Aine Healy

Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese.

Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of the beta-casein, but peptides from alpha s1- and alpha s2-caseins were also identified; the extract also contained alpha-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from alpha s1- and beta-caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysation of alpha s2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.

Proteolytic specificity of plasmin on bovine αs1‐Casein

Abstract The proteolytic specificity of plasmin (fibrinolysin, E. C. 3.4.21.7, from bovine plasma) on bovine αs1‐casein was determined in solution in 50 mM ammonium bicarbonate buffer, pH 8.4, at 37°C. Peptides, isolated by reverse‐phase high performance liquid chromatography on a C18 column or by electroblotting from urea‐polyacrylamide gel electrophoretograms, were identified from their amino acid sequence and mass. The principal plasmin cleavage sites were found at Arg22‐Phe23, Arg90‐Tyr91, Lys102‐Lys103, Lys103‐Tyr104, Lys105‐Val106, Lys124‐Glu125 and Arg151‐Gln152. The initial cleavage sites and the order of production of small (pH 4.6‐soluble) peptides suggest that αs1‐casein was cleaved initially towards the centre of the molecule.

Water-soluble peptides in Cheddar cheese: isolation and identification of peptides in the diafiltration retentate of the water-soluble fraction

The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl. pH 8.6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from beta-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from alpha s1-casein; beta-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.

A scheme for the fractionation of cheese nitrogen and identification of principal peptides

A four-step fractionation scheme for the isolation of peptides from cheese was investigated. The first three steps involved water extraction, ultrafiltration and gel filtration; water was used as solvent which renders the fractions suitable for sensory assessment. In the last step, gel filtration fractions were applied to a Sep-pak reverse phase C18 cartridge from which individual peptides were eluted by using increasing concentrations of acetonitrile. The two principal peptides in the ultrafiltrate permeate of water soluble extract were isolated and identified by amino acid sequencing and mass spectroscopy as αs1-CN f1-9 and αs1-CN f1-13. These peptides may have been produced by the combined action of chymosin and PI-type cell wall-associated proteinase of the Lactococcus spp. starter.

Proteolytic specificity of elastase on bovine αs1-casein

Proteases from polymorphonuclear leukocytes (PMN or neutrophils) and macrophages, the main somatic cells found in milk of healthy cows, may contribute to hydrolysis of caseins at neutral or acid pH in high somatic cell count milks. The objective of this study was to determine the cleavage specificity of elastase, one of the principal PMN proteinases, on αs1-casein. αs1-Casein (5 mg ml−1) was dissolved in phosphate buffer, pH 7.5, and elastase added. Samples were taken over a 24 h period and analyzed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Twenty-five cleavage sites were identified showing that elastase had a broad cleavage specificity on αs1-casein.

Identification of the principal water-insoluble peptides in Cheddar cheese

The water-insoluble fraction (WISF) of a 20 week-old Cheddar cheese was fractionated by anion-exchange FPLC on Mono-Q® 5/5. Those fractions containing peptides were collected and analysed by urea-PAGE. Peptides of interest were isolated by electroblotting from the urea-PAGE gels and identified from their N-terminal amino acid sequence and mass. This study confirmed and completed identification of some of the WIS peptides partially identified by McSweeney et al. (International Dairy Journal 4, 111–122, 1994) and identified a further 13 peptides. Most of the peptides can be attributed to cleavage of αs1- and β-caseins by chymosin, plasmin or proteinase from Lactococcus lactis spp.

Proteolytic specificity of cathepsin D on bovine F-actin

Proteolysis of bovine F-actin by cathepsin D (E.C. 3.4.23.5) in 50 mM Na acetate buffer, pH 5.5, at 37°C was investigated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse-phase high performance liquid chromatography (RP-HPLC). Actin was hydrolyzed by cathepsin D during incubation to peptides detectable by RP-HPLC, although no degradation products were detected by SDS-PAGE. Peptides (2% trichloroacetic acid-soluble) from the hydrolyzate were isolated by RP-HPLC on a C(18) column using an acetonitrile/water gradient and identified from their N-terminal sequence and mass. Cathepsin D cleavage sites were identified at Cys(12)-Asp(13), Gly(22)-Phe(23), Arg(30)-Ala(31), Thr(79)-Asn(80), Ile(87)-Trp(88), Thr(91)-Phe(92), Phe(92)-Tyr(93), Arg(97)-Val(98), His(103)-Pro(104), Leu(107)-Thr(108), Thr(108)-Glu(109), Lys(120)-Met(121), Leu(144)-Tyr(145), Ile(153)-Val(154), Leu(155)-Asp(156), Ile(167)-Tyr(168), Leu(180)-Asp(181), Met(192)-Lys(193), Leu(195)-Thr(196), Arg(208)-Glu(209), Arg(212)-Asp(213), Leu(223)-Asp(224), Lys(240)-Ser(241), Thr(262)-Leu(263), Trp(342)-Ile(343), Arg(349)-Ser(350), Trp(358)-Ile(359), and Lys(375)-Cys(376). In general, cathepsin D preferentially cleaved bonds containing at least one hydrophobic amino acid residue. The results of this study showed that actin was degraded extensively by cathepsin D with peptides released from numerous locations in the protein molecule.