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Massoura, E., J.M. Vereijken, P. Kolster, and J.T.P. Derksen. 1996. Isolation and functional properties of proteins from Crambe abyssinica oil seeds. p. 322-327. In: J. Janick (ed.), Progress in new crops. ASHS Press, Alexandria, VA.

Isolation and Functional Properties of Proteins from Crambe abyssinica Oil Seeds*

E. Massoura, J.M. Vereijken, P. Kolster, and J.T.P. Derksen

    1. Isolation of Proteins
    2. Functional Properties
  5. Table 1
  6. Table 2
  7. Fig. 1
  8. Fig. 2
  9. Fig. 3
  10. Fig. 4
  11. Fig. 5

In recent years a number of new oilseed crops have been considered for introduction in agriculture as a result of the need to broaden the narrow crop rotation schedule and to contribute to a reduction in agricultural surpluses of traditional arable crops. Crambe abyssinica is considered to be a promising new oil crop because of both its agronomic performance (Erickson and Bassin 1990; Lazzeri et al. 1993) and the specific properties of its oil. Crambe oil is one of the richest known sources of erucic acid which makes up 55% to 60% of the oil glycerides (Princen and Rothfus 1984; Lazzeri et al. 1994). The high erucic acid oils have a wide diversity of applications (Nieschlag and Wolff 1971) for products such as rubber and plastic additives, lubricants, coatings, and as raw material for the synthesis of nylon. However, for profitable cultivation, exploration of other major seed components, such as the protein fraction, would be of major importance.

In contrast to the oil, very little is known about the proteins of Crambe. Crude protein levels range from 20% in the whole seed to nearly 50% in the dehulled, defatted meal (Carlson and Tookey 1983). Since crambe protein has a well balanced amino acid pattern (Baker et al. 1977; Steg et al. 1994), most research has been concerned with the use of the meal as feed (Kirk et al. 1971; Mustakas et al. 1976; Baker et al. 1977; Carlson and Tookey 1983; Steg et al. 1994). The suitability of the proteins for applications with a higher added value (food and non-food applications) is not yet known. The use of proteins in food and non-food industries is determined by their functional properties. Therefore, development of a protein isolation procedure and assessment of the functional properties of the isolated proteins is of prime importance to strengthen the economics of crambe cultivation.


Crambe seeds were provided by Cebeco-Handelsraad B.V. (The Netherlands). Dehulling was performed by cracking the seeds in roller mills. Removal of the hulls was obtained by air classification. The ratio of hulls to meal was 35:65. For milling, whole or dehulled seeds were mixed with liquid nitrogen in an analytical grinder and ground for 30 sec. Defatting was performed by stirring a suspension of whole or dehulled meal in petroleum ether (ratio meal to solvent 1:10 w/v) for 45 min at room temperature. After phase separation the supernatant was decanted and the pellet was resuspended in the solvent and the same procedure was repeated. The remaining pellet was left to dry overnight at room temperature.

Fat content was determined by the Soxtec system HT 1043 extraction unit. Protein content of seeds, meal, and of freeze dried materials was determined by Kjeldahl analysis and in solutions by using the Bio-Rad DC protein assay.

Protein extractability was studied as a function of pH. A suspension of defatted meal in water (1:10 w:v) was stirred for 5 min. The pH was then adjusted to the desired value by adding drops of dilute sodium hydroxide or hydrochloric acid solution. Stirring continued for 1 h, while the pH was checked every 10 min and readjusted, if necessary. After centrifugation in a Beckman J-21 C centrifuge at 10.960 g for 45 min, the supernatant was filtered to remove floating particles. Aliquots of the supernatant were freeze dried and their protein content was determined by Kjeldahl analysis.

