Development of a sandwich enzyme-linked immunosorbent kit for reliable detection of milk allergens in processed food.

The inclusion of undeclared cow's milk proteins may cause health complications to milk-allergic consumers and is one of the leading cause of food recall in many countries all over the world. Therefore, to keep control on such incidences in processed products, we established a milk sandwich ELISA test kit by incorporating two polyclonal antibodies against milk proteins obtained from different species. Its analytical effectiveness in terms of sensitivity, specificity, accuracy, trueness, and precision were all analyzed. The limit of detection (LOD) of the test kit was 0.011 ppm, with high specificity for milk protein residues. The test kit was highly specific, apart from considerable cross-reactivity with goat milk and minor cross-reactivity with donkey and horse milk. The coefficient of variation of the test kit for intra-assay ranged from 4.02% to 14.62% and inter-assay ranged from 6.05% to 15.08% respectively. The sandwich ELISA was highly specific in detecting commercial food products. In a limited retail survey, 5/6 of the milk proteins declared on the ingredient labels tested positive for milk proteins. The study offers effective technical support for the sensitive detection of milk products both for food manufacturers and regulatory authorities.

A review on food processing and preparation methods for altering fish allergenicity.

People eat many varieties of food to satiate their hunger. Among them, a few numbers of food cause overreaction of the body's immune system, and fish holds a permanent position on that list. Processing methods, including one treatment or a combination, can have different effects on the allergenic potential of food proteins. An important point to note, however, is that not all of these methods can eliminate the potential for protein allergy. Thus, it is essential to understand the risk involved with the consumption of processed fish and its derivatives. Fish could be prepared in various ways before come to the dining plate. It has shown some of these methods can effectively manipulate the allergenicity owing to the alterations occurred in the protein conformation. This article provides an overview of the impact of fish processing methods (thermal and non-thermal) on the allergenic potential of fish along with possible causative structural modification provokes allergen stability. The article begins with current trends related to fish consumption, proceeds with the prevalence and underlying mechanism of fish allergy. Properties of clinically relevant fish proteins, projected IgE epitopes of PV, cross-reactivity of fish allergens are also addressed in this context to understand and compare the behavioral patterns of PV profiles of different species on processing methods.

Development of a sensitive sandwich-ELISA assay for reliable detection of fish residues in foods.

A new sandwich-type Enzyme-Linked Immunosorbent Assay (ELISA) method was developed based on goat IgG as capturing antibody and rabbit IgG as detecting antibody targeting soluble antigenic fish proteins in foods as detection targets. The assay has provided a relatively lower limit of quantitation (LoQ) for fish proteins with LoQ 0.5 ng/ml and appears highly sensitive. The analysis of 24 different substances, both raw and boiled, revealed no cross-reactivity above the cut-off point of the limit of quantitation. Recoveries of the SB spiked food matrixes were in the range of 83-131%. Assay precision testing proved that repeatability (<5%) and reproducibility (<11%) had an acceptable level of variation. The sandwich ELISA was capable of detecting all tested commercially important fish. As a potential analytical tool, the newly developed immunoenzymatic method is suitable for detecting undeclared fish residues in real food samples available in the market, thereby will help to reduce the incidents of fish allergies.

Whey allergens: Influence of nonthermal processing treatments and their detection methods.

Whey and its components are recognized as value-added ingredients in infant formulas, beverages, sports nutritious foods, and other food products. Whey offers opportunities for the food industrial sector to develop functional foods with potential health benefits due to its unique physiological and functional attributes. Despite all the above importance, the consumption of whey protein (WP) can trigger hypersensitive reactions and is a constant threat for sensitive individuals. Although avoiding such food products is the most successful approach, there is still a chance of incorrect labeling and cross-contamination during food processing. As whey allergens in food products are cross-reactive, the phenomenon of homologous milk proteins of various species may escalate to a more serious problem. In this review, nonthermal processing technologies used to prevent and eliminate WP allergies are presented and discussed in detail. These processing technologies can either enhance or mitigate the impact of potential allergenicity. Therefore, the development of highly precise analytical technologies to detect and quantify the existence of whey allergens is of considerable importance. The present review is an attempt to cover all the updated approaches used for the detection of whey allergens in processed food products. Immunological and DNA-based assays are generally used for detecting allergenic proteins in processed food products. In addition, mass spectrometry is also employed as a preliminary technique for detection. We also highlighted the latest improvements in allergen detection toward biosensing strategies particularly immunosensors and aptasensors.

