CN103308575B - Method for detecting content of glutamic acid by biosensing analyzer and applications of analyzer - Google Patents
Method for detecting content of glutamic acid by biosensing analyzer and applications of analyzer Download PDFInfo
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract description 219
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract description 210
- 239000004220 glutamic acid Substances 0.000 title claims abstract description 210
- 238000000034 method Methods 0.000 title abstract description 50
- 238000000855 fermentation Methods 0.000 claims abstract description 210
- 230000004151 fermentation Effects 0.000 claims abstract description 204
- 238000012937 correction Methods 0.000 claims abstract description 63
- 238000011084 recovery Methods 0.000 claims abstract description 46
- 238000001514 detection method Methods 0.000 claims abstract description 35
- 238000002474 experimental method Methods 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims description 31
- 239000002253 acid Substances 0.000 claims description 26
- 238000012360 testing method Methods 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 14
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000003556 assay Methods 0.000 claims description 7
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 229960000448 lactic acid Drugs 0.000 claims description 6
- 239000012898 sample dilution Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 25
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 abstract description 4
- 235000013923 monosodium glutamate Nutrition 0.000 abstract description 4
- 239000004223 monosodium glutamate Substances 0.000 abstract description 4
- 238000013459 approach Methods 0.000 abstract description 3
- 238000007865 diluting Methods 0.000 abstract 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 178
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- 238000009825 accumulation Methods 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 5
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000007824 enzymatic assay Methods 0.000 description 3
- 150000002307 glutamic acids Chemical class 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for detecting the content of glutamic acid by a biosensing analyzer. The method comprises the following steps of: firstly, diluting a sample to a reasonable ratio; detecting the sample by the biosensing analyzer; implementing calibration by a two-point calibration approach; determining a correction coefficient by a sample recovery rate experiment so as to correct a detected value of the glutamic acid in a fermentation broth sample, wherein the value is detected by the biosensing analyzer; and determining the content of the glutamic acid in an original fermentation broth. By the method, the problem that for a long time in the industry of monosodium glutamate, the detected content of the glutamic acid is lower than the actual content when the glutamic acid is detected by the biosensing analyzer at a later period of fermentation is solved; the detection result from the biosensing analyzer is correct and reliable, can be quickly detected and is identical to a Huabo method result; the detection cost can be lowered and the detection efficiency can be enhanced; the method can be used to substitute for the Huabo method so as to detect the sample during a whole fermentation period and realize whole-process, rapid and correct detection during the production of the glutamic acid.
Description
Technical field
The invention belongs to bioengineering detection technique field, relate to the detection technique of glutamic acid, especially a kind of
Utilize the detection method of bio-sensing analysis-e/or determining content of glutamic acid.
Background technology
Bio-sensing analyser starts from the eighties, and in the nineties, its application obtains large development, uses user to reach 400.Bio-sensing analysis-e/or determining content of glutamic acid, because of analytical approach simple and fast, progressively replaces Hua Bofa and measures glutamic acid, and be used widely in monosodium glutamate industry.Because SBA bio-sensing analysis-e/or determining glutamic acid uses the initial stage, glutamic acid acid production rate is at about 6-7%, the needs of the determination and analysis of content of glutamic acid can be met completely, but along with the development of science and technology, the progress of fermenting and producing level, glutamic acid acid production rate obtains larger raising, the acid yield of biological suboptimal dose bacterial classification reaches 12-14%, temperature sensitivity bacterial classification acid yield 16-18%, more on the low side than actual content of glutamic acid by result during bio-sensing analysis-e/or determining fermentation later stage fermentation liquid content of glutamic acid, so the result obtained often can only be as a reference.
Current mensuration content of glutamic acid generally adopts two kinds of methods:
One is that bio-sensing analyser method combines with Hua Bofa, mainly adopts bio-sensing analyser method in earlier fermentation, mid-term, and the fermentation later stage adopts Hua Bofa.It is utilize glutamate decarboxylase to react with glutamic acid under certain temperature, volume that Hua Bofa measures glutamic acid principle, makes glutamic acid decarboxylase release CO
2, then measure releasing CO with Hua Boshi respirator
2pressure, volume with certain formula scales content of glutamic acid, the method minute is longer, complex operation easily introduces experimental error.
