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Biomedical subjects

F Addeo

Publications and source records attributed to F Addeo.

At least 19 recordsLinked to original sources
PubMed2001

Mass spectrometric characterisation of proteins in rennet and in chymosin-based milk-clotting preparations.

The protein composition of natural rennet and of chromatographic and crystalline chymosin preparations has been defined by on-line reverse-phase high performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS) and by tandem mass spectrometry (MS/MS). Natural rennet was found to consist of six chymosin species, corresponding to chymosin A and B genetic variants, each of which comprised a mixture of two other forms differing at theN-terminal end, with one being three residues longer, and the other two residues shorter, than the mature chymosin. Two main tissue proteins were also identified as lysozyme (isozyme 2 plus a novel isozyme labelled 4) and bovine serum albumin. In addition to the proteins, chymosin fragments 247-323 and 288-323 were consistently present in natural rennet. Conversely, chromatographic and crystalline chymosin preparations lacked bovine serum albumin and/or lysozyme, although they contained the same six chymosin species as natural rennet. Since these tissue-specific contaminating proteins each possess specific functions in terms of stabilising enzyme solutions and protecting proteins from proteolytic enzymes, oxidising agents and bacterial proliferation, the rennet may be considered as a functional enzyme preparation that is effectively and naturally adapted to the purposes of cheesemaking. In practice, the highly complex protein composition inherent to natural rennet provided the possibility to differentiate the natural product from other bovine chymosin-based milk-clotting preparations examined in this work.

Amino Acid Sequence↗
PubMed2001

Mass spectrometry-based procedure for the identification of ovine casein heterogeneity.

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 HTPC 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 alphas1-, eleven alphas2-, seven beta-, and three kappa-casein (CN) components. The alphas1-CN and alphas2-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 kappa-CN and beta-CN. By CE at pH 2.5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLC-derived fractions. The separation of alphas1-CN and alphas2-CN proved to be excellent, with the exception of a co-migration of kappa0-CN with a minor alphas1-CN component and of a glycosylated kappa-CN for with low-phosphorylated = alphas1-CN and beta-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 alphas1-CN variants, the non-allelic alphas1-CN and alphas2-CN forms, dimeric kappa-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

Animals↗
PubMed2001

Characterization of lactic acid bacteria strains on the basis of neutral volatile compounds produced in whey.

AIMS: Seventy-eight strains of lactic acid bacteria belonging to five genera and showing six different phenotype combinations of Lac (lactose fermentation), Prt (proteolytic activity) and Cit (citrate degradation) characters were investigated for their main flavouring properties with the aim to detect variability among and within the groups. METHODS AND RESULTS: High resolution gas chromatography-mass spectrometry analysis of neutral volatile compounds produced in whey showed that, considering both neo-formation compounds and substances quantified in the whey cultures at different concentrations in comparison to the extract from sterile whey, the groups of lactococci, enterococci, thermophilic streptococci and mesophilic lactobacilli produced a higher number of volatiles than thermophilic lactobacilli and leuconostocs. Applying principal component analysis (PCA) to the results, enterococci, mesophilic lactobacilli and thermophilic streptococci showed a broad diversity, while lactococci included rather similar strains as well as strains with special flavouring properties. Applying PCA to thermophilic streptococci and enterococci, to lactococci and enterococci, to lactococci and thermophilic streptococci, or to mesophilic and thermophilic lactobacilli, the strains gathered consistently with their systematic position. CONCLUSION: The study evidenced strains producing some volatile compounds responsible for food flavouring. Flavouring properties were variable among the systematic groups and in some cases different within the same bacterial group. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the development of flavouring adjuncts for the dairy industry.

Cluster Analysis↗
PubMed2000

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry.

Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive fingerprinting of gliadins and glutenins in wheat flour. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic profiles were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. gamma(2)- and gamma(3)-gliadin, were identified through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients.

Amino Acid Sequence↗
PubMed2000

Odour-impact compounds of Gorgonzola cheese.

