**5. Soybean products application in the food industry**

essential for the growth of the sector [34].

ty, responsible for the characteristic flavor and taste [11, 29].

do not differ by Tukey test (p ≤ 0.05).

488 Soybean - Bio-Active Compounds

**Embrapa 48** 4.27 ± 2.52a 5.16 ± 2.50b 4.14 ± 2.28b 4.75 ± 2.39a **BRS 213** 5.24 ± 2.44a 6.44 ± 2.05a 5.61 ± 2.08a 5.76 ± 2.11a **BRS 216** 4.31 ± 2.36a 5.75 ± 2.00ab 4.71 ± 2.32ab 5.12 ± 2.22a **BRS 232** 5.12 ± 2.84a 6.50 ± 2.33a 4.73 ± 2.78ab 5.82 ± 2.65a **BRS 257** 5.26 ± 2.26a 6.05 ± 2.18ab 5.14 ± 2.27ab 5.90 ± 2.11a **BRS 258** 5.17 ± 2.35a 5.87 ± 2.04ab 5.37 ± 2.37ab 5.78 ± 2.26a **BRS 267** 4.74 ± 2.23a 5.80 ± 2.17ab 4.84 ± 2.11ab 5.34 ± 2.16a **BRS 282** 4.97 ± 2.53a 5.98 ± 2.17ab 4.98 ± 2.51ab 5.51 ± 2.63a

> The soybean cultivars currently available in Brazil have different characteristics of produc‐ tivity, production cycle, grain size, climate adaptation, lipoxygenase activity, and others. However, the Brazilian consumption of soybean as a food is still small, due to its exotic fla‐ vor to the palate, since it is an Asian grain and its development was based on the habits and customs of the orientals. These exotic flavors can be assigned to the presence of lipoxyge‐ nase enzymes, saponins and phenolic compounds, responsible for rancid or beany flavors, bitter and astringent, respectively.

> A lot of products can be obtained from the soybean. However, the Brazilian food industry had to adapt them to the consumers habits, like the soymilk applied in soy beverages, which for a better acceptance is developed and commercialized with the addition of flavors or fruit juice. This is the most popular and consumed soy derivative in Brazil.

> Fortunately, with more information published about the benefits of soybean consumption, its nutritional value and functional properties, this scenario is changing. After recognizing the great importance of this legume, several studies on the development of cultivars with better acceptability started, with the goal to insert soybean as an essential part of the human food. To achieve this, however, in addition to the cultivar adaptation, it is also necessary to check if this cultivar is interesting for industrialization.

> The yield is a very important variable in the food industry, but cannot be considered an ex‐ clusion factor, since other important variables, such as composition, functional characteris‐ tics (isoflavones) and antinutritional compounds (trypsin inhibitor) should be observed for the choice of a soybean cultivar. Therefore, the use of cultivars specially developed for soy‐ bean based products, directed to human consumption, may contribute to improve the senso‐ ry quality and increase the soybean acceptability as a food.

## **Author details**

Neusa Fátima Seibel1\*, Fernanda Périco Alves2 , Marcelo Álvares de Oliveira3 and Rodrigo Santos Leite4

\*Address all correspondence to<EMAIL_ADDRESS>
1 Mestrado Profissional em Tecnologia de Alimentos. Universidade Tecnológica Federal do Paraná (UTFPR). Londrina/PR, Brasil

[9] Campelo, G. J. A., Kiihl, R. A. S., & Almeida, L. A. (1999). Características agronôm‐ icas e morfológicas das cultivares de soja desenvolvidas para as regiões de baixas lat‐ itudes. In: Recursos genéticos e melhoramento de plantas para o nordeste brasileiro.

Brazilian Soybean Varieties for Human Use http://dx.doi.org/10.5772/52602 491

Available in http://www.cpatsa.embrapa.br/catalogo/livrorg/temas.html , 15.

brasileira. *In: EMBRAPA SOJA. Documento 113. Londrina: Embrapa Soja*.

women in Hong Kong. *Journal of Nutrition.*, 137, 2451-2455.

*Soja,. CD-ROM.*

122-133.

*da soja*, 41-45.

mentos, 206 , 60.

*istry*, 50(21), 5987-5993.

*pa Soja: Fundação Meridional*, 60.

[10] Carrão-Panizzi, M.C., & Mandarino, J.M.G. (1998). Soja: potencial de uso na dieta

[11] Carrão-Panizzi, M.C., Mandarino, J.M.G., Bordingnon, J. R., & Kikuchi, A. (2000). Al‐ ternativa alimentar na dieta humana. *In: A cultura da soja no Brasil. Londrina: Embrapa*

[12] Cecchi, H.M. (2003). Fundamentos teóricos e práticos em análise de alimentos. 2 ed.. ,

[13] Chan, S., Ho, S. C., Kreiger, N., Darlington, G., So, K. F., & Chong, P. Y. Y. (2007). Dietary sources and determinants of soy isoflavone intake among midlife chinese

[14] Chang, Y. K. (2001). Alimentos Funcionais e AplicaçãoTecnológica: Padaria de Saúde e Centro de Pesquisas em Tecnologia de Extrusão. *In: EMBRAPA Soja. (Org.). Anais do I Simpósio Brasileiro sobre os benefícios da soja para a Saúde Humana. Londrina: Embrapa*

[15] Dutcosky, S. D. (2007). Análise Sensorial de alimentos. *2 ed., Curitiba: Champagnat*.

[16] Embrapa soja. (2010). Cultivares de soja 2010/2011 região centro-sul. *Londrina: Embra‐*

[17] Embrapa soja. (2003). Manual de receitas com soja. Londrina: Embrapa Soja Docu‐

[18] Esteves, E. A., & Monteiro, J. B. R. (2001, jan-abr.) Efeitos benéficos das isoflavonas de

[19] Farias, J. R. B. (1994). Climatic requirements. *In: EMBRAPA-CNPSo. (Ed.) Tropical soy‐*

[20] Friedman, N. M., & Brandon, D. L. (2001). Nutritional and health benefits of soy pro‐

[21] Genovese, M. I., & Lajolo, F.M. (2006, jan./fev.) Fatores antinutricionais da soja. *In‐*

[22] Genovese, M. I., & Lajolo, F. M. (2002). Isoflavones in soy based foods consumed in Brazil: levels, distribution and estimated intake. *Journal of Agricultural and Food Chem‐*

[23] Hodges, T., & FRENCH, V. (1985). Soyphen: soybean growth stages modeled from temperature, day length and water availability. *Agronomic Journal*, 77, 500-505.

soja em doenças crônicas. *Revista de Nutrição,Campinas*, 14(1), 43-52.

teins. *Journal of Agricultural and Food Chemistry*, 49, 1069-1086.

*bean: improvement and production. Rome: FAO*, 13-17.

*forme Agropecuário, Belo Horizonte*, 27(230), 18-33.

4 Mestrando em Tecnologia de Alimentos (UTFPR). Embrapa Soja. Londrina/PR, Brasil

### **References**

[9] Campelo, G. J. A., Kiihl, R. A. S., & Almeida, L. A. (1999). Características agronôm‐ icas e morfológicas das cultivares de soja desenvolvidas para as regiões de baixas lat‐ itudes. In: Recursos genéticos e melhoramento de plantas para o nordeste brasileiro. Available in http://www.cpatsa.embrapa.br/catalogo/livrorg/temas.html , 15.

**Author details**

490 Soybean - Bio-Active Compounds

Rodrigo Santos Leite4

**References**

Neusa Fátima Seibel1\*, Fernanda Périco Alves2

Paraná (UTFPR). Londrina/PR, Brasil

3 Embrapa Soja. Londrina/PR, Brasil

*Nutrition*, 125, 581S-588S.

\*Address all correspondence to<EMAIL_ADDRESS>
2 Tecnóloga em Alimentos (UTFPR). Londrina/PR, Brasil

, Marcelo Álvares de Oliveira3

1 Mestrado Profissional em Tecnologia de Alimentos. Universidade Tecnológica Federal do

4 Mestrando em Tecnologia de Alimentos (UTFPR). Embrapa Soja. Londrina/PR, Brasil

www.cpatsa.embrapa.br/catalogo/livrorg/temas.html , 15.

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[2] Anderson, R. L., & Wolf, W. J. (1995). Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoftavones Related to Soybean Processing. *The Journal of*

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[4] Bedani, R., & Rossi, E. A. (2005, jul.-dez.) Isoflavonas; Bioquímica, fisiologia e impli‐

[5] Behrens, BJ. H., & Silva, M. A. A. P. (2004, jul.-set..) Atitude do consumidor em rela‐ ção à soja e produtos derivados. *Ciênc. Tecnol. Aliment., Campinas*, 24(3), 431-439.

[6] Benassi, V. T., Benassi, M. T., & Prudêncio, S. H. (2011). Cultivares brasileiras de soja: características para a produção de tofu e aceitação pelo mercado consumidor. *Semina:*

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[8] Callou, K. L. A. (2009). Teor de isoflavonas e capacidade antioxidante de bebidas à base de soja. *124 f. Dissertação (Mestrado- Ciência dos Alimentos)- Faculdade de Ciências*

and

[24] Jackson, J. C., Dini, J. P., Lavandier, C., Rupasinghe, H. P. V., Faulkner, H., Poysa, V., Buzzell, D., & DeGrandies, S. (2002). Effects of processing on the content and compo‐ sition of isoflavones during manufacturing of soy beverage and tofu. *Process Biochem‐ istry*, 37, 1117-1123.

newly developed cultivars of Brazilian soybean (Glycine max). *Journal of the Science of*

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U., Melo, V. M. M., Carlini, C. R., & Castelar, L. I. M. (2001). Nutritional study of two Brazilian soybean (Glycine max) cultivars differing in the contents of antinutritional

[39] Vasconcelos, VI. M., Maia, A. A. B., Siebra, E. A., Oliveira, J. T. A., Carvalho, A. F. F.

[40] Vasconcelos, I. M., Campello, C. C., Oliveira, J. T. A., Carvalho, A. F. U., Souza, D. O.

[41] Wilcox, J. R., & Shibles, R. M. (2001). Interrelationships among seed quality attributes

[42] Zava, D. T., & Duwe, G. (1997). Estrogenic and antiproliferative properties of genis‐

[43] Zeller, F. J. (1999). Soybean (Glycine max (L.) Merril): utilization, genetics, biotech‐

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B., & Maia, F. M. M. (2006). Brazilian soybean Glycine max (L.) Merr.cultivars adapt‐ ed to low latitude regions: seed composition and content of bioactive proteins. *Rev.*

and toxic proteins. *Journal of Nutritional Biochemistry*, 12, 1-8.

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[24] Jackson, J. C., Dini, J. P., Lavandier, C., Rupasinghe, H. P. V., Faulkner, H., Poysa, V., Buzzell, D., & DeGrandies, S. (2002). Effects of processing on the content and compo‐ sition of isoflavones during manufacturing of soy beverage and tofu. *Process Biochem‐*

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[34] Sediyama, T. (2009). Tecnologias de produção e usos da soja. *Londrina: Mecenas*, 306.

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**Chapter 22**

**Effects of Soybean Trypsin Inhibitor on Hemostasis**

Soy bean trypsin inhibitor (SBTI) belongs to the family of serpins – serine protease inhibitors widely distributed in the nature [Silverman G.A. et al., 2004; 21]. Serpins participate in the regulation of proteopytic reactions underling very important physiological and pathological processes such as digestion [16], blood clotting [3, 14, 17], immunity [44] apoptosis [36, 42], inflammation [10], dystrophy [31], carcinogenesis [22, 11] and so on. Despite the apparent importance of the correction of imbalances of the proteolysis in pathology, in fact only the pancreatic trypsin inhibitor (aprotinin) is used more or less widely as a protease inhibitor drug (Trasisol, Contrical, Gordox etc) in the treatment of some diseases [24, 13, 40]. Being the animal protein aprotinin possesses substantial disadvantages [23, 12] and attempts to de‐ velop new drug on the base of plant or recombinant proteins or peptidomimetics are under‐

Among proteolytic processes hemostasis is of special importance because of the high fre‐ quency of it's disturbances, accompanied many pathological processes and diseases [20, 18]. At first sight it looks strange to expect that plant protease inhibitor SBTI should influence hemostasis in humans because blood clotting represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43]. However, results of the very early studies sup‐ port such possibility [26, 41]. The present study was aimed to investigate the influence of SBTI and aprotinin on hemostasis using modern bioinformatics approach and classical in vi‐

> © 2013 Borodin et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Borodin et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Eugene A. Borodin, Igor E. Pamirsky,

http://dx.doi.org/10.5772/52600

**1. Introduction**

Alexander V. Korotkikh, Chie Tarumizu,

Mikhail A. Shtarberg, Vladimir A. Dorovskikh,

Kiyoharu Takamatsu and Shigeru Yamamoto

Additional information is available at the end of the chapter

taken [35, 27]. SBTI is one of the candidates for such a role [34].

tro methods. The concrete purposes of the study included:

## **Effects of Soybean Trypsin Inhibitor on Hemostasis**

Eugene A. Borodin, Igor E. Pamirsky, Mikhail A. Shtarberg, Vladimir A. Dorovskikh, Alexander V. Korotkikh, Chie Tarumizu, Kiyoharu Takamatsu and Shigeru Yamamoto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52600

## **1. Introduction**

Soy bean trypsin inhibitor (SBTI) belongs to the family of serpins – serine protease inhibitors widely distributed in the nature [Silverman G.A. et al., 2004; 21]. Serpins participate in the regulation of proteopytic reactions underling very important physiological and pathological processes such as digestion [16], blood clotting [3, 14, 17], immunity [44] apoptosis [36, 42], inflammation [10], dystrophy [31], carcinogenesis [22, 11] and so on. Despite the apparent importance of the correction of imbalances of the proteolysis in pathology, in fact only the pancreatic trypsin inhibitor (aprotinin) is used more or less widely as a protease inhibitor drug (Trasisol, Contrical, Gordox etc) in the treatment of some diseases [24, 13, 40]. Being the animal protein aprotinin possesses substantial disadvantages [23, 12] and attempts to de‐ velop new drug on the base of plant or recombinant proteins or peptidomimetics are under‐ taken [35, 27]. SBTI is one of the candidates for such a role [34].

