Effects of protein variations by different extraction and dehydration approaches on hempseed protein isolate: Protein pattern, amino acid profiling and label-free proteomics.

This study evaluates the effects of alkaline and micellisation extraction methods, alongside freeze-drying and spray-drying, on the protein subunits, amino acid profiles, and proteome data of hempseed protein isolate (HPI). Findings revealed that the extraction methods affect protein profiles more than the drying methods. Micellisation-extracted HPI showed higher albumin, oleosin, and sulphur-containing protein levels than alkaline-extracted HPI. The alkali-extracted undried sample (AU) gave more potentially allergenic proteins, including Hsp70 and triosephosphate isomerase, than its micellization-extracted counterpart (MU). Unique potential allergens were identified, including malate dehydrogenase and enolase in AU, and RuBisCo in MU samples. Both drying processes impacted the HPI proteome and reduced RuBisCo in the micellisation-extracted HPI. These insights highlight the crucial role of method selection in HPI processing for optimising production in the food industry.

Suitability of Selected Apple Varieties for People with Allergies and Diabetes.

The study aimed to select apple varieties suitable for allergy sufferers and people with diabetes. The total polyphenol content, sugar content, acidity, and antioxidant activity of the apple fruit juices were determined using spectrophotometric techniques. The allergenic content in the apple juices was also measured. The strength of sensitisation was assessed using the ELISA method. Given their minimal content of both profilins and Bet v 1 homologues, Koksa Pomaranczowa (4.24 ± 0.08 µg/g Bet v 1 and 4.49 ± 0.82 ng/g profilins) and Ksiaze Albrecht Pruski (5.57 ± 0.07 µg/g Bet v 1 and 3.34 ± 0.09 ng/g profilins) were identified as suitable for people with allergies. For people with diabetes, the most suitable apple variety was found to be Jakub Lebel, providing large doses of antioxidants and polyphenols (41.10 ± 0.20 and 5.16 ± 0.42, respectively) and a relatively low sugar content (9.06 g/100 g).

Effect of Addition of Tannin Extract from Underutilized Resources on Allergenic Proteins, Color and Textural Properties of Egg White Gels.

Tannins, present in numerous plants, exhibit a binding affinity for proteins. In this study, we aimed to exploit this property to reduce the concentration of allergenic egg white proteins. Tannins were extracted, using hot water, from the lyophilized powder of underutilized resources, such as chestnut inner skin (CIS), young persimmon fruit (YPF), and bayberry leaves (BBLs). These extracts were then incorporated into an egg white solution (EWS) to generate an egg white gel (EWG). Allergen reduction efficacy was assessed using electrophoresis and ELISA. Our findings revealed a substantial reduction in allergenic proteins across all EWGs containing a 50% tannin extract. Notably, CIS and BBL exhibited exceptional efficacy in reducing low allergen levels. The addition of tannin extract resulted in an increase in the total polyphenol content of the EWG, with the order of effectiveness being CIS > YPF > BBL. Minimal color alteration was observed in the BBL-infused EWG compared to the other sources. Additionally, the introduction of tannin extract heightened the hardness stress, with BBL demonstrating the most significant effect, followed by CIS and YPF. In conclusion, incorporating tannin extract during EWG preparation was found to decrease the concentration of allergenic proteins while enhancing antioxidant properties and hardness stress, with BBL being particularly effective in preventing color changes in EWG.

In silico evaluation of the potential allergenicity of a fungal biomass from Rhizomucor pusillus for use as a novel food ingredient.

The world's hunger for novel food ingredients drives the development of safe, sustainable, and nutritious novel food products. For foods containing novel proteins, potential allergenicity of the proteins is a key safety consideration. One such product is a fungal biomass obtained from the fermentation of Rhizomucor pusillus. The annotated whole genome sequence of this strain was subjected to sequence homology searches against the AllergenOnline database (sliding 80-amino acid windows and full sequence searches). In a stepwise manner, proteins were designated as potentially allergenic and were further compared to proteins from commonly consumed foods and from humans. From the sliding 80-mer searches, 356 proteins met the conservative >35% Codex Alimentarius threshold, 72 of which shared ≥50% identity over the full sequence. Although matches were identified between R. pusillus proteins and proteins from allergenic food sources, the matches were limited to minor allergens from these sources, and they shared a greater degree of sequence homology with those from commonly consumed foods and human proteins. Based on the in silico analysis and a literature review for the source organism, the risk of allergenic cross-reactivity of R. pusillus is low.

