The protein and peptide fraction of human milk samples collected from mothers of pre- and full-term infants in the first week after parturition was analysed by use of liquid chromatography-mass spectrometry and tandem mass spectrometry. By characterising the peptide sequence, we defined the pathway of casein hydrolysis which leads to the formation of small peptides through intermediate oligopeptides. It was found that the action of a plasmin-like enzyme acting on specific lysine residues is the primary step in casein degradation. This is followed by endopeptidases and/or exopeptidases mediated cleavage of the oligopeptides which, in turn, produces a multiplicity of short peptides differing by one or more amino acid residues. In this process, a series of potentially bioactive peptides (opioid, phosphopeptides) and their precursors are produced.

SummaryThe isolation and identification of low molecular mass peptides formed during the ripening of Parmigiano-Reggiano cheese is described. A strategy was used based on the fractionation of nitrogenous material using chemical methods followed by HPLC to isolate peptides and fast atom bombardment-mass spectrometry to identify them. It was found that the majority of cheese oligopeptides arose from the proteolysis of β-casein. Several phosphopeptides and oligopeptides known in vivo to be biologically active have also been identified during the ripening of cheese.

The primary structures of ovine a sl -casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141-148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for ~ 80% of the entire translated a sl -casein, was as long as its caprine and bovine counterparts, and had a 98 and 89 % degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in a sl -casein A, whereas Ser 115 and Ser 41 were phosphorylated by ~ 50 and ~ 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser 13 -> Pro 13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP 12 ->-Ser 12 . A further substitution, SerP 68 -> Asn 68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser 64 and Ser 66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser 41 .Following the elucidation of the primary structure of bovine caseins (for a review, see Swaisgood, 1992), attention has now focused on the micellar components of other species. The primary structures of ovine K-and /?-casein have been determined on the mature proteins (Jolles et al. 1974;Richardson & Mercier, 1979) and those of a sl -and a s2 -casein on the cDNA precursors (Boisnard et al. 1991;Mercier et al. 1985). The initial translation products of ovine mammary mRNA coding for caseins are larger molecules containing hydrophobic amino terminal extensions. Comparison of ovine with other a sl -casein nucleotide sequences determined from the cDNA (Mercier et al. * This paper is dedicated to M. Bruno Ribadeau Dumas on the occasion of his retirement from INRA. § Present address: Istituto

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A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.

The non-protein nitrogen (NPN) of samples of Parmigiano-Reggiano cheese ripened for 6 and 15 months was fractionated by ion-exchange chromatography on a Cu 2+ -Chelex column to separate oligopeptides from free amino acids. Peptide components were isolated by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry (FAB-MS). Only the NPN fraction of 6 month old cheese samples contained enough peptides to be further characterized. On the basis of FAB-MS spectral results, 39 oligopeptides were identified, the main components being phosphopeptides. Two sets of both intact and partly dephosphorylated peptides, accounting for a total of 19 phosphopeptides, were formed by the hydrolysis of /?-casein and belonged to regions 1-20 and 6-28 of /?-casein. The formation and potential role of these peptides in cheese is discussed.Low molecular mass (LMM) peptides found in mature Parmigiano-Reggiano (PR) cheese, a hard pressed Italian cheese made from raw bovine milk, were isolated from the 120 g/1 trichloroacetic acid (TCA)-insoluble nitrogen fraction (Addeo et al. 1992). They arose from the limited proteolysis of /?-, a sl -and a s2 -casein, and the identification of peptide patterns related to well defined proteolytic mechanisms and ripening stages may contribute to understanding cheese quality, specially in relation to the degree of ripening.The aim of this work was to isolate and identify in the non-protein nitrogen (NPN) the LMM cheese peptides that do not precipitate in 120 g TCA/1. As reported by Panari et al. (1988), the NPN fraction of mature PR cheese (60 samples, 20-24 months) represents ~9 5 % of the pH 4'6-soluble nitrogen fraction and contains mainly free amino acids and ammonia. The composition of this fraction is extremely important because of the potential contribution of LMM peptides both to the formation of typical cheese flavour and to an undesirable bitter flavour (Fiat & Jolles, 1989). A better knowledge of cheese LMM oligopeptides could contribute to understanding the process leading to the formation of free amino acids, the contribution of enzymes to casein degradation and, finally, the stability of LMM peptides during cheese ripening.

The a s1 -casein (a s1 -Cas) locus in the goat is characterized by a polymorphism, the main feature of which is to be qualitative as well as quantitative. A systematic analysis performed in an autochthon southern Italy breed identified a new rare allele (M), which was characterized at both the protein and genomic level. The M protein displays the slowest electrophoretic mobility of the a s1 -Cas variants described so far. MS and automated Edman degradation experiments showed that this behavior was due to the loss of two phosphate residues in the multiple phosphorylation site (64S This was confirmed by sequencing a genomic DNA fragment encompassing exon 9 where the 8th codon (TCG) was shown to be mutated to TTG. Sequencing of amplified genomic DNA segments spanning the 5¢ and 3¢ flanking regions of each exon allowed us to identify 23 single nucleotide polymorphisms and two insertion/deletion events in the coding as well as the noncoding regions. A comparison of specific haplotypes defined for each of the a s1 -CasF, A and M alleles indicates that the M allele probably arises from interallelic recombination between alleles A and B 2 , followed by a C fi T transition at nucleotide 23 of the ninth exon. The region encompassing the recombination break point was putatively located between nucleotide 86 upstream and nucleotide 40 downstream of exon 8. Interallelic recombination therefore appears to be a possible means of generating allelic diversity at the a s1 -Cas locus, at least in the goat. The previously proposed molecular phylogeny must now be revised, possibly starting from two ancestral allelic lineages.

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment–mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, β-CNf(7–28)4P, αs1-CNf(61–79)4P and αs1-CNf(7–21)4P. In total, 45 phosphopeptides were identified: 24 from β-CN, 16 from αs1-CN and 5 from αs2-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed.