The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative -lactoglobulin (-LG) polymorphism may be found in bovine milk; 2) quantitative polymorphisms are of- ten the result of expression gradients in multiple cop- ies of a gene; 3) the -LG gene is duplicated in the dog and bovine genome; 4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine -LG polymorphism checking phe- notypic evidence for copy-number variants of -LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breed- ing Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited beta-LG polymorphism and 4 different quan- titative patterns, which were characterized in detail by a proteomic approach relying on combined chro- matographic and mass spectrometric techniques. The expected figures based on the expression gradient mod- els were compared with well-established α-globin gene arrangements in sheep. The different phenotypes sug- gest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study sup- ports the helpfulness of up-to-date proteomics for in- ferring copy number polymorphisms through the char- acterization of the phenotypic expression.

The assessment of the origin and quality of raw materials is pivotal for the protection and valorization of typical dairy products. Italian high moisture mozzarella cheese, also called “Fiordilatte”, is manufactured by stretching in hot water the curd, which can be produced inside the dairy (fresh curd) or purchased as semi-finished product from specialized companies (pre-made stored curd). The employment of stored curd allows cheese manufacturers to obtain economic profit thanks to the reduction of the production costs. The use of the semi-finished product has not to be mentioned in the label according to the actual EU regulation, and this has given rise to competition between local milk farmers and curd-producing companies causing misinformation among the onsumers, and unfair competition to the disadvantage of the traditional dairies. In the present esearch, a proteomic approach was used to investigate “Fiordilatte” produced with or without the employment of stored curd. The results obtained allowed us to identify a molecular marker which is present in very small amounts in cheese made from fresh curd and at high levels in cheese made from stored curd. The marker is as1-I casein (fragment 24e199), the main product of primary proteolysis formed by the action of chymosin on as1 casein. It can be easily detected by electrophoresis in the presence of urea (urea-PAGE) and is measured by image analysis. Even though the kinetic of formation of this proteolytic product has been well known for several decades, in mozzarella it has a peculiar behavior compared to other cheeses. As a matter of fact, it originates during the first part of cheesemaking, and has a very slow increase during storage of the cheese, due to the denaturation of the clotting enzyme by the hot water stretching process. The results obtained demonstrated that the quantification of as1-I fragment should be recommended to guarantee the production of “Fiordilatte” obtained from fresh milk under normal conditions, and could be the basis for the development of a method able to fully protect the traditional cheesemaking procedure.

The effect of a multiple strain starter (KSL: Kocuria varians, Staphylococcus xylosus and Lactobacillus sakei) on the quality of soppressata molisana was investigated during 50 days of ripening. When compared with control batches (C, traditional production without starter and KS, addition of K. varians and S. xylosus as starter), soppressata produced with KSL showed positive results in terms of proteolysis progression, reduction of biogenic amines (BA) and sensory features. The partial substitution of 50 % NaCl with 50 % KCl further improved the quality of soppressata molisana produced with KSL as starter. In fact, the K+ ions seemed to stimulate the growth of added bacteria, with a resulting additional lowering of the pH and BA formation, and higher proteolysis. Moreover, the addition of KCl did not alter the sensory profile of final products. The analysis of data collected during the fermentation and the drying phases, which followed one other, allowed the individuation of different proteolytic events. Specifically, the proteolysis of the myofibrillar fraction occurred more slowly than that of the sarcoplasmic one. Also, the analysis of fractionated muscle proteins allowed to appreciate that sarcoplasmic proteins were hydrolyzed by both endogenous and microbial enzymes, whereas myofibrillar proteins showed a higher resistance to microbial enzymes, and were primarily hydrolyzed by endogenous ones. In conclusion the combined use of KSL and KCl as partial substitute of NaCl, allowed the production of soppressata molisana having an excellent quality, and features consistent with the guidelines of WHO on the sodium and potassium intake.