Among proteolytic processes hemostasis is of special importance because of the high fre‐ quency of it's disturbances, accompanied many pathological processes and diseases [20, 18]. At first sight it looks strange to expect that plant protease inhibitor SBTI should influence hemostasis in humans because blood clotting represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43]. However, results of the very early studies sup‐ port such possibility [26, 41]. The present study was aimed to investigate the influence of SBTI and aprotinin on hemostasis using modern bioinformatics approach and classical in vi‐ tro methods. The concrete purposes of the study included:

© 2013 Borodin et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Borodin et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**1.** To establish the extent of structural homology, compare functional activities and evalu‐ ate potential targets of SBTI and aprotinin among the human proteases by bioinformat‐ ics (*in silico*) methods.

**Database ID in the Database**

http://www.ncbi.nlm.nih.gov P00974 (Precursor); 1QLQ0 1AVU

To investigate the homology of the primary structures of SBTI and aprotinin we used BLAST (Basic Local Alignment Search Tool) algorithm, sequence alignment editor Bio Edit 5.0.9 and got information on these proteins from Protein Data Bank and Uni Prot using FAS‐ TA (Table 2), MOL and PDB format-files. The extent of homology of the total sequences of SBTI and aprotinin, calculated by the method of the multiple paired alignment, makes up 10% (Figures 1 and 2). The low homology of the total sequences of should be attributed to the different lengths of polypeptide chains these proteins – 58 amino acids in aprotinin [45]

**FASTA fromat** A B C D E F G H I K M

**FASTA fromat** N P Q R S T V W Y Z **-**

**Table 2.** Designation of aminoacids in FASTA format. \*- symbol in FASTA format corresponds to the gap of unlimited

Comparison of the separate domains of SBTI and aprotinin reveals greater sequence similar‐ ity. Thus, the extent of homology of the N-terminal region of SBTI (5-60 amino acid resi‐ dues) and the sequence of aprotonin without last two amino acids makes up 21%. The central region of SBTI (85-116 amino acid residues) and the region of aprotinin within 25-56 amino acids show 24% similarity. The highest extent of homology up to 35% is characteristic

Ala Asn Cys Asp Glu Phe Gly His Ile Lis Met

Asn Pro Gln Arg Ser Tre Val Trp Tyr Glu \*

http://www.pasteur.fr

PDB - Protein Data Bank http://www.rcsb.org

MEROPS (peptidases) http://merops.sanger.ac.uk

and 181 in SBTI [39].

**Traditional abbreviation**

**Traditional abbreviation**

length

MMDB - Molecular Modeling Data Base

**Table 1.** Databases containing the information on aprotinin and SBTI.

**2.1. The homology of the primary structures of SBTI and aprotinin**

**Aprotinin SBTI**

Effects of Soybean Trypsin Inhibitor on Hemostasis

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497

1OA6 1AVU; 1BA7

I02.001 I03.001

## **2. The study of aprotinin and SBTI** *in silico*

*In silico* methods (bioinformatics) entails the creation and advancement of databases, algo‐ rithms, computational techniques to solve formal and practical problems arising from the management and analysis of biological data. One of the potential areas for exploiting these methods is computational drug design. We used *in silico* methods in our study for the com‐ parison of structural homology, functional activities and evaluation of the potential targets of SBTI and aprotinin among the human proteases.

From fifty databases we had browsed in the INTRNET nine contained information on apro‐ tinin and SBTI (Table 1). From these PDB (Protein Data Bank) and UniProt/Swiss-Prot were the most informative and we used these databases in our study.

**Table 1.** Databases containing the information on aprotinin and SBTI.

**1.** To establish the extent of structural homology, compare functional activities and evalu‐ ate potential targets of SBTI and aprotinin among the human proteases by bioinformat‐

**2.** To investigate the influence of trypsin, SBTI and aprotinin on some indexes of blood clotting, fibrilolysis and platelet aggregation as well as on the hemolytic activity of the

**3.** To elucidate the possibility of the regulation of the total proteolytic and tripsin-inhibit‐ ing activity of blood plasma *in vivo* by the consumption of soy foods enriched with soy

*In silico* methods (bioinformatics) entails the creation and advancement of databases, algo‐ rithms, computational techniques to solve formal and practical problems arising from the management and analysis of biological data. One of the potential areas for exploiting these methods is computational drug design. We used *in silico* methods in our study for the com‐ parison of structural homology, functional activities and evaluation of the potential targets

From fifty databases we had browsed in the INTRNET nine contained information on apro‐ tinin and SBTI (Table 1). From these PDB (Protein Data Bank) and UniProt/Swiss-Prot were

http://www.expasy.org BPT1\_BOVIN (P00974) ITRA\_SOYBN (P01070)

**Aprotinin SBTI**

P00974 IPR002160

btau0:ENSBTAG00000017328 ?atha0:At1g17860.1

Precursor P00974; NP\_001001554; -

P00974/BPT1\_BOVIN; PIRSF001621 -

100156830; Bt.32343;BPT1\_BOVIN -

50059016; 3809839; bta:616039;

protein isolate (SPI) possessing thermo-stable fraction of SBTI.

**2. The study of aprotinin and SBTI** *in silico*

of SBTI and aprotinin among the human proteases.

the most informative and we used these databases in our study.

**Database ID in the Database**

ics (*in silico*) methods.

496 Soybean - Bio-Active Compounds

complement *in vitro*.

UniProt-Swiss-Prot

Groups of proteins

Blocks - most highly conserved regions of

COG - the database of Clusters of Ortologous

GTOP - Genomes TO Protein structures and functions http://spock.genes.nig.ac.jp

iProClass - an integrated, comprehensive and annotated Protein Classification database

LIGAND - LIGAND chemical database for

proteins http://www.ebi.ac.uk

http://www.ncbi.nlm.nih.gov

http://pir.georgetown.edu

enzyme reactions

#### **2.1. The homology of the primary structures of SBTI and aprotinin**

To investigate the homology of the primary structures of SBTI and aprotinin we used BLAST (Basic Local Alignment Search Tool) algorithm, sequence alignment editor Bio Edit 5.0.9 and got information on these proteins from Protein Data Bank and Uni Prot using FAS‐ TA (Table 2), MOL and PDB format-files. The extent of homology of the total sequences of SBTI and aprotinin, calculated by the method of the multiple paired alignment, makes up 10% (Figures 1 and 2). The low homology of the total sequences of should be attributed to the different lengths of polypeptide chains these proteins – 58 amino acids in aprotinin [45] and 181 in SBTI [39].

**Table 2.** Designation of aminoacids in FASTA format. \*- symbol in FASTA format corresponds to the gap of unlimited length

Comparison of the separate domains of SBTI and aprotinin reveals greater sequence similar‐ ity. Thus, the extent of homology of the N-terminal region of SBTI (5-60 amino acid resi‐ dues) and the sequence of aprotonin without last two amino acids makes up 21%. The central region of SBTI (85-116 amino acid residues) and the region of aprotinin within 25-56 amino acids show 24% similarity. The highest extent of homology up to 35% is characteristic for C-terminal region of SBTI (132-181 amino acid residues) and aprotenin sequence without six C-terminal amino acids.

We failed to find molecular targets of SBTI and aprotinin because there was no necessary

Effects of Soybean Trypsin Inhibitor on Hemostasis

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499

**Figure 3.** Electronic 3D-structures of SBTI and aprotinin. Left – model with the single surface (Chem Office 5.0). Right-

The exploiting the PASS software shows four potential activities of aprotinin and three for SBTI. Both SBTI and aprotinin may inhibit rennin as well as angiotensin- and endotheleinconverting enzymes according to the revealing of the possible biological activities of these protease inhibitors by ISIS/Draw 2.4 and PASS Professional chemical structure drawing pro‐ grams (Figure 4). The possibility of exerting of such effects (drug-likeness) is nearly the same in both protease inhibitors. Aprotinin may inhibit neutral endopeptidase, also. For all the abovementioned activities probability of the activity (Pa) is higher than the probability of

carcinogenic and teratogenic effects according to in silico, in vitro and in vivo studies.

). SBTI and aprotinin should not reveal serious toxicity, mutagenic,

software in free excess.

ribbon diagram (Yasara 6.2.5).

the lack of activity (Pi

**2.3. Spectra of biological activity of SBTI and aprotinin**

**Figure 1.** The screen of the Bio Edit 5.0.9 with the results of calculation of homology of Aprotinin and SBTI. The identi‐ cal amino acids are marked with black and similar with gray. Amino acid sequence are represented in FASTA format.

**Figure 2.** Homological regions of Aprotinin and SBTI (according to Bio Edit 5.0.9). The identical amino acids are marked with black and similar with gray.

#### **2.2. Tertiary structures of SBTI and aprotinin**

For the visualization of the 3D-structures of SBTI and aprotinin we used Chem Office 5.0 and Yasara 6.2.5 software. The files in MOL format, containing the information on the se‐ quences of these protease inhibitors, were imputed into Chem Office 5.0 software and con‐ verted in PDB format. The obtained PDB files were imputed into Yasara 6.2.5 software. The results of the generation of the electronic 3D-structures of aprotinin and SBTI are presented in Figure 3. Apparently there there is similarity of 3D-structures of these proteins.

We failed to find molecular targets of SBTI and aprotinin because there was no necessary software in free excess.

for C-terminal region of SBTI (132-181 amino acid residues) and aprotenin sequence without

**Figure 1.** The screen of the Bio Edit 5.0.9 with the results of calculation of homology of Aprotinin and SBTI. The identi‐ cal amino acids are marked with black and similar with gray. Amino acid sequence are represented in FASTA format.

**Figure 2.** Homological regions of Aprotinin and SBTI (according to Bio Edit 5.0.9). The identical amino acids are

For the visualization of the 3D-structures of SBTI and aprotinin we used Chem Office 5.0 and Yasara 6.2.5 software. The files in MOL format, containing the information on the se‐ quences of these protease inhibitors, were imputed into Chem Office 5.0 software and con‐ verted in PDB format. The obtained PDB files were imputed into Yasara 6.2.5 software. The results of the generation of the electronic 3D-structures of aprotinin and SBTI are presented

in Figure 3. Apparently there there is similarity of 3D-structures of these proteins.

six C-terminal amino acids.

498 Soybean - Bio-Active Compounds

marked with black and similar with gray.

**2.2. Tertiary structures of SBTI and aprotinin**

**Figure 3.** Electronic 3D-structures of SBTI and aprotinin. Left – model with the single surface (Chem Office 5.0). Rightribbon diagram (Yasara 6.2.5).

#### **2.3. Spectra of biological activity of SBTI and aprotinin**

The exploiting the PASS software shows four potential activities of aprotinin and three for SBTI. Both SBTI and aprotinin may inhibit rennin as well as angiotensin- and endotheleinconverting enzymes according to the revealing of the possible biological activities of these protease inhibitors by ISIS/Draw 2.4 and PASS Professional chemical structure drawing pro‐ grams (Figure 4). The possibility of exerting of such effects (drug-likeness) is nearly the same in both protease inhibitors. Aprotinin may inhibit neutral endopeptidase, also. For all the abovementioned activities probability of the activity (Pa) is higher than the probability of the lack of activity (Pi ). SBTI and aprotinin should not reveal serious toxicity, mutagenic, carcinogenic and teratogenic effects according to in silico, in vitro and in vivo studies.

2-times higher than similar activity of SBTI. However, taking into account that molecular weight of SBTI (20100) is 3-times higher than molecular weight of aprotinin (6514), it should be concluded that the trypsine-inhibitory activity of SBTI, calculated per mole of the inhibi‐ tor, should be nearly 1,5-times higher than trypsine-inhibitory activity of aptotinin. Tryp‐ sine-inhibitory activity of ε-aminocapronic acid and D-lysine when calculated per mole of

The following indexes, characterizing mainly the first and the second phases of blood clot‐ ting, were measured: prothrombin time (reflects activity of coagulation factors YII, V, X and II), activated partial thromboplastin time (reflects activity of coagulation factors YII and VIII), activated clotting time (a measure of the anticoagulation affects of heparin) and throm‐

> **Plasma + trypsin (2)**

13±0,9 Р1-2<0,0001

3±0,9 Р1-2<0,0001

2,5±0,5 Р1-2<0,0001

58±4,7 Р1-2<0.002

182±7

**Table 4.** The influence of trypsin, SBTI and aprotinin on the indexes of hemostasis *in vitro*. The final concentration of

Prothrombin time decreases 33-35% in the presence of trypsin from 20±0,9 to 13±0,9 sec, in‐ creases insignificantly in the presence of aprotinin and increases 1,5-times up to 31±0,9 sec in the presence of SBTI. Activated partial thromboplastin time sharply decreases by trypsin from 36±1,7 sec in the control samples to 3,0+0,9 sec and increases 3-times by aprotinin (113±1,8 sec) and SBTI (105±2,7 sec). The changes of the activated clotting time under the in‐ fluence of trypsin, aprotinin and SBTI are similar. Thrombin time decreases nearly 7-fold in the presence of trypsin from 16±0,9 to 2,5±0.5 sec and increases on 50% by aprotinin to 24±0,9 sec. SBTI completely blocked the formation of fibrin clot at least within 10 min. Simi‐ lar effects trypsin and it's inhibitors exerts on fibrinolysis. Euglobulin lysis time (factor XII–

**Plasma + aprotinin (3)**

21±0,9 Р1-3"/0,05

113±1,8 Р1-3<0,0001

24±0,9

157±12 Р1-4<0.0001

<sup>Р</sup>1-2<0,0001 No lysis of blood clot No lysis of blood clot

<sup>Р</sup>1-3<0,0001 <sup>∞</sup>

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**Plasma + SBTI (4)**

31±0,9 Р1-4<0,0001

105±2,7 Р1-4<0,0001

258±17 Р1-4<0.001

bin time (time of the formation of fibrin clot). Results are summarized in the table 4.

the inhibitor is negligibly low because of the low molecular weight.

