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Resistance of gluten to gastrointestinal digestion is involved in immune-mediated adverse reactions to wheat, since several peptides produced by the incomplete digestion are able to trigger, in predisposed individuals, the immune response responsible, for instance, of celiac disease (CD) and other adverse reactions. Even if several peptides have been identified, an exhaustive description of the peptidome generated by wheat digestion is lacking. To this end, in the present work, durum wheat proteins were fractionated, digested, and then subjected to various proteomic techniques, including single stage and multiple stage mass spectrometry (MS) (SDS-PAGE, UPLC/ESI-MS, UPLC/ESI-MS/MS, and LTQ-Orbitrap). Based on SDS-PAGE, although proteins were severely degraded after in vitro gastrointestinal digestion, some differences were observed among protein profile of the different digests. Through untargeted UPLC techniques, 227 peptide sequences were identified, with only few sequences shared by the different digests. In particular, 9 gluten peptides involved in CD were identified. Based on target proteomic, the quantification of these peptides revealed significant (p ≤ 0.05) differences among the different extracts. Taken together, all the proteomic tools confirmed that gluten digestion is closely related to the matrix regardless of wheat genotype.

Parmigiano Reggiano (PR) is a raw-milk, hard cooked, long-ripened cheese of high quality and nutritional value. Long ripening times allow for extensive proteolysis of milk proteins to yield a number of peptides, some of which have potential healthy bioactive properties. This study aimed to: i) determine the peptide profile of PR cheese subjected to simulated gastrointestinal transit; ii) evaluate in vitro whether the peptides could support growth of beneficial microbial groups of the gut microbiota. PR samples were subjected to in vitro digestion, simulating oral, gastric, and duodenal transit. Liquid chromatography coupled with tandem mass spectrometry revealed that digestion caused the disappearance of the serum proteins and most of the original peptides, while 71 new peptides were found, all ranging from 2 to 24 residues. The digests were given as sole nitrogen source to pure cultures of Bifidobacterium (27 strains) and Lactobacillus (30 strains), and to bioreactor batch cultures of human gut microbiota. Most of bifidobacteria and lactobacilli grew more abundantly on PR digests than on the control peptone, and exhibited strain- or species-specific peptide preferences, as evidenced by principal component analysis. Bifidobacteria generally consumed a greater amount of peptides than lactobacilli, in terms of both the mean peptide consumption and the number of peptides consumed. For bifidobacteria, peptide preferences were very diverse, but a core of 10 peptides with 4 or 5 residues were consumed by all the strains. Lactobacilli behaved more homogenously and consumed nearly only the same 6 peptides, mostly dipeptides. The peptide preferences of the different groups of bifidobacteria and lactobacilli could not be ascribed to features such as the length of the peptide or the abundance of residues with peculiar properties (hydrophobicity, polarity, charge) and likely depend on specific proteases and/or peptide transporters preferentially recognizing specific sequence motifs. The cultures of human colonic microbiota confirmed that PR digest promoted the growth of commensal bifidobacteria. This study demonstrated that peptides derived from simulated gastrointestinal digestion of PR supported the growth of most lactobacilli and bifidobacteria., (Copyright © 2017 Elsevier B.V. All rights reserved.)

It is expected that during the hydrolysis of proteins with specific enzymes only peptides are formed that result from hydrolysis of the specific cleavage sites (i.e. specific peptides). It is, however, quite common to find a-specific peptides (i.e. resulting from a-specific cleavage), which are often ignored, or explained by impurities in the enzyme preparation. In recent work in a whey protein isolate (WPI) hydrolysate obtained with the specific Bacillus licheniformis protease (BLP), 13 peptides of 77 identified were found to be the result of a-specific cleavage. These were formed after degradation of 6 specific peptides, after 5 different types of amino acids. The fact that other peptides were not hydrolyzed after these 5 amino acids suggests that the cleavages were not the result of a contamination with a different enzyme. In other systems, certain peptide sequences have been described to degrade chemically, under relatively mild conditions. This process is referred to as spontaneous cleavage. To test if the a-specific peptides observed in the WPI hydrolysis are the results of spontaneous cleavages, the parental peptides were synthesized. Surprisingly, 4 of the 5 synthesized peptides were indeed spontaneously cleaved under the mild conditions used in this study (i.e. 40°C and pH 8) showing that peptides are less stable than typically considered. The rate of cleavage on the a-specific bonds was found to be enhanced in the presence of BLP. This suggests that the formation of a-specific peptides is not due to side activity but rather an enhancement of intrinsic instability of the peptides., (Copyright © 2015 Elsevier B.V. All rights reserved.)

