Fabrication of an Antifouling Surface Plasmon Resonance Sensor with Stratified Zwitterionic Peptides for Highly Efficient Detection of Peanut Allergens in Biscuits.

Peanut allergen monitoring is currently an effective strategy to avoid allergic diseases, while food matrix interference is a critical challenge during detection. Here, we developed an antifouling surface plasmon resonance sensor (SPR) with stratified zwitterionic peptides, which provides both excellent antifouling and sensing properties. The antifouling performance was measured by the SPR, which showed that stratified peptide coatings showed much better protein resistance, reaching ultralow adsorption levels (<5 ng/cm2). Atomic force microscopy was used to further analyze the antifouling mechanism from a mechanical perspective, which demonstrated lower adsorption forces on hybrid peptide coatings, confirming the better antifouling performance of stratified surfaces. Moreover, the recognition of peanut allergens in biscuits was performed using an SPR with high efficiency and appropriate recovery results (98.2-112%), which verified the feasibility of this assay. Therefore, the fabrication of antifouling sensors with stratified zwitterionic peptides provides an efficient strategy for food safety inspection.

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Novel monoclonal antibodies against house dust mite allergen Der p 21 and their application to analyze allergen extracts.

Background: Allergen extracts and recombinant allergens are used in allergy diagnostics and immunotherapy. Since allergen extracts from different manufacturers lack proper standardization regarding their composition, monoclonal antibodies (MAbs) against specific allergen components can be used for their identification and quantification in allergen extracts. This study aimed to generate MAbs against allergen Der p 21 of Dermatophagoides pteronyssinus for the analysis of allergen extracts. Methods: Recombinant Der p 21 was expressed in E. coli and purified using affinity chromatography. MAbs against Der p 21 were generated using hybridoma technology. House dust mite (HDM) allergen extracts were analyzed using the newly developed sandwich enzyme-linked immunosorbent assay, Western blotting and microarray immunoassay. Results: MAbs raised against recombinant Der p 21 were characterized in detail and proven to be reactive with natural Der p 21. Highly specific sandwich enzyme-linked immunosorbent assay for the quantification of Der p 21 was developed and optimized. The allergen was detected and its concentration was determined in only three of six analyzed HDM allergen extracts from different manufacturers. Conclusion: HDM analysis by MAb-based immunoassays shows their differences in allergen composition. The results demonstrate the importance of allergen-specific MAbs as a tool for the characterization of allergen extracts and the need for their appropriate standardization before their use for allergy diagnostics or immunotherapy.

Effects of different high temperature-pressure processing times on the sensory quality, nutrition and allergenicity of ready-to-eat clam meat.

Investigating technologies to control the allergenicity of seafood is particularly important to safeguard consumer health, but there is currently a dearth of research focused on reducing the allergenicity of clam meat. This study aimed to investigate the effects of high temperature-pressure (HTP) processing times (121 °C, 0.14 MPa; 5, 10, 15, 20 min) on the sensory quality, nutrition, and allergenicity of ready-to-eat clam meat. With the extension of HTP time, the hardness of clam meat gradually decreased, the chewiness decreased initially and then increased, and the meat became tender. HTP processing endowed clam meat with abundant esters and aldehydes. Among all the processing groups, the umami and saltiness were better at 15 min, correlating with the highest overall acceptability. Ready-to-eat clam meat contained high-protein nutritional value. Compared with raw clam meat, the tropomyosin allergenicity of clam meat treated with HTP for 15 and 20 min was significantly reduced by 51.9 % and 56.5 %, respectively (P < 0.05). However, there was no significant difference between these two groups. Appropriate HTP processing time might be an efficient condition to reduce the tropomyosin allergenicity of ready-to-eat clam meat and improve its quality, particularly for the time of 15 min. The results of this study could provide a reliable theoretical basis for the development of hypoallergenic clam foods.

Occurrence of alkyl glucosides in rinse-off cosmetics marketed as hypoallergenic or for sensitive skin.

