Allelic variation of gliadin-encoding genes in a collection of tetraploid wheat genotypes

Wheat is one of the main crops bred worldwide. Durum wheat, specifcally, is a key element of the Mediterranean diet, representing an élite crop grown in Italy. Durum wheat nutritional and technological values are largely due to the grain protein content (GPC), a complex genetic trait strongly afected by environmental factors and management practices. In the last decades, several breeding programs have been focused on improving GPC by both traditional and innovative approaches. Among seed storage proteins, prolamins, including both gliadins and glutenins, represent the major component. These two classes of proteins are indeed responsible of gluten formation and confer the extensibility and elasticity to the dough. Besides being of crucial importance for both technological properties and rheological characteristics, prolamins, and especially gliadins, have been found to be major triggers for human health, as involved in a number of wheat consumption-related conditions, such as the celiac disease, non-celiac gluten sensitivity, defned as the onset of a variety of manifestations related to wheat, rye and barley ingestion, and wheat allergies, both due to wheat ingestion or inhalation (of four or pollen). The identifcation of loci responsible for the gliadin expression, and particularly of polymorphism in the aforementioned genes, which could result in a lower immunogenic/toxic potential, could be of great importance in breeding programs. For this purpose, we screened a collection of tetraploid wheat genotypes for allelic variants of annotated gliadin genes in the durum wheat genome, in order to identify genetic resources available to breeders to improve wheat nutritional and technological properties. Phylogenetic analysis among diferent species of Triticum genus and an in silico expression data analysis may also be useful in the exploitation of the complex scenario of gliadin–glutenin interaction and gluten role in the adverse reactions due to wheat consumption

New insight into microbial degradation of mycotoxins during anaerobic digestion

Abstract Anaerobic digestion represents an interesting approach to produce biogas from organic waste materials contaminated by mycotoxins. In this study a shotgun metagenomic analysis of lab-scale bioreactors fed with mycotoxin-contaminated silage has been carried out to characterize the evolution of microbial community under the operating conditions and the key enzymatic activities responsible for mycotoxin degradation. The study was conducted at two different level of contamination for fumonisins and aflatoxin B1. After 15 days biogas production was not influenced by the presence of mycotoxins. Metagenomic analysis revealed that a high contamination rate of mycotoxins interfere with microbial diversity. Degradation of mycotoxins accounted in about 54% for aflatoxin B1 and 60% for fumonisins. The degradation activity of fumonisins resulted in the presence of partially hydrolyzed forms in both tested contamination levels. Accordingly, metagenomic functional analysis revealed the presence of two new carboxylesterase genes belonging to D. bacterium and P. bacterium putatively involved in fumonisin degradation

Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation

Cow’s milk is considered the best wholesome supplement for children since it is highly enriched with micro and macro nutrients. Although the protein fraction is composed of more than 25 proteins, only a few of them are capable of triggering allergic reactions in sensitive consumers. The balance in protein composition plays an important role in the sensitization capacity of cow’s milk, and its modification can increase the immunological response in allergic patients. In particular, the heating treatments in the presence of a food matrix have demonstrated a decrease in the milk allergenicity and this has also proved to play a pivotal role in developing tolerance towards milk. In this paper we investigated the effect of thermal treatment like baking of cow’s milk proteins that were employed as ingredients in the preparation of muffins. A proteomic workflow was applied to the analysis of the protein bands highlighted along the SDS gel followed by western blot analyses with sera of milk allergic children in order to have deeper information on the impact of the heating on the epitopes and consequent IgE recognition. Our results show that incorporating milk in muffins might promote the formation of complex milk–food components and induce a modulation of the immunoreactivity towards milk allergens compared to milk baked in the oven at 180 °C for ten minutes. The interactions between milk proteins and food components during heating proved to play a role in the potential reduction of allergenicity as assessed by in vitro tests. This would help, in perspective, in designing strategies for improving milk tolerance in young patients affected from severe milk allergies

Peanut digestome: Identification of digestion resistant IgE binding peptides

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

Microwave-Assisted Extraction of Bioactive Compounds from Lentil Wastes: Antioxidant Activity Evaluation and Metabolomic Characterization