During wheat digestion, gluten-derived proteolytic resistant peptides are generated, some of them involved in celiac disease. In vitro digestion models able to mimic the peptides generated in the human gastrointestinal tract are extremely useful to assess the pathogenicity of wheat-derived products. In this paper, samples belonging to three different durum wheat varieties were taken at six different steps of the pasta production chain and two different digestion models present in the literature were assessed on the different samples: a more complex one using artificial fluids simulating the exact composition of digestive juices, and a simplified method based on a peptic-tryptic/chymotryptic treatment of wheat ethanolic extract. An extensive characterization of the peptides generated using two in vitro digestion models was performed through LC-MS/MS techniques and the two methods were compared in order to evaluate qualitative and quantitative differences and their possible implications for varietal screening. Strong differences in the type of peptides produced with the two methods were detected, indicating that the simplified method can still be used for a varietal screening but is not representative of the peptides really generated after physiological human digestion. Results indicate a clear necessity of physiologically accurate models for simulating human gastrointestinal digestion of wheat products.

In this work the antioxidant capacity of water soluble extracts of Parmigiano-Reggiano cheese (Water Soluble Extracts - WSEs) at different aging time was studied, by measuring their radical scavenging capacity with a standard ABTS assay. The WSEs were also fractionated by semi-preparative HPLC-UV and for each fraction the antioxidant capacity and the molecular composition was determined by LC/ESI-MS, in order to identify the most active antioxidant compounds. The antioxidant capacity was also determined after simulated in vitro gastrointestinal digestion of WSEs. The data indicated that antioxidant capacity in WSE from Parmigiano-Reggiano cheese, quite unaffected by ripening time and gastrointestinal digestion, is mostly due to free amino acids, mainly tyrosine, methionine and tryptophan, and only in minimal part to antioxidant peptides.

Food allergy from wheat is triggered by several protein classes, such as LTPs, ω5-gliadins and α-amylase/trypsin inhibitors. The latter proteins, belonging to the prolamin superfamily, are mostly involved in baker's asthma, a form of occupational allergy in which the sensitization occurs through the respiratory tract. α-Amylase/trypsin inhibitors were also found to be involved in wheat-related atopic dermatitis. In this work, the allergen Tri a 30 (the CM3 α-amylase/trypsin inhibitor) was quantified in durum wheat salt soluble extracts using a peptidomic approach. CM3 protein identification was confirmed by using LTQ-OrbiTrap analysis on peptides obtained from the enzymatically digested protein separated by gel electrophoresis. Then, marker peptides derived from the protein after enzymatic cleavage of the full wheat extracts were identified by LC-MS/MS. One of them was used as marker for quantitative determination on an UPLC/ESI-MS system by using its isotopically labelled analogue as internal standard, allowing to assess the protein content in the different samples. The CM3 allergenic proteins were found to greatly vary among different cultivation areas., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A method to detect the presence of common wheat in durum wheat flour samples was developed and tested. Flour samples, or ground wheat samples, were digested by pepsin and chymotrypsin, and the peptide mixture obtained was analyzed by LC/ESI-MS and LC/ESI-MS/MS, which led to the identification of two marker peptides. One peptide was coded only in the DD genome, and thus present only in common wheat; the second was present in all wheat samples (both common and durum), so it was used as marker of the total wheat content. The ratio of the chromatographic areas of these two peptides, as determined by LC/ESI-MS, was related to the proportion of common wheat in the sample using a calibration curve that was constructed with standards of known composition. The proportions of common wheat in samples obtained by mixing different common and durum wheat varieties were accurately determined by this method. Finally, the method was applied in a survey of several durum wheat flour brands present on the Italian market. The results of the survey revealed that contamination of durum wheat flour with common wheat is commonplace.