Two is the scaled values improving bio-sensing analysis-e/or determining content of glutamic acid.The scaled values of this kind of method is calculated by average recovery experiment, fermentation period is different, in sample, composition influence factor content is different, its average recovery is different, the scaled values corrected is also inevitable different, thus measure the content of glutamic acid of whole fermentation period by a scaled values, the content of glutamic acid that earlier fermentation can be caused to measure is more higher than actual content, often truly can not reflect the true rule in glutamic acid fermentation production run and glutamic acid acid production rate; If the different fermentations cycle uses different scaled values, because Workshop Production is that different batches fermentation tank is worked continuously, the same time must have the fermentation broth sample in different fermentations cycle, just need to use different scaled values, and instrument is revised scaled values and is all needed again to start shooting at every turn, again calibrate, add a lot of experimental procedure, be unsuitable for the use of the conventional determining content of glutamic acid of workshop.
The present invention is found by lot of experiments, measures NH in fermentation later stage content of glutamic acid Lower result and sample
4 +the factors such as the stability of concentration, content of glutamic acid, enzyme membrane activity, Linear Camaera Calibrating Method, instrument detection system are closely related, by furtheing investigate these factors to the affecting laws of result and innovative approach, make bio-sensing analysis-e/or determining result more accurately, reliably, reach consistent with Hua Bofa result, to realizing whole process in glutamic acid production run, detecting fast, accurately, instruct actual production significant.
By retrieval, find the following open source literature relevant to present patent application:
1, correction ([1] Zhang Hongjian of SBA bio-sensing instrument enzymatic assays glutamic acid scaled values, Duan Zuoying, the correction of Mao Zhonggui .SBA bio-sensing instrument enzymatic assays glutamic acid scaled values, Wuxi Light Industry Univ.'s journal [J], 2002 (05) 21:529-521), relate to a kind of detection method of glutamic acid, comprise the preparation of sample, average recovery is tested, and correction scaled values is 108-110.The method utilizes average recovery to test and corrects SBA bio-sensing instrument enzymatic assays glutamic acid scaled values, bio-sensing instrument is utilized to measure the result of glutamic acid for twice, the glutamic acid recovery adding known quantity can be calculated, and derive the correct scaled values of bio-sensing instrument with this.The scaled values of this kind of method is calculated by average recovery experiment, fermentation period is different, in fermentation liquor, composition influence factor content is different, its average recovery is different, the scaled values corrected is also inevitable different, thus measure the content of glutamic acid of whole fermentation period by a scaled values, the content of glutamic acid that earlier fermentation can be caused to measure is more higher than actual content, often truly can not reflect the true rule that whole fermentation period content of glutamic acid changes and glutamic acid acid production rate; If the different fermentations cycle uses different scaled values, because Workshop Production is that different batches fermentation tank is worked continuously, the same time must have the fermentation broth sample in different fermentations cycle, just need to use different scaled values, and instrument is revised scaled values and is all needed again to start shooting at every turn, again calibrate, add a lot of experimental procedure, be unsuitable for the use of the conventional determining content of glutamic acid of workshop.Present invention process method is unique, compared to existing technologies, introduce the new ideas of correction coefficient, can the true rule of true rule of actual response whole fermentation period content of glutamic acid change and glutamic acid acid production rate, workshop fortune is simply to use, quick, accurate.
2, NH
4 +ion pair SBA-40 type analysis instrument measure glutamic acid impact ([2] Wei Yaode. NH
4+ ion pair SBA 1 type analysis instrument measures the impact of glutamic acid. and send out scientific and technological communication [J] liquor-saturated, 1998(03) 27:15-16).The method have studied and causes bio-sensing analysis-e/or determining glutamic acid numerical value reason on the low side to be due to NH
4+ continuous increase, but concrete solution is not proposed.The present invention studies discovery by experiment, NH in fermentation liquor
4the continuous accumulation of+ion and glutamic acid, have impact on biochemical enzymes reaction and carries out to the right, and then make measurement result on the low side; Proposition specific solving methods is: expanding rational extension rate to can reduce, and the fermentation later stage is due to content of glutamic acid and NH
4 +the impact that the increase of concentration is reacted biochemical enzymes, reduce the requirement (being greater than 200) to glutamic acid enzyme membrane activity simultaneously, solving the fermentation later stage raises due to content of glutamic acid, and enzymatic activity decline causes content of glutamic acid to exceed the problem of bio-sensing analyzer linear sensing range, make the result of bio-sensing analysis-e/or determining glutamic acid accurately, reliably.
