and can solve effectively that the fermentation liquor foam is not of uniform size, the inconsistent problem of temperature.
Four, embodiment:
Embodiment 1: the maduramicin fermentation liquor is tired and is lower than 6000 o'clock detection method
The detection method that maduramicin fermentation liquor of the present invention is tired, the detailed step of its detection method is following:
A, at first the rifle draught animals with 1mL standard liquid-transfering gun directly enlarges processing; The 1mL liquid-transfering gun rifle draught animals after expansion is handled directly is 3～5 times of 1mL standard liquid-transfering gun rifle draught animals footpath; 1mL liquid-transfering gun rifle head with after handling pipettes the maduramicin fermentation liquor without defoaming treatment, adopts the smart 1.00g of title of electronic balance maduramicin fermentation liquor, the maduramicin fermentation liquor that takes by weighing is put into the volumetric flask of 25mL; The absolute methanol that adds 1～2mL then; Shake volumetric flask fermentation liquor and absolute methanol are mixed, the absolute methanol that adds 20～22mL then mixes, and the mixed liquor after mixing is carried out ultrasound wave lixiviate 8～12min, and (the ultrasound wave model of employing is KQ ﹣ 250B; Frequency of operation is 40KHz, and power is 250W);
In volumetric flask, add the scale mark that absolute methanol is settled to volumetric flask after b, the lixiviate, the mixed liquor behind the constant volume is filtered, obtain filtrating after the filtration;
C, pipette the filtrating and the 2mL vanillic aldehyde solution that obtain after 1mL step b filters with the standard transfer pipet and put into the brown volumetric flask of 10 mL, add the absolute methanol of 5～6mL then, obtain liquid to be measured after shaking up;
D, with the standard transfer pipet pipette 1mL maduramicin standard solution, 2mL vanillic aldehyde solution is put into an other brown volumetric flask of 10mL, adds the absolute methanol of 5～6mL then, obtains titer after shaking up;
E, in the brown volumetric flask of another 10mL, add the absolute methanol of 2mL vanillic aldehyde solution and 5～6mL, obtain reference liquid after shaking up, this reference liquid is as spectrophotometer school zero solution;
F, liquid to be measured, titer and reference liquid that step c, d, e are made respectively are put in water-bath heating 25min in 70 ℃ the water-bath; Take out respectively after the water-bath heating and be cooled to room temperature, add the scale mark that absolute methanol is settled to the brown volumetric flask of 10mL after the cooling respectively;
G, liquid to be measured, titer and reference liquid after step f handled utilize spectrophotometer to survey its absorbance respectively: titer sample absorbance=0.490; Liquid sample absorbance to be measured=0.290; Titer sample size=251.500; Extension rate=25; Fermentation liquor weight=1.00.According to the formula of the tiring calculating of tiring:
Can know through calculating: tire=[(0.290/0.490) * 25 * 251.500]/1.0=3721.2.
The compound method of above-mentioned vanillic aldehyde solution is: take by weighing 15g vanillic aldehyde, 100mL absolute methanol and mass concentration and be 98% concentrated sulphuric acid 7.5mL and join in the 500 mL volumetric flasks and shake up; Shake up back adding absolute methanol and be settled to the volumetric flask scale mark, obtain vanillic aldehyde solution behind the constant volume.
