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Reagent for detecting lipase and method for rapidly detecting lipase

A technology for detecting reagents and lipase, which is applied in the direction of material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of unsuitable food detection, poisonous nitrophenol, low stability, etc., to save detection time, accurately and quickly detect, detect fast effect

Active Publication Date: 2014-08-13
淮安安福科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The copper soap method is based on fatty acids and Cu 2+ Form a green complex, measure the absorbance at a wavelength of 710nm to quantify the enzyme activity, the copper soap method has high accuracy, but the operation is cumbersome and the stability is not high, and because this method uses a large amount of benzene to extract the copper soap, it is easy Cause pollution and harm to the human body; the p-nitrophenol method uses p-nitrophenol ester as a substrate, and after hydrolysis by lipase, colored p-nitrophenol is produced, and the absorbance is measured at a wavelength of 420nm for quantification of enzyme activity. The phenol method has a large deviation in the determination of lipase with low enzyme activity, and p-nitrophenol is toxic, so it is not suitable for food testing

Method used

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  • Reagent for detecting lipase and method for rapidly detecting lipase
  • Reagent for detecting lipase and method for rapidly detecting lipase
  • Reagent for detecting lipase and method for rapidly detecting lipase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the preparation of working solution C

[0048] 1) Weigh 8g of NaCl, 0.2g of KCl, NaCl 2 HPO 4 1.42g and KH 2 PO 4 0.27g, add 800ml of ultrapure water to dissolve, dilute to 1L with ultrapure water, prepare as liquid A after high-temperature sterilization, and store at 4°C;

[0049] 2) Take 2.5ml of 1.5mM 1,2-oxo-dilauryl-(±)glycerol-glutaric acid-6'-methyl-resorufin ester, 150μl of 0.5M sodium acetate pH 4.0 buffer Solution, 25μl Tween 20 and 10μl Proclin 300, add water to make up to 50ml, that is, prepare as B solution, store at 4°C in the dark;

[0050] 3) Mix liquid A and liquid B at a volume ratio of 2:1 to prepare working liquid C.

[0051]

Embodiment 2

[0052] Embodiment 2: standard curve drawing

[0053] 1) Add 5, 10, 15, and 20 μL of standard products with a concentration of 100 μM to the 96-well ELISA plate, and then add 200 μL of the working solution C prepared according to Example 1 to each well, mix well, and the blank control is the corresponding volume working solution C;

[0054] 2) Put the microplate plate into a preheated fluorescent microplate reader at 37°C, excite with an excitation wavelength of 520nm, detect at an emission wavelength of 610nm, and read the fluorescence value as A 标 , the blank control is A 空 , with the fluorescence value of the standard (A 标 -A 空 ) is the ordinate (unit is RFU), and the volume of the standard product is the abscissa (unit is μL), draw the standard curve.

[0055] The obtained standard curve is as figure 1 As shown, the standard curve is very linear, the linear equation is y=2.58x, and the correlation coefficient R2 is 0.998.

[0056]

Embodiment 3

[0057] Embodiment 3: Determination of raw material milk and pasteurized milk lipase enzyme activity

[0058] 1) Take 2 raw milk samples, named 1# and 2#, and take a pasteurized milk sample, named 3#;

[0059] 2) Add 10 μL of samples to the 96-well ELISA plate, add 200 μL of working solution C to each well, mix well, and use the corresponding volume of working solution C as the blank control;

[0060] 3) Put the microplate plate into a fluorescent microplate reader preheated at 37°C, excite with an excitation wavelength of 520nm, detect at an emission wavelength of 610nm, read the fluorescence value, and record it as A1 样 , the blank control is recorded as A1 空 ; After incubation for 40min, read the fluorescence value again and record it as A2 样 , the blank control is recorded as A2 空 ;

[0061] 4) The fluorescence value of the sample [A 样 =(A2 样 -A2 空 )-(A1 样 -A1 空 )] into the standard curve, read the corresponding standard volume (B) of the sample from the standard c...

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Abstract

The invention discloses a reagent for rapidly detecting lipase in a dairy food. The reagent comprises a solution A, a solution B and a standard substance, wherein the standard substance is methoxyl resorufin, the solution A is a PBS, and the solution B contains a fluorogenic substrate, a buffer solution, an emulsifying agent and a preservative. The invention further discloses a method for rapidly detecting lipase in the dairy food by using the reagent. The method comprises the steps: drawing a standard curve, reading a sample fluorescence value and calculating the lipase enzyme. According to the method, the sensitivity is high, the operation is simple, the workload is small, the system errors are small, multiple test repeatability is good, rapidness and convenience are achieved, and a detection result can be obtained within 20-60min.

Description

technical field [0001] The invention belongs to the field of food monitoring, and relates to a lipase detection reagent and a rapid detection method using the lipase detection reagent, in particular to a rapid detection method for lipase in dairy products such as milk. Background technique [0002] Dairy products are rich in nutrition and are ideal nutritious food for human beings, but they are also good culture medium for microorganisms and are high-risk food raw materials. Among many microbial contaminations, psychrophilic bacteria contamination is the main factor affecting the shelf life of dairy products. Psychrophilic bacteria will multiply when milk raw milk is stored at low temperature. Although these bacteria can be killed by pasteurization and UHT sterilization stages, some of them, such as Pseudomonas, can produce extremely heat-resistant bacteria. Lipase, even after 140°C ultra-high temperature sterilization treatment, there will still be a small amount of residu...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 周娟作
Owner 淮安安福科技有限公司
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