National Standard of the People's 
Republic of China 
 
GB xxxxx - xxxx 
 
 
National Standard for Food Safety 
Casein 
(Exposure Draft) 
 
 
    Issued: xxxx-xx-xx Entering into effect:  xxxx-xx-xx   
Issued by National Health and Family Planning Commission 
Of the People’s Republic of China  
 GB xxxxx – xxxx 
 
1  
 
National Standard for Food Safety 
Casein 
 
1. Scope 
 
This standard applies to casein obtained through an acid process, an enzymatic 
process or membrane separation. Casein is a compound composed of alpha-casein, 
beta-casein, kappa-casei n and gamma-casein. 
 
2. Terms and Definitions  2.1 Casein obtained through an acid process  
The product is obtained thr ough degreasing, acid precipitation of casein, followed 
by filtration, washing, drying etc. (with m ilk and/or other dairy products as raw 
materials). 
 
2.2 Casein obtained through an enzymatic process  
The product is obtained through degreasi ng, chymosin precipitation of casein, 
followed by filtration, washing, drying etc. (w ith milk and/or other dairy products as 
raw materials). 
 
2.3 Casein obtained through membrane separation 
 
The product is obtained through membrane separation and enrichment of casein, 
followed by concentration, sterilization, dryi ng etc. (with milk and/or other dairy 
products as raw materials). 
 
3. Technical requirements  3.1 Raw material requirements GB xxxxx – xxxx 
 
2  
Raw materials should comply with relevant standards and (or) relevant provisions. 
 
 
3.2 Sensory requirements 
 
Sensory indexes should comply with requirements specified in Table 1. 
 
Table 1 Sensory indexes 
Item Requirements Test method 
Colour Milky white tocreamy yellow.  Observation of the colour and 
texture of a suitable amount of sample in a white shallow dish 
under natural light, and smelling 
its odour. Taste and odour Neither peculi ar smell nor peculiar taste. 
State Uniform dry powder, allowed to have a small amount 
of dark yellow particles, without visual impurities. 
 
 
3.3 Physicochemical indexes 
 
The physicochemical indexes should comply with Table 2. 
 
Table 2 Physicochemical indexes 
Items Acid 
process Enzymatic 
process Test method 
Protein (dry basis) / (%) ≥ 90.0 84.0 GB 5009.5 Kjeldahl determination or 
spectrophotometry 
Casein (in protein) / (%) ≥ 95.0 95.0 Appendix A 
Moisture / (%) ≤ 12.0 12.0 GB 5009.3 
Fat / (%) ≤ 2.0 2.0 Appendix B 
Ash content/ (%) ≤2.5 ≥7.5 GB 5009.4  Ignition temperature: 825 °C 
Lactose / (%) ≤ 1.0 1.0 Appendix C 
Free acid / (mL 0.1 mol/L NaOH/g) ≤ 0.27 - GB 5413.34 
 
3.4 Contaminant limit 
 
The contaminant limit should comply w ith the requirements for other dairy 
products specified in GB 2762. 
 
3.5 Mycotoxin limit 
 GB xxxxx – xxxx 
 
3 The mycotoxin limit should comply with the requirements for other dairy products 
specified in GB 2761. 
 
 3.6  Microbial limit 
 
The microbial limit shall comply with limits specified in Table 3.  
Table 3 Microbial limit 
Items Sampling plan a and limit 
(unless otherwise specified, all items are expressed as CFU/g) Test method 
n c m M 
Staphylococcus aureus 5 2 10 100 GB 4789.10 
Plate count 
Salmonella 5 0 0/25g - GB 4789.4 
a Samples are analysed and processed according to GB 4789.1 and GB 4789.18. 
 
 
3.7  Food additives  
The use of food additives should comply with the requirements for other dairy 
products specified in GB 2760. GB xxxxx – xxxx 
 
4  
 
Appendix A 
(Normative Appendix) 
Determination of Casein  
A.1 Principle  
After the test sample has been fully dissolved, the obtained solution is regulated 
with acetic acid and sodium acetate until the pH is 4.6, so  as to promote casein 
precipitation. After filtration, casein is collected, and then tested according to GB 
5009.5. 
 