3, bio-sensing analyser combines with Hua Bofa mensuration content of glutamic acid, and earlier fermentation, mid-term mainly adopt bio-sensing analysis-e/or determining, and the fermentation later stage adopts Hua Bofa.It is utilize glutamate decarboxylase to react with glutamic acid under certain temperature, volume that Hua Bofa measures glutamic acid principle, makes glutamic acid decarboxylase release CO
2, then measure releasing CO with Hua Boshi respirator
2pressure, volume with the content of certain formula scales glutamic acid.The Hua Bofa that this technology later stage uses measures content of glutamic acid, and minute is longer, complex operation, and accuracy is relatively poor.Reason: (1) influence factor is many, the impact such as the bath temperature in respirometer, rotating speed, the amplitude of oscillation measures.(2) experimental procedure is many, needs dilution, doses 4 kinds of reagent (sample, glutamate decarboxylase suspension, pH5.0 Acetic acid-sodium acetate damping fluid, distilled water), the consumption of reagent is few, easily introduce error.(3) experiment terminates rear reaction bulb and reaction tube cleaning trouble, need cotton to wipe, washing, the step such as gas and oil is washed, alcohol is washed, wash, washing lotion is washed, wash, distilled water rinse.(4) portion of reagent is poisonous, needs very careful during preparation, wherein for fill inspection pressure pipe inspection hydraulic fluid in containing insalubrious material, for cleaning the washing lotion of inspection pressure pipe and reaction bulb, being prepared by potassium dichromate and the concentrated sulphuric acid, there is strong corrosivity.In the present invention, bio-sensing analysis-e/or determining content of glutamic acid advantage has: (1) adopts immobilized biological activated material to make catalyzer, overcome over enzymatic analysis reagent costly with the shortcoming of chemical analysis very complicated.(2) selectivity is good, only reacts to specific substrate, and not by the impact of color, turbidity.(3) analysis speed is fast, and a sample determination can obtain result at one minute.(4) accuracy is high, relative error about 1%.(5) cost is low, and when using continuously, every example measures only needs Renminbi somewhat.(6) experiment reagent used is nontoxic, harmless, non-corrosiveness, and security is good.
By contrast, there are the different of essence in patented claim of the present invention and above-mentioned open source literature.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, proposition one is simple to operate, control is convenient, testing cost can be reduced, improve detection efficiency, the situation that the phase after fermentation that can effectively solve utilizes bio-sensing analysis-e/or determining content of glutamic acid on the low side, set up the detection method of bio-sensing analysis-e/or determining content of glutamic acid, use the method to record experimental result consistent with Hua Bofa, and the method is more quicker, accurate than Hua Bofa omnidistance detection content of glutamic acid in glutamic acid production run.
The object of the invention is to be achieved through the following technical solutions:
A kind of detection method of bio-sensing analysis-e/or determining content of glutamic acid, step comprises: bio-sensing analyser adopts two-point potentionmetric to measure, by Sample Dilution, carry out average recovery experiment and determine correction coefficient, calculate content of glutamic acid, namely detect and obtain content of glutamic acid, and concrete steps are as follows:
(1) fermentation broth sample preparation: get glutami acid fermentation liquor stoste, is diluted to 100-200 doubly, does three parts of Duplicate Samples, obtain fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then can carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
(3) standard items sample preparation: add glutamic acid standard items in fermentation liquor stoste, be diluted to 100-200 afterwards doubly, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: the measured value of X1-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
The measured value of X2-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL.
1000-Units conversion factor.
And described step (1) middle Sample Dilution is: when glutami acid fermentation liquor stoste is fermented and cultured 6 ~ 12h, fermentation liquor stoste is diluted to 100 times; When glutami acid fermentation liquor stoste is after fermented and cultured 12h, glutami acid fermentation liquor stoste is diluted to 200 times.
And, described step (2) middle bio-sensing analyser model is: SBA-40 type bio-sensing analyser, SBA-40C type bio-sensing analyser, SBA-40E type bio-sensing analyser, SBA-40X type bio-sensing analyser, or SBA-50 type bio-sensing analyser.
