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Amino Acid Composition of Oilseed Meals and Protein Isolates
Amino Acid Composition of Oilseed Meals and Protein Isolates F. W. Sosulski and G. Sarwar Department of Crop Science University of Saskatchewan Saskatoon, Saskatchewan
Abstract Amino acid analyses of oilseed meals indicated that varietal differences in amino acid composition were much greater in soybean, tumip rapeseed, rapeseed and sunflower than in safflower and flax. In genera!, soybean and rapeseed proteins contained high proportions of essential amino acids required for human nutrition such as leucine, lysine and threonine while flax, sunflower and safflower proteins contained more of the nonessential arginine, aspartic acid and glutamic acid. Rapeseed proteins were also rich in methionine cystine and proline while soybean had a higher level of phenylalanine. Because of serious deficiencies in lysine, the oilseed meals and protein isolates of flax, sunflower and safflower rated poorly in essential amino acid indices and protein scores. Higher ratings were obtained for soybean and rapeseed meals and isolates but soybean was deficient in sulfur-containing amino acids while the rapeseed species were low in isoleucine. Since the latter amino acid is rarely deficient in mixed diets, the rapeseed products appeared to have the best protein for the supplementation of human diets. The nitrogen-to-protein conversion factors were calculated from the amino acid -compositions of the oilseed meals and protein isolates. The values for the present oilseed proteins varied between 5.33 and 5.74, indicating that the standard factor of 6.25 caused a substantial overestimation in crude protein content. A common factor of 5.50 is proposed for oilseed meals and, based on literature values, for all seed proteins.
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Resume La determination des acides amines dans les tourteaux d'oleagineux a indique que les differences entre varietes des acides amines sont plus grandes chez la soya, la navette, Ie colza et Ie toumesol que chez Ie carthame et Ie lin. En general, les acides amines essentiels en nutrition humaine tel que leucine, lysine et threonine ont ete plus abondants dans les proteines de soya et de colza tandis que Ie lin, Ie toumesol et Ie carthame contenaient plus des acides non essentiels: arginine, acide aspartique et acide glutamique. Les proteines de colza etaient aussi riches en methionine-cystine et en proline et celles de soya, en phenylalanine. A cause des deficiences serieuses en lysine, les tourteaux d'oleagineux et les isolats proteiques du lin, du tournesol et du carthame ont ete declasses d'apn'ls les indices d'acides amines essentiels et les notaticns proteiques. Des classements meilleurs ont ete obtenus pour les isolats et tourteaux de soya et de colza mais la soya etait carencee en acides amines sulfures tandis que les especes de colza etaient pauvres en isoleucine. Compte tenu que ce demier est rarement carence dans les dietes mixtes, les produits de colza ont semble posseder les meilleures proteines comme supplement dans les dietes de I'homme. Les facteurs de conversion de I'azote en proteine ont ete calcules pour les compositions en acides amines des tourteaux d'oleagineux et des isolats proteiques. Les valeurs pour les proteines d'oIeagineux etudies ont varie entre 5.33 et 5.74 indiquant que Ie facteur norme de 6.25 a cause une surestimation appreciable dans la teneur en proteine brute. Un facteur commun de 5.50 est propose pour les tourteaux d'oleagineux et, d'apres les valeurs rapportees dans la Iitterature, pour toutes les proteines de graines.
Introduction The problem of low protein supplies in many countries has been well documented and the potential role of oilseed meals in balancing the amino acid deficiencies in cereal proteins has been under intensive investigation. High levels of crude fiber, antinutritive factors, and protein damage during oil exJ. lnst. Can. Sci. Techno!. Ailment. Vol. 6, No 1, 1973
traction have restricted the utilization of most oilseed meals to that of livestock and poultry feeds (Altschul, 1958) . When processed under suitable conditions, soybean flours, concentrates and isolates have received widespread acceptance as low-cost vege· table proteins for human nutrition (Meyer, 1967). Most of the oilseed meals produced in Western Canada have not been evaluated as protein sources in human diets. The objective of the present investigation was to determine the nutritive value of these oil· seed meals after oil extraction and desolventization under low temperature conditions. In addition, the proteins were isolated from crude fiber and other nonprotein constituents in the meal to assess their true nutritive value. Initial studies were made on varietal differences in amino acid composition of six oilseed crops. I,arger samples of meal and protein isolate were prepared from a single variety of each crop' for the determination of the essential amino acids (EAA), essential amino acid indices (EAAI) and protein scores. The latter samples were also fed to mice and these biological evaluations are reported in a following publication. In these investigations, casein and the F AO (1965) amino acid requirements for humans were used as comparative standards.
