Brazilian soybean Glycine max (L.) Merr. cultivars adapted to low latitude regions: seed composition and content of bioactive proteins

Cultivares brasileiras de soja Glycine max (L.) Merr. adaptadas ao cultivo em baixas latitudes: composição da semente e conteúdo de proteínas bioativas

Abstracts

Among the goals of the Brazilian soybean improvement programmes, the breeding strategies for cultivars adapted to low latitudes have been included to extend crop areas and to increase production. Seeds of nine Brazilian soybean cultivars adapted to low latitudes were investigated regarding to their composition, and amino acid and antinutritional/toxic protein contents. Protein (394.5 ± 13.1 to 445.3 ± 8.0 g kg-1 dry matter) and oil (200.6 ± 1.2 to 232.3 ± 4.7 g kg-1 dry matter) contents showed low correlation to each other (r = -0.06). The total carbohydrate (141.7 ± 6.1 to 211.1 ± 15.0 g kg-1 dry matter) and ash contents (48.2 ± 4.2 to 52.2 ± 0.5 g kg-1 dry matter) were similar to data available for other soybean cultivars. All soybean cultivars presented low levels of tryptophan and sulphur amino acids. The lectin (1,152 to 147,456 HU kg-1 flour), trypsin inhibitor (34.45 ± 2.28 to 77.62 ± 2.63 g trypsin inhibited kg-1 flour), toxin (6,210 ± 134 to 34,650 ± 110 LD50 kg-1 flour) and urease (0.74 ± 0.02 to 1.22 ± 0.10 g kg¹ flour) presented variations in their contents amongst the cultivars. Compared to other soybean cultivars, urease was higher, the acute toxicity lower and the lectin and trypsin inhibitor contents similar to data available. In general, soybean cultivars showed similar biochemical composition to those developed in different geographic regions. The relevance of these findings to the agronomic features and to choice of soybean cultivars to be used as food or feed is discussed.

amino acid content; antinutritional factors; seed composition; soybean; toxicity

ARTICLES

Brazilian soybean Glycine max (L.) Merr. cultivars adapted to low latitude regions: seed composition and content of bioactive proteins

Cultivares brasileiras de soja Glycine max (L.) Merr. adaptadas ao cultivo em baixas latitudes: composição da semente e conteúdo de proteínas bioativas

Ilka M. VasconcelosII, ¹; Claudio C. Campello^I; José Tadeu A. Oliveira^II; Ana F. Urano Carvalho^III; Daniele O. Bezerra de Souza^II; Fernanda M.M. Maia^IV

^IUniversidade Estadual do Ceará, Faculdade de Veterinária, 60740-000 Fortaleza, CE, Brazil

^IIUniversidade Federal do Ceará, Departamento de Bioquímica e Biologia Molecular, Campus do Pici, Caixa Postal 6020, 60451-970 Fortaleza, CE, Brazil

^IIIUniversidade Federal do Ceará, Departamento de Biologia, 60451-970 Fortaleza, CE, Brazil

^IVUniversidade Estadual do Ceará, Centro de Ciências da Saúde, 60740-000 Fortaleza, CE, Brazil

ABSTRACT

Among the goals of the Brazilian soybean improvement programmes, the breeding strategies for cultivars adapted to low latitudes have been included to extend crop areas and to increase production. Seeds of nine Brazilian soybean cultivars adapted to low latitudes were investigated regarding to their composition, and amino acid and antinutritional/toxic protein contents. Protein (394.5 ± 13.1 to 445.3 ± 8.0 g kg^-1 dry matter) and oil (200.6 ± 1.2 to 232.3 ± 4.7 g kg^-1 dry matter) contents showed low correlation to each other (r = -0.06). The total carbohydrate (141.7 ± 6.1 to 211.1 ± 15.0 g kg^-1 dry matter) and ash contents (48.2 ± 4.2 to 52.2 ± 0.5 g kg^-1 dry matter) were similar to data available for other soybean cultivars. All soybean cultivars presented low levels of tryptophan and sulphur amino acids. The lectin (1,152 to 147,456 HU kg^-1 flour), trypsin inhibitor (34.45 ± 2.28 to 77.62 ± 2.63 g trypsin inhibited kg^-1 flour), toxin (6,210 ± 134 to 34,650 ± 110 LD₅₀ kg^-1 flour) and urease (0.74 ± 0.02 to 1.22 ± 0.10 g kg¹ flour) presented variations in their contents amongst the cultivars. Compared to other soybean cultivars, urease was higher, the acute toxicity lower and the lectin and trypsin inhibitor contents similar to data available. In general, soybean cultivars showed similar biochemical composition to those developed in different geographic regions. The relevance of these findings to the agronomic features and to choice of soybean cultivars to be used as food or feed is discussed.

