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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.

Wheat consumption can represent one of the nutritional factors involved in the onset of diabetes. We specifically investigated the potential diabetogenic effects of Hammurabi, a T. monococcum wheat cultivar, in non-obese diabetic (NOD) mice and analysed the levels of resistant starch in pasta manufactured with Hammurabi after in vitro gastroduodenal digestion. NOD mice were fed with Hammurabi, bread wheat or rice flour to evaluate diabetes incidence and insulitis score. An enzymatic method was applied to compare the content of resistant starch in Hammurabi pasta and durum wheat pasta (control). In NOD mice, the Hammurabi-based diet significantly delayed diabetes onset (p = 0.0042) and reduced insulitis score compared to rice or wheat-based diet. Furthermore, the resistant starch value following in vitro digestion of Hammurabi pasta was significantly higher (4.08%) than that of durum wheat pasta (2.28%). Taken together, these results highlighted the potential positive effects of the Hammurabi-based diet on diabetes incidence.

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.

Aim of this work was to evaluate the impact of bread enrichment with different concentrations of lentil flour (LF) (0 g/100g, 10 g/100g and 20 g/100g) on some physico-chemical, structural, and nutritional features of bread. The addition of LF in formulation affected its properties, leading to a darker appearance and to a more compact structure (e.g., higher density and lower gas bubble area fraction). Besides, it led to a significant rise in the protein, free glucose, and rapidly digestible starch content as well as in the expected glycemic index. Concerning the investigation of nutritional properties, starch and protein digestibility has been evaluated by the combination of different digestion models. Each type of bread was subjected to an in vivo oral phase followed by a semi-dynamic in vitro gastrointestinal digestion. During the oral and the gastric phases of digestion, bread with the greatest LF content (20 g/100g) exhibited an extensive starch digestibility and a partial protein digestibility compared to the other samples, due to its higher protein content. In contrast, during the intestinal phase, control (0 g/100g) showed a stronger starch digestibility compared with the LF samples, while no differences could be observed in terms of protein digestibility investigated by SDS-PAGE., This work has received financial support from project PID2019-107663RB-I00 from the Spanish Ministry of Science and Innovation (MICINN).

Alternative sources of edible proteins are required to feed the world's growing population, such as Moringa oleifera leaves, a protein source with a balanced amino acid composition. Since Moringa leaf proteins is a novel food in the EU and UK, an assessment of their potential allergenicity of is required. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential.

Digestomics can be defined as the systematic characterization of the biomolecular complement produced by food digestion. Protein digestomics, that essentially corresponds to tracking the fate of dietary proteins in human body, assumes special relevance because food-derived peptides are virtually capable of inducing positive (health-promoting) or negative (allergy, adverse reactions) effects. In general, food digestion generates extremely heterogeneous mixtures, which include free amino acids and variously sized (poly) peptides, in addition to products from other macro- and micronutrients. Modern proteomics and peptidomics have enabled the comprehensive study of complex digestomes. On the other hand, several gaps still need to be bridged to define kinetics and dynamics of food-derived peptides, also as a result of food structure, in order to ultimately assess the effects of dietary proteins on human health. This chapter surveys the recent advances in the comprehension of mechanisms of food degradation, highlighting research trends and still unaddressed issues.

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.

In these last years, consumers'choices are being directed towards healthier food and beverages with an increasing demand for functional products. In this study, we investigated the sensory and biochemical properties of novel drinks based on tomato juice. To this purpose, different blends were formulated mixing fresh tomato juice with other fruit juices and nectars in different proportions and then assayed to investigate their sensory (panel test), compositional and biochemical characteristics. Our results indicated that it is possible to formulate tasty drinks based on tomato juice with improved nutritional properties. The combinations of red fruits/tomato (60/40 v/v) and red fruits/orange/tomato (40/30/30 v/v/v) showed a sugar content lower than those of different soft drinks on the market including energy drinks, and suitable lycopene levels as well. Interestingly, the blended red fruits/orange/tomato had a greater number of polyphenols and vitamin C, a softer tomato flavour and high sensory appreciation. High pasteurization (90ºC, 7 min), performed to increase storability, did not significantly affect sensory and biochemical properties of drinks. These achievements may be useful to modulate tomato flavour release and consumer acceptability of novel drinks based on tomato juice.