Rinse-off cosmetic products, primarily shampoos, are frequently implicated in the onset of allergic contact dermatitis (ACD) caused by alkyl glucosides (AGs). AGs are increasingly popular surfactants and known contact allergens. Glucoside-induced ACD was most frequently observed with shampoos and skin-cleansing products in both consumer and occupational settings. Thereby, studies have shown that atopic individuals are the most susceptible to ACD. Also, several investigations have indicated that individuals with sensitive skin might be more prone to skin allergies. This is why the presence of AGs was investigated in shampoos and body cleansers marketed as hypoallergenic or for sensitive skin. For this purpose, the website of Amazon.com was surveyed. Four groups of cosmetics were obtained by using the following keywords: "hypoallergenic shampoo for adults," "sensitive skin shampoo for adults," "hypoallergenic body cleanser for adults," and "sensitive skin body cleanser for adults." The first 30 best-selling cosmetics in each group were investigated for the presence of AGs, by analyzing the product information pages. The results showed that as much as 56.7% of hypoallergenic shampoos contained AGs, as ingredients, whereas the percentage was somewhat lower for other product categories. Even though decyl and lauryl glucoside were nearly ubiquitously used AGs in cosmetics over the past decade, the most commonly present AG in our analysis was coco-glucoside. The results of this study indicated a necessity to include coco-glucoside in the baseline series of patch testing allergens. Industry, regulators, and healthcare providers should be made aware of the frequent presence of AGs in rinse-off cosmetic products marketed as hypoallergenic or for sensitive skin to ensure the safety and well-being of consumers and patients.

[Aerobiological study in Lima, Perú. ¿Tipuana-Tipu, perhaps a new allergen?] / Estudio aerobiológico en Lima, Perú. ¿Tipuana-Tipu, tal vez un nuevo alérgeno?

OBJECTIVE: To report the Tipuana tipu pollen as a new allergen capable of triggering allergic symptoms. METHODS: The pollen counts were made according to standardized technique with a Burkard seven days following the European Aerobiology Society´s Network Group recommendations.1 The trap was installed on the roof of Clinica SANNA, El Golf, San Isidro, which is 20 m high, 12°5'54"S 77°3'6"W in the west-south of the Lima urban area. The sampling period was performed from September 2020 to October 2021. Collection of Tipuana tipu pollens and Preparation of Tipuana tipu pollen extracts 1:20 w/v was done using a previously described method.2 We carried out systematic skin prick testing with Tipuana tipu pollen extract and other aeroallergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), molds (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum), cat and dog danders, Periplaneta americana, grass six mix, weed mix (Inmunotek, Spain) on 80 patients (18 to 50 years old) seen in our allergy center, they suffering from november to january rhinitis and/or conjunctivitis symptoms. The majority living near avenues and large green areas, where Tipuana trees grew. RESULTS: We found a total of 952 grains/m3 of Tipuana tipu pollen between November 2020 to january 2021, with the maximum concentration of 37 grains/m3 on December 10th. We also found other airborne pollen Types: Poaceae, Myrtaceae, Compositae and Betulaceae. 14/80 patients (17,5%) showed positive skin prick test only to Tipuana tipu extract. Most of the patients with positive tests to Tipuana extract presented symptoms of rhinoconjunctivitis during the Tipuana pollination period. Four patients showed positive skin prick test to Tipuana tipu and grass 6 mix extracts, most of the rest of our patients were sensitized to dust mites' extracts (Dermatophagoides pteronyssinus). CONCLUSIONS: The west-south population of Lima urban city is exposed to Tipuana tipu pollen. We do not foud previous publications about Tipuana tipu allergy. Almost 18% of the patients tested in our sample were mono-sensitized to this pollen. The results of this study should be compared with data from the forthcoming years, to identify seasonal and annual fluctuations, extend the traps to other locations in Lima, and of course try to standardize and improve the Tipuana tipu pollen extract.

