Solubility Differences of Major Storage Proteins of Brassicaceae Oilseeds

J Am Oil Chem Soc (2012) 89:869 881 DOI 10.1007/s11746-011-1975-9 ORIGINAL PAPER Solubility Differences of Major Storage Proteins of Brassicaceae Oilseeds Janitha P. D. Wanasundara Sujeema J. Abeysekara Tara C. McIntosh Kevin C. Falk Received: 20 July 2011 / Revised: 26 October 2011 / Accepted: 14 November 2011 / Published online: 13 December 2011 Ó Her Majesty the Queen in Right of Canada 2011 Abstract Seeds of six commercially produced Brassica juncea, Brassica napus and Sinapis alba varieties representing high-glucosinolate condiment-type and lowglucosinolate canola-type were studied for solubility characteristics of the predominant seed storage proteins (SSPs). The non-protein nitrogen components such as glucosinolates, nucleic acids, betaine, choline and sinapine contributed 3.1 5.2% and 7.9 10.8% for the total N content of low- and high-glucosinolate meals, respectively. The cruciferin and napin which are the predominant SSPs of crucifers were purified from these seeds and used to confirm soluble protein types under the conditions provided. The napins were soluble between ph 2 and 4 but not the cruciferins. Strong alkaline ph brought both cruciferin and napin into solution. In general, the SSP solubility was increased due to the presence of NaCl or CaCl 2 salts in the medium. The effect of CaCl 2 on solubility was more positive than NaCl for all the seed types except S. alba at neutral and alkaline ph. Presence of salts indeed reduced solubility of S. alba SSPs at alkaline ph. The medium ph and salt ions and their ionic strength can be manipulated to achieve selective solubility of napin and cruciferin of Brassicaceae seed meals. J. P. D. Wanasundara (&) S. J. Abeysekara T. C. McIntosh K. C. Falk Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada e-mail: janitha.wanasundara@agr.gc.ca Present Address: S. J. Abeysekara BioExx Proteins of Saskatoon Inc., 33, Peters Ave., Site 404 Com 7 RR 4, Saskatoon, SK S7K 3J7, Canada Keywords Cruciferin Napin Brassica juncea Brassica napus Sinapis alba Canola Mustard Seed storage protein Introduction The seed storage proteins (SSPs) constitute a major usable biopolymer of the nonoily fraction of Brassicaceae (Cruciferae) family seeds. In canola (Brassica napus, Brassica rapa and Brassica juncea), the crude protein content of oilfree meal ranges from 36 to 39% [1]. Crucifer seeds have two classes of SSPs; cruciferin a legumin-type globulin of 11S and napin an albumin of 1.7 or 2S [2]. Cruciferin (CRU) is a heteromeric large protein of 300 360 kda and belongs to the cupin superfamily (UniProtKB/TrEmBL; www.uniprot.org [3]). The napin (NAP) found in the Brassicaceae family is classified under prolamin protein superfamily [4] and has molecular weight between 12.7 and 20.3 kda (UniProtKB/TrEmBL; www.uniprot.org [3]). Multigene families are involved in expressing both CRU and NAP therefore several isoforms of these proteins exist in the mature seed. The primary sequence (i.e. amino acid composition) of CRU and NAP is completely different leading to major differences in the secondary and tertiary protein structures which result in diverse functions and properties. Several differences in molecular properties of CRU and NAP that are crucial in determining their utility as functional molecules have been reported [5, 6]. At present, meal from the canola oil extraction process is a competitive animal feed ingredient because of its high protein and energy value [1]. Deriving protein concentrates and isolates from Brassica oilseed for human food applications have been attempted over the past 43 years since canola oilseed crop was developed and is becoming a commercial

870 J Am Oil Chem Soc (2012) 89:869 881 reality (BioExx Specialty Proteins Ltd.; www.bioexx.com [7] and Burcon NutraScience; www.burcon.ca [8]). Feedgrade canola protein products are also available (MCN Bioproducts Inc.; www.mcnbioproducts.com [9]). The processes of feed- and food-grade Brassica protein product preparation use suitable conditions to extract the maximum amount of proteins regardless of the protein type. In Canada, as far as the biofuel industry is concerned, oil rich B. juncea (brown and oriental mustard), S. alba (yellow mustard), Brassica carinata and Camelina sativa have tremendous potential without interfering with the production of edible oil producing B. napus. Co-product development of the oil extraction process of the feed stock (e.g. seeds) is necessary to improve the economic competitiveness of the crucifer biofuel industry. Along this line, as a valuable biopolymer of Brassicaceae oilseed feed stock, proteins warrant investigation beyond food applications. The differences of molecular characteristics of the constituting SSPs can be utilized in generating new protein-based products if obtained separately. Therefore, the knowledge of key characteristics of individual SSPs is necessary to devise suitable recovery methods that are cost effective and simple. The objective of this study was to investigate the solubility characteristics of CRU and NAP of three oil-rich crucifer species under commonly used conditions for wet protein extraction processes from oilseed meals. Experimental Procedures Preparation of Seed Meal Seeds of Brassica napus (var. AC Excel), Brassica juncea (var. AC Vulcan, Duchess) and Sinapis alba (var. AC Pennant and Andante) that represent three crucifer species were obtained from the crucifer breeding programs at the Agriculture and Agri-Food Canada Saskatoon Research Centre and commercial producers in Saskatchewan. The canola-quality mustard var. Dahinda was supplied by Viterra Inc. Saskatoon. First, the seeds were screened through no. 10, 12, 14, and 16 sieves (Tyler, Mentor, OH) to segregate into sizes to facilitate the cracking process. The segregated seeds were frozen, cracked (Morehouse Cowles stone mill, Chino, CA) and then air classified (Agriculex seed cleaner) to obtain seed coat and cotyledons (with embryo) fractions. The cotyledon fraction