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Quantitative detection method for bovine alpha-lactalbumin

A quantitative detection method and technology for lactalbumin, applied in measurement devices, instruments, scientific instruments, etc., can solve the problem of not finding thermally denatured and non-denatured bovine α-lactalbumin, not establishing an accurate and effective evaluation and detection method, and unable to measure bovine α-lactalbumin. α-lactalbumin and other problems, to meet the requirements of large-scale sample detection, fast sample analysis, and simple sample processing.

Inactive Publication Date: 2012-07-18
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are a large number of infant formula milk powder supplemented with bovine α-lactalbumin in the market. Due to different processing techniques and other reasons, the quality of the products varies, but no accurate and effective evaluation and detection methods have been established.
[0003] At present, the detection method for bovine α-lactalbumin at home and abroad is still based on SDS-PAGE, which is a semi-quantitative method and cannot be accurately quantified. It cannot be promoted in general laboratories due to cumbersome operations; someone proposed the application of GPC- The UV detection method can only be used for the detection of high-content raw materials due to poor resolution; Ren Yiping and others have established a method for the determination of bovine α-lactalbumin in infant formula by RPLC-ESI-MS, and the samples are directly extracted. According to the principle that protein will produce multiple charged ions under the condition of electrospray ionization, the selected ion scanning mode (SIR) is used for detection, so it is limited to the quantitative detection of non-denatured bovine α-lactalbumin in the sample, and cannot be determined due to heating. denatured bovine alpha-lactalbumin
After inquiry, so far no accurate method has been found that can simultaneously determine heat-denatured and non-denatured bovine α-lactalbumin in milk and dairy products

Method used

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  • Quantitative detection method for bovine alpha-lactalbumin
  • Quantitative detection method for bovine alpha-lactalbumin
  • Quantitative detection method for bovine alpha-lactalbumin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Optimization of enzymatic hydrolysis conditions

[0033] The enzymolysis conditions refer to the reaction temperature and time after sample dissolution, adding dithiothreitol (DTT), the reaction environment and time after adding iodoacetamide (IAA), and the enzymolysis after adding trypsin Temperature, pH value and time etc.; The invented method utilizes trypsin to only act on the specificity of arginine (R) and lysine (K), and whey protease is cut into molecular weight from tens to thousands of daals Peptide molecules, and then select only the characteristic peptide molecules unique to bovine α-lactalbumin as qualitative and quantitative target peptides. The final optimization method is: take 1-2 g of homogeneity sample, dilute with water to prepare a solution with a total protein content of about 1 mg / mL, then accurately draw 500 μL, add 20 μL of internal standard and 480 μL of NH 4 HCO 3 solution, add 10 μL DTT solution, keep the temperature at 50°C for 30 minut...

Embodiment 2

[0055] Sample type: Raw milk.

[0056] Take 36ml of fresh milk, divide it into 12 tubes×2 groups on average, each tube is 3ml, and then heat it in an 80°C incubator. The heating time of each tube in the same group is 0, 15, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, the heat-treated samples were left to room temperature, and the two groups of samples were processed as follows:

[0057] ① Method in the literature: Accurately weigh 1.0 g heat-treated sample, add 500 μL human milk internal standard solution, dilute to 9 ml with 0.3 mol / L NaCl solution containing 0.2% Triton X-100, adjust pH value to 4.6, fixed solution to 10ml, homogenized extraction for 10min, let stand for 30min, take 2ml solution in a centrifuge tube, centrifuge at 15000r / min for 15min, take the clear night through a 0.22μm filter membrane, inject the sample, and analyze and detect in the selected interval scanning mode , the scanning interval of bovine α-lactalbumin is 2357~2367m / z, the scanning interval ...

Embodiment 3

[0060] Sample type: Commercial infant formula milk powder.

[0061] Weigh 1.0g of the sample into a 100mL volumetric flask, add warm water to dissolve, wait until it cools to room temperature, add water to make up to the mark, accurately draw 500μL, add 20μL 25μmol / L internal standard and 480μL 500mmol / L NH 4 HCO 3 solution, add 10μL 500mmol / L DTT solution, keep the temperature at 50°C for 30 minutes, take it out and cool it to room temperature, add 30μL 500mmol / L IAA solution, let it stand in the dark for 30 minutes, add 10μL 100mmol / L CaCl 2 Solution and 30 μL 100 μg / mL trypsin solution, 37 ℃ constant temperature for overnight enzymolysis, take out the next day and add 20 μL pure formic acid, let it stand at room temperature for 1 hour, and finally dilute the enzymolysis solution to 2mL, take the obtained sample solution according to the method of Example 1 Enter mass spectrometry, and use the internal standard method to calculate the result (linear equation is y=38.36x+209...

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Abstract

The invention relates to a quantitative detection method for thermal-denaturation and non-denaturation bovine alpha-lactalbumin in milk and milk products by applying an enzymolysis-liquid chromatography and mass spectrometry combination technology. The quantitative detection method comprises the steps as follows: taking a certain amount of milk or milk samples, dissolving and diluting the milk or milk samples in water to obtain solution with total protein content being about 1mg / mL; after volume metering, correctly sucking 500 mu L, adding an internal standard substance, reacting disulfide bond with dithiothreitol (DTT), alkylating to protect sulfydryl produced in reaction by iodoacetamide (IAA), and then conducting constant-temperature and constant-time enzymolysis with trypsin; and separating enzymolysis products by reversed phase liquid chromatography, conducting detection with a mass spectrum multiple reaction monitoring (MRM) manner, and calculating the result by an internal standard method. The quantitation limit of the method is 0.001g / 100g; when adding amount is 0.2, 1.7 and 5.0g / 100g, the recovery rate is 98.9-110.8% (n is equal to 6) and repeatability: RSD (Relative Standard Deviation) is smaller than 7.6%; and the quantitative detection method can be applicable to the quantitative detection of samples with different contents of bovine alpha-lactalbumin.

Description

(1) Technical field [0001] The invention relates to a quantitative detection method of heat-denatured and non-denatured bovine α-lactalbumin in milk or milk products. (2) Background technology [0002] Bovine α-Lactalbumin (Bovine α-Lactalbumin) is a compact monomeric globulin protein composed of 123 amino acid residues, containing 4 disulfide bonds, relatively stable structure, and an average molecular weight of 14178 Daltons. Bovine α-lactalbumin is an excellent source of essential and branched-chain amino acids, and it shares 76% amino acid composition similarity with α-lactalbumin in human milk. Its main physiological function lies in its ability to combine with metal ions. It is also rich in tryptophan, which helps to increase the release of serotonin in the blood, promotes nerve development, and has good nutritional value. A large number of infant formula milk powders supplemented with bovine α-lactalbumin appear on the market. Due to different processing techniques a...

Claims

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Application Information

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IPC IPC(8): G01N30/88G01N30/89G01N30/06
Inventor 任一平赖世云张京顺蔡增轩黄百芬韩见龙
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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