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Background: The complexity of food structure is such as to hinder its inclusion in mathematical models predicting food properties and transformations, although a considerable impulse is being determined by using artificial intelligence. As a matter of fact, food definition currently neglects the structural description, even in those fields for which structure is demonstrated to have a decisive role, such as nutrition. Scope and approach: This review aims to analyse the current knowledge about the structure of foods and its potential use to numerically define the sensory and nutritional quality, as well as the stability properties. Starting from this information, a possible methodology is explored to build, even in an automated way, mathematical models for simulating and predicting the properties of food. A model pipeline has been proposed and applied to pasta, in particular exploiting the description of the structural changes occurring upon cooking. Key findings and conclusions: Foods may be designed in silico, based on automated pipelines for direct extraction of information on rheological and sensory properties as derived from structure images and from data on the dynamic state of the water. The ultimate goal of these approaches is to make more limited use of expensive and time-consuming experiments on physically prepared foods to get to use digital twins of foods designed in the laboratory.

The increasing interest of consumers for food with enhanced nutritional characteristics has spurred industry to look for unconventional raw materials for novel food design. Pasta is a staple food consumed all over the world, and therefore represents an ideal matrix for the enrichment with functional ingredients. Despite durum wheat semolina is considered the best raw material for Italian pasta making, due to its physicochemical, rheological and sensory properties, nutritionally enriched pasta is becoming quite popular among specific segments of consumers. Nowadays there is a wide choice of ingredients available to modify and improve the nutritional profile of durum wheat semolina pasta. Among them dietary fibres, legume flours, rice, corn, emmer, cricket flour among others. The novel ingredients and process also allow the production of high quality gluten-free pasta for sensitive and celiac consumers categories. The use of alternative ingredients in pasta formulation results in interesting modifications in quality as well as nutritional and sensory characteristics.

Heat-stable peptidases released in refrigerated raw milk by psychrotrophic bacteria are responsible for UHT milk gelation. K-casein-derived caseinomacropeptides, identified by mass spectrometry, were constantly detected in gelled milk by capillary electrophoresis. Strains of Pseudomonas fluorescens, Ps. poae and Chryseobacterium joostei, selected among aprX-positive strains from raw milk, were incubated in milk up to 6 days at 4 degrees C before sterilization (98 degrees C/4 min). Samples were then stored at 25 or 40 degrees C, visually observed for gelation, and analysed for presence of caseinomacropeptides throughout 90 days of storage. Depending on cold pre-incubation time, caseinomacropeptides accumulated well before gelation onset in milk stored at 25 degrees C. Caseinomacropeptides were successively degraded, especially in milk stored at 40 degrees C, due to extensive proteolysis, and an abundant sediment developed instead of a gel. The caseinomacropeptides are here presented as an early indicator of UHT milk gelation and a mechanism explaining this phenomenon is proposed.

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In this work, we explored the “deep” seed peanut proteome by using both two dimensional electrophoresis (2- DE)-based analysis run under reducing and non-reducing condition (protein-centric) and LC-MS/MS gel-free proteomic (peptide-centric). The former approach allowed to identify high molecular weight disulfide-linked Ara h 1 and Ara h 3 heteroligomers and Ara h 1 homoligomers linked through covalent bonds other than disulfides. The occurrence of these protein complexes revealed natural interactions between Ara(s) subunits with a possible involvement in the allergenic potential of peanut. The second approach, also referred to as shot-gun proteomics, allowed the identification of 149 gene products, including low-abundance proteins escaped the 2-DE detection. Interestingly, we identified 60 proteins never catalogued previously. The complementary exploitation of two proteomic approaches enabled the access to new relevant information about the complexity of the peanut proteome, with special emphasis to the complement of allergens (allergome).