was defatted by screw pressing followed by solvent extraction of the pressed cake with n-hexane. The defatted cotyledon meal was air dried and ground with a coffee grinder to pass through a No. 40 (425 lm, Tyler, Mentor, OH) mesh screen and stored in airtight containers at 4 C until use. The meal so obtained had\1% residual oil content on a dry weight basis. Chemicals All the chemicals used were of ACS grade or better. Preparation of Purified CRU and NAP The chromatographic procedure described by Bérot and group [10] was adopted to obtain purified CRU and NAP of B. juncea (var. Duchess), B. napus (var. AC Excel) and S. alba (var. Andante). The extracts of crucifer meal in 50 mm Tris HCl buffer (containing 750 mm NaCl, 5 mm EDTA and 28 mm sodium bisulphite and at ph 8.5, meal:solvent, 1:10, w:v) was obtained by 1 h extraction and consequent centrifugation at 15,0009g for 10 min to recover the soluble fraction. The resulting pellet of first extraction was re-extracted under the same conditions. The combined supernatant of two extractions was filtered (Whatman No. 1 filter paper) to remove any floating particles. The first step of protein separation was to remove phenolics and other small molecules from the extract by size exclusion chromatography (SEC; Sephadex G-25, mobile phase 50 mm tris HCl ph 8.5, 1 M NaCl). The protein containing fraction of SEC was dialyzed against distilled water and lyophilized before further separation. Separation of CRU and NAP of the SEC fraction was achieved by cation exchange chromatography (CEC; Resource S, mobile phase A: 50 mm tris HCl ph 8.5, 5 mm EDTA, 0.3% NaHSO 3, B; 50 mm tris HCl ph 8.5 containing 1 M NaCl). The very first protein fraction eluted from the CEC was further cleaned by another SEC (Sephacryl S-300, mobile phase 50 mm tris HCl at ph 8.5 containing a 1-M NaCl) step to obtain purified CRU. The NAP-containing fraction that eluted from the CEC at high NaCl concentration ([60%) was further cleaned and polished using hydrophobic interaction chromatography (HIC; Phenyl Sepharose 6, mobile phase buffer A; 50 mm tris HCl ph 8.5, B; 50 mm tris HCl at ph 8.5 containing 0.85 M Na 2 SO 4 ). Both CRU and NAP fractions were dialyzed against deionized water and lyophilized. The purified proteins so obtained were subjected to peptide mass fingerprinting (PMP) to confirm identity and utilized as reference standards in electrophoresis separation. An ÅKTA Explorer system (Amersham Pharmacia, Uppsala, Sweden) was used for all the chromatographic separations and the protein was monitored as UV absorbance at 214 and 280 nm. Identity Confirmation of Proteins Purified CRU, NAP and proteins in question were subjected to mass spectroscopic analysis for identity confirmation by PMP of tryptic digested fragments. The dry protein samples were dissolved in Milli-Q water to a

J Am Oil Chem Soc (2012) 89:869 881 871 concentration of 2 mg/ml. Then 20 ll of the protein solution was placed in a small Eppendorf tube followed by the addition of 20 ll of 0.1 M ammonium bicarbonate. Reducing conditions were provided by adding 40 ll of 10 mm dithiothreitol (DTT) prepared in 50 mm ammonium bicarbonate (to give 5 mm DTT in solution) to the sample and maintaining temperature at 60 C for 30 min. Alkylation was carried out by adding 30.8 ll of 55 mm iodoacetamide (IAA) prepared in 50 mm ammonium bicarbonate (to give 15 mm IAA in solution) for 30 min at ambient temperature. Proteins were hydrolyzed using 20 ll of a solution of 100 ng/ll porcine trypsin (sequencing grade, Promega, Madison WI in 50 mm ammonium bicarbonate) for 5 h at 37 C. The resulting digest was then dried and reconstituted in 40 ll of 0.1% TFA, vortexed vigorously and sonicated before being transferred to an injection plate. When proteins separated on SDS-PAGE gels were used, protein bands separated under non-reducing conditions were excised and placed in a 96-well microtiter plate. The resulting gel pieces were automatically de-stained, reduced with DTT, alkylated with iodoacetamide, and digested with trypsin using a MassPREP protein digest station and recommended procedures of Waters (Manchester, UK). Peptides from tryptic digestion were analyzed using a caplc ternary Waters HPLC system coupled to a Q-ToF Ultima Global Mass Spectrometer. The method used for separation of the peptide digest samples and subsequent analysis using LC MS/ MS and data dependent acquisition (DDA) were similar to the ones described by Sheoran and group [11]. The LC MS/MS data were processed using ProteinLynx software and searched against databases using MASCOT Daemon and Mascot MS/MS ion search performed on a MASCOT server hosted by IBS-NRC (Ottawa, Canada). Taxonomy filters suitable for green plants and Brassicas were used. Analysis of Total Glucosinolate Content Glucosinolate profiles of meal samples were determined according to the method of Landerouin and group [12] with some modifications. First the meal sample was extracted into absolute methanol (1:5, meal:solvent, w:v). To the mixture was added lead barium acetate (0.6 M, 8% of the total volume) and internal standard solution (1 lmol ml -1 allyl glucosinolate for S. alba and B. napus and benzyl glucosinolate for B. juncea) and extracted for 1 h with mixing. The solubles were recovered by centrifugation at 4,000 rpm for 10 min. One milliliter of supernatant was added to a Sephadex A-25 column and each sample was washed with 1.5 ml each of 70% (v/v) methanol, 6% (v/v) acetic acid and distilled water, respectively. A 15-min interval was allowed between washing with each solution. Next, 1 ml of 0.02% pyridine acetate was added to each sample. A purified sulfatase solution (50 ll) was added to each column, covered, and incubated at ambient temperature overnight. The desulfoglucosinolates resulted from the enzyme activity were then eluted with 1 ml of purified water and collected into vials and freeze dried. Then 50 ll of MSTFA (N-methyl-N-TMS-trifluoroacetamide) and TMCS (trimethylchlorosilane) were added to each vial, capped, mixed and heated to 40 C for 15 