The Diverse Potential of Gluten from Different Durum Wheat Varieties in Triggering Celiac Disease: A Multilevel In Vitro, Ex Vivo and In Vivo Approach.

The reasons behind the increasing prevalence of celiac disease (CD) worldwide are still not fully understood. This study adopted a multilevel approach (in vitro, ex vivo, in vivo) to assess the potential of gluten from different wheat varieties in triggering CD. Peptides triggering CD were identified and quantified in mixtures generated from simulated gastrointestinal digestion of wheat varieties (n = 82). Multivariate statistics enabled the discrimination of varieties generating low impact on CD (e.g., Saragolla) and high impact (e.g., Cappelli). Enrolled subjects (n = 46) were: 19 healthy subjects included in the control group; 27 celiac patients enrolled for the in vivo phase. Celiacs were divided into a gluten-free diet group (CD-GFD), and a GFD with Saragolla-based pasta group (CD-Sar). The diet was followed for 3 months. Data were compared between CD-Sar and CD-GFD before and after the experimental diet, demonstrating a limited ability of Saragolla to trigger immunity, although not comparable to a GFD. Ex vivo studies showed that Saragolla and Cappelli activated immune responses, although with great variability among patients. The diverse potential of durum wheat varieties in triggering CD immune response was demonstrated. Saragolla is not indicated for celiacs, yet it has a limited potential to trigger adverse immune response.

Characterization of Defatted Products Obtained from the Parmigiano-Reggiano Manufacturing Chain: Determination of Peptides and Amino Acids Content and Study of the Digestibility and Bioactive Properties.

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.

Occurrence of non-proteolytic amino acyl derivatives in dry-cured ham.

Proteolysis is the most important event occurring during maturation of dry-cured hams: it strongly influences the flavour and the texture of the aged ham by the accumulation of peptides and free amino acids released by protein hydrolysis. Apart from compounds of proteolytic origin, it has been demonstrated that also non-proteolytic amino acyl derivatives (γ-glutamyl amino acids, pyroglutamyl-amino acids and lactoyl-amino acids) may accumulate during ripening of cheese, and they can be also found in fermented soy sauce, where they contribute to the umami taste of the products. Using a semi-quantitative analysis, in this paper we report the occurrence of significant amounts of γ-glutamyl amino acids and, for the first time, pyroglutamyl-amino acids and lactoyl-amino acids, in aged ham. The amino acid counterparts were mainly found to be hydrophobic amino acids. The amount of these compounds was found to increase with time, because they are not degraded by proteolytic activity. They were also found to be stable to simulated gastrointestinal digestion. Angiotensin Converting Enzyme inhibitory activity was also tested, but they were not found to be characterized by significant ACE-inhibitory activity.

Composition of black soldier fly prepupae and systematic approaches for extraction and fractionation of proteins, lipids and chitin.

Black soldier fly (BSF, Hermetia illucens) constitutes an economic way to convert residual biomasses into a valuable source of biomolecules, such as proteins, lipids and chitin. The present investigation was undertaken to evaluate the feasibility of applying different extraction protocols, either chemical extractions or enzymatic assisted extraction, to recover pure fat, protein and chitin fractions. First, exact proximate composition, total amino acids, fatty acids profile, and N-acetylglucosamine content of the prepupae samples were determined. BSF prepupae biomass contained, expressed on dry weight, 32% proteins, 37% lipids, 19% minerals, 9% chitin. The lipid fraction was easily recovered by organic solvents, while the most challenging issue was the separation of protein from chitin. The best separation was obtained by alkali extraction of proteins (96% of protein recovered) albeit with loss in their integrity as indicated by the measurement of the degree of hydrolysis with the o-phthaldialdehyde method. To avoid protein damage in alkali media, a stepwise protein extraction adopting milder conditions was also explored based on Osborne fractionation method, allowing the recovery of >85% of BSF high purity and high quality proteins, and the obtainment of chitin-enriched fraction as well. The possibility of using an enzymatic assisted extraction of proteins was also explored, obtaining a maximum nitrogen solubilisation in the best case (with Bacillus licheniformis protease) of about 60%. In this latter case, the chitin fraction obtained also had a significant residual protein content.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Hybrid in Silico/in Vitro Approach for the Identification of Angiotensin I Converting Enzyme Inhibitory Peptides from Parma Dry-Cured Ham.

