Effect of temperature on structural configuration and immunoreactivity of pH-stressed soybean (Glycine max) agglutinin.

Soybean agglutinin (SBA) was purified using ammonium sulfate precipitation and liquid chromatography. Purified SBA was used to produce monoclonal antibodies through hybridoma technology. SBA secondary structure was studied using circular dichroism. pH-stressed (pHs 3.0, 7.2, 8.5, and 9.6) SBA physical properties (particle size, ζ-potential, and aggregation temperature) were investigated. Gel electrophoresis (non-native and native) was used to study heat-induced structural configuration changes in SBA. The effect of pH and temperature on the immunoreactivity of SBA was analyzed using enzyme-linked immunosorbent assay and immunoblots probed with two anti-SBA monoclonal antibodies with either linear or conformational epitopes. The hemagglutinating activity of heated SBA was measured by hemagglutination assay. Our results indicated that SBA had the least thermostability at pH 3.0 and the highest at pH 8.5. Temperature-induced structural configuration change on pH-stressed SBA led to immunoreactivity change. Heat-induced (70 and 80 °C) soluble SBA aggregation was proportionally related to hemagglutinating activity reduction.

Understanding Hyperuricemia: Pathogenesis, Potential Therapeutic Role of Bioactive Peptides, and Assessing Bioactive Peptide Advantages and Challenges.

Hyperuricemia is a medical condition characterized by an elevated level of serum uric acid, closely associated with other metabolic disorders, and its global incidence rate is increasing. Increased synthesis or decreased excretion of uric acid can lead to hyperuricemia. Protein peptides from various food sources have demonstrated potential in treating hyperuricemia, including marine organisms, ovalbumin, milk, nuts, rice, legumes, mushrooms, and protein-rich processing by-products. Through in vitro experiments and the establishment of cell or animal models, it has been proven that these peptides exhibit anti-hyperuricemia biological activities by inhibiting xanthine oxidase activity, downregulating key enzymes in purine metabolism, regulating the expression level of uric acid transporters, and restoring the composition of the intestinal flora. Protein peptides derived from food offer advantages such as a wide range of sources, significant therapeutic benefits, and minimal adverse effects. However, they also face challenges in terms of commercialization. The findings of this review contribute to a better understanding of hyperuricemia and peptides with hyperuricemia-alleviating activity. Furthermore, they provide a theoretical reference for developing new functional foods suitable for individuals with hyperuricemia.

Investigation of Antioxidant and Cytotoxicity Activities of Chocolate Fortified with Muscadine Grape Pomace.

Muscadine grape pomace and mixed products with chocolate extracts from three muscadine genotypes exhibiting different berry skin colors (black and bronze) were investigated for total phenolic content (TPC), total flavonoid content (TFC), DPPH, FRAP antioxidant activity, and anticancer activity using MDA-MB-468 (MM-468; African American) breast cancer cells. Muscadine berry extracts and mixed products showed cytotoxicity activities of up to 70% against MM-468 breast cancer cells. Cell growth inhibition was higher in 'macerated Floriana' with an IC50 value of 20.70 ± 2.43 followed by 'Alachua' with an IC50 value of 22.25 ± 2.47. TPC and TFC in macerated MGP powder were (1.4 ± 0.14 and 0.45 ± 0.01 GAE/g FW, respectively), which was significantly higher than those in cocoa powder. Data analysis showed a high association between DPPH, FRAP antioxidant activities, and TPC content and a positive high correlation between anticancer activity and antioxidant capacity and between TPC and anticancer activity. The anticancer and antioxidant effects of muscadine grape pomace and chocolate extracts are attributed to the TPC of extracts, which showed a stronger positive correlation with growth inhibition of African American breast cancer cells. This study would be of great value for food industries as well as other manufacturers who are interested in new food blends.

Immunodetection of finfish residues on food contact surfaces.

Finfish is one of the major allergenic foods, whose declaration is required on packages. Undeclared allergenic residues are mainly derived from allergen cross-contact. Swabbing of food contact surfaces helps to detect allergen cross-contamination. This study aimed to establish a competitive enzyme-linked immunosorbent assay (cELISA) to quantify the major finfish allergen, parvalbumin, from swab samples. First, parvalbumin from four finfish species was purified. Its conformation was investigated under reducing, non-reducing and native conditions. Second, one anti-finfish parvalbumin monoclonal antibody (mAb) was characterized. This mAb had a calcium-dependent epitope which was highly conserved in finfish species. Third, one cELISA was established with a working range between 0.59 ppm and 150 ppm. It showed a good recovery of swab samples on food-grade stainless steel and plastic surfaces. Overall, this cELISA could detect a trace amount of finfish parvalbumins on cross-contact surfaces, which is suitable for allergen surveillance in the food industry.