Wheat, an essential ingredient for several bakery preparations, is also responsible for gluten-related diseases in sensitive subjects. The effect of the N fertilization rate (80 vs 160 kg N ha−1) on gluten protein expression profile has been evaluated considering two soft wheats (landrace and modern) and one tritordeum cultivar (cv), grown in the same experimental field in North Italy. The proteins of refined flour were characterized through advanced proteomic approaches, including chromatography (RP-HPLC) and electrophoresis. A static model system was used to simulate in vitro digestion and the digestome peptides were examined by mass spectrometry and in silico approaches, to investigate the celiac and allergenic sequences. The CD-toxic epitopes in the digested samples were quantified by means of a R5 ELISA assay. The N fertilization rate increased the grain protein content, but it did not lead to any difference in gluten composition, with exception of glu/glia ratio in the modern wheat cv. Moreover, the gluten composition and the occurrence of toxic/allergenic epitopes varied to a great extent, according mostly to the genotype. A lower immunoreactivity, determined using R5 ELISA, was detected for the digested tritordeum flours than for the landrace (-51%) or modern (-58%) cvs, while no significant difference was observed for the N rates between each genotype. In silico analysis showed that tritordeum has fewer CD epitopes belonging to the ω-gliadins and a lower LMW-GS than the landrace or modern cv. Tritordeum presented fewer α-gliadin allergenic epitopes than the modern wheat cv. The lower frequency of celiac epitopes in tritordeum, compared to the old and the modern wheat, is probably due to the absence of a D genome.

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The use of lentil flour in bakery (bread, cake, crackers), extruded (pasta, snacks) and other products (dressings, soups, dairy and meat products) is gaining attention of food technologists and of industry as well as popularity among consumers due to an excellent and balanced nutritional composition. This research interest has extended our knowledge of nutritional and functional properties (solubility, emulsification, gelation, foaming) of lentil flour, which, in turn, has disclosed its technological potential for preparation of high-quality foods (gluten free bakery, yogurt and meat products). However, addition of lentil flour may introduce technological problems and novel allergens. This review covers the use of lentil flour in food preparations, with focus on protein and starch digestibility and on the challenges to design lentil-based products with improved quality.

Egg proteins are among the major food allergens. Very often, the same pasta-making plants are used for industrial production of egg-based pasta (EBP) and semolina-only pasta (SP), so that residual egg proteins may be present in SP. This calls for defining the amount of semolina pasta that should be discarded when switching production lines. In this study, the egg proteins content was measured in pasta samples taken at various times after switching production lines from EBP to SP Both long and short pasta shapes were sampled before and after a drying step. Protocols meant to circumvent the difficulties associated with detecting egg proteins in a complex matrix after processing were set up for using commercial ELISA kits to monitor the disappearance of egg proteins from the products. The use of both denaturants and disulphide reductants to solubilise egg proteins was found to be mandatory, as verified by ovalbumin detection by ELISA and by using mass spectrometry to assess residual egg white lysozyme. Appropriate sample preparation protocols were used to monitor the progressive disappearance of egg proteins in the products when shifting production lines in an industrial pasta plant, providing a basis for credible, reliable, and consistent self-control procedures. For lines with a production capacity of 2200-2400 kg h-1, the amount of material to be discarded to ensure that products meet the strictest analytical requirements has been found to be around 2000-3000 kg (for long pasta) and 3000-4000 kg (for short pasta).

The aim of this study was to evaluate the dynamics of proteolysis during dough fermentation started with different lactic acid bacteria species, through the identification of intermediate and small-sized peptides generated during fermentation. Single-strain cultures of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Companilactobacillus alimentarius, and Leuconostoc pseudomesenteroides were assayed as sourdough starters. Assays were carried out at lab-scale for 48 h of fermentation, using both unstarted and yeast-leavened dough as controls. Physicochemical and microbiological analyses were combined with peptidomic and proteomic profiling, identifying several hundreds of peptides mainly released from the water-soluble wheat proteins, including β-amylase, triticin, and serpins. Both α- and γ-gliadins were hydrolyzed, though only at the N-terminal domain, while the central protein region – encrypting celiac disease epitopes– remained unaffected. The bacterial-mediated consumption of sugars and the concomitant hydrolysis of starch degrading β-amylase could underlie improved digestibility and several nutritionally beneficial effects of sourdough baked products.