Hemp (Cannabis sativa L.), traditionally cultivated for industrial use and harvested for fibers and seeds, has raised much interest as a sustainable crop in the last years. Recently, hemp seeds and derived oil have started to be used in a variety of food products. Hemp-based food products are considered less allergenic than those from other edible seeds, although this statement has never been experimentally verified. In this study high purity grade hemp flour (HF) and hemp protein isolate (HPI) were obtained through a fast and cheap process starting from defatted hemp cakes, a residue of hempseed oil extraction. HPI resulted enriched at nearly 86% protein, mainly constituted by the storage protein edestin (accounting for 70% total protein). In vitro protein digestibility was determined using a static model of gastrointestinal digestion (GID), which included a final step with purified brush border membrane (BBM) enzyme preparations. HF and HPI showed a high degree of digestibility. The survival of potential bioactive and/or allergenic peptide sequences in digests was investigated by peptidomic analysis. Only a limited number of sequences survived GID. Among them, fragments from 12 seed proteins. These fragments were precursors of sequences with potential bioactive peptides, which might justify the bioactivity of HPI hydrolysates, reported in previous studies. More importantly, all known hemp allergens, including the major thaumatin-like protein and LTP, were entirely eliminated by the HPI production process, neither fragments of the proteins were present after GID. These data support the use of HPI as an ingredient for hypoallergenic foods.

The demand for sustainably produced proteins is increasing with the world population and is prompting a dietary shift toward plant sourced proteins. Vegetable proteins have lower digestibility and biological value compared to animal derived counterparts. We explored sprouting of chickpea seeds as a strategy for improving digestibility. Protein evolution associated with by the sprouting process was assessed by proteomics. The sprouting induced breakdown of seed storage proteins and doubled the release of free alpha-amino nitrogen in sprouted chickpea flour. During sprouting, several enzymes involved in plant development were newly expressed. An ex vivo model of gastroduodenal and jejunal digestion was applied to assess the bioaccessibility of the protein digests. Proteins from chickpea sprouts showed a greater susceptibility to digestion with a 10% increase in alpha amino nitrogen. Peptides with potential immunoreactivity or bioactivity were catalogued in both digested chickpea sprouts and seeds using an in-silico approach. Peptides belonging to the non-specific transfer proteins, which are allergens in pulses, and peptides belonging to an IgE-binding hemagglutinin protein could only be identified in the digested chickpea sprouts. The observation collected paved the way to immune-based evaluations to assess the effect of germination on the allergenic potential.

Kashk is a fermented dairy product typical of the Middle East, traditionally produced with sour milk and/or dairy waste. The kashk water-soluble peptide fraction was characterized at the molecular level by liquid chromatography-mass spectrometry and its antibacterial and skin healing activity was evaluated. Antibacterial assays showed a significant antibacterial activity against clinical isolates of Staphylococcus aureus (S. aureus) from patients with atopic dermatitis, inhibiting bacterial growth by approximately 45% (500μg/mL). Skin repair activity was evaluated on keratinocytes through scratch tests showing accelerated wound closure in vitro in the presence of TNF-α, by approximately 44% (500μg/mL), compared to control cells. Furthermore, based on the MTT assay, the kashk peptide fraction did not show toxicity on keratinocytes. The results suggested that the peptide kashk extract may be useful in skin care for patients with atopic dermatitis.

The field of food analytical chemistry has greatly evolved over the last years, moving from the descriptive view of raw food composition into the modern instrumental approaches known as the foodomics. The development and optimization of these integrated approaches, based on the use of high resolution techniques for tracing the chemical fingerprint of a food, is arriving to define molecular parameters for the qualification, authentication, and safety of the products. With these new tools, omics are solving some of the new issues of food quality and traceability, including the development of analytical methods to ensure the origin of food, as well as the discovery of biomarkers to identify potential food safety problems. Here we review the main methods used in foodomics from its birth until now, highlighting the use of high throughput instrumentation. This includes the computational problems they generate, but also the significant prospects for development of knowledge that are able to open in important areas such as food technology and biotechnology.

