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## **Meet the editor**

Prof. Hany A. El-Shemy received his two Ph.D. degrees in biochemistry and genetic engineering from the University of Cairo, Egypt and the University of Hiroshima, Japan. He became an Assistant Professor at the Biochemistry Department of Cairo University, Egypt from September 1996, and advanced to Associate Professor in September 2002, as well as full Professor in March 2007.

His research interests are in the fields of plant biotechnology and medicinal plants (Molecular Biology). He received 2 patents, wrote 7 international books, published more than 65 SCI journal papers and 30 conference presentations. He served as the technical committee member and chair in many international conferences as well as editor including PLoS ONE journal, BMC Genomics, and reviewer for more than 10 SCI cited journals. He received several awards, including State prize awarded from Academy of Science, Egypt (2004), Young Arab Researcher prize Awarded from Shuman Foundation, Jordan (2005), State Excellence prize from Academy of Science, Egypt (2011), Cairo University Prizes 2007, 2010.

Contents

**Preface IX**

**Feedstuff 1** Stefano Tavoletti

Chapter 1 **Critical Evaluation of Soybean Role in Animal Production**

Chapter 2 **Advanced Techniques in Soybean Biodiesel 23**

and Danielle Oliveira de Andrade

Chapter 4 **Phytoestrogens and Colon Cancer 75**

**for Multiple Sclerosis 97**

Federica D'Asta

Joanna McFarlane

Chapter 3 **Facilities for Obtaining Soybean Oil in Small Plants 47**

**Chains Based on the Valorization of Locally Produced**

Mauricio G. Fonseca, Luciano N. Batista, Viviane F. Silva, Erica C. G. Pissurno, Thais C. Soares, Monique R. Jesus and Georgiana F. Cruz

Ednilton Tavares de Andrade, Luciana Pinto Teixeira, Ivênio Moreira da Silva, Roberto Guimarães Pereira, Oscar Edwin Piamba Tulcan

B. Pampaloni, C. Mavilia, E. Bartolini, F. Tonelli, M.L. Brandi and

Chapter 5 **Bowman-Birk Protease Inhibitor as a Potential Oral Therapy**

Chapter 6 **Soybean Oil Derivatives for Fuel and Chemical Feedstocks 111**

Chapter 7 **Effect of Dietary Plant Lipids on Conjugated Linoleic Acid (CLA) Concentrations in Beef and Lamb Meats 135**

Farinaz Safavi and Abdolmohamad Rostami

Pilar Teresa Garcia and Jorge J. Casal

## Contents

#### **Preface XIII**



Chapter 17 **Soybean: Non-Nutritional Factors and Their Biological**

Chapter 18 **Approach for Dispersing a Hydrophilic Compound as**

Chapter 19 **Recent Advances on Soybean Isoflavone Extraction and Enzymatic Modification of Soybean Oil 429**

Masakazu Naya and Masanao Imai

Chapter 21 **Brazilian Soybean Varieties for Human Use 475**

Chapter 22 **Effects of Soybean Trypsin Inhibitor on Hemostasis 495**

Chapter 23 **Potential Use of Soybean Flour (Glycine max) in Food**

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

Oliveira and Rodrigo Santos Leite

Takamatsu and Shigeru Yamamoto

Moore-Carrasco and Iván Palomo

**Fortification 513**

Chapter 24 **Food, Nutrition and Health 521**

Chapter 20 **Soybean, Nutrition and Health 453** Sherif M. Hassan

A. Cabrera-Orozco, C. Jiménez-Martínez and G. Dávila-Ortiz

Contents **VII**

Neusa Fátima Seibel, Fernanda Périco Alves, Marcelo Álvares de

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

Eduardo Fuentes, Luis Guzmán, Gilda Carrasco, Elba Leiva, Rodrigo

**Nanoparticles Into Soybean Oil Using Evaporation**

**Functionality 387**

**Technique 411** Kenjiro Koga

Chapter 17 **Soybean: Non-Nutritional Factors and Their Biological Functionality 387**

A. Cabrera-Orozco, C. Jiménez-Martínez and G. Dávila-Ortiz


Chapter 8 **Value - Added Products from Soybean: Removal of Anti-Nutritional Factors via Bioprocessing 161**

Chapter 9 **Soybean and Isoflavones – From Farm to Fork 181**

Chapter 10 **Evaluation of Soybean Straw as Litter Material in Poultry**

**Production and Substrate in Composting of Broiler**

Jaenisch, Taiana Cestonaro and Virginia Santiago Silva

Chapter 11 **Acrylated-Epoxidized Soybean Oil-Based Polymers and Their**

Chapter 13 **Soybean Meal and The Potential for Upgrading Its Feeding Value by Enzyme Supplementation 287**

Chapter 14 **Soybean in Monogastric Nutrition: Modifications to Add Value**

Samuel N. Nahashon and Agnes K. Kilonzo-Nthenge

**Flavonoids in Accessions from a Close Relative of Soybean, Neonotonia wightii (Wright & Arn. J.A. Lackey) in the U.S.**

**and Disease Prevention Properties 309**

Chapter 15 **The Effects of Hydrogenation on Soybean Oil 353**

Chapter 16 **Variability for Phenotype, Anthocyanin Indexes, and**

J.B. Morris, M.L. Wang and B. Tonnis

**Germplasm Collection for Potential Use as a**

Valeria Maria Nascimento Abreu, Paulo Giovanni de Abreu, Doralice Pedroso de Paiva, Arlei Coldebella, Fátima Regina Ferreira