min for derivatization. The derivatized desulfoglucosinolates were separated using a gas chromatograph (Agilent) equipped with a flame ionized detector. Chromatographic conditions were as follows; column 15 9 0.32 mm, 1.0 lm film, DB-1, hydrogen flow rate at 1.0 ml/min for 9 min and ramp to 1 3 ml/min for 9 min, temperature program 70 C for 2 min ramped at 5 C/min to 300 C then maintained for 9 min, detector temperature 310 C. The relative response factor (RRF) was calculated from the area of the internal standard. Area of each was converted to lmol glucosinolate/ml. Nitrogen content contributed by glucosinolate molecules was obtained by considering the number of N atoms and molecular weight of each glucosinolate molecule. Analysis of Total Content of Nucleic Acids and Nucleotides Nucleotides of meal samples were extracted using a commercially available phenol chloroform based reagent (Invitrogen). For each sample, the purity of DNA and RNA was determined as an absorbance ratio at 260 and 280 nm, respectively using a Nano-drop spectrophotometer. The nitrogen content of the purified nucleotide pellet was analyzed by combustion analysis. Analysis of Sinapine, Betaine and Choline The contents of betaine, choline and sinapine of the ground whole meal were determined according to the method developed by M. Reaney, University of Saskatchewan [13]. Defatted meal was extracted with 99.8% methanol (1:20, w:w) in a round-bottom flask for 48 h with stirring. The extracts were filtered through cotton wool placed in 1 9 12 cm glass columns and then concentrated under vacuum. The concentrated sample was added (10 50 mg) internal standard; N,N-Dimethylformamide (DMF), total weight was recorded and re-suspended in 1 3 ml of deuterium oxide (Cambridge Isotope Laboratory). The proton NMR signals of these samples were obtained and recorded at 500 mhz using a Bruker 1 H-NMR system. The singlet peaks recorded at 3.25, 3.17 and 3.11 ppm were identified as phenylpropanoid ester (sinapic acid ester or sinapine), betaine and choline [ N(CH 3 ) 3 ], respectively. Betaine, choline and sinapine contents of the sample were expressed

872 J Am Oil Chem Soc (2012) 89:869 881 on a weight basis. The amount of nitrogen contributed by these compounds was calculated based on the molecular weight and number of N atoms in the molecule. Effect of ph on CRU and NAP Dissolution A series of meal dispersions (meal:solvent, 1:20, w:v) were prepared in ph-adjusted (2.0 12.0) super-q water. The ph of these slurries was adjusted and maintained using HCl or NaOH (0.1, 1 or 6 M as appropriate) while stirring continuously at ambient temperature (22 C) for 60 min. The extraction step was terminated by separating insoluble materials by centrifugation at 10,0009g for 15 min (20 C). The supernatant was filtered (Whatman No. 1 filter paper) under a vacuum to remove floating particles and the total N content of the clear filtrate was determined by combustion analysis. To understand the reversibility of protein dissolution, an extract of B. juncea, B. napus and S. alba meal was prepared at ph 12 as described above. The extract was divided into aliquots of similar volume and the ph was adjusted to different values in the range of 2 10. Precipitated proteins were removed by centrifugation (10,0009g, 15 min, 20 C) and the soluble protein level in the supernatant was determined based on total N content. Effect of Salt Type and Ionic Strength on CRU and NAP Dissolution A series of meal dispersions (meal:solvent, 1:20, w:v) were prepared in NaCl and CaCl 2 solutions (ionic strength 0.1 1.0) and then ph was adjusted to the values between 2.0 and 12.0 similar to the ph study. The ph was maintained using HCl or NaOH (0.1, 1 or 6 M as appropriate) while stirring continuously at ambient temperature for 60 min. The soluble components of the meal were recovered and total N content was determined. Protein Quantification The total nitrogen content of the meals, extracts and remaining residues was determined using combustion N analysis. The nitrogen values resulting from the analysis were expressed as percentages of the total N content of the meal when the percentage soluble N content was reported. A conversion factor of 6.25 was employed to obtain protein values. Characterization of Polypeptide Profile The sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out under reducing (R) [14] and non-reducing (NR) conditions to obtain polypeptide profiles of the seed extracts. When NR conditions were required b-mercaptoethanol (b-me) was not included in the sample buffer. Gradient mini gels (resolving 8 25% T and 2% C, stacking zone 4.5% T and 3% C, 43 9 50 9 0.45 mm, polyacrylamide gels cast on Gel- Bond Ò plastic backing, buffer 0.112 M acetate, 0.112 M Tris, ph 6.4) was used to separate proteins on a Phast- System equipped with separation and development capabilities. Napin standards were separated on 20% T gels. Sample of *1 lg protein was applied into each well and standard proteins of known molecular weight were also applied into a separate well. The molecular weight standards of wide (170 10 kda) and low (26.6 1.06 kda) molecular weight ranges were used. Electrophoretic conditions were 250 V, 10 ma, 15 C and 20 min running time. The running buffer had the composition of 0.2 M Tricine, 0.2 M Tris and 0.55% (w/v) SDS and was at ph 8.1. Following separation, the proteins were fixed and stained using PhastGel blue R (Coomassie R-350) and developed to obtain suitable background color. The gels were scanned and the acquired images were analyzed by the Image Master Ò software. Statistical Analysis All experiments were conducted with a minimum of three replicates. The study of effect of ions on solubility and ph was conducted as a full factor factorial design. Data were analyzed as a general linear model using the SAS program and multiple mean comparison was according to Tukey s multiple comparison test. Results and Discussion Purification of CRU and NAP The aqueous extracts of B. juncea, B. napus and S. alba extracts contained proteins and other UV absorbing components such as phenolics. Separation of extracts on Sephadex-G25 SEC concentrated proteins into a single fraction (fractions Bj 1, Bn 1, Sa 1 of Fig. 1a) and removed small molecular weight non-protein components of the extracts. All other UV absorbing peaks resolved from this separation failed to yield any polypeptide bands. The CEC of the protein peak of SEC was able to provide two protein peaks (Fig. 1b; peaks Bj 1a and -b, Bn 1a and -b, Sa 1a and -b). The unbound peak of CEC (peaks Bj 1a, Bn 1a and Sa 1a) contained proteins that were not positively charged at ph 8.5. The positively charged proteins at ph 8.5 were bound to the strong cation exchanger and eluted when the 1 M NaCl content of elution buffer (buffer B) was at 50% (v/v). The polypeptide profiles of these peaks confirmed