The recovery of industrial by-products is part of the zero-waste circular economy. Lentil seed coats are generally considered to be a waste by-product. However, this low-value by-product is rich in bioactive compounds and may be considered an eco-friendly source of health-promoting phytochemicals. For the first time, a sustainable microwave-assisted extraction technique was applied, and a solvent screening was carried out to enhance the bioactive compound content and the antioxidant activity of green and red lentil hull extracts. With respect to green lentil hull extracts that were obtained with different solvents, the aqueous extract of the red lentil seed coats showed the highest total phenolic and total flavonoid content (TPC = 28.3 ± 0.1 mg GAE/g dry weight, TFC = 1.89 ± 0.01 mg CE/100 mg dry weight, respectively), as well as the highest antioxidant activity, both in terms of the free radical scavenging activity (ABTS, 39.06 ± 0.73 mg TE/g dry weight; DPPH, IC50 = 0.39 μg/mL) and the protection of the neuroblastoma cell line (SH-SY5Y, IC50 = 10.1 ± 0.6 μg/mL), the latter of which has never been investigated so far. Furthermore, a metabolite discovery analysis was for the first time performed on the aqueous extracts of both cultivars using an HPLC separation which was coupled with an Orbitrap-based high-Resolution Mass Spectrometry technique

Critical review on proteotypic peptide marker tracing for six allergenic ingredients in incurred foods by mass spectrometry

Peptide marker identification is one of the most important steps in the development of a mass spectrometry (MS) based method for allergen detection, since the robustness and sensitivity of the overall analytical method will strictly depend on the reliability of the proteotypic peptides tracing for each allergen. The European legislation in place issues the mandatory labelling of fourteen allergenic ingredients whenever used in different food formulations. Among these, six allergenic ingredients, namely milk, egg, peanut, soybean, hazelnut and almond, can be prioritized in light of their higher occurrence in food recalls for undeclared presence with serious risk decision. In this work, we described the results of a comprehensive evaluation of the current literature on MS-based allergen detection aiming at collecting all available information about proteins and peptide markers validated in independent studies for the six allergenic ingredients of interest. The main features of the targeted proteins were commented reviewing all details available about known isoforms and sequence homology particularly in plant-derived allergens. Several critical aspects affecting peptide markers reliability were discussed and according to this evaluation a final short-list of candidate markers was compiled likely to be standardized and implemented in MS methods for allergen analysis

Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1

Effect of thermal/pressure processing and simulated human digestion on the immunoreactivity of extractable peanut allergens

Peanut allergy is one of the most widespread types of food allergies especially affecting developed countries. To reduce the risk of triggering allergic reactions, several technological strategies have been devised to modify or remove allergens from foods. Herein we investigated the combination of high temperature and pressure on the modulation of peanuts immunoreactivity after simulated gastro-duodenal digestion. Extractable proteins of raw and autoclaved peanuts were separated on SDS-PAGE and immunogenicity was assessed by ELISA and Western Blot analyses. Proteins surviving the heat treatment and reacting towards allergic patients' sera were analysed and attributed to Ara h 3 and Ara h 1 proteins by untargeted LC-high resolution-MS/MS. A progressive reduction in the intensity of the major allergen proteins was also highlighted in the protein fraction extracted from autoclaved peanuts, with a total disappearance of the high molecular allergens when samples were preliminary exposed to 2 h hydration although the lower molecular weight fraction was not investigated in the present work. Furthermore, raw and processed peanuts underwent simulated digestion experiments and the IgE binding was assessed by using allergic patients' sera. The persistence of an immunoreactive band was displayed around 20 kDa. In conclusion, the synergistic effects of heat and pressure played a pivotal role in the disappearance of the major peanut allergens also contributing to the significant alteration of the final immunoreactivity. In addition, the surviving of allergenic determinants in peanuts after gastrointestinal breakdown provides more insights on the fate of allergenic proteins after autoclaving treatments

Validation of a MS Based Proteomics Method for Milk and Egg Quantification in Cookies at the Lowest VITAL Levels: An Alternative to the Use of Precautionary Labeling

The prevalence of food allergy has increased over the last decades and consequently the food labeling policies have improved over the time in different countries to regulate allergen presence in foods. In particular, Reg 1169 in EU mandates the labelling of 14 allergens whenever intentionally added to foods, but the inadvertent contamination by allergens still remains an uncovered topic. In order to warn consumers on the risk of cross-contamination occurring in certain categories of foods, a precautionary allergen labelling system has been put in place by food industries on a voluntary basis. In order to reduce the overuse of precautionary allergen labelling (PAL), reference doses and action limits have been proposed by the Voluntary Incidental Trace Allergen Labelling VITAL project representing a guide in this jeopardizing scenario. Development of sensitive and reliable mass spectrometry methods are therefore of paramount importance in this regard to check the contamination levels in foods. In this paper we describe the development of a time-managed multiple reaction monitoring (MRM) method based on a triple quadrupole platform for milk and egg quantification in processed food. The method was in house validated and allowed to achieve levels of proteins lower than 0.2 mg of total milk and egg proteins, respectively, in cookies, challenging the doses recommended by VITAL. The method was finally applied to cookies labeled as milk and egg-free. This method could represent, in perspective, a promising tool to be implemented along the food chain to detect even tiny amounts of allergens contaminating food commodities