High-quality proteins from farmed fish, including sea bream, represent a cheaper and healthier alternative to proteins from other animals, especially for people affected by chronic diseases. Aquaculture has also the possibility to move towards more sustainable growth and production. Therefore, the present study was designed to evaluate the impact of a starvation period before slaughtering in comparison to full feeding on quality attributes of sea bream fillet focused on their health promoting values. SDS-PAGE, Western blotting and high-resolution MS/MS analyses of the protein profiles showed that in the fillets from starved fish, only the amount of myosin light chains (MLCs) was markedly reduced at slaughter, suggesting their higher susceptibility to proteolysis. In the full feeding samples, MLCs degradation was evident only during storage at 4 °C. Conversely, skeletal alpha-actin fragmentation was similar regardless of the pre-slaughter feeding system. In vitro gastric digestibility of the myosin complex from starved fish fillets was much higher at pH 4, a condition that may occur in drug-treated patients affected by the gastroesophageal reflux disease (GERD). Using UHPLC–MS/MS, ACE-inhibitor gastric-resistant bioactive peptides potentially coming from MLCs (VF, MF) were found in all the digested samples, with one of these peptides (MF) being present in larger amount in the starved samples. Pre-slaughter starvation, other than decreases the environmental impact of fish aquaculture, is an effective tool in obtaining good-quality sea bream fillets that provide potential health benefits for hypertensive and GERD consumers. [ABSTRACT FROM AUTHOR]

Cheese microbiota contribute significantly to the final characteristics of cheeses due to the growth and interaction between cheese microorganisms during processing and ripening. For raw milk cheeses, such as Parmigiano Reggiano (PR), the microbiota derive from the raw milk itself, the dairy environment, and the starter. The process of cheese making and time of ripening shape this complex ecosystem through the selection of different species and biotypes that will drive the quality of the final product by performing functions of their metabolism such as proteolysis. The diversity in the final peptide and amino acid composition of the cheese is thus mostly linked to the diversity of this microbiota. The purpose of this study was to get more insight into the factors affecting PR cheese diversity and, more specifically, to evaluate whether the composition of the bacterial community of cheeses along with the specific peptide composition are more affected by the ripening times or by the cheese making process. To this end, the microbiota and the peptide fractions of 69 cheese samples (from curd to cheese ripened 24 months) were analyzed during 6 complete PR production cycles, which were performed in six different dairies located in the PR production area. The relation among microbial dynamics, peptide evolution, and ripening times were investigated in this unique and tightly controlled production and sampling set up. The study of microbial and peptide moieties in products from different dairies – from curd to at least 12 months, the earliest time from which the cheese can be sold, and up to a maximum of 24 months of ripening – highlighted the presence of differences between samples coming from different dairies, probably due to small differences in the cheese making process. Besides these differences, however, ripening time had by far the greatest impact on microbial dynamics and, consequently, on peptide composition. [ABSTRACT FROM AUTHOR]

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI-MS) in order to identify peptidic species-specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high-resolution mass spectrometry, confirming the origin from species-specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI-MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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Peptide nucleic acid (PNA), a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA/DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready® soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin/biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association (kon) and dissociation processes (koff), and the affinity constant (KA) of the PNA/DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA (15mer) target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product (169mer) obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-A resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition. [ABSTRACT FROM AUTHOR]