RATIONALE: In the nuclei of eukaryotic cells, polyamines and phosphate ions self-assemble via ionic interactions and hydrogen bonding, generating three families of supramolecular compounds that have been named large (l-), medium (m-) and small (s-) nuclear aggregates of polyamines (NAPs). In a simulated nuclear environment, polyamines and phosphate ions generate the in vitro NAPs (ivNAPs) that share strict structural and functional analogies with their cellular cognates. Mass spectrometric data are expected to provide important structural details of NAPs/ivNAPs. METHODS: We used both electrospray ionization (ESI) and nitrocellulose (NC) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to support a variety of analytical techniques previously addressed to structurally characterize NAPs/ivNAPs. RESULTS: The dominant m/z values of s-ivNAP (m/z 735, 749, 761) are compatible with a defined set of cyclic or linear aggregates. On the basis of the experimental molecular mass (a cluster centred at m/z 2980), the m-ivNAP corresponds to the supramolecular assembly of four modules of s-ivNAPs. No informative mass spectra were obtained for the l-ivNAP. CONCLUSIONS: MS data support the models of NAPs that have been inferred by using an array of analytical techniques. NC MALDI-MS contributed much more effectively than ESI-MS to the structural characterization of ivNAPs.

Microwave based treatment (MWT) of wet wheat kernels induced a striking reduction of gluten, up to <20 ppm as determined by R5-antibodybased ELISA, so that wheat could be labeled as gluten-free. In contrast, analysis of gluten peptides by G12 antibody-based ELISA, mass spectrometry-based proteomics and in vitro assay with T cells of celiac subjects, indicated no difference of antigenicity before and after MWT. SDS-PAGE analysis and Raman spectroscopy demonstrated that MWT simply induced conformational modifications, reducing alcohol solubility of gliadins and altering the access of R5-antibody to the gluten epitopes. Thus, MWT neither destroys gluten nor modifies chemically the toxic epitopes, contradicting the preliminary claims that MWT of wheat kernels detoxifies gluten. This study provides evidence that R5-antibody ELISA alone is not effective to determine gluten in thermally treated wheat products. Gluten epitopes in processed wheat should be monitored using strategies based on combined immunoassays with T cells from celiacs, G12-antibody ELISA after proteolysis and proper molecular characterization.

The influence of partial replacement of NaCl with KCl on the lipolysis and on the microbial and sensory features of Soppressata molisana is investigated during 50 days of ripening. In addition, the relation between KCl and a multiple strain starter culture is also considered and the effects of their combination on some quality features are examined. The lipolysis, the microbiological and sensorial features and the lipid oxidation of experimental batches, are compared with those exhibited by control batches. The results highlight that the KCl, used as an alternative salt, is able to inhibit undesirable microorganisms equally to NaCl, ensuring the safety of the final products. In addition, a stimulating effect on the growth of added bacteria is detected in presence of KCl, with a resulting better fermentative process. In fact, the addition of KCl, even if produces a decrease in lipolytic activities, improves the nutritional value of soppressata molisana, in terms of higher PUFA/SFA ratio. These advantages are further amplified when the alternative salt is used in combination with the starter culture. Finally, the combined use of KCl and starter culture reduces the lipid oxidation and seems crucial for maintaining the sensorial features. Pratical Applications: The main findings of the present research highlight that the use of KCl in combination with an appropriately selected starter culture allows the production of healthier and safety dry-fermented sausage (soppressata molisana) and consistent with WHO guidelines and modern consumers demand.

Protein compositional data can address nutritional, packaging, origin/authenticity, processing history, safety and other quality questions. Such data has been time-consuming and expensive to generate until recently but "protein analysis on a chip" systems are now available that can analyze a complex food sample in a few minutes and do not require great protein analytical expertise. We review some of the main new approaches with examples of their application and discuss their advantages and disadvantages.

