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.

Effect of magnetic field-assisted fermentation on the in vitro protein digestibility and molecular structure of rapeseed meal.

BACKGROUND: There has been a significant growth in demand for plant-derived protein, and this has been accompanied by an increasing need for sustainable animal-feed options. The aim of this study was to investigate the effect of magnetic field-assisted solid fermentation (MSSF) on the in vitro protein digestibility (IVPD) and functional and structural characteristics of rapeseed meal (RSM) with a mutant strain of Bacillus subtilis. RESULTS: Our investigation demonstrated that the MSSF nitrogen release rate reached 86.3% after 96 h of fermentation. The soluble protein and peptide content in magnetic field feremented rapeseed meal reached 29.34 and 34.49 mg mL-1 after simulated gastric digestion, and the content of soluble protein and peptide in MF-FRSM reached 61.81 and 69.85 mg mL-1 after simulated gastrointestinal digestion, which significantly increased (p > 0.05) compared with the fermented rapeseed meal (FRSM). Studies of different microstructures - using scanning electron microscopy (SEM) and atomic force microscopy (AFM) - and protein secondary structures have shown that the decline in intermolecular or intramolecular cross-linking leads to the relative dispersion of proteins and improves the rate of nitrogen release. The smaller number of disulfide bonds and conformational alterations suggests that the IVPD of RSM was improved. CONCLUSIONS: Magnetic field-assisted solid fermentation can be applied to enhance the nutritional and protein digestibility of FRSM. © 2024 Society of Chemical Industry.

Honey bees and bumble bees may be exposed to pesticides differently when foraging on agricultural areas.

In an agricultural environment, where crops are treated with pesticides, bees are likely to be exposed to a range of chemical compounds in a variety of ways. The extent to which different bee species are affected by these chemicals, largely depends on the concentrations and type of exposure. We quantified the presence of selected pesticide compounds in the pollen of two different entomophilous crops; oilseed rape (Brassica napus) and broad bean (Vicia faba). Sampling was performed in 12 sites in Ireland and our results were compared with the pollen loads of honey bees and bumble bees actively foraging on those crops in those same sites. Detections were compound specific, and the timing of pesticide application in relation to sampling likely influenced the final residue contamination levels. Most detections originated from compounds that were not recently applied on the fields, and samples from B. napus fields were more contaminated compared to those from V. faba fields. Crop pollen was contaminated only with fungicides, honey bee pollen loads contained mainly fungicides, while more insecticides were detected in bumble bee pollen loads. The highest number of compounds and most detections were observed in bumble bee pollen loads, where notably, all five neonicotinoids assessed (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) were detected despite the no recent application of these compounds on the fields where samples were collected. The concentrations of neonicotinoid insecticides were positively correlated with the number of wild plant species present in the bumble bee-collected pollen samples, but this relationship could not be verified for honey bees. The compounds azoxystrobin, boscalid and thiamethoxam formed the most common pesticide combination in pollen. Our results raise concerns about potential long-term bee exposure to multiple residues and question whether honey bees are suitable surrogates for pesticide risk assessments for all bee species.

Effects of fermentation and enzymatic treatment on phenolic compounds and soluble proteins in oil press cakes of canola (Brassica napus).

To develop novel processes for valorizing agro-industry side-streams, canola (Brassica napus) oil press cakes (CPC) were treated with lactic acid bacteria, carbohydrase, and protease. Altogether 29 protein-rich liquid fractions were obtained, of which the composition was analyzed using chromatographic and mass spectrometric methods. A clear association was revealed between the treatments and phenolic profile. Applying certain lactic acid bacteria enhanced the release of sinapic acid, sinapine, glycosylated kaempferols, and other phenolic compounds from CPC. Co-treatment using protease and Lactiplantibacillus plantarum was effective in degrading these compounds. The fraction obtained after 16 h of hydrolysis (with Protamex® of 2% dosage) and 48 h of fermentation (using L. plantarum) contained the lowest phenolic content (0.2 g/100 g DM) and a medium level of soluble proteins (78 g/100 g) among all samples studied. The fractions rich in soluble proteins and low in phenolics are potential food ingredients with improved bioavailability and sensory properties.

