A Review of the Utilization of Canola Protein as an Emulsifier in the Development of Food Emulsions.

Canola is the second-largest cultivated oilseed crop in the world and produces meal consisting of about 35-40% proteins. Despite this, less than 1% of the global plant-based protein market is taken up by canola protein. The reason behind such underutilization of canola protein and its rapeseed counterpart could be the harsh conditions of the industrial oil extraction process, the dark colour of the meal, the presence of various antinutrients, the variability in the protein composition based on the source, and the different properties of the two major protein components. Although academic research has shown immense potential for the use of canola protein and its rapeseed counterpart in emulsion development and stabilization, there is still a vast knowledge gap in efficiently utilizing canola proteins as an effective emulsifier in the development of various emulsion-based foods and beverages. In this context, this review paper summarizes the last 15 years of research on canola and rapeseed proteins as food emulsifiers. It discusses the protein extraction methods, modifications made to improve emulsification, emulsion composition, preparation protocols, and emulsion stability results. The need for further improvement in the scope of the research and reducing the knowledge gap is also highlighted, which could be useful for the food industry to rationally select canola proteins and optimize the processing parameters to obtain products with desirable attributes.

Genetic variation and structural diversity in major seed proteins among and within Camelina species.

MAIN CONCLUSION: Genetic variation in seed protein composition, seed protein gene expression and predictions of seed protein physiochemical properties were documented in C. sativa and other Camelina species. Seed protein diversity was examined in six Camelina species (C. hispida, C. laxa, C. microcarpa, C. neglecta, C. rumelica and C. sativa). Differences were observed in seed protein electrophoretic profiles, total seed protein content and amino acid composition between the species. Genes encoding major seed proteins (cruciferins, napins, oleosins and vicilins) were catalogued for C. sativa and RNA-Seq analysis established the expression patterns of these and other genes in developing seed from anthesis through to maturation. Examination of 187 C. sativa accessions revealed limited variation in seed protein electrophoretic profiles, though sufficient to group the majority into classes based on high MW protein profiles corresponding to the cruciferin region. C. sativa possessed four distinct types of cruciferins, named CsCRA, CsCRB, CsCRC and CsCRD, which corresponded to orthologues in Arabidopsis thaliana with members of each type encoded by homeologous genes on the three C. sativa sub-genomes. Total protein content and amino acid composition varied only slightly; however, RNA-Seq analysis revealed that CsCRA and CsCRB genes contributed > 95% of the cruciferin transcripts in most lines, whereas CsCRC genes were the most highly expressed cruciferin genes in others, including the type cultivar DH55. This was confirmed by proteomics analyses. Cruciferin is the most abundant seed protein and contributes the most to functionality. Modelling of the C. sativa cruciferins indicated that each type possesses different physiochemical attributes that were predicted to impart unique functional properties. As such, opportunities exist to create C. sativa cultivars with seed protein profiles tailored to specific technical applications.

Canola/rapeseed protein - nutritional value, functionality and food application: a review.

Plant-based diet and plant proteins specifically are predestined to meet nutritional requirements of growing population of humans and simultaneously reduce negative effects of food production on the environment. While searching for new sources of proteins, special emphasis should be placed on oilseeds of Brassica family comprising varieties of rapeseed and canola as they contain nutritionally valuable proteins, which have potential to be used in food, but are now rarely or not used as food components. The purpose of the present work is to provide a comprehensive review of main canola/rapeseed proteins: cruciferin and napin, with the focus on their nutritional and functional features, putting special emphasis on their possible applications in food. Technological challenges to obtain rapeseed protein products that are free from anti-nutritional factors are also addressed. As molecular structure of cruciferin and napin differs, they exhibit distinct features, such as solubility, emulsifying, foaming or gelling properties. Potential allergenic effect of 2S napin has to be taken under consideration. Overall, rapeseed proteins demonstrate beneficial nutritional value and functional properties and are deemed to play important roles both in food, as well as, non-food and non-feed applications.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.

N-terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N-end rule pathway.

The N-end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking. To investigate the impact of the Arg/N-end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT-TAILS) for relative quantification of N-terminal peptides in prt6, an Arabidopsis thaliana N-end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6). TMT-TAILS identified over 4000 unique N-terminal peptides representing c. 2000 protein groups. Forty-five protein groups exhibited significantly increased N-terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm α-cruciferin was delayed in prt6 seedlings. N-termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII-dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript. We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action.

AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.

AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and ß-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

Rapeseed napin and cruciferin are readily digested by poultry.