**Use in the Generation of Electrically Conductive Polymer**

Susana Hernández López and Enrique Vigueras Santiago

Casañas Haasis Villavicencio

**Carcasses 203**

**Composites 231**

Chapter 12 **Soybean and Prostate Cancer 265**

Fred A. Kummerow

**Health Forage 375**

Xiaomeng Li, Ying Xu and Ichiro Tsuji

D. Pettersson and K. Pontoppidan

Wang

**VI** Contents

Liyan Chen, Ronald L. Madl, Praveen V. Vadlani, Li Li and Weiqun

Michel Mozeika Araújo, Gustavo Bernardes Fanaro and Anna Lucia


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

Chapter 24 **Food, Nutrition and Health 521** Eduardo Fuentes, Luis Guzmán, Gilda Carrasco, Elba Leiva, Rodrigo Moore-Carrasco and Iván Palomo

Preface

This book provides an overview of the importance of soybean all over the world. The au‐

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, Chapter 1 provides ideas on Critical Evaluation of Soybean Role in Animal

We can also learn that new marketing strategies are necessary to make consumers aware of the importance of the overall characteristics of local production chains in defining the quali‐ ty of a final product and to ensure at the same time a profitable price for the producers.

Chapter 3 brings an overview of the types of equipment used for industrial processing of soybeans for obtaining vegetable oil in small plants and Chapter 4, for example, highlights the international literature which suggests that phytoestrogens have a potentially high clini‐ cal impact and the expansion of knowledge on soy, soy foods, and soy products which will

Other chapters aim at the comprehensive characterization of the antioxidant and antiplatelet activities of bioactive compounds, of soybean and its derivatives, and the extent to which

This book will be useful for soybean researchers and other academic staff and will provide

**Hany A. El-Shemy, Professor** Cairo University, EGYPT

thors contributed with 24 chapters dealing with various topics.

lead to novel future developments in the field of cancer treatment.

its readers with valuable insight into the last developments in the field.

soybean is a health-promoting food.

Production Chains Based on the Valorisation of Locally Produced Feedstuff.

## Preface

This book provides an overview of the importance of soybean all over the world. The au‐ thors contributed with 24 chapters dealing with various topics.

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, Chapter 1 provides ideas on Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorisation of Locally Produced Feedstuff.

We can also learn that new marketing strategies are necessary to make consumers aware of the importance of the overall characteristics of local production chains in defining the quali‐ ty of a final product and to ensure at the same time a profitable price for the producers.

Chapter 3 brings an overview of the types of equipment used for industrial processing of soybeans for obtaining vegetable oil in small plants and Chapter 4, for example, highlights the international literature which suggests that phytoestrogens have a potentially high clini‐ cal impact and the expansion of knowledge on soy, soy foods, and soy products which will lead to novel future developments in the field of cancer treatment.

Other chapters aim at the comprehensive characterization of the antioxidant and antiplatelet activities of bioactive compounds, of soybean and its derivatives, and the extent to which soybean is a health-promoting food.

This book will be useful for soybean researchers and other academic staff and will provide its readers with valuable insight into the last developments in the field.

> **Hany A. El-Shemy, Professor** Cairo University, EGYPT

**Chapter 1**

**Critical Evaluation of Soybean Role in Animal**

**Produced Feedstuff**

http://dx.doi.org/10.5772/52476

Additional information is available at the end of the chapter

be treated as a commodity in the world trade of raw materials.

Stefano Tavoletti

**1. Introduction**

**Production Chains Based on the Valorization of Locally**

Commodities such as soybean and maize are respectively protein and energy concentrates that represent the basic raw materials used by the animal feeding industry and their prices influence the overall market of agricultural products. In animal feeding, soybean is mostly used as soybean meal which is a by-product of oil seed extraction industry and the availabil‐ ity of this raw material on the international market has led to its worldwide diffusion as the main source of protein for animal feed formulation. Soybean can also be used as raw seed due to its high fat content that makes this grain legume a valid feed to increase both protein and energy concentration of animal diets [1]. The close relationship between oil extraction industry and feed industry together with its high nutritional value for human consumption, as reported by several articles included in the present book, made soybean a perfect crop to

However, despite all these positive characteristics, the diffusion of soybean and its by-prod‐ ucts, together with the overall increased importance of the commodities trade, has triggered in agriculture several downstream effects that had deep consequences on the evolution of agricultural practices. After World War II agriculture initiated a course of progressive struc‐ tural changes toward the implementation of more intense production processes due to both the need of increasing world food supply and to the progressive reduction of people em‐ ployed in agriculture. The diffusion of improved varieties, fertilizers, pesticides, advanced agricultural machineries, intensive systems of animal rearing, efficient systems for the stor‐ age and transformation of agricultural products led to the abandonment of traditional crop‐ ping and animal farming systems [2]. Therefore, important agronomic practices such as crop

> © 2013 Tavoletti ; 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,

© 2013 Tavoletti ; 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.