Composition, structural configuration, and antigenicity of Atlantic cod (Gadus morhua) tropomyosin.

Tropomyosin, a myofibrillar muscle protein, has been recognized as a finfish allergen. In this study, tropomyosin from Atlantic cod fillets (Gadus morhua, CTM) was purified using a two-step purification strategy (isoelectric precipitation and anion-exchange chromatography). CTM structural configuration in two sample matrices (impure and pure) were elaborated using different polyacrylamide gel electrophoresis (native, non-reducing, and reducing PAGE). Their corresponding immunoblots were conducted to investigate CTM antigenicity under three conditions. Overall, CTM retained solubility, integrity, and antigenicity after heat treatment. Three CTM monomeric α-type isoforms (33 kDa) were identified using two-dimensional PAGE. Under native condition, the vast majority of CTM existed in the disulfide-reduced dimeric form (66 kDa). Under non-reducing condition, sodium dodecyl sulfate (anionic surfactant) broke CTM dimers, leaving monomers and disulfide-induced tetramers. Under reducing condition, ß-mercaptoethanol (thiol reducing agent) dissociated disulfide-linked CTM tetramers (132 kDa) into monomers (33 kDa). CTM retained antigenicity regardless of structural configuration under different conditions.

Formation and Identification of Six Amino Acid - Acrylamide Adducts and Their Cytotoxicity Toward Gastrointestinal Cell Lines.

Acrylamide (AA) is a food contaminant, and amino acids are suggested to mitigate its toxicity by forming adducts. The emergence of acrylamide adducts may cause underestimation of acrylamide exposure level as well as trigger new safety problems. Based on the acrylamide elimination capability of four amino acids, this study chemically synthesized six amino acid-acrylamide adducts. Their structures were analyzed, followed by content determination in 10 commercially baking foods. The Michael adduct formed by one molecule of γ-aminobutyric acid (GABA) and acrylamide was most abundant in foods among six adducts. Furthermore, it markedly decreased the cytotoxicity of acrylamide in Caco-2 cells and GES-1 cells. This finding suggests that amino acids can be used to reduce acrylamide level in processed foods and mitigate its hazardous effects after intake.

Isolation and Characterization of Chicken Serum Albumin (Hen Egg Alpha-Livetin, Gal d 5).

Chicken serum albumin, i.e., hen egg alpha-livetin, is a recognized food allergen in chicken meat and hen eggs. Currently, there is no immunoassay available for its detection from food matrices. The characterization of chicken serum albumin-specific antibodies and the extraction of the target protein are essential for immunoassay development. One monoclonal antibody (mAb), 3H4, was used in this study due to its selectivity to a linear epitope on avian serum albumin. To study the extraction of chicken serum albumin, phosphate-buffered saline (PBS) with two additives, i.e., sodium dodecyl sulfate (SDS) and dithiothreitol (DTT), was used for its extraction from chicken blood plasma and hen egg yolk. SDS and DTT improved the chicken serum albumin's recovery and enhanced chicken serum albumin's immunodetection. In addition, chicken serum albumin retained the best solubility and immunoreactivity after heat treatment in a neutral condition. It experienced degradation and aggregation in acidic and alkaline conditions, respectively. Overall, PBS containing 0.1% SDS and 1 mM DTT (pH 7.2) was a better extraction buffer for chicken serum albumin. However, the complexity of the food matrix and elevated temperature could reduce its solubility and immunoreactivity.

Magnetic-Core/Gold-Shell Nanoparticles for the Detection of Hydrophobic Chemical Contaminants.