Unravelling the fate of food in the gastrointestinal tract is essential to better understand the health effects of the food and to fight against diet-related pathologies such as cardiovascular diseases and type-2 diabetes. Digestion is the process that transforms food into nutrients. The first step of digestion occurs in the mouth, where mastication transforms solid and semi-solid foods into particles while mixing with saliva allows bolus formation and initiates digestion of carbohydrates.Then the bolus is transferred into the stomach, where acid conditions and specific enzymes (pepsin, gastric lipase) start hydrolyzing macronutrients like proteins and triglycerides. The next step occurs in the small intestine, where other digestive enzymes further degrade macronutrients allowing their absorption. In the small intestine, proteins are hydrolyzed by trypsin, chymotrypsin, elastase, carboxypeptidase etc, lipids by pancreatic lipases, and carbohydrates by pancreatic amylase. Small intestinal digestion is completed by the enzymes of the brush border membrane that release macronutrients, which can be absorbed by enterocytes to reach the bloodstream. Undigested material, fiber for example, reaches the large intestine where it is further metabolized by the intestinal microbiota. Investigating food digestion requires the use of models and a myriad of in vitro (static and dynamic), animal and human models have been described in the literature with the objectives of understanding the fate of food in the gastrointestinal tract. In particular, static in vitro digestion simulations are extremely popular because they are very easy to use and do not require sophisticated equipment. They have been shown to be adapted for screening large series of similar samples in identical conditions or to understand interactions at the molecular scale (Bohn et al.,2017). However, they are too simple to study more complex phenomena and the kinetics of food digestion for which dynamic in vitro models are more appropriate (Dupont et al., 2018). There was a high heterogeneity between the different static in vitro digestion models that were used by the research groups all around the world. Models were differing in the pH used in the different phases (gastric and intestinal), their duration, the digestive enzyme/substrate ratio, etc For that reason, comparing results obtained from one study to another was impossible and there was a crucial need for a harmonized method that could be used by everyone allowing comparison between studies. This was one of the main objectives of the INFOGEST COST Action.

This study aimed at addressing the effect of quinoa sprouting on saponins which are considered the main responsible for quinoa bitterness and astringency, and consequently on quinoa taste. Whole quinoa was sprouted for 12, 24, 48, and 72 h at 22 °C and dried at 55 °C for 6 h. Quinoa taste was assessed by electronic tongue and mainly related to the amount of saponins that were evaluated by thin layer chromatography (TLC) and mass spectrometry (MS). Sprouting decreased quinoa bitterness and astringency, with the main changes occurring within the 48 h of treatment. Both semi-quantitative (by TLC) and quantitative (by MS) analysis revealed that changes in quinoa taste upon sprouting was mainly related to the decrease in the amount of saponins, reaching the value of 0.086 mg/100 g after 48 h of treatment. Finally, changes in phenolic compounds seem to play a minor role in affecting the taste traits of sprouted quinoa.

The intestine presides over a series of vital functions in the human body, among which the digestion/absorption of nutrients. Despite their major digestion role, the impact of the enzymes of the luminal intestinal surface on food components has been considered in relatively few experiments of simulated gastrointestinal digestion. In contrast, the identification of proteolitically stable peptides which survived digestion in multiphasal models that also included a step with small intestinal brush border membrane (BBM) peptidases has provided physiologically consistent results. Herein, we critically review the use of BBM enzymes to simulate the intestinal digestion of dietary polypeptides. Addressing the controversial issue of the in vitro-in vivo correspondence of the digestion models, the review emphasizes the need to establish consensus protocols to simulate the intestinal step, for instance using the BBM hydrolases at least in a selected number of cases. The factors that have limited the development of relevant models of intestinal degradationare discussed together with hints to possible alternatives, forthcoming approaches and future perspectives to reproduce the physiopathology of the human small intestine. (C) 2015 Elsevier Ltd. All rights reserved.

