Trends in Food Science & Technology 22 (2011) 21e39 Review Canola proteins: composition, the chemical composition (amino acids and protein fractions), production and isolation techniques, functional properties, al- lergenicity, food applications and potential uses of canola pro-extraction, functional teins for the production of bioactive compounds are highlighted. properties, Introduction bioactivity, Canola/Rapeseed is an important oilseed crop in many countries and is considered to be the second most abundant source of edible oil in the world. Canola is the rapeseed va-applications as a food riety which was developed in Canada. According to the United States Department of Agriculture, canola production ingredient and exceeds 40 million metric tons per year (USDA, 2004). Canada is a world leader in the large-scale production of high quality canola varieties, which are characterized by allergenicity e A low erucic acid (<2%) and glucosinolate (<30 lmol/g) con- tents (Ghodsvali, Khodaparast, Vosoughi, & Diosady, 2005). practical and critical Canola seeds contain approximately 40% oil and 17e26% protein (Uppstrom, 1995). Canola meal, which review is a by-product of canola oil extraction, is a highly rich raw material and contains up to 50% protein on a dry basis. The major protein constituents of canola meal are napin Mohammed Aidera,* and and cruciferin, which are storage proteins, and oleosin, which is a structural protein associated with the oil fraction Chockry Barbanab (Uppstrom, 1995). This particularity makes canola protein a potential ingredient for use in the food industry. Many a Department of Food Engineering, Universit Laval, e characteristics of canola protein are favorable for human Qubec, Qc, G1K 7P4, Canada e nutrition. The amino acid composition of canola meal is (Tel.: D1 418 656 2131x4051; well balanced and can be used for human nutrition e-mail: email@example.com) (Ohlson Anjou, 1979; Mariscal-Landin, Reis de Souza, b Tecnolog y Bioqu ıa ımica de los Alimentos, Facultad Parra, Aguilera, Mar, 2008). In addition, the protein ef- Veterinaria, Universidad de Zaragoza, C/ Miguel ﬁciency ratio of canola meal is 2.64, which is higher than Servet 177, 50013 Zaragoza, Spain that of soybean (2.19) (Delisle et al., 1984). Canola proteins have shown interesting and promising functional properties and could be potentially used in various foodThere is a well-recognized connection between the use of matrices (Khattab Arntﬁeld, 2009; Xu Diosady,plant proteins in functional foods, nutraceuticals and other 1994a, 1994b). Some properties of canola proteins werenatural health products and health promotion and disease comparable to those of casein and better than those of otherrisk reduction. Plant proteins are largely used in the food in- plant proteins, such as soybean, pea, and wheat (Ghodsvalidustry, and canola/rapeseed proteins are regarded as potential et al., 2005). However, the usefulness of canola protein ex-ingredients that may be used as food additives. In this review, tracts is limited by the presence of some undesirable com- pounds, such as glucosinolates, phytates, and phenols, which are responsible for the toxic, antinutritional and* Corresponding author. undesirable coloration capacity of canola proteins. Also,0924-2244/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2010.11.002
22 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39canola meal contains approximately 20% (w/w) carbohy- signiﬁcantly affected the ﬁnal protein compositiondrates, including soluble sugars (Lacki Duvnjak, 1998). (Chabanon, Chevalot, Framboisier, Chenu, Marc, 2007).However, after protein extraction, the major part of these The amino acid composition of canola meals was ana-saccharides is eliminated. Efforts are focusing to develop lyzed by Shahidi, Naczk, Hall, and Synowiecki (1992).canola protein products for food applications. Recently, The authors reported that the canola meals used hadBurcon NutraScience Corporation has announced that the a high content of glutamic acid (16.77e18.63% w/w pro-Division of Biotechnology and GRAS Notice Review of tein). However, they found that tyrosine, methionine andthe Center for Food Safety and Applied Nutrition of the cysteine were present in lower concentrations. In thestudyFDA has formally acknowledged receipt of Burcon’s of Shahidi et al. (1992), a two-phase solvent extraction pro-GRAS notiﬁcation for its canola protein ingredients. cess was used, and it was shown that this process did not The present review summarizes the chemical and struc- signiﬁcantly alter the amino acid composition of canola/tural compositions of canola proteins, the processes used rapeseed meals. However, a decrease in the proline contentfor canola protein extraction, the functional properties was observed. Of the essential amino acids, cysteine, me-and examples of canola protein applications in different thionine, isoleucine and leucine were also present at lowfood matrices. The potential of use of canola proteins for concentrations. The two-phase solvent extraction processthe production of bioactive peptides is also highlighted. slightly increased the cysteine content in meals obtained from some canola varieties. The authors stated that a slightChemical and structural compositions reduction in the concentration of lysine might be due to theAmino acid proﬁle formation of lysinoalanine in the alkaline solutions used for The amino acid compositions of the canola protein iso- protein extraction. The calculated protein efﬁciency ratioslates have been studied and reported by several researchers; (PER) of rapeseed meals based on the leucine and prolinethese proteins are well balanced and show high glutamine, contents or on the leucine and tyrosine contents wereglutamic acid, arginine and leucine contents and low 2.19e2.64 (Shahidi et al., 1992).amounts of sulfur-containing amino acids, which are prob- Klockeman, Toledo, and Sims (1997) studied the aminoably altered during the industrial oil extraction process acid proﬁle of untreated canola meal and found that it was(Table 1). Indeed, the amino acid composition depends on similar to the published values for high erucic acid rapeseedthe process used for protein extraction from the canola protein extract (Shahidi, 1990) but had lower values for cys-meal residue. Usually, up to 30% of the total protein ini- teine and valine. The canola protein isolate (CMPI)tially present in rapeseed meal is extracted in an alkaline reported in the work of Shahidi (1990) contained signiﬁ-medium, and large-scale puriﬁcation of canola proteins cantly more leucine, phenylalanine, arginine, and asparagine and less isoleucine than a high erucic acid canola reference protein. When compared to defatted canola meal, CMPI con- Table 1. Amino acid composition expressed as mass percent of the tains more isoleucine and arginine and less lysine. The ob- globulins isolate and the albumins isolate: from (Chabanon et al., served differences between the CMPI reported by 2007) Klockeman et al. (1997) and published values for canola (rapeseed) proteins may indicate that the genetic manipula- Amino acid Albumin isolate Globulin isolate tions involved in the development of canola varieties of rape- Asx 5.1 9.5 seed have resulted in changes in seed storage proteins, which Glx 30.4 20.2 Ser 4.1 4.4 are low in both erucic acid and glucosinolate contents. Fur- His 4.7 5.1 thermore, lysine was the only essential amino acid in Gly 1.9 1.7 CMPI and was present in signiﬁcantly lower amounts com- Thr 4.4 4.7 pared with the crude commercial hexane defatted canola Ala 3.4 3.5 meal. Although there was a decrease in the measured lysine, Arg 8.6 9.8 Tyr 3.7 4.5 no signiﬁcant change in the serine or cysteine contents was Cys 0.1 0.0 observed (Klockeman et al., 1997). It is important to note Val 4.3 3.3 that in the work of Klockeman et al. (1997), no lysinoalanine Met 0.5 (0.6)a 1.2 (1.2)a was detected in the studied canola meal protein isolate Phe 3.7 (7.4)b 5.7 (10.2)b (CMPI), which follows previous reports of lysinoalanine Ile 4.3 5.3 Leu 8.5 9.1 levels of 100 ppm in commercial hexane-extracted seeds Lys 6.2 4.7 (Deng, Barefoot, Diosady, Rubin, Tzeng, 1990). The Pro 6.4 6.8 levels of isoleucine, lysine, and aspartic acid present in the Trp n.d. n.d. CMPI were lower than those in soybean protein isolates *Asx: aspartic acid þ asparagine; Glx: glutamic acid þ glutamine. (Nehez, 1985). Protein quality was evaluated using the calcu- n.d.: Not determined. lated protein digestibility corrected amino acid score a Met þ Cys. (PDCAAS) values. It is well recognized that (PDCAAS) b Phe þ Tyr. scores 1.00 indicate an amino acid deﬁciency, while scores
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 23!1.00 are considered to be equivalent when proteins are 2005). Oleosins are low-molecular-weight (15e26 kDa)compared. All (PDCAAS) scores for the present CMPI based alkaline proteins and represent about 2e8% of the totalon the reference values for average infants were 1.00, with canola seed proteins (Huang, 1992). Canola meal also con-the lowest scores for methionine and cysteine. The essential tains minor proteins, such as thionins, trypsin inhibitors andamino acid with the lowest (PDCAAS) score based on the re- lipid transfer proteins (LTP) (Berot, Compoint, Larr, equirements for 2À5-year-olds was lysine. For comparison, Malabat, Guguen, 2005). These different protein frac- ethe lowest score found for soybean protein for this age group tions can be easily separated by chromatography, mem-was methionine plus cysteine (Henley Kuster, 1994). All brane ﬁltration, such as ultraﬁltration, and electrophoretic(PDCAAS) scores calculated for 10À12-year-olds and techniques. In canola seeds, the albumin and globulin frac-adults were 1.00. The (PDCAAS) analysis indicates that tions represent the majority of proteins. The molecularCMPI has a lower protein quality than soy protein for average weight of the albumin fraction is lower than the globulins.infants and 2À5-year-olds. If CMPI is used in products for The relative proportions of these fractions in canolainfants, blending the CMPI with other proteins will be proteins are variable and depend on many factors, such asnecessary to balance the amino acid proﬁle. The limiting the climate environment during maturation and the pres-amino acids in soy protein and CMPI are complementary ence of sulfur compounds. The amount of albumin dependsfor 2À5-year-olds; the two could be blended for nutritional on the contribution of sulfur compounds, which is depen-supplements for this age