And the described step (2) composition of Plays liquid and concentration is: 50 mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
And, described step (2) in bio-sensing analyser calibration time, adopt two-point potentionmetric; When entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measure 5 ~ 8 samples again to calibrate once.
And, described step (3) in fermentation liquor stoste, add glutamic acid standard items quality A be 0.5X
1* V ~ 1.5X
1* V, X1 are that biological sensing assays instrument measures fermentation broth sample content of glutamic acid, and the volume of unit to be mg/dL, V be fermentation broth sample, unit is dL.
The detection method of bio-sensing analysis-e/or determining glutamic acid actual content as above is measuring the application in content of glutamic acid.
The advantage that the present invention obtains and good effect are:
1, the inventive method is simple to operate, it is convenient to control, omnidistance in glutamic acid production run, fast, accurate detection content of glutamic acid, when the phase after fermentation that can effectively solve utilizes bio-sensing analysis-e/or determining glutamic acid, measure the situation that content of glutamic acid is on the low side, solve monosodium glutamate industry for a long time, when phase utilizes bio-sensing analysis-e/or determining glutamic acid after fermentation, measure the situation that content of glutamic acid is on the low side, make bio-sensing analysis-e/or determining result more accurate, reliably, reach consistent with Hua Bofa result, the whole process in glutamic acid production can be realized, fast, accurate detection, reduce testing cost, improve detection efficiency.
2, the inventive method determination two point correction method is the calibrating method that biological sensing assays instrument measures glutamic acid, and the sample test concentration range of two point correction method and linear relationship will significantly better than one-point calibration.
3, the inventive method improves extension rate, due to NH in fermentation liquor
4 +the continuous accumulation of ion and glutamic acid, have impact on biochemical enzymes reaction and carries out to the right, and then make measurement result on the low side.Expand extension rate and can reduce the fermentation later stage due to content of glutamic acid and NH
4 +the increase of concentration, on the impact of enzyme reaction, reduces the requirement to enzymatic activity, solves the fermentation later stage to raise due to content of glutamic acid, and enzymatic activity declines and causes content of glutamic acid to exceed the problem of linear detection range, makes measurement result closer to actual value.
4, the inventive method improves evaluation method, the accuracy of method to measuring result that have employed average recovery is evaluated, and by the data accumulation of a period of time, determines correction coefficient, experimental result is corrected, makes testing result more accurate.
5, the inventive method introduces correction coefficient concept, and when measuring the sample of different cycles, scaled values is identical, and correction coefficient is different, facilitates workshop different fermentations cycle sample determination.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto, and can not limit protection scope of the present invention with following embodiment.
The experimental technique used in following embodiment if no special instructions, is conventional method; Described bio-sensing analyser method and Hua Bofa measure content of glutamic acid, add explanation if not special, and the well-known operations method of content of glutamic acid is consistent with adopting in industry.The material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The bio-sensing analyser used in the present invention is the bio-sensing analyser that can detect content of glutamic acid, such as SBA-40 type bio-sensing analyser, SBA-40C type bio-sensing analyser, SBA-40E type bio-sensing analyser, SBA-40X type bio-sensing analyser, SBA-50 type bio-sensing analyser, when using above-mentioned machine to measure glutamic acid, in machine, glutamic acid enzyme membrane need be loaded by assigned address, after stablizing 12-24 hour, can use.
The detection method of bio-sensing analysis-e/or determining content of glutamic acid of the present invention, step comprises: sample preparation, by Sample Dilution to reasonable multiple, bio-sensing analyser (Shandong Province academy sciences Biology Research Institute, such as SBA-40 type bio-sensing analyser, SBA-40C type bio-sensing analyser, SBA-40E type bio-sensing analyser, SBA-40X type bio-sensing analyser, SBA-50 type bio-sensing analyser, or other can carry out the model of glutamic acid mensuration) adopt two-point potentionmetric to measure, carry out average recovery experiment and determine correction coefficient, correct the fermentation broth sample Glutamic Acid content of bio-sensing analysis-e/or determining, determine the content of glutamic acid of fermentation liquor stoste.Detection method can solve monosodium glutamate industry for a long time, when phase utilizes bio-sensing analysis-e/or determining glutamic acid after fermentation, measure the situation that content of glutamic acid is on the low side, make bio-sensing analysis-e/or determining result more accurately, reliably, reach consistent with Hua Bofa result, testing cost can be reduced, improve detection efficiency, replace Hua Bofa to detect the whole cycle sample of fermentation, realize the whole process in glutamic acid production, detect fast, accurately.