The bioactivity assessment of foodborne peptides is currently a research area of great relevance, and, in particular, several studies are devoted to the antihypertensive effects through the inhibition of angiotensin I converting enzyme (ACE). In the present work, a straightforward workflow to identify inhibitory peptides from food matrices is proposed, which involves a hybrid in vitro/in silico tandem approach. Parma dry-cured ham was chosen as case study. In particular, the advantage of using the hybrid approach to identify active sequences (in comparison to the experimental trials alone) has been pointed out. Specifically, fractions obtained by in vitro gastrointestinal digestion of ham samples of 18 and 24 months of aging have been assessed for ACE inhibition. At the same time, the released peptidomic profiles, which cannot be entirely evaluated by using in vitro assays, have been screened for the inhibition by using an in silico model. Then, to identify novel inhibitory sequences, a series of strong candidates have been synthesized and assessed for their inhibitory activity through in vitro assay. On the one hand, the use of computational simulations appeared to be an effective strategy to find active sequences, as confirmed by in vitro analysis. On the other hand, strong inhibitory sequences were identified for the first time in Parma dry-cured ham (e.g., LGL and SFVTT with IC50 values of 145 and 395 µM, respectively), which is a product of international dietary and economic relevance. Therefore, these findings demonstrate the usefulness of in silico methodologies coupled to in vitro tests for the identification of potentially bioactive peptides, and they give an important contribution to the study of the overall nutritional value of Parma ham.

Isolation and full characterisation of a potentially allergenic lipid transfer protein (LTP) in almond.

Non-specific lipid transfer proteins (nsLTP) were shown to be among the most significant allergens, in particular in several fruits belonging to the Rosaceae family. The molecular features of LTPs, such as the presence of eight cysteine residues forming four disulfide bridges, confer a compact structure, decreasing the probability of degradation due to cooking or digestion, thereby increasing the chance of systemic absorption and severe allergic reactions. Few studies on LTP-induced allergies regarding almond (Prunus dulcis L) are available in the literature. In the present work, we describe for the first time the extraction and purification of an almond LTP, achieving its full characterisation by using liquid chromatography and exact mass spectrometry; the full sequence was identified by means of LC-ESI-Orbitrap-MS applying a bottom-up approach. The characterised protein consists of 92 amino acids and has a calculated exact MW of 9579.0. The presence of four disulfide bridges was confirmed after reduction, as shown by a mass increment of 8 Da. Finally, its potential allergenicity was confirmed via an in silico approach. The results presented here demonstrate the enormous potential of advanced MS techniques for obtaining high-quality structural and functional data of allergenic proteins in a short time.

Electrospray MS and MALDI imaging show that non-specific lipid-transfer proteins (LTPs) in tomato are present as several isoforms and are concentrated in seeds.

Non-specific lipid-transfer proteins (nsLTPs) are major human allergens in many plant species, albeit their role in plant biochemistry is still undefined. They are found in many plant species, either as one or several isoforms according to the species, and usually they are found to concentrate in the outer part of the fruits. In this work, the characterization of tomato nsLTP isoforms was performed on the three main fractions of Piccadilly tomato fruit (peel, pulp and seeds) by using ultracentrifuge devices with molecular cut-off able to retain proteins with molecular weight typical of plant LTPs. The isolated proteins were further analysed by LC-MS, in order to investigate the occurrence and the localization of tomato LTP isoforms. The chromatographic retention times, the molecular masses, the presence of eight cysteine residues in their tertiary structures and the sequence information obtained by MS, although not complete yet, allowed us to identify four different LTP isoforms, not yet reported in the literature, which were found to be concentrated in the seed fractions. None of the molecular masses of these potential LTPs was already present in the UniProtKB/SwissProt database. MALDI imaging experiments confirmed their presence and main localization in seeds, although the actual data hinted at their presence around seeds, rather than exactly in them. These data hint to a complicated scenario concerning LTP proteins in tomato.

Enzymatic production and degradation of cheese-derived non-proteolytic aminoacyl derivatives.

Gamma-glutamyl-amino acids, lactoyl-amino acids and pyroglutamyl-amino acids, collectively named Non-Proteolytic Aminoacyl Derivatives (NPADs) are unusual aminoacyl derivatives of non-proteolytic origins found in consistent amount in several cheeses. Although their enzymatic origin arising from lactic acid bacteria has been demonstrated, the exact enzymes originating them, the ones eventually degrading them and also their resistance to digestive enzymes in the human gastrointestinal tract and in the blood serum after eventual absorption are still unknown. In this paper, pure enzymes and biological media were tested on NPAD and their aminoacidic precursors, for identifying the conditions favoring bioproduction and biodegradation of these compounds. Pure gamma-glutamyl-phenylalanine and its precursor (glutamic acid and phenylalanine), also in the isotopically labeled forms, were tested with Parmigiano-Reggiano extracts, blood serum and different pure enzymes, including typical digestion enzymes (pepsin, trypsin and chymotrypsin), gamma-glutamyl transpeptidase and carboxypeptidase. The data suggested that their production in cheese, and also their partial degradation, might be due to the action of peptidases and gamma-glutamyl transpeptidase. Anyway, under simulated gastrointestinal digestion and in blood serum these compounds turned out to be perfectly stable, suggesting a potential to be absorbed as such and possibly being transported to the body tissues.