Experimental Methods Seeds for the varietal study of amino acid COlllposition were obtained from experimental plots while larger seed samples for protein extraction and feeding trials were purchased from commercial sources. 'Amino acid analyses were conducted on oilseed meals from Portage and Altona soybeans; Polish, Echo and Span turnip rapeseed; Argentine, Target and Oro rapeseed; Redwing and Noralta flax; Commander, Advent and Peredovik sunflower; and US-10 and Gila safflower. Protein extractions, essential amino acids and pl'otein quality indices were determined on Altona soybean, Echo turnip rapeseed, Target rapeseeu, Redwing flax, Commander sunflower, Gila safflower, and a casein sample (Nutritional Biochemicals Inc., Cleveland, Ohio) in preparation for a mouse feeding trial. The seeds were ground and the oil extracted with n-hexane in a Soxhlet apparatus. The meals were desolventized in a vacuum oven at 45°C for 24 hours. Isolated proteins were extracted from the oilseed meals with 0.2% sodium hydroxide and precipitated by adjusting the pH to the isoelectric point of each protein (Sosulski and Bakal, 1969). The pH of minimum solubility for the extracted proteins was 4.5 for soybean, 4.7 for the two rapeseed species, 4.4 for flax, 5.0 for sunflower and 5.0 for safflower. The protein isolates were washed and freeze dried before amino 1
C'l ...... 0 COC'.ll!'l C'l'
acid analyses and feeding to mice. The amino acid analyses were conducted on duplicate hydrolysates of each sample with a Beckman Model 120C analyzer. Sixteen amino acids were determined by the two column procedure of Spackman et al. (1958) after hydrolysis of 35 mg of sample with 7 1111 6N HCl under vacuum in a sealed ampule for 24 hours at 110°C. For the EAA study, cystine and methionine were measured as cysteic acid and methionine sulfone by performic acid oxidation of the samples followed by hydrolysis with 6N HCl as above (Schram et al.) 1954). For tryptophan analysis, about 35 mg of sample were hydrolyzed with 14% barium hydroxide under vacuum at 110°C for 18 hours. After neutralization and Ba++ precipitation (Tkachuk and Irvine, 1969), aliquots of the buffered hydrolysate were analyzed for tryptophan content on a 10 cm column of Type P A-35 resin. The standard error for the determination of each amino acid was calculated.
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The most limiting amino acid, relative to the revised F AO (1965) pattern, was determined for the calculation of protein scores. Arginine and histidine, which are not essential for human nutrition, were not included in the computation of the total EAA, EAAI (Oser, 1959) and protein scores.
M
Results and Discussion Varieties and types of oilseed meals grown commercially in ,Vestern Canada were surveyed for their amino acid compositions. Species differences were relatively large with soybean varieties being particularly high in leucine, lysine, aspartic acid and tyrosine (Table 1). The rapeseed species contained more leucine, lysine and proline than most of the oilseed crops but tended to be low in arginine and aspartic acid. The samples of flax, sunflower and safflower were very low in lysine but contained more of the nonessential glutamic acid. The safflower and flax samples exhibited high levels of arginine while safflower and soybeans were low in methionine. The present results for soybean, rapeseed, flax, and sunflower varieties are in good agreement with the commercial samples evaluated by Tkachuk and Irvine (1969). However, the present arginine levels in sunflower and safflower varieties were higher than the varieties evaluated by Evans and Bandemer (1967) while the lysine contents were lower. 'Within crops, varietal differences in amino acid composition were noted in soybean, turnip rapeseed, rapeseed and sunflower while flax and safflower varieties were relatively uniform in protein composition (Table 1). For example, Altona appeared higher in leucine and arginine content than Portage soybean. Similar differences between Harosoy and Chippewa soybeans were reported by Evans and Bandemer (1967). Varieties witthin each rapeseed species were quite variable in phenylalanine and arginine content. The three rapeseed varieties also differed in valine and proline levels While the turnip rapeseed varieties showed more variation in glutamic acid and tyrosine. 2
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Can. Inst. Fcod Sci. Techno!. J. Vo!. 6, No. I, 1973
1'he sunflower varieties represented the three major bio-types of this crop and large variations in leucine, lysine, methionine, valine and glycine were observed. It appeared that Commander, which has confectionery and food uses, was lower in EAA than the Canadian (Advent) and Russian (Peredovik) oilseed varieties. The latter two varieties were particularly high in methionine while Peredovik contained more lysine than the other sunflower varieties. Evans and Bandemer (1967) evaluated the confectionery varieties and found apparent differences in arginine, lysine, methionine and tryptophan. They also reported large variations in arginine, methionine and tryptophan content among three safflower varieties. Differences in composition of arginine and aspartic acid between US-10 and Gila safflowers were noted in this investigation. The present data demonstrates that varietal differences in amino acid composition may be quite large, particularly among sunflower and rapeseed varieties. However, more inf'ormation on genetic and environmental effects should be obtained before undertaking the breeding and selection for improved amino acid distribution in these crops. The EAA composition of the meals and isolates were compared directly with the optimum dietary requirements f,or the growing child and adult (FAO, 19(5) and indirectly with the EAA composition of egg protein by calcul~tion of the EAAI. The revised FAO (1965) pattern for humans in Table 2 included the recommended decrease in proportions of total sulfur-containing amino acids, methionine cystine (met cys), and tryptophan. This reduced the total requirement from 2016 to 1926 mg of amino acids per g of dietary nitrogen. The FAO (1965) pattern had an EAAI of only 63 because egg proteins contained a much higher level of most EAA - about 3215 mg per g of total nitrogen. Since the F AO (1965) pattern was used as the base level, its protein score was taken as 100.
