Application of casein plate method in quantitative analysis of protease activity

A quantitative analysis and casein technology, which is applied in the field of protease activity detection, can solve the problems such as error-prone detection results, error-prone measurement results, large absorbance value, etc., to avoid errors and inaccurate measurement results, low detection limit, Simple operation effect

Pending Publication Date: 2019-11-22
GUIZHOU UNIV
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Problems solved by technology

The detection results of these methods are accurate, but the detection results are prone to errors when detecting colored foods, mainly because the ultraviolet spectrophotometer uses the principle of light scattering, and when the color of the sample is too dark, the absorbance value is too large, resulting in errors
The principle of measuring protease activity by Folin’s phenol method is: use casein as a substrate, measure the amount of amino acids produced after reacting with the sample for a period of time, and then calculate the protease activity of the sample. The disadvantage of this method is that there are many factors affecting the reaction system. If it contains citric acid, glycerin, glycine, Tris buffer, hyaluronic acid, dithiothreitol, mercaptoethanol, ethylenediaminetetraacetic acid (EDTA) and urea and other substances, it will cause interference; and it is affected by protein The impact of specificity, and the color intensity of different proteins is slightly different due to the content of tyrosine and tryptophan, and the measurement results are prone to errors

Method used

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  • Application of casein plate method in quantitative analysis of protease activity
  • Application of casein plate method in quantitative analysis of protease activity
  • Application of casein plate method in quantitative analysis of protease activity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 Folin phenol method and casein plate method contrast

[0018] (1) Casein plate method: use a 10mL pipette tip to punch the prepared casein plate (three holes on one plate, as parallel), draw 10 μL of enzyme solution into the hole, let it stand for 10 minutes, and then put it into a 37°C incubator Inverted culture for 18 hours, take out to measure the diameter of the hydrolysis circle and calculate the area of ​​the dissolution circle. Properly dilute the commercial trypsin to prepare the solution with the required enzyme activity, respectively punch and culture on the casein plate, take the area of ​​the dissolution circle as the abscissa (x, mm2), and the commercial trypsin activity as the ordinate (y, U · mL-1) Make a commercial trypsin standard curve. The enzyme activity of the sample was measured according to the steps of the standard curve, and the area of ​​the hydrolysis circle of the sample was calculated with the commercial trypsin standard curve ...

Embodiment 2

[0024] Embodiment 2 casein plate method precision

[0025] Accurately weigh 18 parts of 0.100g commercial trypsin, add sterile water to dissolve to 10mL, prepare a 250U / mL trypsin solution, and dilute it into 6 parts of 10U·mL-1, 25U·mL-1, 50U·mL -1 solution. Pipette 10 μL into the casein plate, let it stand for 10 minutes, and incubate it upside down in a 37°C incubator for 18 hours, measure the diameter of the hydrolysis circle, and calculate the enzyme activity and RSD value.

[0026] Table 1 Low concentration, medium concentration and high concentration measurement results and RSD values

[0027]

[0028] The precision results of this method are shown in Table 1. The precision is 1.22%-8.07%, all less than 10%, and the RSD range is 2.92%-8.07% at low concentration, less than 10%; the RSD range is 0.45%-4.86% at medium concentration, less than 10%; high concentration Its RSD range is 1.22%-2.85%, which is less than 10%. Hao Wu used HPLC-UV to determine the content of...

Embodiment 3

[0029] Embodiment 3 casein plate method detection limit

[0030] Accurately weigh 0.1 g of commercial trypsin, dissolve it in 10 mL of water, and prepare a 250 U / mL solution. Then draw 1mL of the above-concentrated solution, dilute it 25 times, and prepare a 10U / mL solution. Then prepare 1U / mL, 2U / mL, 3U / mL, 4U / mL, 5U / mL, 6U / mL, 7U / mL, 8U / mL, 9U / mL, 10U / mL, draw 10μL of casein respectively The plates were left to stand for 10 minutes and then incubated upside down in a 37°C incubator for 18 hours, the diameter of the hydrolysis circle was measured, and the enzyme activity and RSD value were calculated, and parallel triplicate. The results are shown in Table 2.

[0031]Table 2 Detection limit experimental results

[0032]

[0033] According to the experimental results, more accurate results can be detected when the protease activity is 7U / mL, the detection values ​​are 7.115, 7.115, 6.604U / mL, the average value is 6.945U / mL, the standard deviation is 0.295, and the relati...

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Abstract

The invention discloses application of a casein plate method in quantitative analysis of protease activity. According to the application, the casein plate method is utilized for detecting the activityof protease in colored food, it is shown through a result that the casein plate method is good in linear relationship, good in precision and repeatability and low in detection limit, and a result ofdetection for the activity of the protease in a colored sample is accurate, and a new method is provided for detection for the protease activity. The application has the advantages that the situationcan be effectively avoided that due to the sample has a too deep color, errors are caused, and a detection result is inaccurate, there are no requirements for the color and shape of the sample, the casein plate method can be applied to detection for light-color samples or colorless samples and can also be applied to detection for deep-color samples such as red pitaya, and the method is simple to implement, wide in application range and capable of meeting the general detection requirements. The casein plate method can be effectively applied to detection for the activity of enzymes in deep-colorproducts such as soy sauce and tea leaves.

Description

technical field [0001] The invention relates to the technical field of protease activity detection, in particular to a method for preparing perilla sauce by a mixed-bacteria fermentation method. Background technique [0002] Proteolytic enzyme, referred to as protease, is an enzyme that hydrolyzes proteins. It is a group of large and complex enzyme molecules with highly specific proteolytic functions. Catabolism. Protease activity analysis is an important step in the research and application of protease. Based on protease-induced substrate cleavage strategies, although colorimetric and fluorometric methods have been widely developed in protease activity assays, overlapping optical signals and complicated chemical labeling processes limit the application of the methods. Protease activity has been detected using fluorescence methods, but methods based on calorimetric techniques, radioactivity, immunoassays, electrophoresis, amperometric, optical, etc. have also been reported...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/37
CPCC12Q1/37G01N2333/976
Inventor 何腊平帅瑶李翠芹陶菡
Owner GUIZHOU UNIV
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