Protein products and sprouts from flaxseed (Linum usitatissimum L.)

Polysaccharides of flaxseed coat (flax mucilage) were removed by soaking of whole seeds in water or sodium bicarbonate solution or treatment with commercially- available carbohydrases. The extent of polysaccharide removal was determined by monitoring the concentration of total sugars and pentoses as well as viscosity values of the aqueous polysaccharide extracts prepared from treated flaxseed. A direct relationship existed between the amount of seeds dispersed in the extraction medium and the total contents of sugars and pentoses or viscosity values (correlation coefficients of 0.957, 0.974 and 0.921, respectively). These indicators served well in quantitating the content of polysaccharides in aqueous extracts. Soaking in a 0.10 M sodium bicarbonate solution for 12 h removed most of the mucilage from seed coats. Treatment of seeds with Viscozyme® L (22.5 mg protein/100 g seeds) for 12 h removed more polysaccharides from seed coat than Pectinex™ Ultra SP or Celluclast® 1.5L under similar conditions. Scanning electron microscopy provided evidence that polysaccharides were removed from seed coats by pre-treatments employed. Nitrogen solubility and protein recovery from meals of mucilage-reduced seeds was improved since there was less interference from seed coat polysaccharides which were co-extracted with proteins. Sodium hexametaphosphate-assisted protein isolation from mucilage-reduced seeds was optimized under laboratory conditions for pH, meal-to- solvent ratio and concentration of sodium hexametaphosphate using response surface methodology with a central composite rotatable design. Maximum nitrogen solubility and protein recovery were obtained at pH 8.89 and 9.04, meal-to-solvent ratio of 1:33.5 and 1:33 and sodium hexametaphosphate concentration of 2.75 and 2.85%, respectively. The prepared flax protein isolate contained 78% protein. Lysine was the first limiting amino acid of the protein isolate. The computed protein efficiency ratio and biological value indicated a reasonably good quality of protein in the isolate. The isolate contained elevated levels of phytic acid and total phosphorus. Electrophoretic results showed that the isolate was composed of total proteins of flaxseed. -- The isolated flax proteins were acylated with acetic or succinic anhydride. Approximately 84.5 and 56.9% of the free amino groups of protein isolates were acylated when 0.2 g anhydride/g protein was used. The colour and solubility of the protein isolate were improved due to acylation. Emulsifying activity and stability were enhanced by succinylation, but acetylation had a negative influence. Furthermore, foaming ability and fat binding capacity of succinylated products were less than those of their acetylated counterparts. Acylation reduced the in vitro digestibility of flax proteins. -- In another study flaxseeds were germinated for 8 days. Germinated seeds had lowered content of dry matter due to the use of their reserve components as an energy source for the developing seedlings. The crude protein content did not show a significant (p<0.05) change but the content of non-protein nitrogen compounds including free amino acids as well as agmatine and spermidine increased during the germination period. The in vitro digestibility was lowered but the calculated values for protein efficiency ratio and biological value of seeds did not change upon germination. Lipid content of seeds was reduced by 58% during germination but their fatty acid composition did not change. The content of neutral lipids, accounting for 96% of total flax lipids, was significantly (p<0.05) decreased while those of glyco and phospholipids increased. There was also a decrease in the content of triacylglycerols and an increase in the diacylglycerols, monoacylglycerols and free fatty acids of the neutral lipid fraction. The content of phosphatidylcholine, phosphatidyiserine and phosphatidylethanolamine in the phospholipid fraction was decreased while that of phosphatide acid and lysophosphatidylethanolamine increased significantly (p<0.05) due to germination. The quantities of raffinose and sucrose in the seeds were reduced while those of fructose and glucose increased after germination. The mineral composition of the seeds was not affected by germination. The content of known antinutrients of seeds namely, cyanogenic glycosides, phytic acid and trypsin inhibitors were lower in the germinated seeds as compared to those in their ungerminated counterparts