J Am Oil Chem Soc (2012) 89:869 881 873 Fig. 1 Purification and isolation of cruciferin and napin from B. juncea, B. napus and S. alba seed extracts. a Desalting of extracts with size exclusion chromatography. b Cation exchange chromatography of protein peak of step a. c Size exclusion chromatography of unbound protein peak of step b. d Hydrophobic interaction chromatography of bound protein peak of step b. Presence of proteins was monitored as absorbance at 280 nm. Chromatographic conditions were as described in the Materials and methods section. Shaded areas indicate isolated protein fractions and SDS-PAGE separation of these collected protein peaks was on 8 25%T gradient gels along with molecular weight markers (MWM) that the unbound peak contained large molecular weight proteins and the bound proteins (peaks Bj 1b, Bn 1b and Sa 1b) were of small molecular weight. Further separation of the unbound peak on Sephacryl 300 by SEC resulted in elution of a dominant protein peak that was free of polypeptides smaller than 20 kda (Fig. 1c). The bound low molecular weight proteins of CEC went through a polishing step in the HIC and resulted in proteins composed of polypeptides between 6.5 and 20 kda (Fig. 1d). The large molecular weight proteins of Sephacryl 300 SEC were resolved into undissociated cruciferin subunits; 43.0 51.3 and 60.0 kda for B. juncea (Bj 1a-i), 44.5 58.0 kda for B. napus (Bn 1a-i) and 46.3 53.3 kda for S. alba (Sa 1a-i). The CRU protomers of B. napus have molecular weights of 51.3 56.5 kda (UniProtKB/TrEmBL; www.uniprot.org [3]). Under reducing conditions, these cruciferin subunits yielded acidic (a) and basic (b) polypeptides having 18.1 31.2 kda for B. juncea, 17.1 31.8 kda for B. napus and 17.6 33.9 kda for S. alba (Fig. 2a). The presence of free polypeptides (acidic and basic) in SDS-PAGE profiles has been reported for crucifers [15] because of S S interchange reactions. Among all the species of this study, under non-reducing conditions, we observed free polypeptides between 15.9 and 31.0 kda (Fig. 2a). The low molecular weight proteins obtained from this purification process resolved into a single polypeptide band under non-reducing conditions and the molecular weights were between 12.2 and 17.0 kda for the 3 seed types (Fig. 2b). Reduction of S S bonds yielded two polypeptide bands of 7.2 7.6 kda and 4.6 4.8 kda (Bj 1b-i, Bn 1b-i and Sa 1b-i) for all the species (Fig. 2b). The NAP isoforms of B. napus have molecular weights in the range of 12.7 20.3 kda

874 J Am Oil Chem Soc (2012) 89:869 881 Fig. 2 SDS-PAGE profiles of purified cruciferin and napin of B. juncea, B. napus and S. alba. Proteins were separated under nonreducing (NR) and reducing (R) conditions. Cruciferins were separated on 8 25% T gradient gel and napins were separated on 20% T gel and different molecular weight markers as described in materials and methods section was used (UniProtKB/TrEmBL; www.uniprot.org [3]) and the values estimated from SDS-PAGE for these three species were within this range. The mass spectroscopic data analysis of tryptic digests of purified CRU and NAP showed highly probable matches for the published sequences of CRU and NAP of respective species (Table 1). Of the available five CRU sequences for B. napus in the databases, Cruciferin BnC1 (P33523), Cruciferin BnC2 (P33524) and Cruciferin CRU1 (P33525) isoforms were matched. No CRU isoform sequences are available for B. juncea in databases, therefore probable matches to B. napus CRU isoforms is not surprising because of the conserved regions in the protein. Similarly, the only sequence available for S. alba is for the allergenic CRU fragment CRU1_SINAL (Cruciferin, P83908). Due to close homology of conserved regions of CRU sequences among these species, highly probable matching with available CRU proteins can happen in the data analysis of PMP and the same was true for NAP. The low molecular weight proteins of all three species gave matches to NAP sequences available in the databases confirming the identity of low molecular weight proteins as NAP. Because of the unavailability of primary sequences of all CRU and NAP isoforms of each species it was not possible to differentiate between species but PMP confirmed that the isolated purified proteins were indeed CRU and NAP. The procedure first described by Bérot et al. [10] was successfully applied with modifications to isolate CRU and NAP from these three crucifer species with [95% purity. The yield of CRU and NAP was 15.8 21.6% and 5.2 7.0% of meal protein, respectively. These purified CRU and NAP were used to confirm the type of protein extracted under different conditions of the solubility study. Table 1 Major proteins identified for the purified cruciferin and napin from B. juncea, B. napus and S. alba by peptide mass fingerprinting Seed source Purified protein Protein/s matched Swiss prot entry (accession) Score Mr calculated, Da No. of peptides matched Brassica juncea Cruciferin Cruciferin CRU1 (B. napus) CRU3_BRANA (P33525) 260 56,867 5 Cruciferin CRU4 (B. napus) CRU4_BRANA (P33522) 240 51,630 5 Brassica napus Cruciferin Cruciferin CRU1 (B. napus) CRU3_BRANA (P33525) 871 56,867 22 Cruciferin BnC1 (B. napus) CRU1_BRANA (P33523) 462 54,076 12 Cruciferin BnC2 (B. napus) CRU2_BRANA (P33524) 256 54,542 6 Sinapis alba Cruciferin Cruciferin (S. alba) CRU1_SINAL (P83908) 588 56,817 14 Brassica juncea Napin Allergen Bra j 1-E (B. juncea) ALL1_BRAJU (P80207) 1,010 15,090 23 Napin-3 (B. napus) 2SS3_BRANA (P80208) 1,003 21 Napin embryo specific (B. napus) 2SSE_BRANA (P09893) 856 20 Brassica napus Napin Napin-3 (B. napus) 2SS3_BRANA (P80208) 1,281 20,785 25 Napin embryo specific (B. napus) 2SSE_BRANA (P09893) 1,071 27 Allergen Bra j 1-E (B. juncea) ALL1_BRAJU (P80207) 446 11 Sinapis alba Napin Allergen Sin a 1 (S. alba) ALL1_SINAL (P15322) 595 16,713 17 Allergen Bra j 1-E (B. juncea) ALL1_BRAJU (P80207) 427 14

