Cloning and functional characterization of the legumin A gene (EuLEGA) from Eucommia ulmoides Oliver.

Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA3 can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels.

Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Self-assembled poloxamer-legumin/vicilin nanoparticles for the nanoencapsulation and controlled release of folic acid.

Plant-based food proteins are a promising choice for the preparation of nanoparticles (NPs) due to their high digestibility, low cost, and ability to interact with various compounds and nutrients. Moreover, nanoencapsulation offers a potential solution for protecting nutrients during processing and enhancing their bioavailability. This study aimed to develop and evaluate nanoparticles (NPs) based on legumin/vicilin (LV) proteins extracted from fava beans, with the goal of encapsulating and delivering a model nutraceutical compound, folic acid (FA). Specifically, NPs were self-assembled from LV proteins extracted from commercially available frozen fava beans using a pH-coacervation method with poloxamer 188 (P188) and chemically cross-linked with glutaraldehyde. Microscopy and spectroscopy studies were carried out on the empty and FA-loaded NPs in order to evaluate the particle morphology, size, size distribution, composition, mechanism of formation, impact of FA loading and release behavior. In vitro studies with Caco-2 cells also confirmed that the empty and FA-loaded nanoparticles were non-toxic. Thus, the LV-NPs are good candidates as food additives for the delivery and stabilization of nutrients as well as in drug delivery for the controlled release of therapeutics.

Identification, Characterization, Cloning, and Cross-Reactivity of Zan b 2, a Novel Pepper Allergen of 11S Legumin.

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.

Lacticaseibacillus Casei IDCC 3451 Strengthen Digestibility of Plant-based Proteins in Mice.

The demand for plant-based proteins as alternative meat sources continues to increase because of environmental concerns, animal welfare, and religious reasons. However, plant-based proteins have low digestibility than real meat, which should be overcome. In the present study, the effect of co-administration of legumin protein mixture and the probiotic strain on plasma concentration of amino acids was investigated as a strategy of enhancement in protein digestion. First, the proteolytic activity of the four probiotic strains was compared. As a result, Lacticaseibacillus casei IDCC 3451 was identified as an optimal probiotic strain that efficiently digested the legumin protein mixture by forming the largest halo produced by proteolysis. Next, to investigate whether the co-administration of legumin protein mixture and L. casei IDCC 3451 could synergically improve digestibility, mice were fed either a high-protein diet or a high-protein diet with L. casei IDCC 3451 for 8 weeks. Compared to only in the high-protein diet only group, the concentrations of branched chain amino acids and essential amino acids were 1.36 and 1.41 times higher in the co-administered group, respectively. Therefore, co-supplementation of plant-based proteins with L. casei IDCC 3451 can be suggested to improve protein digestibility based on the this study.

Structural properties of pea proteins (Pisum sativum) for sustainable food matrices.

Pea proteins are widely used as a food ingredient, especially in sustainable food formulations. The seed itself consists of many proteins with different structures and properties that determine their structure-forming properties in food matrices, such as emulsions, foams, and gels. This review discusses the current insights into the structuring properties of pea protein mixtures (concentrates, isolates) and the resulting individual fractions (globulins, albumins). The structural molecular features of the proteins found in pea seeds are discussed and based on this information, different structural length scales relevant to foods are reviewed. The main finding of this article is that the different pea proteins are able to form and stabilize structural components found in foods such as air-water and oil-water interfaces, gels, and anisotropic structures. Current research reveals that each individual protein fraction has unique structure-forming properties and that tailored breeding and fractionation processes will be required to optimize these properties. Especially the use of albumins, globulins, and mixed albumin-globulins proved to be useful in specific food structures such as foams, emulsions, and self-coacervation, respectively. These new research findings will transform how pea proteins are processed and being used in novel sustainable food formulations in the future.

Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE.

Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products.

Protein Nanofibrils from Fava Bean and Its Major Storage Proteins: Formation and Ability to Generate and Stabilise Foams.

