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Spectrophotometric method for quickly detecting content of ethyl carbamate

A kind of urethane, content technology, applied in the field of food safety detection, analytical chemistry, can solve the problem of no reported enzyme sensor and so on

Active Publication Date: 2015-01-07
ANHUI HUATENG AGRI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no enzymatic sensor for detecting EC content has been reported so far.

Method used

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  • Spectrophotometric method for quickly detecting content of ethyl carbamate
  • Spectrophotometric method for quickly detecting content of ethyl carbamate
  • Spectrophotometric method for quickly detecting content of ethyl carbamate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Determination of the optimal reaction buffer system for the dual enzyme coupling system

[0026] Before using the dual-enzyme coupling system to detect the content of ethyl carbamate, the optimal reaction buffer system of the dual-enzyme system of ethyl carbamate degrading enzyme and glutamate dehydrogenase was optimized first. The urethane degrading enzyme used in the present invention can maximize its catalytic activity in an acidic environment, while the glutamic acid dehydrogenase has a higher catalytic activity in a moderately alkaline condition. The two catalyze two-step cascade reactions in the same reaction system. The type of buffer and pH are different, and the microenvironment of the enzyme active center and the catalytic reaction rate will change.

[0027] First prepare a concentration of 25mmol L -1 citrate buffer (pH 4.5, 5.5), phosphate buffer (pH 6.0, 6.5, 7.0) and Tris-HCl buffer (pH 7.5, 8.0), and use the corresponding pH buffer to dissolve ...

Embodiment 2

[0028] Example 2 Determination of Optimum Addition of Urethane Degrading Enzyme and Glutamate Dehydrogenase in Dual Enzyme Coupling System

[0029] In the optimal reaction buffer system, when the amount of glutamate dehydrogenase and other reaction substrates was kept constant, the enzyme activities added to the system were 4, 8, 12 and 16 U·mL -1 The urethane degrading enzyme was used, and then the mixed solution was placed at 340 nm to detect the change of absorbance value with time. With the increase of the enzymatic activity of ethyl carbamate degrading enzyme in the reaction system, the rate of decomposing EC to produce ammonia is accelerated, and the rate of glutamate dehydrogenase using ammonia to convert α-ketoglutarate is also accelerated, so the oxidation rate of NADH also increases. Gradually accelerated, manifested as an increase in the change in absorbance at 340 nm. Enzyme activity reaches 16 U·mL -1 After that, the change of the absorbance value at 340 nm has ...

Embodiment 3

[0031] The establishment of embodiment 3 spectrophotometric method ethyl carbamate concentration standard curve

[0032] In the optimal reaction buffer system, in the double-enzyme coupling system with the determined optimal addition of urethane degrading enzyme and glutamate dehydrogenase, 540mmol·L -1 α-ketoglutarate, and 7.5mmol·L -1 In NADH solution, its A was measured 340 is 0.46733, and the concentrations of added urethane are 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 μmol L -1 After the A 340 are 0.46733, 0.48167, 0.48567, 0.49133, 0.493, 0.49767, 0.502, 0.5095, 0.51233, 0.55167, 0.59833, 0.63633, 0.67633, 0.737, thus ΔA 340 0, 0.01434, 0.01834, 0.024, 0.02567, 0.03034, 0.03467, 0.04217, 0.045, 0.08434, 0.131, 0.169, 0.209, 0.26967, respectively, the concentration of ethyl carbamate (μmol L -1 ) as the abscissa, with the corresponding ΔA 340 As the ordinate, the drawing is the spectrophotometric urethane concentration standard curve.

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Abstract

The invention discloses a spectrophotometric method for quickly detecting the content of ethyl carbamate, and belongs to the technical field of analytical chemistry and food safety inspection. Ethyl carbamate degrading enzyme and glutamate dehydrogenase construct a double-enzyme coupled system; the ethyl carbamate degrading enzyme hydrolyzes the ethyl carbamate to generate ethyl alcohol, water and ammonia, the ammonia reacts with a cosubstrate alpha-ketoglutarate under the catalysis of the glutamate dehydrogenase to generate glutamic acid, and reduced coenzyme namely NADH (Nicotinamide Adenine Dinucleotide Hydrogen) is oxidized concomitantly, so that the system changes the content of the ethyl carbamate into the content of the NADH, and the content of the ethyl carbamate in a liquid-state system can be detected by using the change of the light absorption value of the NADH at 340nm. By virtue of the method, the ethyl carbamate in a solution can be quickly detected, the ethyl carbamate in a yellow wine system can be simulated, the detection limit is as low as 0.1mu mol. L<-1>, and an effective way is provided for the detection of the ethyl carbamate in fermented food and beverages.

Description

technical field [0001] The invention relates to a spectrophotometric method for quickly detecting the content of ethyl carbamate, belonging to the technical fields of analytical chemistry and food safety detection. Background technique [0002] Ethyl carbamate (EC for short) is a genotoxic and carcinogenic substance that can cause diseases such as lung cancer, lymphoma, liver cancer and skin cancer in rodents. EC widely exists in fermented food (such as soy sauce, fermented bean curd, etc.), brewed wine (such as rice wine, sake, wine, cider, etc.) in vivo. Studies have shown that about 0.5% of ethyl carbamate is oxidized to acetyl urethane by cytochrome P450 in the organism, and then further forms acetyl urethane epoxide, which can form DNA polyaddition in the organism substances, causing damage to the double strands of the DNA structure, leading to cancerous cells; another 0.1% of ethyl carbamate is oxidized to N-hydroxy ethyl carbamate by cytochrome P450, which induces C...

Claims

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Application Information

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IPC IPC(8): G01N21/31
Inventor 周楠迪卢晓霞田亚平
Owner ANHUI HUATENG AGRI TECH CO LTD
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