High resolution mass spectrometry was employed to study a bloom of the Cyanobacterium Planktothrix rubescens in a dam for drinking water on Lake Occhito, near the town of Foggia, in Southern Italy, to characterise the cyanotoxins produced and to evaluate the related health risks for consumers. The cyanotoxin production in various sites of the contaminated dam was studied by MALDI-TOF mass spectrometry and liquid chromatography coupled to Q/TOF tandem mass spectrometry analysis of water and cyanobacteria samples. MALDI-TOF mass spectrometry was employed for rapid screening over a wide mass range; liquid chromatography coupled to Q/TOF tandem mass spectrometry allowed for molecular structure confirmation of each single cyanotoxin. This analytical strategy allowed for profiling the cyanotoxins produced during the bloom and calculating the concentrations of the main contaminants in drinking water. Microcystins, anabaenopeptins and aeruginosins were the main cyanotoxins identified. A des-methyl variant of the microcystin-RR was recognised as the most abundant compound in all water samples; liquid chromatography coupled to Q/TOF tandem mass spectrometry allowed for molecular structural confirmation of detected substances, and quantitative analysis of microcystins. The contamination levels over 3 weeks were monitored, by sampling from different sites of the dam. This analytical strategy allows for complete characterisation of the cyanotoxins produced during this kind of environmental outbreaks, for time-dependent monitoring of pollution levels and thus represents a powerful tool to prevent intoxication for consumers of contaminated drinking water collected from the dam.

A "virtual two-dimensional (2D)-like" approach based on pH 2.5-6.0 gel isoelectrofocusing coupled to MALDI-TOF mass spectrometry (MS) was addressed for challenging the ovine casein heterogeneity due to polymorphism and post-translational modifications. The procedure enabled the identification of the main casein components including i) the four casein families at different degrees of phosphorylation, ii) nonallelic variants of either alpha(s1)- or alpha(s2)-CN, iii) differently glycosylated kappa-CN, and iv) high molecular mass proteolytic peptides. Protein assignment was confirmed by MALDI-TOF (MS) mass mapping of the tryptic digests, while phosphorylation sites were localized by tandem mass spectrometry. A straightforward identification was hindered by unexpected molecular mass shifts due to the oxidization of alpha(s1)- and the beta-CN. Indeed, using nano-ESI-MS, the casein sub-types were proved to contain a variable number of oxidized methionine residues. Data were additionally substantiated using polyclonal antibodies raised against the single casein families for immunolabelling purposes. The outcomes herein demonstrate the potentiality of the "virtual 2D-like" approach and provide indications useful for the inter-laboratory discrepancies in casein identification. They also greatly facilitate the comparison of data and the establishment of multi-user image-based isoelectrofocusing gels. (C) 2012 Elsevier Ltd. All rights reserved.

The composition of milk has adapted during the evolution of the species to fulfill the specific nutritional needs of the offspring. Currently, it is widely recognized that milk benefits go beyond mere nutrition and serve as a source of a number of functional components to the newborn, particularly host defense effectors. However, the human milk proteome description is still incomplete, primarily because the detection of low-abundance proteins remains challenging. To overcome the limitations of the classical electrophoresis-based approach, previously separated milk fat globule membrane (MFGM) and whey protein fractions were analyzed by nanoflow-high performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). This shotgun strategy showed an as yet unmatched potential to profile low-abundance proteins in human milk. Proteins associated with 301 different gene products were identified, some of which could be clustered into subsets of protein isoforms, thus providing one of the largest protein inventories of human milk. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions. Nevertheless, it appears clear from this study that the overall picture of the human milk proteome is still incomplete, although several protein signatures of milk evolution are emerging.

IgE-mediated allergy to soybean may be connected to an incomplete protein digestion causing an inappropriate immune response in the gut. The aim of this study was to gain insight on the digestibility and immunoreactivity of β-conglycinin and its deglycosylated form. To achieve this goal both proteins were digested mimicking gastrointestinal physiological conditions. Digests were characterised by SDS-PAGE, MALDI-TOF-MS and immunological assays. Data on IgG binding western blot provided key information related to the composition of the digests indicating that whereas deglycosylated β-conglycinin was completely removed by gastrointestinal digestion, α and β subunits of β-conglycinin glycoprotein partially survive the process of digestion. Such differential digestive behaviour might be influenced by glycan moieties, at least to some extent, and should be an additional factor to be considered on its potential allergenicity. Results also support the existence of linear epitopes in the three subunits (α, α′, and β) of both protein forms. © 2011 Elsevier Ltd. All rights reserved., This work has been funded by ERDF-co-financed grant AGL2007-60007/ALI, CONSOLIDER Ingenio 2010 FUN-C-FOOD: CSD 2007-00063, ALIBIRD-CM-S0505/AGR-0153 and IT-2009- 0087 projects. Dr. M.A-B. thanks European Union COST-Action 927 for Short Term Scientific Mission and Danone Institute for her fellowship.