Key words: amino acid content, antinutritional factors, seed composition, soybean, toxicity

RESUMO

Dentre os objetivos dos programas de melhoramento da soja no Brasil, está o desenvolvimento de estratégias para obtenção de cultivares adaptadas às regiões de baixas latitudes, visando a ampliação da área cultivada e o aumento da produção. Sementes de nove cultivares brasileiras de soja adaptadas a estas condições foram investigadas em relação à sua composição proximal e conteúdos de aminoácidos e proteínas tóxicas e/ou antinutricionais. Os conteúdos de proteína (394,5 ± 13,1 a 445,3 ± 8,0 g kg^-1 matéria seca) e óleo (200,6 ± 1,2 a 232,3 ± 4,7 g kg^-1 matéria seca) mostraram baixa correlação (r = -0,06). Os conteúdos de carboidratos totais (141,7 ± 6,1 a 211,1 ± 15,0 g kg^-1 matéria seca) e cinzas (48,2 ± 4,2 a 52,2 ± 0,5 g kg^-1 matéria seca) foram similares aos dados disponíveis para outras cultivares de soja. As cultivares estudadas apresentaram baixos teores de triptofano e aminoácidos sulfurados e ampla variação nos conteúdos de lectina (1.152 a 147.456 HU kg^-1 farinha), inibidores de tripsina (34,45 ± 2,28 a 77,62 ± 2,63 g tripsina inibida kg^-1 farinha), toxina (6.210 ± 134 a 34.650 ± 110 DL₅₀ kg^-1 farinha) e urease (0,74 ± 0,02 a 1,22 ± 0,10 g kg^-1 farinha). Comparados com outras cultivares de soja, os conteúdos de urease verificados foram superiores, a toxicidade aguda observada foi inferior e os conteúdos de lectina e inibidores de tripsina foram semelhantes aos resultados disponíveis na literatura. De um modo geral, as cultivares estudadas mostraram composição bioquímica similar àquela verificada para genótipos desenvolvidos em outras regiões geográficas. A relevância desses achados para as propriedades agronômicas e a seleção de cultivares de soja para a alimentação de seres humanos e animais são discutidas.

Palavras-chave: composição química da semente, conteúdo de aminoácidos, fatores antinutricionais, soja, toxicidade

Introduction

Soybean (Glycine max (L.) Merr.) is currently the most important source of edible oil and high-quality plant protein for feeding both human and animals worldwide (Zeller 1999, Friedman & Brandon 2001). Originated from mid latitude regions, this species is expanding in tropical areas as a result of the development of new genotypes tolerant to the environmental adversities of these localities (Vasconcelos et al. 1997, Campelo et al. 1999). One of the largest soybean producers of the world is Brazil, a tropical country that comprises an extensive ecological region with wide variation in the environmental conditions. In this country, soybean was firstly grown in the South (in mid latitude areas) and more recently next to Equator line, in the Northeastern region, owing to the development of genotypes with high productivity, well adapted to photoperiod effect and resistant to local pathogens and pests (Almeida et al. 1999). Presently, in these places, soybean cultivation has great economic and social importance.

It has been reported that the environmental conditions such as latitude, altitude, temperature and precipitation can affect the seed composition as, for example, the protein and oil contents (Zeller 1999, Van Barneveld 1999). Indeed, Qin et al. (1998) showed that the contents of antinutritional factors were different between raw soybeans produced in two distinct geographical regions. Vollmann et al. (2000) described genetic variations and significant differences in the protein content of soybeans cultivated in distinct climates. Based on these facts, it is reasonable to question whether the soybean cultivars adapted to grow in the Brazilian low latitude regions present differences in their seed composition and nutritional profile when compared to cultivars grown in distinct regions around the world. Furthermore, information on the chemical composition and antinutritional and/or toxic protein contents of soybean cultivars exposed to typical tropical environment is scarce.