Accelerating the identification of the allergenic potential of plant proteins using a stacked ensemble-learning framework.

Plant-allergenic proteins (PAPs) have the potential to induce allergic reactions in certain individuals. While these proteins are generally innocuous for the majority of people, they can elicit an immune response in those with particular sensitivities. Thus, screening and prioritizing the allergenic potential of plant proteins is indispensable for the development of diagnostic tools, therapeutic interventions or medications to treat allergic reactions. However, investigating the allergenic potential of plant proteins based on experimental methods is costly and labour-intensive. Therefore, we develop StackPAP, a three-layer stacking ensemble framework for accurate large-scale identification of PAPs. In StackPAP, at the first layer, we conducted a comprehensive analysis of an extensive set of feature descriptors. Subsequently, we selected and fused five potential sequence-based feature descriptors, including amphiphilic pseudo-amino acid composition, dipeptide deviation from expected mean, amino acid composition, pseudo amino acid composition and dipeptide composition. Additionally, we applied an efficient genetic algorithm (GA-SAR) to determine informative feature sets. In the second layer, 12 powerful machine learning (ML) methods, in combination with all the informative feature sets, were employed to construct a pool of base classifiers. Finally, 13 potential base classifiers were selected using the GA-SAR method and combined to develop the final meta-classifier. Our experimental results revealed the promising prediction performance of StackPAP, with an accuracy, Matthew's correlation coefficient and AUC of 0.984, 0.969 and 0.993, respectively, as judged by the independent test dataset. In conclusion, both cross-validation and independent test results indicated the superior performance of StackPAP compared with several ML-based classifiers. To accelerate the identification of the allergenicity of plant proteins, we developed a user-friendly web server for StackPAP (https://pmlabqsar.pythonanywhere.com/StackPAP). We anticipate that StackPAP will be an efficient and useful tool for rapidly screening PAPs from a vast number of plant proteins.Communicated by Ramaswamy H. Sarma.

The Interplay of Protein Hydrolysis and Ammonia in the Stability of Hevea Rubber Latex during Storage.

Natural rubber (NR) latex derived from Hevea brasiliensis is a complex colloid comprising mainly rubber hydrocarbons (latex particles) and a multitude of minor non-rubber constituents such as non-rubber particles, proteins, lipids, carbohydrates, and soluble organic and inorganic substances. NR latex is susceptible to enzymatic attack after it leaves the trees. It is usually preserved with ammonia and, to a lesser extent, with other preservatives to enhance its colloidal stability during storage. Despite numerous studies in the literature on the influence of rubber proteins on NR latex stability, issues regarding the effect of protein hydrolysis in the presence of ammonia on latex stability during storage are still far from resolved. The present work aims to elucidate the interplay between protein hydrolysis and ammoniation in NR latex stability. Both high- and low-ammonia (with a secondary preservative) NR latexes were used to monitor the changes in their protein compositions during storage. High-ammonia (FNR-A) latex preserved with 0.6% (v/v) ammonia, a low 0.1% ammonia/TMTD/ZnO (FNR-TZ) latex, and a deproteinized NR (PDNR) latex were labeled with fluorescence agents and observed using confocal laser scanning microscopy to determine their protein composition. Protein hydrolysis was confirmed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that protein hydrolysis increased with the storage duration. The change in protein composition accompanying hydrolysis also allows the spatial distribution of allergenic proteins to be estimated in the latex. Concurrently, the latex stability increased with the storage duration, as measured by the latex's mechanical stability time (MST) and the zeta potential of the latex particles. As monitored by AFM, the surface roughness of the NR latex film increased markedly during extended storage compared with that of the DPNR latex, which remained smooth. These results underscore the pivotal role of ammonia in bolstering NR latex stability brought on by protein hydrolysis, which greatly impacts latex film's formation behavior. NR latex stability underpins the quality of latex-dipped goods during manufacturing, particularly those for medical gloves.

Outdoor airborne allergens: Characterization, behavior and monitoring in Europe.