Bovine β-casein and its C-terminal sequence (191–209) were previously shown to possess immunomodulatory properties in studies on partially purified peptides from hydrolysates, where minor amounts of contaminants may affect the cellular response. The inhibitory effect of β-casein and of eight C-terminus synthetic β-casein peptides on mitogen-induced spleen cell proliferation was compared. Use of synthetic peptides allowed the unambiguous and accurate identification that a seven amino acid sequence at the C-terminus, including a PFP motif, was sufficient for immunomodulation. Substitution of the last proline (P206) in the PFP motif with D-Pro had a negative impact on the immunosuppressory activity of all these short peptides, whereas substitution of P206 with structural analogues of proline had almost no impact. A relationship was found between the immunomodulatory properties and the structural features of these peptides, as assessed by various spectroscopic approaches, indicating a role of structure in eliciting the immunomodulatory activity of these peptides.

In this study, the protein and peptide fractions of two commercial Italian barley malt beers, made with different processes by the same producer, have been analysed with a combined immunochemical and mass spectrometry approach. The “gluten” content of beer samples, measured with the R5 monoclonal antibody, was below the caution limit proposed by the Codex Alimentarius for gluten-free foods. The proteomic approach allowed to identify a 17 kDa avenin-like protein partially homologous to hordeins, that was particularly abundant in foam, in addition to the already reported barley albumins (Z 4 -barley and ns-LTPs) and to minor amounts of yeast glycolytic enzymes. No intact hordeins were detected, although fragments derived from γ 3 - and B-hordein were present. In consideration of the many implications of the protein/peptide pattern, these data provide useful information to improve quality and safety of beer.

Ovine α s1 -casein ( CSN1S1 ) allele I ( CSN1S1 ∗ I ) was characterized at the molecular genetic and protein level. Sequencing of CSN1S1 cDNA and mature protein showed the absence of exon 7 from CSN1S1 ∗ I in comparison with the C ″ genetic variant of the C phenotype. This allelic aberration is correlated with a sequence difference in 5′-splice donor sequence of intron 7 (g.656T > A), leading to upstream skipping of exon 7. Consequently mRNA sequence of ovine CSN1S1 ∗ I is 24 bp shorter than complete coding sequence leading to an abbreviation of eight amino acids in the mature protein, resulting in a lower degree of phosphorylation in comparison with CSN1S1 ∗ C ″. However CSN1S1 ∗ I was expressed at a quantitative level similar to that for the C ″ reference variant. Using amplified created restriction site polymerase chain reaction, a DNA-based test for identification of CSN1S1 ∗ I was developed. Altogether six nucleotide substitutions were identified within intron 6 and intron 7 of CSN1S1 variants, forming three different haplotypes.

β-Conglycinin is a functional glycoprotein and one of the most important soybean allergens. The aim of the present research was to investigate the role of the N-glycans moieties of β-conglycinin on its in vitro immunoreactivity. The soy allergen was obtained by isoelectric precipitation from commercial soy protein isolate and was enzymatically deglycosylated by PNGase F (Peptide N-Glycosidase F EC 3.5.1.52). In order to optimize deglycosylation conditions different reaction times and allergen concentrations were tested. The extent of deglycosylation was estimated by SDS–PAGE, CZE, RP-HPLC, and MALDI-TOF MS analyses, which provided information related to changes in protein structure. The antigenicity of both native β-conglycinin and its deglycosylated form was evaluated by western-blotting and indirect ELISA employing polyclonal rabbit anti-soybean sera and horseradish peroxidase-labeled goat anti-rabbit IgG while the in vitro allergenicity was assessed by means of indirect competitive inhibition ELISA employing human sera (IgE) of soy allergics. β-Conglycinin was effectively deglycosylated by PNGase F. Data on immunological tests suggested that glycosyl moieties forming this glycoprotein might be involved in its immunoreactivity.