Embodiment 2: the maduramicin fermentation liquor is tired and is higher than 6000 and is lower than 15000 o'clock detection method
The detection method that maduramicin fermentation liquor of the present invention is tired, the detailed step of its detection method is following:
A, at first the rifle draught animals with 1mL standard liquid-transfering gun directly enlarges processing; The 1mL liquid-transfering gun rifle draught animals after expansion is handled directly is 3～5 times of 1mL standard liquid-transfering gun rifle draught animals footpath; 1mL liquid-transfering gun rifle head with after handling pipettes the maduramicin fermentation liquor without defoaming treatment, and the electronic balance essence is claimed 1.01g maduramicin fermentation liquor, the maduramicin fermentation liquor that takes by weighing is put into the volumetric flask of 50 mL; The absolute methanol that adds 1～2mL then; Shake volumetric flask fermentation liquor and absolute methanol are mixed, the absolute methanol that adds 40～42mL then mixes, and the mixed liquor after mixing is carried out ultrasound wave lixiviate 8～12min, and (the ultrasound wave model of employing is KQ ﹣ 250B; Frequency of operation is 40KHz, and power is 250W);
In volumetric flask, add the scale mark that absolute methanol is settled to volumetric flask after b, the lixiviate, the mixed liquor behind the constant volume is filtered, obtain filtrating after the filtration;
C, pipette the filtrating and the 2mL vanillic aldehyde solution that obtain after 1mL step b filters with the standard transfer pipet and put into the brown volumetric flask of 10mL, add the absolute methanol of 5～6mL then, obtain liquid to be measured after shaking up;
D, with the standard transfer pipet pipette 1mL maduramicin standard solution, 2mL vanillic aldehyde solution is put into an other brown volumetric flask of 10mL, adds the absolute methanol of 5～6mL then, obtains titer after shaking up;
E, in the brown volumetric flask of another 10mL, add the absolute methanol of 2mL vanillic aldehyde solution and 5～6mL, obtain reference liquid after shaking up, this reference liquid is as spectrophotometer school zero solution;
F, liquid to be measured, titer and reference liquid that step c, d, e are made respectively are put in water-bath heating 25min in 70 ℃ the water-bath; Take out respectively after the water-bath heating and be cooled to room temperature, add the scale mark that absolute methanol is settled to the brown volumetric flask of 10mL after the cooling respectively;
G, liquid to be measured, titer and reference liquid after step f handled utilize spectrophotometer to survey its absorbance respectively: titer sample absorbance=0.512; Liquid sample absorbance to be measured=0.470; Titer sample size=251.500; Extension rate=50; Fermentation liquor weight=1.01.According to the formula of the tiring calculating of tiring:
Can know through calculating: tire=[(0.470/0.512) * 50 * 251.500]/1.01=11429.2.
The compound method of above-mentioned vanillic aldehyde solution is: take by weighing 15g vanillic aldehyde, 100mL absolute methanol and mass concentration and be 98% concentrated sulphuric acid 7.5mL and join in the 500 mL volumetric flasks and shake up; Shake up back adding absolute methanol and be settled to the volumetric flask scale mark, obtain vanillic aldehyde solution behind the constant volume.
Embodiment 3: the maduramicin fermentation liquor is tired and is higher than 15000 o'clock detection method
A, at first the rifle draught animals with 1mL standard liquid-transfering gun directly enlarges processing; The 1mL liquid-transfering gun rifle draught animals after expansion is handled directly is 3～5 times of 1mL standard liquid-transfering gun rifle draught animals footpath; 1mL liquid-transfering gun rifle head with after handling pipettes the maduramicin fermentation liquor without defoaming treatment, with the smart 0.98g maduramicin fermentation liquor of claiming of electronic balance, the maduramicin fermentation liquor that takes by weighing is put into the volumetric flask of 100mL; The absolute methanol that adds 1～2 mL then; Shake volumetric flask fermentation liquor and absolute methanol are mixed, add 90～92mL absolute methanol then, the mixed liquor after mixing is carried out ultrasound wave lixiviate 8～12min, and (the ultrasound wave model of employing is KQ ﹣ 250B; Frequency of operation is 40KHz, and power is 250W);
In volumetric flask, add the scale mark that absolute methanol is settled to volumetric flask after b, the lixiviate, the mixed liquor behind the constant volume is filtered, obtain filtrating after the filtration;
C, pipette the filtrating and the 2mL vanillic aldehyde solution that obtain after 1mL step b filters with the standard transfer pipet and put into the brown volumetric flask of 10 mL, add the absolute methanol of 5～6mL then, obtain liquid to be measured after shaking up;
D, with the standard transfer pipet pipette 1mL maduramicin standard solution, 2mL vanillic aldehyde solution is put into an other brown volumetric flask of 10mL, adds the absolute methanol of 5～6mL then, obtains titer after shaking up;
E, in the brown volumetric flask of another 10mL, add the absolute methanol of 2mL vanillic aldehyde solution and 5～6mL, obtain reference liquid after shaking up, this reference liquid is as spectrophotometer school zero solution;
F, liquid to be measured, titer and reference liquid that step c, d, e are made respectively are put in water-bath heating 28min in 68 ℃ the water-bath; Take out respectively after the water-bath heating and be cooled to room temperature, add the scale mark that absolute methanol is settled to the brown volumetric flask of 10mL after the cooling respectively;
G, liquid to be measured, titer and reference liquid after step f handled utilize spectrophotometer to survey its absorbance respectively: titer sample absorbance=0.500; Liquid sample absorbance to be measured=0.328; Titer sample size=251.500; Extension rate=100; Fermentation liquor weight=0.98; Computing formula is calculated according to tiring:
Can know through calculating: tire=[(0.328/0.500) * 100 * 251.500]/0.98=16835.1.