Potential efficacy of processing technologies for mitigating crustacean allergenicity.

Crustacean allergy has become a growing food safety concern at a global scale. In the past decades, various food processing approaches have been employed to develop food products with reduced allergenic potential. Thermal treatment can dramatically influence the allergenicity of crustaceans by either reducing or enhancing their allergenic potential. Maillard reaction, enzymatic and acid treatments have shown to be promising in mitigating crustacean allergenicity. Recently, novel processing technologies, namely high-pressure processing, high-intensity ultrasound, irradiation, pulsed ultraviolet light and hurdle technology have attracted special attention from the researchers and the food industry professionals owing to their benefits over the conventional methods. In this context, this review paper provides an updated overview of the current knowledge on how different food processing methods induce structural changes of crustacean allergens and, subsequently, influence their allergenic potential. Data on prevalence and clinical relevance of crustacean allergy are presented, as well as, the molecular characterization of crustacean allergens and the main analytical methods for their detection in processed foods.

Coronary artery disease pattern: a comparision among different age groups.

BACKGROUND: Angiographic studies have shown that the extent of disease and severity is less in younger population with comparatively fewer number of coronary arteries affected than that of older age groups. This study was conducted to compare the pattern of coronary artery disease (CAD) in young adults under 40 years and those more than 40 years of age with significant coronary artery disease on coronary angiography. METHODS: This cross-sectional study was conducted in Angiography Laboratory of Punjab Institute of Cardiology, Lahore,-from 1st April, 2013 to 1st April, 2014. Patients from Angiography Laboratory of Punjab institute of cardiology Lahore were enrolled in the study. A Joint Committee of consultants reported the coronary lesions (stenosis). Single vessel disease (SVD), Double vessel disease (DVD) and Tripple vessel disease (TVD) was noted down. RESULTS: Out of total 400, patients of age ≤40 years (Group-A) were 48 (12%) and >40 years (Group-B) were 352 (88%). There were 77% male patients and 22.5% females. Mean age of patients was 52.78±10.02. Significant CAD was present in 48 (10.75%) patients of Group-A and in 306 (76.5%) patients of group-B. Frequency of SVD DVD and TVD in Group-A was 24 (50%), 10 (20.8%) and 9 (18%) respectively while frequency of SVD, DVD and TVD in group-B was 98 (27%), 88 (25%) and 120 (34%) respectively with p-value of 0.002, 0.53 and 0.03 among SVD, DVD and TVD between the two groups. CONCLUSION: Young (age <40 years) patients who have coronary artery disease, have less extensive coronary artery disease, with a high incidence of angiographically normal vessels and relative paucity of left main coronary arterial involvement.

Screening of various Brassica species for phytoremediation of heavy metals-contaminated soil of Lakki Marwat, Pakistan.

Natural resources, especially agrarian soils, have been much contaminated with various pollutants including heavy metals since industrial revolution, so it is pertinent to utilize green technology, the so-called phytoremediation technology for reclamation of heavy metal-contaminated soils. A pot experiment was conducted to screen four different species (Brassica juncea, Brassica napus, Brassica rapa, Brassica campestris) of Brassicaceae family for the remediation of HMs contaminated soil of Lakki Marwat city, Pakistan, irrigated with municipal wastewater. Plants were analyzed for various morpho-physiological, biochemical, and phytoextraction factors like bioaccumulation (BAF) and translocation factor (TF). Results showed maximum morpho-physiological responses including seed germination, chlorophyll content, root fresh and dry weights, and shoot fresh and dry weights in B. juncea followed by B. napus, B. campestris, and B. rapa. Plant biochemical analyses of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) also exhibited maximum activity in B. juncea followed by B. napus, B. campestris, and B. rapa, respectively, in both control and contaminated soils. Conversely, plant oxidative stress markers including malondialdehyde (MDA) and hydrogen peroxide (H2O2) showed maximum contents in B. rapa followed by B. campestris, B. napus, and B. juncea in both soils. Plant bioconcentration factors i.e. BAF and TF measured for all species in both soils confirmed that B. juncea accumulated maximum heavy metals. Similarly, enhanced phytoextraction capacity was noticed for all Brassica species in decreasing order i.e. B. napus > B. campestris > B. rapa. Hence, all the results confirmed that B. juncea excelled and can be recommended for phytoremediation purpose in soils of study area.