Thus, the present work was undertaken to evaluate the proximate composition, amino acid content and the presence of trypsin inhibitor, lectin, urease and intraperitoneal acute toxicity of seeds of nine newly developed soybean cultivars adapted to low latitudes in Brazil.

Material and methods

Description of seeds and production fields – Mature seeds of nine soybean (Glycine max (L.) Merr.) cultivars ('M. Soy 108', 'Pati', 'Mirador', '9001', 'Cristalina-RCH', 'Seridó-RCH', 'FT 106', 'FT 107' and 'Sambaíba') adapted to low latitude regions were provided by FAPCEN (Fundação de Apoio à Pesquisa do Corredor de Exportação Norte – Maranhão – Brazil). The seeds were collected in soy culture areas in the Cerrado ecosystem from Balsas Municipality, located on the south of Maranhão State, Brazil, approximately to 7º31'57'' S and 46º02'08" W. Crop fields were characterized by geology consisting of a sedimentary structure composed by sandstones, siltand and claylayers forming a plan geomorphology with altitude from 300 to 500 m. The soils areas are represented for Quartzarenic Neosols (extremely low native fertility, deep, very well drained composed of sand sized quartz grains), Argisols (moderately deep, drained with clay horizon) and Litholic Neosols (very shallow soils, with the parent material less than 0.5 m from the surface), which required extensive modification for viable crop production. The area is characterized by tropical climate, hot and humid, with rainy season lasts from September and August. The annual average rainfall is around 1,400 mm, whilst air temperature varies from 25 to 36 °C, with a mean of 30 ºC. The relative humidity of the air is high, with 40% on the dry season. The Cerrado vegetation is predominant and consists of sparse trees of low to medium height with tortuous stems.

Proximate analysis – Seed moisture, crude fibre, ash and oil contents were determined by AOAC method (1990). Total nitrogen (Baethgen & Alley 1989) was used for determination of seed crude protein contents (N × 5.5). Total carbohydrate contents were calculated by difference from all the other constituents.

Amino acid composition – Defatted seed flours were hydrolysed with 6 M HCl containing 10 g L^-1 phenol at 110 °C for 22 hours in sealed glass tubes under N₂ atmosphere. After removal of HCl/phenol by evaporation, the amino acid compositions were established after chromatography on a Biochrom 20 System (Pharmacia). Tryptophan content was measured colorimetrically (Pintér-Szakács & Molnár-Perl 1990).

Aqueous soybean crude extracts – The seeds were ground in a coffee grinder to a fine powder. The meals were defatted with petroleum ether (100 g L^-1, w/v) under continuous stirring for 72 hours, air-dried at room temperature and stored at 4 °C. For preparation of the aqueous crude extract, the defatted meal was suspended in the extracting buffer (0.025 M Tris-HCl, pH 7.5) in the proportion of 1.0 g of meal to 5 mL of buffer. The suspension was maintained under continuous stirring (400 rpm) during 3 hours at 4 °C and then filtered. The press cake was reextracted during 2 hours under the same conditions. The filtrate was centrifuged at 15,000 g for 30 minutes at 4 °C and the clear supernatant dialysed (cut-off MW 12,000) overnight against the extracting buffer. The crude seed extract from each cultivar was the starting material for the detection of haemagglutinating and toxic activities. The crude extract of each seed was further fractionated by saturation to 20%-55% (F_20-55%) with solid ammonium sulphate. The precipitate was dissolved in and dialyzed exhaustively against 0.025 M Tris-HCl, pH 7.5. Toxic activity of this fraction was also assayed.

Soluble protein determination – The protein content in the seed extracts was determined by the method described by Bradford (1976) using bovine serum albumin (96%, Sigma Chemical Co, St. Louis, MO, USA), as standard.