Celiac disease (CeD) is an autoimmune enteropathy triggered by immunogenic gluten peptides released during the gastrointestinal digestion of wheat. Our aim was to identify T cell epitope-containing peptides after ex vivo digestion of ancestral (einkorn, spelt and emmer) and common (hexaploid) wheat (Fram, Bastian, B&oslash, rsum and Mirakel) using human gastrointestinal juices. Wheat porridge was digested using a static ex vivo model. Peptides released after 240 min of digestion were analyzed by liquid chromatography coupled to high-resolution mass spectrometry (HPLC-ESI MS/MS). Ex vivo digestion released fewer T cell epitope-containing peptides from the ancestral wheat varieties (einkorn (n = 38), spelt (n = 45) and emmer (n = 68)) compared to the common wheat varieties (Fram (n = 72), B&oslash, rsum (n = 99), Bastian (n = 155) and Mirakel (n = 144)). Neither the immunodominant 33mer and 25mer &alpha, gliadin peptides, nor the 26mer &gamma, gliadin peptide, were found in any of the digested wheat types. In conclusion, human digestive juice was able to digest the 33mer and 25mer &alpha, gliadin, and the 26mer &gamma, gliadin derived peptides, while their fragments still contained naive T cell reactive epitopes. Although ancestral wheat released fewer immunogenic peptides after human digestion ex vivo, they are still highly toxic to celiac patients. More general use of these ancient wheat variants may, nevertheless, reduce CeD incidence.

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.

The present study aimed to obtain novel information regarding the pathways of a polyphenol-rich coffee silverskin extract (CSE) for inhibiting the formation of advanced glycation products (AGE). Chlorogenic acid (CGA) is the main phenol present in coffee beans. The contribution of CGA to the antiglycoxidative properties of CSE was also evaluated by analysis of CGA standard. The CGA (90%) and CSE (49%) significantly inhibited (p < 0.001) the formation of fluorescent AGE in a protein–methylglyoxal (MGO) model system (37 °C for 96 h). The lack of MGO-derivative AGE in the presence of CGA and CSE was verified using liquid chromatography–tandem mass spectrometry techniques, which evidenced the almost complete absence of arginine adducts after 24 h of reaction. The inhibitory effect of polyphenols present in CSE may be associated to its carbonyl trapping capacity as well as to its ability to react with side-chains of protein amino residues blocking the reaction sites. In conclusion, aqueous CSE can be considered a natural source of various inhibitors of in vitro formation of AGE acting by different pathways., The present research was supported by SUSCOFFEE project (AGL57239-R, 2014). B. Fernandez-Gomez thanks the FPI Ph.D. program of MINECO for supporting her research career (BES-046827, 2011). This investigation was in collaboration Spain-Italy thanks a mobility grant (EEBB-I-14-08220, 2014).

The use of ingredients based on plant protein isolates is being promoted due to sustainability and health reasons. However, it is necessary to explore the behaviour of plant protein isolates during gastrointestinal digestion including the profile of released free amino acids and the characterization of resistant domains to gastrointestinal digestion. The aim of the present study was to monitor protein degradation of four legume protein isolates: garden pea, grass pea, soybean and lentil, using the harmonized Infogest in vitro digestion protocol. In vitro digests were characterized regarding protein, peptide and free amino acid content. Soybean was the protein isolate with the highest percentage of insoluble nitrogen at the end of the digestion (12%), being this fraction rich in hydrophobic amino acids. Free amino acids were mainly released during the intestinal digestion, comprising 21–24% of the total nitrogen content, while the percentage of nitrogen corresponding to peptides ranged from 66 to 76%. Legume globulins were resistant to gastric digestion whereas they were hydrolysed into peptides and amino acids during the intestinal phase. However, the molecular weight (MW) distribution demonstrated that all intestinal digests, except those from soybean, contained peptides with MW > 4 kDa at the end of gastrointestinal digestion. The profile of free amino acids released during digestion supports legume protein isolates as an excellent source of essential amino acids to be used in protein-rich food products. Peptides released during digestion matched with previously reported epitopes from the same plant species or others, explaining the ability to induce allergic reactions and cross-linked reactivity., This work has received financial support from project AGL2015-66886-R from the Spanish Ministry of Economy and Competitiveness (MINECO) and project PID2019-107663RB-I00 from Ministry of Science and Innovation (MICINN). M. S-H. is the recipient of FPU grant (FPU15-03616) from the Ministry of Education, Culture and Sports.