group. Because all (PDCAAS) dent on the cysteine and methionine metabolism of thescores for the canola protein isolate were !1.00 for both plant. At the same time, the amount of globulins in10À12-year-olds and adults, this protein extract represents the overall protein composition of canola depends on thean excellent source of dietary proteins for products formu- amount of non-nitrogenous compounds. The amount oflated for both of these age groups. CMPI and soy protein each protein fraction in the canola meal depends on thehave equivalent nutritional qualities for these two age extraction and puriﬁcation process (Yew-Min, Levente, groups. Compared to egg albumin, the canola albumin frac- Leon, 1988). Generally, the estimated quantities of globulintion contains more histidine, cysteine, methionine, lysine and and albumin are 60% and 40%, respectively (Mieth et al.,arginine; however, it contains less phenylalanine, tyrosine 1983).and isoleucine (Mieth, Schwenke, Raab, Bruckner, 1983). Chung, Lei, and Li-Chan (2005) conducted a study in which proteins were extracted from dehulled and defattedCanola protein fractions ﬂaxseed/canola (NorMan cultivar) and fractionated by The canola proteins of interest are mainly storage pro- anion exchange chromatography to yield a fraction withteins located in the embryo because they are abundant. a molecular weight of 365 kDa, as determined by SephacrylThey represent approximately 80% of the total protein S-300 gel permeation chromatography. According to their(Hoglund, Rodin, Larsson, Rask, 1992; Mieth et al., results, the authors stated that reducing and non-reducing1983). Canola proteins can be classiﬁed to four groups: al- SDS-PAGE revealed three predominant bands of 20, 23bumins, which constitute the water soluble fraction; globu- and 31 kDa, respectively, and two predominant bands atlins, which are soluble in salt solutions; prolamins, which is 40 and 48 kDa, respectively. In this study, the isoelectricthe ethanol soluble fraction; and glutelins, which is the focusing technique was used to separate three componentsfraction that is insoluble in all of the solvents mentioned with isoelectric points (pI ) of 4.7, 5.1, and 5.6, and acidicabove. Canola proteins can be also divided into various (pI 4.5, 5.9, 6.1) and basic (pI 9.6) components werefractions according to the corresponding sedimentation co- observed under reducing and denaturing conditions. Theefﬁcient in Svedberg units (S). This coefﬁcient indicates the major fraction of canola had high disulﬁde but low sulfhy-speed of sedimentation of a macromolecule in a centrifugal dryl content, high contents of glutamate (or glutamine) andﬁeld. For canola proteins, the following fractions have been aspartate (or asparagine), and a low lysine/arginine ratio.reported: 12 S, 7 S and a split 2 S, 1.7 S or 1.8 S. Cruciferin According to these properties, the cultivar used in this studyand napin are the two major families of storage proteins showed a lower content of the above-mentioned compoundsfound in canola/rapeseed. Napin is a 2 S albumin, and cru- than typical canola globulins. Fourier transform Ramanciferin is a 12 S globulin. They constitute 20% and 60% of spectroscopy (FT-Raman spectroscopy) also indicatedthe total protein content of mature seeds, respectively a high b-sheet content and a strong band near 1065 cmÀ1(Hoglund et al., 1992). Napins are low molecular weight that is typical of inter-molecular sheet interactions, whichproteins (12.5e14.5 kDa) characterized by strong alkalinity supports the oligomeric nature of the protein.that is mainly due to a high level of amidation of amino Canola is well recognized as an economically importantacids. Napin possesses good foaming properties (Schmidt farm-gate crop in many countries, such as Canada and theet al., 2004). Cruciferin is a neutral protein with a high mo- USA; to further explore the potential of canola proteins aslecular weight (300e310 kDa) and several subunits. In its a value-added food ingredient, a better understanding of thenative form, cruciferin acts as a gelling agent. Oleosin, fundamental properties of the two major canola proteins isthe other major type of protein found in canola, is a struc- necessary (Wu Muir, 2008). A study was reported intural protein associated with oil bodies (Ghodsvali et al., which two major protein components, cruciferin and napin,
24 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39were isolated from defatted canola meal by Sephacryl supernatant-derived canola protein isolate, the predominantS-300 gel ﬁltration chromatography. SDS-PAGE showed species was the 2 S protein. These differences lead to dif-that cruciferin consists of more than 10 polypeptides, and ferent behaviors in environments where the canola proteinnoncovalent links are more important than disulﬁde bonds isolates are used because their respective functional proper-for stabilization of the structural conformation. Napin con- ties are different. The different protein content proﬁles ofsists of two polypeptides and is stabilized primarily by canola protein isolates allow the production of any desireddisulﬁde bonds. Puriﬁed cruciferin showed one major 2 S/7 S/12 S protein proﬁle for a speciﬁc application usingendothermic peak at 91 C compared with 110 C for napin mixtures of two different canola protein isolates, such as(Wu Muir, 2008). the PMM-derived isolate and the supernatant-derived It has been reported that low molecular weight proteins isolate.make up 40e50% of the nitrogenous compounds in rape- Yew-Min, Levente, and Leon (1990) described a processseed (canola) (Mieth et al., 1983). According to various au- for the production of canola protein materials by alkalinethors, the molecular masses of these compounds range from extraction followed by precipitation and membrane pro-12 to 18 kDa (Amarowicz, Panasiuk, Pari, 2003b). For cessing. The process consisted of the extraction of oil-the separation of low molecular weight canola proteins, free meal at pH 10.5e12.5, isoelectric precipitation toHPLC methods with ion-exchange columns and capillary recover proteins and ultraﬁltration followed by diaﬁltrationelectrophoresis with SDS as a surfactant have been used to concentrate and purify the remaining acid-soluble(Amarowicz, Kolodziejczyk, Pegg, 2003a). proteins. According to the authors, these steps are comple- mentary and yield three products with excellent proteinCanola protein production recovery. In this process, the isoelectric and soluble protein In the United States patent application number isolates contained 87e100% protein (N Â 6.25). All pro-20100063255 (Logie Milanova, 2008), it was reported tein fractions were free from glucosinolates, and the twothat in addition to the 12 S and 2 S proteins, the procedures types of isolates produced were low in phytate, light inused for the isolation of canola proteins produce signiﬁcant color, and bland in taste. It was reported that the isolatequantities of a 7 S fraction, which appears to contain a new yield depended on the properties of the starting meal. Theprotein. Accordingly, one aspect of the application was the precipitation, ultraﬁltration and diaﬁltration processesisolation and puriﬁcation of the 7 S protein of canola. It was were conducted on hexane-extracted meal (with hulledalso found that the relative proportions of the 12 S, 7 S and and dehulled materials), CH3OH/NH3/H2O-hexane-ex-2 S proteins differ between a protein micellar mass-derived tracted meal and dehulled meal. According to the reportedcanola protein isolate and a supernatant-derived canola pro- information, the meal was extracted with an aqueous NaOHtein isolate, which were prepared using industrial proce- solution at solvent/meal ratio R ¼ 18. The hexane-extracteddures. These procedures involved a multiple step process canola meal and the commercial meal were extracted forof extracting canola oil seed meal using a salt solution, sep- 30 min, while the CH3OH/NH3/H2O-hexane-extractedarating the resulting aqueous protein solution from the re- meal was extracted for 2 h. The solutions were maintainedsidual oil seed meal, increasing the protein concentration either at a pH of 11.0 for hexane-extracted canola mealof the aqueous solution to at least 20% (w/v) while main- and commercial canola meal or at a pH of 12.0 for thetaining a constant ionic strength using a selective mem- CH3OH/NH3/H2O-hexane-extracted canola meal. In addi-brane technique, diluting the resulting concentrated tion, 1% (dry basis) Na2S03 was added to the commercialprotein solution into chilled water to induce the formation canola meal during the aqueous extraction step to preventof protein micelles, settling the protein micelles to form the oxidation of phenolic compounds and to produce pro-an amorphous, sticky, gelatinous gluten-like protein micel- tein products with a lighter color and better ﬂavor. Afterlar mass (PMM), and recovering the protein-rich protein centrifugation and ﬁltration of the liquid phase, the ex-micellar mass from the supernatant. In the US-patent appli- tracted meal was washed twice with pH 11.0 or 12.0 watercation number 20100063255 (Logie Milanova, 2008), at water/meal ratio R ¼ 6, and the washings were added tothe PMM canola protein isolate was characterized by a pro- the original extract. The extracted wet meal was recoveredtein content of 90% (w/w, dry basis) and consisted of about and freeze-dried to produce a residual meal. The pH of the60e98% of the 7 S protein, about 1e15% (w/w) of the 12 S canola protein extracts was adjusted to 3.5 by the additionprotein and 0e25% (w/w) of the 2 S protein. In contrast, of 6 N HCl. After separation, the protein precipitate wasthe supernatant-derived canola protein isolate was charac- washed with pH 3.5 or 4.0 water at a water-to-precipitateterized by a protein content of 90% (w/w) and consisted (wet) ratio of 10. The washed precipitate was then freeze-of 0e5% (w/w) of the 12 S protein, about 5e40% (w/w) dried to produce an isoelectric protein isolate. The proteinof the 7 S protein and about 60e95% (w/w) of the 2 S pro- solution was ﬁrst ultraﬁltered at a concentration factortein fraction. Thus, the protein component proﬁles of the (CF) of 10 and then diaﬁltrated at a diavolume (DV) oftwo canola protein isolates were very different. In the 5. A 10-kD ultraﬁltration membrane was used in both mem-PMM-derived canola protein isolate, the predominant pro- brane processing steps. Finally, the diaﬁltered retentate wastein species was the 7 S protein; however, in the freeze-dried to produce the soluble protein isolate.