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced Feedstuff**

Stefano Tavoletti

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52476

## **1. Introduction**

Commodities such as soybean and maize are respectively protein and energy concentrates that represent the basic raw materials used by the animal feeding industry and their prices influence the overall market of agricultural products. In animal feeding, soybean is mostly used as soybean meal which is a by-product of oil seed extraction industry and the availabil‐ ity of this raw material on the international market has led to its worldwide diffusion as the main source of protein for animal feed formulation. Soybean can also be used as raw seed due to its high fat content that makes this grain legume a valid feed to increase both protein and energy concentration of animal diets [1]. The close relationship between oil extraction industry and feed industry together with its high nutritional value for human consumption, as reported by several articles included in the present book, made soybean a perfect crop to be treated as a commodity in the world trade of raw materials.

However, despite all these positive characteristics, the diffusion of soybean and its by-prod‐ ucts, together with the overall increased importance of the commodities trade, has triggered in agriculture several downstream effects that had deep consequences on the evolution of agricultural practices. After World War II agriculture initiated a course of progressive struc‐ tural changes toward the implementation of more intense production processes due to both the need of increasing world food supply and to the progressive reduction of people em‐ ployed in agriculture. The diffusion of improved varieties, fertilizers, pesticides, advanced agricultural machineries, intensive systems of animal rearing, efficient systems for the stor‐ age and transformation of agricultural products led to the abandonment of traditional crop‐ ping and animal farming systems [2]. Therefore, important agronomic practices such as crop

© 2013 Tavoletti ; 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 Tavoletti ; 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.

rotations including a cereals and legumes, cultivation of forage crops for animal feeding, a close link between animal farming and field productions useful to ensure an adequate con‐ tent of organic matter in the soil were almost completely abandoned due to the diffusion of monocultures [3]. As a consequence, agricultural soil fertility decrease dramatically, as indi‐ cated by the dangerous low levels of organic matter content that at present are generally re‐ corded in most countries that have been characterized by such an intensification of agricultural practices, and the use of chemical fertilizers became an indispensable necessity to reach economically valuable productions [4-7]. At the same time animal farming was based on the use of by-products that were available on the market to reduce the costs of pro‐ duction and simplify the animal production system.

non-standardized food and the attention given to the impact of agricultural activities on the environment, human and animal health [8-10]. Therefore, an increasing number of animal farms adopted more sustainable instead of intensive production systems that were closely linked to the area of production by using locally produced animal feed and reducing their dependence from commodities. Moreover, these farms developed direct commercialization systems trying to make their business more profitable with an emphasis on the quality of

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced

Feedstuff

3

http://dx.doi.org/10.5772/52476

**Figure 2.** Soybean seed production of USA, Brazil, Argentina, China, India and European Union (EU).

tivity both profitable and integrated within local soil and climatic characteristics.

**2. Soybean seed and meal trade**

Aim of the present article is to critically evaluate the effects of the large diffusion of soybean on the international market concerning aspects related to the evolution of soybean trade, the effects on agricultural systems where soybean cannot be cultivated, the present dependence from soybean of intensive animal farming systems and the consequences on small or medi‐ um farms applying not-intensive animal production chains. An experience under way in the Marche Region (Central Italy) will also be illustrated as an attempt to make agricultural ac‐

Data on soybean seed and meal (oil was not considered) production, import, export and pri‐ ces were obtained at the Index Mundi website [11] (data source:USDA) where information on all commodities trade is available. Soybean data were usually available from 1964 to 2011 together with estimates referred to the current year 2012, even though for some countries and also for the European Union data availability covered a shorter period of time. Data

both the final products and the production system.

At present, globalization together with the rapid economic development actually under way in eastern countries and the world economic crisis are jeopardizing the economic feasibility of many agricultural activities, mainly in the European Union. Those farms that restructured their production processes in order to simply satisfy the demand for raw materials by the food and feed industries and by multiple retailers that manage marketing and commerciali‐ zation have recently experienced the negative effects of increased costs of production fol‐ lowed by the low prices of agricultural products paid to the farmers. In particular, the increased costs of commodities such as soybean seed and meal that recently happened sev‐ eral times together with the low price of animal products paid to the farmers, mainly in the beef, pork and dairy production chains, made unprofitable the economic activities of animal farms, especially small or medium size farms.

**Figure 1.** World soybean seed (continuous line) and meal (dashed line) production.

Recently new strategies for agricultural development have emerged due to the interest of consumers toward high quality products and production chains, the increased request of non-standardized food and the attention given to the impact of agricultural activities on the environment, human and animal health [8-10]. Therefore, an increasing number of animal farms adopted more sustainable instead of intensive production systems that were closely linked to the area of production by using locally produced animal feed and reducing their dependence from commodities. Moreover, these farms developed direct commercialization systems trying to make their business more profitable with an emphasis on the quality of both the final products and the production system.

**Figure 2.** Soybean seed production of USA, Brazil, Argentina, China, India and European Union (EU).

Aim of the present article is to critically evaluate the effects of the large diffusion of soybean on the international market concerning aspects related to the evolution of soybean trade, the effects on agricultural systems where soybean cannot be cultivated, the present dependence from soybean of intensive animal farming systems and the consequences on small or medi‐ um farms applying not-intensive animal production chains. An experience under way in the Marche Region (Central Italy) will also be illustrated as an attempt to make agricultural ac‐ tivity both profitable and integrated within local soil and climatic characteristics.