Rapeseed proteins have been considered as being poorly digestible in the gut of non-ruminants. The aim of the study was to assess the digestibility of napin and cruciferin in ileal digesta of broiler chickens, testing sixteen samples of rapeseed co-products with protein levels ranging from 293 g/kg to 560 g/kg dry matter. Each sample was included into a semi-synthetic diet at a rate of 500 g/kg and evaluated with broiler chickens in a randomised design. Dietary and ileal digesta proteins were extracted and identified by gel-based liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three isomers of napin (a 2S albumin) and nine cruciferins (an 11S globulin) were identified in the rapeseed co-products, whereas six endogenous enzymes such as trypsin (I-P1, II-P29), chymotrypsin (elastase and precursor), carboxypeptidase B and α-amylase were found in the ileal digesta. It is concluded that as none of the rapeseed proteins were detected in the ileal digesta, rapeseed proteins can be readily digested by broiler chickens, irrespective of the protein content in the diet.

Enhanced emulsification performance and interfacial properties of Janus-like rapeseed cruciferin through asymmetric acylation modification.

Plant protein emulsifiers, particularly rapeseed protein isolate with its superior amino acid composition and predominantly globular protein, have captured significant interest in the food industry. Nonetheless, the application of these proteins has been stymied by their lackluster emulsification properties. Addressing this challenge, our study implements an innovative asymmetric acylation technique to modify the surface of rapeseed cruciferin (RC), morphing it into a structure resembling Janus nanoparticles. This alteration amplifies the emulsification prowess of RC by a remarkable 2.7 times compared to its natural form, and 1.43 times over its conventionally acylated counterpart. The asymmetrically acylated RC, marked by a distinctive three-phase contact angle of 90.4°, manifests an outstanding amphiphilic character. Moreover, it surpasses both the natural and conventionally acylated RC in terms of diffusion, penetration, and rearrangement rates, as well as protein concentration at the oil-water interface. Compared to commonly used emulsifiers in the food industry, such as lecithin and soy protein, the asymmetrically acylated RC stands out, stabilizing emulsions with the tiniest particle size and effectively staving off emulsion stratification over a longer duration. This study underscores that asymmetric acylation serves as a reliable methodology for producing efficient plant protein emulsifiers, considerably amplifying their utility in the food industry.

Pectin polysaccharide contribution to oleosome extraction after wet milling of rapeseed.

Oleosomes are lipid composites providing energy storage in oilseeds. They possess a unique structure, comprised of a triglyceride core stabilized by a phospholipid-protein membrane, and they have shown potential to be used as ingredients in several food applications. Intact oleosomes are extracted by an aqueous process which includes soaking, milling, and gravitational separation. However, the details of the complexes formed between oleosomes, proteins and pectin polysaccharides during this extraction are not known. It was hypothesized that pectins play an important role during the oleosome separation, and different proteins will be complexed on the surface of the oleosomes, depending on the pH of extraction. Rapeseed extracts were treated with and without pectinase (Pectinex Ultra SP-L) and extracted at pH 5.7 or 8.5, as this will affect electrostatic complexation. Acidic conditions led to co-extraction of storage proteins, structured as dense oleosome emulsions, stabilized by a network of proteins and polysaccharides. Pectinase intensified this effect, highlighting pectic polysaccharides' role in bridging interactions among proteins and oleosomes under acidic conditions. The presence of this dense interstitial layer around the oleosomes protected them from coalescence during extraction. Conversely, under alkaline conditions, the extraction process yielded more purified oleosomes characterized by a larger particle size, most likely due to coalescence. Nevertheless, pectinase addition at pH 8.5 mitigated coalescence tendencies. These results contribute to a better understanding of the details of the colloidal complexes formed during extraction and can be used to modulate the composition of the extracted fractions, with significant consequences not only for yields and purity but also for the functional properties of the ingredients produced.

Oil-water interface and emulsion stabilising properties of rapeseed proteins napin and cruciferin studied by nonlinear surface rheology.

