This standard specifies the method for determining the ratio of casein to whey protein content. This standard is applicable to the determination of the ratio of casein to whey protein content in infant formula and milk powder. GB/T 5413.2-1997 Determination of whey protein in infant formula and milk powder GB/T5413.2-1997 Standard download decompression password: www.bzxz.net

GB/T 5413. 2—1997
This standard refers to the whey protein determination method of Morinaga Co., Ltd. of Japan. This series of standards will replace GB5413--85 from the date of implementation. This standard is proposed by China Light Industry General Association.
This standard is under the jurisdiction of the National Dairy Standardization Center.
The responsible drafting units of this standard are Harbin Morinaga Dairy Co., Ltd. and National Dairy Quality Supervision and Inspection Center. The participating drafting units of this standard are: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Zhejiang Light Industry Research Institute, and Nestle (China) Investment Services Co., Ltd.
The main drafters of this standard are Yu Likun, Zhang Banjie, and Zhao Qin. 225
National Standard of the People's Republic of China
Infant formula foods and milk powder
Determination of whey protein
Milk powder and formula foods for infant and young children-Determination of whey proteinScope
This standard specifies the method for determining the ratio of casein to whey protein. GB/T 5413.2—1997
Replaces GB 5413--85
This standard applies to the determination of the ratio of casein to whey protein in infant formula foods and milk powder. 2 Method Summary
After the sample is subjected to SDS-polysaccharide gel electrophoresis (SDS-PAGE, Laemmli method), the casein and whey protein bands separated in the order of molecular weight are measured by a densitometer to obtain the ratio of casein to whey protein. 3 Reagents
All reagents, if not specified, are of analytical grade; all experimental water, if not specified otherwise, is grade III water. 3.1 Electrophoresis buffer: Dissolve 7.5 g Tris (hydroxymethyl) aminomethane, 36 g glycine, and 2.5 g SDS in distilled water and dilute to 500 mL. Dilute 5 times with distilled water before use. 3.2 SDS sample buffer: 0.125 mol/L Tris-hydrochloric acid (pH 6.8), 20% glycerol by volume, 4% (m/V) SDS, 10% 2-mercaptoethanol by volume, 0.0025% bromophenol blue by mass. 3.3 Dye: 0.1% Coomassie brilliant blue R-250 by mass, 10% methanol by volume, and 7.5% acetic acid by volume. wwW.bzxz.Net
3.4 ​​Decolorizing agent: 10% methanol by volume, 7.5% acetic acid by volume. 3.5 Casein.
3.6 Purified whey protein (WPC).
3.7 SDS-PAGE: Use gradient gel (10% 20%) or equivalent gel. 4 Instruments
Common laboratory instruments and:
4.1 Flat-plate electrophoresis tank.
4.2 Power supply equipment.
4.3 Optical densitometer.
5 Operation steps
5.1 Preparation of sample solution
Dissolve the reagent in distilled water according to the concentration range of 10 to 20 μg/μL of protein. Add the same volume of SDS sample buffer as the sample, and let it stand in boiling water for 5 minutes before using it as the sample solution. 5.2 Preparation of standard solution Before use, the protein concentration of casein and whey protein is accurately determined by Kjeldahl method. Dissolve casein and whey protein according to the above method and treat them as standard solution. In addition, the standard solution of casein and whey protein can be mixed according to any ratio of protein content (such as protein concentration ratio of 75:25.50:50, 25:75) as a mixed standard solution. 5.3 Electrophoresis Set the gel in the electrophoresis tank and add electrophoresis buffer. The amount of sample solution and standard solution added is 10uL per well. The electrophoresis time varies depending on the type of electrophoresis gel. When electrophoresis is performed with a 10%~~20% gradient composite gel, the constant current is 40mA and the time is 60min. The end time of electrophoresis can be marked by the blue band of bromophenol blue reaching 5mm from the bottom of the gel. After the electrophoresis, the gel is placed in the staining solution and oscillated for staining for 1h, then transferred to the decolorizing solution and oscillated for decolorization for 12h and then moved to distilled water. 5.4 Determination
Use a densitometer to measure each colored band and obtain the integral value (Trace value) of its color depth (OD). 6 Expression of analysis results
6.1 Determination of bands
Among the bands after electrophoresis, refer to the molecular weight and the electrophoretic diagram of standard casein and whey protein, and select the following bands for calculation. (1) Casein: α3-casein (molecular weight 23500), β-casein (molecular weight 24000), k-casein (molecular weight 19000); (2) Whey protein: α-lactalbumin (molecular weight 14000), 3-lactoglobulin (molecular weight 18000), serum albumin (molecular weight 66000).
6.2 Calculation method
Add the trace values ​​of the above three casein bands and divide them by the sum of the trace values ​​of all the bands in the same swimming line to obtain the casein ratio (Ci value). In the same way, add the trace values ​​of the above three whey protein bands and divide them by the sum of the trace values ​​of all the bands in the same swimming line to obtain the whey protein ratio (W, value). When the separate solutions of casein and whey protein are used as standard solutions, the C, value (=C value) of the standard casein and the W, value (=W, value) of the standard whey protein are obtained according to the above method. Divide the Cz value and W2 value of the sample by the Cz value and W2 value, respectively, to obtain the provisional casein content ratio (Cz value) and whey protein content ratio (W2). When the Cz value + W2 value is not equal to 1, divide the Cz value and W2 value by the sum of the C2 value and W2 value, respectively, to obtain the ratio of casein and whey protein in the sample. When the mixed solution of casein and whey protein is used as the standard solution, the Cz value of casein and the W2 value of whey protein in each mixing ratio are obtained according to the above method. The Cz value and the casein content ratio, as well as the W2 value and the whey protein content ratio of the mixed standard solution are made into coordinate calibration curves, and the Cz value and W2 value of the sample are obtained using the calibration curves. When the C2 value + W2 value is not equal to 1, divide the C2 value and W2 value by the sum of the C2 value and W2 value, respectively, to obtain the ratio of casein and whey protein in the sample. 227
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