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Like egg protein, casein was a rich source of most EAA, containing 3203 mg of amino acids per g of sample nitrogen (Table 2). The balance of EAA relative to egg protein was better than the F AO (1965) pattern and equalled 80. The levels of the most deficient amino acids, met cys, were nearly the same as the human requirement. However, the ratio of these amino acids to the total EAA in casein was quite high and the protein score became only 57. While l
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J. Inst. Can. Sci. Technol. Aliment. Vol. G. No 1, 1973
of methi'onine and cystine were higher than reported by some investigators because the present analyses were done on the more stable forms of methionine sulfone and cysteic acid. 1'he principal amino acid deficiences in mixed diets are lysine, methionine (+ cystine) and tryptophan (FAO, 19(5), while cereal-based diets may also require threonine (Hegsted, 1969). A supplemental'y protein in the diet should contain surplus quantities of these amino acids and the rapeseed species appeared to be the most suitable protein for this purpose in the present study. While their lysine contents were slightly below soybean, the levels of met cys, tryptophan and threonine in the rapeseed meals and isolates were higher than in most of the other oilseed crops. Other components in the diet would normally supply the requirement for isoleucine and, in practice, the protein scores for the rapeseed products should approach 100. Soybean, flax, sunflower and safflower meals and soybean isolate would not be seriously deficient in EAA when consumed as the sole source of protein in the diet. Low levels of lysine or sulfur-containing amino acids would limit their supplemental value for the proteins in cereals and root crops. Sunflower and safflower isolates were seriously deficient in lysine and would, in themselves, require substantial supplementation.
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The principal effect of protein isolation was to decrease the total EAA in the proteins of four oilseeds but the levels in flax and safflower isolates increased slightly from the very low total EAA in the meals (Table 2). However, the balance of EAA, as measured by the EAAI, was relatively unchanged by protein isolation except in the latter two crops which showea a substantial improvement. The ratios of the most limiting amino acid to the total EAA were altered by the protein extraction process and protein scores differed widely between most meals and isolates. For example, the isolation process improved the protein score for soybean from 67 to 76 while in flax the value decreased from 82 to 61. The relative significance of these changes in protein score could only be assessed in an actual feeding trial. By feeding the meals and is,olates of each crop, the dietary effects of crude fiber and other meal constituents could also be assessed. In the present study, Kjeldahl nitrogen contents of the meals and isolates were converted to protein percentage by use of the factor 6.25. This common practice is based on the incorI'ect assumption that, like animal proteins, plant proteins contain 16% nitrogen. Tkachuk (1969) used quantitative amino acid data to show that the conversion factors for soybean, rapeseed, flax and sunflower meals were 5.69, 5.53, 5.41 and 5.36. The Tkachuk (1969) procedure was applied to the present data on meals and isolates (Table 2) and, since the complete amino acid compositions are not shown, the total amino acid residues and nitrogen are provided in Table 3. The nitrogen-to-protein 3
.... Table 1. Varietal differences in amino acid composition of six oilseed crops (g amino acid/16 g meal nitrogen). ------
Amino acid
Soybean Portage Altona
Turnip rapeseed Polish Echo Span
Rapeseed Argentine Target
Oro
Flax Redwing Noralta
Sunflower Com- Advent Peredovik mander
Safflower Gila US-lO
Standard error
Essential for human nutrition Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
5.0 7.0 6.7 1.3 4.8 3.6 5.1
4.8 7.8 6.2 1.5 4.8 3.8 4.9
4.0 7.1 6.4 1.8 4.5 3.9 5.1
4.0 7.3 6.7 1.9 3.3 3.8 5.0
4.5 7.2 6.6 2.3 3.1 3.6 5.3
4.2 7.6 6.1 2.1 3.9 3.5 5.4
4.3 7.1 6.2 1.9 3.2 3.6 4.6
3.9 7.0 6.3 1.5 3.1 3.6 4.7
3.8 5.4 3.5 1.7 4.0 3.3 4.6
3.7 5.3 3.8 1.7 3.9 3.2 4.7
4.9 5.8 2.9 1.7 4.3 2.9 4.6
5.0 6.5 2.9 2.6 4.5 2.8 6.7
4.4 6.3 3.8 2.3 4.2 2.9 4.8
3.1 5.9 2.3 1.6 3.7 2.4 4.8
3.3 6.0 2.8 1.2 4.0 2.6 4.9
0.2 0.2 0.1 0.1 0.2 0.1 0.3
0.1 0,.4 0.5 O.R 0.2 0.2 0.3 0.3 0.1
Nonessential for human nutrition Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Proline Serine Tyrosine
4.4 7.0 n.5 16.7 4.2 2.3 5.4 4.5 3.4
4.3 8.0 11.1 17.7 4.4 1.8 4.8 3.9 3.6
4.5 5.8 7.1 17.7 5.1 3.2 88 3.0 2.0
4.6 6.8 7.3 19.3 5.5 3.3 8.4 3.1 2.7
4.4 6.0 7.0 18.6 5.3 2.9 8.5 3.0 2.4
4.0 5.6 6.8 18.9 4.8 2.8 6.7 3.0 2.6
4.1 5.8 7.0 18.7 5.2 2.8 7.2 3.0 2.7
4.3 6.4 6.9 18.2 5.4 3.0 7.9 2.8 2.6
4.6 10.5 10.8 20.5 6.6 1.6 3.2 4.5 2.0
4.6 11.2 10.3 21.5 6.6 1.8 3.2 4.4 2.1
3.8 8.7 9.6 22.2 5.4 2.3 4.5 3.1 2.3
4.6 8.3 8.2 19.8 6.2 2.2 5.3 2.8 2.2
4.7 8.5 8.9 20.5 7.0 2.1 4.0 2.7 2.4
4.0 10.0 9.3 22.4 5.7 1.9 4.6 3.8 2.4
4.2 11.0 10.3 22.2 5.7 2.1 4.5 3.6 2.5
Total
92.9
93.4
90.0
93.0
90.7
88.0
87.4
87.6
90.6
92.0
89.0
90.6
89.5
87.9
90.9
Table 3. Calculation of the nitrogen-to-protein factor for oilseed meals and protein isolates by the procedure of Tkachuk (1969).
