Cheddar peptides

Figure 10.23 Water-insoluble and water-soluble peptides derived from asl-casein (A), as2-casein (B) or -casein (C) isolated from Cheddar cheese DF = diafiltration. The principal chymosin, plasmin and lactococcal cell-envelope proteinase cleavage sites are indicated by arrows (data from T.K. Singh...

Hamilton, J. S., Hill, R. D. and Van Leeuwen, H. 1974. A bitter peptide from Cheddar cheese. Agr. Biol Chem. 38, 375-379. 

Bitter peptides have been identified in hydrolyzates of casein (12,13), cheese (13a,b), and soy bean (14,15,15a). The bitter taste has been related to the hydrophobic amino acid content (16-20) and to chain length. Ney and Retzlaff (21) established a formula relating the bitterness of peptides to their amino acid composition and chain length. Too large a proportion of hydro-phobic amino acids gives rise to bitterness yet above a certain molecular weight, bitterness is not perceptible even when there are hydrophobic amino acids (21). Peptides that were responsible for bitterness in Cheddar cheese were rich in Pro, which occurred predominantly in the penultimate position (21a). 

Alii, I., Kokoniewska, M., Gibbs, B. F., and Konishi, Y. (1998). Identification of peptides in Cheddar cheese by electrospray ionization mass spectrometry. Int. Dairy. 8, 643-649. 

Cliffe, A. J., Revell, D., and Law, B. A. (1989). A method for the reverse phase-high performance liquid chromatography of peptides from Cheddar cheese. Food Chem. 34, 147-160. 

Cliffe, A. J., Marks, J. D., and Mulholland, F. (1993). Isolation and characterization of nonvolatile flavors from cheese, peptide profile of flavor fractions from Cheddar cheese, determined by reverse phase high-performance chromatography. Int. Dairy. ]. 3,379-387. 

Gouldsworthy, A. M., Leaver, J., and Banks, J. M. (1996). Application of mass spectrometry sequencing technique for identifying peptides present in Cheddar cheese. Int. Dairy ]. 6, 781-790. 

McSweeney, P. L. H., Pochet, S., Fox, P. F., and Healy, A. (1994). Partial identification of peptides from water-insoluble fraction of Cheddar cheese. J. Dairy Sci. 61, 587-590. 

Broadbent, J.R., Barnes, M., Brennand, C., Strickland, M., Houck, K., Johnson, M.E., Steele, J.L. 2002. Contribution of Lactococcus lactis cell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat Cheddar cheese. Appl. Enviro. Micobiol. 68, 1778-1785. 

Complete characterization of proteolysis in cheese requires isolation and identification of the individual peptides. Various fractionation techniques were compared to Kuchroo and Fox (1983), some of which have been developed further by O Sullivan and Fox (1990), Singh et al. (1994), and Fox et al. (1994). Using these techniques, many of the water-insoluble and water-soluble peptides in Cheddar cheese have been isolated and identified... 

FIG. 14. Identity of peptides isolated from the water-soluble fraction of Cheddar cheese. Peptides derived from aji-casein are shown in A, those from /8-casein in B. The principal chymosin cleavage sites in Oji-casein, the principal plasmin cleavage sites in /8-casein, and the principal cleavage sites of lactococcal cell envelope proteinase on Oji- and -casein are shown by arrows (from Fox et al., 1994,1995, unpublished). 

The N-terminal sequence of asi-CN fl-9 and fl-13 is RPKHPIK therefore, it should be susceptible to Pep X. The accumulation of these peptides in Cheddar and the apparent absence of peptides with a sequence commencing at Lysa of asi-CN suggest that Pep X is not active in cheese isolated Pep X is in fact inactive on srCN fl-23 (W. Bockelmann and P. F. Fox, unpublished). 

The very small peptides in the UF permeate have not yet been identified. A number of authors (Aston and Creamer, 1986 Cliffe et al., 1993 Engels and Visser, 1994) have shown that the very small peptides (<500 Da) make a significant contribution to Cheddar flavor therefore, identification of the small peptides should prove interesting. 

Fractionation and identification of the small water-soluble peptides in cheese varieties is much less advanced than for Cheddar. Although not fractionated systematically, a large number of 12% TCA-soluble and -insoluble peptides were identified in water extracts of Parmesan by Addeo... 

The small peptides in cheese can be fractionated by various forms of chromatography, e.g., gel permeation, ion-exchange, and especially RP-HPLC. Using these techniques, more than 200 peptides have been demonstrated in Cheddar cheese, many of which have been isolated and identified (see Section IVE). Free amino acids are usually quantified by ion-exchange HPLC with post-column derivitization using ninhydrin or by separation of fluorescent amino acid derivatives by RP-HPLC. 

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