Predicting the allergenicity of legume proteins using a PBMC gene expression assay

Background Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. Results PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine). Conclusions The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Characterization and Antibacterial Activity of 7S and 11S Globulins Isolated from Cowpea Seed Protein

The present work was carried out to determine the characteristics and antibacterial activity of 7S and 11S globulins isolated from cowpea seed (Vigna unguiculata (L.) Walp.). The molecular mass of 7S globulin was demonstrated by SDS-PAGE bands to be of about 132, 129 and 95 kDa corresponding the α/, α and β subunits. The molecular mass of 11S globulin was demonstrated by SDS-PAGE bands to be existed between 28 and 52 kDa corresponding the basic and acidic subunits. The minimum inhibitory concentrations MICs of 7S and 11S globulins isolated from cowpea seed were determined against Gram positive bacteria viz: Listeria monocytogenes LMG 10470, Listeria ivanovii FLB 12, Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes ATCC 19615, and Gram negative bacteria such as Klebsiella pneumonia ATCC 43816, Pseudomonas aeruginosa ATCC 26853, Escherichia coli ATCC 25922 and Salmonella ATCC 14028 using disc diffusion assay; they were showed to be in the range 10 to 200 µg/mL. Transmission electron microscope (TEM) examination of the protein-treated bacteria showed the antibacterial action of 11S globulin against S. typhimurium and P. aeruginosa was manifested by signs of cellular deformation, partial and complete lysis of cell components. Adding 11S globulin at both concentrations 50 and 100 µg/g to minced meat showed considerable decreases in bacterial counts of viable bacteria, psychrotrophs and coliforms compared to controls during 15 days storage at 4 °C, reflecting a promising perspective to use such globulin as a meat bio-preservative.

Biochemical characteristics of checkpea (Cicer arietinum L.) seeds for selection of genotypes of food direction

Aim. The goal of research was to study the features of biochemical composition of the chickpea seed for detection the characteristics which can be used to genotype selection of food direction. Methods. Research methods are standard methodologies of plant biochemical analysis and methods developed in the laboratory. 7S and 11S globulins were separated by method, which was developed in the Laboratory of Plant Biochemistry (Patent # 42181, 107671). Results. The features of content of protein, fat, carbohydrates, isoflavones, activity of lectins, lipoxygenase, content and ratio of vicilin and legumin in the chickpea seed of genotypes of Ukrainian and foreign plant breeding were established. The genotype differences of component intensity, presence-absence in the electrophoretic spectrum of chickpea albumins, globulins, glutelins which effect on seed food value were evoluted using electrophoretic and densitometric analyses. Conclusions. The main biochemical characteristics of chickpea which connected with seed quality and food value, content and component composition of main protein fractions, presence in component composition of protein subunits that have negative influence on the man health can be used to selection of chickpea genotypes of food direction. Keywords: сhickpea, breeding, vicilin, legumin, isoflavones.

Analysis of protein content and level of 7S and 11S globulins, their ratio in the soybean seed of F6 - F8 hybrid lines

Aims. The goal of research was to study a character of changes and relationship between level of 7S and 11S globulins, their ratio and protein content in the soybean seed of F6-F8 hybrid lines and their parental forms of different genetic origin in the connection of protein quality breeding. Methods. 7S and 11S globulins were isolated by method, which was developed in the Laboratory of Plant Biochemistry (Patent # 42181). Results. The features of 7S and 11S globulins content and their ratio in the soybean seed of F6 -F8 hybrid lines and their parental forms of different genetic origin were identified. It was established, that varieties and hybrid lines are characterized by considerable distinctions of 7S and 11S globulins contents and their ratio in the soybean protein. A connection between 7S and 11S globulins levels and protein content in the seed of soybean F6- F8 hybrid lines wasn’t established. Conclusions. It was elucidated that the growth conditions influence on the protein content, 11S and 7S globulins level, their ratio and relationship between these parameters in the soybean seeds of F6-F8 hybrid lines and their parental forms of different genetic origin, so that have to be introduced in the selection program of soybean genotypes of food direction soybean breeding as these parameters connected with the food nutritional quality.Key words: Glicine max L., protein, 11S globulin, 7S globulin.