The addition of Glutamic Acid standard items of the present invention will ensure that the content of glutamic acid of standard items sample maintains in the range ability of bio-sensing analysis-e/or determining content of glutamic acid.
Correction coefficient in the present invention: when the content of fermentation liquor stoste Glutamic Acid is less than 7g/dL, its average recovery is general all between 98%-102%, the impact of Related Component content on biochemical reaction in solution is less, its measuring accuracy can meet experiment testing requirement, the glutamic acid fermentation sample measurements of bio-sensing analysis-e/or determining can be corrected without correction coefficient, the fermentation broth sample measured value of bio-sensing analysis-e/or determining is multiplied by corresponding extension rate, carries out base unit conversion and is fermentation liquor stoste content of glutamic acid; When the content of fermentation liquor stoste Glutamic Acid is greater than 7g/dL, when average recovery is less than 98%, just need to carry out linear coefficient correction, the fermentation broth sample measured value of bio-sensing analysis-e/or determining is multiplied by corresponding extension rate, carry out base unit conversion, more namely the correction coefficient being multiplied by corresponding fermentation period obtains glutami acid fermentation liquor stoste content of glutamic acid.
Correction coefficient in the present invention: fermentation period is different, in sample, composition influence factor content is different, and its average recovery is different, and correction coefficient is different.For actual production, because every series-produced process conditions are consistent, the composition of the fermentation broth sample of different fermentations batch same fermentation period and comparision contents are stablized, its average recovery experimental result and correction coefficient substantially close, therefore by the data accumulation of a period of time, determine the correction coefficient being suitable for different fermentations cycle fermentation broth sample, as long as process conditions and acid yield remain unchanged, this correction coefficient can use always; If production technology and fermentation level change to some extent, correction coefficient adjusts accordingly with regard to needs.
Embodiment 1:
A detection method for bio-sensing analysis-e/or determining content of glutamic acid, step is as follows:
(1) fermentation broth sample preparation: getting 1mL fermentation period is that 12 hours fermentation liquid stostes join in 100mL volumetric flask, and adding distil water constant volume, to 100mL, does three parts of Duplicate Samples, obtains fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then can carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
The described step (2) composition of Plays liquid and concentration is: 50 mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
Described step (2) in bio-sensing analyser calibration time, adopt two-point potentionmetric; When entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measuring 5 ~ 8 samples must calibrate once again, and visual equipment service condition can increase calibration number of times.
(3) standard items sample preparation: getting 1mL fermentation period is that 12 hours fermentation liquid stostes join in 100mL volumetric flask, in 100mL volumetric flask, add 40mg glutamic acid standard items simultaneously, adding distil water constant volume is to 100mL, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
X
2the measured value of-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL.
1000-Units conversion factor.
Testing result:
According to the fermentation liquor stoste content of glutamic acid that formulae discovery bio-sensing analyser records, and compare with the fermentation liquor stoste content of glutamic acid that Hua Bofa measures.Experimental result is as shown in table 1:
The measurement result of table 1 content of glutamic acid
Can be found out by testing result in table 1, fermentation period is the fermentation broth sample of 12 hours, average recovery is 98.84% > 98%, can correct without correction coefficient, the fermentation broth sample measured value of bio-sensing analysis-e/or determining is multiplied by corresponding extension rate, carries out base unit conversion and is fermentation liquor stoste content of glutamic acid.Content of glutamic acid (g/dL)=43*100/1000=4.3g/dL, the fermentation liquor stoste content of glutamic acid correcting artifact sensing assays instrument mensuration is 4.3g/dL, to measure fermentation liquor stoste content of glutamic acid 4.33 g/dL basically identical with Hua Bofa, but this method is more quick, convenient, accurately.
