Annatto seed residue (Bixa orellana L.): nutritional quality

Abstract

Considering that annatto seeds are rich in protein, the present work aimed to evaluate the biological quality of this nutrient in the meal residue originating from annatto seed processing. We determined the general composition, mineral levels, amino acid composition and chemical scores, antinutritional factors, and protein quality using biological assays. The following values were obtained: 11.50% protein, 6.74% moisture, 5.22% ash, 2.22% lipids, 42.19% total carbohydrates and 28.45% fiber. The residue proved to be a food rich in fiber and also a protein source. Antinutritional factors were not detected. The most abundant amino acids were lysine, phenylalanine + tyrosine, leucine and isoleucine. Valine was the most limiting amino acid (chemical score 0.22). The protein quality of the seed residue and the isolated protein showed no significant differences. The biological value was lower than that of the control protein but higher than that found in other vegetables. Among the biochemical analyses, only creatinine level was decreased in the two test groups compared to the control group. Enzyme tests did not indicate liver toxicity. The results showed favorable aspects for the use of annatto seed residue in the human diet, meriting further research.

biological value; protein quality; annatto

1 Introduction

With the arrival of the Spanish conquerors in the New World, many plants whose extracts were used by the Mayans and Aztecs became known. One of these plants, annatto, occurring throughout tropical America was used as an extract to color fabrics and as body paint, and besides, it was used together with vanilla in the formulation of drinks made with cocoa (Giuliano, et al., 2003; Sandi & Cuen, 2003; Oliveira, 2005).

Annatto seeds are an important raw material to obtain the pigment bixin (represents more than 80% of the total fat-soluble carotenoids), norbixin and norbixinate, whose levels are variable according to seeds maturation (Dornelas et al., 2015; Mantovani et al., 2013). This is due to their characteristics as a non-toxic product with high dying power and ample color used for coloring food, products, cosmetics, pharmaceuticals and textiles, besides being used in cooking, as with the Brazilian spice colorau. Brazil is one of the largest producers and exporters of natural dye extracted from this plant (Mantovani et al., 2013; Anselmo et al., 2008). Annato seeds have been used to treat various diseases in popular medicine including for high lipid blood levels (Ferreira et al., 2013). Bacterial activity of annatto extracts was proportional to bixin content for various bacteria (Majolo et al., 2013).

The annatto seed contains cellulose, sucrose, oils, fragrances, and alpha-and beta-carotene (Paz et al., 2006). The dry meal residue from annatto seed processing has been found to consist of 13.5% crude protein, 45.7% neutral detergent fiber, 1.5% ether extract, 6.2% mineral matter and 63.8% nitrogen-free extract (Anselmo et al., 2008; Utiyama et al., 2002).

Therefore, since annatto seeds are rich in protein, this study aimed to evaluate the biological quality of this nutrient, the composition of the essential amino acids and other nutrients in the residue of annatto seeds, as well as evaluating the presence of some antinutritional factors.

2 Materials and methods

The annatto seed residue was donated in 2011 by a food industry, CHR HANSEN, located in Valinhos, São Paulo. The waste, consisting of wet annatto seeds and impurities (pieces of branches of the annatto tree), were dried in a forced ventilation oven at 50°C for 24 hours, then cleaned and milled to obtain a meal.

The composition of the annatto seed meal was determined using the methods described by the Adolfo Lutz Institute (Brasil, 2005) for moisture, fixed mineral residue (ash), carbohydrate, fiber, neutral detergent and lipids. Protein was determined by the micro-Kjeldahl method according to the Association of Official Analytical Chemists (1992).

The extract used in the determination of mineral was obtained by the method described by Moraes & Rabelo (1986) and Salinas et al. (1985). The minerals cupper, zinc, manganese, calcium, iron and magnesium were analyzed by flame atomic absorption spectrometry and the calcium in flame photometer, 589nm (Varian model Spectra AA 220FS). The quantification was done by standard curve of each mineral.

Tannins were determined according to the method described in the Association of Official Analytical Chemists (1984). Phytic acid content was analyzed by the method of Latta & Eskin (1980). Trypsin inhibitory activity was evaluated by the method of Erlanger et al. (1961), and lectins were determined according to Witisuwannakul et al. (1998).