Kashk is a typical dairy product of Iran, made from sour milk. It is traditionally produced from buttermilk in a dry, round-shaped form. Today, it is also produced at industrial level in a liquid form starting from fermented milk. We aimed to characterise the kashk proteome and peptidome comparing a traditional product with the industrial using a combination of proteomic approaches including advanced chromatographic and electrophoretic separation technique coupled to tandem mass spectrometry. We identified also phosphorylated casein-derived peptides (CPP) and investigated kashk protein digestibility using a static model of food protein digestion. The molecular characterization, coupled with bioinformatic in silico analysis, allowed the identification of potential bioactive peptides.

Heat treatments induce chemical/physical modifications, which may affect the stability to enzymatic digestion and consequently the allergenicity of food proteins to a varying extent, depending on the time/temperature regimen. Herein, we evaluated the stability to digestion of whole tree nut (walnuts, hazelnuts and almonds) allergens in a food digestion model reflecting the real one by, taking into consideration the allergen-containing processed (roasted) food. To this aim, whole raw and roasted tree nuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. The degradation of allergens was monitored by integrated proteomic/peptidomic and bio-informatic tools. Roasting increased digestibility of tree nuts, since very few peptides were detected in digested samples (6.5 kDa fraction). After BBM digestion step, the degradation of peptides was enhanced in roasted walnuts and hazelnuts compared to the raw counterpart. Conversely, almond allergens showed a different behaviour, since the presence of resistant peptides was more evident for roasted almonds, probably because of the hydrolysis of high molecular weight aggregates generated during roasting. Our results provide new insight into the relationship between thermal processing and metabolic fate of tree nut allergens, highlighting the importance of investigating the digestion stability of whole allergenic food, rather than purified proteins.

This study investigated the simultaneous impact of food matrix and processing on the food allergy eliciting capacity of peanuts in a physiologically relevant context. Whole raw and roasted peanuts were subjected to in vitro digestion combining the harmonized oral-gastric-duodenal digestion models with brush border membrane enzymes (BBM) to simulate the jejunal degradation of peptides. SDS-PAGE and HPLC analysis showed that roasting increased digestibility of peanuts and this trend was even more evident after BBM degradation. The eliciting properties of raw and roasted peanuts were assessed by Rat Basophil Leukemia assay in the presence of sera from peanut-allergic patients. As general features, the BBM digestion reduced allergenicity of roasted peanuts compared to the raw counterpart, suggesting that intestinal peptidases effectively contribute to further destroy specific domains of peanut allergens. These findings provide new and more realistic insights in the stability of peanut allergens within their natural matrix.

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.

The contribution of cysteine and glutathione conjugates to the formation of thiol compounds in Grechetto wine has been evaluated for the first time, by the use of quantitative GC–MS analysis and odour activity values, as a function of the training system (either Royat cordon or simple Guyot) that was used for the vines. The concentration of conjugate precursors in the grape juice, in the case of both training systems, was found to increase significantly until 22°Brix, except for glutathionylated precursor of 4-methyl-4-sulfanyl-pentan-2-one. The amount of both thiol precursor forms was systematically higher for the Royat cordon system that seems to improve the accumulation of thiol precursors. However, this high juice concentration did not reveal an high content of volatile thiols in derived wine. 4-Methyl-4-sulfanyl-pentan-2-one (4MSP), 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) have been identified as aroma compounds in both two monovarietal white wines from Grechetto grape, but only the amounts of 4MSP and 3SHA turned out to be considerably higher than their respective perception thresholds. Considering the growing interest in the varietal wines characteristics on the part of consumers, the results of present work could be useful to improve the scientific knowledge about the relationships between the grape content and wine composition.

This work aimed to study the opioid peptide β-casomorphin-7 (BCM7) degradation or stability during digestion using human gastrointestinal (GI) juices and porcine jejunal brush border membrane (BBM) peptidases. Synthetic BCM7 was subjected to in vitro digestion by GI fluids obtained from human volunteers for 180 min, and to downstream degradation with porcine BBM vesicles. The BCM7 was sampled at 4 time points over 24 h after BBM addition. The digests were profiled by HPLC-electrospray ionization mass spectrometry (ESI/MS) to monitor BCM7 during GI digestion, and intact BCM7 through BBM digestion was quantified by reverse-phase (RP)-HPLC. We found that BCM7 was partly digested with human GI enzymes, as 3 proteolytic fragments in addition to f(60-66) YPFPGPI were detected: f(62-66) FPGPI, f(60-65) YPFPGP and f(61-66) PFPGPI. The RP-HPLC analysis revealed that 42% of the initial peptide was degraded after only 2 h of BBM digestion, and as much as 79% was degraded after 4-h digestion with supplementation of BBM. In conclusion, this study showed that most of BCM7 was degraded during GI and BBM digestion, although a small amount (5%) was still detected after 24-h digestion. It remains to be studied whether the small amount of intact BCM7 detected after in vitro digestion is transported via active transceptors in the human intestinal epithelial cells and enters blood circulation.