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 25 Klockeman et al. (1997) reported the isolation and char- meal as reported in the literature (80e95% reported byacterization of defatted canola meal proteins. According to Ismond Welsh, 1992; Diosady, Tzeng, Rubin, 1984;their study, canola protein was extracted from defatted Tzeng, Diosady, Rubin, 1988a). Protein recovery valuescanola meal using 5% (w/v) extraction solution of 0.4% of 87.5% were obtained as compared to literature values of(w/v) NaOH at room temperature on an orbital shaker at 33e65% (Rohani Chen, 1993; Xu Diosady, 1994a,180À200 rpm for 60 min. The residual solids were dis- 1994b).carded following centrifugation at 3000 g for 20 min at Recently, the preparation of canola protein materials us-5e10 C. Glacial acetic acid was then added to the protein ing membrane technology and the evaluation of the func-extract to adjust the pH to 3.5 for protein precipitation. Af- tional properties of meals were reported (Ghodsvali et al.,terward, the precipitated canola protein was separated by 2005). According to the authors, suitable conditions forcentrifugation at 3000 g for 20 min at 5À10 C. The protein the extraction and precipitation of proteins from Iranian ca-precipitate was washed three times with distilled deionized nola (Brassica napus, cv. Quantum, PF, and Hyola) mealswater and centrifuged at 3000 g for 20 min at 5e10 C be- were determined using a membrane-based process, whichtween each wash. The ﬁnal protein isolate obtained was consisted of extraction of hexane-defatted canola meals atfreeze-dried. This extraction method is summarized in pH 9.5e12.0 and precipitation at pH 3.5 and 7.5 to recoverFig. 1. The reported method consisted of the extraction a precipitated protein isolate (PPI). An acid-soluble proteinand isolation of protein from crude commercial hexane-de- isolate (SPI) was then prepared by ultraﬁltration (UF) fol-fatted canola meal, and the authors stated that this method lowed by diaﬁltration (DF) and drying. The highest proteinhas a signiﬁcantly increased protein extraction capacity and yields were obtained by alkaline extraction at pH 12.0 fora higher protein recovery. In their study, a proximate anal- all meals investigated. The maximum yield of precipitatedysis revealed that the hexane-defatted canola meal con- protein was observed at pH values between 4.5 and 5.5 andtained 12.3% moisture and 32.1% protein, 8.2% ash, depended on the variety and dehulling treatment. Almost4.4% fat, and 55.4% carbohydrate on a dry weight basis. 90% of the proteins were recovered in three fractions: theThe majority of the protein was soluble when dispersed PPI, SPI (81e98% protein, N*6.25), and the meal residuein 0.4% NaOH or 5% NaCl. This solubility proﬁle indicates (35% protein). The glucosinolate contents of all mealsthat the isolated canola proteins are primarily glutelins and tested and the protein fractions were low, and some samplesglobulins. Protein extraction in all concentrations of NaOH were below the detection limit for glucosinolates. Both iso-was signiﬁcantly increased if bafﬂed ﬂasks were used; lates were low in phytic acid content. In the work of95.2e99.6% of the total protein in the meal was extracted Ghodsvali et al. (2005), the alkaline extraction was con-in 0.4% (w/v) NaOH. The maximum protein extraction was ducted by dispersing the canola meal in distilled water atobtained with a 5% (w/v) meal ratio and incubation in 0.4% room temperature for 30 min. The pH of the extraction so-(w/v) NaOH for 60 min at 180e200 rpm. This represents lution was adjusted to a predetermined level and main-an increase in protein extraction from defatted canola tained by the addition of aqueous 5% NaOH as required. The authors reported that an appropriate concentration of 1e6 N NaOH solution was selected to avoid excessive di- lution during pH adjustment. A pH range between 9.5 and 12.0 was examined in increments of 0.5 pH units. The slurry was centrifuged at 5000 rpm for 15 min, and the su- pernatant was ﬁltered. The meal residue was washed twice with an aqueous alkali solution (R ¼ 6) with the same pH as the extraction solution and oven-dried overnight. According to the authors, pH 12.0 was used in subsequent precipita- tion tests. For the isoelectric precipitation of canola pro- teins from the alkaline extract, the isoelectric points were determined. Meals were extracted with 5% NaOH at pH 4.5 and a solvent-to-meal ratio of 18. The alkali solution was acidiﬁed with HCl (6 M) to obtain pH values between 3.5 and 7.5 in increments of 0.5 pH units. After centrifuga- tion at 5000 rpm for 20 min, the supernatant was ﬁltered using ﬁlter paper, and the precipitate was washed with wa- ter acidiﬁed to the precipitation pH at a water-to-precipitate (wet) ratio of 10, centrifuged again and oven-dried over- night. Membrane separation was conducted in an ultraﬁltra- tion unit in the diaﬁltration mode. A built-in peristalticFig. 1. Schematic representation of canola meal protein isolate extrac- pump drew the solution from a sample container and tion method (from Klockeman et al., 1997). pumped it through the hollow ﬁber cartridge. The
26 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39membrane used had a nominal molecular weight cutoff of found in lower proportions in the permeate. The opposite10 kDa and a membrane area of 0.1 m2. The obtained per- trend was observed for basic peptides, whereas neutralmeate consisted of water and dissolved low-molecular peptides were found in the same proportion in the retentateweight components. The retentate was returned to the sam- and permeate. The authors explained this behavior basedple container. A soluble canola protein isolate was prepared on the Donnan theory and the existence of electrostatic in-with the same process described by Tzeng et al., (1988b), teractions (attractive and repulsive forces) at the membra-which consisted of four major operational stages: alkaline neesolution interface. Selectivity between basic and acidextraction and washing, isoelectric precipitation and wash- peptides was as high as 1.90 at pH 9 and low ionic strength.ing, ultraﬁltration and diaﬁltration. Hexane-extracted meals A membrane-based process was proposed for the fraction-were tested, and each meal was extracted at pH 12.0. The ation of rapeseed peptide mixtures (Tessier et al., 2006).extract was combined with all washes, and CaCl2 (15%,by weight of the starting meal) was added to the protein ex-tract. The pH was then reduced to the isoelectric point by Canola protein functional propertiesthe addition of 6 N HCl. The pH was maintained for Water absorption capacity (WAC)15 min to allow protein aggregation, and then the suspen- Khattab and Arntﬁeld (2009) studied the water absorp-sion was centrifuged. The supernatant was ﬁltered, and af- tion capacity of canola proteins and reported that the en-ter separation, the protein precipitate was washed with 10 hanced ability of canola meal to absorb and retain watertimes its weight of distilled water and centrifuged. The su- improved the water binding capacity of the food product,pernatant and all washes were combined and ultraﬁltered at enhanced its ﬂavor retention, improved its mouthfeel anda CF of 10, followed by diaﬁltration at a DV of 5. reduced the moisture of food products. In their study, the A more attractive approach for the valorization of canola water absorption capacities of raw and treated meals were(rapeseed) proteins consists of the production of different reported, and it was shown that the treatment of the mealpeptide fractions by enzymatic hydrolysis followed by had a signiﬁcant effect on the WAC. Canola meal wasmembrane ﬁltration (fractionation). In this context, a recent able to absorb 390% of its initial weight, and its WACstudy reported the selective separation of peptides was higher than soybean meal, which absorbed 303% ofcontained in a rapeseed (Brassica campestris L.) protein its initial weight, but lower than ﬂaxseed meal. This resulthydrolysate using ultraﬁltration/nanoﬁltration (UF/NF) agreed well with Wanasundara and Shahidi (1994), whomembranes (Tessier, Harscoat-Schiavo, Marc, 2006). In found that the WAC of ﬂaxseed meal was considerablythis study, the ability of a charged ultraﬁltration (UF) mem- higher than that of canola. These results agree with thosebrane to fractionate the small peptides found in a rapeseed reported for canola meal by Naczk, Diosady, and Rubinprotein enzymatic hydrolysate based on charge characteris- (1985) and Ghodsvali et al. (2005), who showed that thetics was investigated. Because the peptide mixture obtained water absorption capacities of canola meals vary with ca-after enzymatic hydrolysis was heterogeneous and difﬁcult nola cultivar (variety) and dehulling treatment. For canolato separate, the authors proposed an original approach that meals, values between 218% and 382% have been reported.required the development of technological alternatives for However, it is important to take in consideration that the ca-more efﬁcient separation of the numerous peptide species. nola meals used in the literature contained considerableIn this study, a preliminary step consisted of precipitation amounts of ﬁber, which can enhance the overall water hold-followed by ﬁltration with a 3-kDa molecular weight cutoff ing capacity, and studies on the water holding capacity of(MWCO) membrane to obtain a concentrated solution of canola protein isolates would be more relevant. This agreessmall peptides. The feasibility of fractionating these small with the information reported by Wanasundara and Shahidipeptides with a charged 1-kDa MWCO membrane was (1994), who stated that the higher water adsorption of sol-also investigated. According to this study, this approach al- vent-extracted oil seed meals may be due to the presence oflowed an estimation of the contribution of electrostatic in- hull polysaccharides. Sosulski, Humbert, Bui, and Jonesteractions during membrane fractionation. Moreover, the (1976) studied the water absorption properties of rapeseedeffect of solution pH and ionic strength on peptide trans- ﬂours and isolates and reported that the WHC of these in-mission was studied. The ionic strength contribution was gredients exceeded 200% and compared favorably withconsidered by studying its effect on the selectivity of a de- that of soybean ﬂour. Manamperi, Pryor and Changsalting step by nanoﬁltration on a 0.5-kDa MWCO nanoﬁl- (2007) separated and evaluated canola meal and proteintration membrane. It has been reported that peptide for industrial bioproducts and found that the water absorp-transmission was lower at pH 9 than at pH 4 and the lowest tion capacity of canola meal exceeded 250%; their resultat pH 9 and with low ionic strength. The ionic strength had agrees with values reported for different varieties of canolaa signiﬁcant effect at pH 9 but showed no effect at pH 4. meal, which ranged from 209% (Ghodsvali et al.., 2005) toThis difference could be attributed to the different ionic 382% (Naczk et al., 1985). Mahajan, Dua, and Bhardwajspecies that acted as counter-ions during membrane ﬁltra- (2002) reported a study in which dry and swollen canolation. The amino acid analysis and capillary electrophoresis seeds were defatted with hexane, and the freeze-dried pow-revealed that negatively charged (acidic) peptides were der was analyzed for the functional properties of the meals.
