The authors are solely responsible for the content of this technical presentation. The technical presentation does not necessarily reflect the official position of ASABE, and its printing and distribution does not constitute an endorsement of views which may be expressed. Technical presentations are not subject to the formal peer review process, therefore, they are not to be presented as refereed publications. Citation of this work should state that it is from an ASABE Section Meeting paper. EXAMPLE: Author's Last Name, Initials. Title of Presentation. ASABE Section Meeting Paper No. RRV-07116. St. Joseph, Mich.: ASABE. For information about securing permission to reprint or reproduce a technical presentation, please contact ASABE at rutter@asabe.org or 269-429-0300

(2950 Niles Road, St. Joseph, MI 49085-9659 USA).

An ASABE Section Meeting Presentation

Paper Number: RRV-07116

Separation and Evaluation of Canola Meal and Protein for Industrial Bioproducts

Wajira Asanga Manamperi

Agricultural and Biosystems Engineering, 1221 Albrecht Blvd, Fargo, ND 58105-5626. e-mail – wajira.manamperi@ndsu.edu

Scott W. Pryor

Agricultural and Biosystems Engineering, 1221 Albrecht Blvd, Fargo, ND 58105-5626. e-mail – scott.pryor@ndsu.edu

Sam K.C. Chang

Department of Cereal and Food Sciences, North Dakota State University, Fargo, North Dakota, 58105 kow.chang@ndsu.edu

Written for presentation at the 2007 ASABE/CSBE North Central Intersectional Conference

Sponsored by the Red River Valley Section of ASABE North Dakota State University

Fargo, North Dakota, USA October 12-13, 2007

Abstract. Methods and processing conditions used in the preparation of canola protein materials play an important role in determining the properties of the ultimate products that they are used for. Canola seeds were screw pressed, milled, defatted and the meal was subjected to alkaline solubilization and acid precipitation in order to isolate the proteins. Maximum solubility and precipitabiltiy of proteins were observed at pH 12.0 and pH 5.0, respectively. Biuret method was used in determining protein concentration of the supernatants at each solubilization and precipitation step. Specific proteins were isolated according to the Osborn sequence. Albumins and globulins consist of the majority of the proteins isolated with this procedure.

The authors are solely responsible for the content of this technical presentation. The technical presentation does not necessarily reflect the official position of ASABE, and its printing and distribution does not constitute an endorsement of views which may be expressed. Technical presentations are not subject to the formal peer review process, therefore, they are not to be presented as refereed publications. Citation of this work should state that it is from an ASABE Section Meeting paper. EXAMPLE: Author's Last Name, Initials. Title of Presentation. ASABE Section Meeting Paper No. RRV-07116. St. Joseph, Mich.: ASABE. For information about securing permission to reprint or reproduce a technical presentation, please contact ASABE at rutter@asabe.org or 269-429-0300

(2950 Niles Road, St. Joseph, MI 49085-9659 USA).

Functional properties of canola meal such as water and fat absorption, emulsification, whippability and foaming were determined.

Keywords. Canola/rapeseed meal, Alkaline extraction, Isoelectric precipitation, Osborn sequence, Functional properties.

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Introduction

With the growing interest in green chemistry, the demand for protein-based industrial products is increasing. Soybean, with its high protein content and superior quality, has been used in a variety of products since the early 1900s. Comparatively, canola and rapeseed are a newer introduction to the world as a potential crop for protein-based products.

Canola (Brassica napus L.) was developed in the early 1970s by reducing the anti-nutritional components erucic acid and glucosinolates from rapeseed using traditional plant breeding techniques. The word canola has been derived from “Canadian oil, low in acid”. Rapeseed/canola ranks as the third largest oilseed crop produced worldwide (after soy and palm oil) (USDA, 2007). Canola has an oil content of 45-50% and a protein content of 17-26%; after oil extraction, rapeseed/canola meal contains 30-40% protein. Although canola proteins possess a well balanced amino acid composition, the meal is not used in human food applications due to the presence of glucosinolates (interfere with thyroid function, thus reducing growth), erucic acid (potential to produce toxic effects in heart), phytates (strongly bind polyvalent metal ions such as zinc and iron and make them unavailable for metabolism) and phenolics (bitter flavored and make the protein products darker in color) (Diosady, Rubin et al. 1990) and hence are mainly used for animal feed. However, the usage of canola meal for animal feed has limitations due to the same reasons mentioned above. Industrial uses, however, would not be limited by the antinutritional factors and could have a significant economic impact on the canola biodiesel and food oil industries.

