CN101825576A - Method and kit for rapid detection of ethanol content in microbial fermentation solution - Google Patents
Method and kit for rapid detection of ethanol content in microbial fermentation solution Download PDFInfo
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- CN101825576A CN101825576A CN200910225881A CN200910225881A CN101825576A CN 101825576 A CN101825576 A CN 101825576A CN 200910225881 A CN200910225881 A CN 200910225881A CN 200910225881 A CN200910225881 A CN 200910225881A CN 101825576 A CN101825576 A CN 101825576A
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Abstract
The invention discloses a method for rapid detection of ethanol content and reducing sugar content, which belongs to the technical field of quantitative detection of chemical components. The method comprises the following steps of: pre-processing microbial fermentation solution; establishing a reducing sugar standard curve by using DNS and detecting the reducing sugar content in a sample; establishing the reducing sugar standard curve by using potassium dichromate and detecting the sample; and establishing an ethanol standard curve by using the potassium dichromate and calculating the ethanol content in the sample. The method can directly detect the ethanol content in the microbial fermentation solution without using a precise detector, and has the advantages of high precision, fast, simple and convenient operation, less required sample and reagent quantity, low cost, less required instrument and equipment, low requirement on environment, no pollution generated in the detection process, large quantity of samples detected at one time and the like.
Description
Technical field:
The invention belongs to a kind of ethanol of chemical constitution quantitative measurement technology scope and the method for quick of content of reducing sugar, relate in particular to is in conjunction with 3,5-dinitrosalicylic acid (3,5-dinitrosalicylic Acid is called for short DNS) new method of a kind of quantitative fast detecting ethanol the oxidation reaction of reducing sugar and ethanol set up with the colour developing reflection of reducing sugar and potassium dichromate.
Background technology:
Concentration of alcohol is an important procedure parameter in the microbial fermentations such as alcohol fuel, drinks, directly affect the optimization regulation and control of sweat and the quality and the output of tunning, the rapid and accurate determination of ethanol content is this type of research and prerequisite of producing and basis in the fermentation liquor.
Although the ethanol detection method is more at present,, be difficult to satisfy the needs that concentration of alcohol detects in alcohol fuel, drinks fermentation research and the production run because all there is bigger limitation in every kind of method.The method of measuring at present concentration of alcohol has vapor-phase chromatography, chemical method, and hydrometer method etc., vapor-phase chromatography instrument costliness and incompatibility grass-roots unit popularize.Chemical method has eosin lactone absorbance method, Congo red development process, potassium dichromate method etc., these class methods be utilize between certain material (eosin, Congo red, potassium dichromate) and the ethanol color side takes place should, by measuring the absorbance value of product under certain specific wavelength, utilize external standard method to obtain concentration of ethanol.These methods only are only applicable to the mensuration of sample distillation back ethanol, and composition is comparatively complicated in the microbial fermentation solution, if directly measure in order to last method, other contained in sample materials are with serious interference measurement result.
Domestic food sanitary inspection method adopts hydrometer method more at present.Hydrometer method must distill when analyzing, and takes a lot of work and the sample still-process is time-consuming, needs bigger sample volume, is difficult to a plurality of samples of high throughput testing.And still-process is difficult to the ethanol in the sample is distilled fully on the one hand usually with the volatilization of ethanol on the other hand, and the measured result accuracy is relatively poor.In order to promote the research and development of alcohol fuel fermentation work, for the ease of following the tracks of the effect of sweat, assessment different fermentations condition, press for a kind of directly method of fast detecting ethanol content from microbial fermentation solution at present, and set up corresponding quick detection kit with the method.
Summary of the invention:
Patent of the present invention is at the deficiencies in the prior art, a kind of detection by quantitative new method that directly detects ethanol content from microbial fermentation solution is provided, this method is according to the characteristics that can both carry out oxidation reaction of potassium dichromate to reducing sugar and ethanol, by DNS the oxidation reaction of reducing sugar is calculated content of reducing sugar, deduct its reaction signal in potassium dichromate, thereby draw the content of ethanol in the sample, so the method also is referred to as " reducing sugar deduction alcohol determining method ".