HYPOTHESIS: Two major protein families are present in rapeseed, namely cruciferins and napins. The structural differences between the two protein families indicate that they might behave differently when their mixture stabilises oil-water interfaces. Therefore, this work focuses on elucidating the role of both proteins in interface and emulsion stabilisation. EXPERIMENTS: Protein molecular properties were evaluated, using SEC, DSC, CD, and hydrophobicity analysis. The oil-water interface mechanical properties were studied using LAOS and LAOD. General stress decomposition (GSD) was used as a novel method to characterise the nonlinear response. Additionally, to evaluate the emulsifying properties of the rapeseed proteins, emulsions were prepared using pure napins or cruciferin and also their mixtures at 1:3, 1:1 and 3:1 (w:w) ratios. FINDINGS: Cruciferins formed stiff viscoelastic solid-like interfacial layers (Gs' = 0.046 mN/m; Ed' = 30.1 mN/m), while napin formed weaker and more stretchable layers at the oil-water interface (Gs' = 0.010 mN/m; Ed' = 26.4 mN/m). As a result, cruciferin-formed oil droplets with much higher stability against coalescence (coalescence index, CI up to 10%) than napin-stabilised ones (CI up to 146%) during two months of storage. Both proteins have a different role in emulsions produced with napin-cruciferin mixtures, where cruciferin provides high coalescence stability, while napin induces flocculation. Our work showed the role of each rapeseed protein in liquid-liquid multiphase systems.

Transcription Factor DOF4.1 Regulates Seed Longevity in Arabidopsis via Seed Permeability and Modulation of Seed Storage Protein Accumulation.

Seed longevity is modulated by multiple genetic factors in Arabidopsis thaliana. A previous genome-wide association study using the Elevated Partial Pressure of Oxygen (EPPO) aging assay pinpointed a genetic locus associated with this trait. Reverse genetics identified the transcription factor DOF4.1 as a novel seed longevity factor. dof4.1 loss-of-function plants generate seeds exhibiting higher germination after accelerated aging assays. DOF4.1 is expressed during seed development and RNAseq data show several putative factors that could contribute to the dof4.1 seed longevity phenotype. dof4.1 has reduced seed permeability and a higher levels of seed storage proteins mRNAs (cruciferins and napins) in developing seeds, as compared to wild-type seeds. It has been reported that mutant lines defective in cruciferins or napins present reduced seed longevity. The improved longevity of dof4.1 is totally lost in the quadruple mutant dof4.1 cra crb crc, but not in a dof4.1 line depleted of napins, suggesting a prominent role for cruciferins in this process. Moreover, a negative regulation of DOF4.1 expression by the transcription factor DOF1.8 is suggested by co-inoculation assays in Nicotiana benthamiana. Indeed, DOF1.8 expression anticorrelates with that of DOF4.1 during seed development. In summary, modulation of DOF4.1 levels during seed development contributes to regulate seed longevity.

Extraction, nutrition, functionality and commercial applications of canola proteins as an underutilized plant protein source for human nutrition.

Concerns about sustainability and nutrition security have encouraged the food sector to replace animal proteins in food formulations with underutilized plant protein sources and their co-products. In this scenario, canola protein-rich materials produced after oil extraction, including canola cold-pressed cakes and meals, offer an excellent opportunity, considering their nutritional advantages such as a well-balanced amino acid composition and their potential bioactivity. However, radical differences among major proteins (i.e., cruciferin and napin) in terms of the physicochemical properties, and the presence of a wide array of antinutritional factors in canola, impede the production of a highly pure protein extract with a reasonable extraction yield. In this manuscript, principles regarding the extraction methods applicable for the production of canola protein concentrates and isolates are explored in detail. Alkaline and salt extraction methods are presented as the primary isolation methods, which result in cruciferin-rich and napin-rich isolates with different nutritional and functional properties. Since a harsh alkaline condition would result in an inferior functionality in protein isolates, strategies are recommended to reduce the required solvent alkalinity, including using a combination of salt and alkaline and employing membrane technologies, application of proteases and carbohydrases to facilitate the protein solubilization from biomass, and novel green physical methods, such as ultrasound and microwave treatments. In terms of the commercialization progress, several canola protein products have received a GRAS notification so far, which facilitates their incorporation in food formulations, such as bakery, beverages, salad dressings, meat products and meat analogues, and dairies.

In Silico, Molecular Docking and In Vitro Antimicrobial Activity of the Major Rapeseed Seed Storage Proteins.

