Renata Costa

Abstract Lupine is a legume commonly used in human diet as a functional food due to its high nutritional content and important technological properties. However, its consumption can lead to the manifestation of adverse immunological reactions, posing significant health issues in sensitized/allergic patients. This work aims to investigate the effect of food processing combined with simulated gastrointestinal (GI) digestion on the immunoreactivity of lupine gamma-conglutin. Model foods of wheat pasta containing 35% of lupine flour (Lupinus albus, L. luteus, and L. angustifolius) were prepared and submitted to a boiling process. The proteins were extracted and their profiles characterized by SDS-PAGE. Simulated GI digestion was performed on thermally treated pasta using the INFOGEST harmonized digestion protocol 2.0. The IgG binding capacity of gamma-conglutin was assessed by immunoblotting in non-reducing conditions and indirect ELISA with specific antibodies. Results demonstrate that the boiling treatment affected the immunoreactivity of the three lupine species differently. Simulated GI digestion led to extensive destruction of the protein structure, more significant in the intestinal phase, reducing but not abolishing the IgG affinity to gamma-conglutin and its potential presentation to immunocompetent cells. This information can offer valuable insights to the food industry for developing food formulations with reduced allergenic properties.

Abstract Biosensors, especially those designed for detecting food allergens like Gly m TI in soybean, play a crucial role in safeguarding individuals who suffer from adverse food allergies, extending to both individual well-being and broader public health considerations. Furthermore, their integration into food production and monitoring processes aids in compliance with regulatory standards, reducing the incidence of allergen-related recalls and protecting vulnerable populations. Technological advancements in biosensor development, such as increased portability, real-time monitoring capabilities, and user-friendly interfaces, have expanded their practical applications, making them indispensable in various settings, including manufacturing plants, food service establishments, and even at-home use by consumers. For the first time, a biosensor targeting the Gly m TI allergen based on molecularly imprinted polymer (MIP) technology was developed to detect/quantify soybean in complex food matrices and effectively address the detection challenges of complex and processed foods. The Gly m TI-MIP underwent a thorough validation process using anti-Gly m TI IgG raised as a polyclonal response to the trypsin inhibitor. Gly m TI-MIP was successfully tested across a range of food matrices, including tree nuts (e.g., peanuts, walnuts, and hazelnuts) and legumes (e.g., lentils, beans, and lupine), presenting minimal cross-reactivity with lupine and walnut. The innovative approach provided a linear response in the 1 ag mL(-1) - 10 mu g mL(-1) range, with a LOD<1 ag mL(-1). Applying the Gly m TI-MIP sensor to complex model foods allowed to detect 0.1 mg kg(-1) (0.00001 %) of soybean protein isolate in biscuits, ham, and sausages before and after the respective thermal treatments. The innovative biosensor can significantly improve food safety protocols by addressing the complexities of tracing allergens in processed and unprocessed food products. By ensuring rigorous allergen control, these biosensors may support global food trade compliance with international safety standards, boost consumer confidence, and promote transparency in food labeling, ultimately contributing to a safer food supply chain.

Abstract Artificial receptors that mimic their natural biological counterparts have several advantages, such as lower production costs and increased shelf-life stability/versatility, while overcoming the ethical issues related to raising antibodies in animals. In this work, the proposed tailor-made molecularly imprinted polymer (MIP)-allergen receptors aimed at substituting or even transcending the performance of biological antibodies. For this purpose, a MIP was proposed as an artificial antibody for the recognition of hazelnut Cor a 14-allergen. The target protein was grafted onto the conducting polypyrrole receptor film using gold screen-printed electrodes (Au-SPE). The electrochemical assessment presented a linear response for the dynamic range of 100 fg mL(-1)-1 mu g mL(-1) and a LOD of 24.5 fg mL(-1), as determined by square wave voltammetry from the calibration curves prepared with standards diluted in phosphate buffer. Surface plasmon resonance (SPR) was used as a secondary transducer to evaluate the performance of the Cor a 14-MIP sensor, enabling a linear dynamic range of 100 fg mL(-1) - 0.1 mu g mL(-1) and a LOD of 18.1 fg mL(-1). The selectivity of the tailored-made Cor a 14-MIP was tested against potentially cross-reactive plant/animal species based on the rebinding affinity (Freundlich isotherm-K-F) of homologues/similar proteins, being further compared with custom-made polyclonal anti-Cor a 14 IgG immunosensor. Results evidenced that the MIP mimics the biorecognition of biological antibodies, presenting higher selectivity (only minor cross-reactivity towards walnut and Brazil nut 2S albumins) than the Cor a 14/anti-Cor a 14 IgG immunosensor. The application of electrochemical Cor a 14-MIP sensor to model mixtures of hazelnut in pasta enabled quantifying hazelnut down to 1 mg kg(-1) (corresponding to 0.16 mg kg(-1) of hazelnut protein in the matrix). To the best of our knowledge, Cor a 14-MIP is the first sensor based on an artificial/synthetic biorecognition platform for the specific detection of hazelnut allergens, while presenting high-performance parameters with demonstrated application in food safety management.

Abstract A suitable dispersion of carbon materials (e.g., carbon nanotubes (CNTs)) in an appropriate dispersant media, is a prerequisite for many technological applications (e.g., additive purposes, functionalization, mechanical reinforced materials for electrolytes and electrodes for energy storage applications, etc.). Deep eutectic solvents (DES) have been considered as a promising "green" alternative, providing a versatile replacement to volatile organic solvents due to their unique physical-chemical properties, being recognized as low-volatility fluids with great dispersant ability. The present work aims to contribute to appraise the effect of the presence of MWCNTs and Ag-functionalized MWCNTs on the physicochemical properties (viscosity, density, conductivity, surface tension and refractive index) of glyceline (choline chloride and glycerol, 1:2), a Type III DES. To benefit from possible synergetic effects, AgMWCNTs were prepared through pulse reverse electrodeposition of Ag nanoparticles into MWCNTs. Pristine MWCNTs were used as reference material and water as reference dispersant media for comparison purposes. The effect of temperature (20 to 60 degrees C) and concentration on the physicochemical properties of the carbon dispersions (0.2-1.0 mg cm(-3)) were assessed. In all assessed physicochemical properties, AgMWCNTs outperformed pristine MWCNTs dispersions. A paradoxical effect was found in the viscosity trend in glyceline media, in which a marked decrease in the viscosity was found for the MWCNTs and AgMWCNTs materials at lower temperatures. All physicochemical parameters were statistically analyzed using a two-way analysis of variance (ANOVA), at a 5% level of significance.

Abstract Two immunosensors were advanced to target hazelnut Cor a 14 based on electrochemical and optical transduction. Both approaches were developed with two types of custom-made antibodies, namely anti-Cor a 14 IgG (rabbit) and anti-Cor a 14 IgY (hen's egg) targeting the Cor a 14 allergen. Antibody immobilisation was performed via EDC/NHS onto disposable screen-printed electrodes. The detection limit (LOD) of the electrochemical immunoassay for Cor a 14 was 5-times lower than the optical, being down to 0.05 fg mL-1 with a dynamic range of 0.1 fg mL-1 to 0.01 ng mL-1. Antibody selectivity was verified against non-target 2S albumins (potential crossreactive plant species). Anti-Cor a 14 IgY exhibited the best specificity, presenting minor cross-reactivity with peanut/walnut. Preliminary results of the application of anti-Cor a 14 IgY electrochemical immunosensor to incurred foods established a LOD of 1 mg kg- 1 of hazelnut in wheat (0.16 mg kg- 1 hazelnut protein).