Polyphenolic compounds from rapeseeds (Brassica napus L.): The major types, biofunctional roles, bioavailability, and the influences of rapeseed oil processing technologies on the content.

The rapeseed (Brassica napus L.) are the important oil bearing material worldwide, which contain wide variety of bioactive components with polyphenolic compounds considered the most typical. The rapeseed polyphenols encompass different structural variants, and have been considered to have many bioactive functions, which are beneficial for the human health. Whereas, the rapeseed oil processing technologies affect their content and the biofunctional activities. The present review of the literature highlighted the major types of the rapeseed polyphenols, and summarized their biofunctional roles. The influences of rapeseed oil processing technologies on these polyphenols were also elucidated. Furthermore, the directions of the future studies for producing nutritional rapeseed oils preserved higher level of polyphenols were prospected. The rapeseed polyphenols are divided into the phenolic acids and polyphenolic tannins, both of which contained different subtypes. They are reported to have multiple biofunctional roles, thus showing outstanding health improvement effects. The rapeseed oil processing technologies have significant effects on both of the polyphenol content and activity. Some novel processing technologies, such as aqueous enzymatic extraction (AEE), subcritical or supercritical extraction showed advantages for producing rapeseed oil with higher level of polyphenols. The oil refining process involved heat or strong acid and alkali conditions affected their stability and activity, leading to the loss of polyphenols of the final products. Future efforts are encouraged to provide more clinic evidence for the practical applications of the rapeseed polyphenols, as well as optimizing the processing technologies for the green manufacturing of rapeseed oils.

Fermented rape pollen powder can alleviate benign prostatic hyperplasia in rats by reducing hormone content and changing gut microbiota.

Benign prostatic hyperplasia (BPH) can cause urethral compression, bladder stone formation, and renal function damage, which may endanger the life of patients. Therefore, we aimed to develop plant-based preparations for BPH treatment with no side effects. In this study, the Lactiplantibacillus plantarum 322Hp, Lactobacillus acidophilus 322Ha, and Limosilactobacillus reuteri 322Hr were used to ferment rape pollen. The fermented rape pollen was subsequently converted into fermented rape pollen powder (FRPP) through vacuum freeze-drying technology. After fermenting and drying, the bioactive substances and antioxidant capacity of FRPP were significantly higher than those of unfermented rapeseed pollen, and FRPP had a longer storage duration, which can be stored for over one year. To investigate the therapeutic effect of FRPP on BPH, a BPH rat model was established by hypodermic injection of testosterone propionate. The BPH rats were treated differently, with the model group receiving normal saline, the positive control group receiving finasteride, and the low, medium, and high dose FRPP group receiving FRPP at doses of 0.14 g/kg/d, 0.28 g/kg/d, and 0.56 g/kg/d, respectively. The results indicate that medium dose FRPP reduced the levels of hormone such as testosterone, dihydrotestosterone, and oestradiol in rats with BPH by about 32%, thus bringing the prostate tissue of BPH rats closer to normal. More importantly, medium dose FRPP treatment had a significant effect on the composition of gut microbiota in rats with BPH, increasing the levels of beneficial genera (such as Coprococcus and Jeotgalicoccus), and decreasing the levels of harmful pathogens (such as Turicibacter and Clostridiaceae_Clostridium) in the gut. This study showed that medium dose FRPP reduced the hormone level and regulated the unbalanced gut microbiota in BPH rats, thereby alleviating BPH.

Impact of enzymatic pre-treatment on composition of nutrients and phytochemicals of canola (Brassica napus) oil press residues.

The study aimed to develop a biorefining process to recover proteins and dietary fibres from a food industry side-stream, canola (Brassica napus) oil pressing residues. The materials were treated with commercial protease, carbohydrase, and phytase to obtain protein-rich supernatants and fibre-rich precipitates. The compositions of these fractions were analyzed using LC-MS (glucosinolates and phenolics) and GC-MS (sugars, acids, and amino acids). Compared to raw material, the supernatants were richer in proteins, sugars, acids, amino acids, phenolic acids, and flavonols; the precipitates had higher levels of minerals and dietary fibres. The enzymatic treatment decreased the contents of phytic acid, glucosinolates, and phenolic alkaloids in all fractions. The applied enzymes effectively enhanced solubility of proteins, despite the lower yield of crude proteins compared to the alkaline extraction (40-82 vs 91 g/100 g dry matters). The impact of enzymes on other chemical components was also revealed by using principal component analysis.