For actual production, because every series-produced process conditions are consistent, the composition of the fermentation broth sample of different fermentations batch same fermentation period and comparision contents are stablized, its average recovery experimental result and correction coefficient substantially close, therefore by the data accumulation of a period of time, determine that bio-sensing analysis-e/or determining fermentation period is that the fermentation broth sample measured value of 12 hours is multiplied by corresponding extension rate, carry out base unit conversion and be fermentation liquor stoste content of glutamic acid, can the experiment of it goes without doing average recovery as long as process conditions and acid yield remain unchanged, correction coefficient can not be needed to correct, these computing method can use always, if production technology and fermentation level change to some extent, just need to carry out average recovery experiment, see average recovery experimental result whether between 98%-102%, if average recovery is less than 98%, bio-sensing analysis-e/or determining fermentation period is that the fermentation broth sample measured value of 12 hours is multiplied by corresponding extension rate, carry out base unit conversion, more namely the correction coefficient being multiplied by corresponding fermentation period obtains glutami acid fermentation liquor stoste content of glutamic acid.
Embodiment 2:
A detection method for bio-sensing analysis-e/or determining content of glutamic acid, step is as follows:
(1) fermentation broth sample preparation: getting 0.5mL fermentation period is that 24 hours fermentation liquid stostes join in 100mL volumetric flask, and adding distil water constant volume, to 100mL, does three parts of Duplicate Samples, obtains fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then can carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
The described step (2) composition of Plays liquid and concentration is: 50 mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
Described step (2) in bio-sensing analyser calibration time, adopt two-point potentionmetric; When entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measuring 5 ~ 8 samples must calibrate once again, and visual equipment service condition can increase calibration number of times.
(3) standard items sample preparation: getting 0.5mL fermentation period is that 24 hours fermentation liquid stostes join in 100mL volumetric flask, in 100mL volumetric flask, add 40mg glutamic acid standard items simultaneously, adding distil water constant volume is to 100mL, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
X
2the measured value of-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL.
1000-Units conversion factor.
Testing result:
According to the fermentation liquor stoste content of glutamic acid that formulae discovery bio-sensing analyser records, and compare with the fermentation liquor stoste content of glutamic acid that Hua Bofa measures.Experimental result is as shown in table 2:
The measurement result of table 2 content of glutamic acid
Can be found out by testing result in table 2, fermentation period is the fermentation broth sample of 24 hours, average recovery is 95.23%, correction coefficient=1/95.23%=1.050, fermentation liquor stoste content of glutamic acid (g/dL)=42*1.050*200/1000=8.82, correcting the fermentation liquor stoste content of glutamic acid that artifact sensing assays instrument measures is 8.82g/dL, and to measure fermentation liquor stoste content of glutamic acid 8.79 g/dL basically identical with Hua Bofa, but this method is more quick, convenient, accurate.
For actual production, because every series-produced process conditions are consistent, the composition of the fermentation broth sample of different fermentations batch same fermentation period and comparision contents are stablized, its average recovery experimental result and correction coefficient substantially close, therefore by the data accumulation of a period of time, determine to be suitable for the correction coefficient that fermentation period is the fermentation broth sample of 24 hours, as long as the measured value measuring fermentation broth sample is in actual applications multiplied by corresponding extension rate, carry out base unit conversion, being multiplied by corresponding fermentation period is again that namely to obtain fermentation period be 24 hours glutami acid fermentation liquor stoste content of glutamic acids for the correction coefficient of 24 hours.As long as process conditions and acid yield remain unchanged, this correction coefficient can use always; If production technology and fermentation level change to some extent, correction coefficient adjusts accordingly with regard to needs.
Embodiment 3:
A detection method for bio-sensing analysis-e/or determining content of glutamic acid, step is as follows:
(1) fermentation broth sample preparation: getting 0.5mL fermentation period is that 28 hours fermentation liquid stostes join in 100mL volumetric flask, and adding distil water constant volume, to 100mL, does three parts of Duplicate Samples, obtains fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then can carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
The described step (2) composition of Plays liquid and concentration is: 50 mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
Described step (2) in bio-sensing analyser calibration time, adopt two-point potentionmetric; When entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measuring 5 ~ 8 samples must calibrate once again, and visual equipment service condition can increase calibration number of times.
(3) standard items sample preparation: getting 0.5mL fermentation period is that 28 hours fermentation liquid stostes join in 100mL volumetric flask, in 100mL volumetric flask, add 45mg glutamic acid standard items simultaneously, adding distil water constant volume is to 100mL, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
X
2the measured value of-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL.
1000-Units conversion factor.