Magnetic-core/gold-shell nanoparticles (MAuNPs) are of interest for enabling rapid and portable detection of trace adulterants in complex media. Gold coating provides biocompatibility and facile functionalization, and a magnetic core affords analyte concentration and controlled deposition onto substrates for surface-enhanced Raman spectroscopy. Iron oxide cores were synthesized and coated with gold by reduction of HAuCl4 by NH2OH. MAuNPs were grafted with polyethylene glycol (PEG) and/or functionalized with 4-mercaptobenzoic acid (4-MBA) and examined using a variety of microscopic, spectroscopic, magnetometric, and scattering techniques. For MAuNPs grafted with both PEG and 4-MBA, the order in which they were grafted impacted not only the graft density of the individual ligands, but also the overall graft density. Significant Raman signal enhancement of the model analyte, 4-MBA, was observed. This enhancement demonstrates the functionality of MAuNPs in direct detection of trace contaminants. The magnetic deposition rate of MAuNPs in chloroform and water was explored. The presence of 4-MBA slowed the mass deposition rate, and it was postulated that the rate disparity originated from differing NP-substrate surface interactions. These findings emphasize the importance of ligand choice in reference to the medium, target analyte, and substrate material, as well as functionalization procedure in the design of similar sensing platforms.

Aquatic food animals in the United States: Status quo and challenges.

This review summarizes (1) the U.S. status quo for aquatic food animal production and marketing; (2) major food safety and quality issues/concerns for aquatic food animals in the United States, including fish misbranding, finfish/shellfish allergies, pathogens, toxins and harmful residues, microplastics, and genetically engineered salmon; and (3) various U.S. regulations, guidances, and detection methods for the surveillance of fishery products. Overall, fish misbranding is the biggest challenge in the United States due to the relatively low inspection rate. In addition, due to the regulatory differences among countries, illegal animal drugs and/or pesticide residues might also be identified in imported aquatic food animals. Future regulatory and research directions could focus on further strengthening international cooperation, enhancing aquatic food animal inspection, and developing reliable, sensitive, and highly efficient detection methods.

Non-Specific Binding and Cross-Reaction of ELISA: A Case Study of Porcine Hemoglobin Detection.

Different types of enzyme-linked immunosorbent assays (ELISA) have been widely used to control food safety and quality. To develop an accurate and reproducible ELISA, false immunodetection results caused by non-specific binding (NSB) and cross-reaction must be prevented. During the case study of sandwich ELISA development for the detection of porcine hemoglobin (PHb), several critical factors leading to NSB and cross-reaction were found. First, to reduce the NSB of the target analyte, the selection of microplate and blocker was discussed. Second, cross-reactions between enzyme-labeled secondary antibodies and sample proteins were demonstrated. In addition, the function of (3-aminopropyl)triethoxysilane (APTES) was evaluated. Overall, this study highlights the essence of both antibody and assay validation to minimize any false-positive/negative immunodetection results.

Effect of Processing on Fish Protein Antigenicity and Allergenicity.

Fish allergy is a life-long food allergy whose prevalence is affected by many demographic factors. Currently, there is no cure for fish allergy, which can only be managed by strict avoidance of fish in the diet. According to the WHO/IUIS Allergen Nomenclature Sub-Committee, 12 fish proteins are recognized as allergens. Different processing (thermal and non-thermal) techniques are applied to fish and fishery products to reduce microorganisms, extend shelf life, and alter organoleptic/nutritional properties. In this concise review, the development of a consistent terminology for studying food protein immunogenicity, antigenicity, and allergenicity is proposed. It also summarizes that food processing may lead to a decrease, no change, or even increase in fish antigenicity and allergenicity due to the change of protein solubility, protein denaturation, and the modification of linear or conformational epitopes. Recent studies investigated the effect of processing on fish antigenicity/allergenicity and were mainly conducted on commonly consumed fish species and major fish allergens using in vitro methods. Future research areas such as novel fish species/allergens and ex vivo/in vivo evaluation methods would convey a comprehensive view of the relationship between processing and fish allergy.

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for porcine hemoglobin quantification.

The major objective of this study was to establish a monoclonal antibody (mAb)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of porcine hemoglobin (PHb) in raw meat products. Before assay development, two mAbs immunoreactive to PHb ß subunit with different epitopes were characterized. The optimized immunoassay was specific to PHb and had a wide PHb working range from 15.6 µg/mL to 3,000 µg/mL and high reproducibility with low coefficient of variations (CV < 20%). Through this assay, the estimated PHb residuals in pork loin and shoulder meats were 0.4 mg/g and 1.1 mg/g, respectively. In addition, this immunoassay could effectively quantify PHb in laboratory-spiked meats (pork loin, pork shoulder, and turkey breast) with acceptable recovery. Overall, this is the first mAb-based sandwich ELISA that is suitable for the government, food industry, and third-party authority to monitor PHb residuals or porcine blood adulteration in raw pork and pork-free meat products.

