No abstract

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.

Five ovine a sl -casein variants (A-E) were identified in an Italian population sample using gel electrophoresis at alkaline pH, gel isoelectric focusing, two dimensional gel electrophoresis, and immunoblotting with polyclonal antibodies against a s] -casein. Each casein sample produced two peaks by fast reversed-phase HPLC. Gel isoelectric focusing and electrospray mass spectrometry were used to demonstrate that the first HPLC peak contained the 191 residue a sl -casein molecular species and the second the 199 residue species, in proportions of ~ 20:80. Only in the case of the sample containing a sl -casein CE was the method for the separation of the single short and long forms of each variant unsuccessful. Both two dimensional electrophoresis followed by staining with polyclonal antibodies against

The effects of sheep αs1-casein CC, CD and DD genotypes on milk composition and cheese yield were studied. Processed bulk milk was collected from three groups of 15 ewes, carrying αs1- casein CC, CD and DD genotypes. CC milk was higher in casein content than CD or DD milk (+3·5 and +8·6% respectively), and had a higher protein[ratio ]fat ratio and a smaller casein micelle diameter. In addition, DD milk had a significantly lower αs1-casein content. The main differences were in curd formation: CC milk had better renneting properties. Cheesemaking trials, carried out in a pilot plant, showed that CC milk had better cheesemaking characteristics than DD milk, while CD milk was intermediate. Both 1 d old and fully ripened cheeses had different fat[ratio ]dry matter ratios and αs1-I-casein electrophoretic mobilities: these were lower for DD cheese. As a consequence, these genotypes could be considered as markers of milk and/or cheese quality.

We report a fast and easy-to-use procedure that combines polyacrylamide gel electrophoresis with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF) and nanoelectrospray-tandem mass spectrometry (nES-MS/MS) analysis for the identification of casein components and defined phosphorylated sites. This methodology ensured identification of more than 30 phosphorylated proteins, five beta-, fifteen alpha(s1)-, ten alpha(s2)-, and four kappa-casein (CN) components, including nonallelic, differently phosphorylated, and glycosylated forms. The sugar motif covalently bound to kappa-CN was identified as chains, trisaccharide GalNAc, Gal, NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc. Also identified was a biantennary chain made up of both chains of trisaccharide 1GalNAc, 1Gal, 1NeuGc, and tetrasaccharide 1GalNAc, 1Gal, 2NeuGc moiety on a single kappa-CN component. The phosphate group on site Ser12 of tryptic peptide 8-22 of most phosphorylated alpha(s1)-CN (11 phosphate groups) was localized and the oligosaccharide sequence of the main tryptic glycopeptides of two kappa-CN components was determined by means of MS/MS analysis.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against /?-and a sl -casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4-6-insoluble N fraction of ParmigianoReggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N-and C-terminal ends, as y 3 -casein (/?-casein(fl08-209)) and a sl -PL1 (a sl -casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in /?-and a sl -casein hydrolysis during the ripening of this variety of hard pressed cheese.

Summary -The 0.2 mollL trisodium citrate soluble N fraction of 14-and 38-month-old Grana Padano (GP) chee se diafiltered through 3-and 10-kDa eut-off membranes gave two fractions containing peptides with a molecular mass lower than 3 kDa, and higher than 10 kOa. 91 and 38 oligopeptides lower than 3 kDa were identified by using a combination of fast-atom bombardmentlmass spectrometry and Edman degradation in the 14-and 38-month-old GP cheese samples respectively. Peptides higher th an 10 kDa were submitted to preparative fractionation on Mono Q column with a stepwise ionie strength gradient and then by reverse phase-HPLC on a C4 column. Sorne native caseins and large-derived peptides were identified by using a combination of electrospray mass spectrometry and Edman degradation.

S. The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI-MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography-ESI-MS allowed us to determine each fraction's composition by detecting thirteen α s" -, eleven α s# -, seven β-, and three κ-casein (CN) components. The α s" -CN and α s# -CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences ; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2n5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLCderived fractions. The separation of α s" -CN and α s# -CN proved to be excellent, with the exception of a co-migration of κ ! -CN with a minor α s" -CN component and of a glycosylated κ-CN form with low-phosphorylated α s" -CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI-MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative α s" -CN variants, the non-allelic α s" -CN and α s# -CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.K : Ovine casein, casein heterogeneity, electrospray ionization-mass spectrometry, capillary electrophoresis.Dairy technologists are frequently faced with variations in milk properties such as clotting time and curd consistency. Both factors are strongly linked to the casein