J Am Oil Chem Soc (2012) 89:869 881 875 N-Containing Components of cotyledons The ratio of seed coat weight to kernel weight was 1:2.6 for S. alba (both varieties), 1:2.9 for B. juncea Duchess, 1:4.0 for B. napus (AC Excel), 1:5.0 for B. juncea AC Vulcan and 1:6.2 for B. juncea Dahinda and indicated comparatively low seed coat weight in oriental mustard of both condiment- and canola-quality. The seed coat of all three species contained 14.7 17.5% N-based protein and 7.4 15.6% oil. The proteins of seed coat are not SSPs and may have limited contribution to the extractable protein content of the seed. When the cotyledon protein contents are compared, the sequence of B. napus (AC Excel), B. juncea (Dahinda, AC Vulcan, Duchess) and S. alba (Andante, AC Pennant) (Table 2) followed an increasing order. The same sequence was observed for the decreasing oil content. Among these crucifers, S. alba has comparatively high protein content. Table 2 Levels of protein and other macro components of B. juncea, B. napus and S. alba seeds used in the study a Sample (species and variety) Ash (%) Crude protein, % (%N 9 6.25) Lipids (%) Brassica juncea AC Vulcan Whole seed 4.1 ± 0.1 26.9 ± 1.5 42.5 ± 1.9 Cotyledons 4.1 ± 0.1 28.0 ± 0.5 48.3 ± 0.1 Seed coat 4.3 ± 0.1 17.3 ± 0.3 15.6 ± 0.3 Duchess Whole seed 3.5 ± 0.1 26.9 ± 0.1 43.5 ± 0.1 Cotyledons 3.5 ± 0.1 29.9 ± 1.2 44.5 ± 0.1 Seed coat 4.3 ± 0.4 14.7 ± 0.9 9.2 ± 0.4 Dahinda Whole seed 4.0 ± 0.1 27.6 ± 4.9 46.9 ± 0.4 Cotyledons 3.7 ± 0.1 28.2 ± 1.3 53.7 ± 0.8 Seed coat 6.2 ± 0.1 16.6 ± 0.9 12.3 ± 0.4 Brassica napus AC Excel Whole seed 4.0 ± 0.7 23.9 ± 0.5 49.2 ± 0.6 Cotyledons 3.7 ± 0.5 25.2 ± 1.0 55.2 ± 1.6 Seed coat 6.2 ± 0.1 14.7 ± 2.5 14.3 ± 0.2 Sinapis alba AC Pennant Whole seed 4.4 ± 0.1 32.2 ± 0.7 33.5 ± 0.2 Cotyledons 4.3 ± 0.1 33.5 ± 2.7 29.9 ± 1.5 Seed coat 4.6 ± 0.1 16.9 ± 0.2 9.4 ± 0.3 Andante Whole seed 3.9 ± 0.2 36.7 ± 0.1 30.4 ± 0.4 Cotyledons 3.9 ± 0.2 39.0 ± 0.2 31.8 ± 1.4 Seed coat 4.4 ± 0.1 17.5 ± 0.3 7.4 ± 0.1 a All values are on a dry weight basis, means ± SD are provided The glucosinolates and tri-amine compounds (betaine, choline and sinapine) are the major contributors for nonprotein N in the high-glucosinolate (condiment-quality) seeds (Table 3). The low glucosinolate content (\30 lmol g -1 of dry defatted meal) is one of the quality traits of canola therefore glucosinolates contributed only 0.4 2.0% to the total N content of canola-quality B. juncea and B. napus. The contribution of tri-amine compounds to the total N content was 3.1 4.3% for condiment mustards and 2.0 2.1 for canola-quality seeds. The percentage of total N arising from all three groups of these N-containing compounds was 3.1% for canola-quality B. juncea, 5.2% for B. napus canola, 7.9 and 8.9% for B. juncea brown and oriental mustard, respectively and 9.0 10.8% for S. alba yellow mustard. When calculated based on the N conversion factor of 6.25, the protein content due to these components was 0.87% for Dahinda, 1.3% for AC Excel, 2.5% for AC Vulcan, 2.4% for Duchess, 3.6% for AC Pennant and 3.5% for Andante. The use of the N content to estimate protein levels of seeds and meals has been the norm for the oilseed and seed meal industry. According to this study, when total N content is used to estimate the protein content (6.25 as the conversion factor), a 2.4 3.6% point overestimation of protein content can occur in condiment mustard varieties containing a high level of glucosinolates and this is a 0.8 1.3% point overestimation in canola-quality Brassicas. The reported non-protein N components in this study did not include free amino acids of the seeds. It is reasonable to assume that all protein reported for these crucifer meals cannot be recovered because the N originating from molecules such as structural proteins (e.g. extensins, Gly-rich proteins, Pro-rich proteins and arabinogalactan proteins) in the embryo and endosperm cell walls [16] may not be soluble as SSPs but contribute to the total N content. Solubility Characteristics of Proteins The amount of nitrogenous components soluble at a particular ph varied between the species and the varieties studied (Fig. 3a, b). The ph value of 4 was common for the minimum solubility of nitrogenous compounds of all the samples regardless of the species and variety. The total soluble N content at ph 4 was 19.2 32.1% in B. juncea, 27.6% in B. napus and 21.4 23.2% in S. alba. At ph 4, the soluble proteins resolved into polypeptide bands corresponding to NAP indicating CRU was not soluble at this ph (Fig. 4a d). The solubility of N compounds was increased below and above ph 4 (B. napus had similar N solubility at ph 3) with a considerable increase above ph 8. At ph 2, the soluble N content of these seeds were 34.2 50.5%. The highest value of N solubility was observed at ph 10 and the varieties of B. juncea showed a