Challenges in SERS-based pesticide detection and plausible solutions.

Surface-enhanced Raman spectroscopy (SERS) can be used for the detection of trace amounts of pesticides in foods to ensure consumer safety. In this perspective, we highlight the trends of SERS-based assays in pesticide detection and the various challenges associated with their selectivity, reproducibility, and nonspecific binding. We also discuss and compare the target analyte capture techniques, such as the use of antibodies, aptamers, and molecularly imprinted polymers (MIPs), coupled with SERS to overcome the drawbacks as mentioned above. In addition, issues related to the nonspecific binding of analytes and its potential solution are discussed.

Matrix effect on food allergen detection - A case study of fish parvalbumin.

Two fish parvalbumin models were established to study relationships among matrix effect, extractability, and thermostability during in vitro immunodetection using two parvalbumin-specific monoclonal antibodies (3E1 and PARV19). Our results illustrated that matrix-induced thermal instability of parvalbumin was due mainly to physical (hydrophobic effect) and chemical (thiol-disulfide interchange) interactions. The addition of sodium dodecyl sulfate (SDS, surfactant), ß-mercaptoethanol (reducing agent) or ethylenediaminetetraacetic acid (EDTA, metal chelator) during sample preparation could not only increase the extractability of parvalbumin but also enhanced its immunodetection. Our findings demonstrated excess EDTA completely chelated Ca2+ in parvalbumin and rendered it undetectable using PARV19 (a Ca2+-dependent antibody). Overall, our resulted showed that matrix effect on in vitro analyte quantification cannot be underestimated. Any false negative or positive results could lead to severe or life-threatening allergic reactions.

Can Glycation Reduce Food Allergenicity?

As a naturally occurring reaction during food processing, glycation, also known as non-enzymatic browning or Maillard reaction, can improve food protein physiochemical properties and functionality. In this perspective, three aspects of glycation (terminology confusion between glycation and glycosylation, its current application, and its impact on immunoreactivity) are elaborated. Overall, the immunoreactivity of glycated proteins may decrease, remain unchanged, or even increase after food glycation. Also, it should be noted that the effect of glycation on the immunoglobulin (Ig)E- or IgG-binding capacity of allergens does not necessarily and correctly predict the allergenicity of the glycated protein in the allergic patient population.

Monoclonal antibody-based ELISA for the quantification of porcine hemoglobin in meat products.

Misusage of porcine blood proteins, such as misbranding and substitution, can cause religious objections, law violation, and food quality concerns. These issues highlight the need for detecting unlabeled or overuse of porcine blood in foods. Compared with acidic and neutral pHs, porcine hemoglobin (PHb) at alkaline pH retained the best solubility, molecular integrity, and immunoreactivity after heat treatment. PHb at acidic and alkaline pHs remained stable during storage at 4 °C for 29 days. A monoclonal antibody (mAb) specific to mammalian hemoglobin, 13F7, was developed. A mAb13F7-based indirect competitive ELISA (icELISA) was optimized for the quantification of PHb in meat products. This assay had a wide working range from 0.5 ppm to 1000 ppm. It was sensitive (limit of detection: 0.5 ppm), precise and reproducible with low inter- and intra-coefficient of variances (<20%). This assay is suitable for government, food industry, and third-party authority to surveillance food quality.

Immunoassay for the Detection of Animal Central Nervous Tissue in Processed Meat and Feed Products.

An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the detection of the thermal-stable central nervous tissue (CNT) marker protein, myelin basic protein (MBP), was developed to detect animal CNT in processed meat and feedstuffs. Two meat samples (cooked at 100 °C for 30 min and autoclaved at 133 °C for 20 min) of bovine brain in beef and two feed samples (bovine brain meal in beef meal and in soybean meal) were prepared at levels of 0.0008, 0.0031, 0.0063, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6%. An anti-MBP monoclonal antibody (mAb3E3) was produced using the hybridoma technique and characterized using Western blot. The optimized icELISA was CNT-specific without cross-reactivity with either meat (beef and pork) or soybean meal samples and had low intra-assay (%CV ≤ 3.5) and interassay variability (%CV ≤ 3.3), with low detection limits for bovine MBP (6.4 ppb) and bovine CNT spiked in both meat (0.05%) and feed (0.0125%) samples. This assay is therefore suitable for the quantitative detection of trace amounts of contaminated animal CNT in processed food and feed products.

