Fatty acid profile and coagulating ability of milk from Jersey and Friesian cows fed whole flaxseed.

The experiment was carried out to evaluate the effects of a moderate level of flaxseed administration on milk coagulation properties and fatty acid profile of milk from two different breeds. The experiment was performed on 20 Italian Friesian cows and 20 Jersey cows divided into 2 groups of 10 animals each. The experimental diets were (1) a traditional diet (CON) administrated as unifeed and no supplemental fat and (2) a diet supplemented with 0·5 kg/d of whole flaxseed (FS). Cows were milked twice daily and milk yield was recorded. Milk samples were analysed at 1, 15, and 30 d of the experiment for composition, pH, and milk coagulation properties. To verify the effects of flaxseed administration on the coagulation properties of milk from Friesian and Jersey cows, an electrophoresis study on casein fractions was performed. Milk fatty acid profile can be improved by administrating a moderate level of flaxseed in the diet, however, milk fatty acid profile from Friesian and Jersey cows showed different contents of C18 : 1 trans-11, SFA and MUFA. The results demonstrated that milk coagulating ability can be increased by flaxseed administration in both breeds as a result of different aggregation of casein micelles.

Mass spectrometric analysis of in vitro nuclear aggregates of polyamines.

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.

Effect of chestnut extract and chestnut fiber on viability of potential probiotic Lactobacillus strains under gastrointestinal tract conditions.

The main challenge to probiotics, during their passage through the gastrointestinal tract, are the acidic gastric secretions of the stomach, and the bile salts released into the duodenum. The survival of the strains, in this phase, is strongly influenced by the food used for their delivery. This work is part of a project studying the development of novel food processes, based on the use of chestnuts from cultivar "Castagna di Montella". In detail, the effect of indigestible chestnut fiber and of chestnut extract on the viability of selected lactic acid bacteria strains was evaluated. Among 28 cultures, twelve strains were selected, on the basis of tolerance to low pH values and bile salts, and submitted to exposition to simulated gastric or bile juice in presence of chestnut extract with or without immobilization in chestnut fiber. The presence of chestnut extract proved to play a significant role on the gastric tolerance improvement of lactobacilli. The recorded protective effect could not be simply related to the starch or reducing sugars content. RP-HPLC demonstrated that in the chestnut flour, there are one or more hydrophobic peptides or oligopeptides, which specifically offer a marked resistance to simulated gastric juice, albeit present at low concentration. These beneficial effects proved to be dependent by the cultivar used to produce the flour.

Effect of the Addition of Freeze-Dried Grape Pomace on Fresh Tagliatelle Gluten Network and Relationship to Sensory and Chemical Quality.

The incorporation of 5 and 10% freeze-dried grape pomace powder (GPP) in fresh tagliatelle pasta preparation was evaluated for its effect on chemical composition, gluten protein structure, and sensory properties. The addition of the freeze-dried GPP led to a significant increase (p < 0.05) in polyphenol content in the raw and cooked fortified pasta samples with respect to 100% semolina pasta, although the phenolic content decreased after the cooking process. The opposite phenomenon was observed with the antioxidant activity, which increased significantly (p < 0.05) when switching from raw to cooked pasta samples fortified with GPP. The formation of a proper gluten structure was found in the fortified raw pasta, even if a change in the protein arrangement was shown in the fortified cooked samples, confirmed by a significant reduction (p < 0.05) in both the unextractable polymeric protein percentage (% UPP) and disulfide bond (S-S) formation. These results suggest a possible interaction between the protein sulfhydryl groups (-Cys) and polyphenols of grape pomace during cooking through non-disulfide covalent bonds, which was confirmed by the significant (p < 0.05) decrease in the -SH groups when comparing 100% semolina pasta with fortified pasta sample. Finally, a sensory analysis showed that the highest significant score (p < 0.05) was achieved by the 5% GP-fresh pasta sample.

A proteomic study of "Coppa Piacentina": A typical Italian dry-cured Salami.

