Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties.

Contaminating microplastics can interact with food proteins in the food matrix and during digestion. This study investigated adsorption of chicken egg protein ovalbumin to polystyrene (PS, 110 and 260 µm) and polyethylene terephthalate (PET, 140 µm) MPs in acidic and neutral conditions and alterations in ovalbumin structure. Ovalbumin adsorption affinity depended on MPs size (smaller > larger), type (PS > PET) and pH (pH 3 > pH 7). In bulk solution, MPs does not change ovalbumin secondary structure significantly, but induces loosening (at pH 3) and tightening (at pH 7) of tertiary structure. Formed soft corona exclusively consists of full length non-native ovalbumin, while in hard corona also shorter ovalbumin fragments were found. At pH 7 soft corona ovalbumin has rearranged but still preserved level of ordered secondary structure, resulting in preserved thermostability and proteolytic stability, but decreased ability to form fibrils upon heating. Secondary structure changes in soft corona resemble changes in native ovalbumin induced by heat treatment (80 °C). Ovalbumin is abundantly present in corona around microplastics also in the presence of other egg white proteins. These results imply that microplastics contaminating food may bind and change structure and functional properties of the main egg white protein.

Characterization of Nanoprecipitated PET Nanoplastics by 1H NMR and Impact of Residual Ionic Surfactant on Viability of Human Primary Mononuclear Cells and Hemolysis of Erythrocytes.

Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 µg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.

Ultrasensitive Quantification of Crustacean Tropomyosin by Immuno-PCR.

Tropomyosin is the major and predominant allergen among shellfish. This study developed an ultrasensitive immuno-PCR method for the quantification of crustacean tropomyosin in foods. The method couples sandwich ELISA with the real-time PCR (rtPCR) amplification of marker DNAs. Monoclonal anti-TPM antibody was the capture antibody, polyclonal rabbit anti-shrimp tropomyosin antibody was the detection antibody, while natural shrimp tropomyosin served as the standard. A double-stranded amino-DNA was covalently conjugated to a secondary anti-rabbit antibody and subsequently amplified and quantified via rtPCR. The quantification sensitivity of immuno-PCR was 20-fold higher than analogous ELISA, with LOQ 19.8 pg/mL. The developed immuno-PCR method is highly specific for the detection of crustacean tropomyosin and is highly precise in a broad concentration range. Tropomyosin recovery in the spiked vegetable soup was 87.7-115.6%. Crustacean tropomyosin was also quantified in commercial food products. The reported immuno-PCR assay is the most sensitive method for the quantification of crustacean tropomyosin and is the first immuno-PCR-based assay for the quantification of food allergen and food protein in general. The described method could be easily adapted for the specific and ultrasensitive immuno-PCR-based detection of traces of any food allergen that is currently being quantified with ELISA, which is of critical importance for people with food allergies.

Nano- and Microplastics Migration from Plastic Food Packaging into Dairy Products: Impact on Nutrient Digestion, Absorption, and Metabolism.

The ongoing use of plastic polymers to manufacture food packaging has raised concerns about the presence of nano- and microplastics (NMPs) in a variety of foods. This review provides the most recent data on NMPs' migration from plastic packaging into dairy products. Also discussed are the possible effects of NMPs on nutrient digestion, absorption, and metabolism. Different kinds of dairy products, including skimmed milk, whole liquid milk, powder milk, and infant formula milk, have been found to contain NMPs of various sizes, shapes, and concentrations. NMPs may interact with proteins, carbohydrates, and fats and have a detrimental impact on how well these nutrients are digested and absorbed by the body. The presence of NMPs in the gastrointestinal tract may impact how lipids, proteins, glucose, iron, and energy are metabolized, increasing the risk of developing various health conditions. In addition to NMPs, plastic oligomers released from food packaging material have been found to migrate to various foods and food simulants, though information regarding their effect on human health is limited. Viewpoints on potential directions for future studies on NMPs and their impact on nutrient digestion, absorption, and health are also presented in this review.

Small polystyrene microplastics interfere with the breakdown of milk proteins during static in vitro simulated human gastric digestion.