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 27The results showed that the water absorption capacity of the applications where a high water holding capacity is re-swollen rapeseed meal was higher than dry meal. quired, the situation is quite different because solubility is generally negatively correlated with water holding capacity.Oil/fat absorption As reported by Prinyawiwatkul, Beuchat, McWatters, The fat-adsorption capacity of any food compound is Phillips (1997), protein solubility can be considered asimportant for food applications because it relies mainly the most important property because it affects other proper-on its capacity to physically entrap oil by a complex capil- ties, such as emulsiﬁcation ability, foam-forming capacitylary-attraction process. In many food applications, such as and gel formation. Recently, Khattab and Arntﬁeld (2009)emulsion-type meat products, the ability of a food compo- studied the functional properties of raw and processed ca-nent to entrap oil is an important characteristic because fat nola meals and showed that canola meal had a protein sol-acts as a ﬂavor retainer, a consistency trait and an enhancer ubility (expressed as nitrogen solubility) of 66.42%. At theof mouthfeel (Khattab Arntﬁeld, 2009). The fat absorp- same time, they compared canola meal protein solubilitytion capacity of canola meal was studied and compared with soy and ﬂaxseed meals, which have protein solubilitieswith those of soy and ﬂaxseed meals (Khattab of 74.00% and 56.50%, respectively. The results obtainedArntﬁeld, 2009). It was reported that signiﬁcant differences in this study were comparable with those reported in the lit-in the oil absorption capacities were noted among the erature (John Baize, 1999; Madhusudhan Singh, 1985;above-listed meals and that soy meal had the highest value, Wanasundara Shahidi, 1994). As reported in Khattab andwhich was followed by canola and ﬂaxseed meal, respec- Arntﬁeld (2009), canola meal protein solubility was signif-tively. In general, the fat absorption capacity depends on icantly reduced after heat treatment, which consisted of dryseveral properties, such as powder particle size and surface roasting and boiling in water. The two treatments causedtension. In addition, the fat absorption capacity is nega- 29.07% and 25.61% solubility reductions, respectively.tively correlated with water absorption capacity. This state- The protein solubilities of soy and ﬂaxseed meals were re-ment agrees with the information reported by Naczk et al. duced by 33.69% and 46.27%, respectively, after dry roast-(1985) and Ghodsvali et al. (2005), who reported canola ing treatment and 43.00% and 39.40%, respectively, aftermeal oil absorption and water holding capacity values of boiling. Protein denaturation during heat treatment could188e203 and 265.5e281.5%, respectively. The oil absorp- explain the solubility reduction. Heat treatment could en-tion capacity can be modulated by different treatments, and hance the exposure of hydrophobic residues, which contrib-it was reported that heat treatments increased the oil ab- ute to the reduction in the overall protein solubility.sorption capacities of different oil seed meals, including ca- Electrostatic repulsion and ionic hydration that occur at dif-nola. Among heat treatments, boiling was reported to ferent pHs can also affect protein solubility (Moure,produce the greatest enhancement. The phenomenon of in- Sineiro, Dom ınguez, Paraj, 2006). Klockeman et al. ocreasing fat absorption after heat treatment has been asso- (1997) reported the extraction and isolation of proteinsciated with the heat dissociation of proteins and from canola oil processing waste. They showed that canoladenaturation, which is hypothesized to unmask non-polar proteins had poor solubility between pH 2 and 10 for allresidues in the interior of the protein molecules (Kinsella dispersion solutions used. The solubility of the protein iso- Melachouris, 1976, pp. 219e280). In a report by lates was 60% or less. The low solubility could be attrib-Mahajan et al. (2002), dry and 24-h swollen rapeseeds uted to the extraction conditions used. Radwan and Luwere defatted with hexane, and the freeze-dried powder (1976) studied the solubility of the proteins of the dehulledwas analyzed. It has been reported that the fat absorption and defatted ‘Tower’ variety of rapeseed (canola) in aque-capacity of swollen rapeseed meal is higher than that of ous solutions at 25, 35, 45, and 55 C and at pH 1e13. Ac-the dry meal. Gruener and Ismond (1997) reported the iso- cording to this study, the minimum solubilities occurred atlation of a canola protein concentrate with improved func- pH values of 4.5, 4.8, 7.0, and 7.2, respectively, for the fourtional properties; in their method, the canola 12 S globulin temperatures tested. These differences could be attributedwas isolated by the protein micellar mass procedure (PMM) to the different pHs that were used to precipitate the differ-and modiﬁed by acetylation and succinylation. It was ent protein fractions. Paulson and Tung (1989) studied theshown that the emulsion stability signiﬁcantly increased effects of succinylation (54% and 84% modiﬁcation of freeinitially and then decreased at the highest levels of modiﬁ- amino groups) in the pH interval of 3.5e11.0 and NaClcation. Following acylation, the fat absorption capacity was concentrations up to 0.7 M on the solubility of a canola pro-signiﬁcantly elevated. tein isolate. According to this study, succinylation mark- edly enhanced the protein solubility at alkaline andProtein/nitrogen solubility slightly acidic pH values, while the effect of NaCl concen- For food applications, protein (nitrogen) solubility is an tration depended on the pH value. This might be related toimportant parameter that inﬂuences the extent of applica- the effect of succinylation on surface hydrophobicitytions in different food matrices. In some cases, such as (Paulson Marvin, 1987), which decreases as the levelbeverages, high protein solubility is a determinant for of succinylation increases. This can be conﬁrmed by Zetaapplication as a fortiﬁcation ingredient. In other potential values, which became more electronegative as
28 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39both succinylation and pH increased but decreased with the stability, and textural properties of a protein-fat-water sys-addition of NaCl. Depending on the pH, addition of salt to tem. It is also important to ensure that the technique used toa protein suspension can affect the net charge density of the evaluate emulsifying properties is adequate. In the work ofproteins and thus modify the solubility by enhancing or re- Khattab and Arntﬁeld (2009), the emulsifying capacity ofducing inter-molecular repulsions. In Gruener and Ismond canola proteins (meal) was expressed as the maximum(1997), the canola 12 S globulin was isolated using the pro- amount of oil that the meal solution would emulsify with-tein micellar mass procedure (PMM) and modiﬁed by acet- out losing its emulsion characteristics. According to theylation and succinylation to obtain a canola protein reported results, the investigated canola meal showedconcentrate with improved functional properties. It has signiﬁcantly higher EC values compared with those ofbeen shown that protein solubility below the isoelectric soy and ﬂaxseed meals. However, they also reported thatpoint of the PMM was impaired, but the solubility at neu- roasting and boiling caused signiﬁcant reductions in thetral to alkaline pH values was greatly enhanced. Based on ECs of these meals. They explained that that high proteinthe information on canola protein solubility, it is possible solubility (Kinsella Melachouris, 1976, pp. 219e280)to highlight one parameter that contributes to the solubility and high fat-adsorption capacity were positively correlatedof puriﬁed isolates: the predominance of low molecular with the ability to form and stabilize emulsions. They alsoweight species (Fig. 2). stated that the lower EC values of boiled canola meal might be due to its low nitrogen solubility. Similarly, the degree ofEmulsifying properties heating was also reported to be a determinant for the reduc- In food applications, such as emulsion-type meat prod- tion of the emulsifying capacity of legume proteins in gen-ucts, salad dressings and mayonnaise, emulsifying proper- eral (Eke Akobundu, 1993). Dev and Mukherjee (1986)ties of canola proteins is an important attribute and have reported that rapeseed products generally have lowerlargely deﬁnes the extent of use of this ingredient to stabi- emulsifying capacities but higher emulsifying stabilitieslize food systems. Several studies have been conducted in than soy products, although the processing treatment canthe past two decades on the feasibility of using canola pro- alter this trend. Isolates tend to have improved emulsifyingteins in emulsion-type foods. Recently, Khattab and properties as compared to concentrates (McCurdy, 1990).Arntﬁeld (2009) studied the emulsiﬁcation ability of canola Recently, a fundamental study examined the emulsifyingmeal using two parameters: emulsifying capacity (EC) and properties of the two major canola proteins cruciferin andstability (ES). They showed that both properties are func- napin. It has been reported that the emulsion preparedtions of the protein concentration, pH, and ionic strength. with cruciferin showed a signiﬁcantly higher speciﬁc sur-They also related these properties to the viscosity of the face area and a lower particle size than that of napin. Thesystem, but this parameter (viscosity) was function