For realizing above purpose, the technical scheme that patent of the present invention adopts is:
1. microbial fermentation solution is carried out pre-service: the insoluble substance in the centrifugal removal fermentation liquor, then with the protein in the trichloroacetic acid removal fermentation liquor, utilize hexadecyl trimethyl ammonium bromide (Hexadecyltrimethyl ammonium Bromide again, be called for short CTAB) remove the polysaccharide in the fermentation liquor, the principal ingredient that is contained in the sample after so handling is ethanol and a small amount of reducing sugar.
2. with 3, the 5-dinitrosalicylic acid is set up the content of reducing sugar in reducing sugar typical curve and the test sample: 3, the color reaction of 5-dinitrosalicylic acid and reducing sugar can be by reading to survey A550 light absorption check, 3, the typical curve under the 5-dinitrosalicylic acid is handled is accurately quantitative with the reducing sugar in the sample according to reducing sugar.
3. set up reducing sugar typical curve and test sample with potassium dichromate: potassium dichromate belongs to strong oxidizer, not only can with reducing sugar in the sample but also can with ethanol generation oxidation reaction wherein, its oxidation product can be by reading to survey the check of OD585 optical density.Therefore the DNS method that can at first describe by above-mentioned (2) calculates the content of reducing sugar in the sample, again according to the reducing sugar typical curve of potassium dichromate, calculates reducing sugar shared part in the total oxidation reaction signal of potassium dichromate in the sample.
4, set up ethanol content in ethanol typical curve and the calculation sample with potassium dichromate: from the total oxidation reaction signal of potassium dichromate, deduct the shared part of reducing sugar and potassium dichromate reaction, draw the signal intensity of ethanol synthesis in potassium dichromate and the sample, typical curve according to ethanol and potassium dichromate reaction calculates concentration of ethanol in the sample.
The kit of the method for quick of ethanol content in the said microbial fermentation solution in the inventive method, be used for above-mentioned described application, comprise the required reagent of sample pretreatment, reducing sugar detection reaction reagent, potassium dichromate oxidation reaction reagent, reducing sugar standard items, ethanol standard items, reaction terminating liquid, microwell plate, directions for use etc.
What need a progressive explanation is:
Said microbial fermentation solution mainly is meant any fermentation liquor or the reactant liquor that contains ethanol that is produced by fungi (comprising yeast), bacterium (comprising zymomonas mobilis), fungi or bacterium extract, fungi or bacterial fermentation enzyme system in the inventive method, the used nutriment that ferments mainly is meant agricultural product roughing material, also can be the clear and definite nutrient culture media of component.
Said reducing sugar is meant the sugar that contains hemiacetal or hemiketal structure, can reduce fehling reagent in the inventive method, with glucose is representative, wherein said reducing sugar standard solution adopts the glucose preparation usually, but also can adopt the potpourri preparation of other single reducing sugars or different reducing sugars.
The method of said detection reducing sugar is to utilize reducing sugar to have the characteristics of reductibility and it is detected in the inventive method.
The method of said detection reducing sugar is preferably the DNS method in the inventive method, but also can be additive method.
Said quantitative detecting method in the inventive method, in the application of the aspects such as effect that detect microbial ethanol fermentation result, tracking course of fermentation, evaluation different fermentations condition, application comprises the exploration of alcohol fermentation principle and zymotechnique, the research and development production of drinking alcohol series products, the research and development production of industrial alcohol series products, the research and development production of alcohol fuel series products etc.
The said kit reducing sugar of patent of the present invention detection reaction reagent is 3, the 5-edlefsen's reagent.
Said reducing sugar is meant the sugar that contains hemiacetal or hemiketal structure, can reduce fehling reagent in the inventive method, is representative with glucose.
Patent disclosure of the present invention in microbial fermentation solution the method for quick and the kit of ethanol content, do not need accurate detecting instrument, just can directly from microbial fermentation solution, detect the content of ethanol, method easy highly sensitive, specificity good, fast and convenient, high flux, be particularly useful for detecting simultaneously a plurality of samples.This method is except detecting concentration of alcohol, the also related concentration that obtains reducing sugar in the fermentation liquor of while.This method can be widely used in the aspects such as effect that detect microbial ethanol fermentation result, tracking course of fermentation, identify the different fermentations condition.According to the principle of this method, can develop, produce, allocate and pack the relevant detection product, comprise the ingredient of complete kit and kit.