With the growth of the biodiesel industry, the importance of canola/rapeseed has increased as a potential oil source (National Biodiesel Board, 2007). Utilization of the byproducts of biodiesel production will help strengthen the economies of the canola biodiesel industry. Oilseed meal is the main byproduct of the biodiesel industry both by volume and value (GAIN, 2007). Due to its high content of storage proteins, canola meal has potential to be used in a variety of potential industrial product applications such as adhesives, plastics, and composites.

Storage Proteins

Cruciferin and napin are the two major families of storage proteins found in rapeseed and canola. Seed storage proteins can be classified by the Osborne method according to their solubility in water (albumins), salt solutions (globulins), alcohol (prolamins), and alkali (glutelins) (Chan, 1994). Napin is a 2S albumin and cruciferin is a 12S globulin. They constitute 20% and 60% of the total protein content of mature seeds respectively (Hoglund, 1992). Napins are low molecular weight proteins (12.5-14.5 kDa) characterized by their strong alkalinity, mainly due to high amidation of amino acids. Napin possesses good foaming properties (Schmidt, Renard et al. 2004). Cruciferin is a neutral protein with high molecular weight (300-310 kDa) and several subunits. In its undenatured form, cruciferin acts as a gelling agent. Oleosin, the other major type of protein found in canola, is a structural protein associated with oil bodies (Ghodsvali, 2005). Oleosins are low molecular weight (15-26 kDa) alkaline proteins and represent about 2-8% of the total seed proteins (Huang, 1992). Canola meal also contains some minor proteins, such as thionins, trypsin inhibitors and lipid transfer proteins (LTP) (Bérot et al., 2005).

Canola meal proteins can be purified to varying degrees. Soy proteins in the form of soy flour (40-50% protein), soy protein concentrate (70% protein), and soy protein isolate (>90% protein) have all been used for various applications. Canola meal can also be separated or purified into similar products. When it comes to industrial applications, however, highly refined proteins may be unnecessary or even less useful than crude samples that contain polysaccharides or other fibrous material. Such materials have been shown to enhance the performance characteristics

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for soy-based plastic and composite materials (Chen et al. 2003; Mohanty et al. 2005; Tummala et al. 2004).

Functional properties are of great importance in many applications (Kinsella, 1982). The most important functional properties of protein products include solubility, emulsification, water absorption, fat absorption, whippability, foaming, etc (Holm and Breedon 1983; Hao, Rackovsky et al. 1992; Clarke and Fersht' 1993; Huang and Sun 2000; Chove, Grandison et al. 2002; Agboola, Ng et al. 2005).

Industrial Applications

Soy proteins have been used for industrial products such as paper, ink and paints, adhesives, biocomposites and bioplastics since the early 1900s. However, the attempts to develop commercially viable industrial products derived from canola meal and canola meal proteins are rare. Development of such products requires experience in the extraction and characterization of the protein and other fractions of canola meal. While most protein-based bioproducts research relies on commercial protein products, it is best that advances proceed with full control at all stages of the process – protein extraction, protein modification, and product formulation. Exploring better separation methods and specific functional properties would be very useful in process optimization to produce proteins particularly suited for use in industrial applications.

In this paper we discuss some separation techniques and functional properties of canola proteins that are important for both food and industrial applications.

Materials and Methods

Canola Meal

Canola seeds (Invigor 2573) were cleaned by air classification to remove lighter particles and also manually to remove heavier particles such as grit and seedpods. Crude protein was determined by Kjeldahl method (N*6.25). Moisture content of the seeds was determined by oven drying a sample at 100 °C overnight. Oil was e xtracted at a low speed with a screw press, which was preheated at 100 °C for 30 min prior to f eeding the canola seeds (2kg). Oil and meal was collected separately and weighed. Meal was pressed twice in order to remove most of the oil. The meal was then ground in a Z-mill (Mfr, city) using a 25 mesh size and subjected to solvent extraction using hexane in a Soxhlet apparatus for 24 hours to produce canola meal flour (CMF). The defatted meal flour was desolventized and dried in a fume hood for 24 hours.

