First nanoplate digital PCR method to trace allergenic foods: Improved sensitivity for the detection of sesame.

Sesame (Sesamum indicum L.) is an important allergenic food whose presence can be the cause of severe allergic reactions in sensitised individuals. In this work, nanoplate digital PCR (ndPCR) was used to develop two methods to detect trace amounts of sesame in processed foods and compared with previously proposed real-time PCR assays. Two independent ndPCR approaches were successfully advanced, achieving sensitivities of 5 and 0.1 mg/kg of sesame in dough/biscuits, targeting the CO6b-1 and ITS regions, respectively. The sensitivity using both targets was improved by one order of magnitude comparing with real-time PCR and was not affected by food processing. CO6b-1 system was not influenced by food matrix, exhibiting similar performance regardless the use of complex matrix extracts or serial diluted DNA. Herein, ndPCR was proposed for the first time for the detection of allergenic foods with the advantage of providing better performance than real-time PCR regarding sensitivity and robustness.

Correction: Exploiting Locusta migratoria as a source of bioactive peptides with anti-fibrosis properties using an in silico approach.

Correction for 'Exploiting Locusta migratoria as a source of bioactive peptides with anti-fibrosis properties using an in silico approach' by Carla S. S. Teixeira et al., Food Funct., 2024, 15, 493-502, https://doi.org/10.1039/D3FO04246D.

Exploiting Locusta migratoria as a source of bioactive peptides with anti-fibrosis properties using an in silico approach.

Edible insects have been proposed as an environmentally and economically sustainable source of protein, and are considered as an alternative food, especially to meat. The migratory locust, Locusta migratoria, is an edible species authorised by the European Union as a novel food. In addition to their nutritional value, edible insects are also sources of bioactive compounds. This study used an in silico approach to simulate the gastrointestinal digestion of selected L. migratoria proteins and posteriorly identify peptides capable of selectively inhibiting the N-subunit of the somatic angiotensin-I converting enzyme (sACE). The application of the molecular docking protocol enabled the identification of three peptides, namely TCDSL, IDCSR and EAEEGQF, which were predicted to act as potential selective inhibitors of the sACE N-domain and, therefore, possess bioactivity against cardiac and pulmonary fibrosis.

Quantitative Real-Time PCR for the Detection of Allergenic Species in Foods.

Food allergy is an increasing challenge to public health, with widespread global distribution. With no cure for this pathology, the food-allergic individuals are forced to adopt food eviction measurements, relying on label information to avoid consuming the offending foods. To safeguard these individuals, the analytical methods based on real-time PCR approaches are currently faced as excellent tools to verify labeling compliance, aiding industry and regulatory agencies to efficiently manage food allergen control programs. Therefore, this chapter intends to describe a protocol of real-time PCR to analyze allergenic food species. For method development, the main steps to be considered are (i) in silico sequence analysis and primer/hydrolysis probe design, (ii) preparation of calibrators (model foods containing the allergenic ingredient), (iii) efficient DNA extraction from complex food matrices, (iv) amplification by real-time PCR with hydrolysis probe (90-200 bp) targeting a highly specific DNA region (allergen-encoding gene), (v) sequencing PCR products for identity confirmation, and (vi) validation and application to commercial foods. Herein, a real-time PCR approach for the detection and quantification of cashew nut as an allergenic food is described as an example protocol, including all the steps for method development and validation.

Edible Insects as a Novel Source of Bioactive Peptides: A Systematic Review.

The production of food and feed to meet the needs of the growing world's population will soon become a serious challenge. In search for sustainable solutions, entomophagy is being proposed as an alternative source of proteins, with economic and environmental advantages when compared to meat. Edible insects are not only a valuable source of important nutrients, but their gastrointestinal digestion also originates small peptides with important bioactive properties. The present work intends to provide an exhaustive systematic review on research articles reporting bioactive peptides identified from edible insects, as demonstrated by in silico, in vitro, and/or in vivo assays. A total of 36 studies were identified following the PRISMA methodology, gathering 211 potentially bioactive peptides with antioxidant, antihypertensive, antidiabetic, antiobesity, anti-inflammatory, hypocholesterolemia, antimicrobial, anti-severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), antithrombotic, and immunomodulatory properties, originated from the hydrolysates of 12 different insect species. From these candidates, the bioactive properties of 62 peptides were characterized in vitro and 3 peptides were validated in vivo. Data establishing the scientific basis of the health benefits associated with the consumption of edible insects can be a valuable contribution to overcoming the cultural issues that hinder the introduction of insects in the Western diet.

An in silico approach to unveil peptides from Acheta domesticus with potential bioactivity against hypertension, diabetes, cardiac and pulmonary fibrosis.