OBJETIVO: Reportar al polen de Tipuana tipu como un nuevo alérgeno capaz de desencadenar síntomas alérgicos. MÉTODOS: Los conteos de polen se realizaron según la técnica estandarizada con un equipo colector tipo Hirst, Burkard spore trap for seven days, siguiendo las recomendaciones del grupo de la Red Europea de Sociedades de Aerobiología1. El equipo se instaló en la azotea de la Clínica SANNA El Golf, San Isidro, a 20 m de altura desde el nivel del suelo, 12°5'54"S 77°3'6"O en la zona suroeste del área urbana de Lima. El periodo de captación se llevó a cabo entre septiembre de 2020 y octubre de 2021. La recolección de granos de polen de Tipuana tipu, y la preparación del extracto alergénico (peso/volumen) 1:20 p/v, se realizó usando metodología previamente descrita2. Se realizaron estudios de pruebas cutáneas (skin prick test), en 80 pacientes (entre 18 y 50 años), con sintomatología de rinoconjuntivitis; referían, además, mayor intensidad de sus síntomas entre noviembre y enero. La mayoría de pacientes dijeron vivir cerca a avenidas y parques donde había árboles de Tipuana tipu. Fueron evaluados en el servicio de Alergología de la Clínica SANNA, El Golf, San Isidro. Se aplicaron extractos de polen de Tipuana tipu, y otros aeroalérgenos como ácaros del polvo (Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis), hongos ambientales (Cladosporium herbarum, Alternaria alternata, Aspergillus fumigatus, Penicillium notatum), epitelios de gato y perro, Periplaneta americana, mezclas de seis gramíneas, mezclas de malezas (Inmunotek, España). RESULTADOS: Encontramos un total de 952 granos/m3 de polen de Tipuana tipu entre noviembre de 2020 y enero de 2021; con la máxima concentración de 37 granos/m3 el 10 de diciembre. También identificamos otras familias polínicas: Poaceae, Myrtaceae, Compositae y Betulaceae. 14/80 pacientes (el 17,5%), resultaron positivos solo al extracto de Tipuana tipu, en el skin prick test. La mayoría de los pacientes con resultado positivo al extracto de Tipuana tipu referían síntomas de rinoconjuntivitis durante el periodo de polinización de los árboles de Tipuana. Cuatro pacientes tuvieron positividad al extracto de Tipuana tipu, y al extracto en mezcla de seis gramíneas; la mayoría del resto de pacientes mostraron sensibilidad a ácaros del polvo doméstico (Dermatophagoides pteronyssinus). CONCLUSIONES: Los habitantes de la zona suroeste de la ciudad urbana de Lima están expuestos al polen de Tipuana tipu. No hemos encontrado publicaciones previas sobre alergia a este tipo de polen. Casi un 18% de pacientes estudiados en nuestra muestra, estuvieron monosensibilizados al extracto del polen de Tipuana tipu. Los resultados de este estudio deberían ampliarse y ser comparados con data en los años siguientes, identificar fluctuaciones estacionales y anuales, extender los captadores a otras locaciones en Lima, y por supuesto, intentar estandarizar y mejorar el extracto del polen de Tipuana Tipu.

[Analysis of the electrophoretic profiles of the protein extracts of the species Manihot esculenta (Crantz, 1975), Persea Americana (Mill., 1768) and Actinidia delicious (A. Chevalier., 1984), and their association with the latex-fruit syndrome for searching for potential allergens]. / Análisis de los perfiles electroforéticos de los extractos proteicos de las especies Manihot esculenta (Crantz,1975), Persea americana (Mill.,1768) y Actinidia deliciosa (A. Chevalier., 1984), y su asociación con el síndrome látex-fruta para la búsqueda de alérgenos potenciales.

OBJECTIVE: Determine the electrophoretic profiles of the extracts of Manihot esculenta, Actinidia Deliciosa and Persea Americana and their possible relationship with Latex-Fruit Syndrome. METHODS: Protein extracts of M. esculenta, P. Americana and A. Deliciosa were prepared through the processes of maceration and solvent extraction from plant samples. In the case of the avocado, a prior extraction by soxhlet was carried out to eliminate the fat. The extracts were vacuum filtered, dialyzed and finally lyophilized. Separation of proteins based on molecular weight was performed by SDS PAGE electrophoresis. The electrophoretic profiles obtained were compared with the allergenic proteins previously identified in the latex extract, in order to determine a possible relationship with Latex-Fruit Syndrome, depending on the molecular weight. RESULTS: The extracts of M. esculenta and P. Americana showed a wide range of protein fractions with molecular weights varying from 10 to 250 KD, finding that the region with the highest concentration of bands was between 20 and 89 KD, (60 and 65%), respectively. A 20-band profile was obtained for the M. esculenta extract (Figure 1), with seven bands sharing similar weights with the latex allergens (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 and Hev b 10) (3-5). For the P. Americana extract, 20 bands were also observed (Figure 2), seven of which presented approximate weights to the Latex allergens (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8 , Hev b 10 Hev b 11 Hev b 14). The Kiwi extract showed two bands of 19.1 and 22.9 KD, with weights close to latex proteins (figure 3), (Hev b 3 and Hev b 6.01), and allergens (Act d 2 and Act d 6), reported in the literature for this fruit. CONCLUSIONS: When analyzing the relationship between the separated protein fractions and the latex allergens described in the literature, a possible association of 35% was found for the extracts of M. esculenta and P. Americana, and 10% for A. Delicious, with great relevance being the association found with the allergens Hev b 4, Hev b 2, Hev 8 and Hev b 11, which are involved in Latex-Fruit Syndrome. The electrophoretic profiles of the prepared extracts were determined and compared with the Latex allergens. This information generates a contribution for the development of new research and advances in the standardization of these extracts on a large scale and for their future use in diagnostic tests.