**3.2. The influence of SBTI and aprotinin on coagulation hemostasis**

**Time,sec Plasma (control) (1)**

36±1,7

trypsin in the incubation medium consisted of 0,01%, SBTI and aprotinin - 0,1%..

**Indexes**

Activated partial thromboplastin time

Prothrombin time 20±0,9

Thrombine time 16±0,9

Ativated clotting time 88±9,7

Euglobulin lysis time 400±34,7

**Figure 4.** Spectra of the biological activities of aprotinin and SBTI according to PASS software. Drug-Likeness – proba‐ bility of exerting the effect. Probability of the activity (Pa) and the lack of activity (Pi ). The average accuracy of the prog‐ nosis makes up more than 85%.

## **3. The study of aprotinin and SBTI** *in vitro*

*In silico* methods testify the common features in the primary structure, 3D-structures, func‐ tional activities of t SBTI and aprotinin and allow to propose that SBTI should influence processes of hemostasis similar to aprotinin. To prove this assumption we studied the influ‐ ence of trypsin, SBTI and aprotinin on blood clottting, fibrilolysis and platelets aggregation *in vitro*.

#### **3.1. Trypsin-inhibitory activity of SBTI and aprotinin**

We measured tyipsin-inhibitory activity of aprotinin, SBTI and low molecular weight pro‐ tease inhibitors (ε-aminocapronic acid and D-lysine) by their ability to inhibit BAEE-esterase activity of the trypsin [33]. The results are presented in the table 3.

**Table 3.** Trypsin-inhibitory activity of aprotinin, SBTI, ε-aminocapronic acid and D-lysine.

Trypsin-inhibitory activity aprotinin, SBTI, ε-aminocapronic acid and D-lysine was meas‐ ured as their ability to inhibit BAEE-esterase activity of trypsin.

Tyipsin-inhibitory activity, calculated per mg of inhibitor, decrease down in the order: apro‐ tinin > SBTI > aminocapronic acid > D-lysine. Thus, tyipsin-inhibitory activity of aprotinin is 2-times higher than similar activity of SBTI. However, taking into account that molecular weight of SBTI (20100) is 3-times higher than molecular weight of aprotinin (6514), it should be concluded that the trypsine-inhibitory activity of SBTI, calculated per mole of the inhibi‐ tor, should be nearly 1,5-times higher than trypsine-inhibitory activity of aptotinin. Tryp‐ sine-inhibitory activity of ε-aminocapronic acid and D-lysine when calculated per mole of the inhibitor is negligibly low because of the low molecular weight.

#### **3.2. The influence of SBTI and aprotinin on coagulation hemostasis**

**Figure 4.** Spectra of the biological activities of aprotinin and SBTI according to PASS software. Drug-Likeness – proba‐

*In silico* methods testify the common features in the primary structure, 3D-structures, func‐ tional activities of t SBTI and aprotinin and allow to propose that SBTI should influence processes of hemostasis similar to aprotinin. To prove this assumption we studied the influ‐ ence of trypsin, SBTI and aprotinin on blood clottting, fibrilolysis and platelets aggregation

We measured tyipsin-inhibitory activity of aprotinin, SBTI and low molecular weight pro‐ tease inhibitors (ε-aminocapronic acid and D-lysine) by their ability to inhibit BAEE-esterase

**Trypsin inhibitor Aprotinin SBTI ε-aminocapronic acid D-lysine**

0,922±0,085 1,23±0,02 - -

Trypsin-inhibitory activity (IU/mg) 141±13 61±0,9 1,36±0,04 1,31±0,02

Trypsin-inhibitory activity aprotinin, SBTI, ε-aminocapronic acid and D-lysine was meas‐

Tyipsin-inhibitory activity, calculated per mg of inhibitor, decrease down in the order: apro‐ tinin > SBTI > aminocapronic acid > D-lysine. Thus, tyipsin-inhibitory activity of aprotinin is

). The average accuracy of the prog‐

bility of exerting the effect. Probability of the activity (Pa) and the lack of activity (Pi

**3. The study of aprotinin and SBTI** *in vitro*

**3.1. Trypsin-inhibitory activity of SBTI and aprotinin**

activity of the trypsin [33]. The results are presented in the table 3.

**Table 3.** Trypsin-inhibitory activity of aprotinin, SBTI, ε-aminocapronic acid and D-lysine.

ured as their ability to inhibit BAEE-esterase activity of trypsin.

nosis makes up more than 85%.

500 Soybean - Bio-Active Compounds

Trypsin-inhibitory activity (IU/nmole)

*in vitro*.

The following indexes, characterizing mainly the first and the second phases of blood clot‐ ting, were measured: prothrombin time (reflects activity of coagulation factors YII, V, X and II), activated partial thromboplastin time (reflects activity of coagulation factors YII and VIII), activated clotting time (a measure of the anticoagulation affects of heparin) and throm‐ bin time (time of the formation of fibrin clot). Results are summarized in the table 4.

**Table 4.** The influence of trypsin, SBTI and aprotinin on the indexes of hemostasis *in vitro*. The final concentration of trypsin in the incubation medium consisted of 0,01%, SBTI and aprotinin - 0,1%..

Prothrombin time decreases 33-35% in the presence of trypsin from 20±0,9 to 13±0,9 sec, in‐ creases insignificantly in the presence of aprotinin and increases 1,5-times up to 31±0,9 sec in the presence of SBTI. Activated partial thromboplastin time sharply decreases by trypsin from 36±1,7 sec in the control samples to 3,0+0,9 sec and increases 3-times by aprotinin (113±1,8 sec) and SBTI (105±2,7 sec). The changes of the activated clotting time under the in‐ fluence of trypsin, aprotinin and SBTI are similar. Thrombin time decreases nearly 7-fold in the presence of trypsin from 16±0,9 to 2,5±0.5 sec and increases on 50% by aprotinin to 24±0,9 sec. SBTI completely blocked the formation of fibrin clot at least within 10 min. Simi‐ lar effects trypsin and it's inhibitors exerts on fibrinolysis. Euglobulin lysis time (factor XII– callicrein-dependent fibrinolysis) decreases 60% by trypsin from 400±35 to 182±7 sec. Aptoti‐ nin and SBTI in the abovementioned concentrations entirely inhibit the lysis of fibrin clot (Table 4).

mum aggregation time of irreversible ADP-initiated aggregation). SBTI and aprotinin re‐

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**Figure 5.** ADP-initiated reversible aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 5 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4

**Figure 6.** ADP-initiated two-phase aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 5 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4

– trypsin – 0,1%.

– trypsin – 0,1%.

veals anti-aggregation effect manifested by the decrease of maximum aggregation.

## **3.3 The influence of SBTI and aprotinin on platelet aggregation**

Results of the study of the influence of SBTI and aprotinin on platelets aggregation are pre‐ sented in the table 5 and Figures 5-8.

**Table 5.** The effects of aprotinin, SBTI and trypsin on the aggregation of platelets initiated by ADP (reversible, twophase and irreversible aggregation) and adrenalin (two-phase aggregation). MA – maximum aggregation (transmittance, %), ТМА – time of accomplishment of maximum aggregation (sec).

The effects of trypsin, SBTI and aprotinin on the aggregation of platelets initiated by ADP (reversible, two-phase and irreversible aggregation) and adrenalin (two-phase aggregation) are similar to their effects on blood coagulation and fibrinolysis. Trypsin increases platelet aggregation (the increase of the maximum of all types of aggregation and decrease of maxi‐ mum aggregation time of irreversible ADP-initiated aggregation). SBTI and aprotinin re‐ veals anti-aggregation effect manifested by the decrease of maximum aggregation.

callicrein-dependent fibrinolysis) decreases 60% by trypsin from 400±35 to 182±7 sec. Aptoti‐ nin and SBTI in the abovementioned concentrations entirely inhibit the lysis of fibrin clot

Results of the study of the influence of SBTI and aprotinin on platelets aggregation are pre‐

**Plasma + aprotinin (2)**

14±0,9 Р1-2<0,01 55±4,7

35±0,9 Р1-2<0,01 68±10,5

56±2 Р1-2<0,02 325±9,2

33±0,9 Р1-2<0,02 410±9,3

16±1,8 Р1-2<0,02 125±13,1

44±2 Р1-2"/0,05 430±18

**Table 5.** The effects of aprotinin, SBTI and trypsin on the aggregation of platelets initiated by ADP (reversible, twophase and irreversible aggregation) and adrenalin (two-phase aggregation). MA – maximum aggregation

The effects of trypsin, SBTI and aprotinin on the aggregation of platelets initiated by ADP (reversible, two-phase and irreversible aggregation) and adrenalin (two-phase aggregation) are similar to their effects on blood coagulation and fibrinolysis. Trypsin increases platelet aggregation (the increase of the maximum of all types of aggregation and decrease of maxi‐

**Plasma + SBTI (3)**

15±0,9 Р1-3<0,01 55±4,7

34±1 Р1-3<0,01 70±9

58±1 Р1-3<0,05 320±9,3

29±0,9 Р1-3<0,02 410±9,3

16±1,8 Р1-3<0,02 125±13,1

44±1,8 Р1-3"/0,05 430±18

**Plasma + trypsin (4)**

34±0,9 Р1-4<0,01

57±3 Р1-4<0,01 80±10

99±1 Р1-4<0,02 310±9,2

100 Р1-4<0,001 210±26,4of

32±0,9 Р1-4<0,01 130±9,3

55±0,9 Р1-4<0,02 435±16,2

65±15

**3.3 The influence of SBTI and aprotinin on platelet aggregation**

**Plasma (control) (1)**

 22±1,8 55±4,7

48±1,8 85±4,7

70±2 350±9,2

49±0,9 400±18

23±0,9 110±9,3

47±2,4 390±9,3

(transmittance, %), ТМА – time of accomplishment of maximum aggregation (sec).

(Table 4).

502 Soybean - Bio-Active Compounds

Reversible (ADP)

Two-phase (ADP): Fist phase МА ТМА

Second phase

Irreversible (ADP)

Two-phase (adrenalin):

ТМА

МА ТМА

МА ТМА

МА ТМА

Fist phase МА ТМА

Second phase

sented in the table 5 and Figures 5-8.

**Figure 5.** ADP-initiated reversible aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 5 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4 – trypsin – 0,1%.

**Figure 6.** ADP-initiated two-phase aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 5 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4 – trypsin – 0,1%.

**3.4. The influence of SBTI and aprotinin on the hemolytic activity of the complement**

system. Results are presented in the table 6 and Figures 9-10.

Control (1) 3,5±0,5 8,5±1,5 Aprotinin (1%) (2) 3,2±0,15 Р1-2"/0,05 7,25±0,25 Р1-2"/0,05 SBNI (1%) (3) 3,2±0,15 Р1-3"/0,05 7,25±0,25 Р1-3"/0,05 Trypsin(0,01%) (4) 4,25±0,25 Р1-4<0,02 15,5±0,5 Р1-4<0,02

**Table 6.** The influence of aprotinin, SBTI and trypsin on the hemolytic activity of complement *in vitro*

**Figure 9.** The hemolytic activity of the complement in the presence of aprotinin, SBTI and trypsin in vitro. The lag-

phase is not shown. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4 – trypsin – 0,1%.

Limited cascade proteolysis underlies the activation of the complement system, which in‐ cludes nearly 20 individual proteins [30, 37]. Majority of these proteins belongs to the serine protease family (subunits C1r and C1s, С3/С5-cinvertase of the classical way of activation, factors I and D) [15]. While complement system does not have direct relation to hemostasis and targeted on alien cells and microorganisms, in some conditions complement system may play a role in the disruption of intrinsic cells of the organism and among them blood cells [15, 37]. Because of this we investigated the influence of trypsin, SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard he‐ molysis of red blood cells by inhibiting activation of some components of the complement

**Lag-phase (min) Total time of hemolysis, including lag-**

**phase (min)**

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**Figure 7.** ADP-initiated irreversible aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 25 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4 – trypsin – 0,1%.

**Figure 8.** Adrenalin-initiated two-phase aggregation of platelets in the presence of trypsin, aptotinin and SBTI in vitro. The platelet aggregation was initiated by the addition of the 25 mkM adrenalin. 1 – controle; 2 – trypsin - 0,1%; 3 – aprotinin - 1%; 4 – SBTI - 1%.