Alkaline Extraction of Proteins

To determine the pH at which the maximum solubility of canola proteins occurs, 5 g of CMF was dissolved for 30 min in 90 mL of sodium hydroxide solutions of different pH values (Ghodsvali et al., 2005). Solutions were kept constant at the predetermined pH values using 3N and 0.3N NaOH solutions during the 30 min dispersion period. The solutions were centrifuged at 3000g for 15 min and the supernatant volume was measured after vacuum filtration through Whatman No. 41 filter paper. The protein concentration of the supernatant was obtained using the Biuret method (Hara et al., 1976). The Biuret reagent (blue) turns to pink/violet color depending on the amount of protein present in the solution. Therefore, the protein concentration can be quantified by light absorbance in the visible range using a spectrophotometer. A BSA (bovine serum albumin) standard curve was used to obtain the protein content of the samples by interpolating the absorbance readings (Figure 1).

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R2 = 1

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3

4

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9

0 0.1 0.2 0.3 0.4 0.5 0.6

Absorbance

Co

nce

ntr

atio

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ug

/ml)

Figure 1. BSA Standard Curve

Isoelectric Precipitation

Canola meal flour (20 g) was alkaline-extracted with 360 mL of NaOH at pH 12 according to the method described above. Supernatant was divided and transferred to 9 centrifugetubes (30 ml each). The pH of samples was dropped to between 3.5 and 7.5 in increments of 0.5, using 3N and 6N HCl solutions. Each sample was centrifuged at 3000g for 20 min and the volume of supernatant was measured. The protein content of supernatant was measured using the Biuret method. Isoelectric point was determined by identifying the centrifuge tube with maximum precipitated proteins and the minimum soluble proteins in the supernatant.

Extraction of Proteins According to Osborn Sequence

A conventional Osborn sequence extraction method was used to separate albumins, globulins, glutelins and prolamins (Figure 2). Defatted canola meal flour (100 g) was extracted with 400 mL of distilled water while stirring for 4 hr. The sample was then centrifuged at 3000g for 30 min. The supernatant was vacuum-filtered using Whatman 41 filter paper and the residue (R1) was kept for further solubilization. Supernatant was acidified to pH 4.1 using 1M HCl to precipitate albumins. The acidified solution was centrifuged at 3000g for 30 min. The pellet was washed by resuspension and centrifugation, with water acidified to pH 4.1 (in order to remove residual alkali), lyophilized and weighed (Agboola et al. 2005).

The residue R1 was extracted with 400 mL of 5% NaCl (~ 0.85 M) stirring for 4 hr. The solution was centrifuged at 3000g for 30 min and the supernatant was filtered using Whatman 41 filter paper. The residue (R2) was kept for further solubilization. Then, the supernatant was acidified to pH 4.3 to precipitate globulins. The solution was centrifuged at 3000g for 30 min to pellet the precipitated proteins and the pellet was washed with distilled water, lyophilized and weighed.

The residue R2 was extracted with 400 mL of 0.1 M NaOH for 1 hr. Solution was centrifuged at 3000g for 30 min and the supernatant was filtered using Whatman 41 filter paper. The residue (R3) was kept for further solubilization. Supernatant was then acidified to pH 4.8 using 1 M HCl to precipitate glutelins. The solution was centrifuged at 3000g for 30 min. The pellet was washed with distilled water, lyophilized and weighed.

The residue R3 was extracted with 400 mL of 70% ethanol for 4 hr. Solution was centrifuged at 3000g for 30 min and the supernatant was filtered using Whatman 41 filter paper. The residue was discarded. For the precipitation of prolamins twice the volume of acetone was added to the supernatant and kept at -20 °C for 24 hr (Yamagata et al, 1982). The solution was centrifuged at 3000g for 30 min. The pellet was washed with distilled water, lyophilized and weighed.

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Figure 2. Separation of Proteins According to Osborne Sequence

Functional Properties

The pH value of meal was determined using 5% and 10% (w/v) solutions with distilled water. Water absorption, fat absorption, emulsifying activity, whippability, and foaming properties were determined by the methods followed by Ghodsvali et al. (2005), with minor modifications.

To determine water absorption, 2 g of canola meal was dissolved in 16 ml of distilled water and mixed for 30 sec every 10 min for one hr (7 times). The mixture was centrifuged at 2000g for 15 min. The supernatant was decanted and the tube was inverted and allowed to drain for 30 min. Water absorption was quantified by measuring the weight difference between the initial and final samples.