A.2 Reagents and materials  
Unless otherwise specified, all reagents used in this me thod are analytically pure. 
The tertiary water used complies with GB/T 6682.  A.2.1 Sodium bicarbonate (NaHCO
3). 
A.2.2 Sodium tripolyphosphate (Na 5P3O10). 
A.2.3 Glacial acetic acid (CH 3COOH): Guaranteed reagent. 
A.2.4 Sodium acetate (CH 3COONa·3H 2O). 
A.2.5 Anhydrous sodium acetate (CH 3COONa). 
A.2.6 10% acetic acid solution. A.2.7 Sodium acetate solution (1 mol/L): 41  g of anhydrous sodium acetate (A.2.5) or 
68 g of sodium acetate (A.2.4) is dissolved in water and then diluted to 500 mL. 
A.2.8 Sodium acetate-acetic acid buffer solu tion: 1.0 mL of sodium acetate solution 
(A.2.7) and 1.0 mL of acetic acid solution (A.2.6 ) are diluted to 100 mL with water in a 
volumetric flask. A.2.9 Other reagents and materi als shall comply with GB 5009.5. 
 
A.3 Instruments and devices 
 
A.3.1 Balance: sensitivity of 1 mg.  GB xxxxx – xxxx 
 
5  
A.3.2 Electric-heated thermosta tic water bath: 100 °C + 1 °C. 
A.3.3 The remaining instruments and devices shall comply with GB 5009.5. 
 
A.4  Analysis steps  
A.4.1 Sample treatment 
 
0.2 g of test sample (accurate to 0.001 g) is transferred into a 150 mL dry conical 
flask with a plug. If the test sample is cas ein obtained through an  acid process, 0.02 g + 
0.001 g of sodium bicarbonate is added; if the test sample is casein obtained through an 
enzymatic process, 0.02 g + 0.001 g of s odium tripolyphosphate and then 8 mL of 
water is added. After fully mixing, the mixture is completely dissolved in a water bath 
of 65~67 °C (gently shaken once every 5 mi n, generally for 10~15 mi n). After cooling, 
1 mL of acetic acid solution (A.2.6) is added, fully mixed, a nd left to stand for 5 min. 
Then, 1 mL sodium acetate solution (A.2.7) is added, fully mixed, and left to stand to 
promote casein precipitation, followed by filtra tion with dry filter paper. The conical 
flask and the precipitate are repeatedly washed with a small amount of the buffer 
solution (A.2.8). The filter paper is folded toge ther with the precipita te, and digested in 
a digestive tube. Then the test sample  is treated in compliance with GB 5009.5. 
 
A.4.2 Determination of protein  
Determination complies with the Met hod I or II specified in GB 5009.5. 
 
A.5  Expression of the analysis results 
 
A. 5.1 The results of the casein content an alysis are expressed according to the 
relevant method specified in GB 5009.5. A.5.2 The proportion of casein in the total am ount of protein in the test sample is 
calculated according to Formula (1).  
 GB xxxxx – xxxx 
 
6 Where: 
 
X1 –Proportion of casein in the total amount of protein in the test sample (g/100 g); 
m1 -Casein content in the test sample (g/100 g); 
m2 -Protein content in the test sample (g/100 g); 
 
A.6 Precision  
The absolute difference between the results of two independent measurements 
taken repeatedly shall not be more than 10% of the arithmetic mean. GB xxxxx – xxxx 
 
7  
Appendix B 
(Normative Appendix) 
Determination of Fat in Casein  
 
 
B.1 Principle  
Sulphuric acid is added to casein to da mage the properties of emulsion and the 
outer membrane protein covering the fat globu le. The volume of fat is measured after 
centrifugal separation. 
 
B.2 Reagents and Materials 
 
B.2.1 Sulphuric acid: Spec ific gravity of 1.820~1.825. 
B.2.2 Isoamyl alcohol. B.2.3 Milk centrifuge. B.2.4 Babcock flask. 
 
B.3 Analysis steps  
3 g of test sample accurate to 1 mg is im mersed and preheated in 10 ml of distilled 
water present in a Babcock flask in a wa ter bath of 85 °C + 2 °C for 30 min for 
expansion. After cooling, 10 ml of sulphuric  acid is slowly added along the bottleneck, 
fully mixed, then heated in the water bath for 30 min, and shaken once every 5 min. 
After the test sample is completely dissolv ed, 1 ml of isoamyl alcohol is added, and 
then fully mixed. The mixture is centrifuge d in a cream centrifuge at 1200 r/min for 5 
min. Water above 80 °C is added until th e liquid level reaches the base of the 
bottleneck. The mixture is centr ifuged at 1200 r/min for 10 mi n, and then transferred to 
a water bath of 60 °C + 5 °C . 5 min later, it is taken out to immediately record X. 
 