Within a project aimed at studying the peptide fraction in dry-cured Parma hams, a peptide was purified by means of reversed phase-high performance liquid chromatography (RP-HPLC) and identified by its molecular mass and amino acid sequence analysis. The peptide showed a very high degree of homology with the N-terminal part of different mammalian pyruvate kinases reported in databases and was accordingly identified as the N-terminal part of swine pyruvate kinase, whose sequence had never been reported before. The peptide was determined quantitatively by comparison with a suitable internal standard (Phe-Phe) in commercial ham samples with different age degrees. The peptide was found to be ubiquitous in Parma ham and its amount to increase during ageing even if a large variability was found within each assayed maturing time. The correlation found with the ham proteolysis degree (P<0.01) suggests that this peptide is related to the endopeptidasic activity. The peptide did not show a significant relationship with bitterness perception in assayed dry-cured ham, in agreement with the hypothesis that this taste is more related to free amino acids and low molecular weight peptides. [Copyright &y& Elsevier]

Parmigiano–Reggiano (PR) is a worldwide known Italian, long ripened, hard cheese. Its inclusion in the list of cheeses bearing the protected designation of origin (PDO, EU regulation 510/2006) poses restrictions to its geographic area of production and its technological characteristics. To innovate the Parmigiano–Reggiano (PR) cheese manufacturing chain from the health and nutritional point of view, the output of defatted PR is addressed. Two defatting procedures (Soxhlet, and supercritical CO2 extraction) were tested, and the obtained products were compared in the composition of their nitrogen fraction, responsible for their nutritional, organoleptic, and bioactive functions. Free amino acids were quantified, and other nitrogen compounds (peptides, proteins, and non-proteolytic aminoacyl derivatives) were identified in the extracts and the mixtures obtained after simulated gastrointestinal digestion. Moreover, antioxidant and angiotensin converting enzyme (ACE) inhibition capacities of the digests were tested. Results obtained from the molecular and biofunctional characterization of the nitrogen fraction, show that both the defatted products keep the same nutritional properties of the whole cheese. [ABSTRACT FROM AUTHOR]

Germination is already a well-accepted process by consumers with many products made from sprouted seeds or containing limited amounts of flour form sprouted grains. The present work aimed assessing the usefulness of germination in reducing gluten peptides associated with celiac disease, at the same time evaluating some technological features of the obtained germinated wheat. In the first part of the work, celiac disease (CD)-triggering peptides were tracked as a function of germination kinetics (from day 1 to day 6). Using simulated gastrointestinal digestion and liquid chromatography coupled to mass spectrometry, ten celiac disease triggering peptides were identified: seven peptides presumably involved in the adaptive immune response (TI) and three peptides mainly involved in the innate immune response (TT). All the identified peptides belonged to gliadins. TI track pattern showed three phases: the first two days displayed a significant degradation, a stability phase was observed from day 3 to day 5, and finally a drastic reduction occurred on the 6th day. For TT peptides, important degradation was exclusively observed at the 6th day. In the second part, some techno-functional features of germinated whole wheat flour were assessed to estimate its potential as an alternative to conventional flour. Functionality comparison of the non-germinated versus germinated flours revealed that germination significantly influenced solvents retention capacities as well as swelling and solubility. Thus, with a reduced amount of celiac disease triggering peptides, but also with different technological behavior compared to traditional wheat flour. [ABSTRACT FROM AUTHOR]