Aeroallergens or inhalant allergens, are proteins dispersed through the air and have the potential to induce allergic conditions such as rhinitis, conjunctivitis, and asthma. Outdoor aeroallergens are found predominantly in pollen grains and fungal spores, which are allergen carriers. Aeroallergens from pollen and fungi have seasonal emission patterns that correlate with plant pollination and fungal sporulation and are strongly associated with atmospheric weather conditions. They are released when allergen carriers come in contact with the respiratory system, e.g. the nasal mucosa. In addition, due to the rupture of allergen carriers, airborne allergen molecules may be released directly into the air in the form of micronic and submicronic particles (cytoplasmic debris, cell wall fragments, droplets etc.) or adhered onto other airborne particulate matter. Therefore, aeroallergen detection strategies must consider, in addition to the allergen carriers, the allergen molecules themselves. This review article aims to present the current knowledge on inhalant allergens in the outdoor environment, their structure, localization, and factors affecting their production, transformation, release or degradation. In addition, methods for collecting and quantifying aeroallergens are listed and thoroughly discussed. Finally, the knowledge gaps, challenges and implications associated with aeroallergen analysis are described.

Proteomic characterization of extracellular vesicles released by third stage larvae of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae).

Introduction: Anisakis pegreffii is a sibling species within the A. simplex (s.l.) complex requiring marine homeothermic (mainly cetaceans) and heterothermic (crustaceans, fish, and cephalopods) organisms to complete its life cycle. It is also a zoonotic species, able to accidentally infect humans (anisakiasis). To investigate the molecular signals involved in this host-parasite interaction and pathogenesis, the proteomic composition of the extracellular vesicles (EVs) released by the third-stage larvae (L3) of A. pegreffii, was characterized. Methods: Genetically identified L3 of A. pegreffii were maintained for 24 h at 37°C and EVs were isolated by serial centrifugation and ultracentrifugation of culture media. Proteomic analysis was performed by Shotgun Analysis. Results and discussion: EVs showed spherical shaped structure (size 65-295 nm). Proteomic results were blasted against the A. pegreffii specific transcriptomic database, and 153 unique proteins were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis predicted several proteins belonging to distinct metabolic pathways. The similarity search employing selected parasitic nematodes database revealed that proteins associated with A. pegreffii EVs might be involved in parasite survival and adaptation, as well as in pathogenic processes. Further, a possible link between the A. pegreffii EVs proteins versus those of human and cetaceans' hosts, were predicted by using HPIDB database. The results, herein described, expand knowledge concerning the proteins possibly implied in the host-parasite interactions between this parasite and its natural and accidental hosts.

Proteome Exploration of Human Coronaviruses for Identifying Novel Vaccine Candidate: A Hierarchical Subtractive Genomics and Reverse Vaccinology Approach.

BACKGROUND: The SARS-CoV-2 has been responsible for infecting more than 613,615,658 people in 222 countries by September 11, 2022, of which 6,516,076 have died. COVID-19 was introduced by World Health Organization as a global concern and a pandemic disease due to its prevalence. OBJECTIVE: Developing preventive or therapeutic medications against 2019-nCoV is an urgent need, and has been deemed as a high priority among scientific societies; in this regard, the production of effective vaccines is one of the most significant and high-priority requirements. Because of costly and time-consuming process of vaccine design, different immunoinformatics methods have been developed. METHODS: At the beginning of vaccine design, the proteome study is essential. In this investigation, the whole human coronavirus proteome was evaluated using the proteome subtraction strategy. Out of 5945 human coronavirus proteins, five new antigenic proteins were selected by analyzing the hierarchical proteome subtraction, and then their various physicochemical and immunological properties were investigated bioinformatically. RESULTS: All five protein sequences are antigenic and non-allergenic proteins; moreover, the spike protein group, including spike glycoprotein (E2) (Peplomer protein), spike fragment and spike glycoprotein fragment, showed acceptable stability, which can be used to design new vaccines against human coronaviruses. CONCLUSION: The selected peptides and the other proteins introduced in this study (HE, orf7a, SARS_X4 domain-containing protein and protein 8) can be employed as a suitable candidate for developing a novel prophylactic or therapeutic vaccine against human coronaviruses.