The Brassica Napus Extract (BNE)-Loaded PLGA Nanoparticles as an Early Necroptosis and Late Apoptosis Inducer in Human MCF-7 Breast Cancer Cells.

Loading of the Brassica napus extract (BNE) on PLGA nanoparticle (BNE-PNP) and study its necroptotic activity in human MCF7-breast cancer cells. Double emulsion solvent evaporation methods were used for synthesis of BNE-PNP and DLS, SEM, and surface Zeta-potential analysis were applied for defining the physicochemical properties of BNE-PNP. The cytotoxic impact of BNE-PNP nanoparticles was analyzed by MTT assay and expression of apoptotic (P53 and Cas-3) and necrotic (TNF-α) gene markers were measured by qPCR to evaluate the BNE-PNP-induced cell death type. The stable (-36.07 mV) BNE-PNP were synthesized at 71.07 nm dimension. They significantly decrease the count of metabolically active MCF7 cells (IC50: 170.94 µg/ml after 48 h). The BNE-PNP induced an early programmed necrotic (necroptosis) and late apoptotic death on the MCF7 cancer cells by up-regulating all the P53/TNF-α and Cas-3 gene expression, respectively. The BNE-PNP dose-dependently induced an early cell-selective necroptotic death. Since the necroptotic death is known as a biocompatible cellular death induction, the BNE-PNP have the potential to be used as a safe efficient anticancer compound.

Effect of rapeseed meal degraded by enzymolysis and fermentation on the growth performance, nutrient digestibility and health status of broilers.

The purpose of this study is to investigate the nutritional changes of degraded rapeseed meal and its effects on growth performance, nutrient digestibility and health status of broilers. Raw rapeseed meal (CON), degraded by enzymolysis (protease, ERM), fermentation (Bacillus subtilis, FRM) or both (DRM) were included in diets at 25% and fed to 480 yellow-feathered broilers at 22-63 d of age. Results showed that rapeseed peptide contents (≤1 kDa) were increased (p < 0.05) from 4.13% (CON) to 35.5% (ERM), 24.1% (FRM) and 50.4% (DRM); glucosinolate and erucic acid in DRM were decreased (p < 0.05) by 71.6% and 86.2%, respectively, compared to CON. There were increases (p ≤ 0.029) in feed intake, body weight gain, feed efficiency and precaecal digestibility of dry matter, crude protein, methionine, isoleucine, leucine, lysine, cysteine, phenylalanine, tyrosine, threonine, tryptophan and valine in the three degraded diets. Also, serum immunoglobulin (Ig) A, IgG, glutathione peroxidase, superoxide dismutase and catalase were raised (p ≤ 0.034) in the degraded diets. Additionally, DRM showed more pronounced effects (p < 0.05) on variables related to growth, digestibility and health than ERM and FRM. It is concluded that rapeseed meal degraded by both enzymolysis and fermentation can increase its nutritional values and application in broilers.

Copper sulfate and arginine supplements on performance, thyroid hormones and blood biochemistry of broilers fed with canola meal-based diets / Avaliação da suplementação com sulfato de cobre e arginina sobre o desempenho, hormônios tireoidianos e bioquímica sanguínea de frangos alimentados com dietas à base de canola

The present evaluated the effects of copper sulfate solution (CSS) and arginine powder (Arg) supplements on performance, thyroid hormones and blood biochemistry of broiler chickens fed with canola meal (CM)-based diets. The experimental design was completely randomized with a 3 × 3 factorial and 9 treatments, corresponding to 3 levels of CSS (0, 125 and 250 mg/kg) and 3 levels of Arg (0, 0.1 and 0.2%) (n = 45 per treatment). Feeds were offered ad libitum for 21 days, from 22 to 42 days of age. Feed efficiency was significantly affected by the dietary addition of 250 mg/kg CSS and 0.2% Arg, and by the CSS × Arg interaction. CM supplemented with CSS improved the thyroid gland status and increased the plasma levels of triiodothyronine and thyroxine. Birds fed diets supplemented with 0.2% Arg had lower blood glucose level than the other treatments. The addition of 250 mg/kg CSS and 0.2% Arg reduced the stress caused by the rapid growth of broilers, also increasing the overall bird welfare.