Testing result:
According to the fermentation liquor stoste content of glutamic acid that formulae discovery bio-sensing analyser records, and compare with the fermentation liquor stoste content of glutamic acid that Hua Bofa measures.Experimental result is as shown in table 3:
The measurement result of table 3 content of glutamic acid
Can be found out by testing result in table 3, fermentation period is the fermentation broth sample of 28 hours, average recovery is 92.81%, correction coefficient=1/92.81%=1.077, fermentation liquor stoste content of glutamic acid (g/dL)=47.5*1.077*200/1000=10.23, correcting the fermentation liquor stoste content of glutamic acid that artifact sensing assays instrument measures is 10.23g/dL, and to measure fermentation liquor stoste content of glutamic acid 10.21 g/dL basically identical with Hua Bofa, but this method is more quick, convenient, accurate.
For actual production, because every series-produced process conditions are consistent, the composition of the fermentation broth sample of different fermentations batch same fermentation period and comparision contents are stablized, its average recovery experimental result and correction coefficient substantially close, therefore by the data accumulation of a period of time, determine to be suitable for the correction coefficient that fermentation period is the fermentation broth sample of 28 hours, as long as the measured value measuring fermentation broth sample is in actual applications multiplied by corresponding extension rate, carry out base unit conversion, being multiplied by corresponding fermentation period is again that namely to obtain fermentation period be 28 hours glutami acid fermentation liquor stoste content of glutamic acids for the correction coefficient of 28 hours.As long as process conditions and acid yield remain unchanged, this correction coefficient can use always; If production technology and fermentation level change to some extent, correction coefficient adjusts accordingly with regard to needs.
Embodiment 4:
A detection method for bio-sensing analysis-e/or determining content of glutamic acid, step is as follows:
(1) fermentation broth sample preparation: getting 0.5mL fermentation period is that 32 hours fermentation liquid stostes join in 100mL volumetric flask, and adding distil water constant volume, to 100mL, does three parts of Duplicate Samples, obtains fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then can carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
The described step (2) composition of Plays liquid and concentration is: 50 mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
Described step (2) in bio-sensing analyser calibration time, adopt two-point potentionmetric; When entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measuring 5 ~ 8 samples must calibrate once again, and visual equipment service condition can increase calibration number of times.
(3) standard items sample preparation: getting 0.5mL fermentation period is that 32 hours fermentation liquid stostes join in 100mL volumetric flask, in 100mL volumetric flask, add 50mg glutamic acid standard items simultaneously, adding distil water constant volume is to 100mL, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
X
2the measured value of-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL.
1000-Units conversion factor.
Testing result:
According to the fermentation liquor stoste content of glutamic acid that formulae discovery bio-sensing analyser records, and compare with the fermentation liquor stoste content of glutamic acid that Hua Bofa measures.Experimental result is as shown in table 4:
The measurement result of table 4 content of glutamic acid
Can be found out by testing result in table 4, fermentation period is the fermentation broth sample of 32 hours, average recovery is 85.56%, correction coefficient=1/85.56%=1.155, fermentation liquor stoste content of glutamic acid (g/dL)=52*1.155*200/1000=12.017, correcting the fermentation liquor stoste content of glutamic acid that artifact sensing assays instrument measures is 12.017g/dL, and to measure fermentation liquor stoste content of glutamic acid 12.03g/dL basically identical with Hua Bofa, but this method is more quick, convenient, accurate.
For actual production, because every series-produced process conditions are consistent, the composition of the fermentation broth sample of different fermentations batch same fermentation period and comparision contents are stablized, its average recovery experimental result and correction coefficient substantially close, therefore by the data accumulation of a period of time, determine to be suitable for the correction coefficient that fermentation period is the fermentation broth sample of 32 hours, as long as the measured value measuring fermentation broth sample is in actual applications multiplied by corresponding extension rate, carry out base unit conversion, being multiplied by corresponding fermentation period is again that namely to obtain fermentation period be 32 hours glutami acid fermentation liquor stoste content of glutamic acids for the correction coefficient of 32 hours.As long as process conditions and acid yield remain unchanged, this correction coefficient can use always; If production technology and fermentation level change to some extent, correction coefficient adjusts accordingly with regard to needs.
In summary it can be seen, the detection method of bio-sensing analysis-e/or determining content of glutamic acid of the present invention can be applied in the detection measuring content of glutamic acid aspect.
