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Method for detecting carboxy methyl lysine (CML) in soybean sauce

A carboxymethyl lysine and detection method technology, which is applied in the field of food detection, can solve the problems of no free CML detection pretreatment method, inability to completely obtain the CML content of the soy sauce system, inability to completely precipitate and separate low-molecular-weight polypeptides, etc., to achieve detection The mass spectrometry response signal is good, the detection accuracy is improved, and the cost is low.

Active Publication Date: 2014-06-25
SOUTH CHINA UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, 45% of the total nitrogen in soy sauce exists in low molecular weight polypeptides (1-5kDa), and 45% exists in free amino acids. The above TCA method cannot completely precipitate and separate low molecular weight polypeptides (glycosylated polypeptides), and cannot precipitate and separate free Amino acids (glycosylated amino acids), so the total CML content in the soy sauce system cannot be obtained completely, which seriously affects the accuracy and reliability of the test results
[0006] There is no specific pretreatment method for the detection of free CML in soy sauce, and there is no report on the application of dialysis to the pretreatment technology for the detection of bound CML in soy sauce

Method used

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  • Method for detecting carboxy methyl lysine (CML) in soybean sauce
  • Method for detecting carboxy methyl lysine (CML) in soybean sauce
  • Method for detecting carboxy methyl lysine (CML) in soybean sauce

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Effect test

Embodiment 1

[0053] The pre-processing of CML detection is performed on different brands of Japanese soy sauce, different brands of Chinese soy sauce and mixed soy sauce, including the following steps:

[0054] a. Pre-processing of free CML, including the following steps:

[0055] (1) Take 0.1mL soy sauce sample, dilute to 10mL with 10% methanol solution, and vortex to mix;

[0056] (2) Take 1mL of diluent and use activated C 18 Clean up the solid phase extraction column, add 5mL of 10% methanol solution to elute, and collect the eluate;

[0057] (3) Take 1 mL of the eluate and pass it through a 0.22 μm microfiltration membrane to obtain the desired sample 1 to be tested.

[0058] At the same time as the mixed soy sauce sample is detected, the impurity removal effect analysis is carried out, which specifically includes the following steps:

[0059] Using a UV-visible spectrophotometer (50Conc, VARIAN, USA), measure the absorbance value of the mixed soy sauce sample after the above pretr...

Embodiment 2

[0099] a. Pre-detection treatment of free CML, the specific steps are:

[0100] (1) Take 0.1mL soy sauce sample, dilute to 10mL with 10% methanol solution, and vortex to mix;

[0101] (2) Take 1mL of diluent and use activated C 18 Clean up the solid-phase extraction column, add 8 mL of 10% methanol solution to elute, and collect the eluate;

[0102] (3) Take 1 mL of the eluate and pass it through a 0.22 μm microfiltration membrane to obtain the desired sample 1 to be tested.

[0103] b. Pre-detection treatment of bound CML, the specific steps are:

[0104] (1) Take 7.5mg of soy sauce with equivalent peptide nitrogen content, add boric acid buffer solution (0.5mol / L, pH9.2), after adding, make the final molar concentration of sodium borate in the system reach 0.2mol / L, and add sodium borohydride Solution (2 mol / L sodium borohydride solution dissolved in 0.1 mol / L sodium hydroxide solution), after adding, make the final concentration of sodium borohydride in the system reach ...

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Abstract

The invention relates to a method for detecting carboxy methyl lysine (CML) in soybean sauce. The method comprises the following steps: firstly, carrying out detection pretreatment on CML in a soybean sauce system, namely, detection pretreatment on free-state CML and combined-state CML; carrying out detection pretreatment on the free-state CML, namely sampling, adding a methanol solution to dilute, swirling and evenly mixing, purifying through a C18 solid-phase extraction column, adding a methanol solution to elute, filtering through a micro-filtration membrane to obtain a sample 1 to be detected; carrying out the detection pretreatment on the combined-state CML, namely sampling, reducing through sodium borohydride, separating polypeptide and protein through a dialysis bag, hydrolyzing through hydrochloric acid, disacidifying through vacuum, purifying through the C18 solid-phase extraction column, filtering through the micro-filtration membrane, drying in vacuum, re-dissolving to obtain a sample 2 to be detected; and subsequently, carrying out CML detection on the sample 1 and the sample 2 to be detected respectively. A total content of the CML is sum of the content of the free-state CML and the content of the combined-state CML. The sample pretreatment method has the characteristics of elimination of interference, protection of chromatographic column, safety and reliability, and capability of completely analyzing all the CML in the soybean sauce.

Description

technical field [0001] The invention belongs to the technical field of food detection, and specifically relates to a method for detecting carboxymethyllysine (CML) in food, especially suitable for the detection of CML content in soy sauce. The total content of CML in the system is free CML and bound CML Addition of content. Background technique [0002] CML is a highly oxidized compound produced by the Maillard reaction in foods and organisms. In addition, it can also be produced by complex changes in sugar oxidation and lipid oxidation intermediates. It is divided into free CML (glycosylated amino acids) and bound CML ( Glycosylated peptides and proteins). Existing studies have shown that food-borne CML may be related to the exacerbation of diseases such as renal failure, diabetes, chronic heart failure, atherosclerosis, and Alzheimer's disease, among which free CML is more harmful to the human body. Soy sauce is a condiment made from soybeans, wheat, water, salt and othe...

Claims

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Application Information

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IPC IPC(8): G01N30/14
Inventor 李琳李玉婷李冰苏健裕徐振波卞华伟梁志理
Owner SOUTH CHINA UNIV OF TECH
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