Protein
Weight in meal containing 1.00 g nitrogen residue g amino acid
Q
10
Fl
g amino acid nitrogen
Nitrogen-toprotein factor
Weight in isolate containing 1.00 g nitrogen g amino acid residue
g amino acid nitrogen
Nitrogen-toprotein factor
5.86
0.94
6.23
....
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Casein
P-
ro
il ~
(1) (")
Soybean
5.43
0.95
5.71
5.40
0.94
5.74
Turnip rapeseed
5.21
0.96
5.43
5.02
0.91
5.52
Rapeseed
5.15
0.96
5.36
5.07
0.91
5.57
Flax
5.13
0.95
5.40
5.21
0.96
5.43
Sunflower
5.16
0.96
5.37
5.16
0.95
5.43
Safflower
5.12
0.96
5.33
5.33
0.97
5.49
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factor was equivalent to the sum of the weights of the recovered amino acids in a fixed weight of sample divided by the corresponding weight of nitrogen in these amino acid residues. The conversion factor of 6.23 for casein was equivalent to that of the common 6.25 factor for animal proteins. The nitrogen-toprotein factors for the meals were slightly lower than the isolates with the average values being 5.72 for soybean, 5.47 for turnip rapeseed, 5.46 for rapeseed, 5.42 for flax, 5.40 for sunflower and 5.41 for safflower. Based on the present knowledge of amino acid composition in oilseed proteins and those of other grains (Tkachuk, 1969), a common conversion factor of 5.50 should be adopted for all seed proteins to avoid the substantial overestimation of protein content by use of the factor 6.25.
Conclusions While only a limited number of varieties were analysed, the present study indicated that varietal differences in amino acid composition may be relatively important in several oilseed crops. Variability in amino acid content was particularly large in sunflower and rapeseed varieties and, because of their increasing importance as crops in ",Vestern Canada, the heritability of these differences should be investigated. Species differences in composition of EAA were relatively large. Soybean and rapeseed proteins were more comparable to casein and whole egg in distribution of amino acids than flax, sunflower and safflower proteins. Relative to human requirements, the first limiting amino acids were met cys in soybean; lysine in flax, sunflower and safflower; and isoleucine in rapeseed and turnip rapeseed. Isoleucine dificiency is not common in nm'mal diets and, since rapeseed proteins contained high levels of lysine, met cys, tryptophan and threonine, this oilseed crop
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J. Inst. Can. Sci. Techno!. Aliment. Vol. 6, No 1, 1973
appeared to have the best protein for the supplementation of human diets. The present study has demonstrated that the protein contents of the meals and isolates were overestimated by the use of the nitrogen-to-protein factor of 6.25. 'While the animal protein, casein, had a factor of 6.23, the values for the oilseed proteins varied between 5.74 for soybean to 5.33 for safflower. A common factor of 5.50 should be adopted for these oilseed proteins and probably for all seed proteins.
Acknowledgements The authors are indebted to Mr. H. Braitenbach for technical assistance with the amino acid analyses and to the National Research Council of Canada for financial support.
References Altschul, A. M. 1958. Processed plant protein foodstuffs. Academic Press, New York, N. Y. 942 pp. Evans, R J., and Bandemer, S. L. 1967. Nutritive value of some oilseed proteins. Cereal Chem. 44: 417. F.A.O. 1965. Protein requirements. Nutrition meetings report series No. 37. Food and Agriculture Organization of the United Nations, Rome. 71 pp. Hegsted, D. M. 1969. Nutritional value of cereal proteins In relation to human needs. In Milner, M. (Ed.) Protein-enriched cereal foods for world needs. The Am. Association' of Cereal Chem. Saint PaUl, Minnesota. 343 pp. Meyer, E. W. 1967. Soy protein concentrates and isolates. Proc. Inter. Conf. on soybean protein foods. Peoria, Illinois. 285 pp. Oser. B. L. 1959. An Integrated essential amino acid index for predicting the biological values of proteins. In Albanese, A.A. (Ed.) Protein and amino acid nutrition. Academic Press, New York, N.Y. 281 pp. Schram, E., Moore, S., and Bigwood, E. J. 1954. Chromatographic determination of cystine as cysteic acid. Blochem. J. 57: 33. Sosulski, F. W., and Bakal, A. 1969. Isolated proteins from rapeseed flax and sunflower meals. Can. Inst. Food Techno!. J. 2: 28. Spackman, D. H., Stein, W. H., and Moore, S. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Ana!. Chem. 30: 1190. Tkachuk, R. 1969. Nltrogen-to-protein conversion factor for cereals and oilseed meals. Cereal Chem, 46: 419. Tkachuk, R, and Irvine, G. N. 1969. Amino acid composition of cereals and oilseed meals. Cereal Chem. 46: 206. Received July 26, 1972
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Abstract Amino acid analyses of oilseed meals indicated that varietal differences in amino acid composition were much greater in soybean, tumip rapeseed, rapeseed and sunflower than in safflower and flax. In genera!, soybean and rapeseed proteins contained high proportions of essential amino acids required for human nutrition such as leucine, lysine and threonine while flax, sunflower and safflower proteins contained more of the nonessential arginine, aspartic acid and glutamic acid. Rapeseed proteins were also rich in methionine cystine and proline while soybean had a higher level of phenylalanine. Because of serious deficiencies in lysine, the oilseed meals and protein isolates of flax, sunflower and safflower rated poorly in essential amino acid indices and protein scores. Higher ratings were obtained for soybean and rapeseed meals and isolates but soybean was deficient in sulfur-containing amino acids while the rapeseed species were low in isoleucine. Since the latter amino acid is rarely deficient in mixed diets, the rapeseed products appeared to have the best protein for the supplementation of human diets. The nitrogen-to-protein conversion factors were calculated from the amino acid -compositions of the oilseed meals and protein isolates. The values for the present oilseed proteins varied between 5.33 and 5.74, indicating that the standard factor of 6.25 caused a substantial overestimation in crude protein content. A common factor of 5.50 is proposed for oilseed meals and, based on literature values, for all seed proteins.