5th International Conference on Foodomics - From Data to Knowledge -- JAN 10-12, 2018 -- Cesena, ITALY, WOS: 000456758800042, PubMed ID: 30599953, In this work, the effects of maturation time and simulated gastrointestinal digestion on the molecular and peptide profiles of "Bresaola Valtellina" were assessed through the foodomics approach, in this case food proteomics and peptidomics combined to other analytical and biological assays, aiming at depicting a holistic food quality. Human digestion of this Italian cured meat product was simulated using an in vitro static protocol and the degree of proteolysis and the in vitro bioactivity of the soluble free compounds in the digestates were evaluated by biochemical assays, e.g. SDS-PAGE, size exclusion HPLC, HPLC/MS, 1H NMR, enzymatic and antioxidant activities. The obtained results demonstrated that in vitro gastrointestinal digestion contributed to a considerable release of myofibrillar proteins by the muscle tissue. Data from SDS-PAGE, peptidomic and size exclusion HPLC assays showed that the in vitro digestion largely degraded proteins of muscle tissue to peptides smaller than 250 Da. The released peptides were likely responsible for the inhibitory activity on amylolytic enzymes and for the antioxidant properties elicited by the gastric digestates of Bresaola. Overall, the results demonstrated the negligible role of ripening in making meat proteins more bioaccessible, whereas they confirmed the highly in vitro digestibility of meat proteins from Bresaola. This study represents a new approach merging proteomics and foodomics to evaluate the effect of ripening and in vitro digestion on the bioactivity and bioaccessibility of proteins and peptides of meat products., COST actionEuropean Cooperation in Science and Technology (COST) [FA1005 INFOGEST], The authors participate in the COST action FA1005 INFOGEST (http://www.cost-INFOGEST.eu/).

This work introduces a novel methodological approach to study both the geometry of complex protein networks and the nature of the interacting proteins. This approach is based on the high reactivity of Au + ions on the surface of gold nanoparticles (AuNPs) towards thiols, that allows fast formation of a covalent bond between accessible protein thiols and AuNPs. In the case of the durum wheat semolina used in the exploratory studies reported here, the nature of proteins covalently bound to AuNPs is expected to be affected by both the compactness of the protein network and by the AuNPs size. Simple centrifugation procedures allowed recovery of the protein-loaded AuNPs that remained soluble, and the protein(s) covalently bound on the surface of soluble AuNP were identified by MS analysis of their proteolytic fragments. Gluten-forming proteins were found to be bound to soluble AuNPs only when detergents or chaotropes were added to the semolina/AuNPs suspension at room temperature. AuNPs-bound proteins also included gluten-forming proteins with no reported free thiols, suggesting that they are piggybacked on other thiol-containing gluten-forming proteins via disulfide bonds already present in the otherwise untreated semolina. The potential of this approach is discussed in terms of the possibility of developing a methodology suitable for further clarification of the geometrical features of protein networks, of the nature of the involved proteins, and of the type of interaction they establish, as well as any modifications of these features upon processing.

Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine ?-lactoglobulin (?-Lg, 2 out 6 samples) and one ?s1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely ?-Lg (f42-54), (f42-57) and ?s1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed ?-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response.