Protein nanofibrils (PNFs) have potential for use in food applications as texture inducers. This study investigated the formation of PNFs from protein extracted from whole fava bean and from its two major storage proteins, globulin fractions 11S and 7S. PNFs were formed by heating (85 °C) the proteins under acidic conditions (pH 2) for 24 h. Thioflavin T fluorescence and atomic force microscopy techniques were used to investigate PNF formation. The foaming properties (capacity, stability, and half-life) were explored for non-fibrillated and fibrillated protein from fava bean, 11S, and 7S to investigate the texturing ability of PNFs at concentrations of 1 and 10 mg/mL and pH 7. The results showed that all three heat-incubated proteins (fava bean, 11S, and 7S) formed straight semi-flexible PNFs. Some differences in the capacity to form PNFs were observed between the two globulin fractions, with the smaller 7S protein being superior to 11S. The fibrillated protein from fava bean, 11S, and 7S generated more voluminous and more stable foams at 10 mg/mL than the corresponding non-fibrillated protein. However, this ability for fibrillated proteins to improve the foam properties seemed to be concentration-dependent, as at 1 mg/mL, the foams were less stable than those made from the non-fibrillated protein.

Pulse Globulins 11S and 7S: Origins, Purification Methods, and Techno-functional Properties.

A growing interest in pulse proteins in recent years results from their crucial role in the transition toward sustainable food systems. Consequently, current research is mainly focused on the production of protein ingredients and the evaluation of their nutritional and techno-functional properties for the development of animal product analogues. However, the individual impacts of the major proteins 11S legumin and 7S vicilin on pulse techno-functionalities remains unclear. Thus, this review aims to represent current knowledge on pulse 11S and 7S globulin origins, extraction, separation, and purification methods as well as their techno-functionalities. This paper also discusses the principal challenges related to pulse vicilin and legumin purification methods, such as efficiency and environmental concerns, as well as 11S/7S ratio variability. This review highlights the fact that 11S and 7S fractions serve different purposes in pulse functionality and that more efficient and eco-friendly purification techniques are required to properly assess their respective functional attributes. Such research would allow the determination of optimal 11S/7S ratios for the integration of pulse protein ingredients in various food formulations. Hence, food industries would be able to select species/varieties, agronomical methods, and processing methods to produce ingredients with suitable 11S/7S ratios, catering to consumers' ethical, environmental, and nutritional concerns.

Protein composition of pulses and their protein isolates from different sources and in different isolation pH values using a reverse phase high performance liquid chromatography method.

The objective of this study was to compare the composition of pulse proteins isolated from lentils and green and yellow peas at two isolation pH values (9 and 11) and determine the effect of this variability on protein functionality. Chromatogram peaks obtained from reverse-phase high performance liquid chromatography were identified by isolation of albumin-, vicilin- and legumin-rich fractions for the three pulses. Protein composition was obtained for each isolate and compared against that of the originating pulse flour. Lentil flour showed the highest level of vicilin with a vicilin/legumin ratio of âˆ¼ 2.5, while this ratio was 1.3 and 1.2 for green and yellow pea flour, respectively. Albumin content of yellow pea flour was high (∼36.1 %), which reduced to âˆ¼ 15-19 % in isolated proteins showing a loss in albumins during the isolation. Higher extraction pH increased pea protein yield but led to lower protein solubility with no changes in foaming properties and in-vitro digestibility.

Quantitative In Silico Evaluation of Allergenic Proteins from Anacardium occidentale, Carya illinoinensis, Juglans regia and Pistacia vera and Their Epitopes as Precursors of Bioactive Peptides.

The aim of the study presented here was to determine if there is a correlation between the presence of specific protein domains within tree nut allergens or tree nut allergen epitopes and the frequency of bioactive fragments and the predicted susceptibility to enzymatic digestion in allergenic proteins from tree nuts of cashew (Anacardium occidentale), pecan (Carya illinoinensis), English walnut (Juglans regia) and pistachio (Pistacia vera) plants. These bioactive peptides are distributed along the length of the protein and are not enriched in IgE epitope sequences. Classification of proteins as bioactive peptide precursors based on the presence of specific protein domains may be a promising approach. Proteins possessing a vicilin, N-terminal family domain, or napin domain contain a relatively low occurrence of bioactive fragments. In contrast, proteins possessing the cupin 1 domain without the vicilin N-terminal family domain contain a relatively high total frequency of bioactive fragments and predicted release of bioactive fragments by the joint action of pepsin, trypsin, and chymotrypsin. This approach could be utilized in food science to simplify the selection of protein domains enriched for bioactive peptides.