O objetivo do presente trabalho foi avaliar os efeitos da suplementação com solução de sulfato de cobre (SSC) e arginina em pó (Arg) sobre o desempenho, hormônios tireoidianos e bioquímica sanguínea de frangos de corte alimentados com dietas à base de canola DC. O desenho experimental foi completamente casualizado com fatorial 3 × 3 e nove tratamentos correspondentes a três níveis de inclusão de SSC (0, 125 e 250 mg/kg) e três níveis de Arg (0, 0,1 e 0,2%) (n = 45 para cada tratamento). As rações foram oferecidas ad libitum por 21 dias, de 22 até 42 dias de idade. A eficiência alimentar foi significativamente afetada pela adição de 250 mg/kg de SSC e 0,2% de Arg, assim como pela interação SSC × Arg. A suplementação da DC com SSC melhorou os parâmetros da glândula tireoide e aumentou os níveis plasmáticos de triiodotironina e tiroxina. As aves alimentadas com dietas suplementadas com 0,2% de Arg apresentaram menor nível de glicose sanguínea do que as dos demais tratamentos. A adição de 250 mg/kg de SSC e 0,2% de Arg reduz o estresse causado pelo rápido crescimento dos frangos, além de melhorar as condições gerais de bem estar das aves.

Exploration of physiological and biochemical processes of canola with exogenously applied fertilizers and plant growth regulators under drought stress.

Environmental stresses may alter the nutritional profile and economic value of crops. Chemical fertilizers and phytohormones are major sources which can enhance the canola production under stressful conditions. Physio-biochemical responses of canola altered remarkably with the use of nitrogen/phosphorus/potassium (N/P/K) fertilizers and plant growth regulators (PGRs) under drought stress. The major aim of current study was to evaluate nutritional quality and physio-biochemical modulation in canola (Brassica napus L.) from early growth to seed stage with NPK and PGRs in different water regimes. To monitor biochemical and physiological processes in canola, two season field experiment was conducted as spilt plot under randomized complete block design (RCBD) with four treatments (Control, Chemical fertilizers [N (90 kg/ha), P and K (45 kg ha-1)], PGRs; indole acetic acid (IAA) 15g ha-1, gibberellic acid (GA3) 15g ha-1 and the combination of NPK and PGRs] under different irrigations regimes (60, 100, 120, 150 mm evaporations). Water stress enhanced peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), polyphenol oxidase (PPO), soluble sugar, malondialdehyde (MDA), proline contents as well as leaf temperature while substantially reduced leaf water contents (21%), stomatal conductance (50%), chlorophyll contents (10-67%), membrane stability index (24%) and grain yield (30%) of canola. However, the combined application of NPK and PGR further increased the enzymatic antioxidant pool, soluble sugars, along with recovery of leaf water contents, chlorophyll contents, stomatal conductance and membrane stability index but decreased the proline contents and leaf temperature at different rate of evaporation. There is positive interaction of applied elicitors to the water stress in canola except leaf area. The outcomes depicted that the combination of NPK with PGRs improved the various morpho-physiological as well as biochemical parameters and reduced the pressure of chemical fertilizers cost about 60%. It had also reduced the deleterious effect of water limitation on the physiology and grain yield and oil contents of canola in field experiments.

Valorization Potentials of Rapeseed Meal in a Biorefinery Perspective: Focus on Nutritional and Bioactive Components.