Volatile concentrates were obtained by vacuum distillation from both natural and creamy Gorgonzola cheese and isolated by continuous liquid-liquid extraction. Both were analysed by high resolution gas chromatography (HRGC), HRGC-mass spectrometry and HRGC-olfactometry. A total of 63 components were identified in the neutral extract of the natural type (21 esters, 13 ketones, 14 alcohols, 5 aldehydes, 1 sulphur compound, 7 aromatic compounds and 2 terpenes) and 52 in the creamy type (17 esters, 12 ketones, 10 alcohols, 5 aldehydes, 1 sulphur compound, 5 aromatic compounds and 2 terpenes). Ketones, whose major components were 2-nonanone and 2-heptanone, were the predominant constituents of the neutral fraction. By olfactometric analysis of the neutral extracts, 23 odour-impact compounds were found in the natural and 21 in the creamy Gorgonzola cheese. 1-Octen-3-ol, ethyl hexanoate, 2-nonanone, 2-heptanone, 2-heptanol, ethyl butanoate, 2-nonanol and 4-methylanisole were the key odorants of the natural cheese, whereas 2-heptanone, 2-heptanol, ethyl butanoate, 3-methyl thiopropanal and an unidentified constituent with a fruity odour were characteristic of the creamy Gorgonzola cheese. On the basis of high odour unity values, 2-nonanone, 1-octen-3-ol, 2-heptanol, ethyl hexanoate, methylanisole and 2-heptanone were the most important odorants of natural and creamy Gorgonzola cheese aroma.

Animals↗
PubMed2000

Immunochemical evaluation of bovine beta-casein and its 1-28 phosphopeptide in cheese during ripening.

Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.

Amino Acid Sequence↗
PubMed2000

Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4.

Two fermented milks containing angiotensin-I-converting-enzyme (ACE)-inhibitory peptides were produced by using selected Lactobacillus delbrueckii subsp. bulgaricus SS1 and L. lactis subsp. cremoris FT4. The pH 4.6-soluble nitrogen fraction of the two fermented milks was fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest ACE-inhibitory indexes were further purified, and the related peptides were sequenced by tandem fast atom bombardment-mass spectrometry. The most inhibitory fractions of the milk fermented by L. delbrueckii subsp. bulgaricus SS1 contained the sequences of beta-casein (beta-CN) fragment 6-14 (f6-14), f7-14, f73-82, f74-82, and f75-82. Those from the milk fermented by L. lactis subsp. cremoris FT4 contained the sequences of beta-CN f7-14, f47-52, and f169-175 and kappa-CN f155-160 and f152-160. Most of these sequences had features in common with other ACE-inhibitory peptides reported in the literature. In particular, the beta-CN f47-52 sequence had high homology with that of angiotensin-II. Some of these peptides were chemically synthesized. The 50% inhibitory concentrations (IC(50)s) of the crude purified fractions containing the peptide mixture were very low (8.0 to 11.2 mg/liter). When the synthesized peptides were used individually, the ACE-inhibitory activity was confirmed but the IC(50)s increased considerably. A strengthened inhibitory effect of the peptide mixtures with respect to the activity of individual peptides was presumed. Once generated, the inhibitory peptides were resistant to further proteolysis either during dairy processing or by trypsin and chymotrypsin.

Angiotensin I↗
PubMed2000

Pyroglutamic acid in cheese: presence, origin, and correlation with ripening time of Grana Padano cheese.

Pyroglutamic acid is present in many cheese varieties and particularly in high amounts (0.5 g/100 g of cheese) in extensively ripened Italian cheeses (Grana Padano and Parmigiano Reggiano) that are produced with thermophilic lactic acid bacteria as starters. The mechanism of pyroglutamic acid formation in cheese seems to be mostly enzymatic, as demonstrated by the presence of only L-pyroglutamic acid enantiomer. Thermophilic lactobacilli are involved in pyroglutamic acid production, as suggested by the low pyroglutamic acid content found in Bagos, a ripened Italian mountain cheese produced without addition of starter. Because milk pasteurization did not influence the pyroglutamic acid content in the ripened Grana Padano cheese, the formation of pyroglutamic acid mainly depends on the whey starter microflora rather than that of raw milk. Pyroglutamic acid concentration is linearly correlated (R2 = 0.94) with the age of Grana Padano cheese.