So, the results of *in vitro* experiments proves the ability of SBTI to influence the hemostatsis

predicted by the *in silico* studies.

#### **3.4. The influence of SBTI and aprotinin on the hemolytic activity of the complement**

Limited cascade proteolysis underlies the activation of the complement system, which in‐ cludes nearly 20 individual proteins [30, 37]. Majority of these proteins belongs to the serine protease family (subunits C1r and C1s, С3/С5-cinvertase of the classical way of activation, factors I and D) [15]. While complement system does not have direct relation to hemostasis and targeted on alien cells and microorganisms, in some conditions complement system may play a role in the disruption of intrinsic cells of the organism and among them blood cells [15, 37]. Because of this we investigated the influence of trypsin, SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard he‐ molysis of red blood cells by inhibiting activation of some components of the complement system. Results are presented in the table 6 and Figures 9-10.

**Table 6.** The influence of aprotinin, SBTI and trypsin on the hemolytic activity of complement *in vitro*

**Figure 7.** ADP-initiated irreversible aggregation of platelets in the presence of aptotinin, SBTI and trypsin in vitro. The platelet aggregation was initiated by the addition of the 25 mkM ADP. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4

**Figure 8.** Adrenalin-initiated two-phase aggregation of platelets in the presence of trypsin, aptotinin and SBTI in vitro. The platelet aggregation was initiated by the addition of the 25 mkM adrenalin. 1 – controle; 2 – trypsin - 0,1%; 3 –

So, the results of *in vitro* experiments proves the ability of SBTI to influence the hemostatsis

– trypsin – 0,1%.

504 Soybean - Bio-Active Compounds

aprotinin - 1%; 4 – SBTI - 1%.

predicted by the *in silico* studies.

**Figure 9.** The hemolytic activity of the complement in the presence of aprotinin, SBTI and trypsin in vitro. The lagphase is not shown. 1 – controle; 2 – aprotinin - 1%; 3 – SBTI - 1%; 4 – trypsin – 0,1%.

sumption of soy foods will follow anti-proteolytic effect. Because of this it was interesting to elucidate whether prolonged consumption of the cookies enriched with soy protein isolate (SPI) will influence total proteolytic and trypsin-inhibiting activity of the blood plasma in

According to the characteristics, specified by manufacturer, protein content of SPI consists of 90%. From this 10% belongs to protease inhibitors [32]. Within the processing of soy beans into SPI the former are expoused to high temperatures, which inactivates the major part of soy proteins. However, about 5-20 % of SBTI are represented by the thermostable fraction [19, 22]. The measurement of the trypsin-inhibiting activity of SPI shows that tryp‐ sin-inhibiting activity of SPI consists of 1,4±0,1 IU/mg of SPI. For example 1 mg of pure SBTI possesses activity equal nearby 60 IU. Thus, the active SBTI makes up 2,7% from SPI mass.

**4.2. The influence of the consumption of SPI by healthy persons on the total proteolytic**

30 adult people aged 35-67 years without the expressed signs of chronic diseases consumed cookies, enriched with SPI (30% protein content), for two months [7]. Fasting blood samples were drawn before and after the dietary treatment. Serum samples were frozen and ana‐ lyzed for total proteolytic (BAEE-esterase activity) and tripsin-inhibiting activity [33]. Daily intake of 100g of cookies corresponds to consumption of 30g of SPI and about 0,85g of the active SBTI (nearly 52 00 IU). The total consumption of SPI for two months consists of 1,8 kg or 50 g of an active SBTI. Twenty-eight participants (19 females and 9 males) could complete

**(relative units) Trypsin-inhibitory activity (IU/ml)**

137±5,3 р<0,01

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0,343±0,010 113±3,6

humans or not?

Before the consumption

After the consumption

**4.1. Tripsin-inhibiting activity of SPI**

**and trypsin inhibiting activity of blood serum**

the trial. Results are presented in the table 7.

months consumption of soy protein isolate.

**Total proteolytic activity**

0,282±0,008 р<0,05

**Table 7.** Total proteolytic and trypsin-inhibitory activity of blood serum of healthy persons before and after two

Total proteolytic activity of blood serum was measures as serum BAEE-esterase activity and trypsin-inhibitory activity by inhibiting by serum of BAEE-esterase activity of trypsin.

Two months long SPI consumption was followed by 18% decrease of total proteolytic activi‐ ty of blood serum from 0,343±0,010 to 0,282±0,008 relative units (р<0,05) and by 21% increase of trypsin-inhibiting activity from 113±3,6 to 137±5,3 IU/ml ( р<0,01). The results obtained show possibility of the regulation of proteolysis *in vivo* by the consumption of soy foods.

**Figure 10.** The hemolytic activity of the complement in the presence of different concentrations of trypsin in vitro. The lag-phase is not shown.

SBTI and aprotinin in the final concentrations 0,001-0,1% do not influence hemolysis time and the only statistically insignificant effect consists of small decrease of lag-phase from 3,5±0,3 to 3,2±0,15 min (p>0,05) and total time of hemolysis from 8,5±1,5 to 7,25±0,15 min (p>0,05). Trypsin suppresses the hemolytic activity of the complement in the concentrationdependent manner. In the presence of 0.0001% and 0,001% trypsin the lag-phase of the he‐ molysis increases to 4,25±0,25 min (p<0,02) and total time of hemolysis to 11,5±0,5 min (p<0,02) and 15,5±1,6 min (p<0,01), respectively. 0,01% trypsin entirely blocks the hemolysis. The inhibiting effect of trypsin should be attributed to the disruption of С3 component of the complement system.

Thus, the results obtained show that aprotinin and SBTI in the abovementiioned concentra‐ tions do not influence the hemolytic activity of the complement.

## **4. The influence of the consumption of soy priotein isolate by healthy persons on the indexes of proteolysis in the blood serum**

Soya is cultivated as a foodstuff, having favorable effect on human health, more than 3000 years [28]. Within the last decades soy foods have started to be used in Europe, USA and in Russia as dietotherapy means at a number of diseases [6, 29]. In particular, we have shown the cholesterol-lowering effect of soy protein, resulting from the consumption of SPI en‐ riched cookies by persons with modern hypelipidemia [7, 9]. The consumption of soy foods is followed by the antioxidant effect [Santana et al., 2008, 8, 9] due to the high antioxidant content in these foods [2, 9]. Antioxidant effect is important for the treatment of diseases fol‐ lowed by the development of the oxidative stress [1, 38].

The other possible use of soy foods in dietotherapy may be the correction of disturbances of proteolytic processes. Activation of the tissue proteolysis accompanies different diseases and pathological processes [34]. Soy protein foods contain Bouman-Birk type trypsin inhibi‐ tor – SBTI [4]. This protease inhibitor has been officially recognized as a component of food‐ stuff [25]. There are data showing the possibility of absorbtion of SBTI in the intestin after the consumption of soy protein foods [22]. So, it seams reasonable to assume that the con‐ sumption of soy foods will follow anti-proteolytic effect. Because of this it was interesting to elucidate whether prolonged consumption of the cookies enriched with soy protein isolate (SPI) will influence total proteolytic and trypsin-inhibiting activity of the blood plasma in humans or not?

#### **4.1. Tripsin-inhibiting activity of SPI**

**Figure 10.** The hemolytic activity of the complement in the presence of different concentrations of trypsin in vitro. The

SBTI and aprotinin in the final concentrations 0,001-0,1% do not influence hemolysis time and the only statistically insignificant effect consists of small decrease of lag-phase from 3,5±0,3 to 3,2±0,15 min (p>0,05) and total time of hemolysis from 8,5±1,5 to 7,25±0,15 min (p>0,05). Trypsin suppresses the hemolytic activity of the complement in the concentrationdependent manner. In the presence of 0.0001% and 0,001% trypsin the lag-phase of the he‐ molysis increases to 4,25±0,25 min (p<0,02) and total time of hemolysis to 11,5±0,5 min (p<0,02) and 15,5±1,6 min (p<0,01), respectively. 0,01% trypsin entirely blocks the hemolysis. The inhibiting effect of trypsin should be attributed to the disruption of С3 component of

Thus, the results obtained show that aprotinin and SBTI in the abovementiioned concentra‐

**4. The influence of the consumption of soy priotein isolate by healthy**

Soya is cultivated as a foodstuff, having favorable effect on human health, more than 3000 years [28]. Within the last decades soy foods have started to be used in Europe, USA and in Russia as dietotherapy means at a number of diseases [6, 29]. In particular, we have shown the cholesterol-lowering effect of soy protein, resulting from the consumption of SPI en‐ riched cookies by persons with modern hypelipidemia [7, 9]. The consumption of soy foods is followed by the antioxidant effect [Santana et al., 2008, 8, 9] due to the high antioxidant content in these foods [2, 9]. Antioxidant effect is important for the treatment of diseases fol‐

The other possible use of soy foods in dietotherapy may be the correction of disturbances of proteolytic processes. Activation of the tissue proteolysis accompanies different diseases and pathological processes [34]. Soy protein foods contain Bouman-Birk type trypsin inhibi‐ tor – SBTI [4]. This protease inhibitor has been officially recognized as a component of food‐ stuff [25]. There are data showing the possibility of absorbtion of SBTI in the intestin after the consumption of soy protein foods [22]. So, it seams reasonable to assume that the con‐

tions do not influence the hemolytic activity of the complement.

lowed by the development of the oxidative stress [1, 38].

**persons on the indexes of proteolysis in the blood serum**

lag-phase is not shown.

506 Soybean - Bio-Active Compounds

the complement system.

According to the characteristics, specified by manufacturer, protein content of SPI consists of 90%. From this 10% belongs to protease inhibitors [32]. Within the processing of soy beans into SPI the former are expoused to high temperatures, which inactivates the major part of soy proteins. However, about 5-20 % of SBTI are represented by the thermostable fraction [19, 22]. The measurement of the trypsin-inhibiting activity of SPI shows that tryp‐ sin-inhibiting activity of SPI consists of 1,4±0,1 IU/mg of SPI. For example 1 mg of pure SBTI possesses activity equal nearby 60 IU. Thus, the active SBTI makes up 2,7% from SPI mass.

#### **4.2. The influence of the consumption of SPI by healthy persons on the total proteolytic and trypsin inhibiting activity of blood serum**

30 adult people aged 35-67 years without the expressed signs of chronic diseases consumed cookies, enriched with SPI (30% protein content), for two months [7]. Fasting blood samples were drawn before and after the dietary treatment. Serum samples were frozen and ana‐ lyzed for total proteolytic (BAEE-esterase activity) and tripsin-inhibiting activity [33]. Daily intake of 100g of cookies corresponds to consumption of 30g of SPI and about 0,85g of the active SBTI (nearly 52 00 IU). The total consumption of SPI for two months consists of 1,8 kg or 50 g of an active SBTI. Twenty-eight participants (19 females and 9 males) could complete the trial. Results are presented in the table 7.

**Table 7.** Total proteolytic and trypsin-inhibitory activity of blood serum of healthy persons before and after two months consumption of soy protein isolate.

Total proteolytic activity of blood serum was measures as serum BAEE-esterase activity and trypsin-inhibitory activity by inhibiting by serum of BAEE-esterase activity of trypsin.

Two months long SPI consumption was followed by 18% decrease of total proteolytic activi‐ ty of blood serum from 0,343±0,010 to 0,282±0,008 relative units (р<0,05) and by 21% increase of trypsin-inhibiting activity from 113±3,6 to 137±5,3 IU/ml ( р<0,01). The results obtained show possibility of the regulation of proteolysis *in vivo* by the consumption of soy foods.

#### **5. Discussion**

The disturbances of the hemostasis accompany many pathological processes and diseases [20, 18]. Both blood clotting and fibrinolysis represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43]. To correct disturbances of the fibrinolysis as well as imbalances of the other proteolytic processes the animal trypsin inhibitor aprotinin is used as a drug (Trasisol, Contrical, Gordox etc) [24, 13, 40]. The effectiveness of the aprotinin in the treatment of some diseases has been questioned recently because of it's substantial disadvantages [23, 12]. Following consultation with the German Federal Institute for Drugs and Medical Devices, the U.S. Food and Drug Administration, Health Canada, and other health authorities, the producer of Trasilol - Bayer announced in 2007 that it has elected to temporarily suspend worldwide marketing of Trasylol® (aprotinin injection) until final re‐ sults from the Canadian BARTtrial can be compiled, received and evaluated. Information re‐ garding the decision has been posted to Bayer's websites [5]. Because of this attempts to develop new protease inhibitor drug are undertaken [35, 27]. One of the candidates for such a role is plant protease inhibitor - SBTI.

ten for a long time. Our study represent new attempt to revive interest to SBTI as possible

2 Institute of Geology and Natural Management of the Far Easteren Branch of Russia Acade‐

[1] Aivatidi, C., Vourliotakis, G., Georgopoulos, S., Sigala, F., Bastounis, E., & Papalam‐ bros, E. (2011, March). Oxidative stress during abdominal aortic aneurysm repair--bi‐ omarkers and antioxidant's protective effect: a review. *Eur. Rev. Med. Pharmacol. Sci.*,

[2] Anderson, R. L., & Wolf, W. J. (1995, March). Compositional changes in trypsin in‐ hibitors, phytic acid, saponins and isoflavones related to soybean processing. *J Nutr.*,

[3] Bernstein, J. A. (2008). Hereditary angioedema: a current state-of-the-art review, VIII: current status of emerging therapies. *Ann. Allergy Asthma Immunol* [1, 2], S41-S46,

[4] Birk, Y. (1985). The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor

[5] Bayer (2007). Available from http://www.trasylol.com http://www.phar‐ ma.bayer.com http://www.bayerscheringpharma.de/trasylol/en http://www.bayer‐

[6] Borodin, E. A., Aksyonova, T. V., & Anishchenko, N. I. (2000). Soy foods. New role.