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Resume La determination des acides amines dans les tourteaux d'oleagineux a indique que les differences entre varietes des acides amines sont plus grandes chez la soya, la navette, Ie colza et Ie toumesol que chez Ie carthame et Ie lin. En general, les acides amines essentiels en nutrition humaine tel que leucine, lysine et threonine ont ete plus abondants dans les proteines de soya et de colza tandis que Ie lin, Ie toumesol et Ie carthame contenaient plus des acides non essentiels: arginine, acide aspartique et acide glutamique. Les proteines de colza etaient aussi riches en methionine-cystine et en proline et celles de soya, en phenylalanine. A cause des deficiences serieuses en lysine, les tourteaux d'oleagineux et les isolats proteiques du lin, du tournesol et du carthame ont ete declasses d'apn'ls les indices d'acides amines essentiels et les notaticns proteiques. Des classements meilleurs ont ete obtenus pour les isolats et tourteaux de soya et de colza mais la soya etait carencee en acides amines sulfures tandis que les especes de colza etaient pauvres en isoleucine. Compte tenu que ce demier est rarement carence dans les dietes mixtes, les produits de colza ont semble posseder les meilleures proteines comme supplement dans les dietes de I'homme. Les facteurs de conversion de I'azote en proteine ont ete calcules pour les compositions en acides amines des tourteaux d'oleagineux et des isolats proteiques. Les valeurs pour les proteines d'oIeagineux etudies ont varie entre 5.33 et 5.74 indiquant que Ie facteur norme de 6.25 a cause une surestimation appreciable dans la teneur en proteine brute. Un facteur commun de 5.50 est propose pour les tourteaux d'oleagineux et, d'apres les valeurs rapportees dans la Iitterature, pour toutes les proteines de graines.
Introduction The problem of low protein supplies in many countries has been well documented and the potential role of oilseed meals in balancing the amino acid deficiencies in cereal proteins has been under intensive investigation. High levels of crude fiber, antinutritive factors, and protein damage during oil exJ. lnst. Can. Sci. Techno!. Ailment. Vol. 6, No 1, 1973
traction have restricted the utilization of most oilseed meals to that of livestock and poultry feeds (Altschul, 1958) . When processed under suitable conditions, soybean flours, concentrates and isolates have received widespread acceptance as low-cost vege· table proteins for human nutrition (Meyer, 1967). Most of the oilseed meals produced in Western Canada have not been evaluated as protein sources in human diets. The objective of the present investigation was to determine the nutritive value of these oil· seed meals after oil extraction and desolventization under low temperature conditions. In addition, the proteins were isolated from crude fiber and other nonprotein constituents in the meal to assess their true nutritive value. Initial studies were made on varietal differences in amino acid composition of six oilseed crops. I,arger samples of meal and protein isolate were prepared from a single variety of each crop' for the determination of the essential amino acids (EAA), essential amino acid indices (EAAI) and protein scores. The latter samples were also fed to mice and these biological evaluations are reported in a following publication. In these investigations, casein and the F AO (1965) amino acid requirements for humans were used as comparative standards.