The compound method of above-mentioned vanillic aldehyde solution is: take by weighing 15g vanillic aldehyde, 100mL absolute methanol and mass concentration and be 98% concentrated sulphuric acid 7.5mL and join in the 500 mL volumetric flasks and shake up; Shake up back adding absolute methanol and be settled to the volumetric flask scale mark, obtain vanillic aldehyde solution behind the constant volume.
Claims (7)
1. the detection method that the maduramicin fermentation liquor is tired is characterized in that, said detection method may further comprise the steps:
A, at first the rifle draught animals with 1mL standard liquid-transfering gun directly enlarges processing; The 1mL liquid-transfering gun rifle draught animals after expansion is handled directly is 3～5 times of 1mL standard liquid-transfering gun rifle draught animals footpath; 1mL liquid-transfering gun rifle head with after handling pipettes the maduramicin fermentation liquor without defoaming treatment; And take by weighing 1.0g with electronic balance, and the maduramicin fermentation liquor that takes by weighing is put into volumetric flask, in volumetric flask, add the absolute methanol of 1～2 mL then; Shaking volumetric flask mixes fermentation liquor and absolute methanol; And then add an amount of absolute methanol and mix, add that total addition of absolute methanol accounts for 85～90% of volumetric flask volume behind an amount of absolute methanol, the mixed liquor after mixing is carried out ultrasound wave lixiviate 8～12min or static placement;
B, lixiviate or static back add the scale mark that absolute methanol is settled to volumetric flask in volumetric flask, the mixed liquor behind the constant volume is filtered, and obtain filtrating after the filtration;
C, pipette the filtrating and the 2mL vanillic aldehyde solution that obtain after 1mL step b filters with the standard transfer pipet and put into the brown volumetric flask of 10 mL, add the absolute methanol of 5～6mL then, obtain liquid to be measured after shaking up;
D, with the standard transfer pipet pipette 1mL maduramicin standard solution, 2mL vanillic aldehyde solution is put into an other brown volumetric flask of 10mL, adds the absolute methanol of 5～6mL then, obtains titer after shaking up;
E, in the brown volumetric flask of another 10mL, add the absolute methanol of 2mL vanillic aldehyde solution and 5～6mL, obtain reference liquid after shaking up, reference liquid is as spectrophotometer school zero solution;
F, liquid to be measured, titer and reference liquid that step c, d, e are made respectively are put in water-bath heating 22～28min in 68～72 ℃ the water-bath; Take out respectively after the water-bath heating and be cooled to room temperature, add the scale mark that absolute methanol is settled to the brown volumetric flask of 10mL after the cooling respectively;
G, liquid to be measured, titer and reference liquid after step f handled utilize spectrophotometer to survey its absorbance respectively, and be last according to the formula of the tiring calculating of tiring.
2. the detection method that maduramicin fermentation liquor according to claim 1 is tired is characterized in that, volumetric flask described in the step a is 25mL volumetric flask or 50mL volumetric flask or 100mL volumetric flask.
3. the detection method that maduramicin fermentation liquor according to claim 1 is tired is characterized in that, the ultrasound wave model that the lixiviate of ultrasound wave described in the step a is adopted is KQ ﹣ 250B, and frequency of operation is 40KHz, and power is 250W; Be 20～30min the standing time during said static place.
4. the detection method that maduramicin fermentation liquor according to claim 1 is tired is characterized in that: described in the step b mixed liquor behind the constant volume is removed by filter the large granular impurity in the solution, obtain filtrating.
5. the detection method that maduramicin fermentation liquor according to claim 1 is tired; It is characterized in that; The compound method of vanillic aldehyde solution is described in step c, d and the e: take by weighing 15g vanillic aldehyde, 100mL absolute methanol and mass concentration and be 98% concentrated sulphuric acid 7.5mL and join in the 500 mL volumetric flasks and shake up; Shake up back adding absolute methanol and be settled to the volumetric flask scale mark, obtain vanillic aldehyde solution behind the constant volume.
6. the detection method that maduramicin fermentation liquor according to claim 1 is tired is characterized in that: liquid to be measured, titer and the reference liquid of described in the step f step c, d, e being made respectively be put in 70 ℃ water-bath cross in water-bath heating 25min.
7. the detection method that maduramicin fermentation liquor according to claim 1 is tired is characterized in that: utilizing spectrophotometer to survey its absorbance respectively described in the step g is to survey its absorbance respectively in the 515nm wavelength.
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Application publication date: 20120125 |