Human ingestion of microplastics (MPs) is common and inevitable due to the widespread contamination of food items, but implications on the gastric digestion of food proteins are still unknown. In this study, the interactions between pepsin and polystyrene (PS) MPs were evaluated by investigating enzyme activity and conformation in a simulated human gastric environment in the presence or absence of PS MPs. The impact on food digestion was also assessed by monitoring the kinetics of protein hydrolysis through static in vitro gastric digestion of cow's milk contaminated with PS. The binding of pepsin to PS showed that the surface chemistry of MPs dictates binding affinity. The key contributor to pepsin adsorption seems to be π-π interactions between the aromatic residues and the PS phenyl rings. During quick exposure (10 min) of pepsin to increasing concentrations (222, 2219, 22188 particles/mL) of 10 µm PS (PS10) and 100 µm PS (PS100), total enzymatic activities were not affected remarkably. However, upon prolonged exposure at 1 and 2 h, preferential binding of pepsin to the small, low zeta-potential PS caused structural changes in the protein which led to a significant reduction of its activity. Digestion of cow's milk mixed with PS10 resulted in transient accumulation of larger peptides (10-35 kDa) and reduced bioavailability of short peptides (2-9 kDa) in the gastric phase. This, however, was only observed at extremely high PS10 concentration (0.3 mg/mL or 5.46E+05 particles/mL). The digestion of milk peptides, bound preferentially over pepsin within the hard corona on the PS10 surface, was delayed up to 15 min in comparison to bulk protein digestion. Intact caseins, otherwise rapidly digested, remained bound to PS10 in the hard corona for up to 15 min. This work presents valuable insights regarding the interaction of MPs, food proteins, and pepsin, and their dynamics during gastric digestion.

Exploring and strengthening the potential of R-phycocyanin from Nori flakes as a food colourant.

This study aimed to purify, characterise and stabilise the natural food colourant, R-phycocyanin (R-PC), from the red algae Porphyra spp. (Nori). We purified R-PC from dried Nori flakes with a high purity ratio (A618/A280 ≥ 3.4) in native form (α-helix content 53%). SAXS measurements revealed that R-PC is trimeric ((αß)3) in solution. The thermal denaturation of α-helix revealed one transition (Tm at 52 °C), while the pH stability study showed R-PC is stable in the pH range 4-8. The thermal treatment of R-PC at 60 °C has detrimental and irreversible effects on R-PC colour and antioxidant capacity (22 % of residual capacity). However, immobilisation of R-PC within calcium alginate beads completely preserves R-PC colour and mainly retains its antioxidant ability (78 % of residual capacity). Results give new insights into the stability of R-PC and preservation of its purple colour and bioactivity by encapsulation in calcium alginate beads.

Food Antioxidants and Their Interaction with Human Proteins.

Common to all biological systems and living organisms are molecular interactions, which may lead to specific physiological events. Most often, a cascade of events occurs, establishing an equilibrium between possibly competing and/or synergistic processes. Biochemical pathways that sustain life depend on multiple intrinsic and extrinsic factors contributing to aging and/or diseases. This article deals with food antioxidants and human proteins from the circulation, their interaction, their effect on the structure, properties, and function of antioxidant-bound proteins, and the possible impact of complex formation on antioxidants. An overview of studies examining interactions between individual antioxidant compounds and major blood proteins is presented with findings. Investigating antioxidant/protein interactions at the level of the human organism and determining antioxidant distribution between proteins and involvement in the particular physiological role is a very complex and challenging task. However, by knowing the role of a particular protein in certain pathology or aging, and the effect exerted by a particular antioxidant bound to it, it is possible to recommend specific food intake or resistance to it to improve the condition or slow down the process.

Regioselective protein oxidative cleavage enabled by enzyme-like recognition of an inorganic metal oxo cluster ligand.

Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K8[Cu2+(H2O)(α2-P2W17O61)], (CuIIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic CuIIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.

Comparative digestion of thermally treated vertebrates and invertebrates allergen pairs in real food matrix.

The digestion stability of allergen pairs, tropomyosin, TM (fish and seafood allergen), and myosin light chain, MLC (chicken meat allergen) is compared among vertebrates and invertebrates in raw and cooked food matrix under standardized simulated in vitro gastrointestinal (GI) digestion. SDS-PAGE followed by multiple TM and MLC-specific antibodies in semidry WB revealed pepsin resistance of invertebrate TMs (abalone, oyster, shrimp) under diet-relevant conditions (raw, cooked). Vertebrate TMs (chicken, pork, beef) were less stable to digestion except that the raw chicken TM was observed pepsin resistant (not diet-relevant). Vertebrate (chicken) MLC was thermally stable. A new 28 kDa protein bound to anti-MLC antibody in cooked chicken and pork; could be the aggregates of MLC. Raw shrimp MLC showed pepsin resistance among invertebrates. A good correlation was observed between combined resistance of TM and MLC to GI digestion following the diet-relevant thermal treatment and reported protein allergenicity among vertebrates and invertebrates.

Sandwich ELISA for the Quantification of Nucleocapsid Protein of SARS-CoV-2 Based on Polyclonal Antibodies from Two Different Species.