The solubility of the protein the annatto seed residue meal according to pH was determined by the methods described by Glória & Regitano-D’Arce (2000). Proteins of the residue meal extract were assayed according to Macedo & Damico (2000), and amino acid analysis was performed by the method of Heinrikson & Meredith (1984), using the analyzer Pico-Tag (Waters System) and identification in HPLC reverse phase column, comparing the retention times of the amino acids of the sample with the standards (Pierce).

The biological assay was carried out in 40 rats, males, age of 21 days, (10 per group), Wister, Rattus norvegicus, in a clean environment with controlled temperature of 25^oC and appropriate light, with a 12-hour light/dark cycle, lasting 28 days. The use of the animals was approved in the ethics committee, n. 267, by the Ethic Committee in Animal use CEUA/UFMS.

The diets offered to the animals were prepared in accordance with AIN93-G (Reeves et al., 1993) modified for the following groups: control (casein), non-protein, test 1 (annatto seed residue) and test 2 (isolated protein). During the experiment, feces and urine were collected from the animals in each group for quantification of excreted nitrogen by the Kjeldahl method as described by the Association of Official Analytical Chemists (1992). From these results, the indices that provide information on the quality of the protein were calculated. Protein efficiency rate (PER) and biological value (BV) were determined according to the methods described by Hiane et al. (2006) and Tirapegui et al. (2012). Coefficient of feeding efficiency (CFE) was calculated according to Souza (2003) and true digestibility (TD) using the method of Cruz et al. (2005) and Tirapegui et al. (2012). Nitrogen balance (NB) was calculated according to the method of Hiane et al. (2006). The animals’ weight was monitored weekly.

At the end of the experiment, after fasting for 12 hours, blood was collected and glucose, total cholesterol, creatinine, triglycerides, very low density lipoprotein (VLDL), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using specific kits for each sample and using an automated 400 Plus Cobas Integra System (Roche) according to methods described in the instrument manual.

3 Results and discussion

The composition of the seed residue meal showed a high levels of protein (11.5%), fixed mineral residue (5.22%), carbohydrates (42.2%) and fiber (28.48%) (Table 1). The protein content here was slightly higher than that recorded by Carvalho et al. (1991) (10.8%) and similar to that obtained by Pedrosa et al. (1999) (12.67%) for annatto seeds, compared to 11.56% for wheat flour reported by Pires et al. (2006).

Table 1
Proximate composition of the annatto seed residue expressed in g.100 g^–1 of whole sample^*.

Parameter	Means ± SD*	%DRI	DRI (g)**
Moisture	6.75 ± 0.089	-	-
Fixed mineral residue	5.22 ± 0.35	-	-
Total lipids	2.23 ± 0.11	4.05	60
Protein	11.50 ± 0.06	15.33	75
Total carbohydrate	42.2 ± 0.69	14.06	300
Sucrose	11.78 ± 0.02	-	-
Starch	30.42 ± 0.58	-	-
Fiber	28.48 ± 0.91	113.92	25
Total caloric value (kcal/100g**)	234.46	11.72	2000

• ^*
  Mean values of 3 determinations ± standard deviation.
• ^**
  Dietary reference intake based on a diet of 2000 kcal (Brasil, 2003).

The percentage of fiber, however, was lower than the 36.8% obtained by Utiyama et al. (2002) and much higher than the 16% found by Bressani et al. (1983). Comparing the protein and fiber levels with the daily reference intake values ​​established by Brazilian Resolution RDC No. 360/2003 (Brasil, 2003) and with classification of food as rich or sources of nutritious food when providing 30 or 15% of the recommended intake in 100 g, respectively, according to current Brazilian law (Brasil, 1998), the annatto seed residue can be classified as rich in fiber and a protein source.

The total carbohydrate was high (42.2%), but lower than that obtained by Moraes (2007) (78.11%). The percentage of minerals was similar to the 5.05%, found by Moraes (2007) and comparable to the 6.32 and 6.2%, obtained respectively by Pereira et al. (2009) and Utiyama et al. (2002).

