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A liquid chromatography tandem mass spectrometry method for the detection of royal jelly major protein 5 in Italian honey and Chinese honey

A royal jelly main protein and tandem mass spectrometry technology, which is applied in the field of food detection, can solve problems such as amino acid sequence differences, and achieve the effects of strong specificity, good accuracy and precision, high specificity and sensitivity

Active Publication Date: 2021-01-15
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

Royal jelly major proteins (MRJPs) are the main components of protein in honey. Although the honey produced by Zhongfeng and Italian bees both contain MRJPs, the amino acid sequences of the two are different

Method used

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  • A liquid chromatography tandem mass spectrometry method for the detection of royal jelly major protein 5 in Italian honey and Chinese honey
  • A liquid chromatography tandem mass spectrometry method for the detection of royal jelly major protein 5 in Italian honey and Chinese honey
  • A liquid chromatography tandem mass spectrometry method for the detection of royal jelly major protein 5 in Italian honey and Chinese honey

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Sample source

[0053] A total of 20 samples of real honey were purchased from the market or beekeepers.

[0054] 2. Experimental steps

[0055] 2.1 Solution preparation

[0056] 5M Urea solution: Weigh 4.5 g of Urea, and dilute to 15 mL with ultrapure water;

[0057] 100 mM DTT: Weigh 308.5 mg DTT, 40 mM NH 4 HCO 3 Dilute the solution to 20 mL, dispense 1 mL into each tube, and store in a -20°C refrigerator until use.

[0058] 40 mM NH 4 HCO 3 Solution: weigh 0.316 g NH 4 HCO 3 , dilute to 100 mL with ultrapure water, and store in a 4°C refrigerator until use.

[0059] 100 mM IAA solution: Weigh 0.39 g of IAA, add 40 mM NH 4 HCO 3 The solution was fixed to 20 mL, and stored in a -20°C refrigerator until use.

[0060] Activation solution: 500 μL ACN, 3 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.

[0061] Equilibrium solution: 1 μL TFA, dilute to 1 mL with ultrapure water, store at 4°C.

[0062] Eluent: 800 μL ACN, 1 μL TFA, dilute to 1 mL...

Embodiment 2

[0080] Example 2 Detection of honey adulteration

[0081] 1. Sample source

[0082] Buy real samples of Chinese or Italian honey from the market or beekeepers.

[0083] 2. Experimental steps

[0084] (1) Solution preparation

[0085] With embodiment 1.

[0086] (2) Blending of honey: Italian honey is mixed into medium honey in different proportions.

[0087] Mix Italian honey into the honey according to the ratio of 5%, 10%, 20%, 30%, 40%, 50%.

[0088] (3) Pretreatment of honey samples

[0089] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new 2 mL centrifuge tube.

[0090] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. 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 solut...

Embodiment 3

[0096] Example 3 Detection of honey in commodities

[0097] 1. Sample source

[0098] 30 bottles of medium-sized honey from different manufacturers purchased from the mall.

[0099] 2. Experimental steps

[0100] (1) Solution preparation

[0101] With embodiment 1.

[0102] (2) Pretreatment of samples to be tested

[0103] ① Accurately weigh 10 g of honey to be tested evenly into a 50 mL centrifuge tube, add 10 mL of deionized water, and vortex until the honey is fully dissolved. Centrifuge at 12000 rpm for 10 min at 4°C. Collect the supernatant in a new centrifuge tube.

[0104] ② Pipette 200 μL of protein solution and 800 μL of 40 mM NH 4 HCO 3 mix. 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 and react in dark at room temperature for 60 min.

[0105] ③Add 30 μL of trypsin solution to each sample, and digest at 37°C overnight. When the digestion reaction is complete, ...

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Abstract

The invention provides a method for detecting major royal jelly protein 5 (MRJP5) of apis cerana honey and apis mellifera honey by liquid chromatography-tandem mass spectrometry. The method comprisesthe following steps: screening characteristic peptide fragments respectively belonging to apis cerana MRJP5 and apis mellifera MRJP5, establishing a liquid chromatography tandem mass spectrometry detection method, pretreating a honey sample, carrying out liquid chromatography separation, carrying out tandem mass spectrometry detection, carrying out data analysis and the like. The characteristic peptide fragments comprises an apis mellifera MRJP5 characteristic peptide fragment: YLDYDFGSDER, and an apis cerana MRJP5 characteristic peptide fragment: NLANSMNVIHEWK. The method for detecting MRJP5in honey has high accuracy, precision and sensitivity and strong stability, is suitable for identifying the apis mellifera honey and apis cerana honey, and is especially suitable for identifying adulteration of the apis cerana honey mixed with theapis mellifera 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 royal jelly main protein 5 in honey by liquid chromatography tandem mass spectrometry, and the method is suitable for distinguishing middle honey and Italian honey. Background technique [0002] Honey is a natural sweet substance that is collected by bees from the nectar of nectar source plants, mixed with their own secretions, fully brewed and stored in the hive. Honey is rich in amino acids, phenolic acids, flavonoids, trace elements and other substances that are beneficial to people, which makes it have the effects of moisturizing the intestines, beautifying and health care, and is very popular among people. According to the different nectar source plants, honey can be divided into single-flower honey varieties such as acacia honey, linden honey, jujube honey, wattle honey, litchi honey, and longan honey. According to the difference of honeybee species, it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N30/72G01N30/02
CPCG01N30/02G01N30/72G01N33/6848
Inventor 杨术鹏李熠丛晓蕾张金震周金慧杨宇晖
Owner BEE RES INST CHINESE ACAD OF AGRI SCI
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