To evaluate process-induced protein modifications in cooked ham and emulsion sausages, proteomes of whole-cut (Parma and "Praga" cooked hams) and comminuted pork (mortadella and würstel) products were compared to raw pork using two-dimensional gel electrophoresis (2-DE) coupled to image analysis and mass spectrometry (MS). Other than heat-induced breakdown of part of the myosin heavy chains, the 2-DE pattern of cooked ham was substantially similar to that of raw pork. However, the MS-based analysis showed minor modifications, including the extensive oxidation of methionines. In contrast, likely due to emulsification, comminuted sausages were characterized by an abundant insoluble protein fraction(IPF). Interestingly, tropomyosin and myosin light chains in comminuted sausages were exclusively found in the IPF. Our results indicate that the protein aggregation systems of cooked hams and emulsion sausages reflect the processing conditions and are definitely different, the former being characterized mainly by disulphide bridges and the latter by additional covalent inter-protein links. Our results indicate that the protein aggregation systems of cooked hams and emulsion sausages reflect the processing conditions and are definitely different, the former being characterized mainly by disulphide bridges and the latter by additional covalent inter-protein links. Thus, specific signature protein patterns have the potential to be monitored for the a posteriori evaluation of the quality of meat products as well as for authentication purposes.

A study was undertaken on Cacioricotta, a traditional Italian goat’s cheese obtained from overheated milk (90 °C) without use of starter. The profile of proteolysis in the artisanal type, made with vegetable coagulant (latex released from caprifig branches) as milk clotting agent, was compared to that of the “industrial” one, manufactured with calf rennet. Particular aim of the investigation was to study the diVerences and, possibly, establish a useful tool for distinguishing the two types of cheese. The study was based on the quantiWcation of the water soluble, 15% TCA soluble and amino acid nitrogen fractions, RP-HPLC separation of low molecular weight peptides and their identification by mass spectrometry (MALDI-ToF MS). The use of Wg latex was associated to higher amounts of the nitrogen fractions and to RP-HPLC chromatograms very rich in peptides, in contrast to an almost complete lack of peptides in the industrial counterpart. These results confirm the strong proteolyitic activity exerted by the caprifig clotting enzymes in spite of the intense overheating of the milk, which is considered to cause reduction of the rate of casein degradation in cheese. The MS-based identiWcation of several peptides provided a support at the molecular level for the characterization of Cacioricotta made with this vegetable coagulant and could be useful for “tracing back” purposes. In conclusion, the peptide pattern determined by the use of capriWg for milk coagulation can be considered a particular feature of the artisanal Cacioricotta, giving conWrmation of its vocation to EU protection as typical product.

The guarantee of the origin and quality of raw material is essential for the protection and valorization of Campana buffalo mozzarella cheese. The risk of utilization of semifinished products and stored milk in substitution for fresh milk is increasing, due to the continuous desire to reduce production costs. A proteomics approach and electrophoresis survey of retail mozzarella cheeses indicated different rates of proteolysis in the production of dairy industries. The use of fresh milk and correct cheesemaking protocol yielded only γ-caseins, which are derived from β-casein by plasmin, and para-κ-casein, which is derived from κ-casein by chymosin. The detection of abnormal hydrolysis resulting in β- and αS1-casein fragments, identified by mass spectrometry, indicates the use of stored milk or stored and pressed curd, or the reuse of unsold mozzarella cheese, to produce mozzarella. The formation of γ-caseins and other fragments during a long storage of raw materials at room or refrigeration temperature was ascribed to plasmin (endogenous milk enzyme), whereas formation of αS1-casein fragments, mainly αS1-I(6P)- and αS1-I(7P)-casein during the storage of curd was ascribed to the action of chymosin (exogenous enzyme) from rennet. Sodium dodecyl sulfate-PAGE and alkaline urea-PAGE permitted us to evaluate the freshness of the raw materials used in the manufacturing of buffalo mozzarella cheese and to reveal possible inappropriate preservation.

L'invenzione propone un metodo per la detossificazione delle proteine del glutine dalle granaglie dei cereali, in particolare dalla granella di frumento, volto ad ottenere farine detossificate atte alla preparazione di prodotti della panificazione e pasta in particolare dal frumento, preferibilmente idonei all’alimentazione dei pazienti affetti da celiachia, ma anche adeguati, per le caratteristiche organolettiche ed aspetto, all’alimentazione di tutta la popolazione