Experimental Methods Seeds for the varietal study of amino acid COlllposition were obtained from experimental plots while larger seed samples for protein extraction and feeding trials were purchased from commercial sources. 'Amino acid analyses were conducted on oilseed meals from Portage and Altona soybeans; Polish, Echo and Span turnip rapeseed; Argentine, Target and Oro rapeseed; Redwing and Noralta flax; Commander, Advent and Peredovik sunflower; and US-10 and Gila safflower. Protein extractions, essential amino acids and pl'otein quality indices were determined on Altona soybean, Echo turnip rapeseed, Target rapeseeu, Redwing flax, Commander sunflower, Gila safflower, and a casein sample (Nutritional Biochemicals Inc., Cleveland, Ohio) in preparation for a mouse feeding trial. The seeds were ground and the oil extracted with n-hexane in a Soxhlet apparatus. The meals were desolventized in a vacuum oven at 45°C for 24 hours. Isolated proteins were extracted from the oilseed meals with 0.2% sodium hydroxide and precipitated by adjusting the pH to the isoelectric point of each protein (Sosulski and Bakal, 1969). The pH of minimum solubility for the extracted proteins was 4.5 for soybean, 4.7 for the two rapeseed species, 4.4 for flax, 5.0 for sunflower and 5.0 for safflower. The protein isolates were washed and freeze dried before amino 1
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acid analyses and feeding to mice. The amino acid analyses were conducted on duplicate hydrolysates of each sample with a Beckman Model 120C analyzer. Sixteen amino acids were determined by the two column procedure of Spackman et al. (1958) after hydrolysis of 35 mg of sample with 7 1111 6N HCl under vacuum in a sealed ampule for 24 hours at 110°C. For the EAA study, cystine and methionine were measured as cysteic acid and methionine sulfone by performic acid oxidation of the samples followed by hydrolysis with 6N HCl as above (Schram et al.) 1954). For tryptophan analysis, about 35 mg of sample were hydrolyzed with 14% barium hydroxide under vacuum at 110°C for 18 hours. After neutralization and Ba++ precipitation (Tkachuk and Irvine, 1969), aliquots of the buffered hydrolysate were analyzed for tryptophan content on a 10 cm column of Type P A-35 resin. The standard error for the determination of each amino acid was calculated.
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The most limiting amino acid, relative to the revised F AO (1965) pattern, was determined for the calculation of protein scores. Arginine and histidine, which are not essential for human nutrition, were not included in the computation of the total EAA, EAAI (Oser, 1959) and protein scores.
M
Results and Discussion Varieties and types of oilseed meals grown commercially in ,Vestern Canada were surveyed for their amino acid compositions. Species differences were relatively large with soybean varieties being particularly high in leucine, lysine, aspartic acid and tyrosine (Table 1). The rapeseed species contained more leucine, lysine and proline than most of the oilseed crops but tended to be low in arginine and aspartic acid. The samples of flax, sunflower and safflower were very low in lysine but contained more of the nonessential glutamic acid. The safflower and flax samples exhibited high levels of arginine while safflower and soybeans were low in methionine. The present results for soybean, rapeseed, flax, and sunflower varieties are in good agreement with the commercial samples evaluated by Tkachuk and Irvine (1969). However, the present arginine levels in sunflower and safflower varieties were higher than the varieties evaluated by Evans and Bandemer (1967) while the lysine contents were lower. 'Within crops, varietal differences in amino acid composition were noted in soybean, turnip rapeseed, rapeseed and sunflower while flax and safflower varieties were relatively uniform in protein composition (Table 1). For example, Altona appeared higher in leucine and arginine content than Portage soybean. Similar differences between Harosoy and Chippewa soybeans were reported by Evans and Bandemer (1967). Varieties witthin each rapeseed species were quite variable in phenylalanine and arginine content. The three rapeseed varieties also differed in valine and proline levels While the turnip rapeseed varieties showed more variation in glutamic acid and tyrosine. 2
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Can. Inst. Fcod Sci. Techno!. J. Vo!. 6, No. I, 1973
1'he sunflower varieties represented the three major bio-types of this crop and large variations in leucine, lysine, methionine, valine and glycine were observed. It appeared that Commander, which has confectionery and food uses, was lower in EAA than the Canadian (Advent) and Russian (Peredovik) oilseed varieties. The latter two varieties were particularly high in methionine while Peredovik contained more lysine than the other sunflower varieties. Evans and Bandemer (1967) evaluated the confectionery varieties and found apparent differences in arginine, lysine, methionine and tryptophan. They also reported large variations in arginine, methionine and tryptophan content among three safflower varieties. Differences in composition of arginine and aspartic acid between US-10 and Gila safflowers were noted in this investigation. The present data demonstrates that varietal differences in amino acid composition may be quite large, particularly among sunflower and rapeseed varieties. However, more inf'ormation on genetic and environmental effects should be obtained before undertaking the breeding and selection for improved amino acid distribution in these crops. The EAA composition of the meals and isolates were compared directly with the optimum dietary requirements f,or the growing child and adult (FAO, 19(5) and indirectly with the EAA composition of egg protein by calcul~tion of the EAAI. The revised FAO (1965) pattern for humans in Table 2 included the recommended decrease in proportions of total sulfur-containing amino acids, methionine cystine (met cys), and tryptophan. This reduced the total requirement from 2016 to 1926 mg of amino acids per g of dietary nitrogen. The FAO (1965) pattern had an EAAI of only 63 because egg proteins contained a much higher level of most EAA - about 3215 mg per g of total nitrogen. Since the F AO (1965) pattern was used as the base level, its protein score was taken as 100.