Seed Development and Protein Accumulation Patterns in Faba Bean (Vicia faba, L.).

A major objective in faba bean breeding is to improve its protein quality by selecting cultivars with enhanced desirable physicochemical properties. However, the protein composition of the mature seed is determined by a series of biological processes occurring during seed growth. Thus, any attempt to explain the final seed composition must consider the dynamics of the seed proteome during seed development. Here, we investigated the proteomic profile of developing faba bean seeds across 12 growth stages from 20 days after pollination (DAP) to full maturity. We analyzed trypsin-digested total protein extracts from the seeds at different growth stages by liquid chromatography-tandem mass spectrometry (LC-MS/MS), identifying 1217 proteins. The functional clusters of these proteins showed that, in early growth stages, proteins related to cell growth, division, and metabolism were most abundant compared to seed storage proteins that began to accumulate from 45 DAP. Moreover, label-free quantification of the relative abundance of seed proteins, including important globulin proteins, revealed several distinct temporal accumulation trends among the protein classes. These results suggest that these proteins are regulated differently and require further understanding of the impact of the different environmental stresses occurring at different grain filling stages on the expression and accumulation of these seed storage proteins.

Comparative structural and emulsifying properties of ultrasound-treated pea (Pisum sativum L.) protein isolate and the legumin and vicilin fractions.

The structural properties, interfacial behavior, and emulsifying ability of ultrasound-treated pea protein isolate (PPI) and the legumin (11S) and vicilin (7S) globulin fractions prepared with a salt-solubilization procedure were investigated. Of the three protein groups, PPI was strongly responsive to ultrasound perturbation (20 kHz, 57-60 W·cm-2) showing the greatest solubility increase, particle size reduction, structure destabilization, and conformational change. Similar but less remarkable effects were observed on 11S globulins; 7S proteins, already highly soluble (>99%), were generally less sensitive to ultrasound. The ultrasound treatment significantly improved emulsifying activity, which resulted in greater emulsifying capacity and stronger interfacial adsorption for all protein samples. PPI exhibited the higher activity increase (70.8%) compared to approximately 30% for 11S and 7S. For both control and ultrasound treated proteins, the emulsifying capacity was in the order of 7S > 11S > PPI, inversely related to the trend of protein loading at the interface, indicating efficiency differences. The latter was attributed to emulsion clusters formed through protein-protein interaction in PPI and 11S emulsions which were visibly absent in 7S emulsions.

Identification of vicilin, legumin and antimicrobial peptide 2a as macadamia nut allergens.

Macadamia nut is an increasingly popular food item of a healthy diet. However, macadamia nut is also a potent allergenic food. To date, there is little information about the allergenic proteins involved. In this study, using sera from macadamia nut allergic individuals, four IgE-binding proteins were detected. Their identities were determined by tandem mass spectrometry with de novo sequencing. Three IgE-reactive proteins, the vicilin Mac i 1, the legumin Mac i 2 and the antimicrobial peptide 2a/Mac i 1 (28-76) were purified from the nut while the non-specific lipid transfer protein was produced as a recombinant in Pichia pastoris. IgE-binding assays using sera from well-characterized groups of tree nut and/or peanut allergic patients revealed that the allergens were mainly recognized by sera from macadamia nut allergic individuals. Hence, these newly discovered allergens will enable molecular diagnostics to identify patients at high risk of macadamia nut allergy.

Mechanisms behind protein-protein interactions in a ß-lg-legumin co-precipitate.

Interactions between pea protein and whey protein isolates in co-precipitates and blends consist of a combination of disulphide bonds, hydrophobic and electrostatic interactions. The present study aims to clarify if the two proteins with free thiols, ß-lactoglobulin (ß-lg) and legumin, played a significant role for these interactions. This study used different reagents to modify the conditions of interactions: N-ethylmaleimide (NEM) was used to block reactive thiols, while NaCl and SDS were used to prevent electrostatic or hydrophobic interactions, respectively. The effects of treatments were studied on protein solubility, structure and stability. SDS had no effect, while NEM and NaCl both had great effect, especially in combination. The results showed that interactions of ß-lg and legumin in both co-precipitates and blends are a synergism of electrostatic interactions and disulphide bonds. Thus, ß-lg and legumin are the main proteins responsible for previously observed interactions in protein isolates of whey and pea.