Rapeseed meal (RSM), a by-product of oilseed extraction connected to the agri-food and biofuel sectors, is currently used as animal feed and for other low-value purposes. With a biorefinery approach, RSM could be valorized as a source of bio-based molecules for high-value applications. This study provides a chemical characterization of RSM in the perspective of its valorization. A qualitative study of main functional groups by fourier transform infrared (FTIR) spectroscopy was integrated with a chemical characterization of macronutrients, minerals by inductively coupled plasma optical emission spectrometry (ICP-OES), phenolic acids and lipid components by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), HPLC-diode-array detector (HPLC-DAD) and gas chromatography-mass spectrometry/flame ionization detector (GC-MS/FID). The study, conducted on different lots of RSM collected over a one-year period from an oil pressing factory serving a biofuel biorefinery, highlighted a constant quality over time of RSM, characterized by high protein (31-34%), fiber (33-40%) and mineral (5.5-6.8%) contents. Polyphenol extracts showed a significant antioxidant activity and a prevalence of sinapic acid, accounting for more than 85% of total phenolic acids (395-437 mg kg-1 RSM). Results highlight the potentialities of RSM for further valorization strategies that may lead to the creation of new cross-sector interconnections and bio-based value chains with improvement of the economics and sustainability of the bioeconomy sectors involved.

Comparison of Different d-SPE Sorbent Performances Based on Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) Methodology for Multiresidue Pesticide Analyses in Rapeseeds.

Pesticide extraction in rapeseed samples remains a great analytical challenge due to the complexity of the matrix, which contains proteins, fatty acids, high amounts of triglycerides and cellulosic fibers. An HPLC-MS/MS method was developed for the quantification of 179 pesticides in rapeseeds. The performances of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method were evaluated using different dispersive solid-phase extraction (d-SPE) sorbents containing common octadecylsilane silica/primary-secondary amine adsorbent (PSA/C18) and new commercialized d-SPE materials dedicated to fatty matrices (Z-Sep, Z-Sep+, and EMR-Lipid). The analytical performances of these different sorbents were compared according to the SANTE/12682/2019 document. The best results were obtained using EMR-Lipid in terms of pesticide average recoveries (103 and 70 of the 179 targeted pesticides exhibited recoveries within 70-120% and 30-70%, respectively, with low RSD values). Moreover, the limits of quantification (LOQ) range from 1.72 µg/kg to 6.39 µg/kg for 173 of the pesticides. Only the recovery for tralkoxydim at 10 µg/kg level was not satisfactory (29%). The matrix effect was evaluated and proved to be limited between -50% and 50% for 169 pesticides with this EMR-Lipid and freezing. GC-Orbitrap analyses confirmed the best efficiency of the EMR-Lipid sorbent for the purification of rapeseeds.

Identification and Capture of Phenolic Compounds from a Rapeseed Meal Protein Isolate Production Process By-Product by Macroporous Resin and Valorization Their Antioxidant Properties.

In this study, phenolic compounds from an aqueous protein by-product from rapeseed meal (RSM) were identified by HPLC-DAD and HPLC-ESI-MS, including sinapine, sinapic acid, sinapoyl glucose, and 1,2-di-sinapoyl gentibiose. The main phenolic compound in this by-product was sinapine. We also performed acid hydrolysis to convert sinapine, and sinapic acid derivatives present in the permeate, to sinapic acid. The adsorption of phenolic compounds was investigated using five macroporous resins, including XAD4, XAD7, XAD16, XAD1180, and HP20. Among them, XAD16 showed the highest total phenolic contents adsorption capacities. The adsorption behavior of phenolic compounds was described by pseudo-second-order and Langmuir models. Moreover, thermodynamics tests demonstrated that the adsorption process of phenolic compounds was exothermic and spontaneous. The highest desorption ratio was obtained with 30% (v/v) and 70% (v/v) ethanol for sinapine and sinapic acid, respectively, with a desorption ratio of 63.19 ± 0.03% and 94.68 ± 0.013%. DPPH and ABTS tests revealed that the antioxidant activity of the hydrolyzed fraction was higher than the non-hydrolyzed fraction and higher than the one of vitamin C. Antioxidant tests demonstrated that these phenolic compounds could be used as natural antioxidants, which can be applied in the food industry.

