Standard number: GB/T 38578-2020
Standard name: Rapid determination
of the allergenic protein from aquatic animals origin- ||tt
||Capillary electrophoresis
Standard format: PDF
Release time: 2020-03-31
Implementation time: 2020-03-31 ||
tt||Standard size: 1.31M
Standard introduction: 1 Scope
This standard specifies the method for determining allergenic proteins from aquatic origin by capillary electrophoresis.
This standard is applicable to the rapid detection of crustacean tropomyosin, crustacean arginine kinase and fish parvalbumin content.
2 Normative reference documents
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) applies to this document.
GB/T6682 Specifications and test methods for water used in analytical laboratories
GB/T30891 Sampling specification for aquatic products
This standard specifies the method for determining allergenic proteins from aquatic products by capillary electrophoresis. This standard is applicable to the rapid detection of crustacean tropomyosin, crustacean arginine kinase and fish parvalbumin content.

ICS07.080
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National Standard of the People's Republic of China
GB/T38578—2020
Rapid determination of the allergenic protein from aquatic animals origin-Capillaryelectrophoresis
Published on 2020-03-31
State Administration for Market Regulation
National Standardization Administration
Implementation on 2020-03-31
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This standard was drafted in accordance with the rules given in GB/T1.1—2009. This standard was proposed and managed by the China National Institute of Standardization. iHKAa~cJouaKA
GB/T38578—2020
Drafting units of this standard: China National Institute of Standardization, Greentown Agricultural Science Testing Technology Co., Ltd., Zhejiang Gongshang University, Beijing Technology and Business University, Beijing Samber Technology Co., Ltd., Jimei University, Hunan Agricultural University, Wenzhou University, Rongcheng Huatong Marine Biotechnology Co., Ltd. The main drafters of this standard: Ma Aijin, Wang Chuanbu, He Xiaoming, Zhou Jinru, Fu Linglin, Jia Yingmin, Wu Qi, Hao Shuai, Liu Guangming, Zhou Hui, Chen Xiaojing, Wang Rong, Liu Fenxiang, Liu Zhongshi, Gong Yanbo. iKAa-cJouaKA
1 Scope
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GB/T38578—2020
Rapid detection of allergenic proteins from aquatic products
Capillary electrophoresis
This standard specifies the method for determining allergenic proteins from aquatic products by capillary electrophoresis. This standard applies to the rapid detection of crustacean tropomyosin, crustacean arginine kinase and fish parvalbumin content. 2 Normative references
The following references are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. GB/T6682 Specifications and test methods for water for analytical laboratories GB/T30891 Sampling specifications for aquatic products
3 Terms and definitions
The following terms and definitions apply to this document. 3.1
Allergenic protein
Allergenic protein
Protein that can cause allergic reaction in the body. 4 Principle
The allergenic protein in the sample is extracted with protein extract and purified by anion exchange chromatography, and determined by capillary electrophoresis, with migration time for qualitative analysis and external standard method for quantitative analysis.
5 Reagents or materials
Unless otherwise specified, only analytically pure reagents shall be used. 5.1 Water
First-grade water as specified in GB/T6682.
5,2 Methanol
Chromatographically pure.
Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer
1.21g of tris(hydroxymethyl)aminomethane, add 900mL of double distilled water to dissolve, adjust pH to 7.5 with 6mol/L hydrochloric acid solution, dilute to 1000mL with distilled water, filter with 0.45μm filter membrane, ultrasonically vibrate (power 200W) for 30min, and store at room temperature. 1
GB/T38578—2020
5.4 Sodium chloride solution
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14.61g of sodium chloride, dissolve with appropriate amount of deionized water, dilute to 500mL, filter with 0.45um filter membrane, ultrasonically vibrate (power 200W) for 30min, and store at room temperature.
5.5 Phosphate buffer
Sodium chloride 8.00g, potassium chloride 0.20g, disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, add the above reagents to a quantitative container, dissolve in distilled water and make up to 1000mL, adjust the pH to 7.4 with 6mol/L hydrochloric acid solution, filter with a 0.45um filter membrane, ultrasonically vibrate (power 200W) for 30min and store at room temperature.
5.6 Shrimp tropomyosin standard sample
Purity ≥ 98%.
5.7 Shrimp arginine kinase standard sample
Purity ≥ 98%
5.8 Fish parvalbumin
Purity 9%
5.9 Standard reserve
Allergenic egg standard (shrimp tropomyosin, shrimp arginine kinase
dissolve in a 1.5-well centrifuge tube and homogenize to obtain
0.1 me/L sodium hydroxide solution
Albumin
, add
mL sodium chloride buffer
Each bottle is divided into
mg/mL egg point
standard reserve
tubes for every 100L, and frozen in
potassium hydroxide 4.00g. After appropriate amount of double distilled water, the volume is adjusted to: 190ml: filter with 0.46μm filter membrane, super shock power 200W) 30min, and store at room temperature.