Coppa Piacentina is considered a peculiar dry cured salami, since it is manufactured by the entire neck muscles stuffed and matured in natural casings, the same as dry cured ham and fermented dry cured sausages. In this work the proteolysis of external and internal portions was investigated by a proteomic approach and by amino acids analysis. Samples of "Coppa Piacentina" were analyzed at 0 days and after 5 and 8 months of ripening through mono- and two-dimensional gel electrophoresis. Image analysis of 2D electrophoretic maps indicated a more intense enzyme activity on the external part, mainly due to endogenous enzymes. They favored, respectively, myofibrillar or sarcoplasmic proteins at 5 or 8 months of ripening. Free amino acids determination proved that lysine and glutamic acid were the most represented ones, followed by a free amino acids sequence like that of dry cured ham. The peculiarities of "Coppa Piacentina" were characterized by a slow proteolysis, due to sacking and binding of the whole cut of the pork neck.

DNA is wrapped by the nuclear aggregates of polyamines: the imaging evidence.

In the cell nucleus, putrescine, spermidine, and spermine self-assemble with phosphate ions to generate three forms of compounds, named nuclear aggregates of polyamines (NAPs), which may interact with DNA. In an in vitro setting mimicking the cell nucleus milieu, this molecular aggregation occurs within well-defined ratios. Structural and functional analogies exist between the in vitro NAPs (ivNAPs) and their extractive homologues. The present Article reports images of ivNAPs at different resolution levels. Independent of the DNA template, ivNAPs become hierarchically stacked to produce ultimately macroscopic filamentous structures. The ivNAP-DNA complexes arranged in long and repetitive structures that displayed the self-similar features of natural fractals when dehydrated onto glass slides. Atomic force microscopy showed that ivNAPs have a cyclic structure and dispose around the DNA in a tube-like arrangement. Overall, the images indicate that these aggregates envelope the genomic DNA, thus proving that NAPs play a crucial role in DNA compaction and functioning.

Protein aggregation in cooked pork products: New details on the supramolecular organization.

The molecular weight distribution of protein aggregates from raw meat and cooked pork products was assessed by size exclusion-high performance liquid chromatography (SE-HPLC). Electrophoretic analysis under reducing conditions showed that the high molecular weight SE-HPLC peak (peak 1) of the cooked products contained protein aggregates in addition to high molecular weight muscle proteins, while the second peak (peak 2) still contained aggregates and <50 kDa proteins. The protein aggregates composition was investigated by HPLC-tandem mass spectrometry. Different classes of proteins were identified and the cooked products showed a more complex composition and organization, according to the muscle structure and the technological procedures, respectively. The key role of actin in the building of the protein networks was also confirmed. The different multi-protein systems found in the cooked products suggest protein re-organization in heat-induced supramolecular structures, which might be responsible for the texture and the structural properties of the final products.

Effects of microwave treatment of durum wheat kernels on quality characteristics of flour and pasta.

The influence of microwave treatment of hydrated durum wheat kernels of two different cultivars (cv Aureo and Sfinge), on wholemeal flour and pasta quality was addressed. Size exclusion-HPLC and electrophoresis analysis were used to investigate changes in the gluten network arrangement as affected by the microwave treatment. Rheological properties of dough, cooking quality and sensory properties of pasta were also assessed. Results suggested that the microwave treatment on hydrated durum wheat kernels blocks gluten protein conformation through SS bonds formation and the free -SH are no longer able to create a strong network during pasta processing, due to the conformational changes. Rheological study of dough confirmed that the modifications induced by microwave treatment greatly affected pasta making characteristics of wheat flour, with significant negative consequences on product quality, especially for pasta cooking quality. Pasta from treated durum wheat showed low sensory quality, mainly due to high bulkiness and adhesiveness.

Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease.

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.

Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis.

In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate comparison of 2D maps to define the events occurring during their ripening and individuate candidate molecular markers of the characteristic proteolytic processes. Considering that, essentially, muscle endogenous enzymic activity, calpains and cathepsins, occur in the ripening process of dry-cured ham, whereas a combined action between endogenous and microbial enzymes takes place in the case of sausage ripening, these results provide deeper insight into the respective role of endogenous and microbial enzymes in performing proteolysis. Finally, image analysis and creation of data bank could be achieved to quickly identify and protect typical products.