[7] Borodin, E. A., Menshikova, I. G., Dorovskikh, V. A., Feoktistova, N. A., Shtarberg, M. A., Yamamoto, T., Takamatsu, K., Mori, H., & Yamamoto, S. (2009, December). Ef‐

from soybeans. *Int J Pept Protein Res.*, 25(2), 113-31, 0367-8377.

3 Asian Nutrition and Food Culture Research Center, Jumonji University, Saitama, Japan

, Mikhail A. Shtarberg1

, Kiyoharu Takamatsu4

, Vladimir A. Dorovskikh1

Effects of Soybean Trypsin Inhibitor on Hemostasis

http://dx.doi.org/10.5772/52600

and Shigeru Yamamoto3

,

509

protease inhibiting drug.

Alexander V. Korotkikh1

Eugene A. Borodin1\*, Igor E. Pamirsky2

my of Sciences, Russian Federation

4 Fuji Oil Co., Ltd Osaka, Japan

15(3), 245-252, 1128-3602.

125(3), 581S-588S, 0022-3166.

healthcare.com/trasylol/en

*Vestnik DVO RAN* [3], 72-85.

1081-1206.

**References**

\*Address all correspondence to<EMAIL_ADDRESS>
, Chie Tarumizu3

1 Amur State Medical Academy, Blagoveshchensk, Russian Federation

**Author details**

In the present study we exploited the advantages of the modern bioinformatics for establish‐ ing the extent of structural homology, comparing functional activities and evaluating poten‐ tial targets of SBTI and aprotinin among the human proteases. The results of *in silico* study testifies apparent homology of SBTI and aprotinin manifested by the common features in the primary structure, 3D-structures, functional activities of these proteins and allow us to propose that SBTI should influence processes of hemostasis similar to aprotinin. The investi‐ gation of the influence of SBTI and aprotinin on coagulation and thrombocyte hemostasis by *in vitro* methods prove this assumption and show that both proteins inhibit blood clotting, fibrinolusis and platelet aggregation which is evident from the increase of prothrombin time, activated partial thromboplastin time, activated clotting time, thrombin time and in‐ hibition of fibrinolysis. We investigated the influence of SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard hemolysis of red blood cells by inhibiting activation of some components of the complement system. Howev‐ er, both inhibitors do not influence hemolysis time.

While the major part of SBTI is disrupted by heating, nearly 20% of inhibitor are thermosta‐ ble and remains active in soy foods [19]. Part of the consumed SBTI are absorbed in the intestine after the consumption of soy foods [22]. Because of this we elucidated whether prolonged consumption of the cookies, enriched with SPI, will influence total proteolytic and trypsininhibiting activity of the blood plasma in humans or not? Daily intake of 100g of cookies corresponds to consumption of about 0,85g of the active SBTI. The total consumption of SBTI for the study period consists of 50 g of an active inhibitor. The consumption of 100 g of cookies daily for two months was followed by 18% decrease of total proteolytic activity of blood serum and by 21% increase of trypsin-inhibiting activity. The results obtained testify possibility of the soft regulation of proteolysis *in vivo* by the consumption of soy foods.

At first, the ability of SBTI to inhibit blood clotting was shown more than half a centaury ago [26, 41]. However, these results had no any practical consequences and in fact were forgot‐ ten for a long time. Our study represent new attempt to revive interest to SBTI as possible protease inhibiting drug.

## **Author details**

**5. Discussion**

508 Soybean - Bio-Active Compounds

a role is plant protease inhibitor - SBTI.

er, both inhibitors do not influence hemolysis time.

The disturbances of the hemostasis accompany many pathological processes and diseases [20, 18]. Both blood clotting and fibrinolysis represents the cascade of proteolytic reactions catalyzed by highly specific proteases [43]. To correct disturbances of the fibrinolysis as well as imbalances of the other proteolytic processes the animal trypsin inhibitor aprotinin is used as a drug (Trasisol, Contrical, Gordox etc) [24, 13, 40]. The effectiveness of the aprotinin in the treatment of some diseases has been questioned recently because of it's substantial disadvantages [23, 12]. Following consultation with the German Federal Institute for Drugs and Medical Devices, the U.S. Food and Drug Administration, Health Canada, and other health authorities, the producer of Trasilol - Bayer announced in 2007 that it has elected to temporarily suspend worldwide marketing of Trasylol® (aprotinin injection) until final re‐ sults from the Canadian BARTtrial can be compiled, received and evaluated. Information re‐ garding the decision has been posted to Bayer's websites [5]. Because of this attempts to develop new protease inhibitor drug are undertaken [35, 27]. One of the candidates for such

In the present study we exploited the advantages of the modern bioinformatics for establish‐ ing the extent of structural homology, comparing functional activities and evaluating poten‐ tial targets of SBTI and aprotinin among the human proteases. The results of *in silico* study testifies apparent homology of SBTI and aprotinin manifested by the common features in the primary structure, 3D-structures, functional activities of these proteins and allow us to propose that SBTI should influence processes of hemostasis similar to aprotinin. The investi‐ gation of the influence of SBTI and aprotinin on coagulation and thrombocyte hemostasis by *in vitro* methods prove this assumption and show that both proteins inhibit blood clotting, fibrinolusis and platelet aggregation which is evident from the increase of prothrombin time, activated partial thromboplastin time, activated clotting time, thrombin time and in‐ hibition of fibrinolysis. We investigated the influence of SBTI and aprotinin on the hemolytic activity of the complement assuming that SBTI and aprotinin may retard hemolysis of red blood cells by inhibiting activation of some components of the complement system. Howev‐

While the major part of SBTI is disrupted by heating, nearly 20% of inhibitor are thermosta‐ ble and remains active in soy foods [19]. Part of the consumed SBTI are absorbed in the intestine after the consumption of soy foods [22]. Because of this we elucidated whether prolonged consumption of the cookies, enriched with SPI, will influence total proteolytic and trypsininhibiting activity of the blood plasma in humans or not? Daily intake of 100g of cookies corresponds to consumption of about 0,85g of the active SBTI. The total consumption of SBTI for the study period consists of 50 g of an active inhibitor. The consumption of 100 g of cookies daily for two months was followed by 18% decrease of total proteolytic activity of blood serum and by 21% increase of trypsin-inhibiting activity. The results obtained testify possibility of

At first, the ability of SBTI to inhibit blood clotting was shown more than half a centaury ago [26, 41]. However, these results had no any practical consequences and in fact were forgot‐

the soft regulation of proteolysis *in vivo* by the consumption of soy foods.

Eugene A. Borodin1\*, Igor E. Pamirsky2 , Mikhail A. Shtarberg1 , Vladimir A. Dorovskikh1 , Alexander V. Korotkikh1 , Chie Tarumizu3 , Kiyoharu Takamatsu4 and Shigeru Yamamoto3

\*Address all correspondence to<EMAIL_ADDRESS>
1 Amur State Medical Academy, Blagoveshchensk, Russian Federation

2 Institute of Geology and Natural Management of the Far Easteren Branch of Russia Acade‐ my of Sciences, Russian Federation

3 Asian Nutrition and Food Culture Research Center, Jumonji University, Saitama, Japan

4 Fuji Oil Co., Ltd Osaka, Japan

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with moderate hyperlipidemia. *J. Nutr. Sci. Vit*, 50(6), 492-497, 0301-4800.

and in vivo. *Far Eastern Medical Journal (Russian)* [4], 26-30, 1994-5191.

978-9-53307-535-8.

510 Soybean - Bio-Active Compounds

*Surg*, 13, 1344-4964.

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**Chapter 23**

**Potential Use of Soybean Flour (***Glycine max***) in Food**

Protein-energy malnutrition (PEM) results from prolonged deprivation of essential amino acids and total nitrogen and/or energy substrates. Growth which is a continuous process re‐ sults from the complex interaction between inheritance and environment. Protein-energy malnutrition (PEM) also results from food insufficiency as well as from poor social and eco‐ nomic conditions [6, 20]. Malnutrition originates from a cellular imbalance between nu‐ trient/energy supply and the body's demand to ensure growth and maintenance [22]. Dietary energy and protein deficiencies usually occur together, although one sometimes predominates the other and if severe enough, may lead to the clinical syndrome of kwa‐

shiorkor (predominant protein deficiency) or marasmus (mainly energy deficiency).

gy, macro- and micronutrients they need to enjoy a healthy and productive life [3].

Legumes are one of the world's most important sources of food supply especially in the de‐ veloping countries in terms of food, energy as well as nutrients. It has been recognized as

> © 2013 Adelakun et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Adelakun et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The origin of PEM can be primary, when it is the result of inadequate food intake, or secon‐ dary, when it is the result of other diseases that lead to low food ingestion, inadequate nu‐ trient absorption or utilization, increased nutritional requirements and/or increased nutrient losses [13]. In as much as protein-energy malnutrition applies to a group of related disorders that develop in children and adults whose consumption of protein and energy is insufficient to satisfy the bodies nutritional needs, about one third of children worldwide suffer from malnutrition [8]. This continues to be an important problem mostly in developing countries. One way to curb the global menace of PEM is through food fortification of plant origin. Food fortification is broadly aimed to allow all people to obtain from their diet all the ener‐

**Fortification**

B. F. Olanipekun

**1. Introduction**

http://dx.doi.org/10.5772/52599

O. E. Adelakun, K. G. Duodu, E. Buys and

Additional information is available at the end of the chapter

## **Potential Use of Soybean Flour (***Glycine max***) in Food Fortification**

O. E. Adelakun, K. G. Duodu, E. Buys and B. F. Olanipekun

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52599

## **1. Introduction**

combinant aprotinin and a monoclonal antibody product using plant-based transient

[36] Radović, N., Cucić, S., & Altarac, S. (2008). Molecular aspects of apoptosis. *Acta Med.*

[37] Sarma, J. V., & Ward, P. A. (2011, January). The complement system. *Cell Tissue Res.*,

[38] Shi, Y. C., & Pan, T. M. (2012, April). Red mold, diabetes, and oxidative stress: a re‐

[39] Sigma. (2006). Catalog reagents ang labware. Sigma-Aldrich, Inc. 0013-7227 , 1440.

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[41] Tagnon, H. J., & Soulier, J. P. (1946). Anticoagulant activity of the trypsin inhibitor

[42] Tang, M., Asamoto, M., Ogawa, K., Naiki-Ito, A., Sato, S., Takahashi, S., & Shirai, T.

[43] Walsh, P. N., & Ahmad, S. S. (2002). Proteases in blood clotting. *Essays Biochem.*, 38,

[44] Wouters, D., Wagenaar-Bos, I., van Ham, M., & Zeerleder, S. (2008). C1 inhibitor: just

[45] Zhirnov, O. P., Klenk, H. D., & Wright, P. F. (2011, July). Aprotinin and similar pro‐

tease inhibitors as drugs against influenza. *Antiviral Res.*, 92(1), 27-36, 16635429-9999.

of C1 inhibitor. *Expert. Opin. Biol. Ther*, 8(8), 1225-1240, 1744-7682.

a serine protease inhibitor? New and old considerations on therapeutic applications

(2009). Induction of apoptosis in the LNCaP human prostate carcinoma cell line and prostate adenocarcinomas of SV40T antigen transgenic rats by the Bowman-Birk in‐

from soy bean flour. *Proc. Soc. Exper. Biol. & Med*, 61, 440, 0003-4819.

expression systems. *Plant Biotechnol J.*, 8(5), 638-654, 1467-7644.

view. *Appl Microbiol Biotechnol.*, 94(1), 47-55, 0175-7598.

pancreatitis. *J. Clin. Pract.*, 4(1), 84-92, 1368-5031.

hibitor. *Pathol Int.*, 59(11), 790-796, 13205-4630.

95-111, 0071-1365.

*Croatica.*, 62(3), 249-256, 1330-0164.

343(1), 227-235, 0030-2766X.

512 Soybean - Bio-Active Compounds

Protein-energy malnutrition (PEM) results from prolonged deprivation of essential amino acids and total nitrogen and/or energy substrates. Growth which is a continuous process re‐ sults from the complex interaction between inheritance and environment. Protein-energy malnutrition (PEM) also results from food insufficiency as well as from poor social and eco‐ nomic conditions [6, 20]. Malnutrition originates from a cellular imbalance between nu‐ trient/energy supply and the body's demand to ensure growth and maintenance [22]. Dietary energy and protein deficiencies usually occur together, although one sometimes predominates the other and if severe enough, may lead to the clinical syndrome of kwa‐ shiorkor (predominant protein deficiency) or marasmus (mainly energy deficiency).

The origin of PEM can be primary, when it is the result of inadequate food intake, or secon‐ dary, when it is the result of other diseases that lead to low food ingestion, inadequate nu‐ trient absorption or utilization, increased nutritional requirements and/or increased nutrient losses [13]. In as much as protein-energy malnutrition applies to a group of related disorders that develop in children and adults whose consumption of protein and energy is insufficient to satisfy the bodies nutritional needs, about one third of children worldwide suffer from malnutrition [8]. This continues to be an important problem mostly in developing countries. One way to curb the global menace of PEM is through food fortification of plant origin. Food fortification is broadly aimed to allow all people to obtain from their diet all the ener‐ gy, macro- and micronutrients they need to enjoy a healthy and productive life [3].