876 J Am Oil Chem Soc (2012) 89:869 881 Table 3 Levels of N resulted from glucosinolates, tri-amines (betaine, choline and sinapine) and nucleotides of B. juncea, B. napus and S. alba meals a Seed Total N content (%) Glucosinolate N(% b ) Nucleic acid N (%) Choline, betaine and sinapine N (%) Contribution of non-protein N to the total N content (%) Brassica juncea AC Vulcan 4.48 ± 0.08 0.23 0.03 0.14 8.9 Duchess 4.78 ± 0.19 0.18 0.02 0.18 7.9 Dahinda 4.50 ± 0.20 0.02 0.03 0.09 3.1 Brassica napus AC Excel 4.03 ± 0.16 0.08 0.04 0.09 5.2 Sinapis alba AC Pennant 5.36 ± 0.43 0.32 0.03 0.23 10.8 Andante 6.24 ± 0.03 0.32 0.04 0.20 9.0 a Mean values are presented b AC Vulcan and Duchess contained allyl, butenyl, pentenyl OH-pentenyl, OH-benzyl, indolyl and OH-indolyl glucosinolates and Dahinda variety contained all except OH-pentenyl and OH-benzyl, AC Pennant and Andante contained OH-pentenyl, benzyl, OH-benzyl, indolyl and OHindolyl glucosinolates, AC Excel contained allyl, butenyl, pentenyl, OH-pentenyl, indolyl and OH-indolyl glucosinolates Fig. 3 Nitrogen solubility of B. juncea, B. napus and S. alba meals under different phs of the solvation medium wide range; 56.3% for AC Vulcan to 84.1% for Dahinda. At this ph, B. napus showed 72.7% solubility while S. alba had 69.4% soluble N for AC Pennant and 71.8% for Andante. The solubility of N-containing compounds of B. napus showed a greater increase in dissolution above ph 4.0 than others as well as not showing a second solubility minimum at ph 8.0 like B. juncea or S. alba seeds. When all the seed types were compared for soluble N values, S. alba showed lower values than B. juncea or B. napus throughout this ph range (except ph 10) although yellow mustard contained the highest content of total protein (Table 2) among these seeds. The canola quality B. juncea variety had higher solubility values at ph 10 than B. napus but showed depressed solubility at ph 7 and 8. The soluble proteins under different ph conditions showed a similar solubility pattern of CRU and NAP regardless of the species or the variety of crucifer (Fig. 4). At ph 7 and 10, both CRU and NAP were soluble as the extracts resolved into almost all the polypeptide bands corresponding to these two proteins. Extracts of ph 2 showed intensely staining polypeptide band with higher molecular weight than bands corresponding to NAP and minor bands corresponding to some polypeptides of CRU (Fig. 4). When these extracts were treated with the reducing agent for a longer time (15 min as opposed to 5 min at 95 C with 2.0%, v/v b-mercaptoethanol), separate polypeptide bands corresponding to large and small chains of NAP appeared and the high molecular weight band disappeared upon SDS- PAGE separation (Fig. 5). Mass spectroscopic analysis of the tryptic digest of this polypeptide band of B. juncea, B. napus and S. alba showed peptide mass ions predominantly matching with NAP proteins of the respective species (data not shown). This confirmed that the protein extracted at ph 2 was mainly composed of NAP. The CRU and NAP proteins are stored in the protein storage vacuoles (PSV) of embryonic tissues of crucifer seeds [2] and become the predominant proteins of the seed cotyledon extracts. In B. napus seed, nearly 40% of meal N

J Am Oil Chem Soc (2012) 89:869 881 877 Fig. 4 SDS-PAGE separation of soluble components of B. juncea, B. napus and S. alba recovered at different ph values of the solvation medium. Samples were prepared under reducing conditions and Fig. 5 SDS-PAGE separation of B. juncea and S. alba extracts at ph 2. Different conditions were used for reducing S S bonds as labeled in each lane remained soluble at ph 4 where the N-component solubility was at its minimum [17]. Another solubility minimum was observed at ph 8, where phytate-p solubility was cruciferin and napin of the respective species were separated along with molecular weight markers (MWM) at the lowest. This second solubility minimum at ph 8 was observed for all B. juncea varieties. The B. napus AC Excel in the present study did not show a reduced solubility but S. alba varieties showed a depression in N solubility at ph 8. Since no salts were added to the dissolving medium, the soluble proteins at a particular ph are the ones that carry a net charge. NAP is a basic protein with a pi close to 11 [18]. Therefore it is not surprising that charge ionization at acidic ph keeps NAP soluble. The insoluble proteins may be charge neutral or have formed insoluble salts at this ph. CRU may not have sufficient ionizable groups to become soluble at ph as low as 4.0 because it has isoelectric ph around 7.25 [19]. The carboxyl groups of CRU that originate from the abundant Glu and Asp residues may be charge neutralized at ph 4 and may have decreased the solubility of CRU. The present study shows that in these crucifers NAP is the predominant protein soluble at low ph, especially around ph 4.0. It is also clear that all protein estimated based on total N do not become soluble even extreme alkaline phs were used. In B. napus only about 70% N is soluble at neutral ph and ph 12 is needed to solubilize more than 90% total N [17]. In this study, the Dahinda showed exceptionally high levels of soluble N at

878 J Am Oil Chem Soc (2012) 89:869 881 ph 10 (87.6%) than other varieties of B. juncea (56.3 66.4%), B. napus (72.7%) or S. alba (69.2 71.8%). Although we did not analyze the levels of CRU and NAP of these seeds, we showed that at ph 4, in the absence of salt, only NAP was soluble therefore the level of soluble protein at this ph may be related to the total NAP content. Among the European genotypes, the ratio of CRU to NAP was highly variable and ranged from 0.6 to 2.0 in the lowto high-erucic acid and glucosinolate containing germplasm [20]. The same study concluded that intensive breeding to reduce glucosinolate content and to increase oil content has concomitantly increased the CRU content in double-low varieties (e.g., canola quality). When the values of B. juncea high- and low-glucosinolate varieties in the present study are compared, canola quality Dahinda had 25.5%, condiment mustard AC Vulcan and Duchess had 19.2% and 32.1% N solubility, respectively at ph 4. If the soluble N content at ph 4 can extrapolate to NAP content, our observation does not support the assumption that the reduction of glucosinolate content through breeding decreased NAP content of the germplasm. The solubility values at ph 4 did not relate well with the glucosinolate content either. The lowest glucosinolate levels were found in Dahinda (10.8 lmol g -1 total