Clonagem, expressão recombinante e validação por microarray de proteínas alimentares potencialmente alergênicas / Cloning, recombinant expression and validation by microarray of potentially allergenic food proteins

A maioria das respostas alérgicas a alimentos é mediada por IgE, que pode ser detectada para fins de diagnóstico da alergia alimentar. No entanto, para isso é necessário que alérgenos purificados estejam disponíveis para a elaboração dos diferentes formatos de ensaio, inclusive por microarray, que se constitui em uma ferramenta bastante útil para análise simultânea, e também para a identificação de reatividade cruzada. A esse respeito, é imprescindível ampliar a plataforma de alérgenos que possam ser empregados para a confecção de microarrays. Atualmente, alguns alimentos que constituem objeto de interesse na clínica em função do número de casos de alergia, e sobre os quais as informações a respeito dos alérgenos são escassas, são: abacaxi, mamão, mandioca e manga. Assim, o objetivo desse trabalho foi clonar, expressar e purificar proteínas potencialmente alergênicas de alimentos de importância regional. Após confirmadas por ensaios imunológicos, essas proteínas foram utilizadas na construção e validação de um microarray através de ensaios com os soros de pacientes alérgicos aos alimentos selecionados. Para atingir esse objetivo, foram selecionadas proteínas potencialmente alergênicas coincidentes, apontadas tanto pela similaridade com espécies taxonomicamente mais próximas, quanto pela técnica 2D Western Blotting acoplada à espectrometria de massas. Dezenove proteínas, sendo 4 de abacaxi, 5 de mamão, 6 de mandioca e 4 de manga, foram expressas em Pichia pastoris, purificadas e impressas em um microarray. Após incubar essas proteínas com os soros dos pacientes alérgicos aos alimentos estudados, 18 proteínas mostraram-se potencialmente alergênicas. Além disso, foi observada reatividade cruzada entre proteínas dos alimentos estudados e também em relação ao látex e outros frutos

The majority of allergic reactions to foods is IgE-mediated, which can be detected for the diagnosis of food allergy. However, purified allergens are necessary to produce different kinds of allergy tests, including microarray, which is a useful tool for simultaneous analysis, as well as for the identification of cross-reactivity. In this respect, it is essential to expand the platform of allergens to include them on microarrays. Nowadays, some foods that are object of interest in the clinical area in Brazil and it is necessary a further evaluation about their potential allergens, since there is a limited information about them, are: pineapple, papaya, cassava and mango. Therefore, the aim of this study was cloning, expressing and purifying potentially allergenic proteins of important Brazilian foods. After confirmed by immunological tests, these proteins were used in microarray production and validation by assays with sera from allergic patients to the selected foods. Achieving this goal, matching potentially allergenic proteins were selected, which were identified by comparison among taxonomically closer species (in silico) and 2D Western Blotting coupled with Mass Spectrometry. Nineteen proteins: 4 from pineapple, 5 from papaya, 6 from cassava and 4 from mango were expressed in Pichia pastoris, purified and printed on a microarray. After incubating those proteins with sera from allergic patients to the selected foods, 18 proteins were detected as potentially allergenic. In addition, cross-reactivity was observed among the proteins from the studied foods, and also regarding to the latex and other fruits

Effect of Sample Preparation on the Detection and Quantification of Selected Nuts Allergenic Proteins by LC-MS/MS.

The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0-15 kDa, 15-35 kDa, 35-70 kDa and 70-250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.

Changes in qualitative and quantitative traits of birch (Betula pendula) pollen allergenic proteins in relation to the pollution contamination.

Birch (Betula pendula) pollen causes inhalant allergy in about 20% of human population in Europe, most of which is sensitive to the main birch allergen, Bet v1. The aim of the study was to find out (i) whether and how the analysed birch individuals differ in regard to composition of individual subunits of pollen proteins and to protein content in these subunits; (ii) whether the level of particulate matter relates to concentration of Bet v1 allergen. Study was performed in Southern Poland, in 2017-2019. Pollen material was collected at 20 sites, of highly or less polluted areas. Protein composition was analysed by SDS-PAGE, while the concentration of Bet v1 was evaluated by ELISA. The obtained results were estimated at the background of the particulate matter (PM10) level and the birch pollen seasons in Kraków. The electrophoregrams of pollen samples collected at different sites showed huge differences in staining intensities of individual protein subunits, also among important birch allergens: Bet v1, Bet v2, Bet v6 and Bet v7. The level of Bet v1 was significantly higher in the pollen samples collected at the more polluted sites. While the birch pollen allergenic potential is determined, the both pollen exposure and the content of the main allergenic components should be considered, as factors causing immunological response and clinical symptoms manifestation in sensitive individuals.