A sensitive sandwich enzyme-linked immunosorbent assay (sELISA) targeted multiple wheat protein fractions for the detection of several cereal grains in processed foods.

Wheat allergy has become a global public health and food safety concern; however, there is no accessible cure for wheat allergy. The complete exclusion of wheat-containing foods and environmental exposure is the most efficient allergy management to avoid the adverse reactions, which can be severe and occasionally life threatening. Therefore, the assay for accurate detection of wheat residues is demanded urgently for appropriate labeling guidelines and consumer safety. Thus, a sandwich enzyme-linked immunosorbent assay (sELISA) targeting multiple wheat protein fractions was fabricated in the present study. The results showed that the limit of detection (LOD) and limit of quantitation (LOQ) of the constructed sELISA were 0.25 and 0.5 µg/g with high specificity for wheat. No cross-reactivity was observed in 32 foods or food ingredients tested, except barley and rye. The developed sELISA can also discriminate against many commercial foods containing declared or undeclared wheat residues except for Chinese yellow wine. Furthermore, high heat also can obtain a higher level of proteins extracted with corresponding enhanced detectability up to 100°C from heated samples and 160 °C in baked samples. PRACTICAL APPLICATION: Wheat is the most common food ingredient and wildly applied in various processed foods. However, wheat can cause severe and life-threatening symptoms in some allergic patients and must be labeled and tested accurately to protect those with a wheat allergy. Developing a new test assay can serve as a powerful tool for food manufacturers and regulatory agencies to accurately quantify wheat residues in processed foods and ensure their absence due to unintended contamination.

Identification and Amino Acid Analysis of Allergenic Epitopes of a Novel Allergen Paramyosin (Rap v 2) from Rapana venosa.

Besides tropomyosin (TM) that is widely recognized as a major allergen in molluscs, a 99-kDa novel allergen (Rap v 2) was recently found in the sea snail Rapana venosa and identified as paramyosin (PM). However, the allergenic epitopes of PM in any molluscs have not been identified yet. In the present study, seven allergenic epitopes of Rap v 2 were identified by immunoinformatics tools, dot-blot inhibition assay, and basophil degranulation assay. Based on the analysis of PM and allergenic epitope amino acids, it was found that highly hydrophobic and positively charged amino acid residues play an important role in the formation of Rap v 2 epitopes. In addition, three potential critical amino acids that may account for TM and PM cross-reactivity in molluscs were found by sequence- and structure-based methods. These findings could be of major importance for improving the understanding of relevant paramyosin epitopes and the prevention and therapy of mollusc allergy.

Extraction of total wheat (Triticum aestivum) protein fractions and cross-reactivity of wheat allergens with other cereals.

A one-step mild extraction of total wheat protein fractions was developed in this study, and the allergic cross-reactivity among dietary cereals were assessed by SDS-PAGE, western blotting, indirect ELISA, and inhibition ELISA using sera from 12 wheat allergic patients. The fractions of albumin, globulin, gliadin and glutenins in wheat flour can be obtained by a one-step extraction with Na2CO3-NaHCO3 (20 mM, pH 9.6, 0.5 M NaCl, 40% ethanol, 1 mM PMSF) in comparison to sequential extractions. Results showed high cross-reactivity in wheat, barley and rye due to close resemblance and high sequence identity (>50%), whereas nearly negligible cross-reactivity among rice, buckwheat, and quinoa was observed. Our research findings suggest that people with wheat allergy should rely primarily on the use of rice, quinoa and non-grain buckwheat, which is an effective substitute for wheat, while those with hypersensitivity should avoid the use of barley and rye in their diet.