B.4 Result calculation  
Milk fat content is expressed as X
2 in %, and calculated according to Formula (2): GB xxxxx – xxxx 
 
8  
 
Where: 
 
X – Value measured as per the steps in B.3 
 
B.5 Precision 
 
The absolute difference between the results of two independent measurements 
taken repeatedly shall not be more than 5% of the arithmetic mean. GB xxxxx – xxxx 
 
9  
Appendix C 
(Normative Appendix) 
Determination of Lactose in Casein  
 
C.1 Reagent 
 
C.1.1 Hydrochloric acid: 0.1 mol/L. C.1.2 Glacial acetic acid (CH
3COOH): 10% (m/v) solution. 
C.1.3 Sodium acetate (CH 3COONa·3H 2O): 1 mol/L solution. 
C.1.4 Phenol (C 6H5OH): 80% (m/m) solution: 8 g of phenol and 2 g of water are fully 
mixed while heating. C.1.5 Sulphuric acid. C.1.6 Lactose (C
12H22O11): 2.00% (m/v) solution:  2.105 + 0.001 g of lactose 
monohydrate (equivalent to 2.00 g of anhydrous lactose) is diluted to 100 ml with 
water in a volumetric flask, fully mixed, and then kept at 0 °C. 
 
 C.2 Instrument 
 
C.2.1 Analytical balance C.2.2 100 mL conical flask C.2.3 1 mL, 2 mL, 10 mL pipette C.2.4 50 mL beaker C.2.5 Water bath 
C.2.6 100 mL volumetric flask 
C.2.7 Ultraviolet spectrophotometer 
 
C.3 Analysis steps  C.3.1 Sample treatment  
1 g test sample accurate to 1 mg is placed  in a 100 ml conical flask. If the test 
sample is casein obtained through an acid process, 0.1 + 0.001 g of sodium bicarbonate GB xxxxx – xxxx 
 
10 is added; if the test sample is casein obtained through an enzymatic process, 0.1+0.001 
g of sodium tripolyphosphate and then 25 mL of water is added. The mixture is 
completely dissolved in a water bath of  65°C ± 2°C (generally 10~15 min). After 
cooling, 15 mL of water, 8 mL of 0.1 mol/L  HCL solution and 1 mL  of 10% acetic acid 
solution (fully mixed after adding each soluti on) are added and left to stand for 5 min. 
Then, 1 mL of 1 mol/L sodium acetate solu tion is added, and fully mixed to promote 
fast casein precipitation, followed by filtration with dry filter paper. The filtrate is collected after discarding the first few mL of the filtrate. 
 
C.3.2 Determination of lactose  
2.0 mL of filtrate is transferred into a 50 mL  beaker with a pipette. Then, 0.2 mL of 
80% phenol solution is added with a micro-pi pette, and the mixture is fully mixed on a 
magnetic stirrer. 5 mL of concentrated sul phuric acid is added within 1 s. The mixture 
is fully mixed, left to stand for 15 min, and th en cooled in a water bath at 20 °C for 5 
min. The absorbance of the solution is determ ined at 490 nm with blank solution as the 
control solution. If the absorbance exceeds th e upper limit of the standard curve, the 
filtrate is properly diluted, and then the operati on is repeated (note: If diluted solution 
is used, the calculation formula should be changed accordingly). 
 
C.4 Preparation of Blank Solution  
The blank solution is prepared using th e same instruments, devices and same 
amount of reagents, the same  way desribed above, but the sample dissolving and 
filtering operation should be omitted. 
 
C.5 Plotting of the Standard Curve 
 
C.5.1 10 mL of 2.00% lactose is diluted to 100 mL in a volumetric flask (solution 
A). 1 mL, 2 mL and 3 mL of solution A are respectively diluted to 100 mL in three 
volumetric flasks to get three standard solutions. The concen tration of anhydrous 
lactose in the obtained standard so lutions is respectively 20, 40 and 60 μg/mL. 
C. 5.2 2 mL of each of the three standard solutions mentioned above is respectively 
added to three 50 mL beakers, while 2 mL of  water is added to another beaker. Then GB xxxxx – xxxx 
 
11 the absorbance of other three standard solutio ns is respectively determined with the 
same operation as the determination method for the sample filtrate, and with the fourth 
one as the reference solution. Finally, a curve between th e absorbance and 
concentration of anhydrous lactose ( μg/mL) is obtained. 
 
C.6 Result calculation  
Lactose content is expressed as X
3 in %, and calculated according to Formula (3): 
 
Where: 
 
C1- Concentration of anhydrous lactose in the sample solution obtained from the 
standard curve; 
m3 –Sample mass, g; 
 
C.7 Precision 
 
The absolute difference between the results of two independent measurements 
taken repeatedly shall not be more than 5% of the arithmetic mean.