Trypsin inhibitor activity – The protease inhibitor assay was carried out by a slight modification of the method originally described by Kakade et al. (1974) and Hamerstrand et al. (1981). Defatted soybean meal (0.02 g) was suspended in 1 mL of 0.01 M NaOH in an microcentrifuge tube and the suspension was stirred magnetically for 3 hours. The mixture was then left for 30 minutes without stirring and then 0.5 mL of the supernatant was mixed with 0.5 mL of 0.01 M NaOH. This solution was centrifuged 5 minutes at 10,000 g. After centrifugation, 0.1 mL of the alkaline extract was mixed with 1.6 mL 0.05 M Tris-HCl, pH 8.2, containing 0.02 M CaCl₂, with 0.1 mL of trypsin solution (from a stock solution of 0.4 mg in 10 mL 0.001 M HCl) plus 0,1 mL of N-a-benzoyl-L-arginine pnitroanilide solution (10 mg mL^-1 in 97% dimethyl sulfoxide plus H₂O, 1:3 v/v). The mixture was incubated for 45 minutes at 37 °C and then 0.2 mL 30% acetic acid solution was added. The absorbance was measured at 410 nm. Activity was expressed as the amount of trypsin inhibited calculated from a calibration curve using purified soybean trypsin inhibitor.

Haemagglutination assay – Haemagglutination activity was assessed by serial twofold dilution of the aqueous extracts (Moreira & Perrone 1977, Vasconcelos et al. 1991). The extracts were diluted with 0.15 M NaCl in glass tubes and mixed with rabbit red cells (20 mg mL^-1 suspension prepared in 0.15 M NaCl) with or without previous proteolytic enzyme treatment. The degree of agglutination was monitored visually after the tubes had been left at 37 °C for 30 minutes and then left at room temperature for further 30 minutes. The enzyme (trypsin, bromelain, subtilisin or papain) treated cells were obtained by incubating 25 mL 20 mg mL^-1 red cell suspension with the enzyme (0.1 mg) for 60 minutes at 4 °C, followed by washings, six times, with 0.15 M NaCl. The results were expressed as haemagglutinating titre (HU), which is the reciprocal of the highest dilution giving visible agglutination.

Acute toxicity assay – The acute toxicity of the Brazilian soybean crude extracts and F_(20-55%) was measured using 2-3 month-old adult male Swiss mice, reared in the animal house of the Universidade Federal do Ceará according to Vasconcelos et al. (1994). The animals were housed under the optimum temperature condition for mice (22 ± 2 °C) and light was set at 12 hours light/dark. The mice had unrestricted access to a commercial mouse diet and tap water. The acute toxic activity was defined as the mortality observed in mice within 24 hours after intraperitoneal injections of the crude extracts. One LD₅₀ unit was taken as the amount of protein (in g protein kg^-1 mouse body weight) producing convulsions and death of 50% animals tested (six doses; six mice per dose). The trial was conducted under official welfare guidelines and protocols covering animal experimentation at the university.

Urease assay – The crude extract was obtained by suspension of the meals in 0.01 M monobasic sodium phosphate buffer, pH 7.0, containing 0.15 M NaCl, using the same protocol previously described. Urease assay was carried out by minor modifications of the procedure described by Kaplan (1969). To 0.1 mL of 0.5 M urea solution was added 0.7 mL of 20 mg mL^-1 EDTA buffered with 0.2 M potassium phosphate, pH 6.5, and 0.1 mL of crude extract (containing 0.2 mg protein mL^-1). This mixture was incubated for 15 minutes at 37 °C followed by the addition of 1 mL of phenol plus sodium nitroprusside solution (62.0 g phenol with 0.25 g of sodium nitroprusside L^-1) and 1 mL of sodium hypochlorite plus alkali solution (43.0 mL of 52.5 g l^-1 NaClO with 20.0 g of NaOH L^-1). The mixture was incubated for further 5 minutes at 37 °C. After that, 7 mL of distilled water was added. The tubes were shaken vigorously and the absorbance measured at 625 nm. The enzyme activity was calculated from a calibration curve using urease (41H7008, 870000 units g^-1 Sigma Chemical Co, St. Louis, MO, USA).

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) – SDS-PAGE was performed according to the method of Laemmli (1970). It was carried out in a 3.5-mm vertical slab gel (18,5 × 16,5 cm) consisting of stacking gel mix, 5% total acrylamide, and main running gel mix, 15% acrylamide, prepared in 1.5 M Tris-HCl, pH 8.8. Samples were dissolved in Tris-HCl 0.125 M, pH 6.8, containing 1% SDS and 1% 2mercaptoethanol and incubated at 100 °C for 10 minutes. Electrophoresis was carried out at 40 mA constant electric current for 6 hours. Protein bands were visualized by staining with 0.05% Coomassie brilliant blue R-250. Protein markers employed were bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.2 kDa).

Statistical analysis – The results were subjected to analysis of variance and the significance between means determined by Tukey's test (P < 0.05).