The pH at which the highest protein solubility was seen in the studied sample (pH 12) was similar to that found by Glória & Regitano-D’Arce (2000). According to the used assays, tannins, phytic acid and protease inhibitors were not detectable in the sample.

The amino acid composition of the sample showed high levels of lysine (76.7 mg.g^–1), isoleucine (59.3 mg.g^–1), leucine (83.9 mg.g^–1) and phenylalanine + tyrosine (100.2 mg.g^–1) (Table 2). These levels higher than intake levels were for adults recommended by FAO/WHO (Food and Agriculture Organization of the United Nations, 2007), which are 45, 30, 59 and 30 mg.g^–1 protein, respectively. The amino acid valine was the most limiting with a chemical score of 0.22.

Table 2
Composition and the chemical score (CS) of the essential amino acids of the isolated protein (IP) of the annatto seed residue and the comparison with the FAO/WHO requirements for adults.

	IP (mg.g–1 protein)	CS	FAO* (mg.g–1 protein)
Lysine	76.7	1.70	45
Histidine	7.2	0.48**	15
Threonine	10.9	0.47**	23
Valine	8.5	0.22**	39
Methionine	12.8	0.8**	16
Methionine + cysteine	12.8	0.58**	22
Isoleucine	59.3	1.98	30
Cystine	Nd	Nd	6
Leucine	83.9	1.42	59
Phenylalanine + Tyrosine	100.2	3.34	30

• ^*
  Food and Agriculture Organization of the United Nations (2007).
• ^**
  Limiting amino acids; nd: not determined.

The minerals in annatto seed showing the highest values were sodium (35.61 mg.g^–1) and potassium (70.77 mg.g^–1), followed by manganese (0.25 mg.g^–1), calcium (0.11 mg.g^–1) and copper, iron and magnesium (0.03 mg.g^–1). The values found for ​potassium, zinc, copper and manganese were higher than those found by Ferreira & Falesi (1989), respectively, 19.2, 0.0354, 0.0046 and 0.023 mg.g^–1, while these authors reported a higher magnesium level (2.22 mg.g^–1). Calcium and iron levels here were higher than those reported for annatto seeds from EMEPA (0.07 and 0.008 mg.g^–1, respectively) by Anselmo et al. (2008), but calcium was lower than that found by Ferreira & Falesi (1989) (1.82 mg.g^–1).

During the 28-day experiment, it was observed that there was a decrease in weight of the animals in the group without protein; the casein and test 2 (isolated protein) groups showed higher weight gain, 64.87 and 65.74 g, respectively; although, there was no statistically significant difference (p > 0.05) between these groups and test 1 (annatto seed residue) (55.08 g). As to the food that was consumed, there was no significant difference between the casein (392 g), test 1 (384.74 g) and test 2 (415.27 g) groups, indicating that the protein quality of the three diets was the same.

Considering the protein quality indices (Table 3), there was no significant difference between the annatto residue group and the isolated protein group in relation to PER (p > 0.05), but both differed from the casein group. Although protein quality is low in vegetables, the PER found for both groups was higher than that found by Pires et al. (2006) in wheat flour (0.98 g) and soy flour (1.75 g) and also the values ​​obtained by Hafez et al. (2009) in bread with 10% salt (0.94 g) and 15% (1.45 g) linseed flour. Tirapegui et al. (2008), obtained a PER value of 2.1 g for soybeans and 1.5 g for wheat.

Table 3
Protein quality indices of the casein (CA), annatto residue (test 1) and isolated protein (test 2) groups.

Protein quality index	CA*	Test 1*	Test 2*
Protein efficiency rate (PER) (g)	2.57 ± 0.25a	1.71 ± 0.41b	2.10 ± 0.33b
Coefficient of feeding efficiency (CFE) (g)	0.17 ± 0.02a	0.14 ± 0.03a	0.16 ± 0.02a
Biological value (BV) (%)	99.01 ± 0.22a	94.89 ± 1.24b	95.95 ± 0.52b
Nitrogen balance (NB) (g)	3.64 ± 0.67a	4.08 ± 0.94a	4.39 ± 0.82a
True digestibility (TD) (%)	96.43 ± 0.76a	87.99 ± 2.53c	92.63 ± 1.14b

• ^*
  Values followed by the same letter in a row are not significantly different by Tukey’s test at 5% probability.

