Isolation and identification of peptides from the diafiltration permeate of the water-soluble fraction of cheddar cheese

The water-soluble extract of a mature Cheddar cheese was fractionated by diafiltration using 10 kDa nominal molecular weight cutoff membranes. The permeate had a savory, cheesy taste, whereas the retentate was bland. The permeate was resolved into nine fractions by gel permeation chromatography on Sephadex G-25. Fractions I-III contained only peptides, whereas fractions IV-IX comprised mainly free amino acids. Fraction IV contained a mixture of all amino acids except Phe (fraction V), Tyr (fraction VI), and Trp (fraction IX). Fraction III, which had the savory cheesy taste of the permeate, was dominated by one major peak with several minor ones. Fraction III was rechromatographed on a Sephadex G-25 column, and a number of peptides were isolated from subfractions thereof by reversed-phase high-performance liquid chromatography and characterized by N-terminal amino acid sequencing and mass spectrometry. The results showed that; starter bacteria cell-envelope proteinase, endopeptidases, and aminopeptidases play an important role in the degradation of the primary proteolytic products produced by chymosin and plasmin from alpha(s1)-, alpha(s2)-, and beta-caseins.