Type-2 diabetes is continuously increasing worldwide. Hence, there is a need to develop functional foods that efficiently alleviate damage due to hyperglycaemia complications while meeting the criteria for a sustainable food processing technology. Inhibition of mammalian alpha-amylase and alpha-glucosidase was studied for white grape skin samples recovered from wineries and found to be higher than that of the drug acarbose. In white grape skins, quercetin and kaempferol derivatives, analysed by UPLC-DAD-MS, and the oligomeric series of catechin/epicatechin units and their gallic acid ester derivatives up to nonamers, analysed by MALDI-TOF-MS were identified. White grape skin was then used for enrichment of a tomato puree (3%) and a flat bread (10%). White grape skin phenolics were found in the extract obtained from the enriched foods, except for the higher mass proanthocyanidin oligomers, mainly due to their binding to the matrix and to a lesser extent to heat degradation. Proanthocyanidin solubility was lower in bread, most probably due to formation of binary proanthocyanin/protein complexes, than in tomato puree where possible formation of ternary proanthocyanidin/protein/pectin complexes can enhance solubility. Enzyme inhibition by the enriched foods was significantly higher than for unfortified foods. Hence, this in vitro approach provided a platform to study potential dietary agents to alleviate hyperglycaemia damage and suggested that grape skin phenolics could be effective even if the higher mass proanthocyanidins are bound to the food matrix.

Wheat beer is a traditional light-colored top-fermenting beer brewed with at least 50% malted (e.g., German Weissbier) or unmalted (e.g., Belgian Witbier) wheat (Triticum aestivum) as an adjunct to barley (Hordeum vulgare) malt. For the first time, we explored the proteome of three Weissbier samples, using both 2D electrophoresis (2DE)-based and 2DE-free strategies. Overall, 58 different gene products arising from barley, wheat, and yeast (Saccharomyces spp.) were identified in the protein fraction of a representative Weissbier sample analyzed in detail. Analogous to all-barley-malt beers (BMB), barley and wheat Z-type serpins and nonspecific lipid transfer proteins dominated the proteome of Weissbier. Several alpha-amylase/trypsin inhibitors also survived the harsh brewing conditions. During brewing, hundreds of peptides are released into beer. By liquid chromatography-electrospray tandem mass spectrometry (LC-ESI MS/MS) analysis, we characterized 167 peptides belonging to 44 proteins, including gliadins, hordeins, and high- and low-molecular-weight glutenin subunits. Because of the interference from the overabundant yeast-derived peptides, we identified only a limited number of epitopes potentially triggering celiac disease. However, Weissbier samples contained 374, 372, and 382 ppm gliadin-equivalent peptides, as determined with the competitive G12 ELISA, which is roughly 10-fold higher than a lager BMB (41 ppm), thereby confirming that Weissbier is unsuited for celiacs. Western blot analysis demonstrated that Weissbier also contained large-sized prolamins immunoresponsive to antigliadin IgA antibodies from the pooled sera of celiac patients (n = 4).

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastroduodenal hydrolysis, while other digestion-stable allergens such as alpha-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastroduodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

The effects of fining with various plant proteins were assessed on Aglianico red wine, using both the young wine and wine aged for twelve and twenty-four months, and including wine unfined or fined with gelatin as controls. Color traits and fining efficiency were considered, along with the content of various types of phenolics and of aroma-related compounds of either varietal or fermentative origin. All agents had comparable fining efficiency, although with distinct kinetics, and had similar effects on wine color. Individual plant proteins and enzymatic hydrolyzates differed in their ability to interact with some anthocyanins, with specific proanthocyanidins complexes, and with some aroma components of fermentative origin. Changes in varietal aroma components upon fining were very limited or absent. Effects of all the fining agents tested in this study on the anthocyanidin components were most noticeable in young red wine, and decreased markedly with increasing wine ageing.

The kinetics of phosphopeptide release from gum fortified with a commercial casein hydrolysate in artificial and human saliva was studied. Using mass spectrometry techniques, the caseinophosphopeptide (CPP) composition of the commercial casein hydrolysate compared with a standard tryptic casein digest from whole raw cow milk was determined. In in vitro trials, the amount of CPPs released was always the same for each chewing time; in contrast, during in vivo chewing, a decrease of peptide levels was detected, from 10% after 5 min to 0.1% at 20 min, due to physiological swallowing. It is concluded that gums are a useful vehicle for CPP intake, owing to their functional role in bone health. However, their use is less satisfactory for prevention of dental caries, due to the brief contact with the enamel surface. Moreover, more calcium would need to be added to enrich the CPP fraction of the commercial hydrolysate.

The study is aimed at developing a new cereal-based product, with increased nutritional quality, by using quinoa flour. The effect of the use of transglutaminase (TGase) and proteolytic enzymes on the microstructure, properties and in vitro digestion of gluten-free bakery products based on quinoa flour was evaluated. Microstructural results evaluated by means of Scanning Electron Microscopy (SEM) showed that the quinoa starch granules are rather small (0.4–2 μm) and the presence of TGase induced significantly changes in dough and baked samples microstructures. The overall acceptability of the breads was improved by TGase addition. The results achieved showed that these enzymes have different effects on the bread characteristics and may improve properties of formulations, setting the basis for development of baked quinoa products.