In this study, a cost-effective sandwich ELISA test, based on polyclonal antibodies, for routine quantification SARS-CoV-2 nucleocapsid (N) protein was developed. The recombinant N protein was produced and used for the production of mice and rabbit antisera. Polyclonal N protein-specific antibodies served as capture and detection antibodies. The prototype ELISA has LOD 0.93 ng/mL and LOQ 5.3 ng/mL, with a linear range of 1.52-48.83 ng/mL. N protein heat pretreatment (56 °C, 1 h) decreased, while pretreatment with 1% Triton X-100 increased analytical ELISA sensitivity. The diagnostic specificity of ELISA was 100% (95% CI, 91.19-100.00%) and sensitivity was 52.94% (95% CI, 35.13-70.22%) compared to rtRT-PCR (Ct < 40). Profoundly higher sensitivity was obtained using patient samples mostly containing Wuhan-similar variants (Wuhan, alpha, and delta), 62.50% (95% CI, 40.59 to 81.20%), in comparison to samples mostly containing Wuhan-distant variants (Omicron) 30.00% (6.67-65.25%). The developed product has relatively high diagnostic sensitivity in relation to its analytical sensitivity due to the usage of polyclonal antibodies from two species, providing a wide repertoire of antibodies against multiple N protein epitopes. Moreover, the fast, simple, and inexpensive production of polyclonal antibodies, as the most expensive assay components, would result in affordable antigen tests.

Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting.

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

MP-Net: Deep learning-based segmentation for fluorescence microscopy images of microplastics isolated from clams.

Environmental monitoring of microplastics (MP) contamination has become an area of great research interest, given potential hazards associated with human ingestion of MP. In this context, determination of MP concentration is essential. However, cheap, rapid, and accurate quantification of MP remains a challenge to this date. This study proposes a deep learning-based image segmentation method that properly distinguishes fluorescent MP from other elements in a given microscopy image. A total of nine different deep learning models, six of which are based on U-Net, were investigated. These models were trained using at least 20,000 patches sampled from 99 fluorescence microscopy images of MP and their corresponding binary masks. MP-Net, which is derived from U-Net, was found to be the best performing model, exhibiting the highest mean F1-score (0.736) and mean IoU value (0.617). Test-time augmentation (using brightness, contrast, and HSV) was applied to MP-Net for robust learning. However, compared to the results obtained without augmentation, no clear improvement in predictive performance could be observed. Recovery assessment for both spiked and real images showed that, compared to already existing tools for MP quantification, the MP quantities predicted by MP-Net are those closest to the ground truth. This observation suggests that MP-Net allows creating masks that more accurately reflect the quantitative presence of fluorescent MP in microscopy images. Finally, MAP (Microplastics Annotation Package) is introduced, an integrated software environment for automated MP quantification, offering support for MP-Net, already existing MP analysis tools like MP-VAT, manual annotation, and model fine-tuning.

Comparative profiling and exposure assessment of microplastics in differently sized Manila clams from South Korea by µFTIR and Nile Red staining.

The accumulation of microplastics in marine organisms is an emerging concern. Due to trophic transfer, the safety of seafood is under investigation in view of the potential negative effects of microplastics on human health. In this study, market samples of Manila clams (Ruditapes philippinarum) from South Korea were segregated into two groups of considerably different size (p < 0.05), namely small clams with shell length of 40.69 ± 3.97 mm, and large clams of shell length 51.19 ± 2.86 mm. Comparative profiling of the number, size, shape, and polymer type of microplastics were performed using µFTIR imaging and Nile red staining. Overall, µFTIR detected only 1559 microplastics while 1996 microplastics were counted based on staining from 61 Manila clams (30 small and 31 large), leading to an overestimation of 18 to 75 %. Comparable microplastics concentration, based on µFTIR, were observed at 2.70 ± 1.66 MP/g or 15.64 ± 9.25 MP/individual for the small samples, and 3.65 ± 1.59 MP/g or 41.63 ± 16.90 MP/individual for the large ones (p > 0.05). Particle diameters of 20-100 µm was the most dominant, accounting for 44.6 % and 46.5 % of all microplastics from the small and large groups, respectively. Particles, with a circularity (resemblance to a circle) value between 0.6 and 1.0, were the most prevalent, followed by fragments and fibers. At least 50 % of microplastics from the small and large samples were polystyrene, making it the most abundant polymer type. Despite the substantial difference in the size of the animals, only a weak to moderate correlation was observed between microplastics content and the physical attributes of the clams such as shell length and weight, (soft) tissue weight, and total weight (Spearman's coefficient < 0.5). The estimated intake of microplastics by the Korean population was 1232 MP/person/year via small clams, 1663 MP/person/year via large clams, and 1489 MP/person/year via clams independent of size.

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.

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.

Probing the stability of the food colourant R-phycoerythrin from dried Nori flakes.