5.110.1mul/L sodium hydroxide solution
Sodium hydroxide 0.400 appropriate amount of double distilled water, after dissolving, the volume is adjusted to 100mL, filter with 0.45um filter membrane, sonication (power 200W) 30min, and store at room temperature.
Boric acid-borax buffer solution
Precisely weigh 0.9525 sodium tetraborate (NaBO,·10HO) and add 80mL double distilled water to dissolve, adjust pH to 9.2 with 1mol/L sodium hydroxide solution. Make up to 100mL with double distilled water, filter with 0.45μm filter membrane, ultrasonically vibrate (power 200W) for 30min, and store at room temperature.
5.13 Standard working solution
Pick 5uL, 10ul25L, 40L, 50L of standard stock solution, place them in 5 1.5m plastic centrifuge tubes respectively, add 1mL phosphate buffer, vortex and shake to mix, the mass concentration of each standard working solution is 5μg/mL10μg/ml, 25μg/mL, 40μg/mL.50μg/mL, store at 4℃ for 2 weeks. 5.14 Benzenemethylsulfonyl fluoride solution
Dissolve 0.174 μg of phenylmethylsulfonyl fluoride in 10 mL of isopropanol, vortex to mix, and store at -20°C. 2
5.15 Protein extract
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GB/T38578—2020
Add 1 μL of protease inhibitor, 10 μL of phosphatase inhibitor and 5L of 0.1 mol/L phenylmethylsulfonyl fluoride solution to 1 mL of pre-cooled lysate, mix and store on ice for later use. 6 Instruments
6.1 High-performance capillary electrophoresis instrument: equipped with a UV detector with a wavelength range of 214 nm to -220 nm, a positive high-voltage component, and a quartz capillary (total length 45 cm, inner diameter 75 μm).
Low-temperature centrifuge: 13000g.
pH meter: precision 0.01.
Microporous filter membrane: 0.45μm, aqueous phase.
001g001g and 0.0001g.
Analytical balance: sensitivity
Protein purifier: two self-dividing collectors. 7 Experimental conditions
Capillary column treatment method
Columnization. Sequentially rinse the capillary column with distilled water for 30min, rinse with distilled water for 5min, and finally rinse with acid-adjusted solution. Before each sample is separated, rinse with boric acid
sodium hydroxide solution and rinse with distilled water
Test operating conditions
Test operating conditions see experiment 1.
The capillary
n1molL sodium hydroxide solution was rinsed
The mass was rinsed with distilled water for 3min,
Table 1 Test operating conditions
Wavelength/nm
Coverage/℃
Injection conditions
Analysis conditions
8 Sample preparation
Injection pressure/Pa
Injection time/s
Separation voltage/kV
Detection time/min
Fish parvalbumin
Crustacean tropomyosin
Crustacean arginine kinase
According to GB/T30891, at least 500g of representative aquatic source samples were extracted and reduced to 100g by quartering; placed on ice and cut into thin pieces. Grind into powder with liquid nitrogen and store at low temperature for later use. GB/T38578—2020
9Test steps
9.1 Protein extraction
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Quickly weigh 0.100g of the ground sample and put it into 1.5ml. pre-cooled centrifuge tube, add 1.0mL protein extract, vortex and mix well, then simmer at 4℃ for 2h, centrifuge at 10000r/min, 4℃ for 5min, take the supernatant, divide and store at -80℃, and avoid repeated freezing and thawing. After extracting the protein supernatant, purify the protein supernatant by anion exchange chromatography (1mLDEAEscpharascF.F. pre-packed column), load 2mL protein supernatant at a flow rate of 0.5mL/min, and then linearly elute with 40mL0.01mol/L tris(hydroxymethyl)aminomethane hydrochloric acid buffer and 0.5mol/L sodium chloride solution, the elution rate is 1mL/min, collect the protein eluate according to the peak situation, and freeze the obtained sample at -80℃ for later use.