Antimicrobial activity of poultry bone and meat trimmings hydrolyzates in low-sodium turkey food.

This research was aimed at the evaluation of the antimicrobial activity exerted by poultry protein hydrolyzates derived from industrial leftovers added to minced turkey meat, intended for the production of burgers for human consumption. Hydrolyzates were obtained through enzymatic hydrolysis from poultry bone and meat trimmings, as by-products from the poultry industry. Colony forming unit assays, under both laboratory and industrial conditions, were performed to assess microbial growth. Poultry protein hydrolyzates inhibited microbial growth occurring in semi-finished turkey meat during the normal retention period because of their water holding capacity resulting in a decreased water activity. Overall, the findings demonstrated that poultry protein hydrolyzates could decrease mesophilic, psychrophilic, and thermophilic bacterial growth for the entire product shelf-life. Bacterial growth inhibition obtained in minced turkey meat by addition of poultry protein hydrolyzates (1.5%), hygroscopic amino acids mixture (1.5%) or sodium chloride (1%) was similar. It is suggested that the use of hydrolyzates could allow the reduction of salt content in poultry meat based products leading to the production of low-sodium turkey food still maintaining acceptable sensory characteristics.

Antioxidant capacity of water soluble extracts from Parmigiano-Reggiano cheese.

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.

LC/ESI-MS/MS analysis outlines the different fumonisin patterns produced by F. verticillioides in culture media and in maize kernels.

Fumonisins are a family of food-borne mycotoxins with a wide spectrum of toxicological activities, produced by Fusarium verticillioides. Twenty-eight fumonisin analogues have been characterised so far, which can be separated into four main groups, identified as fumonisin A, B, C and P, being fumonisin B the most widely occurring in maize and corn-based food. In this work, major and minor fumonisin analogues produced by F. verticillioides have been determined by the development of a suitable tandem mass spectrometry procedure for target compound identification and quantification. The method has been applied to the determination of the major fumonisins in culture media of F. verticillioides and in mouldy maize. In addition to the main fumonisins produced by F. verticillioides, also secondary compounds such as FB4, FB5, FAs and FCs have been detected in both fungal liquid cultures and contaminated maize samples. The use of this method for quantification of major and minor fumonisins may be useful for an exhaustive evaluation of their occurrence and toxicological relevance in food; moreover, it may be applied for a better definition of the fumonisin biosynthetic pathways in different growing media as well as in maize.

Tolerability of a fully maturated cheese in cow's milk allergic children: biochemical, immunochemical, and clinical aspects.

BACKGROUND: From patients' reports and our preliminary observations, a fully maturated cheese (Parmigiano-Reggiano; PR) seems to be well tolerated by a subset of cow's milk (CM) allergic patients. OBJECTIVE AND METHODS: To biochemically and immunologically characterize PR samples at different maturation stage and to verify PR tolerability in CM allergic children. Seventy patients, with suspected CM allergy, were enrolled. IgE to CM, α-lactalbumin (ALA), ß-lactoglobulin (BLG) and caseins (CAS) were tested using ImmunoCAP, ISAC103 and skin prick test. Patients underwent a double-blind, placebo-controlled food challenge with CM, and an open food challenge with 36 months-maturated PR. Extracts obtained from PR samples were biochemically analyzed in order to determine protein and peptide contents. Pepsin and trypsin-chymotrypsin-pepsin simulated digestions were applied to PR extracts. Each PR extract was investigated by IgE Single Point Highest Inhibition Achievable assay (SPHIAa). The efficiency analysis was carried out using CM and PR oral challenges as gold standards. RESULTS: The IgE binding to milk allergens was 100% inhibited by almost all PR preparations; the only difference was for CAS, mainly α(S1)-CAS. Sixteen patients sensitized to CM tolerated both CM and PR; 29 patients tolerated PR only; 21 patients, reacted to both CM and PR, whereas 4 patients reactive to CM refused to ingest PR. ROC analysis showed that the absence of IgE to BLG measured by ISAC could be a good marker of PR tolerance. The SPHIAa using digested PR preparations showed a marked effect on IgE binding to CAS and almost none on ALA and BLG. CONCLUSIONS: 58% of patients clinically reactive to CM tolerated fully maturated PR. The preliminary digestion of CAS induced by PR maturation process, facilitating a further loss of allergenic reactivity during gut digestion, might explain the tolerance. This hypothesis seems to work when no IgE sensitization to ISAC BLG is detected.

Applications of liquid chromatography-mass spectrometry for food analysis.