BACKGROUND: In addition to their use as an edible oil and condiment crop, mustard and rapeseed (Brassica napus L., B. juncea (L.) Czern., B. nigra (L.) W.D.J.Koch, B. rapa L. and Sinapis alba L.) have been commonly used in traditional medicine for relieving pain, coughs and treating infections. The seeds contain high amounts of oil, while the remaining by-product meal after oil extraction, about 40% of seed dry weight, has a low value despite its high protein-content (~85%). The seed storage proteins (SSP) 2S albumin-type napin and 12S globulin-type cruciferin are the two predominant proteins in the seeds and show potential for value adding to the waste stream; however, information on their biological activities is scarce. In this study, purified napin and cruciferin were tested using in silico, molecular docking, and in vitro approaches for their bioactivity as antimicrobial peptides. MATERIALS AND METHODS: The 3D-structure of 2S albumin and 12S globulin storage proteins from B. napus were investigated to predict antimicrobial activity employing an antimicrobial peptide database survey. To gain deeper insights into the potential antimicrobial activity of these SSP, in silico molecular docking was performed. The purified B. napus cruciferin and napin were then tested against both Gram-positive and Gram-negative bacteria for in vitro antimicrobial activity by disc diffusion and microdilution antimicrobial susceptibility testing. RESULTS: In silico analysis demonstrated both SSP share similar 3D-structure with other well studied antimicrobial proteins. Molecular docking revealed that the proteins exhibited high binding energy to bacterial enzymes. Cruciferin and napin proteins appeared as a double triplet and a single doublet, respectively, following SDS-PAGE. SDS-PAGE and Western blotting also confirmed the purity of the protein samples used for assessment of antimicrobial activity. Antimicrobial susceptibility testing provided strong evidence for antimicrobial activity for the purified napin protein; however, cruciferin showed no antimicrobial activity, even at the highest dose applied. DISCUSSION: In silico and molecular docking results presented evidence for the potential antimicrobial activity of rapeseed cruciferin and napin SSP. However, only the in vitro antimicrobial activity of napin was confirmed. These findings warrant further investigation of this SSP protein as a potential new agent against infectious disease.

Subunit composition affects formation and stabilization of o/w emulsions by 11S seed storage protein cruciferin.

The 11S globulin cruciferin is the major storage protein in Brassicaceae/Cruciferae seeds and exists as a hexamer in its natural configuration. Arabidopsis thaliana cruciferin is composed of CRUA, CRUB and CRUC subunits. Wild type (WT) cruciferin and cruciferins composed only of identical CRUA, CRUB and CRUC subunits were examined for their ability to form and stabilize oil-in-water (o/w) emulsions. All proteins (0.9% at pH 7.4 and 2.0), except CRUC, formed stable canola oil or triolein emulsions with a dispersed phase volume fraction of 22-23%. A fine emulsion was formed by CRUB at pH 7.4 with droplet sizes of 6.8 and 8.6 µm for canola oil and triolein, respectively. The presence of 0.5 M NaCl reduced the level of adsorbed protein and protein load at the interface at pH 7.4, and resulted in emulsions that were less stable. Emulsions of CRUA and CRUB (pH 7.4, zero ionic strength, canola oil or triolein) had higher stability than emulsions with WT cruciferin up to 15 days after formation. CRUC formed a stable emulsion only at pH 2.0. The low solubility, low surface hydrophobicity and compact structure of the CRUC protein may contribute to its inferior emulsifying properties at neutral pH; however, acidic pH-induced dissociation of the hexameric assembly improved these properties. The abundance and exposure of hydrophobic residues in the hypervariable regions, extended loop regions, and solvent exposed surfaces of cruciferin are critical factors affecting o/w interface stabilization.

Effect of endogenous reference genes on digital PCR assessment of genetically engineered canola events.

Droplet digital PCR (ddPCR) has been used for absolute quantification of genetically engineered (GE) events. Absolute quantification of GE events by duplex ddPCR requires the use of appropriate primers and probes for target and reference gene sequences in order to accurately determine the amount of GE materials. Single copy reference genes are generally preferred for absolute quantification of GE events by ddPCR. Study has not been conducted on a comparison of reference genes for absolute quantification of GE canola events by ddPCR. The suitability of four endogenous reference sequences (HMG-I/Y, FatA(A), CruA and Ccf) for absolute quantification of GE canola events by ddPCR was investigated. The effect of DNA extraction methods and DNA quality on the assessment of reference gene copy numbers was also investigated. ddPCR results were affected by the use of single vs. two copy reference genes. The single copy, FatA(A), reference gene was found to be stable and suitable for absolute quantification of GE canola events by ddPCR. For the copy numbers measured, the HMG-I/Y reference gene was less consistent than FatA(A) reference gene. The expected ddPCR values were underestimated when CruA and Ccf (two copy endogenous Cruciferin sequences) were used because of high number of copies. It is important to make an adjustment if two copy reference genes are used for ddPCR in order to obtain accurate results. On the other hand, real-time quantitative PCR results were not affected by the use of single vs. two copy reference genes.

Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2/HT29-MTX co-cultures.

The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery. STATEMENT OF SIGNIFICANCE: The long-term objective of this research is to investigate the potential of edible and safe biopolymer in enhanced oral delivery of drugs and/or vaccines. Here, we investigated the potential application of nanoparticles based on a protein extracted from Canola seeds, i.e., cruciferin, for oral delivery of a model small molecule, i.e., coumarin, through cells representing gastrointestinal epithelium, Caco-2 and Caco-2/HT29 cell monolayer. This study was completed for intact cruciferin nanoparticles and cruciferin coated chitosan nanoparticles, before and after digestion with gastric or intestine simulating fluids. This comparison was useful to understand the fate the cruciferin based particles in digestive mucosal tissues and their potential mucoadhesive and/or mucus-penetrating property.

Identification and In Silico Analysis of Major Redox Modulated Proteins from Brassica juncea Seedlings Using 2D Redox SDS PAGE (2-Dimensional Diagonal Redox Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis).

The thiol-disulphide exchange regulates the activity of proteins by redox modulation. Many studies to analyze reactive oxygen species (ROS), particularly, hydrogen peroxide (H2O2) induced changes in the gene expression have been reported, but efforts to detect H2O2 modified proteins are comparatively few. Two-dimensional diagonal redox sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used to detect polypeptides which undergo thiol-disulphide exchange in Brassica juncea seedlings following H2O2 (10 mM) treatment for 30 min. Eleven redox responsive polypeptides were identified which included cruciferin, NLI [Nuclear LIM (Lin11, Isl-1 & Mec-3 domains)] interacting protein phosphatase, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) large subunit, and myrosinase. Redox modulation of RuBisCO large subunit was further confirmed by western blotting. However, the small subunit of RuBisCO was not affected by these redox changes. All redox modulated targets except NLI interacting protein (although it contains two cysteines) showed oxidation sensitive cysteines by in silico analysis. Interestingly, interactome of cruciferin and myrosinase indicated that they may have additional function(s) beside their well-known roles in the seedling development and abiotic stress respectively. Cruciferin showed interactions with stress associated proteins like defensing-like protein 192 and 2-cys peroxiredoxin. Similarly, myrosinase showed interactions with nitrilase and cytochrome p450 which are involved in nitrogen metabolism and/or hormone biosynthesis. This simple procedure can be used to detect major stress mediated redox changes in other plants.

Canola/Rapeseed Protein: Future Opportunities and Directions-Workshop Proceedings of IRC 2015.

At present, canola meal is primarily streamlined into the animal feed market where it is a competitive animal feed source owing to its high protein value. Beyond animal feed lies a potential game-changer with regards to the value of canola meal, and its opportunity as a high quality food protein source. An economic and sustainable source of protein with high bioavailability and digestibility is essential to human health and well-being. Population pressures, ecological considerations, and production efficiency underscore the importance of highly bioavailable plant proteins, both for the developed and developing world. Despite decades of research, several technologies being developed, and products being brought to large scale production, there are still no commercially available canola protein products. The workshop entitled "Canola/Rapeseed Protein-Future Opportunities and Directions" that was held on 8 July 2015 during the 14th International Rapeseed Congress (IRC 2015) addressed the current situation and issues surrounding canola meal protein from the technological, nutritional, regulatory and genomics/breeding perspective. Discussions with participants and experts in the field helped to identify economic barriers and research gaps that need to be addressed in both the short and long term for the benefit of canola industry.

Structural Properties of Cruciferin and Napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature.

The two major storage proteins identified in Brassica napus (canola) were isolated and studied for their molecular composition, structural characteristics and the responses of structural features to the changes in pH and temperature. Cruciferin, a complex of six monomers, has a predominantly ß-sheet-containing secondary structure. This protein showed low pH unstable tertiary structure, and distinctly different solubility behaviour with pH when intact in the seed cellular matrix. Cruciferin structure unfolds at pH 3 even at ambient temperature. Temperature-induced structure unfolding was observed above the maximum denaturation temperature of cruciferin. Napin was soluble in a wider pH range than cruciferin and has α-helices dominating secondary structure. Structural features of napin showed less sensitivity to the changes in medium pH and temperature. The surface hydrophobicity (S0) and intrinsic fluorescence of tryptophan residue appear to be good indicators of cruciferin unfolding, however they were not the best to demonstrate structural changes of napin. These two storage proteins of B. napus have distinct molecular characteristics, therefore properties and functionalities they provide are contrasting rather than complementary.