Entomophagy is a sustainable alternative source of proteins for human nutrition. Acheta domesticus is one of the three insect species that complies with the European Union Regulation on novel foods, but to date, there are no reports on their potential bioactive peptides. In this study, an in silico approach was applied to simulate the gastrointestinal (GI) digestion of six A. domesticus proteins and identify new peptides with potential anti-hypertensive and/or anti-diabetic properties, resulting from their capability to inhibit the somatic Angiotensin-I converting enzyme (sACE) and/or dipeptidyl peptidase 4 (DPP-4), respectively. A molecular docking protocol was applied to evaluate the binding interactions between the 43 peptides ranked with high probability of being bioactive and three drug targets: DPP-4 and two catalytic domains (N- and C-) of sACE. Five peptides (AVQPCF, CAIAW, IIIGW, DATW and QIVW) showed high docking scores for both enzymes, suggesting their potential to inhibit the DPP-4 and both catalytic domains of sACE, thus possessing multifunctional bioactive properties. Two peptides (PIVCF and DVW) showed higher docking scores for the N-domain of sACE, indicating a potential action as selective inhibitors and consequently with anti-cardiac and pulmonary fibrosis bioactivities. This is the first study identifying peptides originated from the simulated GI digestion of A. domesticus with potential activities against hypertension, diabetes, cardiac and pulmonary fibrosis.

Plasmonic genosensor for detecting hazelnut Cor a 14-encoding gene for food allergen monitoring.

A plasmonic nanostructure was constructed as a biorecognition element coupled to an optical sensing platform in sandwich format, targeting the hazelnut Cor a 14 allergen-encoding gene. The analytical performance of the genosensor presented a linear dynamic range between 100 amol L-1 and 1 nmol L-1, a limit of detection (LOD) < 19.9 amol L-1, and a sensitivity of 13.4 ± 0.6 m°. The genosensor was successfully hybridized with hazelnut PCR products, tested with model foods, and further validated by real-time PCR. It reached a LOD <0.001% (10 mg kg-1) of hazelnut in wheat material (corresponding to 1.6 mg kg-1 of protein) and a sensitivity of -17.2 ± 0.5 m° for a linear range of 0.001%-1%. Herein, a new genosensing approach is proposed as a highly sensitive and specific alternative tool with potential application in monitoring hazelnut as an allergenic food, protecting the health of sensitized/allergic individuals.

Monitoring Yellow Mealworm (Tenebrio molitor) as a Potential Novel Allergenic Food: Effect of Food Processing and Matrix.

The consumption of insects has increased in western countries, raising concerns about their potential to induce food allergic reactions in sensitized/allergic individuals. This work intended to develop a real-time PCR approach for the detection/quantification of yellow mealworm (Tenebrio molitor) as a potential allergenic food in complex matrices. For this purpose, reference mixtures simulating the production of pork sausages and wheat biscuits containing known amounts of mealworm were used. Real-time PCR with TaqMan probe targeting the cytochrome b gene of T. molitor was able to detect up to 2 fg of insect DNA, and 1.0 and 0.1 mg/kg of mealworm flour in autoclaved sausages and baked biscuits, respectively. Generally, the method showed acceptable analytical performance parameters, confirming its suitability/applicability for a wide range of foods. However, real-time PCR data showed significant differences among food matrix and processing, highlighting the importance of using appropriate calibration models for quantitative analysis. Finally, the real-time PCR approach was successfully validated with blind mixtures and applied to commercial samples, demonstrating its efficacy and reliability in the quantification of mealworm in processed foodstuffs.

Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

Sesame as a source of food allergens: clinical relevance, molecular characterization, cross-reactivity, stability toward processing and detection strategies.

Sesame is an allergenic food with an increasing allergy prevalence among the European/USA population. Sesame allergy is generally life-persisting, being the cause of severe/systemic adverse immune responses in sesame-allergic individuals. Herein, clinical data about sesame allergy, including prevalence, diagnosis, relevance, and treatments are described, with focus on the molecular characterization of sesame allergens, their cross-reactivity and co-sensitization phenomena. The influence of food processing and digestibility on the stability/immunoreactivity of sesame allergens is critically discussed and the analytical approaches available for their detection in foodstuffs. Cross-reactivity between sesame and tree nuts or peanuts is frequent because of the high similarities among proteins of the same family. However, cross-reactivity phenomena are not always correlated with true clinical allergy in sensitized patients. Data suggest that sesame allergens are resistant to heat treatments and digestibility, with little effect on their immunoreactivity. Nevertheless, data are scarce, evidencing the need for more research to understand the effect of food processing on sesame allergenicity modulation. The demands for identifying trace amounts of sesame in foods have prompted the development of analytical methods, which have targeted both protein and DNA markers, providing reliable, specific, and sensitive tools, crucial for the effective management of sesame as an allergenic food.

Authentication of Argan (Argania spinosa L.) Oil Using Novel DNA-Based Approaches: Detection of Olive and Soybean Oils as Potential Adulterants.