Protein precipitation with and without the addition of carboxylmethylcellulose (CMC) was studied as a function of pH. First, proteins were extracted at pH 11 from defatted-dehulled meal. Two aliquots of this extract were diluted with water to give a concentration of approximately 1.5 mg protein per ml. The pH was then readjusted to 11. In one of the two solutions, CMC was added to a ratio of 0.166 CMC/protein (w/w); this ratio was found to yield the highest precipitation of rapeseed proteins (Gillberg and Törnell 1976). Samples of varying pHs were prepared from both solutions and kept at ambient temperature for 1 h, after which they were centrifuged for 15 min at 16,000 g. The protein content of the supernatants was quantified using the Bio-Rad assay. Solubility at pH 11 was assumed to be 100% and the results were expressed relative to this value. Proteins that remained soluble after isoelectric precipitation were concentrated by ultrafiltration. The protein solution (pH 5.5) was circulated through a membrane (Nephross Andante HF/Organon Technica) of molecular weight cut-off (MWCO) 5000 at a pressure of 0.5 Bar. The solution was ultrafiltrated to a concentration factor (initial volume/final volume) of 20. The retentate was then freeze dried.

In order to determine protein solubility freeze-dried protein concentrates were dissolved in water at pH 11 (concentration of approximately 1.5 mg protein per ml). Samples of varying pHs were prepared from this solution by acidification. After standing 1 h at room temperature they were centrifuged for 15 min at 16,000 g. The protein content of the supernatants was determined by the Bio-Rad assay.

To assess emulsifying properties a protein sample was dispersed in water (0.1% w/v). After stirring for 1 h the protein dispersion was mixed with commercial soybean oil. The mixture was homogenised for 1 min by means of an ultra-turrax at 13,500 rpm. An aliquot of the emulsion (1 ml) was diluted in water (250 ml) and the absorbance was measured at 500 nm. This value was taken as a measure of emulsifying activity. For assessment of the emulsifying stability, the emulsion was left to rest for 30 min. An aliquot (1 ml) of the bottom half of the emulsion was diluted in water (250 ml) and the absorbance of this solution was measured at 500 nm. The emulsifying stability was expressed as a percentage of the emulsifying activity.

Foaming properties were determined according to Patel et al. (1988) with a slight modification. The protein sample was stirred for 30 min in 0.1 M phosphate buffer (pH 7) instead of distilled water.


Isolation of Proteins

Table 1 shows the crude fat and crude protein content of crambe meals. Crude protein levels ranged from 18.3% in the whole seed to nearly 40% in the dehulled, defatted meal. The efficiency of oil extraction was 85.7% and 87.8% for the whole and dehulled meal, respectively. This result indicates that the presence of hulls did not interfere with oil removal, at least at a laboratory scale. Defatting was not complete, probably due to the procedure being performed at room temperature to avoid protein denaturation.

Fig. 1 shows protein extractability as a function of pH for dehulled and non-dehulled meal. The shape of both curves was similar with three solubility minima namely at pH 4, 6.5, and around 8. This could be an indication of the presence of proteins with different isoelectric points. For both types of meal, the extraction efficiency was higher at alkaline than at acid pH values. In comparison with the whole meal the extraction efficiency for the dehulled meal was higher at almost all pH values tested.

Highest protein extractability was obtained from dehulled meal at pH 11. Double extraction at this pH increased protein yield to 78%. This extract was used in further experiments. Its protein content was 50% (dry wt basis). Therefore, further treatment was necessary in order to decrease the amount of non-protein components present in the extract. Precipitation of proteins, with or without addition of precipitating agent, from alkaline extracts by acidification is a procedure often applied for the preparation of protein concentrates/isolates from oilseeds (Gillberg and Törnell 1976; Lásztity et al. 1993; Mansour et al. 1993; Xu and Diosady 1994). To determine the optimal pH value for precipitation, the protein precipitation efficiency was studied as a function of pH with and without the presence of CMC (Fig. 2). Addition of CMC clearly affected protein precipitation. Without CMC, maximum precipitation (55%) occurred at pH 5-6. Addition of CMC resulted in a more narrow curve, with a maximum precipitation (75%) at pH 4.4. This displacement of the precipitation maximum towards lower pH values is in agreement with the results of Gillberg and Törnell (1976). The protein contents of the precipitates (dry wt basis) recovered at pH 4.4 (with CMC) and pH 5.5 (without CMC) were 64% and 60%, respectively. Since this difference was rather small and because CMC might affect the functional properties of the proteins, precipitation was performed in further experiments at pH 5.5 without addition of CMC.