Results and Discussion

As expected, the nine soybean cultivars studied in this present work were rich in protein, varying from 394.5 ± 13.1 to 445.3 ± 8.0 g kg^-1 dry matter, and oil, varying from 200.6 ± 1.2 to 232.3 ± 4.7 g kg^-1 dry matter (table 1). Although the protein contents of the cultivars are comparable to those of available data on other soybean cultivars (Vasconcelos et al. 1997, Wilcox 1998, Vollman et al. 2000, Ridley et al. 2004), the results evidenced some variability among the nine cultivars. For example, cv. Pati, which presented the highest protein content, had around 11% more protein than cv. FT 107 (table 1). Regarding to the oil contents, the values found for the nine Brazilian soybean cultivars are close to those reported for other soybean cultivars (Vasconcelos et al. 1997, Fernández-Quintela et al. 1998). The sum of protein and oil contents represents around 636.3 g kg^-1 dry matter, i.e., more than 50% of the whole seed (table 1). This value is higher than that reported by Vieira et al. (1999). Although the protein contents were nearly twofold higher than the oil, their amounts in the seeds had no correlation (r = -0.06). Hence these current data and those reported by Armour et al. (1998) did not support the negative association between the seed protein and the oil contents suggested before (Wilcox 1998, Vieira et al. 1999). This information may be important in soybean improvement programmes. Although the upper limit of increasing simultaneously protein and lipid contents is not yet predicted, these results show that the genetic improvement in the content of one of these two components is not necessarily accompanied by a reduction in the amount of the other seed compound and vice-versa.

The ash contents varied from 48.2±4.2 to 52.2±0.5 g kg^-1 dry matter (table 1) and no significant variation was observed among the cultivars. Furthermore, they are comparable to data reported by Férnandez-Quintela et al. (1997), slightly higher than those observed by Vasconcelos et al. (1997), but a little lower when compared to results obtained by Vieira et al. (1999). The carbohydrate contents varied from 141,7±6,1 a 211,1±15,0 g kg^-1 dry matter (table 1). According to Bau et al. (1997), mature soybeans contain only traces of starch, but are rich sources of dietary fibre particularly in hemicellulose and cellulose, and are poor in lignin, features that have been shown to produce beneficial effects on glucose and lipid metabolism.

The amino acid profiles of the Brazilian soybean cultivars and that of the hen egg, together with the minimal requirements of essential amino acids established for 2-5 and 10-12-year-old children (FAO/WHO/UNU 1985) are presented in table 2. Overall, the seed meal amino acid compositions were very similar. Regardless the cultivar analysed there was prevalence of the glutamine/glutamic acid (209.0 to 228.3 g kg^-1 protein) and asparagine/aspartic acid (112.2 to 149.6 g kg^-1 protein). Both glutamine and asparagine, which are derived from glutamic acid and aspartic acid, respectively, constitute important reservoirs of amino groups for the body. The total contents of essential amino acids in the studied cultivars ranged from 377.8 to 415.0 g kg^-1 protein, which are very close to those reported by Vasconcelos et al. (1997), Vieira et al. (1999) and Ridley et al. (2004). Analysis of the chemical scores (CS) of the essential amino acids revealed that most of them have contents below to those of the corresponding amino acids in the hen egg protein (FAO/WHO/UNU 1985). As reported for other legumes, the soybean cultivars presented methionine + cysteine and tryptophan as the first and second limiting amino acids, respectively. When these figures were compared to FAO/WHO/UNU (1985) scoring patterns of amino acid requirements for children (2-5 and 10-12 years old), the cultivars showed to be also deficient in methionine + cysteine and tryptophan. Tryptophan was the first limiting amino acid for six out of the nine studied soybean cultivars (Glycine max (L.) Merr. 'M. Soy', 'Pati', 'Cristalina-RCH', 'Seridó-RCH', 'FT 106' and 'FT 107') whereas the sulphur-containing amino acids were the first limiting ones for the remaining cultivars (Glycine max 'Mirador', '9001' and 'Sambaíba'). All other essential amino acids met the children's needs, although the contents of most of them were lower than those for hen egg. Deficiency of the sulphur amino acids is common in the great majority of leguminous seeds (Friedman et al. 1991, Duranti & Gius 1997). It is well known that cystine, cysteine and methionine are destroyed to varying degrees during hydrochloric acid hydrolysis (Gehrke et al. 1985). In this work, it was used 6 M HCl + 10 g L^-1 phenol to prevent degradation of some amino acids (Gehrke et al. 1985). In this condition, loss of methionine and cysteine may have occurred, but to a lesser extension. The insignificant values of methionine + cysteine need to be considered when the proteins are used for nutritional purpose. Nevertheless, these low contents must not be seen only as a nutritional limitation. Messina (1999) showed that low contents of sulphur amino acids might actually provide an advantage, since hydrogen ions produced from the sulphur amino acid metabolism can cause demineralisation of bone and excretion of calcium in urine.