The structure and stability analysis of the pea seed legumin glycosylated by oligochitosan.

BACKGROUND: The functionality of pea proteins is relatively weak relative to that of soybean proteins, which limits the application of pea proteins in food and nutritional applications. Glycosylation is a promising approach to influence the protein structure and in turn change the functional properties of pea proteins. RESULTS: In this study, the effect of transglutaminase-induced oligochitosan glycosylation on the structural and functional properties of pea seed legumin was studied. Different oligochitosan-modified legumin complexes (OLCs) were prepared by applying different molar ratios of legumin to oligochitosan (1:1 to 1:4) induced by transglutaminase (10 U g-1 protein). Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glucosamine, and free amino analysis showed that the legumin could be covalently bonded with the oligochitosan and were influenced by the applying dose of the oligochitosan. Infrared spectroscopy, fluorescence, and scanning electron microscopy analysis indicated that the structure of the different OLC samples could be changed to different extents. Moreover, although the emulsifying activity decreased, the emulsification stability, thermal stability, and in vitro digestive stability of the OLCs were remarkably improved relative to that of the untreated legumin. CONCLUSION: Oligochitosan glycosylation could change the structure of the legumin and consequently improve its emulsification stability, thermal stability, and in vitro digestive stability. This study will facilitate the legumin functionalization by the glycosylation approach to fabricate protein-oligochitosan complex for potential food and nutritional applications. © 2020 Society of Chemical Industry.

Identification and Quantification of Major Faba Bean Seed Proteins.

Faba bean (Vicia faba L.) holds great importance for human and animal nutrition for its high protein content. However, better understanding of its seed protein composition is required in order to develop cultivars that meet market demands for plant proteins with specific quality attributes. In this study, we screened 35 diverse Vicia faba genotypes by employing the one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) method, and 35 major protein bands obtained from three genotypes with contrasting seed protein profiles were further analyzed by mass spectrometry (MS). Twenty-five of these protein bands (MW range: ∼ 9-107 kDa) had significant (p ≤ 0.05) matches to polypeptides in protein databases. MS analysis showed that most of the analyzed protein bands contained more than one protein type and, in total, over 100 proteins were identified. These included major seed storage proteins such as legumin, vicilin, and convicilin, as well as other protein classes like lipoxygenase, heat shock proteins, sucrose-binding proteins, albumin, and defensin. Furthermore, seed protein extracts were separated by size-exclusion high-performance liquid chromatography (SE-HPLC), and percentages of the major protein classes were determined. On average, legumin and vicilin/convicilin accounted for 50 and 27% of the total protein extract, respectively. However, the proportions of these proteins varied considerably among genotypes, with the ratio of legumin:vicilin/convicilin ranging from 1:1 to 1:3. In addition, there was a significant (p < 0.01) negative correlation between the contents of these major fractions (r = -0.83). This study significantly extends the number of identified Vicia faba seed proteins and reveals new qualitative and quantitative variation in seed protein composition, filling a significant gap in the literature. Moreover, the germplasm and screening methods presented here are expected to contribute in selecting varieties with improved protein content and quality.

Antitumor Features of Vegetal Protein-Based Nanotherapeutics.

The introduction of nanotechnology into pharmaceutical application revolutionized the administration of antitumor drugs through the modulation of their accumulation in specific organs/body compartments, a decrease in their side-effects and their controlled release from innovative systems. The use of plant-derived proteins as innovative, safe and renewable raw materials to be used for the development of polymeric nanoparticles unlocked a new scenario in the drug delivery field. In particular, the reduced size of the colloidal systems combined with the peculiar properties of non-immunogenic polymers favored the characterization and evaluation of the pharmacological activity of the novel nanoformulations. The aim of this review is to describe the physico-chemical properties of nanoparticles composed of vegetal proteins used to retain and deliver anticancer drugs, together with the most important preparation methods and the pharmacological features of these potential nanomedicines.