## **2. Soybean seed and meal trade**

rotations including a cereals and legumes, cultivation of forage crops for animal feeding, a close link between animal farming and field productions useful to ensure an adequate con‐ tent of organic matter in the soil were almost completely abandoned due to the diffusion of monocultures [3]. As a consequence, agricultural soil fertility decrease dramatically, as indi‐ cated by the dangerous low levels of organic matter content that at present are generally re‐ corded in most countries that have been characterized by such an intensification of agricultural practices, and the use of chemical fertilizers became an indispensable necessity to reach economically valuable productions [4-7]. At the same time animal farming was based on the use of by-products that were available on the market to reduce the costs of pro‐

At present, globalization together with the rapid economic development actually under way in eastern countries and the world economic crisis are jeopardizing the economic feasibility of many agricultural activities, mainly in the European Union. Those farms that restructured their production processes in order to simply satisfy the demand for raw materials by the food and feed industries and by multiple retailers that manage marketing and commerciali‐ zation have recently experienced the negative effects of increased costs of production fol‐ lowed by the low prices of agricultural products paid to the farmers. In particular, the increased costs of commodities such as soybean seed and meal that recently happened sev‐ eral times together with the low price of animal products paid to the farmers, mainly in the beef, pork and dairy production chains, made unprofitable the economic activities of animal

duction and simplify the animal production system.

2 Soybean - Bio-Active Compounds

farms, especially small or medium size farms.

**Figure 1.** World soybean seed (continuous line) and meal (dashed line) production.

Recently new strategies for agricultural development have emerged due to the interest of consumers toward high quality products and production chains, the increased request of Data on soybean seed and meal (oil was not considered) production, import, export and pri‐ ces were obtained at the Index Mundi website [11] (data source:USDA) where information on all commodities trade is available. Soybean data were usually available from 1964 to 2011 together with estimates referred to the current year 2012, even though for some countries and also for the European Union data availability covered a shorter period of time. Data were organized in an excel data sheet and elaborated to obtain information concerning sin‐ gle countries involved in the soybean international trade. The total worldwide value of seed and meal production, import and export, expressed as Million Metric Tons (MMT), were then calculated by summing the data available for each country for each year. Results were summarized by graphics concerning the overall soybean trade, the characteristics of single countries significantly involved in the international market of soybean and the comparisons among different countries.

Soybean seed production progressively increased from 1964 (28,3 MMT) to 2010 (264,7 MMT) and, although followed by a slight decrement in 2011 (236,4 MMT), soybean seed US‐ DA 2012 estimated production (referred to june 2012) is 266,8 MMT confirming the positive trend for this commodity (Figure 1).

USDA data identified 42 countries characterized by an estimated soybean seed production of at least 0.001 MMT. However since 1964 more than 90% of total soybean seed production was concentrated in 5 countries (USA, Brazil, Argentina, China and India) and USA, Brazil and Argentina covered about 80% of worldwide soybean production (Figure 2).

European Union (Figure 2) produced between 0.6 MMT (year 2008) and 1,4 MMT (year 1999) of soybean seeds and in 2011 EU27 ranked twelfth with a production of 1,1 MMT (0.47% of world production). These data confirm the almost complete dependence of Europe from non-EU and mainly American countries to satisfy the needs of protein concentrates of European animal production chains.

**Figure 3.** Soybean meal production of USA, Brazil, Argentina, China, India and European Union (EU).

da) compared to the progressive overall world increase of soybean meal production.

commodity by several new countries.

Moreover, in the 1994-2011 time period the relative contribution to the worldwide soybean meal production of Japan decreased from 8% (1994) to 0.82% (2011) and Canada was also characterized by the same negative trend (from 3.12% in 1994 to 0.62% in 2011); this behav‐ iour could be attributed to the low increase in meal production over time that characterized these two countries (from 1.07 to 1.45 MMT for Japan and from 0.42 to 1.10 MMT for Cana‐

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced

Feedstuff

5

http://dx.doi.org/10.5772/52476

Therefore, the international scenario concerning soybean clearly shows that 3 countries (USA, Brasil and Argentina) handle almost all the world production of soybean seed, where‐ as China must be added to USA, Brasil and Argentina concerning the production of soybean meal. Conversely European Union has a negligible level of soybean seed production and a low level of soybean meal production as a by-product of oil extraction from imported soy‐ bean seeds. This determines that European Union animal production chains rely completely on imported soybean and this situation generates an almost total dependence of European farms from the international trade of these commodities. Data on the import/export of soy‐ bean seeds and meal also confirm that EU animal farming is suffering the effects of globali‐ zation of the markets, mostly because the dynamics of the international market of soybean is changing as a consequence of the new scenario due to the increased interest toward this

Figure 4 and Figure 5 summarize the world scenario of soybean seed and meal trade, re‐ spectively. World seed production increase was also followed by an increase in the amount, expressed as percentage of total world production value, of overall exported seed that was about 25% until 1995, then raised to about 30% between 1996 and 2005 and finally reached

the value of 35 % in 2010 with an estimated amount of 36% for year 2012 (Figure 4).

Figure 1 also shows that the production of soybean meal had almost the same trend of world soybean seed production, with a steady increase from 1964 (13,5 MMT) to 2011 (177,4 MMT). However, the relative contribution of each country is different than what has been described for seed production. USA was the highest soybean meal producer until 2009 when it was exceeded by China that, based on the 2012 estimate, at present seems to be the world leader in soybean meal production. China showed a relatively low meal production until 1997 when it started a progressive increase in the production of this by-product of oil extrac‐ tion (Figure 3).

