Unit 2

• Analytical Fractionation of Non-Protein Nitrogen (NPN) in Milk:
• Standard Definition (Rowland Fractionation Protocol):
• Non-Protein Nitrogen (NPN) is chemically defined as the nitrogen fraction that remains soluble in 12% trichloroacetic acid (TCA) solution.
• 12% TCA quantitatively precipitates all proteins (caseins and native/denatured whey proteins), leaving only low-molecular-weight nitrogenous compounds (urea, creatine, creatinine, free amino acids, peptides) in the filtrate.
• Conclusion: (A) only (Option 2).

Correct option: (2) (A) only,

• Post-Translational Phosphorylation Site on Caseins:
• Phosphoserine Residues:
• Caseins ($\alpha_{s1}, \alpha_{s2}, \beta$-casein) are phosphoproteins where orthophosphate groups are monoesterified to the hydroxyl group of serine residues (as O-phosphoserine).
• Phosphorylation occurs within the recognition sequence motif $\text{Ser-X-Glu/SerP}$.
• These phosphoserine clusters bind calcium ions, forming nanoclusters of colloidal calcium phosphate that stabilize casein micelles.
• Conclusion: Serine (Option 3).

Correct option: (3) Serine

• Role of Potassium Sulphate in Kjeldahl Nitrogen Digestion:
• Boiling Point Elevation:
• Concentrated sulfuric acid boils at $338^\circ\text{C}$, which is insufficient for rapid organic matter destruction.
• Adding potassium sulphate ($\text{K}_2\text{SO}_4$) raises the boiling point of the digestion mixture to $370 - 380^\circ\text{C}$, accelerating oxidative digestion without causing nitrogen volatilization losses.
• Conclusion: Increasing the boiling point of sulphuric acid (Option 1).

Correct option: (1) Increasing the boiling point of sulphuric acid