The formation of adducts between acrylamide and hemoglobin in vitro was investigated by using mass spectrometric methodologies to identify the amino acid residues sensitive to alkylation. Liquid chromatography-electrospray ionisation mass spectrometry analysis of either intact or trypsin-digested alpha- and beta-globin chains isolated from hemolysate samples incubated in vitro with acrylamide at different molecular ratios allowed us to identify Cys93 of beta-globin as the most reactive site in hemoglobin, according to a Michael-type addition reaction between acrylamide and the sulphydryl group of cysteine. The only other reactive sites were Cys104 of alpha-globin and the N-terminal amino groups of both chains. The method developed, based on electrospray ionisation quadrupole time-of-flight tandem mass spectrometry analysis of intact globin chains was able to specifically detect low levels of adducts. In this way, rapid identification of alkylated portion of Hb was achieved to be potentially used as a biomarker for high-sensitivity biological monitoring.

Molecular approaches by means of a combined use of mass spectrometric techniques can be important in order to open new possibilities in the differentiation and defense of typical products; in this study, a possible approach to the analysis of varietal volatile compounds and some precursors of a non-aromatic grape variety (Falanghina cv., Vitis vinifera L.) was traced through a combined use of techniques based on mass spectrometry (GC/MS, LC/ESI-MS, MALDI-TOF-MS). Dominant terpene compounds (limonene, cis-furanlinalool oxide, geraniol, 4-carene, myrcene, linalool, a-terpineol), terpene-derivatives (bornyl acetate, menthol), terpene glycosides (glucosides, arabinosylglucosides and rhamnosylglucosides of linalool and geraniol), and norisoprenoids (b-damascenone) were identified in grapes and monovarietal wines, overcoming the analytical difficulties deriving from the low concentration of these compounds strictly related to the variety. The potential release of varietal volatile compounds from the grapes was also explored by enzyme hydrolysis.

The main peptides produced by hydrolysis of water buffalo beta-casein with plasmin were characterized by capillary electrophoresis and mass spectrometry and compared with their bovine homologous. A novel breakdown product arising from the hydrolysis of water buffalo beta-casein, originated by the presence of a plasmin-sensitive Lys bond at position 68 was identified, which was not present in bovine beta-casein. On the basis of this evidence, an improved procedure for the detection and the differentiation of the products of plasmin hydrolysis of bovine and water buffalo beta-casein by capillary isoelectric focusing was set-up. In the experimental conditions, the gamma-casein from the two species was efficiently separated. Comparison of the capillary electropherograms with those obtained by ultra-thin-layer isoelectric focusing, the reference method for routine analysis of plasmin digests of casein, suggests that capillary electrophoresis isoelectric focusing may constitute a successful alternative to the traditional slab gel electrophoresis analysis of plasmin digests of casein either for basic structural studies or for applications in the quality assessment of dairy products.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their d-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 ′-triiodothyronine (T3) (171%) and 3,5,3′,5′-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.

Food Digestion is the process that lies between the foodwe consume and any health benefits that it may impart. In a world where food related disease is already a significant and increasing burden on health services, it is important to understand the link between food and health. This requires a range of approaches including the use of in vitro simulations of the digestive process. However, if experimental data is to be comparable and useful it needs to make use of harmonised and physiologically relevant methods. The Infogest static model [9] offers such a method and has been cited in more than 100 times.The success of this article confirms the interest in understanding the links between food structure and health.

Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.

A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T3), and tetraiodothyronine (thyroxine, T4) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T4 of thyroglobulin (0.30 mol of T4/mol of 660-kDa thyroglobulin), and 8% of total T3 (0.08 mol of T3/mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.