Although the biological values ​​observed in the annatto residue group (94.89%) and isolates protein group (95.95%) were lower than in the casein group (99.01%), they were higher than those obtained by Silva et al. (2006) for soybean grain (87%) and soybean residue (88,1%).

In relation to the biochemical parameters (Table 4), only the data obtained for the total cholesterol for the casein group (75 mg.dL^–1) was similar to that found by Santos et al. (2010) (75 mg.dL^–1), while both test groups (test 1 and test 2) had higher values ​​when compared to the data from the same authors (82.5 and 94.83 mg.dL^–1, respectively).

Table 4
Biochemical parameters (mean ± sd, N = 10/group) of the casein (CA), annatto seed residue (test 1) and protein isolate (test 2) groups at the end of the biological assay.

Parameter (mg.dL–1)	CA*	Test 1*	Test 2*
Glucose	72.40 ± 14.64a	59.33 ± 12.72a	70.20 ± 15.29a
Total cholesterol	75.00 ± 10.65b	82.5 ± 5.96ab	94.83 ± 9.43a
Creatinine	0.3 ± 0.00a	0.2 ± 0.00b	0.23 ± 0.05b
Triglycerides	47.60 ± 19.48a	48.83 ± 16.58a	68.17 ± 17.89a
VLDL1	12.17 ± 7.68a	9.83 ± 3.37a	13.67 ± 3.72a
ALT2	24.17± 6.82a	32.17± 5.49a	27.67 ± 5.65a
AST3	155.5 ± 24.94a	149.33 ± 45.22a	152.33 ± 48.19a

• ^*
  Values followed by the same letter within a row are not significantly different by the Tukey’s test at 5% probability.
• ¹
  VLDL-very low density lipoprotein.
• ²
  ALT-alanine aminotransferase.
• ³
  AST-aspartate aminotransferase.

The triglyceride levels showed no significant differences between the different groups studied, although the test groups were slightly higher than the casein and also above the reference value for BALB/c and C57BL6 mice, from 21.11 to 29.68 mg.dL^–1 as evaluated by Almeida et al. (2008) but close to that found by Santos et al. (2010) (47.6 mg.dL^–1). There were statistically significant differences in glucose levels in both groups (Test 1 and Test 2); however, they were lower than that found by Santos et al. (2010) (85 mg.dL^–1). The creatinine values showed a significant difference only with the control casein group; in the test groups the values were lower, indicating that the proteins in the annatto seed residue caused a decrease in creatinine levels.

4 Conclusion

Annatto seed residue was found to have high levels of protein, carbohydrates, fiber and minerals, and considered to be a food rich in fiber and protein. The most abundant minerals found were potassium and sodium. Substantial concentrations of the amino acids lysine, isoleucine, leucine and phenylalanine + tyrosine were found, while valine was the limiting amino acid with a chemical score of 0.22. The antinutritional factors tannins, phytic acid and protease inhibitors were not detected.

The proteins of the annatto seed residue and from the isolated protein showed a lower biological value compared to the control protein. Among the biochemical parameters evaluated in the blood samples of the animals at the end of the experiment, only total cholesterol showed a significant difference between the groups; the creatinine level in the test groups was reduced, and the enzyme assays did not indicate liver toxicity.

Although the results showed favorable aspects for the use of the seed residue in food, further studies are needed to confirm these findings.

• Practical Application: The evaluation of biological quality of the annatto seeds’ protein showed favorable aspects for the use in the human diet.