It is interesting to compare soybean seed and meal amounts produced over time in China; soybean seed production in this country was always lower than 20 MMT, ranging from 6.14 MMT in 1995 to 17.4 MMT in 2004 (Figure 2), whereas soybean meal production was lower than 10 MMT until 1997 but increased from 10 MMT in 1998 to 46.9 MMT in 2011 with an estimated 50.2 MMT for the year 2012 (Figure 3). Starting from 1997 also Brasil and Argenti‐ na began to increase their soybean meal production reaching about 28 MMT in 2011, where‐ as USA, after a constant increase from 1964 to 1996, was characterized by and almost constant meal production of about 35 MMT/year in the 1997-2011 time period (Figure 3).

India, which was the fifth producer of soybean seeds, also showed a constant increase in soybean meal production from 1987 to 2011 although remaining below the 10 MMT level of meal production (Figure 3). On the contrary in the 2001-2011 time period the European Un‐ ion was characterized by a negative trend of soybean meal production that decreased from 14 MMT in 2001 to less than 10 MMT after 2008 (Figure 3).

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced Feedstuff http://dx.doi.org/10.5772/52476 5

**Figure 3.** Soybean meal production of USA, Brazil, Argentina, China, India and European Union (EU).

were organized in an excel data sheet and elaborated to obtain information concerning sin‐ gle countries involved in the soybean international trade. The total worldwide value of seed and meal production, import and export, expressed as Million Metric Tons (MMT), were then calculated by summing the data available for each country for each year. Results were summarized by graphics concerning the overall soybean trade, the characteristics of single countries significantly involved in the international market of soybean and the comparisons

Soybean seed production progressively increased from 1964 (28,3 MMT) to 2010 (264,7 MMT) and, although followed by a slight decrement in 2011 (236,4 MMT), soybean seed US‐ DA 2012 estimated production (referred to june 2012) is 266,8 MMT confirming the positive

USDA data identified 42 countries characterized by an estimated soybean seed production of at least 0.001 MMT. However since 1964 more than 90% of total soybean seed production was concentrated in 5 countries (USA, Brazil, Argentina, China and India) and USA, Brazil

European Union (Figure 2) produced between 0.6 MMT (year 2008) and 1,4 MMT (year 1999) of soybean seeds and in 2011 EU27 ranked twelfth with a production of 1,1 MMT (0.47% of world production). These data confirm the almost complete dependence of Europe from non-EU and mainly American countries to satisfy the needs of protein concentrates of

Figure 1 also shows that the production of soybean meal had almost the same trend of world soybean seed production, with a steady increase from 1964 (13,5 MMT) to 2011 (177,4 MMT). However, the relative contribution of each country is different than what has been described for seed production. USA was the highest soybean meal producer until 2009 when it was exceeded by China that, based on the 2012 estimate, at present seems to be the world leader in soybean meal production. China showed a relatively low meal production until 1997 when it started a progressive increase in the production of this by-product of oil extrac‐

It is interesting to compare soybean seed and meal amounts produced over time in China; soybean seed production in this country was always lower than 20 MMT, ranging from 6.14 MMT in 1995 to 17.4 MMT in 2004 (Figure 2), whereas soybean meal production was lower than 10 MMT until 1997 but increased from 10 MMT in 1998 to 46.9 MMT in 2011 with an estimated 50.2 MMT for the year 2012 (Figure 3). Starting from 1997 also Brasil and Argenti‐ na began to increase their soybean meal production reaching about 28 MMT in 2011, where‐ as USA, after a constant increase from 1964 to 1996, was characterized by and almost constant meal production of about 35 MMT/year in the 1997-2011 time period (Figure 3).

India, which was the fifth producer of soybean seeds, also showed a constant increase in soybean meal production from 1987 to 2011 although remaining below the 10 MMT level of meal production (Figure 3). On the contrary in the 2001-2011 time period the European Un‐ ion was characterized by a negative trend of soybean meal production that decreased from

14 MMT in 2001 to less than 10 MMT after 2008 (Figure 3).

and Argentina covered about 80% of worldwide soybean production (Figure 2).

among different countries.

4 Soybean - Bio-Active Compounds

trend for this commodity (Figure 1).

European animal production chains.

tion (Figure 3).

Moreover, in the 1994-2011 time period the relative contribution to the worldwide soybean meal production of Japan decreased from 8% (1994) to 0.82% (2011) and Canada was also characterized by the same negative trend (from 3.12% in 1994 to 0.62% in 2011); this behav‐ iour could be attributed to the low increase in meal production over time that characterized these two countries (from 1.07 to 1.45 MMT for Japan and from 0.42 to 1.10 MMT for Cana‐ da) compared to the progressive overall world increase of soybean meal production.

Therefore, the international scenario concerning soybean clearly shows that 3 countries (USA, Brasil and Argentina) handle almost all the world production of soybean seed, where‐ as China must be added to USA, Brasil and Argentina concerning the production of soybean meal. Conversely European Union has a negligible level of soybean seed production and a low level of soybean meal production as a by-product of oil extraction from imported soy‐ bean seeds. This determines that European Union animal production chains rely completely on imported soybean and this situation generates an almost total dependence of European farms from the international trade of these commodities. Data on the import/export of soy‐ bean seeds and meal also confirm that EU animal farming is suffering the effects of globali‐ zation of the markets, mostly because the dynamics of the international market of soybean is changing as a consequence of the new scenario due to the increased interest toward this commodity by several new countries.