Legumes are one of the world's most important sources of food supply especially in the de‐ veloping countries in terms of food, energy as well as nutrients. It has been recognized as

© 2013 Adelakun et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Adelakun et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

an important source of protein and in some cases oil. As a legume, soybeans are an impor‐ tant global crop that provides oil and protein for users. It is the richest sources of protein among the plant foods. Features that motivate researchers to explore its utility in a wide range include the good quality and functionality of its proteins, surplus availability and low cost. Soybeans in the form of full fat flour, concentrate, isolate and texturised proteins have been used in a wide range of food products. These attributes made soybeans to be consid‐ ered as an ideal food for meeting the protein needs of the population.

wide range of soils and climate. Because it contains high amount of protein and oil, the soy‐ bean was considered one of the five sacred grains along with rice, wheat, barley and millet [4]. The protein and oil component of soybean are not only high in terms of quantity but al‐ so in quality. For instance, soy oil has a highest proportion of unsaturated fatty acids such as linoleic and linolenic acid making it a healthy oil to use. Soybeans are known to be typical of such crops that contain all three of the macro-nutrients required particularly for human nu‐ trition. They also contain protein which provides all the essential amino acids in the amounts needed for human health. Most of the essential amino acid present in soybean is available in an amount that is close to those required by animals and humans. The protein – digestibility – correlated amino acid score is close to 1, a rating that is the same for animal proteins such as an egg white and casein. The profile of various nutrients in raw matured seeds of soybeans as highlighted in USDA Nutrient database is as indicated in Table 1 be‐ low. Additionally, soybean contains phytochemicals which have been shown to offer unique health benefit. Soybean also has versatile end uses which include human food, animal feed

Potential Use of Soybean Flour (*Glycine max*) in Food Fortification

http://dx.doi.org/10.5772/52599

515

Soybean can be processed to give soy milk, a valuable protein supplement in infant feeding, soycurds and cheese [23]. It is also used to produce soysauce used extensively in cooking and as a sauce. Soybeans are also used for making candies and ice cream and soybean flour which could be mixed with wheat flour to produce a wide variety of baked goods such as bread and biscuits [16]. Soybean oil is used for edible purposes, particularly as a cooking, and salad oil and, for manufacture of margarine [16]. The oil can also be used industrially in the processing of paints, soap, oil, cloth and printing inks. The meal and soybean proteins are used in the manufacture of synthetic fibre (artificial wool) adhesives and textile [14]. Soybeans could be made into such products as tempeh, miso and natto which may include

Soybean protein fibre has been reportedly produced from bean dregs that are produced when extracting oil [14]. From these, globular protein is extracted, made into a spinning sol‐ ution of a consistent concentration with the addition of a functional auxiliary, and spun into yarn by the wet method [21]. Effect of fermentation on soybean has the tendency of altering the features of the arising dregs when oil is so extracted from soybean. This thus has tenden‐ cy of either skewing up or otherwise the various arrays of benefits known to accrue from the development of soybean fiber blends with other fibers [21]. [7] has also reported on the chemical composition and total digestible nutrients (TDN) of fermented soybean paste resi‐ due. This is usually exploited for the utilization of such residues in livestock rations. Fur‐ thermore, [9] had suggested the likelihood of the use of fermented soybean paste residue for livestock feed in the near future as a form of turning waste to wealth and thus serving as

and industrial materials [10].

**4. Utilization of soybeans**

other sub-products [16].

anchor for many other accruing benefits.

The objective of this chapter is to briefly describe the quality attributes of soybean and the potential use of its flour in food fortification.

## **2. Soybeans**

Soybeans *(Glycine max)* belonging to the family *leguminosae* constitute one of the oldest culti‐ vated crops of the tropics and sub-tropical regions, and one of the world's most important sources of protein and oil. Soybeans are probably the most important oil seed legume which has its origin in Eastern Asia, mainly China. The cultivar *Glycine max* is thought to be de‐ rived from *Glycine ussuriensis* and *Glycine tomentosa* which grow wild in China, and can be found in great quantities in Asian countries such as Japan and Indonesia [16].

The seeds vary in shape and colour depending on the cultivar. In shape, they can be spheri‐ cal to flatten while the colour varies from white, yellow and brown to black. Also, the chem‐ ical composition of each variety of soybeans differs from each other. Due to the long and tedious processing technique of soybeans, Japan which is one of the largest suppliers of soy‐ bean has developed highly advanced processing technologies in the processing and manu‐ facturing of highly acceptable and palatable soya products [14]. As a result of high protein content in soybean, it can be used as a substitute for expensive meat and meat products [5].

Soybean is particularly very unique for different reason and hence classify as a valuable and economical agricultural commodity. In the first instance, it possesses agronomic char‐ acteristics with its ability to adapt to a wide range of soil and climate; and its nitrogen fixing ability. This makes it to be a good rotational crop for use with high nitrogen – consuming crops such as corn and rice. Secondly, soybean unique chemical composition on an aver‐ age dry matter basis is about 40% of protein and 20% of oil. This composition makes it to rank highest in terms of protein content among all food crops and second in terms of oil content after peanut (48%) among all food legumes. Furthermore, soybean is a very nutri‐ tious food crop.

### **3. Nutritional content of soybean**

Soybean (*Glycine max*) first emerged as a domesticated crop in the eastern half of North Chi‐ na around the 11th century B.C of Zhou Dynasty. It is easy to grow and has adaptability to a wide range of soils and climate. Because it contains high amount of protein and oil, the soy‐ bean was considered one of the five sacred grains along with rice, wheat, barley and millet [4]. The protein and oil component of soybean are not only high in terms of quantity but al‐ so in quality. For instance, soy oil has a highest proportion of unsaturated fatty acids such as linoleic and linolenic acid making it a healthy oil to use. Soybeans are known to be typical of such crops that contain all three of the macro-nutrients required particularly for human nu‐ trition. They also contain protein which provides all the essential amino acids in the amounts needed for human health. Most of the essential amino acid present in soybean is available in an amount that is close to those required by animals and humans. The protein – digestibility – correlated amino acid score is close to 1, a rating that is the same for animal proteins such as an egg white and casein. The profile of various nutrients in raw matured seeds of soybeans as highlighted in USDA Nutrient database is as indicated in Table 1 be‐ low. Additionally, soybean contains phytochemicals which have been shown to offer unique health benefit. Soybean also has versatile end uses which include human food, animal feed and industrial materials [10].

## **4. Utilization of soybeans**

an important source of protein and in some cases oil. As a legume, soybeans are an impor‐ tant global crop that provides oil and protein for users. It is the richest sources of protein among the plant foods. Features that motivate researchers to explore its utility in a wide range include the good quality and functionality of its proteins, surplus availability and low cost. Soybeans in the form of full fat flour, concentrate, isolate and texturised proteins have been used in a wide range of food products. These attributes made soybeans to be consid‐

The objective of this chapter is to briefly describe the quality attributes of soybean and the

Soybeans *(Glycine max)* belonging to the family *leguminosae* constitute one of the oldest culti‐ vated crops of the tropics and sub-tropical regions, and one of the world's most important sources of protein and oil. Soybeans are probably the most important oil seed legume which has its origin in Eastern Asia, mainly China. The cultivar *Glycine max* is thought to be de‐ rived from *Glycine ussuriensis* and *Glycine tomentosa* which grow wild in China, and can be

The seeds vary in shape and colour depending on the cultivar. In shape, they can be spheri‐ cal to flatten while the colour varies from white, yellow and brown to black. Also, the chem‐ ical composition of each variety of soybeans differs from each other. Due to the long and tedious processing technique of soybeans, Japan which is one of the largest suppliers of soy‐ bean has developed highly advanced processing technologies in the processing and manu‐ facturing of highly acceptable and palatable soya products [14]. As a result of high protein content in soybean, it can be used as a substitute for expensive meat and meat products [5].

Soybean is particularly very unique for different reason and hence classify as a valuable and economical agricultural commodity. In the first instance, it possesses agronomic char‐ acteristics with its ability to adapt to a wide range of soil and climate; and its nitrogen fixing ability. This makes it to be a good rotational crop for use with high nitrogen – consuming crops such as corn and rice. Secondly, soybean unique chemical composition on an aver‐ age dry matter basis is about 40% of protein and 20% of oil. This composition makes it to rank highest in terms of protein content among all food crops and second in terms of oil content after peanut (48%) among all food legumes. Furthermore, soybean is a very nutri‐

Soybean (*Glycine max*) first emerged as a domesticated crop in the eastern half of North Chi‐ na around the 11th century B.C of Zhou Dynasty. It is easy to grow and has adaptability to a

found in great quantities in Asian countries such as Japan and Indonesia [16].

ered as an ideal food for meeting the protein needs of the population.

potential use of its flour in food fortification.

**2. Soybeans**

514 Soybean - Bio-Active Compounds

tious food crop.

**3. Nutritional content of soybean**

Soybean can be processed to give soy milk, a valuable protein supplement in infant feeding, soycurds and cheese [23]. It is also used to produce soysauce used extensively in cooking and as a sauce. Soybeans are also used for making candies and ice cream and soybean flour which could be mixed with wheat flour to produce a wide variety of baked goods such as bread and biscuits [16]. Soybean oil is used for edible purposes, particularly as a cooking, and salad oil and, for manufacture of margarine [16]. The oil can also be used industrially in the processing of paints, soap, oil, cloth and printing inks. The meal and soybean proteins are used in the manufacture of synthetic fibre (artificial wool) adhesives and textile [14]. Soybeans could be made into such products as tempeh, miso and natto which may include other sub-products [16].

Soybean protein fibre has been reportedly produced from bean dregs that are produced when extracting oil [14]. From these, globular protein is extracted, made into a spinning sol‐ ution of a consistent concentration with the addition of a functional auxiliary, and spun into yarn by the wet method [21]. Effect of fermentation on soybean has the tendency of altering the features of the arising dregs when oil is so extracted from soybean. This thus has tenden‐ cy of either skewing up or otherwise the various arrays of benefits known to accrue from the development of soybean fiber blends with other fibers [21]. [7] has also reported on the chemical composition and total digestible nutrients (TDN) of fermented soybean paste resi‐ due. This is usually exploited for the utilization of such residues in livestock rations. Fur‐ thermore, [9] had suggested the likelihood of the use of fermented soybean paste residue for livestock feed in the near future as a form of turning waste to wealth and thus serving as anchor for many other accruing benefits.

**5. Antinutritional factors in soybeans**

by leading to elimination of flatulence.

**6. Soybean in food fortification**

tion in developing countries [17].

Flatulence is characterized by stomach cramps, nausea, diarrhoea, intestinal and gastric dis‐ comfort resulting from the production of large amounts of gas in the gastrointestinal tract. Although all the causative factors in flatulence formation are still unknown, it has been sug‐ gested and generally accepted that low-molecular weight oligosaccharides, primarily raffi‐ nose, stachyose and verbascose present in most legume seeds are linked to flatulence [2]. Researchers have suggested practical procedures for the reduction of flatulence in cooked and processed soybeans. These include fermentation, removal of seed coat prior to cooking or processing, soaking in water, germination and cooking with a mixture of sodium carbo‐ nate and bicarbonate [11]. Oligosaccharides can also be reduced through fermentation there‐

Potential Use of Soybean Flour (*Glycine max*) in Food Fortification

http://dx.doi.org/10.5772/52599

517

In many developing nations, cereal based foods are widely utilized as food and as dietary staples for adults and weaning foods for infants including Africa where it accounts for up to 77% of total caloric consumption. The major cereal based foods in these regions are derived mainly from maize, sorghum, millet, rice, or wheat. Oilseeds are the largest single source for production of protein concentrates. Of these, soybeans, in terms of tonnage produced, are the most important source. Properly defatted soybean flour will contain 50% or more of pro‐ tein. By removing soluble carbohydrates and minerals, concentrates containing up to 70% protein can be prepared, and dispersible isolates containing 90% or more of protein are be‐ ing made. The isolates are of interest as highly concentrated fortification media and also as bases for a variety of high-protein beverages, desserts, and similar products. Soybean con‐ centrates have the virtue of low cost and good nutritive value. Fortification of staple cereals with soybean can help improves their nutritive value and may aid in alleviating malnutri‐

[12] determined the effect of germination and drying on the functional and nutritional prop‐ erties of common red bean flour and evaluated the effect of incorporating different levels of cowpea and a constant level of soybean into red bean flour on the functional properties of the composite. Incorporation of soybean and cowpea flour into germinated bean flour at lev‐ els of 10 and 30%, respectively, produced a composite with higher functional properties. [15] studied the effect of fermentation with *Rhizopus oligosporus* on some physico-chemical prop‐ erties of starch extracts from soybean flour. Their study revealed that the length of fermenta‐ tion with *R. oligosporus* within the period of 0–72 h on soybean (G. max) affected many physico-chemical properties of starch extract as well as pasting characteristics of extracted starch. The physico-chemical attributes of starch 'extract' from fermented soybean flour re‐ vealed that water binding capacity, water absorption capacity, swelling capacity and solu‐ bility power decreased slightly with increases in fermentation period. Also, while the starch yield and amylose content decreased and amylopectin contents increased with fermentation

**Table 1.** Source: USDA Nutrient database, Percentage relative to US recommendations for adultsNutritional content of raw mature seeds of soybean

## **5. Antinutritional factors in soybeans**

**Nutrient value per 100g** Energy 1,866kJ (446kcal)

**Table 1.** Source: USDA Nutrient database, Percentage relative to US recommendations for adultsNutritional content

of raw mature seeds of soybean

Carbohydrates 30.16g Sugars 7.33g Dietary fiber 9.3g Fat 19.94g Saturated 2.884g Monounsaturated 4.404g Polyunsaturated 11.255g Protein 36.49g Tryptophan 0.591g Threonine 1.766g Isoleucine 1.971g Leucine 3.309g Lysine 2.706g Methionine 0.547g Phenylalanine 2.122g Tyrosine 1.539g Valine 2.029g Arginine 3.153g Histidine 1.097g Alanine 1.915g Aspartic acid 5.112g Glutamic acid 7.874g Glycine 1.880g Proline 2.379g Serine 2.357g Water 8.54g Vitamin A equiv. 1μg (0%) Vitamin B6 0.377mg (29%) Vitamin B12 0μg (0%) Vitamin C 6.0 mg (10%) Vitamin K 47 μg (45%) Calcium 277mg (28%) Iron 15.70 mg (126%) Magnesium 280 mg (76%) Phosphorus 704 mg (101%) Potasium 1797 mg (38%) Sodium 2 mg (0%) Zinc 4.89 mg (49%)

516 Soybean - Bio-Active Compounds

Flatulence is characterized by stomach cramps, nausea, diarrhoea, intestinal and gastric dis‐ comfort resulting from the production of large amounts of gas in the gastrointestinal tract. Although all the causative factors in flatulence formation are still unknown, it has been sug‐ gested and generally accepted that low-molecular weight oligosaccharides, primarily raffi‐ nose, stachyose and verbascose present in most legume seeds are linked to flatulence [2]. Researchers have suggested practical procedures for the reduction of flatulence in cooked and processed soybeans. These include fermentation, removal of seed coat prior to cooking or processing, soaking in water, germination and cooking with a mixture of sodium carbo‐ nate and bicarbonate [11]. Oligosaccharides can also be reduced through fermentation there‐ by leading to elimination of flatulence.