9.2 Determination of protein extraction rate
Add 1 mg of allergenic protein standard to the sample processed according to Chapter 8 and grind it into powder with liquid nitrogen. Quickly weigh 0.100 g of the grinded spiked sample and put it into a 1.5 mL pre-cooled centrifuge tube, add 1.0 mL of protein extract, vortex mix well, let stand at 4°C for 2 h, centrifuge at 10000 r/min, 4°C for 5 min, take the supernatant, and store it in aliquots at -80°C. Repeated freezing and thawing should be avoided. After extracting the protein supernatant, purify the protein supernatant with anion exchange chromatography (1mL DEAE sepharose F.F. pre-packed column), load 2mL of protein supernatant at a flow rate of 0.5mL/min, and then linearly elute with 40mL 0.01mol/L dihydroxymethylaminomethane-hydrochloric acid buffer and 0.5mol/L sodium chloride solution at a rate of 1mL/min. Collect the protein eluate according to the peak, and freeze the spiked sample at -80C for later use. The protein content of the sample and the spiked sample was determined by the BCA method. The protein extraction rate is calculated according to formula (1):
Wherein,
Protein extraction rate, %;
X=-PixV
PoXV+m
Protein mass concentration of spiked sample solution, in micrograms per milliliter (μug/mL); P
V. Volume of spiked sample solution, in milliliter (mL): po—protein mass concentration of sample solution, in micrograms per milliliter (μg/mL); V. —volume of sample solution, in milliliter (mL)); mass of allergenic protein standard, in milligrams (mg), m
9.3 Determination
9.3.1 Take 200 ml of sample solution and place it in a sample bottle. After pressure injection for 5 seconds, separate and determine it by capillary electrophoresis. The analysis time is 10min~15min.
9.3.2 Quantitative determination is carried out by external standard calibration curve method. Take the mass concentration of the series of allergenic protein standard solutions as the coordinates and the capillary electrophoresis peak area as the ordinate to make a linear regression equation for the standard curve. Compare the peak area of ​​the sample with the standard curve for quantification. The response values ​​of the allergenic proteins in the standard solution and the sample extract should be within the linear range of the instrument. See Appendix A for the capillary electrophoresis diagram of the standard solution. 9.3.3 If the mass concentration in the sample exceeds the highest point of the standard curve, further dilute the sample. 9.3.4 At least 2 electrophoresis diagrams for each test solution. 9.3.5 After testing 3 to 5 samples, new boric acid-xenazolate buffer solution should be replaced at the inlet and outlet of the instrument. 10 Calculation of results
The allergenic protein content was calculated according to formula (2):
Wherein:
p=euxv
—the content of allergenic protein, in milligrams per gram (mg/g): p
pThe content of allergenic protein in the sample solution, in micrograms per milliliter (μ8/mL); the volume of the sample solution, in milliliters (mL): the mass of the sample, in grams (g);
xProtein extraction rate, %.
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GB/T38578—2020
The calculation results are expressed as the arithmetic mean of the parallel determination values, retaining two significant figures. 10.2
GB/T38578—2020
Appendix A
(Informative Appendix)
Capillary electrophoresis of standard solutions
A.1 The capillary electrophoresis of tropomyosin is shown in Figure A.1. 0.05-
0,00 cal
2 The capillary electrophoresis of arginine kinase is shown in Figure A.2. A.2
A.3 The capillary electrophoresis of parvalbumin is shown in Figure A.3. D.040
Migration time/min
Capillary electrophoresis of tropomyosin
Migration time/min
Capillary electrophoresis of arginine kinasebzxz.net
Migration time/min
Capillary electrophoresis of parvalbumin
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GB/T38578-2020
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People's Republic of China
National Standard
Rapid detection of allergenic proteins from aquatic products
Capillary electrophoresis method
GB/T385782020
Published and distributed by China Standards Press
Peace, Chaoyang District, Beijing No. 2, Lixi Street (100029) No. 16, Sanlihe North Street, Xicheng District, Beijing (100045) Website: spc.net.cn
Editorial Office: (010) 68333533 Distribution Center: (010) 51780238 Reader Service Department: (Q10) 68523946
Printed by Qinhuangdao Printing Factory of China Standard Press Distributed by Xinhua Bookstores in various places
Format 880×12301/16
Printing Sheet 0.75 Word Count 14 Words
First Edition in March 2020 First Printing in March 2020 Book Number: 155066·1-64026 Price 16.00 yuan If there is any printing error, the distribution center of our company will replace it. Copyright infringement will be investigated
Report Phone: (010685101072 Quantitative determination is performed using the external standard calibration curve method. With the mass concentration of a series of allergenic protein standard solutions as the coordinates and the capillary electrophoresis peak area as the ordinate, a linear regression equation for the standard curve is drawn, and the peak area of ​​the sample is compared with the standard curve for quantification. The response values ​​of the allergenic proteins in the standard solution and the sample extract should be within the linear range of the instrument. See Appendix A for the capillary electrophoresis diagram of the standard solution. 9.3.3 If the mass concentration in the sample exceeds the highest point of the standard curve, further dilute the sample. 9.3.4 At least 2 electrophoresis diagrams for each test solution. 9.3.5 After testing 3 to 5 samples, new boric acid-xenazolate buffer solution should be replaced at the inlet and outlet of the instrument. 10 Calculation of results
The allergenic protein content was calculated according to formula (2):
Wherein:
p=euxv
—the content of allergenic protein, in milligrams per gram (mg/g): p
pThe content of allergenic protein in the sample solution, in micrograms per milliliter (μ8/mL); the volume of the sample solution, in milliliters (mL): the mass of the sample, in grams (g);
xProtein extraction rate, %.