Proteome-wide identification of S-sulphenylated cysteines in Brassica napus.

Deregulation of reduction-oxidation (redox) metabolism under environmental stresses results in enhanced production of intracellular reactive oxygen species (ROS), which ultimately leads to post-translational modifications (PTMs) of responsive proteins. Redox PTMs play an important role in regulation of protein function and cellular signalling. By means of large-scale redox proteomics, we studied reversible cysteine modification during the response to short-term salt stress in Brassica napus (B. napus). We applied an iodoacetyl tandem mass tags (iodoTMT)-based proteomic approach to analyse the redox proteome of B. napus seedlings under control and salt-stressed conditions. We identified 1,821 sulphenylated sites in 912 proteins from all samples. A great number of sulphenylated proteins were predicted to localize to chloroplasts and cytoplasm and GO enrichment analysis of differentially sulphenylated proteins revealed that metabolic processes such as photosynthesis and glycolysis are enriched and enzymes are overrepresented. Redox-sensitive sites in two enzymes were validated in vitro on recombinant proteins and they might affect the enzyme activity. This targeted approach contributes to the identification of the sulphenylated sites and proteins in B. napus subjected to salt stress and our study will improve our understanding of the molecular mechanisms underlying the redox regulation in response to salt stress.

Replacing fish meal with rapeseed meal: potential impact on the growth performance, profitability measures, serum biomarkers, antioxidant status, intestinal morphometric analysis, and water quality of Oreochromis niloticus and Sarotherodon galilaeus fingerlings.

The aim of this study was to assess the impact of using rapeseed meal as a partial replacement for fish meal in the diet of farmed tilapia. We evaluated the effect of this replacement on growth performance, profitability, serum biomarkers, antioxidant status, gut morphology, and water quality. A total of 960 apparently healthy Oreochromis niloticus (O. niloticus) and Sarotherodon galilaeus (S. galilaeus) fingerlings were randomly distributed into four dietary treatment groups for each tilapia species (triplicate design, 120 fish/group, and 40 fish/replicate). The diets consumed by these groups were formulated to replace fish meal (FM) with rapeseed meal (RM) at 0%, 10%, 20%, and 30%, respectively, for 12 consecutive weeks. Results indicated that replacing RM in the diet of S. galilaeus (up to 20%) and O. niloticus (up to 10%) resulted in increased growth performance parameters, including final weight, weight gain, length, length gain, weight gain rate, and specific growth rate (SGR), and return parameters such as a total return and relative return compared to the control group. Moreover, an increase in RM up to 30% improved net profit and increased the mucosal length, intestinal villi length, and the number of goblet cells compared with results in its relative control groups. Additionally, we observed a significant increase in serum and liver AST and ALT with increased RM replacement. With respect to water parameters, we observed a significant difference in the ammonia levels, turbidity, and conductivity with the changes to the percentage of RM in the diets. As for the effect on each species, O. niloticus showed a more significant increase in all examined parameters compared to results in S. galilaeus. In summary, up to 10% RM can be used to replace FM without any adverse effects on the growth performance, profitability measures, intestinal morphometric analysis, or water quality.

Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties.

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

Evaluation of a quick one-step sample preparation method for the determination of the isotopic fingerprint of rapeseed (Brassica napus): Investigation of the influence of the use of 2,2-dimethoxypropane on compound-specific stable carbon and hydrogen isotope analyses by gas chromatography combustion/pyrolysis isotope ratio mass spectrometry.