Argan oil is a traditional product obtained from the fruits of the argan tree (Argania spinosa L.), which is endemic only to Morocco. It is commercialized worldwide as cosmetic and food-grade argan oil, attaining very high prices in the international market. Therefore, argan oil is very prone to adulteration with cheaper vegetable oils. The present work aims at developing novel real-time PCR approaches to detect olive and soybean oils as potential adulterants, as well as ascertain the presence of argan oil. The ITS region, matK and lectin genes were the targeted markers, allowing to detect argan, olive and soybean DNA down to 0.01 pg, 0.1 pg and 3.2 pg, respectively, with real-time PCR. Moreover, to propose practical quantitative methods, two calibrant models were developed using the normalized ΔCq method to estimate potential adulterations of argan oil with olive or soybean oils. The results allowed for the detection and quantification of olive and soybean oils within 50-1% and 25-1%, respectively, both in argan oil. Both approaches provided acceptable performance parameters and accurate determinations, as proven by their applicability to blind mixtures. Herein, new qualitative and quantitative PCR assays are proposed for the first time as reliable and high-throughput tools to authenticate and valorize argan oil.

Single-tube nested real-time PCR versus normalised real-time PCR for the quantification of allergenic cashew nut in foods: Impact of thermal processing and matrix.

In this work, three allergen-encoding genes (Ana o 1, Ana o 2, Ana o 3) were investigated for the detection of cashew nut as an allergenic food. Normalised and single-tube nested real-time PCR approaches targeting the Ana o 2 or Ana o 3 genes are proposed and compared. Normalised real-time PCR detected 10 pg, while single-tube nested real-time PCR achieved 1 pg of cashew nut DNA. Single-tube nested real-time PCR targeting Ana o 3 allowed the best relative sensitivities (10 mg/kg cashew nut in dough/biscuit), being successfully validated regarding precision/accuracy. The normalised real-time PCR did not show acceptable accuracy for both targets. Sensitivity of single-tube nested real-time PCR was affected by the matrix (pasta), but not by thermal processing (dough/biscuit). Herein, two highly sensitive and specific single-tube nested real-time PCR targeting allergen-encoding genes are proposed for the first time as quantitative/validated tools for cashew nut analysis as an allergenic food.

Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

New applications of advanced instrumental techniques for the characterization of food allergenic proteins.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Are Physicochemical Properties Shaping the Allergenic Potency of Animal Allergens?

Key determinants for the development of an allergic response to an otherwise 'harmless' food protein involve different factors like the predisposition of the individual, the timing, the dose, the route of exposure, the intrinsic properties of the allergen, the food matrix (e.g. lipids) and the allergen modification by food processing. Various physicochemical parameters can have an impact on the allergenicity of animal proteins. Following our previous review on how physicochemical parameters shape plant protein allergenicity, the same analysis was proceeded here for animal allergens. We found that each parameter can have variable effects, ranging on an axis from allergenicity enhancement to resolution, depending on its nature and the allergen. While glycosylation and phosphorylation are common, both are not universal traits of animal allergens. High molecular structures can favour allergenicity, but structural loss and uncovering hidden epitopes can also have a similar impact. We discovered that there are important knowledge gaps in regard to physicochemical parameters shaping protein allergenicity both from animal and plant origin, mainly because the comparability of the data is poor. Future biomolecular studies of exhaustive, standardised design together with strong validation part in the clinical context, together with data integration model systems will be needed to unravel causal relationships between physicochemical properties and the basis of protein allergenicity.

The first post-natal clinical description of true mosaic complete tetrasomy 21: A case report.

Tetrasomy 21 is a rare occurrence. Only 14 cases have been reported in the literature, 8 of which are partial tetrasomy cases and 6 which are complete tetrasomy cases. Of the incidences, no proband with true complete tetrasomy 21 has survived the neonatal period. We report complete mosaic tetrasomy 21 in a female infant with the typical Down syndrome phenotype, including Hirschsprung's disease and atrioventricular (AV) canal defect. This is in contrast to cases of partial tetrasomy 21, which often have an atypical trisomy 21 presentation and multiple nonspecific traits, including short stature, microcephaly, and developmental delays. This case demonstrates the difference in clinical presentation between the partial and complete subtype of tetrasomy 21 and provides the first postnatal clinical picture of an infant with true mosaic complete tetrasomy 21.

Milk Ingredients in Meat Products: Can Autoclaving and In Vitro Gastroduodenal Digestion Mitigate Their IgE-Binding Capacity?