References

• Almeida, A. S., Faleiros, A. C. G., Teixeira, D. N. S., Cota, U. A., & Chica, J. E. L. (2008). Valores de referência de parâmetros bioquímicos no sangue de duas linhagens de camundongos. Jornal Brasileiro de Patologia e Medicina, 44(6), 429-432. http://dx.doi.org/10.1590/S1676-24442008000600006.
  » http://dx.doi.org/10.1590/S1676-24442008000600006
• Anselmo, G. C. S., Mata, M. E. R. M. C., & Rodrigues, E. (2008). Comportamento Higroscópico do extrato seco de urucum ( L.). Bixa orellanaCiência Agrotecnica, 32(6), 1888-1892. http://dx.doi.org/10.1590/S1413-70542008000600030.
  » http://dx.doi.org/10.1590/S1413-70542008000600030
• Association of Official Analytical Chemists - AOAC. (1984). Official Methods of Analysis: Tannin in cloves and all spice (14th ed., pp. 364). Washington. (Method 30.018; 30.019).
• Association of Official Analytical Chemists - AOAC. (1992). Official Methods of Analysis of AOAC International (12th ed. Washington.).
• Brasil, Ministério da Saúde, Agencia Nacional de Vigilância Sanitária - ANVISA. (1998). Regulamento Técnico Referente a Informação Nutricional Complementar (Portaria nº 27 de 13/01/1998). Diário Oficial da União.
• Brasil, Ministério da Saúde, Agencia Nacional de Vigilância Sanitária - ANVISA. (2003). Regulamento técnico sobre rotulagem nutricional de alimentos embalados. (Resolução RDC no 360, de 23 de dezembro de 2003). Diário Oficial da União.
• Brasil, Ministério da Saúde, Agência Nacional de Vigilância Sanitária - ANVISA. (2005). Métodos físico-químicos para análise de alimentos. Brasília.
• Bressani, R., Porta-España de Barneón, F., Braham, J. E., Elías, L. G., & Gómez-Brenes, R. (1983). [Chemical composition, amino acid content and nutritive value of the protein of the annatto seed (Bixa orellana, L.)]. Archivos Latinoamericanos de Nutricion, 33(2), 356-376. PMid:6673674.
• Carvalho, P. R. N., Carvalho, C. R. L., & Mantovani, D. M. B. (1991). Estudo da composição de sementes, cachopas, folhas e galhos do urucuzeiro. In Seminário Internacional de Corantes Naturais para alimento, Campinas. Campinas: ITAL.
• Cruz, G. A. D. R., Oliveira, M. G. A., Costa, N. M. B., Pires, C. V., Cruz, R. S., & Moreira, M. (2005). Comparação entre a digestibilidade protéica in vitro e in vivo de diferentes cultivares de feijão ( l.) armazenados por 30dias. Phaseolus vulgarisAlimentos e Nutrição, 16(3), 265-271.
• Dornelas, C. S. M., Almeida, A., Figueiredo Neto, A., Sousa, D. M. M., & Evangelista, A. P. (2015). Desenvolvimento na maturação de frutos e sementes de Urucum (Bixa orellana L.). Scientia Plena, 11(01), 1-8.
• Erlanger, B. F., Kokowsky, N., & Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Archives of Biochemistry and Biophysics, 95(2), 271-278. http://dx.doi.org/10.1016/0003-9861(61)90145-X. PMid:13890599
  » http://dx.doi.org/10.1016/0003-9861(61)90145-X
• Food and Agriculture Organization of the United Nations – FAO, World Health Organization – WHO, United Nations University – UNU. (2007) Evaluation on protein and amino acid requirements in Human Nutrition: report of the joint FAO/WHO/UNU Expert Consultation (WHO Tech Rep Ser., No. 935). Geneva.
• Ferreira, J. M., Sousa, D. F., Dantas, M. B., Fonseca, S. G. C., Menezes, D. B., Martins, A. M. C., & Queiroz, M. G. (2013). Effects of Bixa orellana L. seeds on hyperlipidemia. Phytotherapy Research, 27(1), 144-147. http://dx.doi.org/10.1002/ptr.4675. PMid:22451331