OBJETIVO: Determinar los perfiles electroforéticos de los extractos de Manihot esculenta, Actinidia deliciosa y Persea americana y su posible relación con el Síndrome de Látex ­ Fruta. MÉTODOS: Se prepararon extractos proteicos de M. esculenta, P. Americana y A. Deliciosa, a través de los procesos de macerado y extracción con solventes a partir muestras vegetales. En el caso del aguacate, se realizó una extracción previa por soxhlet, para eliminar la grasa. Los extractos se filtraron al vacío, se sometieron a diálisis y por último se liofilizaron. La separación de las proteínas en función del peso molecular se realizó mediante electroforesis SDS PAGE. Se compararon los perfiles electroforéticos obtenidos con las proteínas alergénicas previamente identificadas en el extracto de látex, con el fin de determinar una posible relación con el Síndrome de Látex-Fruta, en función del peso molecular. RESULTADOS: Los extractos de M. esculenta y P. americana mostraron una amplia gama de fracciones proteicas con pesos moleculares que varían desde 10 a 250 KD, encontrando que la región con mayor concentración de bandas se situó entre 20 y 89 KD, (60 y 65 %), respectivamente. Se obtuvo un perfil de 20 bandas para el extracto de M. esculenta (figura 1), con siete bandas que comparten pesos similares con los alérgenos del látex (Hev b 1, Hev b 2, Hev b3, Hev b 4, Hev b 5, Hev b 6.03, Hev b 8 y Hev b 10) (3-5). Para el extracto de P. americana, también se observaron 20 bandas (figura 2), siete de las cuales presentaron pesos aproximados a los alérgenos de Látex (Hev b 1, Hev b 2 Hev b 4 Hev b 6.01 Hev b 6.03 Hev b 8, Hev b 10 Hev b 11 Hev b 14). El extracto de Kiwi mostró dos bandas de 19,1 y 22,9 KD, con pesos cercanos a proteínas de látex (figura 3), (Hev b 3 y Hev b 6.01), y los alérgenos (Act d 2 y Act d 6), reportados en la literatura para esta fruta. CONCLUSIONES: Al analizar la relación existente entre las fracciones proteicas separadas y los alérgenos de los látex descritos en la literatura, se encontró una posible asociación del 35% para los extractos de M. esculenta y P. Americana, y del 10% para A. Deliciosa, siendo de gran relevancia la asociación encontrada con los alérgenos Hev b 4, Hev b 2, Hev 8 y Hev b 11, los cuales se encuentran implicados en el Síndrome de Látex-Fruto. Se lograron determinar los perfiles electroforéticos de los extractos elaborados y se compararon con los alérgenos del Látex. Está información genera un aporte para el desarrollo de nuevas investigaciones y avances en la estandarización de estos extractos a gran escala y para su uso futuro en pruebas diagnósticas.

Variation in Shrimp Allergens: Place of Origin Effects on Food Safety Assessment.