D-amino acids can affect the action of digestive enzymes, hence the protein digestion. In this work the behaviour of the main stomach and gut digestive enzymes (pepsin, trypsin, and chymotrypsin) in the presence of D-amino acids in the protein chain was monitored over time using a model peptide, Ac-LDAQSAPLRVYVE-NH 2 (belonging to β-lactoglobulin, position 48–60), where L-amino acids were systematically substituted by D-amino acids. The results showed several changes in the behaviour of digestive enzymes, not only when the D-amino acids are inserted at the specific cleavage sites (after Val-57), but in some cases also when in distant positions. The effect seemed more pronounced in the case of pepsin rather than the gut enzymes, possibly indicating a better resilience of the upper gut phase of digestion to racemization. These results demonstrated that racemization could impair nutritional value by slowing down digestibility and has different effects according to the enzyme/amino acids involved. [Display omitted] • Racemization reduced the proteolytic process impairing the nutritional value • D-amino acid appears to affect more pepsin activity than trypsin/chymotrypsin • The slowing down of cleavage is not only observed at the preferred sites • Protein cleavability changed depending on the enzyme and/or amino acid involved [ABSTRACT FROM AUTHOR]

[Display omitted] • The essential amino acids profile is comparable for both analogue and beef burgers. • Protein integrity of plant- and animal-based products was not affected by treatments. • INFOGEST in vitro model showed good protein solubilisation for both protein sources. • High prevalence of collagen-deriving peptides was observed in beef digestates. • The quality of raw material affects digestibility regardless of the protein source. Nowadays, consumers are increasingly inclined toward plant-based meat analogues for sake of food security, safety, and sustainability. This growing interest, not only from consumers but also from food companies, brought the offer on the market to be wide and vast. From our previous study it emerged that the market supply, especially the Italian one, is diversified both in terms of protein sources and nutrient content. Although these products are increasingly consumed, for most of the meat analogues today on the market, little is still known about their actual protein quality and digestibility. To fill this gap, in this study different commercial plant-based burgers (2 soy-based and 2 pea-based) were selected and compared to two beef burgers, as controls, in terms of protein quality and digestibility. The findings of this study demonstrated the essential amino acidic profile lacks lysine for almost all burgers (including the meat-based ones) compared to the amino acid scoring pattern set by FAO/WHO (for older children and adults), even if the sum of essential amino acids was within the range of sufficiency. All samples showed good initial protein integrity with low hydrolysis (above 6%) and percentage of D-enantiomers (above 15%). The study of the digestibility, performed by the validated INFOGEST in vitro model, showed better protein solubilisation in the case of meat burgers (63 ± 3% and 61 ± 8%), but a good digestibility also in the case of plant-based ones (from 55% to 40%). The degree of hydrolysis of the solubilised proteins was very high in all samples (from 65% to 40%) indicating a very good protein accessibility to digestive enzymes. The analysis of the peptide fraction of digestates indicated a high prevalence of collagen proteins in beef burgers and of reserve proteins in plant-based burgers. This study showed that the differences between these products are mostly dependent on the quality of the raw materials used, rather than on the vegetal or animal protein source. Therefore, to have a product with a good protein quality and digestibility, independently from the protein origin, the consumer needs to make an accurate choice, carefully reading the ingredient list. [ABSTRACT FROM AUTHOR]

Old wheat genotypes are perceived by consumers as healthier than modern ones. The release of gluten peptides with in vitro digestion and the content of potentially prebiotic carbohydrates (i.e. resistant fraction of starch and cell-wall associated dietary fiber) were evaluated in tetraploid wheats, namely 9 old and 3 modern Triticum turgidum ssp. genotypes. Simulated digestion of wholemeal flours yielded 152 major peptides, 59 of which were attributed a sequence. Principal component analysis revealed that peptide profiles were variable in old genotypes, unlike in modern ones. Digestion of old genotypes generally yielded peptides in greater concentration. In particular, 5 peptides of γ-gliadin, known to trigger the adaptive immune reaction, and two peptides of α-gliadin, known to be toxic to celiac patients, were particularly abundant in some old varieties. Resistant starch (RS) was negligible in modern genotypes (<0.6%), but it was remarkably abundant in some old varieties, reaching the highest value in Dauno III (8.5%, P < 0.05). Dauno III also presented the highest amount of soluble fiber (4.2%, P < 0.05). Pasta was made with an old and a modern genotype (Dauno III and PR22D89, respectively) with opposite RS content. Pasta making and cooking affected starch digestibility, overtaking differences between genotypes and yielding the same amount of RS for both the varieties (approx. 1.7%). The data herein presented suggest that the wholemeal flours of old tetraploid wheat genotypes could not boast particular claims associated to a lower exposure to gluten peptides and, if cooked, to a prebiotic potential. Unlabelled Image • Gluten peptides, fiber, and starch were analyzed in old and modern wheat genotypes. • Peptides associated to celiac disease were released abundantly in some old genotypes. • Resistant starch is abundant in the wholemeal of some old wheat genotypes. • Pasta cooking overtook differences in resistant starch content between genotypes. • Old wheats cannot claim prebiotic potential or low gluten compared with the new ones. [ABSTRACT FROM AUTHOR]