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Fat absorption was measured by dispersing 2 g of canola meal in 12 mL of pure canola oil. The mixture was stirred (using a glass rod) for 30 sec every 5 min for 30 min (7 times). The mixture was centrifuged at 1600g for 25 min. Free oil was decanted and the tube was inverted and allowed to drain for 1 hr. Oil absorption was quantified by measuring the weight difference of the dry sample and the oil-absorbed samples.

Emulsifying activity was determined by homogenizing 7 g of canola meal in 100 ml of distilled water for 30 sec. Then canola oil (50 mL) was added to the solution and homogenized for 30 sec. An additional 50 mL of canola oil was added and homogenized (using a Waring blender) again for 90 sec. The emulsion was centrifuged at 1100g for 5 min at 25 °C. Volume of the emulsified layer after centrifugation was measured. Emulsifying activity was expressed as a percentage volume of emulsified layer after centrifugation to the volume of emulsion before centrifugation.

To determine the whippability, 3 g of canola meal was dispersed in total of 100 mL of solution with distilled water and 50 mL of solution was taken and homogenized for 90 sec. Mixture was transferred into a 250-mL graduated cylinder and the foam volume was measured. Final volume as a percentage of initial volume was expressed as whippability.

Foaming properties were analyzed by preparing 200 mL of 3% dispersion of canola meal in distilled water. The mixture was homogenized for 90 sec and transferred immediately into a 500-mL graduated cylinder. The foam volume remaining after 20, 40, 60 and 120 min was measured in order to determine the foam stability.

Results and Discussion

The moisture content of canola seeds was 6.84% on wet basis. On a 100% dry matter basis, the nitrogen content was 7.57% and the total canola protein content was 47.32%.This indicates that the canola variety Invigor 2573 has a significantly higher crude protein content compared to other varieties reported before (39 -43%) (Ghodsvali et al., 2005).

Oil removal efficiency at various stages (1st and 2nd stage screw pressing, and solvent extraction) is tabulated in Table 1.

Table 1. Percentage of Oil Removed at Different Stages

Oil removed from the first stage screw-pressing 72.2%

Oil removed from the second stage screw-pressing 10.2%

Oil removed from the solvent extraction 17.6%

This shows that screw pressing alone is not effective in extraction of total oil content from the canola seeds. The residual oil content may or may not have an impact on the yield or quality of protein extracted from the canola meal. However, the efficiency of protein recovery with the residual oil content in the meal has not been studied in this paper.

Extractability of Canola Proteins

Both extractability and precipitability of canola proteins are important in large scale purification of proteins to be used in industrial products. Determination of maximum extractability and maximum precipitability of proteins can be used to optimize the process conditions.

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Solution pH has a significant effect on the yield during both extraction and precipitation of proteins. Figure 3 shows the influence of pH on the recovered protein content from defatted canola meal flour. Solubility of proteins was tested in the range between pH 11.5 and 12.6. It was difficult to detect any difference in extractability within this range. However, others have obtained maximum solubility within this pH range (Tzeng et al., 1990, Ghodsvali et al., 2005).

0

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11.4 11.6 11.8 12 12.2 12.4 12.6 12.8

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Figure 3. Effect of pH on Protein Extractability

Precipitability of Canola Proteins

The amount of protein precipitated was followed by varying the pH between 3.5 and 7.5, in order to find the optimal conditions for the recovery of proteins dissolved in NaOH aqueous solutions. Minimum amount of protein in the supernatant solution after pelleting the precipitated proteins indicated the pH at which the maximum precipitability occurred. Figure 4 shows the soluble protein content of the supernatant. Soluble protein content decreases with the increasing pH until 5.0 and starts increasing beyond that. In this case pH 5.0 is considered as the isoelectric point of the proteins since it is indicative of the minimum charge condition of canola proteins within this range.

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Figure 4. Effect of pH on Protein Precipitability

The values represented in Figure 4 are not consistent with the previously reported values by Diosady et al. (1990) and Tzeng et al (1990). They have shown that pH 3.5 was a more suitable condition to obtain the maximum precipitability. However, our results agree with values obtained by Ghodsvali et al (2005), where they achieved maximum precipitability at pH values between 4.5 and 5.5 for three canola varieties.

