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Method for detecting apis mellifera and apis cerana glucose dehydrogenase in honey through liquid chromatography-tandem mass spectrometry

A technology of glucose dehydrogenase and tandem mass spectrometry, which is applied in the field of detection of glucose dehydrogenase in honey by liquid chromatography tandem mass spectrometry, achieving the effects of strong specificity, high specificity and sensitivity, and high sensitivity

Inactive Publication Date: 2020-07-10
BEE RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of this technique for the quantitative detection of glucose dehydrogenase in honey

Method used

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  • Method for detecting apis mellifera and apis cerana glucose dehydrogenase in honey through liquid chromatography-tandem mass spectrometry
  • Method for detecting apis mellifera and apis cerana glucose dehydrogenase in honey through liquid chromatography-tandem mass spectrometry
  • Method for detecting apis mellifera and apis cerana glucose dehydrogenase in honey through liquid chromatography-tandem mass spectrometry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] 1. Sample source

[0052] Purchase a total of 20 samples of real honey from the market or beekeeper.

[0053] 2. Experimental procedure

[0054] 2.1 Solution preparation

[0055] 5M Urea solution: Weigh 4.5 g of Urea and dilute the volume of ultrapure water to 15 mL;

[0056] 100 mM DTT: Weigh 308.5 mg of DTT, 40 mM NH 4 HCO 3 The volume of the solution is adjusted to 20 mL, each tube is 1 mL aliquoted, and stored in a refrigerator at -20°C until use.

[0057] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , Use ultrapure water to make a constant volume of 100 mL, and store in a refrigerator at 4°C until use.

[0058] 100 mM IAA solution: weigh 0.39 g of IAA and use 40 mM NH 4 HCO 3 The solution is dissolved to 20 mL, and stored in a refrigerator at -20°C until use.

[0059] Activation solution: 500 μL ACN, 3 μL TFA, dilute ultrapure water to 1 mL, and store at 4°C.

[0060] Equilibrium solution: 1 μL TFA, dilute ultrapure water to 1 mL, and store at 4°C.

[0061] Eluent: 800 μL ...

Embodiment 2

[0078] Example 2 Detection of honey adulteration

[0079] 1. Sample source

[0080] Buy real Chinese honey, Italian honey samples and syrup from the market or beekeeper.

[0081] 2. Experimental steps

[0082] (1) Solution preparation

[0083] The same as in Example 1.

[0084] (2) Mixing of honey: Add Italian honey to the middle honey in different proportions.

[0085] The honey is mixed with Italian honey in proportions of 5%, 10%, 20%, and 50%.

[0086] (3) Pretreatment of honey samples

[0087] ①Accurately weigh 10 g of uniform honey to be tested into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm and 4°C for 10 min. Collect the supernatant in a new 2 mL centrifuge tube.

[0088] ②Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mixing. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution to react in t...

Embodiment 3

[0094] Example 3 Detection of honey in commodities

[0095] 1. Sample source

[0096] 50 bottles of Chinese honey from different manufacturers purchased from the mall.

[0097] 2. Experimental steps

[0098] (1) Solution preparation

[0099] The same as in Example 1.

[0100] (2) Pre-processing of samples to be tested

[0101] ①Accurately weigh 10 g of uniform honey to be tested into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm and 4°C for 10 min. Collect the supernatant in a new centrifuge tube.

[0102] ②Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mixing. Add 100 μL of 30 mM DTT solution to the above mixed solution, react at room temperature for 60 min, and then add 500 μL of 100 mM IAA solution to react in the dark at room temperature for 60 min.

[0103] ③Add 30 μL of trypsin solution to each sample and digest at 37°C overnight. When the digestion reaction is complete, add 1 μL of ...

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Abstract

The invention relates to a method for detecting apis mellifera and apis cerana honey glucose dehydrogenase through liquid chromatography-tandem mass spectrometry. The method comprises the following steps: selecting characteristic peptide fragments respectively belonging to glucose dehydrogenase of apis mellifera honey and apis cerana honey, establishing a liquid chromatography-tandem mass spectrometry detection method, pretreating a sample, performing liquid chromatography separation, performing tandem mass spectrometry detection and performing data analysis, wherein the characteristic peptidefragments comprise an apis mellifera glucose dehydrogenase characteristic peptide fragment, namely ITLDNQSVQVR, and an apis cerana glucose dehydrogenase characteristic peptide fragment, namely FPYQPPFAWEILK. The method for detecting glucose dehydrogenase in honey has high accuracy, precision and sensitivity and strong stability and is suitable for distinguishing apis mellifera honey from apis cerana honey and is especially suitable for identifying apis mellifera honey doped in apis cerana honey. The method has important practical significance for protecting rights and interests of honey consumers and maintaining healthy development of the bee product industry.

Description

Technical field [0001] The invention relates to the field of food detection, in particular to a method for detecting glucose dehydrogenase in honey by liquid chromatography tandem mass spectrometry, and the method is suitable for identifying Chinese honey and Italian honey. Background technique [0002] Honey is rich in nutrition, has a sweet taste, and has certain functions of beauty, intestine moisturizing and health care. It is deeply loved by people. In addition, honey is rich in nutrients and active substances, such as phenolic acid, flavonoids, etc., which makes honey have certain medicinal value, has anti-inflammatory, antibacterial, and antioxidant effects, and is used in medicine and cosmetics. Compared with Italian honey, Chinese honey has a better taste and is more popular with consumers. However, the average output of Chinese honey per colony is only about one-fifth of Italian honey, which causes the market to be in short supply. Currently, the price of Chinese honey...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88
CPCG01N30/88G01N2030/8831
Inventor 杨术鹏李熠周金慧丛晓蕾张金震杨宇晖黄京平金玥王鹏赵文
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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