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Enzymatic method for quantitative determination of lactoperoxidase activity in milk and its kit

A lactoperoxidase and kit technology, applied in the field of enzyme chemical analysis and detection, can solve the problems of complex protein components, high sensitivity and specificity requirements, and distorted results

Active Publication Date: 2020-12-08
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, milk contains a wide variety of enzymes, and ELISA is used to quantitatively detect lactoperoxidase, and cross-reaction between enzymes is inevitable; on the other hand, the protein components in milk are complex, and lactoperoxidase Compared with other proteins, the content of the enzyme is extremely low, and the sensitivity and specificity of the method are very high for the determination of the ELISA method; and because the antibody of lactoperoxidase used is usually unable to distinguish the active enzyme from the inactivated enzyme The enzymes that cause the method to have a certain degree of reaction to the inactivated enzymes, so that the results of the ELISA method are seriously distorted

Method used

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  • Enzymatic method for quantitative determination of lactoperoxidase activity in milk and its kit
  • Enzymatic method for quantitative determination of lactoperoxidase activity in milk and its kit
  • Enzymatic method for quantitative determination of lactoperoxidase activity in milk and its kit

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0080] 1) Preparation of LP standard solution: put the LP standard with accurate content after activity determination into a sterilized 50mL volumetric flask, add the weighed trehalose and CaCl 2 Afterwards, adjust the pH to 6.2 and set the volume to the scale to prepare a standard solution containing LP400U / L, trehalose 6w / w%, and calcium chloride 0.05w / w% calculated as calcium. Pipette 1 mL into 2 mL brown reagent bottles and store at 4°C.

[0081] Before detection, the standard solution was diluted to obtain a concentration gradient of 25, 50, 100, 200, and 400 U / L, which was used to establish a standard curve.

[0082] 2) Preparation of LP substrate solution: take citric acid monohydrate (C 6 h8 o 7 ·H 2 O) 21.014g, disodium hydrogen phosphate (Na 2 HPO 4 ) 28.392g and 0.0721g of 3,3`,5,5`-tetramethylbenzidine (TMB) were put into a 1L beaker, and 900mL of deionized water was added, and the pH was adjusted to 5.4 after dissolution. Transfer the solution into a 1L volu...

Embodiment 1

[0087] The mensuration of lactoperoxidase in the raw milk of embodiment 1

[0088] 1) Sample processing:

[0089] Pipette 10ml of milk sample into a centrifuge tube and return to room temperature. Slowly add 0.6ml of sample treatment solution to the centrifuge tube while shaking slowly. Let stand at room temperature for 5-10 minutes. Centrifuge at 3000 g for 10 minutes and filter the supernatant with filter paper. Collect the filtrate for testing, filter it with a 0.45 μm filter membrane, pipette an appropriate amount of filtrate into a 10 mL finger tube, dilute it with sample diluent 20 times, 40 times, 80 times and measure it.

[0090] 2) Drawing of standard curve

[0091] Take a 96-well microwell plate, add 50 μl LP standard solution to the microwells, the concentrations are 0, 25, 50, 100, 200, 400 U / L), add 50 μl substrate solution to each microwell, mix , incubate at room temperature (20-25°C) for 10 minutes. Then add 100 μl of stop solution to each microwell, shak...

Embodiment 2

[0102] Example 2: Determination of Lactoperoxidase Content in Pasteurized Milk

[0103] The determination process was carried out as in Example 1, and the dilution after sample treatment was determined by 5 times, 10 times and 20 times.

[0104] Six simultaneous experiments were carried out on the same pasteurized milk sample, and the results are shown in Table 3.

[0105] Table 3 Determination of lactoperoxidase content in the same pasteurized milk

[0106]

[0107]

[0108] From the results in Table 3, it can be seen that for pasteurized milk, the absorbance value of the 5-fold dilution exceeds the range of the standard curve, and the determination result is invalid.

[0109] The RSDs of the two dilutions of 10 times and 20 times were 0.71% and 1.53%, indicating that there was no significant difference between the 6 simultaneous determinations of the same dilution, which met the methodological requirements. Moreover, the RSD between the 10-fold and 20-fold dilutions ...

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Abstract

A method for determining LP (lactoperoxidase) in milk with an enzyme method is improved, a stable standard solution is prepared, a pretreatment method and a posttreatment method of a milk sample are improved, the technical problems of unstable standard curves and low sample detection specificity and sensitivity in quantitative detection of the activity of the LP in milk with the enzyme method aresolved, a complete kit applicable to the detection method is provided, and detection of the activity of the LP in the milk in conventional laboratories is facilitated.

Description

technical field [0001] The invention belongs to the field of enzyme chemical analysis and detection, and specifically relates to a kit for quantitative detection of lactoperoxidase activity in milk by an enzyme chemical method, a preparation method thereof and a corresponding determination method. Background technique [0002] Lactoperoxidase (EC 1.11.1.7, LP for short) is a member of the peroxidase family, a heme protein present in milk, consisting of a polypeptide chain, including 612 amino acids The residue, with a molecular weight of about 78kD and an isoelectric point of 9.6, is one of the very common components in human and bovine milk. [0003] Lactoperoxidase and hydrogen peroxide and thiocyanate (SCN) can form the "lactoperoxidase system (LPS)". This enzyme system has antibacterial activity, can inhibit the growth of Gram-positive bacteria and negative bacteria without refrigeration, prolong the shelf life of fresh milk, and has the effect of "cold sterilization". ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96G01N21/31G01N1/38
CPCG01N1/38G01N21/31
Inventor 郑楠文芳李松励张养东李慧颖王加启
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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