Extraction of protein variants
1. On the day of the analysis, if the samples were frozen, thaw these at room temperature (25 °C).
2. Take a 1 ml aliquot of milk and transfer it to a 2.2 mL Eppendorf tube using an automatic pipet.
3. Add 1 mL of solution A to the 2.2 mL Eppendorf tube containing the milk sample (1:1 - sample: solution A).
4. Shake each mixture in a vortex for 10 s at medium speed.
5. Incubate the mixture at room temperature (25 °C) for 1 h.
Note: you can climate the room (25 °C) in which incubation is running by using an air conditioning unit. Standardization of the incubation conditions contributes to low variations across different processing days.
6. Centrifuge at 14200 x g for 5 min.
7. An upper fat layer is formed after centrifugation. Then, carefully remove it using a micro-spatula.
8. Take 450 μl of the supernatant and transfer it to a new 2.2 mL Eppendorf tube. To this same tube, add 1350 μl of solution B (1:3 - supernadant: solution B).
9. Transfer 1 mL of the mixture to a vial for HPLC analysis.
10. Analyze the samples by RP-HPLC-DAD. Note: storage the samples under refrigeration at 4 °C until analysis.
Chromatographic analysis (conditions)
- Column temperature: 45 °C.
- Injection volume: 5 µL.
- Stationary phase: Hypersil GOLD C8 column (3 µm, 150 x 4.6 mm) equipped with Hypersil GOLD C8 guard column (5 µm, 10 mm).
- Working wavelength: 214 nm.
- Mode: gradient, constant flow: 0.500 mL/min (see the elution gradient in table 1, supplementary files).
NIRS analysis
Determine the total protein and casein contents in g/100 g milk in the sample using a NIRS equipment. Additionally, determine the milk sample density.
Calculations
1. Determine the peak area for each protein in the chromatogram, including caseins (κ-CN X2, αs2-CN, κ-CN X1, αs1-CN B, αs1-CN A, and βγ-CN) and whey proteins (α-La and β-Lg) variants (Figure 1). Take in main that some casein variants could be absent in the sample.
2. For calculation of the quantities of casein variants (κ-CN X2, αs2-CN, κ-CN X1, αs1-CN B, αs1-CN A, and βγ-CN), use the following procedure (use the supplementary material as well):
a. Calculate the total area of casein variant peaks (sum of peak areas).
b. Calculate the proportion of each casein variant according to the following equation:
Proportion of casein variant = Peak area of casein variant/sum of peak areas of casein variants
c. Calculate the concentration of each casein variant in g/100 g milk:
Concentration of casein variant (g/100 g milk) = Casein content (g/100 g milk) x Proportion of casein variant
d. Optional: calculation of the concentration of casein variant in g/L milk:
Concentration of casein variant (g/L milk) = (Concentration of casein variant (g/100 g milk)) * (milk density (g/mL)) * 10
3. For calculation of the quantities of whey proteins variants (α-La and β-Lg), use the following procedure (use the supplementary material as well):
a. Calculate the total area of whey proteins variants (sum of peak areas).
b. Calculate the proportion of each whey protein variant according to the following equation:
Proportion of whey protein variant = Peak area of whey protein variant/sum of peak areas of whey proteins
c. Calculation of total whey protein: assuming that total milk protein = casein + whey protein, then:
Whey protein content (g/100 g milk) = total milk protein (g/100 g milk) – casein (g/100 g milk).
d. Calculate the concentration of each whey protein variant in g/100 g milk:
Concentration of whey protein variant (g/100 g milk) = Whey protein content (g/100 g milk) x Proportion of whey protein variant
e. Optional: calculation of the concentration of whey protein variants in g/L milk:
Concentration of whey protein variant (g/L milk) = (Concentration of whey protein variant (g/100 g milk)) * (milk density (g/mL)) * 10