The presence of lectin is depicted in table 3. Untreated or enzyme-treated rabbit erythrocytes were agglutinated in the presence of aqueous crude extract from all studied soybean cultivars. Enzyme-treated cells were more sensitive to agglutination than the untreated cells probably owing to a greater exposure of the carbohydrate moieties present in the cell membrane for which the lectin has higher affinity. Both total (1,152 to 147,456 HU kg^-1 flour) and specific (2,920 to 373,779 HU g^-1 protein) haemagglutinating activities showed a wide variation amongst the cultivars. The crude extracts from Cristalina-RCH and FT 107 cultivars presented the more noticeable total haemagglutinating activity against enzyme-treated rabbit erythrocytes. It has been long known that some lectins are considered important antinutritional factors since they are resistant to digestion by proteolytic enzymes and so, when present in diets, may cause alterations in gut and systemic metabolism by interacting with glycoconjugates on the gut cell membrane. These interactions may induce disruption of the brush border, atrophy of microvilli, hyperplasia of crypt cells, loss of viability of epithelial cells and also macroscopical alterations of key organs (Pusztai et al. 1995, Lajolo & Genovese 2002).

The trypsin inhibitor activity is shown in table 4. All the nine studied soybean crude extracts were able to inhibit trypsin. The amounts of trypsin inhibited in the studied cultivars varied from 34.45 ± 2.28 to 77.62 ± 2.63 g kg^-1 flour. The G. max 'M. Soy' and G. max 'Paty' presented the highest contents of trypsin inhibitor, whereas G. max 'Mirador' and G. max 'Sambaíba' showed the lowest ones. Variation in the protease inhibitor contents had already been observed in several soybean cultivars (Vasconcelos et al. 1997, Bau et al. 1997). Nevertheless, the contents of trypsin inhibitor found for the nine cultivars of the present study showed to be similar or slightly higher than those reported for other soybean cultivars (Vasconcelos et al. 1997, Sousa 2001, Brasil 2002, Olguin et al. 2003). Trypsin inhibitors interfere with the physiological functions of the trypsin, which consist of both pro-enzyme activation and hydrolysis of dietary proteins in the gut. Because of this double action, trypsin inhibitors are the main components claimed to be responsible for the poor digestibility of dietary proteins (Evenepoel et al. 2000, Bajpai et al. 2005). On the other hand, it is known that trypsin inhibitors are sulphur amino acid rich proteins and accordingly contribute substantially to the methionine content of soybeans (Clarke & Wiseman 2000). As the seeds are consumed after heat processing, which causes, at least in part, denaturation and inactivation of these proteins, trypsin inhibitors may even play a positive nutritional role (Duranti & Gius 1997).

The crude extracts of the nine Brazilian soybean cultivars exhibited urease activity (table 4). The values varied from 0.74 ± 0.02 (FT 106) to 1.22 ± 0.10 g kg^–1 flour (G. max 'Seridó-RCH'), corresponding to 643,000 and 1, 061,400 units kg^-1 flour, respectively. These data show that these nine cultivars could be regarded as high-urease genotypes when compared to some other Brazilian soybean cultivars (G. max 'Rio Balsas', G. max 'Tropical', G. max 'BR-10' and G. max 'Bays') which showed values varying from 0.123 to 0.252 g kg¹ flour (Vasconcelos et al. 1997). Apparently this variation in the urease content is a common feature within the soybean cultivars and might reflect both genetic and environmental variability (Vasconcelos et al. 1997). Nevertheless, it has been suggested that urease may act as a plant chemical defence against predators rather than have a urea assimilatory role (Polacco & Holland 1993, Follmer et al. 2002).

