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Method for screening potential allergens in food

A screening method and allergen technology, applied in the field of inspection and quarantine, can solve the problems of limited accuracy and application, incomplete and reliable conclusions, large workload, etc., achieving easy high-throughput screening, simple and easy method, and repeatability strong effect

Inactive Publication Date: 2016-07-06
DALIAN OCEAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large workload of this method (need to purify a certain amount of target protein and determine its amino acid sequence), and there are only about 500 known allergens, the accuracy and application of this method are limited.
In addition, in vitro testing using sera from patients with known allergies, the conclusions obtained are not entirely reliable due to the limited number of serum samples from allergic cases
Similarly, the same problem exists with allergen screening tests in other foods such as eggs, milk, nuts, etc.

Method used

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  • Method for screening potential allergens in food
  • Method for screening potential allergens in food
  • Method for screening potential allergens in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Extraction of protein from large yellow croaker

[0031] Take 300g of washed large yellow croaker muscle, grind the large yellow croaker muscle with a blender, then homogenize the ground fish meat with a homogenizer, add 7 times the volume of water, and adjust the pH value to about 2.4 with hydrochloric acid , to dissolve most of the protein, centrifuge at 5000r / min for 15min, and collect the supernatant. Then adjust the pH value of the supernatant to about 4.75 with sodium hydroxide solution to precipitate the protein, centrifuge at 5000r / min for 15min, collect the protein precipitate, and freeze-dry it for future use.

[0032] 2. In vitro simulated gastric juice digestion

[0033] Simulated gastric juice: Weigh 0.1g NaCl in 50ml distilled water, adjust the pH to 1.2 with HCl, add pepsin to make the mass concentration of pepsin 150ug / ml.

[0034] The control solution was prepared in the same way, and the difference from the simulated gastric juice was that it only...

Embodiment 2

[0049] Using a method similar to that of Example 1, Spanish mackerel protein was extracted and digested. After digestion with simulated gastric juice, two enzyme-resistant protein bands were found, with molecular weights around 35kDa and 45kDa, as shown in the attached figure 2 shown.

[0050] The enzymolysis-resistant protein bands of Spanish mackerel were hydrolyzed in gel, analyzed by liquid chromatography-mass spectrometry, and identified in combination with the protein database. The results are shown in Table 2:

[0051] Table 2: General-purpose liquid chromatography-mass spectrometry identification results of enzymatic hydrolysis-resistant bands of Spanish mackerel proteins

[0052]

[0053] The NCBI / blast database was used to compare the identified Spanish mackerel protein with known allergen or epitope sequences, and the following results were obtained:

[0054] 1. The homology of enolase in Spanish mackerel and known allergen enolase in Wuchang fish (Megalobram...

Embodiment 3

[0057] Using the method of Example 1, the allergens of egg white protein were screened.

[0058] The results of SDS-PAGE electrophoresis analysis of digestion products under different digestion time conditions are shown in the attached image 3 shown.

[0059] Table 3 shows the identification and analysis results of the peptides obtained by enzymolysis-resistant bands by liquid chromatography-mass spectrometry after in-gel enzymolysis.

[0060] Table 3. General-purpose liquid chromatography-mass spectrometry identification results of enzymatic hydrolysis-resistant bands of egg white proteins

[0061]

[0062] The enzyme-resistant protein in egg white was identified as ovalbumin, which is the main allergen reported in eggs. In summary, the enzymolysis-resistant proteins obtained by in vitro digestion with simulated gastric juice are highly allergic, so this method is feasible.

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Abstract

A method for screening potential allergens in food includes: mixing food proteins with in-vitro digestive juice, and analyzing digestion products under different digestion time conditions by means of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); selecting enzymolysis-resistant bands to perform in-gel digestion, identifying and analyzing obtained peptides by liquid chromatography-mass spectrography, and determining types of allergenic proteins and allergenicity thereof according to a protein data bank and an allergen database.The method avoids pre-purification of target proteins and is simple in operation, remarkable in result, high in reliability and high in repeatability.By the method, not only can the allergens in the food be detected, but also allergenicity of the enzymolysis-resistant proteins can be assessed.In addition, the method is simple, convenient and feasible, realizes simultaneous detection of multiple samples and high-throughput screening and is in accordance with the requirements of accuracy and high efficiency in the field of food detection.

Description

technical field [0001] The invention relates to a method for evaluating and detecting allergens in food, belonging to the field of inspection and quarantine. Background technique [0002] A food allergy is when the body's immune system mounts an immune response to a substance in food, including the release of antibodies, which in turn trigger the release of certain chemicals in the body, such as histamine, which can cause itchy skin, runny nose, Coughing, dyspnea, and even death (Zhang Ruihao et al., China Inspection and Quarantine, 2009, 3:22). In addition to common terrestrial food allergens such as milk, eggs, and nuts, fish and other aquatic product allergens also account for a very large proportion. In developed countries, people allergic to fish account for about 0.1-0.3% of the total population (Zhang W., etc, Food Chemistry 2015, 174:547.). In my country, aquatic products, eggs, milk, etc. are the main causes of food allergies. Epidemiological studies on food aller...

Claims

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Application Information

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IPC IPC(8): G01N30/88
CPCG01N30/88
Inventor 祁艳霞高庆历于洋赵前程李智博宋杨
Owner DALIAN OCEAN UNIV
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