The term “FoodOmics” first popped up in the scientific literature around 2009. In the same year, the first International Conference on FoodOmics took place in Cesena. Probably neither us, as the scientific organizers of the conference, nor Alejandro Cifuentes, who first used the term in a scientific paper, thought that somany scientists would have been interested in the field. Since then, FoodOmics, like all the other Omics, rapidly evolved due to the development of novel tools and technologies. This iswitnessed by the many FoodOmics articles that have been published on scientific journals, and professor Cifuentes has been one of the invited speakers in the Second International Conference on FoodOmics, held in Cesena in 2011. At the end of 2012,we asked ourselves “is it timefor the third edition of the Conference?” What is new in this third edition? Themost important news is that we don't need to explain what FoodOmics is anymore, since everybody knows. And it is a big step ahead!Whatwe need nowis to share the possibility of the application of the FoodOmics concept (and techniques) to the different research fields. So, the subtitle of the Conference “A Science for Nutrition, Health and Wellness in the Post- Genomic Era” used in the first two editions was turned to “FoodOmics, the science for discovering” in 2013. The abstract book of the Conference, and this special issue of Food Research International, testify that the FoodOmics vision is a reality shared by many researchers. In this special issue of FRI dedicated to the Conference, critical reviews as well as descriptions of new approaches for use in the analysis and manipulation of food genomes, proteomes and glycomes are presented. The issue startswith three research papers each describing the use of themost recentNMR andMS instrumental advances to analyze in depth food metabolomes. These three articles are followed by a section of five papers investigating the process of food digestion – the digestome– and the fate of food constituents across it. The next article extends the field of applications to peptidomics and glycomics through the characterization of milk colostrum. The final section in this issue presents six articles facing the impact of food bioactive components from the nutritional, clinical and diagnostic sides. What about 2015? Difficult to say, even using “omics” techniques… One thing is certain: the field of FoodOmics will continue to grow in both application and scope and this will assist research to reshape our current understanding of the complex issues related to food, nutrition and health.We are planning the Fourth FoodOmics Conference, hoping to involve an increasing number of participants. At present, we wish to thank all Colleagues who contributed to the 2013 Conference, and to this special issue devoted to it.

A new silent hemoglobin variant, Hb Ozieri (alpha 71(E20)Ala-->Val), was observed in five apparently unrelated newborn babies during a screening for hemoglobinopathies on the island of Sardinia. This asymptomatic variant was detected by means of isoelectric focusing (IEF), isolated using IEF in an immobilized ultranarrow pH-gradient and characterized at the structural level using FAB- and electrospray-mass spectrometric techniques. A Val for Ala substitution was unambiguously detected at position 71 of the alpha-globin chain. This substitution indicates that a C to T transition occurred in the GCG codon for Ala which contains one of the 35 unmethylated CpG dinucleotides of the alpha-globin gene. This observation brings the number of variants due to a mutation in the alpha-globin gene CpGs (the third instance of a silent mutation) to 13 and raises the possibility that unmethylated CpGs might be hotspots for mutations as the methylated ones.

1. 1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains. 2. 2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins. 3. 3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987). 4. 4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β2-globin) involve uncharged substitutions.

1. 1. Hemoglobins from three phenotypes of Italian water buffalo ( Bubalus bubalis ), named AA, AB and BB, were selected by starch gel electrophoresis at alkaline pH and analyzed using polyacrylamide gel isoelectric focusing and subsequent analysis of titration curves to reveal differences between two types of hemoglobin identified as Hb fast and Hb slow. 2. 2. Globins from Hb fast and Hb slow were purified by fast protein liquid chromatography (FPLC). Electrophoretic differences were found in the respective α-chains using polyacrylamide gel disc-electrophoresis at acid pH, polyacrylamide gel isoelectric focusing and by subsequently analyzing titration curves. 3. 3. The results suggest that the α chains of Hb fast and Hb slow, called II α and II α , respectively, differ in at least two aminoacid residues. Subsequently, these amino acids were identified as lysine and cysteine.