Method and kit for quantitatively detecting lactulose in liquid milk by using microplate reader enzymatic method
A quantitative detection and microplate reader enzyme technology, applied in the field of enzyme chemical analysis and detection, can solve the problems of low detection efficiency, cumbersome determination steps, and high detection cost
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Embodiment 1
[0087] The preparation of embodiment 1 detection reagent
[0088] 1. Glucose oxidase and catalase mixture lyophilized powder, NADP + Preparation of the mixture lyophilized powder with ATP
[0089] (1) Preparation of the mixture freeze-dried powder of glucose oxidase and catalase: get the glucose oxidase with activity as 40KU and the catalase with activity as 5000KU in 10 milliliter sterile test tubes, add 6mL sterile water to make It is completely dissolved, immediately draw 100 μL with a liquid dispenser and divide it into 50 4mL brown reagent bottles, put it in a freeze dryer, and keep the enzyme solution at a temperature of -20°C to -35°C under a low vacuum environment of 200 Torr. Freeze under low conditions and then dry by sublimation for 24-72 hours. Seal the freeze-dried enzyme reagent and store it in a -20°C refrigerator.
[0090] Before the detection, the lyophilized powder of the mixture is prepared into a suspension with sterile water for use.
[0091] (2) NADP ...
Embodiment 2
[0114] Determination of lactulose in embodiment 2 sterilized milk
[0115] 1) Sample processing:
[0116] Draw 0.5mL of the milk sample into a 2mL polypropylene centrifuge tube, add 1.3mL of distilled water, 0.1mL of 168g / L zinc sulfate solution, and 0.1mL of 130g / L potassium ferrocyanide solution, vortex and mix after fastening the cap, 5000rpm Centrifuge for 10 minutes.
[0117] Hydrolyze lactose and lactulose: Take out each sample and carefully pipette two supernatants, each 500 μL into two 2 mL polypropylene screw-capped microtubes, add 200 μL of buffer A and β- Galactosidase (2.0×10 3 U / mL) 20 μL was used as a test sample; 200 μL of buffer A and 20 μL of distilled water were added to another tube as a blank control. After mixing by vortexing, place in a water bath at 50 °C for 1 h.
[0118] Glucose oxidation: 200 μL of buffer C, 50 μL of 0.33M NaOH solution, 10 μL of octanol, and glucose oxidase (4.0×10 3 U / mL) and catalase (5.0×10 5 U / mL) mixed solution 20μL, 30%H ...
Embodiment 3
[0143] The rate of recovery of embodiment 3 assay methods
[0144] Utilize liquid milk to add standard as matrix, adopt the same detection method of embodiment 2, check the recovery rate of assay method of the present invention, the result is as shown in table 2:
[0145] Table 2 recovery rate determination result
[0146]
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