Claims (7)
1. the detection method of a bio-sensing analysis-e/or determining content of glutamic acid, it is characterized in that: step comprises: bio-sensing analyser adopts two-point potentionmetric to measure, by Sample Dilution, carry out average recovery experiment and determine correction coefficient, calculate content of glutamic acid, namely detect and obtain content of glutamic acid;
Concrete steps are as follows:
(1) fermentation broth sample preparation: get glutami acid fermentation liquor stoste, is diluted to 100-200 doubly, does three parts of Duplicate Samples, obtain fermentation broth sample;
(2) bio-sensing analyser calibration: after the start of bio-sensing analyser, treat that instrument cleans automatically complete, after sample introduction green light, draw 25 μ L titers and inject reaction tank, after 2 ~ 4 times to be determined, instrument auto-scaling, then draw 12.5 μ L titers injection reaction tanks, reaction terminates, linearly check key, correct and completely to calibrate again, then carry out test sample, the measured value of bio-sensing analysis-e/or determining fermentation broth sample glutamic acid is X
1;
(3) standard items sample preparation: add glutamic acid standard items in fermentation liquor stoste, be diluted to 100-200 afterwards doubly, do three parts of Duplicate Samples, obtain standard items sample, the measured value of bio-sensing analysis-e/or determining standard items sample glutamic acid is X
2;
(4) result calculates: according to X
1and X
2calculate average recovery and correction coefficient; According to the content of glutamic acid that following formulae discovery bio-sensing analyser records, namely detect and obtain fermentation liquor stoste content of glutamic acid;
Wherein, computing formula: average recovery (%)=(X
2-X
1) * V/A*100;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
X
2the measured value of-bio-sensing analysis-e/or determining standard items sample glutamic acid, mg/dL;
The volume of V-fermentation broth sample, dL;
The quality of A-interpolation glutamic acid standard items, mg;
Correction coefficient=1/ average recovery;
Fermentation liquor stoste content of glutamic acid (g/dL)=X
1* correction coefficient * extension rate/1000;
In formula: X
1the measured value of-bio-sensing analysis-e/or determining fermentation broth sample glutamic acid, mg/dL;
1000-Units conversion factor.
2. the detection method of bio-sensing analysis-e/or determining content of glutamic acid according to claim 1, it is characterized in that: described step (1) middle Sample Dilution is: when glutami acid fermentation liquor stoste is fermented and cultured 6 ~ 12h, glutami acid fermentation liquor stoste is diluted to 100 times; When glutami acid fermentation liquor stoste is fermented and cultured >12h, glutami acid fermentation liquor stoste is diluted to 200 times.
3. the detection method of bio-sensing analysis-e/or determining content of glutamic acid according to claim 1, it is characterized in that: described step (2) middle bio-sensing analyser is: SBA-40 type bio-sensing analyser, SBA-40C type bio-sensing analyser, SBA-40E type bio-sensing analyser, SBA-40X type bio-sensing analyser, or SBA-50 type bio-sensing analyser.
4. the detection method of bio-sensing analysis-e/or determining content of glutamic acid according to claim 1, is characterized in that: the described step (2) composition of Plays liquid and concentration is: 50mg/dL lactic acid, 100mg/dL glutamic acid and 100mg/dL glucose.
5. the detection method of bio-sensing analysis-e/or determining content of glutamic acid according to claim 1, it is characterized in that: described step (2) in bio-sensing analyser calibration time, when entering 25 μ L titer, scaled values is 100, and when entering 12.5 μ L titer, scaled values is 50; Often measure 5 ~ 8 samples again to calibrate once.
6. the detection method of bio-sensing analysis-e/or determining content of glutamic acid according to claim 1, is characterized in that: described step (3) in fermentation liquor stoste, add glutamic acid standard items quality be: 0.5X
1* V ~ 1.5X
1* V, wherein, X
1for biological sensing assays instrument measures the measured value of fermentation broth sample glutamic acid, the volume of unit to be mg/dL, V be fermentation broth sample, unit is dL.
7. the detection method of the bio-sensing analysis-e/or determining content of glutamic acid as described in any one of claim 1 to 6 is measuring the application in content of glutamic acid.
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| 杨海麟,吕霞付等.发酵过程在线检测系统在谷氨酸发酵中的应用研究.《中国调味品》.2003,(第10期),16-20. * |
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