Figure 4 and Figure 5 summarize the world scenario of soybean seed and meal trade, re‐ spectively. World seed production increase was also followed by an increase in the amount, expressed as percentage of total world production value, of overall exported seed that was about 25% until 1995, then raised to about 30% between 1996 and 2005 and finally reached the value of 35 % in 2010 with an estimated amount of 36% for year 2012 (Figure 4).

observed between 1980 and 1995 and subsequently the percentage of exported meal went back to the value of 33% for year 2010, the same value estimated for year 2012 (Figure 5). Therefore, about one third of total world soybean seed and meal production is exported to

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced

Feedstuff

7

http://dx.doi.org/10.5772/52476

The relative importance of the import/export of soybean seed and meal compared with the internal production is also shown separately for each of the 4 most important countries (USA, China, Brazil and Argentina) together with the situation characterizing the European

At present, USA is the highest producer of soybean seed in the world and in 2010 45% of total USA internal production was exported, this level being maintained in 2012 estimate.

**Figure 6.** USA: Soybean seed and meal production and export; imported amounts are negligible and therefore are

Together with USA, also Brazil and Argentina (data available from 1978 to 2012) are charac‐ terized by a high amount of internal production which is exported followed by a negligible import of soybean products. In particular, Brazil is getting close to USA levels of production for both soybean seed and meal (Figures 2 and 3) and seed export, that ranged between 12 and 20% in the 1980-1995 time period, steadily increased from 30% in 1996 to 55% in 2011 with an estimate of 45% for year 2012 (Figure 7). A different trend characterized soybean meal Brazilian export that was about 76% from 1978 to 1990 and then decreased to 50% in 2011 with an estimated level of 48% for year 2012. On the whole, about 50% of both seed and

meal internal Brazilian productions is exported (Figure 7).

About 20-25% of internal USA soybean meal has always been exported (Figure 6).

countries that show a deficit in the internal production of these commodities.

Union (Figures 6-10).

not shown.

**Figure 4.** Soybean seed: comparison between world production and export; every five year export values expressed as percentage of total world production are shown (2012 data are estimated values).

**Figure 5.** Soybean meal: comparison between world production and export; every five year export values expressed as percentage of total world production are shown (2012 data are estimated values).

A similar trend was shown by global soybean meal production. An increase from 17% to 35% in the percentage of exported meal, expressed as percentage the total world meal pro‐ duction, was registered in the 1965-1980 time period, a further slight increase until 38% was observed between 1980 and 1995 and subsequently the percentage of exported meal went back to the value of 33% for year 2010, the same value estimated for year 2012 (Figure 5). Therefore, about one third of total world soybean seed and meal production is exported to countries that show a deficit in the internal production of these commodities.

The relative importance of the import/export of soybean seed and meal compared with the internal production is also shown separately for each of the 4 most important countries (USA, China, Brazil and Argentina) together with the situation characterizing the European Union (Figures 6-10).

At present, USA is the highest producer of soybean seed in the world and in 2010 45% of total USA internal production was exported, this level being maintained in 2012 estimate. About 20-25% of internal USA soybean meal has always been exported (Figure 6).

**Figure 4.** Soybean seed: comparison between world production and export; every five year export values expressed as

**Figure 5.** Soybean meal: comparison between world production and export; every five year export values expressed

A similar trend was shown by global soybean meal production. An increase from 17% to 35% in the percentage of exported meal, expressed as percentage the total world meal pro‐ duction, was registered in the 1965-1980 time period, a further slight increase until 38% was

as percentage of total world production are shown (2012 data are estimated values).

percentage of total world production are shown (2012 data are estimated values).

6 Soybean - Bio-Active Compounds

**Figure 6.** USA: Soybean seed and meal production and export; imported amounts are negligible and therefore are not shown.

Together with USA, also Brazil and Argentina (data available from 1978 to 2012) are charac‐ terized by a high amount of internal production which is exported followed by a negligible import of soybean products. In particular, Brazil is getting close to USA levels of production for both soybean seed and meal (Figures 2 and 3) and seed export, that ranged between 12 and 20% in the 1980-1995 time period, steadily increased from 30% in 1996 to 55% in 2011 with an estimate of 45% for year 2012 (Figure 7). A different trend characterized soybean meal Brazilian export that was about 76% from 1978 to 1990 and then decreased to 50% in 2011 with an estimated level of 48% for year 2012. On the whole, about 50% of both seed and meal internal Brazilian productions is exported (Figure 7).

ure 8). On the other end the amount of soybean meal internally produced increased approximately four times and almost 100% of the meal was exported, as clearly shown by Figure 9. Therefore these data suggest that Argentina is essentially growing soybean to ex‐

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**Figure 9.** China: soybean seed and meal production and import; exported amounts are negligible and therefore are

**Figure 10.** European Union: soybean seed and meal production and import; exported amounts are negligible and

port soybean meal.

not shown.

therefore are not shown.