Nuclear aggregates of polyamines are supramolecular structures that play a crucial role in genomic DNA protection and conformation.

In a previous study we showed that natural polyamines interact in the nuclear environment with phosphate groups to form molecular aggregates [nuclear aggregates of polyamines (NAPs)] with estimated molecular mass values of 8000, 4800 and 1000 Da. NAPs were found to interact with genomic DNA, influence its conformation and interfere with the action of nucleases. In the present work, we demonstrated that NAPs protect naked genomic DNA from DNase I, whereas natural polyamines (spermine, spermidine and putrescine) fail to do so. In the context of DNA protection, NAPs induced noticeable changes in DNA conformation, which were revealed by temperature-dependent modifications of DNA electrophoretic properties. In addition, we presented, for NAPs, a structural model of polyamine aggregation into macropolycyclic compounds. We believe that NAPs are the sole biological forms by which polyamines efficiently protect genomic DNA against DNase I, while maintaining its dynamic structure.

The alpha chains of goat hemoglobins: old and new variants in native Apulian breeds.

Blood samples were collected from 324 goats belonging to the native Apulian breeds Garganica and Jonica; 60 Alpine goats were also sampled to serve as a comparison. Hemoglobin phenotypes were analyzed with isoelectric focusing in a pH range of 6.7-7.7. Heterogeneity of globin chains was evidenced both by AUT-PAGE and RP-HPLC. The primary structure of four alpha globins was analyzed by combined mass spectrometry approaches. Two of these globins had never been sequenced before. One was a new alpha variant, an allele of the HBA1A gene from which it differed for the mutation A26T and has been registered with a low frequency only in Apulian breeds; the other was a globin encoded by the HBA2 locus, whose primary structure was previously derived from the corresponding gene. The two alleles recorded at the HBA2 locus presented a different frequency in the three breeds but may be considered to be generally rather common. Notwithstanding the sample size no goat was found to exhibit HbA1B. The authors discuss their findings in the light of the results reported by other researchers and argue that, in spite of what had been inferred in pioneer works on goat hemoglobins, HBA1B is not a common allele.

A proteomic approach to study protein variation in GM durum wheat in relation to technological properties of semolina.

Genetic manipulation of durum wheats by tobacco rab-1 genes influence the trafficking of gluten proteins through the secretory system by up- or down-regulating the transport step from the ER to the Golgi apparatus which may in turn modify functional performance of the grain. Gluten proteins were extracted from two genetically manipulated lines - Svevo B730 1-1 and Ofanto B688 1-2 - and their control lines and were analyzed by two dimensional gel electrophoresis. When the two-dimensional maps were compared by image analysis no significant differences between the GM line with an up-regulated trafficking containing the wild type tobacco rab1 (Svevo B730 1-1) and its control (Svevo control). By contrast, significant differences were found between the GM line with a down-regulated trafficking due to the tobacco rab1 mutant form (Ofanto B688 1-2) and its control (Ofanto control). Of the new protein spots detected in the down-regulated Ofanto B688 1-2 map, only a beta-amylase was identified. The remaining spots were susceptible to chymotripsin action but not to trypsin one, as in the case of the gluten protein. Rheological measurements showed that gluten quality was enhanced in the down-regulated Ofanto B688 1-2 without an increase in the amount of gluten. Proteomics is a useful and powerful tool for investigating protein changes in GMOs and in understanding events in food science and technology.

Protein modifications in cooked pork products investigated by a proteomic approach.

To evaluate process-induced protein modifications in cooked ham and emulsion sausages, the 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.

Cereal-based gluten-free food: how to reconcile nutritional and technological properties of wheat proteins with safety for celiac disease patients.