Due to the widespread use of shellfish ingredients in food products, accurate food labelling is urgently needed for consumers with shellfish allergies. Most crustacean allergen detection systems target the immunorecognition of the allergenic protein tropomyosin. However, this mode of detection may be affected by an origin-dependent protein composition. This study determined if the geographic location of capture, or aquaculture, influenced the allergenic protein profiles of Black Tiger Shrimp (Penaeus monodon), one of the most farmed and consumed shrimp species worldwide. Protein composition was analysed in shrimp from nine different locations in the Asia-Pacific by SDS-PAGE, immunoblotting, and mass spectrometry. Ten of the twelve known shrimp allergens were detected, but with considerable differences between locations. Sarcoplasmic calcium-binding protein, myosin light chain, and tropomyosin were the most abundant allergens in all locations. Hemocyanin-specific antibodies could identify up to six different isoforms, depending on the location of origin. Similarly, tropomyosin abundance varied by up to 13 times between locations. These findings suggest that allergen abundance may be related to shrimp origin and, thus, shrimp origin might directly impact the readout of commercial crustacean allergen detection kits, most of which target tropomyosin, and this should be considered in food safety assessments.

An emerging aeroallergen in Europe: Tree-of-Heaven (Ailanthus altissima [Mill.] Swingle) inventory and pollen concentrations - Taking a metropolitan region in Germany as an example.

Urban areas are often hotspots for the dissemination of non-native (invasive) plant species, some of which release (potentially) allergenic pollen. Given the high population density in cities, a considerable number of people can be regularly and potentially intensively exposed to the pollen from these plants. This study delves into the Tree-of-Heaven (Ailanthus altissima, [Mill.] Swingle), native to East Asia, which is known for its high invasiveness in temperate regions worldwide, particularly favoring urban colonization. This study explores the botanical and aerobiological dimensions of this species using the central European metropolitan region of Berlin, Germany, as a case study, and provides a comprehensive global overview of allergological insights. The number of Ailanthus trees decreased markedly from the center to the periphery of Berlin City, following a temperature gradient. The same spatial trend was mirrored by airborne Ailanthus pollen concentrations measured with volumetric spore traps (Hirst-type) at five sites using seven traps. Ailanthus pollen was most abundant around midday and in the afternoon, with concentrations tenfold higher at street level than at roof level. The Ailanthus flowering period in June and July coincided well with the pollen season. To the best of our knowledge this is the first study to investigate Ailanthus altissima pollen production. On average, 5539 pollen grains were found per anther. A literature review on the allergy relevance of Ailanthus altissima pollen indicates the high allergenic potential of pollen from this species. Considering the anticipated expansion of suitable habitats for Ailanthus owing to global warming and the allergological significance of its pollen, it is recommended to include Ailanthus pollen in routine pollen monitoring, particularly in areas colonized by this species. This comprehensive study provides new insights into a pollen taxon whose significance as an emerging aeroallergen should be factored into plant selection and greenspace management in all temperate regions.

Advancing High-Throughput MS-Based Protein Quantification: A Case Study on Quantifying 10 Major Food Allergens by LC-MS/MS Using a One-Sample Multipoint External Calibration Curve.

The LC-MS-based method has emerged as the preferred approach for quantifying food allergens. However, the preparation of a traditional calibration curve (MSCC) is labor-intensive and error-prone. Here, a sensitive and robust LC-MS/MS method for quantifying 10 major food allergens was developed and validated, where the one-sample multipoint external calibration curve (OSCC) was employed instead of MSCC. By employing the multiple isotopologue reaction monitoring (MIRM) technique with only one spiked level in the blank, OSCC can be effectively established. Results demonstrate that the proposed method exhibits excellent performance in selectivity, sensitivity, accuracy, and precision, comparable to that of the traditional MSCC. Additionally, this strategy allows for isotope sample dilution by monitoring the less abundant MIRM channel. Moreover, the developed method was successfully applied to investigate the contamination of 10 food allergens in commercial food products. With its high throughput and robustness, the MIRM-OSCC-LC-MS/MS methodology has many potential applications, especially in the MS-based protein quantification analysis.

Mass-Spectrometry-Based Research of Cosmetic Ingredients.

Cosmetic products are chemical substances or mixtures used on the skin, hair, nails, teeth, and the mucous membranes of the oral cavity, whose use is intended to clean, protect, correct body odor, perfume, keep in good condition, or change appearance. The analysis of cosmetic ingredients is often challenging because of their huge complexity and their adulteration. Among various analytical tools, mass spectrometry (MS) has been largely used for compound detection, ingredient screening, quality control, detection of product authenticity, and health risk evaluation. This work is focused on the MS applications in detecting and quantification of some common cosmetic ingredients, i.e., preservatives, dyes, heavy metals, allergens, and bioconjugates in various matrices (leave-on or rinse-off cosmetic products). As a global view, MS-based analysis of bioconjugates is a narrow field, and LC- and GC/GC×GC-MS are widely used for the investigation of preservatives, dyes, and fragrances, while inductively coupled plasma (ICP)-MS is ideal for comprehensive analysis of heavy metals. Ambient ionization approaches and advanced separation methods (i.e., convergence chromatography (UPC2)) coupled to MS have been proven to be an excellent choice for the analysis of scented allergens. At the same time, the current paper explores the challenges of MS-based analysis for cosmetic safety studies.