**Figure 7.** Brazil: Soybean seed and meal production and export; imported amounts are negligible and therefore are not shown.

**Figure 8.** Argentina: Soybean seed and meal production and export; imported amounts are negligible and therefore are not shown.

A large amount of Argentina's seed production in 1978-1980 period was exported, but since 1981 the amount of exported seed dropped drastically to 33-55% until 1985 and there‐ after it decreased even more reaching 19% in 2011 with an estimate of 20% in 2012 (Fig‐

ure 8). On the other end the amount of soybean meal internally produced increased approximately four times and almost 100% of the meal was exported, as clearly shown by Figure 9. Therefore these data suggest that Argentina is essentially growing soybean to ex‐ port soybean meal.

**Figure 7.** Brazil: Soybean seed and meal production and export; imported amounts are negligible and therefore are

**Figure 8.** Argentina: Soybean seed and meal production and export; imported amounts are negligible and therefore

A large amount of Argentina's seed production in 1978-1980 period was exported, but since 1981 the amount of exported seed dropped drastically to 33-55% until 1985 and there‐ after it decreased even more reaching 19% in 2011 with an estimate of 20% in 2012 (Fig‐

not shown.

8 Soybean - Bio-Active Compounds

are not shown.

**Figure 9.** China: soybean seed and meal production and import; exported amounts are negligible and therefore are not shown.

**Figure 10.** European Union: soybean seed and meal production and import; exported amounts are negligible and therefore are not shown.

On the other hand, an important soybean importer is China (Figure 9) whose inner soybean seed production only slightly increased over time whereas this country has become the larg‐ est importer of soybean seed (61 Mt estimated for 2012). All the internal production and im‐ port of soybean seed is used for oil extraction and meal by-product production. The large amount of soybean meal produced is almost completely used within the country to support internal animal productions, since China export of meal is negligible. Due to this large vol‐ ume of import China is getting a predominant role influencing the worldwide exchange of soybean products, sometimes competing with other strong importers such as the European Union (Figure 10). As a matter of fact estimated levels of 2012 EU import of soybean seed and meal are 11 and 21.9 MMT respectively, against an internal production of 1.2 and 8.8 MMT of seed and meal, respectively.

in highly intensive farms trying to maximize animal growth rate, to simplify the manage‐ ment of feedstuff and to standardize meat or milk production systems. The same trend oc‐ curred for maize which is currently the most important cereal for animal production chains. Moreover, most of the soybean is produced by using new varieties obtained by genetic engi‐

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On the whole, globalization of the market of animal products favoured the development of animal farms rearing large numbers of animals determining a high concentration of animals in a reduced number of farms which is typical of intensive farming systems, followed by a progressive reduction in the number of small farms that were not able to compete in such a situation of high costs of production coupled with low prices of products for the farmers.

Therefore, even though both soybean and maize have extremely interesting nutritional char‐ acteristics as feed intended for almost all farm animals, an alternative strategy was necessa‐ ry to reduce this complete dependence of European animal production chains from the market of the commodities. However, environmental and climatic conditions in most Euro‐ pean countries hinder the cultivation of soybeans and maize because they are warm-season crops growing during the spring-summer season making irrigation a fundamental need for the success of these crops. Therefore the limited amount of lands suitable for cultivation of these crops, the high water request making irrigation a fundamental component of the pro‐ duction cost, the low levels of soybean grain production per hectare suggested that attention should have been addressed toward other sources of proteins to reduce dependence on im‐

This is particularly true for European countries characterized by a Mediterranean climate, where rainfall is mainly concentrated during winter until late spring whereas summer is usually characterized by high temperatures coupled with very low and often irregular rain‐ fall. Moreover, the use of water for irrigation is often very expensive and therefore, where it is available, it is devoted to other crops such as orchards and vegetables. As a consequence, in these areas agriculture is essentially based on cool-season crops that could be sown in au‐ tumn-end of winter and harvested at the end of spring or during summer. Therefore, these agricultural systems are typically based on rotations between winter cereals and grain le‐ gumes such as faba bean (*Vicia faba* var. *minor L.*), field pea (*Pisum sativum* L.), chick pea (*Cicer arietinum* L.) and white lupin (*Lupinus albus* L.). For animal feeding the most common‐ ly used grain legume was faba bean. Recently, genetic selection of field pea new varieties stimulated the diffusion of this crop as protein source for animal feeding. Also chickpea has been proposed as a possible alternative to soybean together with sweet lupin, which is char‐ acterized by the highest protein content among the grain legumes alternative to soybean. However, since chickpea is mainly cultivated for human consumption and lupin can be cul‐ tivated only in locations with specific soil pH conditions, the main grain legume crops on which to focus attention as possible replacements of soybean are field pea and faba bean.

neering [12] whose acceptance by European consumers is strongly debated.

**3. Strategies for partial or total soybean replacement.**

ported soybean.

Therefore, costs of production of European animal farms strictly depended on soybean pri‐ ces that are set by the international market. As shown in Figure 11 monthly price of soybean seeds and meal in the 1983-2011 time period showed a marked change in part due to the trend of world soybean production. In particular, after 2007 seed and meal prices showed a clear average increase compared to the previous years. The unpredictable variation in the price of the basic protein concentrate for animal feeding strongly influenced the incomes of European farmers since it was also related to a general increase in the prices of other com‐ modities and production factors (fertilizers, pesticides, seed, fuels etc.) whereas the prices of animal products to the farmers did not follow the same positive trend.

