Detection of casein phosphopeptides as food nutritional enhancers

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Unless otherwise specified in this standard, the purity of the reagents used should be analytical pure or higher. The standard titration solution used, impurity determination standard solution, formulation, and product should be prepared in accordance with the provisions of GB/T601, GB/T602, and GB/T603. The test water should comply with the provisions of Grade III water in GB/T6682. The solution used in the experiment refers to aqueous solution when no specific solvent is specified for preparation

A.2 Identification Test

A.2.1 Reagents and Materials

A.2.1.1  Sulfuric acid

A.2.1.2; Copper sulfate (CuSO4 · 5H2O)

A.2.1.3; Potassium sulfate (K2SO4)

A.2.1.4 Mixed reagent: sulfuric acid solution (mass fraction 25%)+ammonium molybdate solution (25g/L)+water+vitamin C solution (100g/L), 1+1+2+1

A.2.2 Analysis Step

Transfer the dried precipitate (A.3.4) obtained from the determination of casein phosphopeptide content to a Kjeldahl digestion tube, add 10mL of sulfuric acid and 1g of catalyst (0.1g of copper sulfate and 0.9g of potassium sulfate), and heat for complete digestion. Take 1mL of digestive solution into a test tube, add 1mL of water, and soak in boiling water for 10 minutes. Take out and cool to room temperature. Take 100uL and transfer it to another test tube. Add 1.9mL of water, then 3mL of mixed reagent, and incubate in a 45 ℃ water bath for 20 minutes. Take it out and the solution should appear blue

A.3 Determination of Casein Phosphopeptide Content

A.3.1 Method Summary

Add a certain concentration of barium ions to the sample solution. The barium ions bridge between the casein phosphopeptide molecules, and at appropriate temperature and pH, add a certain concentration of ethanol to precipitate the bridged casein phosphopeptide molecules. Dry and weigh the precipitate to obtain the content of casein phosphopeptide in the product

A.3.2 Reagents and Materials

A.3.2.1 Anhydrous ethanol: Refrigerate to 4 ℃ before use

A.3.2.2; Barium chloride solution: 100g/L

A.3.2.3 Hydrochloric acid solution: Take 18mL of hydrochloric acid and slowly add it to 100mL of water

A.3.2.4 Sodium hydroxide solution: 80g/L

A.3.3 Instruments and Equipment

Frozen Centrifuge

A.3.4 Analysis Steps

Weigh 0.5g to 1.5g of the sample, accurate to 0.0002g, and place it in a 50mL centrifuge tube A. Add 15mL of water to completely dissolve it.

. Adjust the pH of the sample solution to 4.6 with hydrochloric acid solution, and then place it in a refrigerated centrifuge. Centrifuge at 5000r/min to 7000r/min at about 4 ℃ for 30 minutes. Take the supernatant and place it in a 50mL centrifuge tube B that has been pre dried to constant weight at 105 ℃. Adjust the pH of the supernatant to 6.8 with sodium hydroxide solution, and control the total amount of the solution to within 20mL. Add 1.5mL of barium chloride solution, then add 50mL of anhydrous ethanol, shake well, and place it in a refrigerator at 4 ℃ for at least 12 hours, Remove from the refrigerator and place in a refrigerated centrifuge. Centrifuge at 5000r/min to 7000r/min at approximately 4 ℃ for 30 minutes, and discard the precipitate from the supernatant. The precipitate is first dried in a drying oven at 60 ℃ to 70 ℃ for 1 hour, and then raised to 105 ℃ and dried to constant weight

A.3.5 Result Calculation

The mass fraction w of casein phosphopeptide content is calculated according to equation (A.1):

In the equation:

m1- the mass of centrifuge tube B and sediment after constant weight, in grams (g)

m2- mass of centrifuge tube B, in grams (g)

m - mass of the sample, in grams (g)

w0- Drying loss of the sample,%

The experimental results shall be based on the arithmetic mean of the parallel measurement results. The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean

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