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Like egg protein, casein was a rich source of most EAA, containing 3203 mg of amino acids per g of sample nitrogen (Table 2). The balance of EAA relative to egg protein was better than the F AO (1965) pattern and equalled 80. The levels of the most deficient amino acids, met cys, were nearly the same as the human requirement. However, the ratio of these amino acids to the total EAA in casein was quite high and the protein score became only 57. While l
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J. Inst. Can. Sci. Technol. Aliment. Vol. G. No 1, 1973
of methi'onine and cystine were higher than reported by some investigators because the present analyses were done on the more stable forms of methionine sulfone and cysteic acid. 1'he principal amino acid deficiences in mixed diets are lysine, methionine (+ cystine) and tryptophan (FAO, 19(5), while cereal-based diets may also require threonine (Hegsted, 1969). A supplemental'y protein in the diet should contain surplus quantities of these amino acids and the rapeseed species appeared to be the most suitable protein for this purpose in the present study. While their lysine contents were slightly below soybean, the levels of met cys, tryptophan and threonine in the rapeseed meals and isolates were higher than in most of the other oilseed crops. Other components in the diet would normally supply the requirement for isoleucine and, in practice, the protein scores for the rapeseed products should approach 100. Soybean, flax, sunflower and safflower meals and soybean isolate would not be seriously deficient in EAA when consumed as the sole source of protein in the diet. Low levels of lysine or sulfur-containing amino acids would limit their supplemental value for the proteins in cereals and root crops. Sunflower and safflower isolates were seriously deficient in lysine and would, in themselves, require substantial supplementation.
+
The principal effect of protein isolation was to decrease the total EAA in the proteins of four oilseeds but the levels in flax and safflower isolates increased slightly from the very low total EAA in the meals (Table 2). However, the balance of EAA, as measured by the EAAI, was relatively unchanged by protein isolation except in the latter two crops which showea a substantial improvement. The ratios of the most limiting amino acid to the total EAA were altered by the protein extraction process and protein scores differed widely between most meals and isolates. For example, the isolation process improved the protein score for soybean from 67 to 76 while in flax the value decreased from 82 to 61. The relative significance of these changes in protein score could only be assessed in an actual feeding trial. By feeding the meals and is,olates of each crop, the dietary effects of crude fiber and other meal constituents could also be assessed. In the present study, Kjeldahl nitrogen contents of the meals and isolates were converted to protein percentage by use of the factor 6.25. This common practice is based on the incorI'ect assumption that, like animal proteins, plant proteins contain 16% nitrogen. Tkachuk (1969) used quantitative amino acid data to show that the conversion factors for soybean, rapeseed, flax and sunflower meals were 5.69, 5.53, 5.41 and 5.36. The Tkachuk (1969) procedure was applied to the present data on meals and isolates (Table 2) and, since the complete amino acid compositions are not shown, the total amino acid residues and nitrogen are provided in Table 3. The nitrogen-to-protein 3
.... Table 1. Varietal differences in amino acid composition of six oilseed crops (g amino acid/16 g meal nitrogen). ------
Amino acid
Soybean Portage Altona
Turnip rapeseed Polish Echo Span
Rapeseed Argentine Target
Oro
Flax Redwing Noralta
Sunflower Com- Advent Peredovik mander
Safflower Gila US-lO
Standard error
Essential for human nutrition Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine
5.0 7.0 6.7 1.3 4.8 3.6 5.1
4.8 7.8 6.2 1.5 4.8 3.8 4.9
4.0 7.1 6.4 1.8 4.5 3.9 5.1
4.0 7.3 6.7 1.9 3.3 3.8 5.0
4.5 7.2 6.6 2.3 3.1 3.6 5.3
4.2 7.6 6.1 2.1 3.9 3.5 5.4
4.3 7.1 6.2 1.9 3.2 3.6 4.6
3.9 7.0 6.3 1.5 3.1 3.6 4.7
3.8 5.4 3.5 1.7 4.0 3.3 4.6
3.7 5.3 3.8 1.7 3.9 3.2 4.7
4.9 5.8 2.9 1.7 4.3 2.9 4.6
5.0 6.5 2.9 2.6 4.5 2.8 6.7
4.4 6.3 3.8 2.3 4.2 2.9 4.8
3.1 5.9 2.3 1.6 3.7 2.4 4.8
3.3 6.0 2.8 1.2 4.0 2.6 4.9
0.2 0.2 0.1 0.1 0.2 0.1 0.3
0.1 0,.4 0.5 O.R 0.2 0.2 0.3 0.3 0.1
Nonessential for human nutrition Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Proline Serine Tyrosine
4.4 7.0 n.5 16.7 4.2 2.3 5.4 4.5 3.4
4.3 8.0 11.1 17.7 4.4 1.8 4.8 3.9 3.6
4.5 5.8 7.1 17.7 5.1 3.2 88 3.0 2.0
4.6 6.8 7.3 19.3 5.5 3.3 8.4 3.1 2.7
4.4 6.0 7.0 18.6 5.3 2.9 8.5 3.0 2.4
4.0 5.6 6.8 18.9 4.8 2.8 6.7 3.0 2.6
4.1 5.8 7.0 18.7 5.2 2.8 7.2 3.0 2.7
4.3 6.4 6.9 18.2 5.4 3.0 7.9 2.8 2.6
4.6 10.5 10.8 20.5 6.6 1.6 3.2 4.5 2.0
4.6 11.2 10.3 21.5 6.6 1.8 3.2 4.4 2.1
3.8 8.7 9.6 22.2 5.4 2.3 4.5 3.1 2.3
4.6 8.3 8.2 19.8 6.2 2.2 5.3 2.8 2.2
4.7 8.5 8.9 20.5 7.0 2.1 4.0 2.7 2.4
4.0 10.0 9.3 22.4 5.7 1.9 4.6 3.8 2.4
4.2 11.0 10.3 22.2 5.7 2.1 4.5 3.6 2.5
Total
92.9
93.4
90.0
93.0
90.7
88.0
87.4
87.6
90.6
92.0
89.0
90.6
89.5
87.9
90.9
Table 3. Calculation of the nitrogen-to-protein factor for oilseed meals and protein isolates by the procedure of Tkachuk (1969).