Description of drawings:
Fig. 1 is equipped with the glucose canonical plotting for the DNS legal system;
Fig. 2 decides the glucose canonical plotting for the potassium dichromate legal system;
Fig. 3 decides the ethanol canonical plotting for the potassium dichromate legal system;
Embodiment:
Specify this patent with embodiment below and detect step:
1, preparation DNS and potassium bichromate solution: DNS solution: with 3 of 6.3g, the 2mol NaOH solution of 5-dinitrosalicylic acid and 262mL is added to 500mL and contains in the hydrothermal solution of 185g sodium potassium tartrate tetrahydrate, add 5g crystallization phenol and 5g sodium sulphite again, stirring and dissolving, cooling back adding distil water is settled to 1000mL, be stored in the brown bottle stable, standby after 1 week.Potassium bichromate solution: the sulfuric acid with 8N makes potassium dichromate saturated.
2, preparation ethanol and glucose standard sample liquid: ethanol standard sample liquid: will the 4mL absolute ethyl alcohol add in the 96mL pure water and be mixed with 4% solution, with pure water it is carried out 2 times of serial dilutions to 2%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.007813% ethanolic solution then.Glucose standard sample liquid: take by weighing 80 ℃ and dry, add dissolved in purified water and be settled to 100mL, be mixed with 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625% standard glucose solution then with pure water to the pure glucose 100mg of the analysis of constant weight.
3, production standard curve:
(1) DNS method glucose typical curve: get each concentration 200 μ L of above-mentioned standard glucose solution, add 600 μ L DNS, 100 ℃ of heating 10min, to cooling, get 200 μ L with the flowing water flushing in 96 orifice plates, under 550nm, survey the OD value, with the optical density value is ordinate, concentration of glucose (mg/mL) is a horizontal ordinate, draws the glucose typical curve, calculates regression equation and related coefficient.The results are shown in Figure 1:
(2) potassium dichromate method glucose typical curve: get each concentration solution 60 μ L of above-mentioned standard glucose solution, the potassium bichromate solution that adds 120 μ L, behind the room temperature reaction 30min, under 585nm, read to survey the OD value, with the optical density value is ordinate, concentration of glucose (mg/mL) is a horizontal ordinate, draws the glucose typical curve, calculates regression equation and related coefficient.The results are shown in Figure 2.
(3) potassium dichromate method ethanol typical curve: get each concentration 60 μ L of above-mentioned standard ethanolic solution, the potassium bichromate solution that adds 120 μ L, behind the room temperature reaction 30min, under 585nm, read to survey the OD value, with the optical density value is ordinate, concentration of alcohol (%) is a horizontal ordinate, draws the ethanol typical curve, calculates regression equation and related coefficient.The results are shown in Figure 3.
4, preparation microbial fermentation solution: the preparation fermentation medium, the inoculum concentration according to 10% behind the high pressure steam sterilization is inoculated.Get the 1mL fermentation liquor later at suitable temperature bottom fermentation 24h.
5, pre-service fermentation broth sample: with the centrifugal 5min of 1mL fermentation liquor room temperature 12000rpm, get the 0.5mL supernatant, adding isopyknic 20% trichloroacetic acid left standstill 30 minutes, the centrifugal 10min of room temperature 13000r/min, get supernatant, if there is suspended material in supernatant, can filter once more by bacterial filter.The CTAB solution that adds 200 μ L then, 65 ℃ of water-bath 10min, the centrifugal 10min of room temperature 13000r/min gets supernatant, and with 10 times of the diluted samples that obtain, detects then.
6, the reducing sugar in the DNS method working sample:, add 600 μ L DNS solution, mixing from handling well to such an extent that get 200 μ L the sample through diluting 10 times supernatant in centrifuge tube, 100 ℃ are heated 10min down, to cooling, get 200 μ L with the flowing water flushing in 96 orifice plates, under 550nm, survey the OD value.Adopt real data, the concentration of reduced sugar in the calculation sample.
7, potassium dichromate oxidation working sample: get 60 μ L through diluting 10 times of supernatants in 96 orifice plates from the sample of handling well, add 120 μ L potassium bichromate solutions, mixing reacts 30min under the room temperature, surveys absorbance under 585nm.Adopt real data, the potassium dichromate oxidation of calculation sample reacts overall OD585 numerical value.