glucosinolates) and AC Excel (74.8 lmol g -1 total glucosinolates) and their N solubility at ph 4 was 25.2% and 27.0%, respectively. These solubility values were significantly (p \ 0.05) higher than the high glucosinolate containing AC Vulcan (162.6 lmol g -1 total glucosinolates, 19.4% N solubility), Andante (263.0 lmol g -1 total glucosinolates, 20.2% N solubility), AC Pennant (263.7 lmol g -1 total glucosinolates, 23.7% N solubility). Only the Duchess variety which had 130.0 lmol g -1 total glucosinolates exhibited 32.1% N component solubility at ph 4. A study on water leaching of B. napus glucosinolates at ph 4, 6 and 8 at 25 C showed that the highest glucosinolate dissolution occurred at ph 6 [21] therefore, a comparatively low contribution from glucosinolates can be expected to the soluble N components at phs as low as 4. Quinn and Jones [22] have also suggested that in B. napus, the lowest N component extractability occurred between ph 3.7 and 4.0 and also between 7.7 and 8.0. A similar observation was reported for B. juncea [23] and a protein solubility of 33 35% between ph 7 and 9 has been reported [24]. The abundance of NAP may be different among these species and varieties and may contribute to the differences in soluble protein levels at ph 4. The amount of soluble N compounds that remained in solution of the ph 12 extract when the ph was progressively lowered to 1.9 showed a similar pattern among the seed types (Fig. 6a). In B. juncea and S. alba extracts, decrease of ph beyond 9.8 caused a drastic drop in the soluble N content. The decrease in soluble N content continued until ph 6.2 for B. juncea (31.6%) and S. alba (20.1%) indicating considerable accumulation of insoluble protein. This accumulated insoluble protein resembles the isoelectric protein recovery process commonly used in seed protein recovery studies and industries. Further reduction of ph below 5.2 tend to increase the soluble protein level and at ph 1.9 solubility was at 69.5% for B. napus AC Excel, 60.4% for B. juncea AC Vulcan and 50.9% for S. alba AC Pennant (Fig. 6a). Although B. napus followed the same trend, the solubility drop was not as pronounced as the other two species. More N-containing compounds remained soluble in B. napus through out the ph range and the minimum soluble N content (37.2%) was observed at ph 5.2. This observation shows that the ph of the lowest N solubility (Fig. 3, ph 4.0) of meal has less relevance when meal protein is solubilized at an alkali ph. When the ph of a rapeseed extract prepared at ph 6.2 was adjusted beyond 5, a decrease in the precipitate dry matter and its total N content has been reported [17]. The SDS-PAGE analysis showed that soluble proteins remained at ph 6.2 and 4.3 were composed of CRU and NAP and the same composition was found in the precipitate (Fig. 6b). Interestingly, at ph 4.3, NAP was partitioned between solubles and insolubles therefore changing the ph of an alkali extract of crucifer proteins to ph * 4.0 may not give a good separation of CRU from NAP. In this study, the three crucifer species showed different ph values of minimum N-solubility for the alkali extracted proteins than meal intact proteins. Both B. juncea and S. alba extracts gave minimum soluble protein level (maximum protein precipitation) at ph 6.2 and showed that formation of insoluble salts or aggregates of alkali ph soluble proteins followed a different pattern than protein solubilization from cellular matrix in this ph range. Compared to B. juncea and S. alba a poor protein recovery was observed for B. napus as 37.2% protein remained soluble at ph 5.2. The charge reversal of proteins is ph dependant but in these crucifer proteins the number of charges reversed was different than what was ionized at alkaline ph. Precipitation of alkali ph soluble protein by lowering ph to acidic may occur either due to acid neutralization of the base previously combined with the protein and forming insoluble proteins or by forming protein salt that is insoluble in water. Further addition of acid beyond the least solubility point of the insoluble species may change the amount of salt in the solution. The reduction of ph of the medium in combination with the level of NaCl formed (HCl and NaOH was used to adjust ph) may have resulted in more soluble proteins. This may be the reason that alkali extracted crucifer proteins does not follow the same solubility pattern as meal intact protein during ph changes. When alkali extracted (ph 11, with NaOH) rapeseed meal proteins was precipitated using HCl at ph 6.6, the precipitate

J Am Oil Chem Soc (2012) 89:869 881 879 Fig. 6 Titration of B. juncea, B. napus and S. alba extracts at ph 12 a Nitrogen solubility and b SDS-PAGE separation of the extract predominantly contained high molecular weight ([150 kda) acidic proteins (pi ranging from 4 to 6) and low and intermediate molecular weight (13 to 50 kda) proteins remained in the solution [25]. A complete precipitation of alkali extracted rapeseed protein can occur with precipitation aids such as carboxy methyl cellulose or hexametaphosphate but with a poor resolubilization capability even at ph 11 [25]. The practical implication of this incomplete charge reversal behavior of CRU and NAP of crucifer meals is the incomplete recovery of proteins solubilized at basic phs by reversing ph to acidic. Solubility Changes with Salts The soluble nitrogen content of crucifer seed kernels at ph 4, 7 or 10 was significantly (p \ 0.05) different when NaCl or CaCl 2 ions were present (Table 4). The quantitative effect of the ionic strength on solubility was larger at ph 4 and 7 than at ph 10. Among the B. juncea varieties, the effect of ionic strength at ph 4 was much larger for both ion types than other varieties. The presence of CaCl 2 (l = 0.25 1.0) at ph 4 and 7 resulted in higher solubility values than NaCl for all the samples except S. alba AC Pennant at ph 7. At ph 4, the presence of CaCl 2 at ionic strength[0.5 resulted in 45 55% N solubility in B. juncea and B. napus while in S. alba the soluble N content