of the study reported by Wu and Muir (2008) indicated that thementioned above conditions. Indeed, emulsion is a complex presence of napin could detrimentally affect the emulsionsystem that involves a multitude of chemical and physical stability of canola protein isolates (Wu Muir, 2008).phenomena, which play different roles in the formation, The emulsifying ability of canola proteins was studied in combination with hydrocolloids. A study investigated the properties of commercial canola protein isolatee hydrocolloid-stabilized emulsions under varied conditions, such as different canola protein isolate concentrations, amounts of salt and hydrocolloid added, pH values of the me- dium and the presence of denaturants (Uruakpa Arntﬁeld, 2005). In this study, the emulsifying activity index (EAI) and emulsion stability (ES) were determined by a turbidimet- ric method. According to the authors, the obtained results showed that under conditions that promote complex formation between the proteins and hydrocolloids, which included pH 6 and the addition of 1% (w/v) k-carrageenan, the EAI of CPI- stabilized emulsions increased from 162 to 201 m2/g and the ES increased from 68% to 95%. Under conditions that pro- mote incompatibility between canola proteins and the hydro- colloid (pH 10), the use of 1% (w/v) guar gum increased the EAI of CPI-stabilized emulsions from 68 to 177 m2/g and the ES from 66% to 100%. The lower EAI and ES values ob- served in the hydrocolloid-stabilized CPI emulsions treatedFig. 2. SDS-PAGE of canola protein (from Ebrahimi, Nikkhah, Sadeghi. with sodium salts and denaturants support the involvementRaisali, 2009). Lane 1 is the standards and Lane 2 is the canola/ of hydrophobic interactions, hydrogen bonds and disulﬁde rapessed proteins. linkages in the emulsiﬁcation of these systems. It was also
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 29shown that the interfacial properties of CPIehydrocolloid degree of hydrolysis (DH) increased. The authors con-complexes were improved by electrostatic complex cluded that hydrolysates with an increased degree of hydro-formation and thermodynamic incompatibility, making lysis are apparently capable of foaming but lack thethese systems suitable for stabilizing food emulsions, such strength to maintain the foam as a result of the reductionas salad dressings and mayonnaise (Uruakpa Arntﬁeld, in protein molecular weight. The foam stability of the pro-2005). tein isolates dropped to 0% after 15 min of hydrolysis (Vioque, Snchez-Vioque, Clemente, Pedroche, Milln, a aFoaming properties 2000). In many food applications, because the surface area inthe liquid/air interface increases, proteins denature and ag- Gelling abilitygregate during whipping. Air entrapment plays a major role In general, all proteins can form a gel, but differencesin different food matrices and is important for ﬂours used in exist in the gel strengths. The ability of proteins to formmany leavening food products, such as breads, cakes and gels can be measured by the determination of the least ge-cookies (Sreerama, Sasikala, Pratape, 2008). The foam- lation concentration, which is deﬁned as the minimal pro-ing capacity of canola meal was studied and compared tein concentration needed to produce a gel that does notwith soy and ﬂaxseed (Khattab Arntﬁeld, 2009). They slide down the walls of an inverted tube (Moure et al.,showed that the foam capacities and stabilities of raw and 2006). Khattab and Arntﬁeld (2009) studied the least gela-treated canola meal were higher than those of soy and ﬂax- tion concentrations of raw and heat-treated canola mealsseed meals. They expressed the foaming capacity as a per- and reported that neither roasting nor boiling caused a sig-centage and reported values of 56.44%, 44.56% and niﬁcant increase in the least gelation concentration of dif-17.82%, for canola, soy and ﬂaxseed meal, respectively. ferent canola meals. This is supported by reports in theIt was shown that heat treatment signiﬁcantly reduced scientiﬁc literature, which show that the gelation of proteinsboth the foaming capacity and foam stability of the differ- increases with molecular weight (size) because large mole-ent meals, including canola meal. The authors explained cules form extensive networks by cross-linking in threethat this reduction was mainly related to protein denatur- dimensions (Oakenfull, Pearce, Burley, 1997). Comparedation; this conclusion agrees with the data reported by with soy and ﬂaxseed meals, canola meal required the high-Lin, Humbert, Sosulski, 1974, who stated that a native est concentration for gelation regardless of the treatmentprotein gives a higher foam stability than a denatured used.one. Gruener and Ismond (1997) conducted a study in To improve the function of canola proteins in gel-likewhich the canola 12 s globulin was isolated by the protein food systems, different additives can be combined with thesePMM and modiﬁed by acetylation and succinylation to im- proteins to make convenient gels. In this context, a study hasprove the functional properties of the canola protein con- been reported in which the thermogelation properties of a ca-centrate. The PMM foaming capacity was signiﬁcantly nola protein isolate in a mixed system with k-carrageenan (k-increased by acylation, and the foam stability decreased CAR) were studied using dynamic rheological testing. Thesigniﬁcantly after acylation. They concluded that in general gel properties were evaluated under different conditions,the acylated concentrates possessed improved functionality such as pH, NaCl, and k-carrageenan and canola protein iso-as compared to the PMM, which makes them more suitable late concentrations. The factorial and response surface opti-as a food ingredient. Xu and Diosady (2002) studied the mization models were used to identify the processingfunctional properties of Chinese rapeseed meals and re- conditions that would result in CPIek-CAR gels with max-ported that Chinese rapeseed meals foams were more stable imized G0 values (!44,000 Pa) and minimized tan d valuesthan those of the canola meals prepared by Naczk et al. (0.01e0.11). According to the results, it was found that(1985). Moreover, they showed that Chinese rapeseed pro- canola proteinecarrageenan gel formation was stronglytein isolates were characterized by excellent whippability. pH-, salt (NaCl)- and k-carrageenan concentration-depen-They found that the foaming properties of the soybean iso- dent (Uruakpa Arntﬁeld, 2004). The reported results indi-late were between those of the rapeseed meals and rapeseed cated that the optimum conditions for CPIek-CAR gelsprotein isolates. The difference between a meal and an iso- were pH 6, 0.05 M NaCl, 3% k-CAR and 15% CPI. Sampleslate is the protein content, which is obviously higher in an prepared at pH 6 showed high G0 (95,465 Pa) and lowisolate. All foams were stable and lasted for more than 2 h. tan d (0.15) values. High G0 values indicate stronger inter-The foaming properties of canola protein were evaluated as molecular networks and increased proteineprotein and pro-a function of degree of protein hydrolysis. Limited canola teinepolysaccharide interactions, while low tan d valuesprotein hydrolysates ranging from 3.1% to 7.7% hydrolysis indicate a more elastic network. A synergistic behavior be-were produced from an isoelectrically precipitated protein tween CPI and k-CAR was observed with superior networkisolate. It has been shown that all canola protein hydroly- strength (high G0 values) for the mixed gels at temperaturessates have lower foam stabilities than those reported in above 80 C. Furthermore, the gels showed improved net-the literature for other rapeseed protein products work structure (low tan d values) during the heating and(Frokjaer, 1994), and the foam stability decreased as the cooling phases. The canola proteinek-carrageenan mixtures
30 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39exhibited very strong and elastic networks, indicating that 1998). Paulson and Tung (1989) studied the thermally in-CPI can serve as a structuring agent in mixed food systems duced gelation of a rapeseed (canola) protein isolate heated(Uruakpa Arntﬁeld, 2004). to 72 C. Gels were formed only at high pH (9.5). Lger e Canola proteins have been considered as potential ingre- and Arntﬁeld (1993) studied the gelation of 12 S canola glob-dients for food applications where a gel-like structure is de- ulin. Gels prepared with 6% protein under alkaline condi-sired. In a recent study, enzymatic modiﬁcation with tions were superior to gels prepared from acidic solutions.transglutaminase (TG) was used to enhance the gelation The effects of pH, salts, and denaturing and reducing agentsof a canola protein isolate and thus improve its potential on the gelation properties led the authors to conclude thatas a food ingredient. Different parameters, such as the ef- hydrophobic forces and electrostatic interactions were re-fects of canola protein isolate concentration, transglutami- sponsible for establishing the gel network, while gel stabili-nase (TG) concentration, and treatment temperature and zation and strengthening were attributed to disulﬁde bonds,time, have been studied to determine their effects on canola electrostatic interactions, and hydrogen bonding. Succinyla-protein isolate gelation properties. Different techniques, tion was used to improve the gel-forming properties of a can-such as texture analysis, sodium dodecyl sulfate-polyacryl- ola protein isolate by Paulson and Tung (1989). In this study,amide gel electrophoresis (SDS-PAGE) and scanning elec- the pH range of gel formation