8, calculate reducing sugar shared signal intensity (OD585 numerical value) in the reaction of fermentation liquor potassium dichromate oxidation:, the concentration of reduced sugar in the sample is converted to the OD585 numerical value of potassium dichromate oxidation reaction according to potassium dichromate method glucose typical curve.(adopting real data, the potassium dichromate OD585 numerical value of reducing sugar in the calculation sample)
9, the concentration of alcohol in the calculation sample: the potassium dichromate oxidation with sample reacts the potassium dichromate OD585 numerical value that overall OD585 numerical value deducts reducing sugar in the sample, draws the potassium dichromate OD585 numerical value of ethanol in the sample.According to potassium dichromate method ethanol typical curve, calculate the concentration of alcohol in the sample then.
10, checking this method result: detect the content of ethanol in this sample, comparative result by the way of distillation or other aspects.
In order to verify the accuracy of this detection method, we carry out pre-service with the fermentation liquor of sweet potato alcohol fermentation according to the method for this patent, (being labeled as No. 0).Get the absolute ethyl alcohol of No. 0 liquid 2mL adding different volumes, obtain the sample (being labeled as 1-13) of 13 different ethanol concentration, use this method to detect wherein concentration of alcohol then, the results are shown in Table 1, as seen from table the RSSEA method be used for detecting fermentation liquor concentration of alcohol accurately and reliably.
The comparison of table 1 alcohol measurement value and actual value
The implementation process of patent of the present invention is traditional DNS to be detected reducing sugar method, potassium dichromate detect the reducing sugar method, potassium dichromate detects the ethanol method and carried out technological improvement, makes it have that the sample size of use is few, working sample number is many, measure advantages such as accurate quick.
Claims (6)
1. the method for quick of ethanol content and kit in the microbial fermentation solution, it is characterized in that: described detection method is:
(1) microbial fermentation solution is carried out pre-service: the insoluble substance in the centrifugal removal fermentation liquor, then with the protein in the trichloroacetic acid removal fermentation liquor, utilize the polysaccharide in the hexadecyl trimethyl ammonium bromide removal fermentation liquor again, the principal ingredient that is contained in the sample after so handling is ethanol and a small amount of reducing sugar.
(2) with 3, the 5-dinitrosalicylic acid is set up the content of reducing sugar in reducing sugar typical curve and the test sample: 3, the color reaction of 5-dinitrosalicylic acid and reducing sugar can be by reading to survey A550 light absorption check, 3, the typical curve under the 5-dinitrosalicylic acid is handled is accurately quantitative with the reducing sugar in the sample according to reducing sugar.
(3) set up reducing sugar typical curve and test sample with potassium dichromate: utilize content of reducing sugar in the sample,, calculate reducing sugar shared part in the total oxidation reaction signal of potassium dichromate in the sample according to potassium dichromate reducing sugar typical curve.
(4) set up ethanol content in ethanol typical curve and the calculation sample with potassium dichromate: from the total oxidation reaction signal of potassium dichromate, deduct the shared part of reducing sugar and potassium dichromate reaction, draw the signal intensity of ethanol synthesis in potassium dichromate and the sample, typical curve according to ethanol and potassium dichromate reaction calculates concentration of ethanol in the sample.
Patent kit reagent of the present invention comprises the required reagent of sample pretreatment, reducing sugar detection reaction reagent, potassium dichromate oxidation reaction reagent, reducing sugar standard items, ethanol standard items, reaction terminating liquid, microwell plate.
2. the method for quick and the kit of ethanol content in the microbial fermentation solution according to claim 1, it is characterized in that: described microbial fermentation solution mainly is meant any fermentation liquor or the reactant liquor that contains ethanol that is produced by fungi, bacterium, fungi or bacterium extract, fungi or bacterial fermentation enzyme system, the used nutriment that ferments mainly is meant agricultural product roughing material, also can be the clear and definite nutrient culture media of component.
3. the method for quick and the kit of ethanol content in the microbial fermentation solution according to claim 1, it is characterized in that: described reducing sugar is meant the sugar that contains hemiacetal or hemiketal structure, can reduce fehling reagent, with glucose is representative, wherein said reducing sugar standard solution adopts the glucose preparation usually, but also can adopt the potpourri preparation of other single reducing sugars or different reducing sugars.
4. the method for quick and the kit of ethanol content in the microbial fermentation solution according to claim 1 is characterized in that: the method for described detection reducing sugar is 3,5-dinitrosalicylic acid method.