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Protein Fractions from Osborn Method

Weights of fractions albumins, globulins, glutelins and prolamins obtained from 100 g of dry defatted canola meal flour are listed in Table 2. All the proteins in canola meal can not be isolated within these fractions. Protein recovery efficiency of this method was 63% compared to the total protein content determined by the Kjeldahl method. The highest weight fraction was obtained from albumins, followed by globulins and glutelins. Prolamins represented a small percentage (1.5%) of total meal proteins (figure 5).

Table 2. Weights of Osborne Sequence Fractions

Fraction Weight (g) Albumins 20.53 Globulins 4.56 Glutalins 4.20 Prolamins 0.70

Wt % to total meal protein

43.38

9.6 8.91.5

0

10

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30

40

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Albumins Globulins Glutalins Prolamins

Per

cent

age

(%)

Figure 5. Percentage of Proteins to the Total Meal Proteins

A large difference was observed in the yields of albumin and globulin fractions (20.53 g vs 4.56 g, respectively). Usually the albumin and globulins consist of the majority of the canola proteins. The difference observed in the data could be due to the sequence of separation carried out in this experiment. Due to the fact that canola meal contains some salts, a large part of the globulins could be dissolved during the solubilization step carried out for albumins. Therefore, the albumin fraction is somewhat higher and the globulin fraction is lower than the expected values. However, an alternative sequence (solubilization of globulins first followed by solubilization of albumins) is expected to address this issue but is not discussed in this paper.

Functional Properties of Canola Meal

Canola meal was slightly acidic with pH values for 5% and 10% dispersions of 6.29 and 6.11, respectively. Water absorption, fat absorption, emulsifying activity, whippability and foaming properties are listed in table 3.

Table 3. Functional Properties of Canola Meal

Functional Property Value Water absorption (g water /g) 2.5 Fat absorption (g oil /g) 1.2 Emulsifying activity (ml emulsified layer / ml) 0.5 Whippability (mL /mL) 1.5

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0.5 min 20 min 40 min 60 min 120 min

Foam stability 285 mL 198 mL 188 mL 185 mL 163 mL

All the functional properties are comparable with previously reported values (Ghodsvali et al, 2005, Naczk et al., 1985). Water absorption of canola meal exceeded 250%. For different varieties of canola meal, water absorption values from 209% (Ghodsvali et al, 2005) to 382% (Naczk et al., 1985) have been reported. Fat absorption and emulsifying activity of Invigor 2573 variety were lower than the reported values for Quantum, PF and Hyola varieties (Ghodsvali et al., 2005).

Conclusion

Canola meal proteins showed high solubility at all pHs tested between 11.5 and 12.6. Maximum precipitability (Isoelectric point) was obtained at pH 5.0. Most of the canola proteins recovered under the Osborne sequence separation method comprised of albumins. The globulin fraction was considerably lower than albumins and may be due to the dissolution of globulins by salts already present in canola meal during albumin extraction. This can be corrected by extracting globulins prior to albumins. A dialysis step could be added between these two steps to remove salts. Functional properties of meal are comparable with previously reported values.

Acknowledgements

We are thankful to Mr. Tom Borden for providing the canola seeds. We also thank Dr. Dennis Wiesenborn, Kristi Tostenson, Dr. Darren Hagensen, Dr. Zhisheng Liu, and Mary Niehaus for their assistance with various portions of this project.

References Agboola, S., D. Ng, and D. Mills. 2005. Characterization and functional properties of Australian

rice protein isolates. Journal of Cereal Science 41(3): 283-290. Berot, S., J.P. Compoint, C. Larre, C. Malabat, and J. Gueguen. 2005. Large scale purification

of rapeseed proteins (Brassica napus L.). Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 818(1): 35-42.

Chen, Y., L. Zhang , Y. Lu , C. Ye, and L. Du. 2003. Preparation and Properties of Water-

Resistant Soy Dreg/Benzyl Konjac Glucomannan Composite Plastics. Journal of Applied Polymer Science 90(14):3790-3796.

Chove, B. E., A. S. Grandison, and M.J. Lewis. 2002. Emulsifying properties of soy protein

isolates obtained by microfiltration. Journal of the Science of Food and Agriculture 82(3): 267-272.

Clarke, J. and A. R. Fersht' .1993. Engineered Disulfide Bonds as Probes of the Folding

Pathway of Barnase: Increasing the Stability of Proteins against the Rate of Denaturation. Biochemistry 32: 4322-4329.