was extended from the alkalinetron microscopy, were used to characterize the resulting region (pH 9.5) to slightly acidic pHs (5.0). Although sev-canola protein isolate networks. It has been reported that eral studies have analyzed the gelling ability of canolathe protein concentration, amount of transglutaminase proteins, it is well recognized that comparison of the gel-(TG), and treatment temperature signiﬁcant affected gel forming properties of the different canola protein prepara-strength. According to the authors (Pinterits Arntﬁeld, tions is difﬁcult. This is principally because there are major2008), gelation was improved by increasing the amounts differences the compositions and purities of the proteins.of protein and TG while keeping the treatment temperature Therefore, studies in which pure fractions and knownclose to 40 C. SDS-PAGE showed that subunit cross-link- combinations must be conducted bettering increase theing occurred during TG treatment, which thus explained the understanding of the canola protein behavior in relation toincrease in gel strength observed during texture analysis. gel formation. In this context, the study reported byThe above-mentioned effects were also conﬁrmed by Gruener and Ismond (1997), which the canola 12 S globulinmicroscopy (Pinterits Arntﬁeld, 2008). was isolated by the protein PMM and modiﬁed by acetylation Schwenke, Dahme, and Wolter (1998) reported the gel- and succinylation, showed that the gelation properties of ca-forming abilities of a rapeseed (canola) protein isolate, which nola proteins were generally improved by acylation. Further-was composed of 70% globulin (cruciferin) and 30% albu- more, the acylated concentrates were signiﬁcantly lighter inmin (napin), and their individual protein components. In color than the original PMM.this study, the inﬂuence of acetylation upon the gelationproperties was also studied. The highest gel strength (mea- Bioactive compounds from canola proteinssured as shear modulus) of the isolate was obtained at pH The increase in consumer awareness about healthy foodsvalues around 9, which is between the isoelectric points of has encouraged researchers to identify bioactive naturalthe major proteins. Moreover, puriﬁed cruciferin gave the components in different products (Murty, Pittaway, highest shear modulus values, with maxima at pH 6 and 8. Ball, in press). Canola proteins are considered to be attrac-Weak and poorly stable gels that exhibited strong hysteresis tive and promising sources of bioactive compounds.were obtained with isolated napin. The authors also reported Recently, a number of research works have focused onthat acetylation resulted in a pH shift of the shear modulus the investigation of different methods to produce activemaximum of the protein isolate to about 6. The gelation tem- peptides from the enzymatic hydrolysis of canola proteins.perature of the acetylated isolate was more dependent on pH Angiotensin-I Converting Enzyme (ACE) inhibitory activ-and concentration compared with the other proteins ity, antioxidant activity, bile acid-binding capacity, anti-(Schwenke et al., 1998). Because of the increasing demand thrombotic activity and cell growth effects have beenfor plant proteins in gel-like products, the ability to form identiﬁed as bioactive characteristics of canola/rapeseedgels, which is a key functional property of plant proteins, proteins (Table 2).has been extensively studied in the past two decades. Gilland Tung (1978) ﬁrst demonstrated the ability of a highly ACE inhibitory activityglycosylated 12 S rapeseed (canola) protein to form gels dur- Blood pressure is controlled by a regulatory hormonaling heating at pH 4. The strongest gels were formed at high mechanism known as the “renin angiotensin system.” ThepH and ionic strength conditions. The high carbohydrate regulatory mechanism takes place in the kidneys, wherecontents of the canola meal (12.9%) led the authors to pro- the hydrolytic enzyme renin is secreted. Hence, plasma an-pose that proteinecarbohydrate interactions occurred during giotensinogen is hydrolyzed to a decapeptide called Angio-gel formation. It was also reported that the viscosity of a hex- tensin I, which is subsequently hydrolyzed by Angiotensinametaphosphate-extracted rapeseed protein isolate heated to Converting Enzyme (ACE) to form Angiotensin II, which is80 C increased, but it did not form a gel (Schwenke et al., a vasoconstricting octapeptide that elevates the blood
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 31 Table 2. Bioactivity of rapeseed proteins and hydrolysates Bioactivity Rapeseed proteins/hydrolysates References ACE inhibitory activity Subtisilin hydrolysates of the protein isolate (puriﬁcation of Yamada et al., 2010. rapakinin Arg-Ile-Tyr) Hydrolysates of the defatted meal obtained by enzymatic treatment Wu et al., 2009. with: Umamizyme; Proteases A, P, R, M and S Amano; Proleather FG-F; Alcalase 2.4L; Enzeco alkaline protease L-FG; Enzeco neutral protease NBP-L; Pepsin, Trypsin and Chymotrypsin-TLCK Alcalase 2.4L hydrolysate of the protein isolate Megias et al., 2006,Wu Muir, 2008. Alcalase 2.4L hydrolysate of the defatted meal (puriﬁcation of Val- Wu et al., 2008 Ser-Val and PheeLeu located in napin and cruciferin, respectively) Alcalase 2.4L hydrolysates of the protein isolate, napin and Wu Muir, 2008. cruciferin Protein isolate Yoshie-Stark et al., 2008. Pepsin and Pepsin/Pancreatin hydrolysates of the protein isolate Antioxidant properties Alcalase/Flavourzyme hydrolysate of the albumin isolate Xue et al., 2009. Alcalase 2.4L hydrolysate of the ﬂour (puriﬁcation of Pro-Ala-Gly- Zhang et al., 2008; Zhang et al., 2009. Pro-Phe corresponding to the sequence 38e42 of napin). Protein isolate Yoshie-Stark et al., 2008. Pepsin and Pepsin/Pancreatin hydrolysates of the protein isolate Bile acid-binding capacity Protein isolate Yoshie-Stark et al., 2008. Pepsin and pepsin/pancreatin hydrolysates of the protein isolate Anti-thrombotic activity Alcalase 2.4L hydrolysate of the rapeseed slurry from a wet-milling Zhang et al., 2008 Effect on cell growth Alcalase, Esperase, Neutrase, Orientase and Pronase hydrolysates Chabanon et al., 2008; of the protein isolate Farges-Haddani et al., 2006; Farges et al., 2008.pressure (Chen et al., 2009). Furthermore, ACE also con- 18.1 to 82.5 mg protein/mL. The differences in the ACE-in-tributes to vasoconstriction by the degradation of bradyki- hibitory activities of the various protein hydrolysates re-nin, a vasodilator. Consequently, the inhibition of ACE ﬂected the different enzyme speciﬁcities. In this study,could be an alternative method to lower blood pressure ion-exchange chromatography was used to purify speciﬁc(Chen et al., 2009). fractions of canola protein hydrolysate, and this approach Defatted canola meals from seeds that were processed yielded an increase in the protein content to more thanwith different methods were hydrolyzed by Alcalase to pro- 95% without loss of ACE-inhibitory activity. Accordingduce hydrolysates that inhibited ACE activity (Megias to this study, this fraction was resistant to degradation byet al., 2006; Wu, Aluko, Muir, 2009). Heat-treated meals gastrointestinal enzymes and ACE during in vitro incuba-yielded protein hydrolysates with 50% ACE-inhibitory con- tion (Wu, Aluko, Muir, 2008). Speciﬁc canola proteincentrations of 27.1 and 28.6 mg protein/mL compared with fractions were also used to produce hydrolysates. Cruci-35.7 and 44.3 mg protein/mL for the non-heat treated meals. ferin and napin hydrolysis yielded peptide fractions thatIn this study, separation of the hydrolysate on a Sephadex showed potent angiotensin I-converting enzyme inhibitoryG-15 gel permeation column (GPC) yielded a fraction activity in vitro (IC50 0.035 and 0.029 mg/mL, respec-with an IC50 value of 2.3 mg protein/mL. From a fundamen- tively), but these activities were weaker than that of the ca-tal point of view of the mechanism of action, amino acid nola protein isolate hydrolysate (IC50 0.015 mg/mL) (Wu analysis showed that the GPC fraction contained 45% aro- Muir, 2008). This behavior can be attributed to the syner-matic amino acids in comparison to 8.5% in the raw hydro- getic effect of different fractions of raw canola proteinlysate. In particular, two peptides with the primary hydrolysate.compositions Val-Ser-Val and PheeLeu were puriﬁed and On the other hand, the in vivo anti-hypertensive proper-located in the primary structures of the napin and cruciferin ties of rapeseed proteins have been also reported bynative proteins. It has thus been suggested that the canola Yamada et al. (2010). Thus, an ACE-inhibitory peptideprotein hydrolysate should be considered as a potential in- called rapakinin (Arg-Ile-Tyr), which had an IC50 ofgredient for the formulation of hypotensive functional 28 mM, was isolated from the subtisilin-digested rapeseedfoods (Wu et al., 2009). To simplify the method of ACE-in- proteins. Rapakinin induced vasorelaxation with an EC50hibitory peptide production, defatted canola meal was sub- of 5.1 mM in the mesenteric artery of spontaneously hyper-jected to enzymatic proteolysis with different enzymes, and tensive rats (SHRs). Hence, the mechanism of vasorelaxa-it was found that Alcalase 2.4 L and protease M “Amano” tion was elucidated, and it has been suggested that thewere the most effective enzymes for the production of anti-hypertensive activity of rapakinin might be mediatedACE-inhibitory peptides from canola proteins. The IC50 by the PGI(2)-IP receptor, followed by CCKeCCK(1)values of the canola protein hydrolysates ranged from receptor-dependent vasorelaxation.