Abstract Food frauds are a critical issue in the field of food safety and quality. Given the high added value, and the complexity of the matrix, processed meat products are among those most susceptible of adulteration. Despite all the efforts made by the official control authorities and by the food industry to counteract these frauds, the undeclared replacement of meat species with cheaper ones is still widespread. The meat species allowed for food consumption are many, and their specific and accurate detection in highly processed food products requires very sensitive and selective analytical methods. In this work, a LC-MS method was developed to identify and quantify eight different meat species (duck, rabbit, chicken, turkey, buffalo, equine, deer and sheep) in a complex food matrix, such as Bolognese sauce. After protein extraction and trypsin digestion, a species-specific peptide marker for each species was chosen for qualification and quantification. The method was validated on real Bolognese sauce samples prepared in an industrial environment, showing a good sensitivity (LOD 0.2–0.8% on whole finished product) and the possibility, using specifically defined calibration lines, to quantify the amount of meat present coming from different species. Highlights • Species-specific marker peptides were identified for eight meat species. • A LC-MS based method was developed for the detection of the marker peptides. • Bolognese sauces were prepared with increasing amount of each species. • Using proper calibration curves the method can quantify the meat species present. • A validation was performed analyzing blind Bolognese sauce samples. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simplified whey protein-catechins system was prepared in the same ratio as in milk chocolate. • Interaction between whey proteins and catechins determined by (chiro-)optical spectroscopy. • Protein hydrolysis/solubilisation of whey proteins during digestion unaffected by catechins. • Milk chocolate proteins completely digested. • Interactions of catechins with milk proteins does not impair in vitro protein digestibility. Flavonoids are largely present in plant food such as cocoa and derived products. These compounds can interact with proteins inherently contained in the food matrix and/or the proteolytic enzymes involved in gastrointestinal digestion. The flavonoid/protein interaction might hamper protein bioaccessibility and digestibility, affecting the nutritional quality. However, information on the digestion fate of proteins in food matrices containing both proteins and flavonoids is limited. The aim of this work was to evaluate the interaction between proteins and flavonoids and verify the potential effects of this interaction on protein digestibility. Taking milk chocolate as model, first a simple whey proteins/catechins mixed system was evaluated, and then the effects on digestibility were also verified in a real sample of commercial milk chocolate. The effects of the catechins/whey proteins interaction in the model system were evaluated by optical and chiro-optical spectroscopy, outlining a slight protein structure modification upon interaction with catechins. The digestibility of the protein fraction both in the model system, with and without catechins, and also in milk chocolate, was then determined by the application of INFOGEST in vitro digestion method: the bioaccessibility was evaluated in terms of protein hydrolysis and protein solubilisation, and major peptides generated by the digestion were also determined by LC/HR-MS. Despite the slight interaction with proteins, flavonoids were found to not hinder nor modify protein solubilization, protein hydrolysis and peptide profile by digestive enzymes. Also protein digestibility in milk chocolate, evaluated by SDS-PAGE, was found to be complete. The present data clearly indicate that the interaction of the proteins with the flavonoids present in the cocoa matrix does not to affect protein bioaccessibility during digestion. [ABSTRACT FROM AUTHOR]