Investigation of fennel protein extracts by shot-gun Fourier transform ion cyclotron resonance mass spectrometry.

A rapid shot-gun method by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is proposed for the characterization of fennel proteins. After enzymatic digestion with trypsin, few microliters of extract were analyzed by direct infusion in positive ion mode. A custom-made non-redundant fennel-specific proteome database was derived from the well-known NCBI database; additional proteins belonging to recognized allergenic sources (celery, carrot, parsley, birch, and mugwort) were also included in our database, since patients hypersensitive to these plants could also suffer from fennel allergy. The peptide sequence of each protein from that derived list was theoretically sequenced to produce calculated m/z lists of possible m/z ions after tryptic digestions. Then, by using a home-made Matlab algorithm, those lists were matched with the experimental FT-ICR mass spectrum of the fennel peptide mixture. Finally, Peptide Mass Fingerprint searches confirmed the presence of the matched proteins inside the fennel extract with a total of 70 proteins (61 fennel specific and 9 allergenic proteins).

Response to stress and allergen production caused by metal ions (Ni, Cu and Zn) in oregano (Origanum vulgare L.) plants.

Heavy metals are the cause of one of the most significant biosphere contamination problems worldwide, as they can be highly reactive and toxic according to their oxidation levels. Their toxic effects are correlated with the elevated production of reactive oxygen species (ROS) and oxidative cellular damage occurring in plants. The aim of the present study was the investigation of the effects of three heavy metals (Ni, Cu, Zn) applied to the soil in biochemical defense-related responses and allergen production in the aromatic plant oregano (Origanum vulgare L.) from the Lamiaceae family. The concentrations of the three heavy metals used, were based on the 2002 Regulation of the Polish Ministry of the Environment on Soil Quality Standards [(i) agricultural land (group B): Ni 100 ppm, Ni 210 ppm, Cu 200 ppm, Cu 500 ppm, Zn 720 ppm and (ii) industrial land (group C): Ni 500 ppm, Cu 1000 ppm, Zn 1500 ppm, Zn 3000 ppm]. The investigated plants accumulated heavy metal ions in aerial parts to a variable extent. For plants grown in soil contaminated with Zn, phenotypic representation of the growth and development were strongly limited and dependent on zinc concentration. Phenotypic representation of plants grown in soil contaminated with Ni and Cu were characterized by normal growth, slightly lower or equal to that of the control plants. All tested metals (Ni, Cu, Zn) caused a concentration-dependent decrease in photosynthetic pigments especially in total chlorophyll content. Highest cellular damage levels were observed in plants treated with Cu and Zn. Increasing concentration of these metals (especially Zn) caused a further increase in cellular damage. 3000 ppm Zn caused highest increase in the concentration of proline compared with control plants, suggesting osmotic stress imposition. Treatment with 1000 ppm Cu led to increased concentration of the allergenic protein profilin in relation to control plants by profilin ELISA analysis, while increasing concentrations of Cu and Zn led to a decrease in the concentration of phenolic compounds and total antioxidant capacity. On the basis of these findings, Ni stress in oregano plants appears to be less damaging (in relation to Cu and Zn) and with lower allergenic potential, compared with 1000 ppm Cu. The present study provides novel biochemical insight in the defense and allergenic response of aromatic plants to metal ions present in the rhizosphere; however, more comprehensive research under realistic field conditions is needed to fully decipher this interaction.

Could Global Intensification of Nitrogen Fertilisation Increase Immunogenic Proteins and Favour the Spread of Coeliac Pathology?