32 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39Antioxidant properties W€sche (2008) have shown that rapeseed protein isolates, a The production of free radicals leads to many health dis- which were obtained either by iso-precipitation or ultraﬁltra-orders due to the damage they cause to biological macro- tion, and their hydrolysates, which were obtained by pepsinmolecules, especially DNA. Therefore, many natural and pepsin/pancreatin digestions, were able to bind to bileantioxidants have been used to prevent peroxidation pro- salts. In that study, the concentration of the bile salts usedcesses (Kim Wijesekara, 2010). Xue et al. (2009) inves- was of 1.5 mM, which corresponds to the physiological con-tigated the possible conversion of insoluble rapeseed meal centrations of bile acids in the human body (1.5e7 mM). Theprotein into functionally active ingredients for food appli- results showed that 5.77%e12.6% of sodium cholate and so-cations. The rapeseed meal protein isolates were digested dium deoxycholate were bound by the rapeseed precipitatedwith Alcalase and Flavourzyme, and the resultant rapeseed protein isolate and hydrolysates, whereas ultraﬁltered rape-crude hydrolysate (RSCH) exhibited dose-dependent reduc- seed protein isolate and hydrolysates bound 5.81% toing antioxidant activities and hydroxyl radical scavenging 22.8% of the bile salts (Yoshie-Stark et al., 2008). Neverthe-abilities. The RSCH also inhibited malonyldialdehyde less, neither the sodium cholate- nor the sodium deoxycho-(MDA) generation by 50% in blood serum at 150 mg/ late-binding capacities were signiﬁcantly affected bymL. The RSCH was further separated into three fractions hydrolysis with pepsin and pepsin/pancreatin, suggesting(RSP1, RSP2, and RSP3) by Sephadex gel ﬁltration accord- that some large molecular weight protein fractions and undi-ing to molecular weight. The amino acid compositions and gested polypeptides are also able to bind the ligands. A sim-antioxidant potentials of the fractions were assessed, and it ilar lack of a direct association between hydrolysis and bilehas been reported that all three fractions showed inhibition acid-binding capacity has been observed in other plantof superoxide anion generation to various extents. They proteins and hydrolysates (Yoshie-Stark W€sche, 2004; aalso inhibited the autohemolysis of rat red blood cells and Ma Xiong, 2009).MDA formation in a rat liver tissue homogenate (Xueet al., 2009). In another study, the antioxidative capacity Anti-thrombotic activitywas observed for rapeseed peptides obtained by Alcalase Thrombosis is an anomaly in blood coagulation that ishydrolysis (Zhang, Wang, Xu, 2008). The median effec- generally caused by blood hyperviscosity, platelet hyper-re-tive dose (ED50) values of the three peptide fractions for activity, a high level of hemostatic proteins, such as ﬁbrin-a,a-diphenyl-b-picrylhydrazyl (DPPH) radical scavenging ogen, and defective ﬁbrinolysis. Thus, antithromboticsactivity were between 41 and 499 mg/mL; for the inhibition reduce the risk of thrombosis mainly by the reduction ofof lipid peroxidation in a liposome model system, the ED50 platelet aggregation and the enhancement of ﬁbrinolysisvalues were between 4.06 and 4.69 mg/mL, which are com- (Erdmann, Cheung, Schr€der, 2008). oparable to that of ascorbic acid at 5 mg/mL (Zhang et al., The anti-thrombotic activity of crude rapeseed peptide2008; Zhang, Wang, Xu, Gao, 2009). Moreover, the fractions prepared from an aqueous extraction of rapeseedmost antioxidative peptide was identiﬁed by LC-MS/MS proteins digested with Alcalase 2.4 L has been observedas Pro-Ala-Gly-Pro-Phe, which corresponds to amino acid (Zhang et al., 2008). The anti-thrombotic peptide fractionsresidues 38e42 of napin (Zhang et al., 2009). were analyzed in terms of their amino acid content, and the The results suggest that rapeseed protein hydrolysates results showed that they were rich in His, Pro, Trp, Tyr,could be useful as a human food additive and a source of Met, Cys, and Phe. Furthermore, the rapeseed bioactivebioactive peptides with antioxidant properties (Xue et al., peptide fractions had signiﬁcant inhibitory activities on2009). the thrombin-catalyzed coagulation of ﬁbrinogen, which is considered to be a key step in the formation of ﬁbrin clotsBile acid-binding capacity and therefore thrombosis. Although the inhibitory effect Hypercholesterolemia is characterized by the accumula- was not dose dependent, 90% inhibition was observedtion of LDL (low-density lipoprotein) cholesterol in the with peptide concentrations between 30 and 50 mg/mL.blood vessels and is considered as a major cause of heart However, the anti-thrombotic effects observed were weakerdiseases and atherosclerosis (World Health Organization, than that of heparin, which possessed a dose-dependent ef-2009). However, hypercholesterolemia can be prevented fect and an ED50 value of 0.07 mg/mL (Zhang et al., 2008).by exercise, a healthy diet and the consumption of bileacid sequestrants, which are better known as hypolipidemic Effects on cell growthagents (Anderson Siesel, 1990). Plasma cholesterol Rapeseed protein hydrolysates, which were obtained af-serves as a substrate for the biosynthesis of the bile acids ter digestion with Alcalase, Esperase, Neutrase, Orientasein the liver. Hence, the sequestration of the bile acids leads and Pronase and characterized by degrees of hydrolysis be-to degradation of cholesterol, which therefore reduces the tween 24.7% and 36.3% and contained low molecular sizelevel of cholesterol in the blood (Anderson Siesel, 1990). peptides (under 1 kDa), have been shown to increase the The in vitro hypocholesterolemic properties of rapeseed maximal cell density of Chinese Hamster Ovary (CHO)proteins have been evaluated through the determination of C5 cells (Chabanon et al., 2008). Cell growth in the pres-the bile acid-binding capacity. Yoshie-Stark, Wada, and ence of hydrolysates reached 120e150% of the reference
M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39 33cells (grown in media without supplementation with the bonding. Ionic bonding is very strong, and thus the separa-rapeseed hydrolysates) in serum-free medium, which con- tion of protein/phenolic complexes is extremely difﬁcult. Insisted of a simple basal medium supplemented with trans- this way, some studies have attempted to understand theferrin, insulin, albumin and trace elements. In addition to types of interactions between canola proteins and phenolicthe nutritional effect of the rapeseed protein hydrolysates, compounds and identify ways to effectively and economi-which are rich in free amino acids, the presence of peptides cally remove these coloring materials. Xu and Diosadythat affect growth or survival, including anti-apoptotic ac- (2000) developed a technique for the quantitative character-tivity, have been also suggested to explain the positive ization of canola proteinephenolic bonding in aqueous so-growth effects of the rapeseed protein hydrolysates lutions. The proposed approach combined chemical(Chabanon et al., 2008; Farges, Chenu, Marc, Goergen, treatments, which disrupted speciﬁc types of proteinephe-2008). nolic bonds, with membrane separation to remove the re- A fraction of Alcalase-hydrolyzed rapeseed proteins that leased phenolic compounds. It has been reported that upwas puriﬁed by ultraﬁltration (3 and 1 kDa) and nanoﬁltra- to 50% of the extracted phenolic compounds formed com-tion (500 Da), which produced a mixture of both large plexes with canola proteins by different mechanisms of in-(500e5000 Da) and small peptides (500 Da), signiﬁ- teraction, among which ionic bonding accounted for aboutcantly stimulated the growth rates of CHO and other animal 30%. It is interesting that this kind of interaction is consid-cells, including NS0, CHO K1 and Vero cells, and a maxi- ered to be the strongest among the interaction types. Xu andmal cell density of 1.7Â was obtained by addition of 4 g/l Diosady (2000) concluded that the amount of phenolicof hydrolysate. Moreover, the puriﬁed fraction reduced the compounds bound by canola proteins through hydrophobicdeath rate of CHO cells, increased the secretion of g-inter- interactions, hydrogen bonding, and covalent bonding didferon and accelerated cell adaptation to serum-free condi- not exceed 10% of the total extractable phenolic com-tions (Farges-Haddani et al., 2006; Farges et al., 2008). pounds. A combination of chemical treatments and mem- brane processes is one of the most promising ways toColor of canola protein solutions remove the phenolic compounds (Tzeng, Diosady, Like many plant materials, canola contains a consider- Rubin, 1990).able quantity of phenolic compounds. Most phenolic com- According to the United States Patent 7678392 (Green,pounds identiﬁed in canola are phenolic acids and Milanova, Segall, Xu, 2003), a process for the prepara-condensed tannins, which are ﬂavonoid-based polymeric tion of a canola protein isolate with improved color fromphenolic compounds. The major phenolic component in ca- canola meal was proposed. The process comprises the ex-nola was reported to be sinapine, the choline ester of si- traction of the canola meal to solubilize canola proteinsnapic