RATIONALE: Gas chromatographic analyses for vegetable oils require transesterification, which generally involves multiple steps, mainly to generate fatty acid methyl esters (FAMEs). A quick method based on acid-catalyzed transesterification using 2,2-dimethoxypropane (DMP) enables the conversion in one step, in a single reactor. For compound-specific stable carbon and hydrogen isotope analyses (C- and H-CSIA) of individual fatty acids (FAs) in oil, the verification of this one-step method has not yet been reported. METHODS: In this study, we evaluated the feasibility of the one-step method for C- and H-CSIA of individual FAMEs in rapeseed samples. The focus was on the investigation of the influence of methanol, which was produced from the reactions of DMP with glycerol and water during transesterification, on the accuracy of isotope composition of FAMEs, consequently of the FAs. The reproducibility of the one-step method was assessed by the measurement of the FAMEs from rapeseed and rapeseed oil. For the C- and H-CSIA of individual FAMEs, a gas chromatography combustion/pyrolysis isotope ratio mass spectrometry system was used. RESULTS: Our results showed that no significant differences arise in the carbon and hydrogen isotope compositions of the selected main FAMEs produced with and without DMP except for the H-CSIA value of C18:3. The reproducibility of the one-step method for rapeseed was in the range of ±0.1 mUr to ± 0.3 mUr for C-CSIA and ±1 mUr to ±3 mUr for H-CSIA of the main FAMEs. CONCLUSIONS: DMP improves the transesterification efficiency without influencing the accuracy of the C- and H-CSIA of FAMEs. The performance of the one-step method for rapeseed samples for the determination of C- and H-CSIA values of FAMEs is satisfactory. Thus, the applicability of the one-step method for isotopic fingerprint analyses of FAs in oilseeds is reported for the first time.

A Novel Reversibly Glycosylated Polypeptide-2 of Bee Pollen from Rape (Brassica napus L.): Purification and Characterization.

BACKGROUND: Reversibly glycosylated polypeptide (RGP), a kind of hydrosoluble and plasmodesmal-associated protein found in plants, plays a crucial role in the development of pollen. OBJECTIVE: A novel RGP 2 was isolated and identified from rape (Brassica napus L.) bee pollen. METHODS: RGP2 was isolated and purified by ion-exchange column and gel filtration chromatography, and characterized by MALDI-TOF-MS, LC-MS, immunological histological chemistry, and transmission electron microscope. RESULTS: Our results indicated that the RGP2 is an acidic protein (pI=5.46) with the molecular weight 42388 Da. It contained 17 kinds of amino acids, among which aspartic acid had the highest amount (71.56 mg/g). Homologous alignment of amino acid sequence results showed that RGP2 was 80.33%, 85.02%, 86.06%, and 88.93% identical to Arabidopsis thaliana RGP2 (AtRGP2), Oryza sativa RGP (OsRGP), Triticum aestivum RGP (TaRGP), and Zea maize RGP (ZmRGP), respectively. The localization results showed that RGP2 in rape anther existed in exine and intine of anther cells of rape flower by immunological histological chemistry and the subcellular localization identified that RGP2 appeared around the Golgi apparatus in cytoplasm by transmission electron microscope. CONCLUSION: RGP2 has a highly conserved sequence of amino acid residues and potential glycosylation sites.

Proteomic analysis of oilseed cake: a comparative study of species-specific proteins and peptides extracted from ten seed species.

BACKGROUND: In recent years there has been a visible trend among consumers to move away from consuming meat in favor of plant products. Meat producers have therefore been trying to meet the expectations of consumers by introducing new products to the food market with a greater proportion of plant ingredients. Meat products are enriched not only by the addition of vegetable oils but also by ground or whole oilseeds or their preparation. In this study, we present in-solution tryptic digestion and an ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS)-based proteomics approach to investigate specific proteins and peptides of ten oilseed cakes, by-products of cold pressing oil from coconut, evening primrose, hemp, flax, milk thistle, nigella, pumpkin, rapeseed, sesame, and sunflower seeds, for authentication purposes. RESULTS: We identified a total of 229 unique oilseed proteins. The number of specific proteins varied depending on the sample, from 4 to 48 in evening primrose and sesame. Moreover, we identified approximately 440 oilseed unique peptides in the cakes of all the analyzed oilseeds; the largest amounts were found in sesame (107 peptides), sunflower (100), pumpkin, hemp (42), rapeseed (36), and flax cake (35 peptides). CONCLUSIONS: We provide novel information on unique / species-specific peptide markers that will extend the scope of testing the authenticity of a wide range of foods. The results of this peptide discovery experiment may further contribute to the development of targeted methods for the detection and quantification of oilseed proteins in processed foods, and thus to the improvement of food quality. © 2020 Society of Chemical Industry.