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPβ transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD. [ABSTRACT FROM AUTHOR]

Coeliac disease is an autoimmune enteropathy that develops in genetically predisposed subjects after the ingestion of gluten or related proteins. Coeliac disease has an increasing incidence in the last years in western countries and it has been suggested that wheat breeding might have contributed to select more toxic forms of gluten. In this work, we analysed gluten peptides generated by in vitro digestion of different old and modern Triticum varieties, using LC-MS. We concluded that old varieties analysed produced a higher quantity of peptides containing immunogenic and toxic sequences than modern ones. Thus old wheat lines are not to be considered “safer” for subjects that are genetically predisposed to celiac disease. [ABSTRACT FROM AUTHOR]

Abstract: Gliadin content of wheat is highly variable, both qualitatively and quantitatively, in function of the plant genetic and of the growing conditions. The gliadins are among the major triggers of celiac disease: peptides derived from the gastrointestinal digestion of these proteins and absorbed from the lamina propria cause the immunological reactions that damage the villous structure in affected subjects. In the present work, the peptide mixtures generated by simulated gastrointestinal digestion of the prolamin fraction extracted from different wheat varieties (Triticum turgidum subsp. durum and Triticum aestivum) were characterized by LC/MS and LC-MS/MS techniques. Peptides related to the amount of α-gliadin, as well as toxic and immunogenic peptides for celiac patients have been identified and quantified using an isotopically labeled internal standard. The quantification demonstrated strong differences among the varieties tested. Some samples, belonging to the same varieties and/or cultivation area, showed a lower α-gliadin content, and a smaller amount of toxic and immunogenic peptides. [Copyright &y& Elsevier]

Abstract: Lys-based ‘chiral box’ Peptide Nucleic Acids (PNAs with three adjacent 2D-Lys-based chiral monomers) have shown unsurpassed specificity in DNA recognition. In this Letter, the binding performances of arginine-based chiral PNAs were evaluated for PNAs containing in the middle part of the strand either a 2D,5L-Arg monomer or three adjacent 2D-; 2D,5L-; 5L-Arg monomers (‘Extended Chiral Box’), a combination never studied before. The binding performances of the PNAs were studied by evaluating the melting temperatures of fullmatch and mismatch PNA–DNA and PNA–RNA hybrids and by studying their structure by circular dichroism (CD). The data indicated that the arginine side chains inserted in the PNA structure are perfectly equivalent to lysine side chains as far as oligonucleotide recognition is concerned. The insertion of an ‘Extended Chiral Box’ into PNA differently influences the binding properties to DNA and RNA: the additional side chains had no observable effect on binding affinity and selectivity toward DNA, whereas, seemed to slightly disturb the binding affinity to RNA but at the same time highly enhancing the recognition selectivity. [ABSTRACT FROM AUTHOR]

• Insects are proposed in Europe as protein sources for feed and food application. • Methods for insect-containing ingredient authentication needed. • Marker peptides were identified for Lesser Mealworm and Black Soldier Fly species. • Three species- specific markers were identified as most suitable for authentication by untargeted and targeted LC/MS methods. • The species-specific marker peptides were detected in insect-enriched (down to 1%) feed formulations. Insects have been proposed as new source of proteins to meet the growing demand connected to the increasing world population. In the EU the inclusion of insect proteins in feed and food is strictly regulated. Hence, analytical methods able to discriminate and identify different insect species in food and feed are a necessity. In this work, a peptidomic approach was applied to determine peptide biomarkers for two edible insect species: lesser mealworm and black soldier fly. Three species specific peptide biomarkers were identified using LC-MS/MS. The two insects were mixed with fish standard feed at different concentrations, to evaluate the feasibility of their use as markers in complex matrices. The detection of marker peptides was confirmed down to 1% insect amount. The data here reported constitutes the first proof of concept for the potential application of the peptide marker approach for the identification and quantification of insect ingredients. [ABSTRACT FROM AUTHOR]