Storage Stability of Food Protein Hydrolysates-A Review.

In recent years, mainly due to the specific health benefits associated with (1) the discovery of bioactive peptides in protein hydrolysates, (2) the reduction of protein allergenicity by protein hydrolysis, and (3) the improved protein digestibility and absorption of protein hydrolysates, the utilization of protein hydrolysates in functional foods and beverages has significantly increased. Although the specific health benefits from different hydrolysates are somewhat proven, the delivery and/or stability of these benefits is debatable during distribution, storage, and consumption. In this review, we discuss (1) the quality changes in different food protein hydrolysates during storage; (2) the resulting changes in the structure and texture of three food matrices, i.e., low moisture foods (LMF, aw < 0.6), intermediate moisture foods (IMF, 0.6 ≤ aw < 0.85), and high moisture foods (HMF, aw ≥ 0.85); and (3) the potential solutions to improve storage stability of food protein hydrolysates. In addition, we note there is a great need for evaluation of biofunction availability of bioactive peptides in food protein hydrolysates during storage.

Storage stability of a commercial hen egg yolk powder in dry and intermediate-moisture food matrices.

Quality loss in intermediate-moisture foods (IMF) such as high-protein nutrition bars (HPNB) in the form of hardening, nonenzymatic browning, and free amino group loss is a general concern for the manufacturers. To measure the extent of quality loss over time in terms of these negative attributes, through changing the ratio by weight between two commercial spray-dried hen egg powders, egg white (DEW) and egg yolk (DEY), the storage stability of 10 IMF systems (water activity (aw) ∼ 0.6) containing 5% glycerol, 10% shortening, 35% protein, and 50% sweetener (either maltitol or 50% high-fructose corn syrup/50% corn syrup (HFCS/CS)) were studied. Additionally, the storage stability of the DEY powder itself was investigated. Overall, during storage at different temperatures (23, 35, and 45 °C), the storage stability of DEY in dry and IMF matrices was mainly controlled by the coaction of three chemical reactions (disulfide bond interaction, Maillard reaction, and lipid oxidation). The results showed that by replacing 25% of DEW in an IMF model system with DEY, the rate of bar hardening was significantly lower than that of the models with only DEW at all temperatures due to the softening effect of the fat in DEY. Furthermore, the use of maltitol instead of HFCS/CS in all bar systems not only resulted in decreased hardness but also drastically decreased the change in the total color difference (ΔE*). Interestingly, there was no significant loss of free amino groups in the maltitol systems at any DEW/DEY ratio.

Storage stability of hen egg white powders in three protein/water dough model systems.

In recent years, due to the specific health benefits associated with bioactive peptides and the reduction of protein allergenicity by enzymatic hydrolysis, the utilisation of protein hydrolysates in the intermediate-moisture food (IMF) market, such as high protein nutrition bars (HPNB), has significantly increased. Currently, no reported study is related to the storage stability of dried hen egg white (DEW) and its hydrolysates (HEW) in an IMF matrix. Therefore, three DEW/HEW dough model systems (100%HEW+0%DEW, 75%HEW+25%DEW and 50%HEW+50%DEW) were established using two commercial spray-dried egg white powders to study the effect of temperature and fraction of HEW on these IMF models (water activity (a(w)): ∼0.8). During storage at three different temperatures (23, 35 and 45°C) for 70 days, the selected physicochemical properties of the dough systems were compared. Overall, kinetic analysis showed an apparent zero-order model fit for the change in the colour (L(∗)), fluorescence intensity (FI) and hardness, as a function of time, for different dough model systems. As expected, the L(∗), FI and hardness increased as a function of time mainly due to the Maillard reaction. The amount of free amino groups decreased, with an increase in rate of loss, as temperature increased in the 100%HEW+0%DEW model. When DEW was substituted for some HEW, the regeneration of the free amino groups after loss was observed as a function of time. Furthermore, when the percentage of HEW was decreased, the incidence of mouldy samples occurred sooner, which indicates that HEW has some antimicrobial ability, especially in the 100%HEW+0%DEW system where mould growth did not occur.