This study aimed to characterise the stability of R-phycoerythrin (R-PE), a vivid natural colourant with emerging potential for application in the food industry. High-quality (A560/A280 ≥ 5), native (α-helix content 75%) R-PE was purified from commercial dried Nori (Porphyra sp.) flakes. Thermal unfolding revealed two transitions (at 56 and 72 °C), ascribed to different protein subunits. Contrary to elevated temperature, high-pressure (HP) treatment showed significant advantages: The R-PE unfolding was partly reversible and the colour bleaching was minimal. Binding of Cu2+ (6.3 × 105 M-1) and Zn2+ (1.7 × 103 M-1) influenced conformational changes in the protein tetrapyrrole chromophore without affecting R-PE structure and stability (colour). The results give new insights into the stability of R-PE suggesting its usefulness for the replacement of toxic synthetic dyes. Preservation of the red colour of R-PE could be considered in fortified food and beverages by HP processing. R-PE may act as a biosensor for Cu2+ in aquatic systems.

Molecular Mechanisms of Possible Action of Phenolic Compounds in COVID-19 Protection and Prevention.

The worldwide outbreak of COVID-19 was caused by a pathogenic virus called Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Therapies against SARS-CoV-2 target the virus or human cells or the immune system. However, therapies based on specific antibodies, such as vaccines and monoclonal antibodies, may become inefficient enough when the virus changes its antigenicity due to mutations. Polyphenols are the major class of bioactive compounds in nature, exerting diverse health effects based on their direct antioxidant activity and their effects in the modulation of intracellular signaling. There are currently numerous clinical trials investigating the effects of polyphenols in prophylaxis and the treatment of COVID-19, from symptomatic, via moderate and severe COVID-19 treatment, to anti-fibrotic treatment in discharged COVID-19 patients. Antiviral activities of polyphenols and their impact on immune system modulation could serve as a solid basis for developing polyphenol-based natural approaches for preventing and treating COVID-19.

Life cycle assessment of edible insects (Protaetia brevitarsis seulensis larvae) as a future protein and fat source.

Because it is important to develop new sustainable sources of edible protein, insects have been recommended as a new protein source. This study applied Life Cycle Assessment (LCA) to investigate the environmental impact of small-scale edible insect production unit in South Korea. IMPACT 2002 + was applied as the baseline impact assessment (IA) methodology. The CML-IA baseline, EDIP 2003, EDP 2013, ILCD 2011 Midpoint, and ReCiPe midpoint IA methodologies were also used for LCIA methodology sensitivity analysis. The protein, fat contents, and fatty acid profile of the investigated insect (Protaetia brevitarsis seulensis larvae) were analyzed to determine its potential food application. The results revealed that the studied edible insect production system has beneficial environmental effects on various impact categories (ICs), i.e., land occupation, mineral extraction, aquatic and terrestrial ecotoxicity, due to utilization of bio-waste to feed insects. This food production system can mitigate the negative environmental effects of those ICs, but has negative environmental impact on some other ICs such as global warming potential. By managing the consumption of various inputs, edible insects can become an environmentally efficient food production system for human nutrition.

Role of Resveratrol in Prevention and Control of Cardiovascular Disorders and Cardiovascular Complications Related to COVID-19 Disease: Mode of Action and Approaches Explored to Increase Its Bioavailability.

Resveratrol is a phytoalexin produced by many plants as a defense mechanism against stress-inducing conditions. The richest dietary sources of resveratrol are berries and grapes, their juices and wines. Good bioavailability of resveratrol is not reflected in its high biological activity in vivo because of resveratrol isomerization and its poor solubility in aqueous solutions. Proteins, cyclodextrins and nanomaterials have been explored as innovative delivery vehicles for resveratrol to overcome this limitation. Numerous in vitro and in vivo studies demonstrated beneficial effects of resveratrol in cardiovascular diseases (CVD). Main beneficial effects of resveratrol intake are cardioprotective, anti-hypertensive, vasodilatory, anti-diabetic, and improvement of lipid status. As resveratrol can alleviate the numerous factors associated with CVD, it has potential as a functional supplement to reduce COVID-19 illness severity in patients displaying poor prognosis due to cardio-vascular complications. Resveratrol was shown to mitigate the major pathways involved in the pathogenesis of SARS-CoV-2 including regulation of the renin-angiotensin system and expression of angiotensin-converting enzyme 2, stimulation of immune system and downregulation of pro-inflammatory cytokine release. Therefore, several studies already have anticipated potential implementation of resveratrol in COVID-19 treatment. Regular intake of a resveratrol rich diet, or resveratrol-based complementary medicaments, may contribute to a healthier cardio-vascular system, prevention and control of CVD, including COVID-19 disease related complications of CVD.