5. the method for quick and the kit of ethanol content in the microbial fermentation solution according to claim 1 is characterized in that: described kit reducing sugar detection reaction reagent is 3, the 5-edlefsen's reagent.
6. the method for quick and the kit of ethanol content in the microbial fermentation solution according to claim 1 is characterized in that: described reducing sugar is meant the sugar that contains hemiacetal or hemiketal structure, can reduce fehling reagent, is representative with glucose.
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| CN103884667A (en) * | 2014-04-18 | 2014-06-25 | 湖北工业大学 | Method for measuring polysaccharide content of medlar extracting solution |
| CN104155253A (en) * | 2014-09-09 | 2014-11-19 | 中国石油化工股份有限公司 | Method for fast detecting yield of biological surfactants |
| CN107490573A (en) * | 2017-10-02 | 2017-12-19 | 黄海霞 | 1 to 10 degree wine alcoholic strength simplicity rapid assay methods |
| CN107576627A (en) * | 2017-10-02 | 2018-01-12 | 黄种山 | 60 to 70 degree wine alcoholic strength simplicity rapid assay methods |
| CN107655843A (en) * | 2017-10-02 | 2018-02-02 | 黄种山 | 40 to 50 degree wine alcoholic strength simplicity rapid assay methods |
| CN107677669A (en) * | 2017-10-02 | 2018-02-09 | 黄海霞 | 20 to 30 degree wine alcoholic strength simplicity rapid assay methods |
| CN107677668A (en) * | 2017-10-02 | 2018-02-09 | 黄海霞 | 10 to 20 degree wine alcoholic strength simplicity rapid assay methods |
| CN107727588A (en) * | 2017-10-02 | 2018-02-23 | 黄海霞 | 30 to 40 degree wine alcoholic strength simplicity rapid assay methods |
| CN107807103A (en) * | 2017-11-06 | 2018-03-16 | 洪佳欣 | 18 to 20 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
| CN107817222A (en) * | 2017-11-06 | 2018-03-20 | 洪佳欣 | 16 to 18 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
| CN107843570A (en) * | 2017-11-06 | 2018-03-27 | 陈荷冰 | 12 to 14 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
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| CN112485249A (en) * | 2020-11-27 | 2021-03-12 | 无锡市疾病预防控制中心 | Kit for visual rapid detection of ethanol content in disinfectant and detection method |
| CN114058671A (en) * | 2020-07-31 | 2022-02-18 | 北京安普生化科技有限公司 | Klebsiella pneumoniae detection method and detection reagent device for producing ethanol |
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| CN103884667A (en) * | 2014-04-18 | 2014-06-25 | 湖北工业大学 | Method for measuring polysaccharide content of medlar extracting solution |
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| CN107677669A (en) * | 2017-10-02 | 2018-02-09 | 黄海霞 | 20 to 30 degree wine alcoholic strength simplicity rapid assay methods |
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| CN107655843A (en) * | 2017-10-02 | 2018-02-02 | 黄种山 | 40 to 50 degree wine alcoholic strength simplicity rapid assay methods |
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| CN107677668A (en) * | 2017-10-02 | 2018-02-09 | 黄海霞 | 10 to 20 degree wine alcoholic strength simplicity rapid assay methods |
| CN107727588A (en) * | 2017-10-02 | 2018-02-23 | 黄海霞 | 30 to 40 degree wine alcoholic strength simplicity rapid assay methods |
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| CN107817222A (en) * | 2017-11-06 | 2018-03-20 | 洪佳欣 | 16 to 18 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
| CN107843570A (en) * | 2017-11-06 | 2018-03-27 | 陈荷冰 | 12 to 14 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
| CN107860734A (en) * | 2017-11-06 | 2018-03-30 | 陈荷冰 | 14 to 16 degree yellow rice wine non-sugar solidity simplicity rapid assay methods |
| CN114058671A (en) * | 2020-07-31 | 2022-02-18 | 北京安普生化科技有限公司 | Klebsiella pneumoniae detection method and detection reagent device for producing ethanol |
| CN112485249A (en) * | 2020-11-27 | 2021-03-12 | 无锡市疾病预防控制中心 | Kit for visual rapid detection of ethanol content in disinfectant and detection method |
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Application publication date: 20100908 |