Animals↗
PubMed1999

Alternative nonallelic deletion is constitutive of ruminant alpha(s1)-casein.

Multiple forms of alpha(s1)-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While alpha(s1)-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141-148, 110-117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in alpha(s1)-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.

Alleles↗
PubMed1999

Presence of peptidase activities in different varieties of cheese.

Lactic acid bacteria (LAB) counts, PepX activity towards H-Phe-Pro-beta NA, and aminopeptidase activity towards H-Arg-beta NA. HCl, H-Lys-beta NA, H-Leu-beta NA, H-Pro-beta NA, H-Glu-beta NA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo-peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo-peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.

Cheese↗
PubMed1998

Copresence of Deleted Protein Species Generates Structural Heterogeneity of Ovine alpha(s1)-Casein.

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

Journal Article↗
PubMed1998

Antipeptide Antibodies as Analytical Tools To Discriminate among Bovine alpha(s1)-Casein Components.

Polyclonal antibodies raised against synthetic peptides reproducing sequence stretches of bovine alpha(s1)-casein were used as probes to discriminate within the alpha(s1)-casein fraction of bovine milk and cheese. A minor alpha(s1)-casein component, selectively recognized by an antisera directed against the bovine 139-149 alpha(s1)-casein sequence, was found to be a C-terminally truncated alpha(s1)-casein form. This component coeluted with the main alpha(s1)- and alpha(s2)-casein by anion-exchange chromatography of whole casein, whereas by RP-HPLC it eluted with alpha(s2)-casein only. Similarly to the main alpha(s1)-casein, the C-terminally truncated form was hydrolyzed in vitro by chymosin and early in the cheese-making.

Journal Article↗
PubMed1998

Identification of C-terminally truncated forms of beta-lactoglobulin in whey from Romagnola cows' milk by two dimensional electrophoresis coupled to mass spectrometry.

Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass profile allowed us to establish a correlation between beta-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated beta-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100-123 and 136-147 respectively. Two of the minor components were related to beta-lactoglobulin A and two to beta-lactoglobulin B.

Amino Acid Sequence↗
PubMed1998

The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.

Amino Acid Sequence↗
PubMed1997

Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein.

The identity of multiple forms of caprine alpha(s1)-casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat alpha(s1)-casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199-residues form already described. The other three, in lesser amounts, were shorter forms of alpha(s1)-casein and differed for the deleted peptides 141-148, as shown previously for ovine alpha(s1)-casein, peptide 110-117, or Gln78. Analysis of alpha(s1)-casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 110-117) and 16 (codifying for peptide 141-148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of alpha(s1)-casein in goat. A further source of heterogeneity of caprine alpha(s1)-casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77-->Gln which prevents phosphorylation of Ser75). Conversely, Ser115 and Ser41 are phosphorylated only to about 50% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine alpha(s1)-casein variants. These results confirm that there are stabilised mechanisms of simultaneous synthesis of alpha(s1)-casein at different length and of post-translational modification in both caprine and ovine species.

Alternative Splicing↗
PubMed1997

Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening.

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, beta-CNf(7-28)4P, alpha s1-CNf(61-79)4P and alpha s2-CNf(7-21)4P. In total, 45 phosphopeptides were identified: 24 from beta-CN, 16 from alpha s1-CN and 5 from alpha s2-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.

Amino Acid Sequence↗
PubMed1996

Occurrence of five alpha s1-casein variants in ovine milk.

Five ovine alpha s1-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 alpha s1-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 alpha s1-casein molecular species and the second the 199 residue species, in proportions of approximately 20:80. Only in the case of the sample containing alpha s1-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 alpha s1-casein and electrospray mass spectrometry showed a heterogeneity consistent with that expected from a protein chain with three levels of phosphorylation and two different lengths. However, especially for alpha s1-caseins D and E, a further uncharacterized heterogeneity was detected.

Animals↗