Fertilisation of cereal crops with nitrogen (N) has increased in the last five decades. In particular, the fertilisation of wheat crops increased by nearly one order of magnitude from 1961 to 2010, from 9.84 to 93.8 kg N ha-1 y-1. We hypothesized that this intensification of N fertilisation would increase the content of allergenic proteins in wheat which could likely be associated with the increased pathology of coeliac disease in human populations. An increase in the per capita intake of gliadin proteins, the group of gluten proteins principally responsible for the development of coeliac disease, would be the responsible factor. We conducted a global meta-analysis of available reports that supported our hypothesis: wheat plants growing in soils receiving higher doses of N fertilizer have higher total gluten, total gliadin, α/ß-gliadin, γ-gliadin and ω-gliadin contents and higher gliadin transcription in their grain. We thereafter calculated the per capita annual average intake of gliadins from wheat and derived foods and found that it increased from 1961 to 2010 from approximately 2.4 to 3.8 kg y-1 per capita (+1.4 ± 0.18 kg y-1 per capita, mean ± SE), i.e., increased by 58 ± 7.5%. Finally, we found that this increase was positively correlated with the increase in the rates of coeliac disease in all the available studies with temporal series of coeliac disease. The impacts and damage of over-fertilisation have been observed at an environmental scale (e.g., eutrophication and acid rain), but a potential direct effect of over-fertilisation is thus also possible on human health (coeliac disease).

High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis Affect Mealworm Allergenic Proteins.

Edible insects have garnered increased interest as alternative protein sources due to the world's growing population. However, the allergenicity of specific insect proteins is a major concern for both industry and consumers. This preliminary study investigated the capacity of high hydrostatic pressure (HHP) coupled to enzymatic hydrolysis by Alcalase® or pepsin in order to improve the in vitro digestion of mealworm proteins, specifically allergenic proteins. Pressurization was applied as pretreatment before in vitro digestion or, simultaneously, during hydrolysis. The degree of hydrolysis was compared between the different treatments and a mass spectrometry-based proteomic method was used to determine the efficiency of allergenic protein hydrolysis. Only the Alcalase® hydrolysis under pressure improved the degree of hydrolysis of mealworm proteins. Moreover, the in vitro digestion of the main allergenic proteins was increased by pressurization conditions that were specifically coupled to pepsin hydrolysis. Consequently, HHP-assisted enzymatic hydrolysis represents an alternative strategy to conventional hydrolysis for generating a large amount of peptide originating from allergenic mealworm proteins, and for lowering their immunoreactivity, for food, nutraceutical, and pharmaceutical applications.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays.

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic (CMC) method based on human mast cells (HMC-1) for screening potential allergens in infant formula milk powders (IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns (10 mm × 2 mm i.d., 5 µm). Under the conditions of 0.2 mL/min flow rate and 214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, ß-casein, α-lactalbumin, and ß-lactoglobulin A, were identified. Furthermore, these proteins and ß-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and ß-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and ß-hexosaminidase. Also, we established a reversed phase liquid chromatographic (RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and ß-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more ß-casein allergic risk than other proteins. This conclusion was consistent with other studies.

Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays / 药物分析学报

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on po-sitive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic (CMC) method based on human mast cells (HMC-1) for screening potential allergens in infant formula milk powders (IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns (10 mm × 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and 214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lacto-globulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic (RPLC) method for the determination of the five proteins in IFMP and the results showed that 90％ proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This con-clusion was consistent with other studies.

LC⁻Q⁻TOF⁻MS/MS Identification of Specific Non-Meat Proteins and Peptides in Beef Burgers.

Beef burgers are a popular food choice, due to their taste and convenience. The extensive range of beef burgers with different flavours currently offered on the market is adding to their growing consumption. This study detected and identified specific non-meat proteins and peptide markers originating from functional preparations, i.e., powdered mixes of protein additives and spices, used as meat substitutes in the production of ready-to-cook beef burgers. Twenty-eight soy proteins, including isoforms (nine milk-, three pea- and one beetroot-specific protein) were found concurrently with a set of peptide markers unique to soy glycinin and ß-conglycinin, pea vicilin and provicilin, milk αS1-casein, ß-lactoglobulin, as well as beetroot elongation factor 2. Soy and beetroot proteins and peptides were observed in all burgers containing additives. Milk and pea proteins were included in powdered mixes but were not detected in burgers, indicating that their content was below the limit of detection. The study demonstrates that the proposed method can be implemented to analyse protein additives in cooked burgers; however, the presence of low amounts of additives, below 1⁻2%, should be further confirmed by using a more sensitive triple quadrupole instrument.