  » http://dx.doi.org/10.1002/ptr.4675
• Ferreira, W. A., & Falesi, I. C. (1989). Características nutricionais do fruto e teor de bixina em urucum (Bixa orellana L.). Belém: CPATU.
• Giuliano, G., Rosati, C., & Bramley, P. M. (2003). To dye or not to dye: biochemistry of annatto unveiled. Trends in Biotechnology, 21(12), 513-516. http://dx.doi.org/10.1016/j.tibtech.2003.10.001. PMid:14624856
  » http://dx.doi.org/10.1016/j.tibtech.2003.10.001
• Glória, M. M., & Regitano-D’Arce, M. A. B. (2000). Concentrado e isolado protéico de torta de castanha do Pará: obtenção e caracterização química e funcional. Ciência e Tecnologia de Alimentos, 20(2), 240-245. http://dx.doi.org/10.1590/S0101-20612000000200019.
  » http://dx.doi.org/10.1590/S0101-20612000000200019
• Hafez, V. C. B., Oliveira, G. P. da C., & Gonçalves, R. C. C., Guaitoli, M. R., Ferrini, M. T., Bottoni, A. & Waitzberg, D. L. (2009). Eletrólitos, minerais, elementos traços e elementos ultratraços. In D. L. Waitzberg, Nutrição oral, enteral e parenteral na prática clínica (cap. 10, pp. 183-208). São Paulo: Atheneu.
• Heinrikson, R. L., & Meredith, S. C. (1984). Amino acid analysis by reverse-phase high-performance liquid chromatography: precolumn derivatization with phenylisothiocyanate. Analytical Biochemistry, 136(1), 65-74. http://dx.doi.org/10.1016/0003-2697(84)90307-5. PMid:6711815
  » http://dx.doi.org/10.1016/0003-2697(84)90307-5
• Hiane, P. A., Macedo, M. L. R., Silva, G. M., & Braga Neto, J. A. (2006). Avaliação nutricional da proteína de amêndoas de bocaiúva, (Jacq.) Lodd., em ratos wistar em crescimento. Acrocomia aculeataBoletim do Centro de Processamento de Alimentos, 24(1), 191-206.
• Latta, M., & Eskin, M. (1980). A simple and rapid colorimetric method for phytate determination. Journal of Agricultural and Food Chemistry, 28(6), 1313-1315. http://dx.doi.org/10.1021/jf60232a049.
  » http://dx.doi.org/10.1021/jf60232a049
• Macedo, M. L. R., & Damico, D. C. S. (2000). Effects of protein fractions from Zea mays L. on development and survival of Mexican bean weevil (Boh.). Zabrotes subfasciatusInsect Science, 24(1), 129-130.
• Majolo, C., Carvalho, H. H., & Wiest, J. M. (2013). Atividade antibacteriana “in vitro” de diferentes acessos de urucum (Bixa orellana L.) e sua relação com o teor de bixina presente nas sementes. Boletim do Centro de Pesquisa e Processamento de Alimentos, 31(1), 115-124.
• Mantovani, N. C., Grando, M. F., Xavier, A., & Otoni, W. C. (2013). Avaliação de genótipos de urucum ( L.) por meio da caracterização morfológica de frutos, produtividade de sementes e teor de bixina. Bixa orellanaCiência Florestal, 23(2), 355-362.
• Moraes, J. F. V., & Rabelo, N. A. (1986). Um método simples para a digestão de amostras de plantas. Brasília: EMBRAPA–DDT.
• Moraes, S. A. (2007). Subprodutos da agroindústria e indicadores externos de digestibilidade aparente em caprinos (Tese de doutorado). Universidade Federal de Minas Gerais, Belo Horizonte.
• Oliveira, J. S. (2005). Caracterização, extração e purificação por cromatografia de compostos de urucum (Bixa orellana L.) (Tese de doutorado). Universidade Federal de Santa Catarina, Florianópolis.
• Paz, J., Baldochi, M. R., Paz, K., Prado, F. O., Martins, C. H. G., Lopes, R. A., Ribeiro, A. F., & Sala, M. A. (2006). Estudo hematológico em ratos sob ação de plantas medicinais. XXXVIII. Ação da bixina, um corante alimentar natural extraído de L. Bixa orellanaRevista Brasileira de Plantas Medicinais, 8(4), 157-161.