Enzymatic dough improvers (DIs) are increasingly used as baking co-adjuvants. Herein, an array of techniques, including Western blotting, PCR, electrophoresis-based and shotgun proteomics, was addressed to identify the enzymes in six commercial DI preparations. In particular, this work sought to exclude the possible undeclared use of amylolytic enzymes from porcine (or other animal origin) pancreas in DIs. PCR-amplified mitochondrial cytochrome b (mt cyt b) gene region and porcine pancreatic α-amylase were the targets of DNA-based and protein methods, respectively, both assuring a limit of detection lower than 0.5-0.1% (w/w). Aspergillum oryzae α-amylase and Hordeum vulgare (barley) β-amylase were the most represented enzymes in all DI samples. Although one sample was PCR-positive, none among the DIs contained porcine pancreatic enzymes. Comparative gas chromatographic analysis of fatty acids suggested that the porcine contamination might arise from hard fats of porcine origin (lard), emphasizing the need of performing analyses at the protein level when the targets are enzymes or proteins.

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.

Plant-derived insoluble proteins (wheat gluten, and isolates from pea, lentil, and soybean) were used as fining agents in model white wine (made from Catalanesca grapes) after cold stabilization. Plant proteins were effective in giving a fast and remarkable decrease in turbidity. GC/MS and HPLC/MS approaches indicated that individual proteins had a different impact on the levels of compounds relevant to wine stability. Protein stability of wine was not affected by fining with plant proteins. Lentil proteins and gluten gave the best removal of monomeric and dimeric flavonol. Both caused a decrease in the total content of fermentative aroma compounds, such as ethyl esters, acetate esters, and alcohols. Lentil proteins had the highest impact on the aroma components, giving a marked decrease in aroma components. Gluten may thus be regarded as giving the best balance between fining efficacy and retention of aroma compounds. Also, gluten in the treated wines remained well below the suggested threshold for gluten-free foods. This study provides a methodological frame for thorough characterization of the impact of specific interventions on key wine components.

Proteins determine several key sensory and technological properties of beer. Most of the protein components of beer are barley pathogenesis–related proteins, which endure the harsh brewing treatments. Due to their intrinsic resistance, they can also survive gastrointestinal digestion in humans, evoking immune responses in predisposed individuals. For these reasons, beer proteins have been studied for several decades. However, only the recent development of the mass spectrometry–based proteomic techniques is disclosing the “deep” proteome of beer. Several dozens of barley- and yeast-derived proteins constitute the current “core” proteome of beer, while many others are specific to beer brands or styles. Proteolytic peptides of beer include a multitude of sequences derived from prolamins that are potentially harmful for celiacs. In this chapter, we survey the compositional and functional aspects of the beer proteome/peptidome emphasizing the most recent findings obtained using the newest proteomic approaches.

Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases. Immunogenicity was evaluated by Western Blot using N=8 pooled sera of peanut allergic pediatric subjects. Immunoreactive, large-sized and fragments of Ara h 2, Ara h 6 and Ara h 3 survived hydrolysis as assessed by SDS-PAGE. Smaller resistant peptides mainly arising from Ara h 3 and also Ara h 1 were detected and further identified by LC-high resolution-MS/MS. RP-HPLC purification followed by dot-blot analysis and MS/MS-based identification demonstrated that stable IgE-binding peptides derived from Ara h 3. These results provide a more realistic picture of the potentially allergenic determinants of peanuts that survived the human digestion, taking into account the role of the food matrix, which may significantly affect gastrointestinal breakdown of peanut allergens. (C) 2017 Elsevier Ltd. All rights reserved.

Hazelnut (Corylus avellana) is widely used in food production in raw, roasted, salted, and paste form. Proteins are key components conferring favorable sensory, technological, and functional properties to hazelnut. For these reasons, the protein fraction of hazelnut has been the subject of extensive research over the last years. In addition, the complete annotation of the hazelnut proteome at the molecular level is of great interest, in particular in clinical research because hazelnut proteins can elicit even severe allergic reactions in sensitive subjects. This chapter provides an overview of the current knowledge on the hazelnut proteome.

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.