## **6. Soybean in food fortification**

In many developing nations, cereal based foods are widely utilized as food and as dietary staples for adults and weaning foods for infants including Africa where it accounts for up to 77% of total caloric consumption. The major cereal based foods in these regions are derived mainly from maize, sorghum, millet, rice, or wheat. Oilseeds are the largest single source for production of protein concentrates. Of these, soybeans, in terms of tonnage produced, are the most important source. Properly defatted soybean flour will contain 50% or more of pro‐ tein. By removing soluble carbohydrates and minerals, concentrates containing up to 70% protein can be prepared, and dispersible isolates containing 90% or more of protein are be‐ ing made. The isolates are of interest as highly concentrated fortification media and also as bases for a variety of high-protein beverages, desserts, and similar products. Soybean con‐ centrates have the virtue of low cost and good nutritive value. Fortification of staple cereals with soybean can help improves their nutritive value and may aid in alleviating malnutri‐ tion in developing countries [17].

[12] determined the effect of germination and drying on the functional and nutritional prop‐ erties of common red bean flour and evaluated the effect of incorporating different levels of cowpea and a constant level of soybean into red bean flour on the functional properties of the composite. Incorporation of soybean and cowpea flour into germinated bean flour at lev‐ els of 10 and 30%, respectively, produced a composite with higher functional properties. [15] studied the effect of fermentation with *Rhizopus oligosporus* on some physico-chemical prop‐ erties of starch extracts from soybean flour. Their study revealed that the length of fermenta‐ tion with *R. oligosporus* within the period of 0–72 h on soybean (G. max) affected many physico-chemical properties of starch extract as well as pasting characteristics of extracted starch. The physico-chemical attributes of starch 'extract' from fermented soybean flour re‐ vealed that water binding capacity, water absorption capacity, swelling capacity and solu‐ bility power decreased slightly with increases in fermentation period. Also, while the starch yield and amylose content decreased and amylopectin contents increased with fermentation period. The pasting characteristics of starch 'extract' from the fermented soybean flour re‐ vealed the potential use of the flours in weaning food formulation due to reduce viscosity trend as fermentation period increased.

1 Department of Food Science, University of Pretoria, Pretoria 0002, South Africa

Nigeria

**References**

2 Department of Food Science and Engineering, Ladoke Akintola University of Technology,

Potential Use of Soybean Flour (*Glycine max*) in Food Fortification

http://dx.doi.org/10.5772/52599

519

[1] Adelakun, O. E., Adejuyitan, J. A., Olajide, J. O., & Alabi, B. K. (2005). Effect of Soy‐ bean Substitution on some Physical, Compositional and Sensory Properties of Ko‐ koro (A Local Maize Snack). *European Food Research and Technology.*, 220, 79-82.

[2] Akpapunan, M. A., & Markakis, P. (1979). Oligosaccharides of 13 Cultivars of Cow‐

[3] Allan, L., Benoist, B., Dary, O., & Hurrell, R. (2006). Guidelines on food fortification with micronutrients. *A Report: Word Health Organization and Food and Agriculture Or‐*

[4] Ang, C. Y. W., Liu, K., & Huang, Y. W. (1999). Asian Foods Science and Technology.

[5] Charles, A., & Guy, L. (1999). Food Biochemistry. *Aspen Publishers Inc. Gaitheasburg,*

[6] Dulger, H., Arik, M., Sekeroglu, M. R., Tarakcioglu, M., Noyan, T., & Cesur, Y. (2002). Proinflammatory cytokines in Turkish children with protein energy malnutri‐

[7] FFTC. (2002). Food and Fertilizer Technology. *An International Center for Farmers in*

[8] Gray, V. B., Cossman, J. S., & Powers, E. L. (2006). Stunted growth is associated with physical indicators of malnutrition but not food insecurity among rural school chil‐

[9] Jong-Kyu, Ha., Kim, S. W., & Kim, W. Y. (1996). Use of Agro-Industrial. By-products a Animal Feeds in Korea. *A publication of FFTC. An International Centre for Farmers in*

[11] Ndubuaku, V. O., Nwaegbule, A. C., & Nnanyeluyo, D. O. (1989). Flatulence and Other Discomfort Associated with Consumption of Cowpea (Vigna uguiculata). *Ap‐*

[12] Njintang, N. Y., Mbofung, C. M. F., & Waldron, K. W. (2001). In Vitro Protein Digesti‐ bility and Physicochemical Properties of Dry Red Bean (Phaseolus vulgaris) Flour:

[10] Liu, K. (2000). Expanding soybean food utilization. *Food Technol*, 54(7), 46-47.

pea (Vigna Sinensis). *J. Food Science*, 44, 317-318.

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*Maryland*, 39-41.

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*the Asia Pacific Region. Taiwan.*

*the Asia Pacific Region.*

*petite*, 13, 171-181.

[1] also worked on the effect of soybean substitution on some physical, compositional and sensory properties of kokoro (a local maize snack). Kokoro which is a finger-shaped snack made from maize is widely acceptable and consumed by children and adults, especially in the southwestern part of Nigeria in the Oyo and Ogun states. Soybean (*Glycine max*), which has high quality protein with high contents of sulphur-containing amino acids, is a good supplement in maize products. Because Snacks provide an avenue for introducing plant proteins such as soybeans to people who normally resist trying any unfamiliar food, kokoro was prepared from maize–soybean flour mixtures in ratios of 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50. The physical, compositional, and sensory characteristics of kokoro were evaluat‐ ed. Protein and fat contents increased, while carbohydrate content decreased as the soy flour proportion of the flour mixture used in the kokoro was increased.

The bulk density and water-holding capacity increased with increasing proportion of soy‐ bean flour, while the swelling capacity was found to decrease. High soy-substitution signifi‐ cantly reduced the sensory acceptance of kokoro. Sensory evaluation indicated that maize:soybean

flour mixture ratios of 100:0 and 90:0 were the most acceptable to the panellists. Several studies have been conducted to improve the protein quality of food products by fortification with plant proteins such as soybean, which is less expensive. Amino acid fortification was suggested by [18]. [19] also determined the acceptability of fermented maize meal fortified with defatted soy-flour in traditional Ghanaian foods.

## **7. Conclusion**

Soybean flour has huge potentials of being used to enrich foods in order to provide ade‐ quate nutrients for individuals not meeting daily needs. Based on the available information on the nutrients profile of soybean including the amino profiles, human consumption of soybean flour can be promoted because of its positive effect on nutritional enhancement on different fortified food products. However, more efforts need to be directed at addressing associated technological issue which is flatulence to further increase effective utilization of the food product in food fortification.

## **Author details**

O. E. Adelakun1\*, K. G. Duodu1 , E. Buys1 and B. F. Olanipekun2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Department of Food Science, University of Pretoria, Pretoria 0002, South Africa

2 Department of Food Science and Engineering, Ladoke Akintola University of Technology, Nigeria

#### **References**

period. The pasting characteristics of starch 'extract' from the fermented soybean flour re‐ vealed the potential use of the flours in weaning food formulation due to reduce viscosity

[1] also worked on the effect of soybean substitution on some physical, compositional and sensory properties of kokoro (a local maize snack). Kokoro which is a finger-shaped snack made from maize is widely acceptable and consumed by children and adults, especially in the southwestern part of Nigeria in the Oyo and Ogun states. Soybean (*Glycine max*), which has high quality protein with high contents of sulphur-containing amino acids, is a good supplement in maize products. Because Snacks provide an avenue for introducing plant proteins such as soybeans to people who normally resist trying any unfamiliar food, kokoro was prepared from maize–soybean flour mixtures in ratios of 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50. The physical, compositional, and sensory characteristics of kokoro were evaluat‐ ed. Protein and fat contents increased, while carbohydrate content decreased as the soy flour

The bulk density and water-holding capacity increased with increasing proportion of soy‐ bean flour, while the swelling capacity was found to decrease. High soy-substitution signifi‐ cantly reduced the sensory acceptance of kokoro. Sensory evaluation indicated that

flour mixture ratios of 100:0 and 90:0 were the most acceptable to the panellists. Several studies have been conducted to improve the protein quality of food products by fortification with plant proteins such as soybean, which is less expensive. Amino acid fortification was suggested by [18]. [19] also determined the acceptability of fermented maize meal fortified

Soybean flour has huge potentials of being used to enrich foods in order to provide ade‐ quate nutrients for individuals not meeting daily needs. Based on the available information on the nutrients profile of soybean including the amino profiles, human consumption of soybean flour can be promoted because of its positive effect on nutritional enhancement on different fortified food products. However, more efforts need to be directed at addressing associated technological issue which is flatulence to further increase effective utilization of

and B. F. Olanipekun2

, E. Buys1

\*Address all correspondence to<EMAIL_ADDRESS>
proportion of the flour mixture used in the kokoro was increased.

with defatted soy-flour in traditional Ghanaian foods.

trend as fermentation period increased.

518 Soybean - Bio-Active Compounds

maize:soybean

**7. Conclusion**

**Author details**

the food product in food fortification.

O. E. Adelakun1\*, K. G. Duodu1

Effect of Processing and Incorporation of Soybean and Cowpea Flour. *J. Agric. Food Chem.*, 49, 2465-2471.

**Chapter 24**

**Food, Nutrition and Health**

Eduardo Fuentes, Luis Guzmán, Gilda Carrasco,

Elba Leiva, Rodrigo Moore-Carrasco and

Additional information is available at the end of the chapter

Botanically, soybean belongs to the order *Rosaceae*, family *Leguminosae* or *Papillonaceae* or *Fa‐ baceae*, subfamily *Papilionoidae*, the genus *Glycine* and the cultivar Glycine max. It is an annu‐ al plant that measures up to 1.5 m tall, with pubescent leaves and pods; the stems are erect and rigid. In its primary and secondary roots, are located a variable number of nodes. One of the characteristics of the root system development is its sensitivity to variations in the supply and distribution of inorganic nutrients in the soil. The root system has a main root which can reach a meter deep, with an average being between 40 and 50 centimeters [1].

The soybean is a traditional oriental food, a leguminous plant native to eastern Asia, espe‐ cially in China. Soybean is cultivated worldwide, the United States is the country that grows more than 50% of the world production of this important food, which has been utilized in the diet of humans around the world, due to its high content of essential amino acids (Table 1) and calcium. It is consumed as cooked beans, soy sauce, soymilk and tofu (soybean curd). Also, a vegetable oil is obtained from soybeans, rich in polyunsaturated fatty acids [2].

Soybean is an annual plant, whose seeds are the edible organ. Soybean grains are rich in protein, and also a good source of various phytochemicals such as isoflavones and lignans, molecules with antioxidant and antiplatelet activities, among other effects; also may help fight and prevent various diseases, so constitute a useful source of food. For these reasons,

Soybean consumption benefits, especially in several chronic diseases, have been related to its important protein content, high levels of essential fatty acids, vitamins and minerals. Consequently, the present chapter aimed at the comprehensive characterization of the anti‐

> © 2013 Fuentes et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 Fuentes et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

these compounds have been intensively studied at basic and clinical level [3].