The gluten-free diet is, to date, the only efficacious treatment for patients with Celiac Disease. In recent years, the impressive rise of Celiac Disease incidence, dramatically prompted changes in the dietary habit of an increasingly large population, with a rise in demand of gluten-free products. The formulation of gluten-free bakery products presents a formidable challenge to cereal technologists. As wheat gluten contributes to the formation of a strong protein network, that confers visco-elasticity to the dough and allows the wheat flour to be processed into a wide range of products, the preparation of cereal-based gluten-free products is a somehow difficult process. This review focuses on nutritional and technological quality of products made with gluten-free cereals available on the market. The possibility of using flour from naturally low toxic ancient wheat species or detoxified wheat for the diet of celiacs is also discussed.

Transport across Caco-2 monolayers of peptides arising from in vitro digestion of bovine milk proteins.

The entire panel of peptides produced from caseins (CN) and whey proteins (WP) that survive in vitro sequential gastro-pancreatic digestion and translocate across monolayers of Caco-2 cells, used as a model of the intestinal epithelium, has been characterised by HPLC and mass spectrometry. Among the milk-derived bioactive peptides, only minor amounts of mono-phosphorylated peptides arising from αs1- and ß-CN were detected. The absorption behaviour of two resistant ß-lactoglobulin (ß-Lg) domains, ß-Lg 125-135 and ß-Lg 40-60, was studied in detail using synthetic peptides. The IgE-binding properties of the digests recovered from the apical and basolateral monolayer compartments were evaluated by dot-blot, using the sera of milk allergic children (N=5). Outcomes indicated ß-Lg 127-135 as a possible "immune sensitising factor"in vivo. The almost complete loss of the IgE-affinity of CN and WP after digestion points out the need to design in vivo experiments to track the metabolic fate of dietary proteins.

Protein analysis-on-chip systems in foodomics.

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.

Biochemical traits of Ciauscolo, a spreadable typical Italian dry-cured sausage.

Ciauscolo is a short-ripened fermented sausage manufactured in the Marche region (central Italy) that has recently received a protected geographical indication product classification (PGI). The aim of this study was the exploration of the biochemical traits of this traditional Italian salami, with a special focus on protein and lipid composition. Ciauscolo salami was characterized by pH of 5.1 and 0.91 water activity. A prevalence of lactic acid bacteria in the microbiota was found. The free amino acids and biogenic amines average content was 2657 and 255 mg/kg, respectively. With regards to lipids composition unsaturated fatty acids represented 63% and 72% of total and free fatty acids. Despite these results had wide statistical variability, attributable to differences in the processing parameters and raw matter used, some peculiar traits were found: (1) structural muscular proteins underwent to less proteolysis than sarcoplasmic ones; (2) glycogen phosphorylase, enolase, and aldolase were the most proteolyzed among the sacoplasmic proteins; (3) there was inverse correlation between histamine content and yeasts population, and a direct correlation between the gly-pro content and lactic acid bacteria counts; (4) the content of aspartic acid and methyonine seem to be a possible molecular marker able to distinguish between double and single milling.

Peptides surviving the simulated gastrointestinal digestion of milk proteins: biological and toxicological implications.

Resistance to proteases throughout the gastrointestinal (GI) tract is a prerequisite for milk-derived peptides to exert biological activities. In this work an in vitro multi-step static model to simulate complete digestion of the bovine milk proteins has been developed. The experimental set-up involved the sequential use of: (i) pepsin, (ii) pancreatic proteases, and (iii) extracts of human intestinal brush border membranes, in simulated gastric, duodenal and jejuneal environments, respectively. Enzymatic concentrations and reaction times were selected in order to closely reproduce the in vivo conditions. The aim was to identify the peptide candidates able to exhibit significant bioactive effects. Casein and whey protein peptides which survived the in vitro GI digestion have been identified by the combined application of HPLC and mass spectrometry techniques. While the permanence of the main potentially bioactive peptides from both casein and whey proteins was found of limited physiological relevance, the high resistance to proteolysis of specific regions of beta-lactoglobulin (beta-Lg), and especially that of the peptide beta-Lg f125-135, could have implications for the immunogenic action of beta-Lg in the insurgence of cow's milk allergy.

Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain.

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS-PAGE electrophoresis, (32)P labelling and immunodetection show that "in vitro" PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. (32)P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.