acid. The overall concentration of these compounds in an aqueous solution at pH 5 to 6.8, the separation ofis not negligible; it is estimated to be about 1% (w/w) of the canola aqueous protein solution from the residual oilthe meal. Condensed tannins may cause astringency due seed meal, the concentration of the obtained canola proteinto their ability to precipitate proteins in the mouth. After while maintaining a constant ionic strength of the aqueousoxidation, phenolic compounds induce the development canola protein solution by ultraﬁltration, diaﬁltration of theof dark colors in canola proteins. Phenolic compounds concentrated canola protein solution until no signiﬁcantare highly reactive molecules, and under alkaline condi- further quantities of phenols and color were present in thetions, they can undergo enzymatic as well as non-enzymatic permeate, dilution of the diaﬁltered protein solution intooxidation to form quinones, which can react with proteins water chilled to below 15 C to form discrete canola proteinand produce dark green or brown colors in canola protein micelles, the formation of an amorphous, sticky, gelatinous,solutions (Leung, Fenton, Mueller, Clandinin, 1979; gluten-like canola protein micellar mass, and ﬁnally the re-Xu Diosady, 2000). In most processes used to produce covery of the canola protein micellar mass (protein isolate)canola proteins, the color of the isoelectrically precipitated with a protein content of at least 90% (w/w).canola proteins cannot be washed or enhanced without sig-niﬁcant protein losses and increasing the overall process Undesirable compoundscost. One of the limiting factors for the use of these proteins Phenolics are generally considered to be responsible forin food applications is related to the presence of undesirable the dark color, undesirable ﬂavour and lower nutritionalcolors; thus, to produce canola protein concentrates and value of rapeseed products. Rapeseed/canola meal containsisolates that can be used in food formulations, these color- free phenolic acids which constitute up to 24% of the totaling compounds must be effectively and economically re- phenolic acids present in rapeseed/canola meal and ﬂours.moved. The interactions between phenolic compounds These free phenolic acids represent approximately 15% ofand canola proteins are complex (Rubino, Arntﬁeld, the total phenolics present in rapeseed/canola mealsNadon, Bernatsky, 1996). It has been established that (Krygier, Sosulski, Hogge, 1982). Rapeseed/canola pro-these compounds in canola proteins interact through a vari- tein products contain different phenolics acids such as si-ety of mechanisms in aqueous media, including hydrogen napic, p-hydroxybenzoic, vanillic, gentisic, protocatechuic,bonding, covalent and hydrophobic interactions, and ionic syringic,p-coumaric, cis- and tran-ferulic, caffeic and
34 M. Aider, C. Barbana / Trends in Food Science Technology 22 (2011) 21e39chlorogenic acids in the free form (Naczk, Amarowicz, Erucic acid in the oil may cause heart lesion in certain ex-Shahidi, 1998). These phenolic acids are derivatives of ben- perimental animals. However, even if the new varieties arezoic and cinnamic acids. It was found that sinapic acid is the signiﬁcantly improved, they still contain too high levels ofpredominant phenolic acid in rapeseed/canola cultivars. glucosinolates. Different approaches such as chemicalPhenolic acids are present in canola protein products in the modiﬁcations, microbial and physical treatments and theirfree, esteriﬁed and bound forms. One of the limiting factors combinations have been used to reduce the content of glu-of the use of canola meal residue and derivatives (protein cosinolates in meals or seeds to negligible levels. Recently,concentrates and isolates) is that the content of phenolic the use of membrane ﬁltration seems to be promising to re-acids in these meals is up to ﬁve times higher than in soybean duce the glucosinolates content in canola protein isolates.meals and the content of phenolic acids in rapeseed/canola Another limiting factor is due to the fact that rapeseed/ca-ﬂours is 10e30 times higher than in ﬂours from other oleag- nola proteins contain up to 4% phytates (Naczk, Diosady, inous seeds such as ﬂaxseed. It has been reported that free Rubin, 1986). Phytates are responsible for the decrease inand esteriﬁed phenolic acids are the principal contributors the bioavailability of divalent cations such as Ca, Mg, Zn,to the undesirable taste of rapeseed/canola products. All Cu and Fe. This is a result complex formation (chelating ef-the followings phenolics are found in rapessed/canola meals fect). Phytates are also known to inhibit the digestion ofand derivatives: protocatechuic, vanillic, syringic, gallic, starch. Because of this, a number of methods have been de-p-hydroxybenzoic, p-coumaric, caffeic,ferulic, sinapic veloped to remove phytic acid from rapeseed productsacids. Of these phenolics, sinapic acids constitute 70e85% (Naczk et al., 1998). On the other hand, some recently pub-of the total phenolic acids present (Naczk et al., 1998). On lished studies indicate that phytates, at low concentrations,the other hand, the ﬂavour of phenolic acids was described may possess antioxidative and anticarcinogenic effectsas sour, astringent, bitter and phenol-like. This taste is also (Rickard Thompson, 1997). Rapeseed meal contains upfound in canola meals and proteins. Condensed tannins are to 20e30% indigestible ﬁbres on a dry basis. The rapeseedalso found in rapessed/canola meal residue and proteins ex- ﬁbres are low-molecular-weight carbohydrates, polysaccha-tracted from it. They are dimers, oligomers and polymers of rides, pectins, cellulose and lignin. Generally, they are com-ﬂavan-3-ols. The consecutive units of condensed tannins are plexed to proteins, polyphenols, glucosinolates andlinked through inter-ﬂavanoid bonds between C-4 and C-8 or minerals. High levels of ﬁbres limit the use of meal asC-6 atoms. Condensed tannins upon acidic hydrolysis pro- food ingredient. Dehulling of rapeseed/canola has been pro-duce anthocyanidins and therefore are also known as proan- posed but it is still not efﬁcient and therefore dehulling isthocyanidins. Rapeseed/canola meals may contain up to 3% not a standard practice in canola processing (Naczk et al.,tannins (Clandinin Heard, 1968; Shahidi Naczk, 1989). 1998).Proteins are macromolecules and may interact with ﬂavour-ing compounds such as phenolics. The character of these in- Food applicationsteractions inﬂuences the ﬂavour release and its perception. Canola proteins are characterized by interesting func-Phenolic compounds may form soluble and insoluble com- tional properties that allow them to be used as ingredientsplexes with proteins. The phenol-protein complexes may in different food formulations. Native and partially hydro-be stabilized by covalent bonds, ionic bonds, hydrogen bond- lyzed canola proteins have been extensively studied for po-ing and/or hydrophobic interactions (Shahidi Naczk, tentials uses in the food industry to replace classical1995). Studies on the complexations of polyphenols with ingredients, such as milk whey and egg yolk. This approachproteins mainly concentrated on the evaluation of factors is mainly justiﬁed by economic considerations and possibleinﬂuencing these interactions and on the impact of formation whey/egg allergies in customers. In this context, an alkalineof phenol-protein complexes on nutritive value of proteins. extract of canola meal was hydrolyzed using a protease toThe phenol-protein interactions are affected both by the obtain protein hydrolysates with 7% and 14% degrees ofsize, conformation and charge density (zeta-potential) of hydrolysis (DH), respectively. The protein hydrolysatesthe proteins and by the size, and ﬂexibility of phenol mole- were used to replace up to 50% (w/w) of the egg yolk incule (Hagerman Butler, 1981; Naczk et al., 1998). a model mayonnaise preparation, and the effects on the Even if the proximate composition, nutritive value and physicochemical properties of the end product were deter-functional properties of rapeseed/canola meal and deriva- mined. It was found that unhydrolyzed canola proteinstives (protein concentrates and isolates) are comparable to could only be substituted in mayonnaise for a maximumsoybean products, the use of rapeseed/canola protein prod- 15% (w/w) of the egg yolk without emulsion breakdown.ucts as food ingredient is limited by the presence of unde- According to the authors, at 7% DH, the canola proteinsirable components such as glucosinolates, phytates, and could be used to substitute up to 20% (w/w) of the eggﬁbres. The composition of rapeseed has been signiﬁcantly yolk, while at 14% DH, the maximum level of substitutionimproved by developing low glucosinolate and low erucic was up to 50% (w/w). However, some problems, includingacid rapeseed/canola cultivars. Glycosinolates upon hydro- the dark color of the canola protein solution, still need to belysis produce nitriles, hydroxynitrites, isothiocyanates and solved before the use of canola protein can be extended tothiocyanates which are responsible for goitrogenic effects. food formulations. Aluko and McIntosh (2005) reported