• Pedrosa, J. P., Cirne, L. E. M. R., & Neto, J. M. M. (1999). Teores de bixina e proteína em sementes de urucum em função do tipo e do período de armazenagem. Revista Brasileira de Engenharia Agrícola e Ambiental, 1(1), 121-123.
• Pereira, E. S., Regadas Filho, J. G. L., Freitas, E. R., Neiva, J. N. M., & Cândido, M. J. D. (2009). Valor energético de subprodutos da agroindústria brasileira. Archivos de Zootecnia, 58(223), 455-458. http://dx.doi.org/10.4321/S0004-05922009000300015.
  » http://dx.doi.org/10.4321/S0004-05922009000300015
• Pires, C. V., Oliveira, M. G. de A., Rosa, J. C., & Costa, N. M. B. (2006). Qualidade nutricional e escore químico de aminoácidos de diferentes fontes proteicas. Ciência e Tecnologia de Alimentos, 26(1), 179-187. http://dx.doi.org/10.1590/S0101-20612006000100029.
  » http://dx.doi.org/10.1590/S0101-20612006000100029
• Reeves, P. G., Nielsen, F. H., & Fahey, G. C. Jr (1993). AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. The Journal of Nutrition, 123(11), 1939-1951. PMid:8229312.
• Salinas, J., Garcia, R., & Garcia, R. (1985). Métodos químicos para el análisis de suelos ácidos y plantas forrajeras. Cali: Centro Internacional de Agricultura Tropical, Programa de Pastos Tropicalis.
• Sandi, M. P. & Cuen, B. R. (2003). Biodiversitas. El Achiote: Boletín Bimestral de la Comisíon Nacional para el Conocimiento y Uso de la Biodiversidad, 7(46), 7-11.
• Santos, M. R. V., Souza, V. H., Menezes, I. A. C., Bitencurt, J. L., Rezende Neto, J. M., Barreto, A. S., Cunha, F. A., Marçal, R. M., Teixeira-Silva, F., Quíntans Júnior, L. J., & Barbosa, A. P. O. (2010). Parâmetros bioquímicos, fisiológicos e morfológicos de ratos ( linghagem Wistar) produzidos pelo biotério central da Universidade Federal de Sergipe. Rattus novergiusScientia Plena, 6(10), 1-6.
• Silva, M. S., Naves, M. M., Oliveira, R. B., & Leite, O. S. M. (2006). Composição química e valor protéico do resíduo de soja em relação ao grão de soja. Ciência e Tecnologia de Alimentos., 26(3), 571-576. http://dx.doi.org/10.1590/S0101-20612006000300014.
  » http://dx.doi.org/10.1590/S0101-20612006000300014
• Souza, A. V. C. (2003). Interpretando os índices de conversão alimentar (I.C.A) e de eficiência alimentar (I.E.A).Osasco: Poli-Nutri Alimentos. Artigo técnico.Retrieved from http://www.polinutri.com.br/
  » http://www.polinutri.com.br/
• Tirapegui, J., Castro, I. A., & Rossi, L. (2012). Biodisponibilidade de proteínas. In S. M. F. Cozzolino, Biodisponibilidade de Nutrientes (cap. 6, pp. 131-192). Barueri: Manole.
• Tirapegui, J., Rogero, M. M., & Lajolo, F. M. (2008). Proteínas e aminoácidos. In J. E. Dutra-de-Oliveira & J. S. Marchini, Ciências nutricionais: aprendendo a aprender (cap. 3, pp. 53-91). São Paulo: Sarvier.
• Utiyama, C. E., Miyada, V. S., Figueiredo, A. N., & Oetting, L. (2002). Digestibilidade de nutrientes do resíduo de semente processadas de urucum (Bixa orellana) para suinos. In Reunião Anual da Sociedade Brasileira de Zootecnia, Recife. Recife: Sociedade Brasileira de Zootecnia.
• Witisuwannakul, R., Witisuwannakul, C., & Sakulborirug, A. (1998). Lectin from the bark of the rubber tree (Hevea brasiliensis). Phytochemistry, 36, 899-905.

Publication Dates

• Publication in this collection
  June 2015

History

• Received
  15 Dec 2014
• Accepted
  08 Mar 2015