remained between 36 and 42%. The ionic strength [0.25 did not cause a significant increase in the N solubility for B. juncea at ph 10. The same is true for B. napus across all the phs with CaCl 2. Presence of salts actually decreased the solubility of S. alba AC Pennant N-compounds at ph 10. Throughout the ph and ionic strength combinations, S. alba proteins had lower solubility values than B. napus or B. juncea similar to the observation of ph alone study (Table 4; Fig. 3). Comparatively high solubility of alkali extracted S. alba proteins at 0.75 M Ca 2? has been reported [26]. It is clear that at ph 4 or 7 more protein of crucifer seeds were soluble when ionic strength was maintained at 0.25 or 0.5 either with NaCl or CaCl 2 ions (Table 4). Exceptionally high amount of protein was solubilized from B. juncea AC Vulcan at ph 10 when ionic strength of 0.25 was provided with NaCl. Determination of the types of protein soluble under different ionic strengths was difficult because the salt ions caused streaky protein lanes in SDS-PAGE separation of the extracts. Removal of salt by dialyzing seed extracts changes soluble protein composition and does not represent the soluble proteins when salts are present. The stabilizing effect of neutral salts towards protein molecules in solution is a well known phenomenon and this is through the salt affect on bulk water structure. At low concentration (\0.2 M), ions interact with proteins by nonspecific electrostatic interactions and this electrostatic neutralization of protein molecule charges may stabilize the protein structure. The added NaCl binds very weakly to the protein surface and enhances preferential hydration of

880 J Am Oil Chem Soc (2012) 89:869 881 Table 4 Nitrogen solubility of B. juncea (AC Vulcan and Dahinda), B. napus (AC Excel) and S. alba (AC Pennant) at different phs and different ionic strengths provided by NaCl and CaCl 2 Seed and salt type ph 4.0 A ph 7.0 A ph 10.0 A 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 B. juncea AC Vulcan NaCl 19.4 a 35.9 b 41.2 d 39.5 c 40.8 cd 45.9 a 50.3 b 64.5 d 54.8 c 63.9 d 54.8 a 83.6 e 74.4 d 68.6 c 64.6 b CaCl 2 19.4 a 48.7 b 47.3 b 49.4 b 55.1 c 45.9 a 63.2 b 93.2 c 65.8 b 64.0 b 54.8 a 66.7 c 54.9 a 63.6 b 55.9 a Dahinda NaCl 25.3 a 47.9 e 43.7 d 35.8 b 38.9 c 46.5 a 76.9 c 90.7 e 72.2 b 84.4 d 83.6 b 87.6 c 84.1 b 73.3 a 82.2 b CaCl 2 25.3 a 55.8 d 47.8 b 50.0 c 48.6 b 46.5 a 88.7 c 82.0 b 80.6 b 78.4 b 83.6 d 86.0 c 69.3 b 73.6 c 66.3 a B. napus AC Excel NaCl 27.3 a 36.1 b 40.9 c 38.8 c 41.2 c 60.6 a 74.4 b 73.7 b 70.2 b 71.4 b 72.7 a 76.0 b 76.6 b 99.3 d 80.8 c CaCl 2 27.3 a 45.6 bc 44.7 b 47.0 c 46.1 bc 60.6 a 81.4 c 76.5 b 81.0 c 79.4 c 72.7 b 78.3 c 73.0 ab 70.7 a 74.4 b S. alba AC Pennant NaCl 23.7 a 32.5 c 31.3 c 29.6 b 31.7 c 35.4 a 52.0 c 53.9 c 46.0 b 56.5 d 68.8 d 57.8 c 55.1 b 52.0 a 68.2 d CaCl 2 23.7 a 42.4 d 36.4 b 39.1 bcd 38.4 bc 35.4 a 39.3 b 42.9 c 42.6 c 42.7 c 68.8 b 44.2 a 37.5 a 38.3 a 39.3 a A For a given ph value and a salt type, the mean solubility value followed by different superscript indicate statistically significant (p \ 0.05) effect of ionic strength of that salt at the particular ph the protein surface and the ion concentration near the protein becomes lower than the bulk solution. The concentration gradient near the protein molecule and the bulk solution creates an osmotic pressure gradient around the protein molecule. This effect was clearly seen in B. juncea, B. napus and S. alba varieties when ionic strength was changed from 0 to 0.25 at ph 4 and 7. Across the species studied, different solubility behavior was observed for CRU and NAP. The differences of solubility behavior of CRU and NAP with the changing ph were similar among the species. High solubility values can be achieved with NaCl or CaCl 2 but the effect is salt concentration and seed species specific. The preferential solubility of NAP between ph 3 and 4 is advantageous in separating CRU and NAP of Brassicaceae seeds [27]. The high ph and thermal stability of NAP [5, 6] may resist any ph-induced structural changes of the protein during low ph solubilization of the protein. The differences in amino acid composition, digestibility, immunogenic characteristics and techno-functional properties of CRU and NAP [28] makes it necessary to obtain these proteins separately in order to obtain maximum uses of them. Conclusions The solubility of major seed storage proteins of crucifer seeds showed distinct characteristics; NAP is highly soluble at low phs while CRU is not. The N-based protein values of high glucosinolate containing seeds may overestimate protein content by about 3.5%. The combination of NaCl or CaCl 2 at moderate ionic strength and low ph can enhance the solubility of NAP leaving CRU intact in the meal. This selective solubility behavior of CRU and NAP makes it possible to develop processes to obtain protein products from crucifer oilseeds and consequently utilizing the unique properties of these molecules in different applications. Acknowledgments This work is supported by the Agriculture and Agri-Food Canada funded project Optimized Crucifers for Canadian Bioeconomy (ID no. 170). SaskCanola is greatly acknowledged for the Dr. Roger Rimmer Graduate scholarship provided to S. Abeysekara. Dr. Randy Purves and Steve Ambrose of NRC-PBI Saskatoon are appreciated for their invaluable help in PMP work. References 1. Newkirk RW (2009) Canola Meal Feed Industry guide, 4th edn. Canola Council of Canada, Winnipeg. MB 2. Crouch ML, Sussex IM (1981) Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta 153:64 74 3. http://www.uniprot.org. Accessed 01 Nov 2010 4. Shewry PR, Napler JA, Tatham AS (1995) Seed storage proteins: structure and biosynthesis. Plant Cell 7:945 956 5. Krause JP, Schwenke KD (2001) Behaviour of a protein isolate from rapeseed (Brassica napus) and its main protein components globulin and albumin at air/solution and solid interfaces, and in emulsions. Colloids Surf B: Biointerfaces 21:29 36

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