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GB/T38578—2020
The calculation results are expressed as the arithmetic mean of the parallel determination values, retaining two significant figures. 10.2
GB/T38578—2020
Appendix A
(Informative Appendix)
Capillary electrophoresis of standard solutions
A.1 The capillary electrophoresis of tropomyosin is shown in Figure A.1. 0.05-
0,00 cal
2 The capillary electrophoresis of arginine kinase is shown in Figure A.2. A.2
A.3 The capillary electrophoresis of parvalbumin is shown in Figure A.3. D.040
Migration time/min
Capillary electrophoresis of tropomyosin
Migration time/min
Capillary electrophoresis of arginine kinase
Migration time/min
Capillary electrophoresis of parvalbumin
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GB/T38578-2020
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People's Republic of China
National Standard
Rapid detection of allergenic proteins from aquatic products
Capillary electrophoresis method
GB/T385782020
Published and distributed by China Standards Press
Peace, Chaoyang District, Beijing No. 2, Lixi Street (100029) No. 16, Sanlihe North Street, Xicheng District, Beijing (100045) Website: spc.net.cn
Editorial Office: (010) 68333533 Distribution Center: (010) 51780238 Reader Service Department: (Q10) 68523946
Printed by Qinhuangdao Printing Factory of China Standard Press Distributed by Xinhua Bookstores in various places
Format 880×12301/16
Printing Sheet 0.75 Word Count 14 Words
First Edition in March 2020 First Printing in March 2020 Book Number: 155066·1-64026 Price 16.00 yuan If there is any printing error, the distribution center of our company will replace it. Copyright infringement will be investigated
Report Phone: (010685101072 Quantitative determination is performed using the external standard calibration curve method. With the mass concentration of the series of allergenic protein standard solutions as the coordinates and the capillary electrophoresis peak area as the ordinate, a linear regression equation for the standard curve is drawn, and the peak area of ​​the sample is compared with the standard curve for quantification. The response values ​​of the allergenic proteins in the standard solution and the sample extract should be within the linear range of the instrument. See Appendix A for the capillary electrophoresis diagram of the standard solution. 9.3.3 If the mass concentration in the sample exceeds the highest point of the standard curve, further dilute the sample. 9.3.4 At least 2 electrophoresis diagrams for each test solution. 9.3.5 After testing 3 to 5 samples, new boric acid-xenazobium buffer solution should be replaced at the inlet and outlet of the instrument. 10 Calculation of results
The allergenic protein content was calculated according to formula (2):
Wherein:
p=euxv
—the content of allergenic protein, in milligrams per gram (mg/g): p
pThe content of allergenic protein in the sample solution, in micrograms per milliliter (μ8/mL); the volume of the sample solution, in milliliters (mL): the mass of the sample, in grams (g);
xProtein extraction rate, %.
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GB/T38578—2020
The calculation results are expressed as the arithmetic mean of the parallel determination values, retaining two significant figures. 10.2
GB/T38578—2020
Appendix A
(Informative Appendix)
Capillary electrophoresis of standard solutions
A.1 The capillary electrophoresis of tropomyosin is shown in Figure A.1. 0.05-
0,00 cal
2 The capillary electrophoresis of arginine kinase is shown in Figure A.2. A.2
A.3 The capillary electrophoresis of parvalbumin is shown in Figure A.3. D.040
Migration time/min
Capillary electrophoresis of tropomyosin
Migration time/min
Capillary electrophoresis of arginine kinase
Migration time/min
Capillary electrophoresis of parvalbumin
TTiKAa-cJouakA
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GB/T38578-2020
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People's Republic of China
National Standard
Rapid detection of allergenic proteins from aquatic products
Capillary electrophoresis method
GB/T385782020
Published and distributed by China Standards Press
Peace, Chaoyang District, Beijing No. 2, Lixi Street (100029) No. 16, Sanlihe North Street, Xicheng District, Beijing (100045) Website: spc.net.cn
Editorial Office: (010) 68333533 Distribution Center: (010) 51780238 Reader Service Department: (Q10) 68523946
Printed by Qinhuangdao Printing Factory of China Standard Press Distributed by Xinhua Bookstores in various places
Format 880×12301/16
Printing Sheet 0.75 Word Count 14 Words
First Edition in March 2020 First Printing in March 2020 Book Number: 155066·1-64026 Price 16.00 yuan If there is any printing error, the distribution center of our company will replace it. Copyright infringement will be investigated
Report Phone: (01068510107
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