Seafood product safety: A hybrid graphene/gold-based electrochemical immunosensor for fish allergen analysis.

Seafood product labels with accurate allergen contents can avoid and/or minimize allergic reactions. Therefore, an electrochemical immunosensor for the analysis of ß-parvalbumin (ß-PV, a major fish allergen) was developed. Screen-printed carbon electrodes were nanostructured with reduced graphene oxide and gold nanoparticles. The platform was characterized by scanning electron microscopy and elemental analysis. In a sandwich-type assay (∼75 min), the antigen-antibody interaction was detected by chronoamperometry using horseradish peroxidase and TMB-H2O2. A linear range of 25-3000 ng/mL, a sensitivity of 2.99 µA.mL/ng, and a limit of detection of 9.9 ng/mL (corresponding to 0.40 ng in the analysed aliquot) were obtained. The selectivity and possible interferences were assessed by analysing several other food allergens and a marine toxin. The sensor was applied to the analysis of 17 commercial foods and the effect of culinary processing (e.g., grilled, canned, smoked) on the ß-PV concentration was assessed. Traces of ß-PV were successfully quantified and ELISA was used to assess the results.

Botanical Impurities in the Supply Chain: A New Allergenic Risk Exacerbated by Geopolitical Challenges.

BACKGROUND: The supply chains of food raw materials have recently been heavily influenced by geopolitical events. Products that came from, or transited through, areas currently in conflict are now preferentially supplied from alternative areas. These changes may entail risks for food safety. METHODS: We review the potential allergenicity of botanical impurities, specifically vegetable contaminants, with particular attention to the contamination of vegetable oils. We delve into the diverse types of botanical impurities, their sources, and the associated allergenic potential. Our analysis encompasses an evaluation of the regulatory framework governing botanical impurities in food labeling. RESULTS: Unintended plant-derived contaminants may manifest in raw materials during various stages of food production, processing, or storage, posing a risk of allergic reactions for individuals with established food allergies. Issues may arise from natural occurrence, cross-contamination in the supply chain, and contamination at during production. The food and food service industries are responsible for providing and preparing foods that are safe for people with food allergies: we address the challenges inherent in risk assessment of botanical impurities. CONCLUSIONS: The presence of botanical impurities emerges as a significant risk factor for food allergies in the 2020s. We advocate for regulatory authorities to fortify labeling requirements and develop robust risk assessment tools. These measures are necessary to enhance consumer awareness regarding the potential risks posed by these contaminants.

Development of a Mass Spectrometry-Based Method for Quantification of Total Cashew Protein in Roasting Oil.

BACKGROUND: Food allergen cross-contact during food preparation and production is one of the causes of unintentional allergen presence in packaged foods. However, little is known about allergen cross-contact in shared frying or roasting oil, which prevents the establishment of effective allergen controls and may put allergic individuals at risk. To better understand the quantity of allergen transferred to frying oil and subsequent products, an analytical method is needed for quantifying protein in oil that has been exposed to frying/roasting conditions. OBJECTIVE: The goal of this study was to develop a parallel reaction monitoring LC-MS/MS method to quantify the amount of cashew protein in shared roasting oil. METHODS: The sample preparation method was evaluated to improve protein extractability and peptide performance. Four quantitative peptides representing cashew 2S and 11S proteins were selected as targets based on their sensitivity, heat stability, and specificity. A calibration strategy was developed to quantify the amount of total cashew protein in oil. Method performance was evaluated using a heated cashew-in-oil model system. RESULTS: The method showed high recovery in oil samples spiked with 100 or 10 parts per million (ppm) total cashew protein heated at 138 or 166°C for 2-30 min. Samples (100 ppm total cashew protein) heated for 30 min had more than 90% recovery when treated at 138°C and more than 50% when heated at 166°C. CONCLUSION: The method is fit-for-purpose for the analysis of cashew allergen cross-contact in oil. HIGHLIGHTS: A novel MS-based method was developed that can accurately quantify the amount of cashew protein present in heated oil.