Protein
Weight in meal containing 1.00 g nitrogen residue g amino acid
Q
10
Fl
g amino acid nitrogen
Nitrogen-toprotein factor
Weight in isolate containing 1.00 g nitrogen g amino acid residue
g amino acid nitrogen
Nitrogen-toprotein factor
5.86
0.94
6.23
....
D
(J>
:+ "!l 0 0
Casein
P-
ro
il ~
(1) (")
Soybean
5.43
0.95
5.71
5.40
0.94
5.74
Turnip rapeseed
5.21
0.96
5.43
5.02
0.91
5.52
Rapeseed
5.15
0.96
5.36
5.07
0.91
5.57
Flax
5.13
0.95
5.40
5.21
0.96
5.43
Sunflower
5.16
0.96
5.37
5.16
0.95
5.43
Safflower
5.12
0.96
5.33
5.33
0.97
5.49
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factor was equivalent to the sum of the weights of the recovered amino acids in a fixed weight of sample divided by the corresponding weight of nitrogen in these amino acid residues. The conversion factor of 6.23 for casein was equivalent to that of the common 6.25 factor for animal proteins. The nitrogen-toprotein factors for the meals were slightly lower than the isolates with the average values being 5.72 for soybean, 5.47 for turnip rapeseed, 5.46 for rapeseed, 5.42 for flax, 5.40 for sunflower and 5.41 for safflower. Based on the present knowledge of amino acid composition in oilseed proteins and those of other grains (Tkachuk, 1969), a common conversion factor of 5.50 should be adopted for all seed proteins to avoid the substantial overestimation of protein content by use of the factor 6.25.
Conclusions While only a limited number of varieties were analysed, the present study indicated that varietal differences in amino acid composition may be relatively important in several oilseed crops. Variability in amino acid content was particularly large in sunflower and rapeseed varieties and, because of their increasing importance as crops in ",Vestern Canada, the heritability of these differences should be investigated. Species differences in composition of EAA were relatively large. Soybean and rapeseed proteins were more comparable to casein and whole egg in distribution of amino acids than flax, sunflower and safflower proteins. Relative to human requirements, the first limiting amino acids were met cys in soybean; lysine in flax, sunflower and safflower; and isoleucine in rapeseed and turnip rapeseed. Isoleucine dificiency is not common in nm'mal diets and, since rapeseed proteins contained high levels of lysine, met cys, tryptophan and threonine, this oilseed crop
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J. Inst. Can. Sci. Techno!. Aliment. Vol. 6, No 1, 1973
appeared to have the best protein for the supplementation of human diets. The present study has demonstrated that the protein contents of the meals and isolates were overestimated by the use of the nitrogen-to-protein factor of 6.25. 'While the animal protein, casein, had a factor of 6.23, the values for the oilseed proteins varied between 5.74 for soybean to 5.33 for safflower. A common factor of 5.50 should be adopted for these oilseed proteins and probably for all seed proteins.
Acknowledgements The authors are indebted to Mr. H. Braitenbach for technical assistance with the amino acid analyses and to the National Research Council of Canada for financial support.
References Altschul, A. M. 1958. Processed plant protein foodstuffs. Academic Press, New York, N. Y. 942 pp. Evans, R J., and Bandemer, S. L. 1967. Nutritive value of some oilseed proteins. Cereal Chem. 44: 417. F.A.O. 1965. Protein requirements. Nutrition meetings report series No. 37. Food and Agriculture Organization of the United Nations, Rome. 71 pp. Hegsted, D. M. 1969. Nutritional value of cereal proteins In relation to human needs. In Milner, M. (Ed.) Protein-enriched cereal foods for world needs. The Am. Association' of Cereal Chem. Saint PaUl, Minnesota. 343 pp. Meyer, E. W. 1967. Soy protein concentrates and isolates. Proc. Inter. Conf. on soybean protein foods. Peoria, Illinois. 285 pp. Oser. B. L. 1959. An Integrated essential amino acid index for predicting the biological values of proteins. In Albanese, A.A. (Ed.) Protein and amino acid nutrition. Academic Press, New York, N.Y. 281 pp. Schram, E., Moore, S., and Bigwood, E. J. 1954. Chromatographic determination of cystine as cysteic acid. Blochem. J. 57: 33. Sosulski, F. W., and Bakal, A. 1969. Isolated proteins from rapeseed flax and sunflower meals. Can. Inst. Food Techno!. J. 2: 28. Spackman, D. H., Stein, W. H., and Moore, S. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Ana!. Chem. 30: 1190. Tkachuk, R. 1969. Nltrogen-to-protein conversion factor for cereals and oilseed meals. Cereal Chem, 46: 419. Tkachuk, R, and Irvine, G. N. 1969. Amino acid composition of cereals and oilseed meals. Cereal Chem. 46: 206. Received July 26, 1972
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