Characteristics of solvent extracted flaxseed (Linum usitatissimum L.) meals

Flaxseed meal was prepared by a two-phase solvent extraction system consisting of alkanol, ammonia, water and hexane. Methanol, ethanol and isopropanol were used as the alkanol and the prepared meals were evaluated with respect to their effect on nutrients, antinutrients and functional characteristics. Commercially available flaxseed meal was also studied. -- Approximately 46 to 49% meal and 46 to 50% oil were recovered from dry seeds depending on the extraction system employed. The removal of 4.2 to 5.7% polar substances from the seeds resulted in an increase of 13 and 10% in the content of crude protein and ash, respectively. The presence of ammonia in the polar phase had little effect on the non-protein nitrogen content. -- A method was developed to isolate and quantify individual cyanogenic glycosides of linseed using chromatographic techniques. The cultivar used for this study was free of linamarin and contained 4.42±0.08 mg/g of linustatin and 1.90±0.03 mg/g of neolinustatin in the defatted meal on a dry basis. The extraction system, consisting of 10% (w/w) ammonia in 95% (v/v) methanol, removed 57% of linustatin and neolinustatin present in the original samples. A higher content of water, up to 15% in the methanol-ammonia-water phase, removed 67 to 68% of cyanogenic glycosides but resulted in a sticky, dark-coloured meal. Increased contact time (30 min) and solvent-to-seed ratio (R, 13.3) were more effective as 78% to 8l% of cyanogenic glycosides were removed by this process. A two stage extraction with methanol-ammonia-water/hexane gave similar results, but, a three stage extraction removed approximately 92.5% of cyanogenic glycosides present in flaxseed. -- The content of total phenolic acids (220±13 mg/lOOg), condensed tannins (136±13 mg/lOOg) and soluble sugars (7.69±0.16%) of defatted meals were reduced by 10-48%, 26-74% and 5-46%, respectively. Defatted flaxseed meals contained 2.4±0.13 to 2.8±0.37% of phytic acid and solvent extraction resulted in a slight increase in its content in the products. Flaxseed meal was low in methionine, lysine and tryptophan compared to the FAO/WHO reference values. Methanol-ammonia-water/hexane extraction had little effect on the content of amino acids but resulted in lowering of the content of some of the fatty acids possibly due to the removal of some phopholipids by the polar phase. -- Flaxseed meal had a very high water absorption (9.7g H₂O/g) and water hydration capacity (5.2 g H₂O/g) and they were not altered by extraction with the two-phase solvent processing. The presence of ammonia in the extraction system enhanced the fat absorption of the meals 2.6-3.2 fold and increased the pH by almost one unit. Nitrogen solubility of the meals was fairly high (46-65%) and extraction with methanol-ammonia improved the nitrogen solubility of the products at their natural pH. Minimum value of the nitrogen solubility in the meals was observed at pH between 3.0-3.5. Emulsifying capacities of the meals were 64.5% to 80.6% and they were fairly stable to heat and retained 95 to 100% of the emulsifying activity. Whippability of the meals was between 55 to 70% and the foams were stable

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 β-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

Health Beneficial Bioactivities of Faba Bean Gastrointestinal (In Vitro) Digestate in Comparison to Soybean and Pea

Faba beans are a promising emerging plant-based protein source to be used as a quality alternative to peas and soy. In this study, the potential health beneficial activities of three Canadian faba bean varieties (Fabelle, Malik and Snowbird) were investigated after in vitro gastrointestinal digestion and compared to two commonly used legumes (peas and soy). The results revealed that the faba beans had a higher antioxidant activity than peas when assessed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays, except for the Fabelle variety. In the oxygen radical absorbance capacity (ORAC) and the iron chelating assays, the faba beans had a lower antioxidant activity than soy. Interestingly, Fabelle and Snowbird showed a higher antioxidant effect than the peas and soy at the cellular level. The antihypertensive properties of Fabelle and Malik varieties were significantly higher than peas but lower than soy. The in vitro antidiabetic activity was higher for soy, but no differences were found at the cellular level. The faba bean peptides were further fractionated and sequenced by mass spectrometry. Eleven peptides with in silico predicted bioactivities were successfully identified in the faba bean digestate and support validating the health-promoting properties of peptides. The results demonstrate the bioactive potential of faba beans as a health-promoting food ingredient against non-communicable diseases

Canola/rapeseed protein-functionality and nutrition

Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S) and napin (2S) dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed

Canola/rapeseed protein-functionality and nutrition

Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S) and napin (2S) dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed

Structural and Physicochemical Property Relationships of Cruciferin Homohexamers

Heteromeric cruciferin from wild type (WT) <i>Arabidopsis thaliana</i> and homomeric cruciferin CRUA, CRUB, and CRUC composed of identical subunits obtained from double-knockout mutant lines were investigated for their structural and physicochemical properties. A three-step chromatographic procedure allowed isolation of intact cruciferin hexamers with high purity (>95%). FT-IR and CD analysis of protein secondary structure composition revealed that all cruciferins were folded into higher order structures consisting of 44–50% β-sheets and 7–9% α-helices. The structural and physicochemical properties of homohexameric CRUC deviated from that of CRUA and CRUB and exhibited a compact, thermostable, and less hydrophobic structure, confirming the predictions made using 3D homology structure models

Characterization of Arabidopsis thaliana Lines with Altered Seed Storage Protein Profiles Using Synchrotron-Powered FT-IR Spectromicroscopy

Arabidopsis thaliana lines expressing only one cruciferin subunit type (double-knockout; <i>CRUAbc</i>, <i>CRUaBc</i>, or <i>CRUabC</i>) or devoid of cruciferin (triple-knockout; <i>CRU-</i>) or napin (napin-RNAi) were generated using combined T-DNA insertions or RNA interference approaches. Seeds of double-knockout lines accumulated homohexameric cruciferin and contained similar protein levels as the wild type (WT). Chemical imaging of WT and double-knockout seeds using synchrotron FT-IR spectromicroscopy (amide I band, 1650 cm^–1, νCO) showed that proteins were concentrated in the cell center and protein storage vacuoles. Protein secondary structure features of the homohexameric cruciferin lines showed predominant β-sheet content. The napin-RNAi line had lower α-helix content than the WT. Lines entirely devoid of cruciferin had high α-helix and low β-sheet levels, indicating that structurally different proteins compensate for the loss of cruciferin. Lines producing homohexameric CRUC showed minimal changes in protein secondary structure after pepsin treatment, indicating low enzyme accessibility. The Synchrotron FT-IR technique provides information on protein secondary structure and changes to the structure within the cell