HPLC-MS applications in the agrifood sector are among the fastest developing fields in science and industry. The present tutorial mini-review briefly describes this analytical methodology: HPLC, UHPLC, nano-HPLC on one hand, mass spectrometry (MS) and tandem mass spectrometry (MS/MS) on the other hand. Analytical results are grouped together based on the type of chemicals analyzed (lipids, carbohydrates, glycoproteins, vitamins, flavonoids, mycotoxins, pesticides, allergens and food additives). Results are also shown for various types of food (ham, cheese, milk, cereals, olive oil and wines). Although it is not an exhaustive list, it illustrates the main current directions of applications. Finally, one of the most important features, the characterization of food quality (including problems of authentication and adulteration) is discussed, together with a future outlook on future directions.

Common wheat determination in durum wheat samples through LC/MS analysis of gluten peptides.

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.

Growth promotion of Bifidobacterium species by poultry bone and meat trimming hydrolyzate.

UNLABELLED: The growth of bifidobacteria that are employed in the production of functional food is often slow or limited, even on synthetic media. In this study, we investigated whether a peptide hydrolyzate (functional animal protein [FAP]), from poultry bones and meat trimmings, could be a potential source of growth stimulators. The bifidogenic activity of FAP on 18 strains of Bifidobacterium species was assessed via 2 different techniques: turbidimetric measurements and a direct count by fluorescence microscopy. Growth experiments were performed in B12 broth as the basal medium, B12 broth supplemented with N-acetylglucosamine, and B12 broth supplemented with FAP. FAP supplementation yielded the highest maximum optical density (OD) and count values. The use of the microscopic fluorescence counts allowed for better evaluation of the extent of growth and assessment of the viability of cells. FAP from poultry bones and meat trimmings has potential as a growth stimulator for different bifidobacteria of human origin. FAP is a promising ingredient for inclusion in industrial media that are used to culture probiotic strains, including bifidobacteria, because it supports growth very well and maintains cells at a high level of viability. PRACTICAL APPLICATION: Proteinaceous hydrolyzate can be considered a promising ingredient for industrial media that are used to culture probiotic strains, including bifidobacteria, because it improves bacterial growth and maintains cells at a high level of viability.

In vitro digestion assay for determination of hidden fumonisins in maize.

Hidden fumonisins have received great attention in the last years as they have been frequently found in maize products in addition to the free forms. Several papers have shown that interaction with macromolecular components such as protein and starch is at the base of the phenomenon: although the nature of the interaction (covalent or not) is still not clarified, the occurrence of hidden forms is generally revealed by the application of an alkaline hydrolysis procedure. In this study, an in vitro digestion model has been applied to raw maize to evaluate the possible release of hidden fumonisins under gastrointestinal conditions. Upon digestion of the food matrix, an increased amount of total detectable fumonisins was observed in comparison with the analysis on the nondigested matrix, an amount even higher than that calculated through the application of the hydrolysis procedure. Besides the analytical issues, our data have serious implications, since consumers may be exposed to a systematic higher risk than that estimated by conventional techniques.

In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: molecular characterization of the products and assessment of their IgE binding abilities.

A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.

A multiresidual method for the simultaneous determination of the main glycoalkaloids and flavonoids in fresh and processed tomato (Solanum lycopersicum L.) by LC-DAD-MS/MS.

A new multiresidual LC-DAD-MS/MS method for the simultaneous detection of the major flavonoids and glycoalkaloids occurring in tomatoes has been developed and applied to the characterization of seven varieties of raw tomatoes grown in Italy and to their processed products. The new method allowed to determine rapidly the concentration of these secondary metabolites in a single analysis, showing that their content can be quite variable among the different varieties considered. On the contrary, a great depletion in glycoalkaloids and flavonoids is observed after processing, probably due to the harsh thermal treatment, which prevents the recognition of the varieties in the processed products.

Difficulties in fumonisin determination: the issue of hidden fumonisins.

In this paper, the results obtained by five independent methods for the quantification of fumonisins B(1), B(2), and B(3) in raw maize are reported. Five naturally contaminated maize samples and a reference material were analyzed in three different laboratories. Although each method was validated and common calibrants were used, a poor agreement about fumonisin contamination levels was obtained. In order to investigate the interactions among analyte and matrix leading to this lack of consistency, the occurrence of fumonisin derivatives was checked. Significant amounts of hidden fumonisins were detected for all the considered samples. Furthermore, the application of an in vitro digestion protocol to raw maize allowed for a higher recovery of native fumonisins, suggesting that the interaction occurring among analytes and matrix macromolecules is associative rather than covalent. Depending on the analytical method as well as the maize sample, only 37-68% of the total fumonisin concentrations were found to be extractable from the samples. These results are particularly impressive and significant in the case of the certified reference material, underlying the actual difficulties in ascertaining the trueness of a method for fumonisin determination, opening thus an important issue for risk assessment.