Tyrosinase/caffeic acid cross-linking alleviated shrimp (Metapenaeus ensis) tropomyosin-induced allergic responses by modulating the Th1/Th2 immunobalance.

In this study, the effect of enzymatic cross-linking of shrimp tropomyosin (TM) with tyrosinase and caffeic acid (TM-Tyr/CA) on the allergic response were assessed using in vitro and in vivo models. The RBL-2H3 and KU812 cell lines were employed to evaluate the changes in the stimulation abilities of TM-Tyr/CA that showed significant inhibition of mediators and cytokines. The digestibility of cross-linked TM was improved and the recognitions of IgG/IgE were markedly reduced, as revealed by western blotting. TM-Tyr/CA decreased anaphylactic symptoms, and hindered the levels of IgG1, IgE, histamine, tryptase and mouse mast-cell protease-1 (mMCP-1) in mice sera. Cross-linked TM downregulated the production of interleukin (IL)-4, IL-5, and IL-13 by 51.36, 12.24 and 20.55%, respectively, whereas, IL-10 and IFN-γ were upregulated by 20.71 and 19.0%. TM-Tyr/CA showed reduced allergenicity and may have preventive effect in relieving TM induced allergic response via immunosuppression and positive modulation of T-helper (Th)1/Th2 immunobalance.

A Tagetes minuta based eco-benign synthesis of multifunctional Au/MgO nanocomposite with enhanced photocatalytic, antibacterial and DPPH scavenging activities.

In this research work, facile, economical and eco-benign experimental procedure were adopted to synthesize Au/MgO nanocomposite with the help of Tagetes minuta leaves extract. Phytochemicals present in the leaves of Tagetes minuta were acting as reducing and stabilizing agents to avoid aggregation of nanomaterials during the preparation of Au/MgO nanocomposite. The biologically synthesized nanocomposite were systematically characterized by UV-vis spectroscopy, Scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared microscopy (FTIR), High resolution transmission electron microscopy (HRTEM), Thermogravimetric analysis (TGA), dynamic light scattering (DLS) and elemental mapping. UV-visible spectrum confirmed the presence of MgO and Au due to the presence of two SPR peaks at 315 nm and 528 nm, respectively. Moreover, the Au/MgO nanocomposite exhibited superior photocatalytic, antibacterial, hemolytic, and antioxidant activities. Photocatalytic performance tests of Au/MgO nanocomposite were- appraised by the rapid degradation of the methylene blue (MB) under UV light illumination. More importantly, after four successive cycles of MB degradation, the photocatalytic efficacy remained unchanged, which ensures the stability of the Au/MgO nanocomposite. Furthermore, the antibacterial tests showed that the advanced nanocomposite inhibited the growth of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus with zones of inhibition 18 (±0.3), 21 (±0.5), and 19 (±0.4) mm, respectively. The cytotoxicity study revealed that Au/MgO nanocomposite is nontoxic to ordinary healthy RBCs. Interestingly, the Au/MgO nanocomposite also possesses an excellent antioxidant activity, whereby effectively scavenging 82% stable and harmful DPPH. Overall, the present study concludes that eco-benign Au/MgO nanocomposite has excellent potential for the remediation of bacterial pathogens and degradation of MB.

Purification, Characterization, and Three-Dimensional Structure Prediction of Paramyosin, a Novel Allergen of Rapana venosa.

Paramyosin (PM) is an important structural protein in molluscan muscles. However, as an important allergen, there is a little information on PM in the molluscs. In this study, a 99 kDa molecular weight allergen protein was purified from Rapana venosa and confirmed as PM by mass spectrometry. The results of immunoglobulin E (IgE)-binding activity and physicochemical characterization showed that R. venosa PM could react with a specific IgE of the sera from sea snail-allergic patients, and the IgE-binding activity could be reduced by thermal treatment. The full-length cDNA of R. venosa PM was cloned, which encodes 859 amino acid residues, and it has a higher homology among molluscan species. According to the circular dichroism results, Fourier transform infrared, and 2D and 3D structure analysis, both PM and tropomyosin are conserved proteins, which are mainly composed of the α-helix structure. These results are significant for better understanding the anaphylactic reactions in sea snail-allergic patients and allergy diagnosis.