Surprisingly none of the crude extracts from the nine soybean cultivars were toxic to mice by intraperitoneal route, even when administered at the highest dose (0.3 mL10 g^-1 mouse body weight). These results differ from those reported by Vasconcelos et al (1997) who verified toxicity to mice in the crude aqueous extracts of four out of five other soybean cultivars when injected intraperitoneally. As the absence of toxicity was not expected to occur in all the nine cultivars, their crude extracts were fractionated by precipitation with ammonium sulphate at 20%-55% saturation as it was successful done in a similar study conducted with other soybean cultivar (Vasconcelos et al. 1994). Indeed, all the F_20-55% obtained from the nine cultivars were able to kill mice within 12 hours when injected by intraperitoneal route, producing acute symptoms, such as dyspnoea, tonic-clonic convulsions and death. The LD₅₀ values varied amongst the F_20-55% from 136 to 509 mg protein kg¹ mouse body weight (data not shown), which correspond to 6,210 ± 134 to 34,650 ± 110 LD₅₀ kg^-1 flour, respectively (table 4). These figures are close to the values reported for other soybean cultivars (Vasconcelos et al. 1994, Vasconcelos et al. 1997, Sousa 2001, Brasil 2002). The general toxic effects observed were similar to those observed for soyatoxin (LD₅₀ of 7.4 ± 0.3 mg kg¹ mouse body weight, according to Vasconcelos et al. 1994) and soybean toxin (LD₅₀ 5.6 ± 0.1 mg kg^-1 mouse body weight) (our unpublished results). As the F_20-55% did not represent the pure toxic component free of other seed constituents as were soyatoxin (Vasconcelos et al. 1994) and soybean toxin, their LD₅₀ were very much higher than those of the former soybean proteins. However, it seems important to monitor the levels of toxic proteins in soybean cultivars as nutritional studies showed that they contribute with biological alterations in mice, in addition to soyabean agglutinin (SBA) and trypsin inhibitors (Vasconcelos et al. 2001). Moreover, it is relevant to gather more information related to their physiological role since preliminary studies have revealed that these toxins display inhibitory activity against phytopathogenic fungi (our unpublished results).

SDS-PAGE did not reveal differences in the distribution of protein bands amongst the nine soybean cultivars. Exceptions are the protein bands with molecular mass around 27 to 28 kDa (figure 1). Four (G. max 'Mirador', G. max 'FT 106', G. max 'FT 107' and G. max '9001') of the nine soybean cultivars analyzed showed the presence of a protein band of M_r 27 kDa. On the other hand, in other cultivars (G. max 'M. Soy', G. max 'Pati', G. max 'Seridó-RCH', G. max 'Cristalina-RCH' and G. max 'Sambaíba') this protein band could not be detected, predominating another one of M_r 28 kDa. In addition to the protein bands of M_r 27 and 28 kDa, the cultivars showed several protein bands with M_r characteristics of b-conglycinin (three major subunits, namely, a' - 72 kDa, a - 68 kDa and b - 52 kDa) and glycinin (acid and basic subunits of ~ 35 kDa and 20 kDa, respectively) (Thanh & Shibasaki 1977, Nielsen 1985).

In conclusion, the data obtained in the present study for the nine Brazilian soybean cultivars confirmed that variations exist among several biochemical parameters depending on the cultivar considered. Thus it is relevant to gather more information on the proximate composition, amino acid content and the presence and levels of antinutritional and/or toxic proteins of the various genetically improved cultivars released for consumption. These results can allow future correlations of data to possibly predict which cultivars possess better nutritional quality and the reasons that explain these differences: whether of genetic origin or derived from plant nutrition status or features of the production area. Furthermore, this information is very relevant to support studies or possible correlations between the levels of antinutritional/toxic factors and the quality of the soybean derived food/feed as well as studies on the resistance of this crop to disease and pests.

Acknowledgements – This work was supported by "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq/PADCT), "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" (Capes/Procad), and "Fundação de Amparo à Pesquisa do Estado do Ceará" (Funcap). The authors are grateful to Dr. Gisela Introvini (Fapcen) for providing the soybean cultivars.

(received: November 03, 2005; accepted: October 19, 2006)

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Publication Dates

• Publication in this collection
  02 Apr 2007
• Date of issue
  Dec 2006

History

• Accepted
  19 Oct 2006
• Received
  11 Mar 2005