A proportion of the oil originally present in the dehulled meal was not extracted. It was found that most, if not all, of the unextracted oil was present in the precipitate. Therefore, the precipitate was cold-defatted which increased its protein content to 70% (dry wt basis). This protein concentrate is denoted as fraction 1.

Protein content of the supernatant after precipitation at pH 5.5 was 42% (dry wt basis). To concentrate the proteins present in this solution, ultrafiltration was applied. In this way, a concentrate of 84% protein (dry wt basis) was produced (fraction 2). The functional properties of both fractions were determined.

Functional Properties

Fig. 3 shows that there were marked differences in the solubility profiles of fraction 1 and 2. Proteins present in fraction 1 showed minimal solubility at pH 5.5-6, where approximately 80% of the proteins were insoluble whereas high solubility occurred at pH values <3.5 and >8. The solubility of proteins from fraction 2 was high (>80%) over the whole range of pH tested. Similar results were obtained by Xu and Diosady (1994), who used comparable preparation techniques (precipitation-ultrafiltration) for rapeseed protein isolates.

Fig. 4 and 5 show the emulsifying activity and stability of fractions 1 and 2. Both properties were markedly affected by the pH. For both fractions, lowest emulsifying activity and stability occurred near pH 6. For fraction 1, this could be due to the low solubility at this pH; for fraction 2 the reason is unclear. Both fractions were more efficient in emulsifying the oil at alkaline than at acid pH values. Similar pH dependence of emulsification properties has been reported for other proteins (Crenwelge et al. 1974; Ramanatham et al. 1978).

The foaming properties of fraction 1 and 2 are presented in Table 2. In comparison with hen's egg white, the foam expansion of both fractions was higher and foam volume stability was similar. Proteins from fraction 1 exhibited foam liquid stability comparable to that of hen's egg white while, proteins of fraction 2 exhibited lower values.


The proteins from crambe seeds were efficiently recovered by alkaline extraction followed by isoelectric precipitation/ultrafiltration. The fractions obtained have distinct solubility characteristics and very interesting foaming properties. At alkaline pH, crambe proteins show good emulsifying properties.


*Financial support by the Commission of the European Communities (DG XII) is gratefully acknowledged.
Table 1. Crude fat and protein content of crambe meals (calculated on a dry basis).

Constituent Crude fat (%) Protein (Nx6.25) (%)
Whole meal 34.6 18.3
Dehulled meal 45.4 25.1
Defatted whole meal 7.0 26.2
Defatted dehulled meal 9.2 39.6

Table 2. Foaming properties at pH 7 0.1 M phosphate buffer.

Type of protein Foam expansion (%) Foam volume stability (%) Liquid drainage (%) Foam liquid stability (%)
Hen egg white 420 80 62 37
Fraction 1 500 90 59 40
Fraction 2 620 80 80 20

Fig. 1. Protein extractability as a function of pH: (m) whole meal; (l) dehulled meal.

Fig. 2. Protein precipitation as a function of pH: (m) without CMC; (l) with CMC.

Fig. 3. Solubility of both fractions: (m) fraction 1; (l) fraction 2.

Fig. 4. Emulsifying activity as a function of pH: (m) fraction 1; (l) fraction 2.

Fig. 5. Emulsifying stability as a function of pH: (m) fraction 1; (l) fraction 2.

Last update August 19, 1997 aw