The food industry commonly uses milk ingredients as technological aids in an uncounted number of products. On the other hand, milk contains allergenic proteins causing adverse allergic reactions in sensitized/allergic individuals. This work intends to evaluate the effect of autoclaving and in vitro digestion on the allergenicity of milk proteins incurred in meat products. Protein profiles of raw and autoclaved sausages without and with the addition of 10% of milk protein concentrates were analyzed by gel electrophoresis and liquid chromatography-mass spectrometry. Additionally, residual IgE-reactivity was evaluated by immunoblot analysis using pooled sera of cow's-milk-allergic individuals followed by bioinformatic analysis. Results showed that autoclaving led to an increase in protein fragmentation (higher number of short peptides) and consequently to a higher digestion rate, that was found to be more pronounced in ß-casein. The IgE-binding capacity of milk proteins seems to be reduced after autoclaving prior to digestion, with a residual reactivity in caseins, but was eliminated following digestion. This study highlights the importance of autoclaving as a processing strategy to produce hypoallergenic formulas.

Lupine allergens: Clinical relevance, molecular characterization, cross-reactivity, and detection strategies.

Lupine is commonly utilized as a technological food and ingredient in a great variety of processed products (snacks, bakery, meat, and dairy products) principally owing to its nutritional value and technological properties. However, its ingestion, even at trace amounts (in the range of mg protein per kg of food), can lead to severe adverse reactions in allergic individuals. Lupine belongs to the Leguminosae family, having the conglutins (α-, ß-, δ-, and γ-) as allergens, among other proteins. Cross-sensitization of lupine-sensitized individuals with other legume species, mainly peanut, can occur, but the associated clinical reactivity is still unclear. The protection of the sensitized individuals should depend on an avoidance diet, which should rely on the compliance of food labeling and, as such, on their verification by analytical methods. Food processing, such as heat treatments, has an important influence on the structural properties of lupine proteins, altering their detectability and allergenicity. In this review, different aspects related with lupine allergy are described, namely, the overall prevalence, clinical relevance, diagnosis, and treatment. The characterization of lupine allergens and their potential cross-reactivity with other legumes are critically discussed. The effects of food matrix, processing, and digestibility on lupine proteins, as well as the available analytical tools for detecting lupine at trace levels in foods, are also herein emphasized.

Effects of ohmic heating on the immunoreactivity of ß-lactoglobulin - a relationship towards structural aspects.

ß-Lactoglobulin (ß-LG) encompasses important biological functions, but it is also a major milk allergen, being responsible for the high incidence of cow's milk allergy in childhood. Despite numerous studies addressing the effect of thermal processing on the immunoreactivity of ß-LG, little is known about the impact of emergent food processing technologies, such as ohmic heating (OH), on its immunoreactivity. This study describes, for the first time, the effects of thermal and electric effects of OH on the immunoreactivity of ß-LG. The influence of high-temperature short time (HTST; 72.5 °C for 15 and 90 °C for 1 s) and low-temperature long time (LTLT; 65 °C for 30 min) pasteurization binomials combined with different electrical variables (electric field intensity and electrical frequency) was evaluated and compared with that of conventional heating. HTST at 90 °C for 1 s extensively affects the protein secondary structure and increases the levels of reactive aggregates, resulting in increased total immunoreactivity (assessed by ELISA and immunoblotting). OH at 4 V cm-1 and a frequency of 25 kHz reduced the immunoreactivity of monomeric ß-LG and its resultant aggregates, compared with conventional heat treatments. In contrast, the presence of electric fields (4 V cm-1 and 25 kHz) during LTLT increased the immunoreactivity of monomeric ß-LG. The thermal and electrical variables of OH change the balance between monomeric and aggregated forms of ß-LG, affecting their ability to bind specific antibodies. These outcomes bring novel insights into how to modulate OH aiming at reducing the allergenic potential of ß-LG based products.

Immunoreactivity of Lupine and Soybean Allergens in Foods as Affected by Thermal Processing.

Lupine and soybean are important technological aids for the food industry. However, they are also capable of inducing severe allergic reactions in food-sensitized/allergic individuals. In this context, this work intended to study the combined effects of thermal processing and food matrix on the immunoreactivity of lupine and soybean proteins used as ingredients in bakery and meat products, respectively. For this purpose, the effects of baking, mild oven cooking, and autoclaving on the protein profiles were evaluated, using model mixtures simulating the production of lupine-containing breads and soybean-containing cooked hams/sausages, by native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting using specific antibodies. The results showed that lupine gamma-conglutin immunoreactivity was slightly decreased in wheat flour mixtures compared to rice, but it was more pronounced in baked products. In meat mixtures, substantial protein fragmentation was noted after autoclaving, with decreased immunoreactivity of soybean trypsin inhibitor. The analysis of 22 commercial products enabled the identification of lupine gamma-conglutin in four bakery samples and soybean trypsin-inhibitor in five sausages, and further differentiated autoclaved from other milder thermally treated products. Generally, the immunoreactivity of target proteins was reduced by all the tested thermal treatments, though at a higher extent after autoclaving, being slightly altered by the food matrix.