10

Diosady, L. L., L. J. Rubin, and B.K. Chen. 1990. Membrane Based Processes for the

Production of Rapeseed Protein Isolates. International Congress on Engineering and Food 3.

GAIN. 2007. Impacts on Oilseed Industry following Biofuel Boom, GAIN Report No. FR7009,

USDA Foreign Agricultural Service Ghodsvali, A., M. H. H. Khodaparast, M. Vosoughi, and L.L. Diosady. 2005. Preparation of

canola protein materials using membrane technology and evaluation of meals functional properties. Food Research International 38(2): 223-231.

Gruener, L. and M. A. H. Ismond. 1997. Effects of acetylation and succinylation on the

physicochemical properties of the canola 12S globulin. Part I. Food Chemistry 60(3): 357-363.

Hao, M., S. Rackovsky, A. Llwo, M.R. Pincust and H.A. Scheraga . 1992. Effects of compact

volume and chain stiffness on the conformations of native proteins. Proc. Natl. Acad. Sci. USA 89: 6614-6618.

Hara I., K. Wada and H. Matsubara. 1976. Pumpkin (Cucurbita sp.) seed globulin II. Alterations

during germination. Plant and Cell Physiology 17(4) 815-823. Heywood A.A. , D.J. Myers, T.B. Bailey, and L.A. Johnson. 2002. Functional Properties of

Extruded-Expelled Soybean Flours from Value-Enhanced Soybeans. JAOCS 79(7). Hoglund, A. S., J. Rodin, E. Larsson, and L. Rask. 1992. Distribution of napin and cruciferin in

developing rape seed embryos. Plant Physiology 98(2): 509-515. Holm, E. T. and C. H. Breedon. 1983. Comparison of the Whippability of Defatted Meals from

Three Hybrid Varieties of Sunflower Seed. Journal of Food Science 48 (4): 1131. Huang, W. and X. Sun. 2000. Adhesive properties of soy proteins modified by sodium dodecyl

sulfate and sodium dodecylbenzene sulfonate. Journal of the American Oil Chemists' Society 77(7): 705-708.

Kinsella J.E., 1982. Structure and functional properties of food proteins. Food proteins. 72–85. Klockeman, D. M., R. Toledo, and K.A. Sims. 1997. Isolation and Characterization of Defatted

Canola Meal Protein. Journal of Agricultural and Food Chemistry 45(10): 3867-3870. Mohanty A. K, P. Tummala, W. Liu, M. Misra, P.V. Mulukutla, L.T. Drzal. 2005. Injection molded

biocomposites from soy protein based bioplastic and short industrial hemp fiber. Journal of Polymers and the Environment 13(3):279-285.

11

National Biodiesel Board. 2007. U.S. Biodiesel Production Capacity. Available at: http://www.biodiesel.org/pdf_files/fuelfactsheets/Production_Capacity.pdf. Accessed 30 April 2007

Naczk, M., L.L Diosady, & L. J. Rubin. 1985. Functional properties of canola meals produced by

a two-phase solvent extraction system. Journal of Food Science, 50, 1685–1692. Schmidt, I., D. Renard, D. Rondeau, P. Richomme, Y. Popineau, and M.A.V. Axelos. 2004.

Detailed physicochemical characterization of the 2S storage protein from rape (Brassica napus L.). Journal of Agricultural and Food Chemistry 52(19): 5995-6001.

Tummala P., A.K. Mohanty, M. Misra, and L.T. Drzal. 2004. Eco-composite materials from novel

soy protein-based bio-plastics and natural fibers. ICCM-14:61-70. San Diego, CA. USDA. 2007. Economics statistics and market information system, Oil crops yearbook: 2006.

Washington, D.C.: USDA National Agricultural Statistics Service. Available at: http://usda.mannlib.cornell.edu/ers/89002/2005/tab25.xls. Accessed 26 April 2007

Vioque, J., R. Sánchez-Vioque, A. Clemente, J. Pedroche, J. Bautista and F. Millan. 2000.

Partially hydrolyzed rapeseed protein isolates with improved functional properties. Journal of the American Oil Chemists' Society. 77(4): 447-450.

Wang H., L. A. Johnson, and T.Wang. 2004. Preparation of soy protein concentrate and isolate

from extruded-expelled soybean meals. Journal of the American Oil Chemists' Society 81(7):713-717.