Barbara Prandi

Qualitative and quantitative determination of peptides related to celiac disease in mixtures derived from different methods of simulated gastrointestinal digestion of wheat products

AbstractDuring 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. FigureIn depth determination of peptides related to celiac disease after simulated gastrointestinal digestion of wheat products

LC/MS analysis of proteolytic peptides in wheat extracts for determining the content of the allergen amylase/trypsin inhibitor CM3: Influence of growing area and variety

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 bakers 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.

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.

Polar Lipid Profile of Nannochloropsis oculata Determined Using a Variety of Lipid Extraction Procedures

Lipid compositions obtained from microalgae species are affected by both the cultivation conditions and the extraction method used. In this study, the extraction of lipids from Nannochloropsis oculata using traditional and modern extraction technologies with several solvents has been compared. Because important polyunsaturated fatty acids are bound to polar lipids, these polar lipids were the main focus of this study. The dominant compounds in the glycolipid fractions were monogalactosyldiglycerides and digalactosyldiglycerides bearing fatty acid chains containing at least one site of unsaturation. Phosphatidylcholine and trimethylhomoserines were detected in the phospholipid fractions. The fatty acid profile comprised large fractions of C16:0, C16:1, C20:5, and C18:3. Extraction of specific compounds was determined by extraction efficiency as well as differences in the selectivity of the method used. The composition derived from a glycolipid fraction was observed to be affected by the method used to a greater extent than the phospholipid fraction.

Enzymatic production of pectic oligosaccharides from onion skins

Onion skins are evaluated as a new raw material for the enzymatic production of pectic oligosaccharides (POS) with a targeted degree of polymerization (DP). The process is based on a two-stage process consisting of a chelator-based crude pectin extraction followed by a controlled enzymatic hydrolysis. Treatment of the extracted crude onion skins pectin with various enzymes (EPG-M2, Viscozyme and Pectinase) shows that EPG-M2 is the most appropriate enzyme for tailored POS production. The experiments reveal that the highest amount of DP2 and DP3 is obtained at a time scale of 75-90min with an EPG-M2 concentration of 26IU/mL. At these conditions the production amounts 2.5-3.0% (w/w) d.m for DP2 and 5.5-5.6% (w/w) d.m for DP3 respectively. In contrast, maximum DP4 production of 5.2-5.5% (w/w) d.m. is obtained with 5.2IU/mL at a time scale of 15-30min. Detailed LC-MS analysis reveals the presence of more methylated oligomers compared to acetylated forms in the digests.

Influence of fermentation level and geographical origin on cocoa bean oligopeptide pattern.

Peptides and amino acids generated during cocoa bean fermentation are the most important precursors for the development of cocoa aroma, however cocoa oligopeptide fraction is under-investigated. In this study, we perform a deep investigation of the presence of oligopeptides in unfermented, under fermented, and well-fermented cocoa beans from all of the main producing countries, with the aim to obtain a better definition of cocoa quality and a deeper comprehension of biochemical changes occurring during fermentation. Oligopeptides were determined by UPLC/ESI-MS and 35 low-molecular weight peptides were identified and subjected to semi-quantitative analysis. Results showed that fermented cocoas can be differentiated from unfermented cocoas by their possession of a higher total amount of oligopeptides and a lower ratio of vicilin to 21kDa cocoa seed albumin peptides. A variability in the peptide pattern was observed also among well-fermented cocoa samples of different geographical origin, suggesting diversified proteolytic activities.

Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: assessment of allergen resistance and characterization of the peptides by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry.

RATIONALE Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. METHODS Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. RESULTS The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. CONCLUSIONS An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

Gliadin-mediated production of polyamines by RAW264.7 macrophages modulates intestinal epithelial permeability in vitro

Celiac disease (CD) is an immune-mediated enteropathy sustained by dietary gluten in susceptible individuals, and characterized by a complex interplay between adaptive and innate responses against gluten peptides (PTG). In a recent contribution we have demonstrated that the treatment with PTG induces the expression and activity of arginase in both murine macrophages and human monocytes from healthy subjects, thus suggesting a role for arginine and its metabolites in gluten-triggered response of these cells. Here we further explore this field, by addressing the effects of PTG on polyamine synthesis and release in murine RAW264.7 macrophages, and how they affect epithelial permeability of Caco-2 monolayers. Results obtained show a massive production and release of putrescine by macrophages upon incubation with gluten peptides; this, in turn, causes a decrease in TEER in epithelial cells, indicating that PTG-driven secretion of polyamines by macrophages has a role in the modulation of intestinal permeability in vitro. At a molecular level, putrescine production appears referable to the activation of C/EBPβ transcription factor, which is known to be responsible for arginase induction in activated macrophages and is a crucial mediator of inflammation. Whether these pathways are stimulated also in vivo deserves to be further investigated, as well as their role in gluten-driven cellular and intestinal defects typical of CD patients.

Identification and quantification of different species in animal fibres by LC/ESI‐MS analysis of keratin‐derived proteolytic peptides

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.

Current Trends in Ancient Grains-Based Foodstuffs: Insights into Nutritional Aspects and Technological Applications

For centuries, ancient grains fed populations, but due to their low yield, they were abandoned and replaced by high-yielding species. However, currently, there is a renewed interest in ancient wheat and pseudocereal grains from consumers, farmers, and manufacturers. Ancient wheat such as einkorn, emmer, spelt, and Kamut®, are being reintegrated because of their low fertilizer input, high adaptability and important genetic diversity. New trends in pseudocereal products are also emerging, and they are mostly appreciated for their nutritional outcomes, particularly by the gluten-free market. Toward healthier lifestyle, ancient grains-based foodstuffs are a growing business and their industrialization is taking 2 pathways, either as a raw ingredient or a functional ingredient. This paper deals with these grain characteristics by focusing on the compositional profile and the technological potential.