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.

A rapid immunomagnetic beads-based sELISA method for the detection of bovine αs1-casein based on specific epitopes.

Although αs1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine αs1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 µg/mL-2.000 µg/mL, with a limit of detection of 0.099 µg/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting αs1-CN in real food.

Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system.

Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.

Variation in the Allergenicity of Scrambled, Boiled, Short-Baked and Long-Baked Egg White Proteins.

BACKGROUND: Hen's egg white (HEW) is the most common cause of food allergy in children which induces mild to fatal reactions. The consultation for a proper restriction is important in HEW allergy. We aimed to identify the changes in HEW allergenicity using diverse cooking methods commonly used in Korean dishes. METHODS: Crude extract of raw and 4 types of cooked HEW extracts were produced and used for sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition assays using 45 serum samples from HEW allergic and tolerant children. Extracts were prepared; scrambled without oil for 20-30 seconds in frying pan without oil, boiled at 100°C for 15 minutes, short-baked at 180°C for 20 minutes, and long-baked at 45°C for 12 hours with a gradual increase in temperature up to 110°C for additional 12 hours, respectively. RESULTS: In SDS-PAGE, the intensity of bands of 50-54 kDa decreased by boiling and baking. All bands almost disappeared in long-baked eggs. The intensity of the ovalbumin (OVA) immunoglobulin E (IgE) bands did not change after scrambling; however, an evident decrease was observed in boiled egg white (EW). In contrast, ovomucoid (OM) IgE bands were darker and wider after scrambling and boiling. The IgE binding reactivity to all EW allergens were weakened in short-baked EW and considerably diminished in long-baked EW. In individual ELISA analysis using OVA+OM+ serum samples, the median of specific IgE optical density values was 0.435 in raw EW, 0.476 in scrambled EW, and 0.487 in boiled EW. Conversely, it was significantly decreased in short-baked (0.406) and long-baked EW (0.012). Significant inhibition was observed by four inhibitors such as raw, scrambled, boiled and short-baked HEW, but there was no significant inhibition by long-baked HEW (IC50 > 100 mg/mL). CONCLUSION: We identified minimally reduced allergenicity in scrambled EW and extensively decreased allergenicity in long-baked EW comparing to boiled and short-baked EW as well as raw EW. By applying the results of this study, we would be able to provide safer dietary guidence with higher quality to egg allergic children.

Identifying key environmental factors to model Alt a 1 airborne allergen presence and variation.

Fungal spores, commonly found in the atmosphere, can trigger important respiratory disorders. The glycoprotein Alt a 1 is the major allergen present in conidia of the genus Alternaria and has a high clinical relevance for people sensitized to fungi. Exposure to this allergen has been traditionally assessed by aerobiological spore counts, although this does not always offer an accurate estimate of airborne allergen load. This study aims to pinpoint the key factors that explain the presence and variation of Alt a 1 concentration in the atmosphere in order to establish exposure risk periods and improve forecasting models. Alternaria spores were sampled using a Hirst-type volumetric sampler over a five-year period. The allergenic fraction from the bioaerosol was collected using a low-volume cyclone sampler and Alt a 1 quantified by Enzyme-Linked ImmunoSorbent Assay. A cluster analysis was executed in order to group days with similar environmental features and then analyze days with the presence of the allergen in each of them. Subsequently, a quadratic discriminant analysis was performed to evaluate if the selected variables can predict days with high Alt a 1 load. The results indicate that higher temperatures and absolute humidity favor the presence of Alt a 1 in the atmosphere, while time of precipitation is related to days without allergen. Moreover, using the selected parameters, the quadratic discriminant analysis to predict days with allergen showed an accuracy rate between 67 % and 85 %. The mismatch between daily airborne concentration of Alternaria spores and allergen load can be explained by the greater contribution of medium-to-long distance transport of the allergen from the major emission sources as compared with spores. Results highlight the importance of conducting aeroallergen quantification studies together with spore counts to improve the forecasting models of allergy risk, especially for fungal spores.