Iván Palomo

**1. Introduction**

http://dx.doi.org/10.5772/52601

## **Chapter 24**

## **Food, Nutrition and Health**

Eduardo Fuentes, Luis Guzmán, Gilda Carrasco, Elba Leiva, Rodrigo Moore-Carrasco and Iván Palomo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52601

## **1. Introduction**

Effect of Processing and Incorporation of Soybean and Cowpea Flour. *J. Agric. Food*

[13] Nnakwe, N. (1995). The effect and causes of protein-energy malnutrition in Nigerian

[14] NSRL. (2002). A Publication of China National Soybean Research Laoratory on Soy‐

[15] Olanipekun, B. F., Otunola, E. T., Adelakun, O. E., & Oyelade, O. J. (2009). Effect of fermentation with Rhizopus oligosporus on some physico-chemical properties of

[16] Onwueme, I. C., & Sinha, T. D. (1999). Field crop production in tropical Africa. *Eng‐*

[17] Parman, G. K. (1968). Fortification of Cereals and Cereal Products with Proteins and

[19] Plahar, W. A., & Leung, H. K. (1983). Composition of Ghanian fermented maize meal and the effect of soya fortification on sensory properties. *J Sci Food Agric*, 34, 407-411.

[20] Sereebutra, P., Solomons, N., Aliyu, M. H., & Jolly, P. E. (2006). Sociodemographic and environmental predictors of childhood stunting in rural Guatemala. *Nutr Res*, 26,

[21] Senshoku, K. S. (2002). Physical Characteristics and Processing method of Chinese

[22] Shils, M. E., Olson, J. A., & Moshe, S. (1999). Modern Nutrition in Health and Dis‐

[23] Tunde-Akintunde, T. Y. (2000). Predictive Models for Evaluating the Effect of Proc‐ essing Parameters or Yield and Quality of Some Soybean (Glycine max (L) Merri)

Soybean Fiber. *Home Technology Jianghe Tianrongbi. Fiber Co. Ltd. (China).*

starch extracts from soybean flour. *Food and Chemical Toxicology*, 1401-1405.

*Chem.*, 49, 2465-2471.

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children. *Nutrition Research*, 15(6), 785-794.

*land, Michael Health Ltd.*, 402-414.

65-70.

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ease. *Philadelphia Lippincott Williams & Wilkins*.

Products. *Ph.D Thesis- Universityof Ibadan, Nigeria.*

bean Procesing:. *From Field to Consumer*, http://www/NSRL.

[18] Potter, N. N. (1978). Food Science. *3rd Edition, Ilhiala, New York*, 356.

Botanically, soybean belongs to the order *Rosaceae*, family *Leguminosae* or *Papillonaceae* or *Fa‐ baceae*, subfamily *Papilionoidae*, the genus *Glycine* and the cultivar Glycine max. It is an annu‐ al plant that measures up to 1.5 m tall, with pubescent leaves and pods; the stems are erect and rigid. In its primary and secondary roots, are located a variable number of nodes. One of the characteristics of the root system development is its sensitivity to variations in the supply and distribution of inorganic nutrients in the soil. The root system has a main root which can reach a meter deep, with an average being between 40 and 50 centimeters [1].

The soybean is a traditional oriental food, a leguminous plant native to eastern Asia, espe‐ cially in China. Soybean is cultivated worldwide, the United States is the country that grows more than 50% of the world production of this important food, which has been utilized in the diet of humans around the world, due to its high content of essential amino acids (Table 1) and calcium. It is consumed as cooked beans, soy sauce, soymilk and tofu (soybean curd). Also, a vegetable oil is obtained from soybeans, rich in polyunsaturated fatty acids [2].

Soybean is an annual plant, whose seeds are the edible organ. Soybean grains are rich in protein, and also a good source of various phytochemicals such as isoflavones and lignans, molecules with antioxidant and antiplatelet activities, among other effects; also may help fight and prevent various diseases, so constitute a useful source of food. For these reasons, these compounds have been intensively studied at basic and clinical level [3].

Soybean consumption benefits, especially in several chronic diseases, have been related to its important protein content, high levels of essential fatty acids, vitamins and minerals. Consequently, the present chapter aimed at the comprehensive characterization of the anti‐

© 2013 Fuentes et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Fuentes et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

oxidant and antiplatelet activities of bioactive compounds, of soybean and its derivatives, and the extent to which soybean is a health-promoting food.

served in buckets. Also, from soybeans is possible to obtain vegetable oil, which is character‐

Food, Nutrition and Health http://dx.doi.org/10.5772/52601 523

Soy is a source not only of proteins, vitamins and minerals, but also of many bioactive com‐ pounds, such as isoflavones, protease inhibitors, saponins, and phytates. The great impor‐ tance of these compounds is based on their biochemical activity, which results in health

Soybeans contain a variety of bioactive phytochemicals such as phenolic acids, flavonoids, isoflavones, saponins, phytosterols and sphingolipids; being the phenolic compounds with the highest antioxidant capacity. The key benefits of soy are related to their excellent protein content, its high content of essential fatty acids, numerous vitamins and minerals, their iso‐

Polyphenolic compounds are a class of secondary metabolites biosynthesized by the vegetal kingdom [6] and involve a wide range of substances that possess one or more aromatic rings with at least one hydroxyl group. Among them, can be mentioned flavonoids, isoflavones, anthraquinones, anthocyanins, xanthones, phenols, hydroxycinnamic acids, lignin and others.

All of them act as scavengers or stabilizers of free radicals, and can produce chelation of metals, those having carboxyl groups at its end. Works have also been reported that its antioxidant

A study by Xu (2008) in 30 samples of soybean from different regions of North Dakota, Min‐ nnesota (USA), found that some cultivars of black soybean had a higher antioxidant capacity measured as ORAC, FRAP and DPPH than yellow soybeans and that the phenolic acid con‐ tent, isoflavones and antiochians was different, suggesting that some selected cultivars can be used as producers of high quality soy, because it provides a high content of phenolic phy‐

*Isoflavones and equol.* Of all plants, soybean contains the highest amount of different isofla‐ vones, a variety of phytoestrogens that have a structure similar to estrogen (figure 1). The interest in soybean isoflavones has gained importance since the 90's to today, there are a lot of evidence that these phytoestrogens possess a powerful and wide range of biological activ‐ ities. Isoflavones are not a steroid structure, however, has a phenolic ring than is capable of binding to the estrogen receptor (ER) and according to Makela (1995) can act as either an

The discovery of high concentrations of isoflavones in urine of adults who consume soy pro‐ tein, in addition to the evidence supporting its biological action, elevate the soybean to the category of functional food. The FDA in 1999 gave approval to give foods containing 6.25 g of soy protein the seal of protector of cardiovascular health, increasing significantly the sales

action can be attributed to the inhibition of prooxidants enzymes as lipoxygenase [7].

promotion and disease prevention, by their antioxidant, and antiplatelet activities.

ized by high polyunsaturated fatty acids [5].

flavones and their higher fiber content.

tochemicals and antioxidant properties [8].

of foods fortified with soy and isoflavone constituent.

agonist or an antagonist [9].

*Antioxidant Activity*

**Table 1.** Amino acid composition of soybeans seeds. Source: Adapted by authors from FAO (1970) and FAO/WHO (1973).

### **2. Soybean: foods and bioactive compounds**

Soybeans are consumed as cooked beans, which previously should be boiled for at least three hours. With these grains are prepared meals, salads and soups, which in turn, are a source of preparation of other foods. From soybean grains also is possible to obtain soy sauce, which is used especially in oriental foods, such as sushi. The soy sauce is usually made by fermenting soya grains with cracked roasted wheat, which are arranged in blocks and immersed in a cold salt water, the process takes about a year in pots mud, sometimes dried mushrooms are added as mushrooms. In Japan, it is illegal to produce or import artifi‐ cial soy sauce and therefore all Japanese soy sauces are made by the traditional way [4].

Another food derived from soy is tofu, which is a widely used food in the East as well as vegetarian meals around the world. Required for preparing tofu soybeans are water and a coagulant. Initially, you get the coagulated soymilk, then is pressed and separated the liquid portion from the solid. Tofu has a firm texture similar to cheese milk; the color is cream and served in buckets. Also, from soybeans is possible to obtain vegetable oil, which is character‐ ized by high polyunsaturated fatty acids [5].

Soy is a source not only of proteins, vitamins and minerals, but also of many bioactive com‐ pounds, such as isoflavones, protease inhibitors, saponins, and phytates. The great impor‐ tance of these compounds is based on their biochemical activity, which results in health promotion and disease prevention, by their antioxidant, and antiplatelet activities.

### *Antioxidant Activity*

oxidant and antiplatelet activities of bioactive compounds, of soybean and its derivatives,

**Amino acid g/16 g Nitrogen**

**Table 1.** Amino acid composition of soybeans seeds. Source: Adapted by authors from FAO (1970) and FAO/WHO

Soybeans are consumed as cooked beans, which previously should be boiled for at least three hours. With these grains are prepared meals, salads and soups, which in turn, are a source of preparation of other foods. From soybean grains also is possible to obtain soy sauce, which is used especially in oriental foods, such as sushi. The soy sauce is usually made by fermenting soya grains with cracked roasted wheat, which are arranged in blocks and immersed in a cold salt water, the process takes about a year in pots mud, sometimes dried mushrooms are added as mushrooms. In Japan, it is illegal to produce or import artifi‐ cial soy sauce and therefore all Japanese soy sauces are made by the traditional way [4].

Another food derived from soy is tofu, which is a widely used food in the East as well as vegetarian meals around the world. Required for preparing tofu soybeans are water and a coagulant. Initially, you get the coagulated soymilk, then is pressed and separated the liquid portion from the solid. Tofu has a firm texture similar to cheese milk; the color is cream and

4.54 7.78 6.38 1.26 1.33

4.94 3.14

3.86 1.28 4.80

7.23 2.53 4.26 11.70 18.70 4.18 5.49 5.12

and the extent to which soybean is a health-promoting food.

522 Soybean - Bio-Active Compounds

Isoleucine Leucine Lysine Methionine Cysteine

Phenylalanine Tyrosine

Threonine Tryptophan Valine

Arginine Histidine Alanine Aspartic acid Glutamic acid Glycine Proline Serine

**2. Soybean: foods and bioactive compounds**

(1973).

Soybeans contain a variety of bioactive phytochemicals such as phenolic acids, flavonoids, isoflavones, saponins, phytosterols and sphingolipids; being the phenolic compounds with the highest antioxidant capacity. The key benefits of soy are related to their excellent protein content, its high content of essential fatty acids, numerous vitamins and minerals, their iso‐ flavones and their higher fiber content.

Polyphenolic compounds are a class of secondary metabolites biosynthesized by the vegetal kingdom [6] and involve a wide range of substances that possess one or more aromatic rings with at least one hydroxyl group. Among them, can be mentioned flavonoids, isoflavones, anthraquinones, anthocyanins, xanthones, phenols, hydroxycinnamic acids, lignin and others.

All of them act as scavengers or stabilizers of free radicals, and can produce chelation of metals, those having carboxyl groups at its end. Works have also been reported that its antioxidant action can be attributed to the inhibition of prooxidants enzymes as lipoxygenase [7].

A study by Xu (2008) in 30 samples of soybean from different regions of North Dakota, Min‐ nnesota (USA), found that some cultivars of black soybean had a higher antioxidant capacity measured as ORAC, FRAP and DPPH than yellow soybeans and that the phenolic acid con‐ tent, isoflavones and antiochians was different, suggesting that some selected cultivars can be used as producers of high quality soy, because it provides a high content of phenolic phy‐ tochemicals and antioxidant properties [8].

*Isoflavones and equol.* Of all plants, soybean contains the highest amount of different isofla‐ vones, a variety of phytoestrogens that have a structure similar to estrogen (figure 1). The interest in soybean isoflavones has gained importance since the 90's to today, there are a lot of evidence that these phytoestrogens possess a powerful and wide range of biological activ‐ ities. Isoflavones are not a steroid structure, however, has a phenolic ring than is capable of binding to the estrogen receptor (ER) and according to Makela (1995) can act as either an agonist or an antagonist [9].

The discovery of high concentrations of isoflavones in urine of adults who consume soy pro‐ tein, in addition to the evidence supporting its biological action, elevate the soybean to the category of functional food. The FDA in 1999 gave approval to give foods containing 6.25 g of soy protein the seal of protector of cardiovascular health, increasing significantly the sales of foods fortified with soy and isoflavone constituent.

vone glycoside [16] at intestinal level and subsequent bacterial biotransformation in the co‐

Food, Nutrition and Health http://dx.doi.org/10.5772/52601 525

Equol does not originate in plants, but is the product of degradation of the isoflavone glyco‐ side in the intestine [21], situation that was confirmed in infants of 4 months who were fed with formulations containing soy [22, 23]. All mammals can biotransform isoflavone glyco‐ side permanently, except the man who for reasons still unexplained, only 20-35% of adults produces equol after eating foods made from soybeans or that have been enriched with pure

Several studies have suggested that those mammals who are equol producers show a great‐ er response to isoflavone-enriched diets, leading to the conclusion that equol is a more po‐ tent isoflavone than genistein and is the only one that has a chiral carbon at position 3 of the furan ring, making two enantiomeric forms, S and R that differ significantly in their confor‐

Gopaul (2012) investigated the effect of equol on gene expression of proteins in the skin, us‐ ing a cellular model of human dermis and found that equol significantly increased gene ex‐ pression of collagen, elastin (ELN), and tissue factor inhibitor of metalloproteinases and decreased metalloproteinases (MMPs), causing positive changes in the skin's antioxidant and anti-aging genes. The same occurred in cultured human fibroblasts (hMFC), in which equol significantly increased type I collagen (COL1A1), while 5a-dihydrotestosterone (5a-DHT) significantly decreased cell viability. These findings suggest that equol has great po‐ tential for topical applications to the skin, for the treatment and prevention of aging of the