**Figure 11.** Trend of soybean seed and meal prices (US\$ per Metric Ton) compared to the world total seed production (MMT) of each year.

Furthermore, the worldwide diffusion of soybean as protein feed component has almost completely replaced any other protein source for animal feeding and this happened mainly in highly intensive farms trying to maximize animal growth rate, to simplify the manage‐ ment of feedstuff and to standardize meat or milk production systems. The same trend oc‐ curred for maize which is currently the most important cereal for animal production chains. Moreover, most of the soybean is produced by using new varieties obtained by genetic engi‐ neering [12] whose acceptance by European consumers is strongly debated.

On the whole, globalization of the market of animal products favoured the development of animal farms rearing large numbers of animals determining a high concentration of animals in a reduced number of farms which is typical of intensive farming systems, followed by a progressive reduction in the number of small farms that were not able to compete in such a situation of high costs of production coupled with low prices of products for the farmers.

## **3. Strategies for partial or total soybean replacement.**

On the other hand, an important soybean importer is China (Figure 9) whose inner soybean seed production only slightly increased over time whereas this country has become the larg‐ est importer of soybean seed (61 Mt estimated for 2012). All the internal production and im‐ port of soybean seed is used for oil extraction and meal by-product production. The large amount of soybean meal produced is almost completely used within the country to support internal animal productions, since China export of meal is negligible. Due to this large vol‐ ume of import China is getting a predominant role influencing the worldwide exchange of soybean products, sometimes competing with other strong importers such as the European Union (Figure 10). As a matter of fact estimated levels of 2012 EU import of soybean seed and meal are 11 and 21.9 MMT respectively, against an internal production of 1.2 and 8.8

Therefore, costs of production of European animal farms strictly depended on soybean pri‐ ces that are set by the international market. As shown in Figure 11 monthly price of soybean seeds and meal in the 1983-2011 time period showed a marked change in part due to the trend of world soybean production. In particular, after 2007 seed and meal prices showed a clear average increase compared to the previous years. The unpredictable variation in the price of the basic protein concentrate for animal feeding strongly influenced the incomes of European farmers since it was also related to a general increase in the prices of other com‐ modities and production factors (fertilizers, pesticides, seed, fuels etc.) whereas the prices of

**Figure 11.** Trend of soybean seed and meal prices (US\$ per Metric Ton) compared to the world total seed production

Furthermore, the worldwide diffusion of soybean as protein feed component has almost completely replaced any other protein source for animal feeding and this happened mainly

animal products to the farmers did not follow the same positive trend.

MMT of seed and meal, respectively.

10 Soybean - Bio-Active Compounds

(MMT) of each year.

Therefore, even though both soybean and maize have extremely interesting nutritional char‐ acteristics as feed intended for almost all farm animals, an alternative strategy was necessa‐ ry to reduce this complete dependence of European animal production chains from the market of the commodities. However, environmental and climatic conditions in most Euro‐ pean countries hinder the cultivation of soybeans and maize because they are warm-season crops growing during the spring-summer season making irrigation a fundamental need for the success of these crops. Therefore the limited amount of lands suitable for cultivation of these crops, the high water request making irrigation a fundamental component of the pro‐ duction cost, the low levels of soybean grain production per hectare suggested that attention should have been addressed toward other sources of proteins to reduce dependence on im‐ ported soybean.

This is particularly true for European countries characterized by a Mediterranean climate, where rainfall is mainly concentrated during winter until late spring whereas summer is usually characterized by high temperatures coupled with very low and often irregular rain‐ fall. Moreover, the use of water for irrigation is often very expensive and therefore, where it is available, it is devoted to other crops such as orchards and vegetables. As a consequence, in these areas agriculture is essentially based on cool-season crops that could be sown in au‐ tumn-end of winter and harvested at the end of spring or during summer. Therefore, these agricultural systems are typically based on rotations between winter cereals and grain le‐ gumes such as faba bean (*Vicia faba* var. *minor L.*), field pea (*Pisum sativum* L.), chick pea (*Cicer arietinum* L.) and white lupin (*Lupinus albus* L.). For animal feeding the most common‐ ly used grain legume was faba bean. Recently, genetic selection of field pea new varieties stimulated the diffusion of this crop as protein source for animal feeding. Also chickpea has been proposed as a possible alternative to soybean together with sweet lupin, which is char‐ acterized by the highest protein content among the grain legumes alternative to soybean. However, since chickpea is mainly cultivated for human consumption and lupin can be cul‐ tivated only in locations with specific soil pH conditions, the main grain legume crops on which to focus attention as possible replacements of soybean are field pea and faba bean.

As a consequence, scientific research was directed to increase knowledge on cultivation, plant breeding and utilization as animal feed of these grain legumes [13-23]. Results showed that in ruminant, monogastric and avian animals at least a partial replacement of soybeans is feasible in intensive animal farming systems, whereas in low input or organic farming systems soybean can be completely replaced by grain legumes that can be grown on farm where soybean cultivation is not feasible [24]. However, despite these encouraging results, grain legume cultivation has suffered a clear reduction in Europe. This trend was related to the development of feed industry that, due to the large quantity of raw materials handled, rely mostly on commodities available on the international market and on the use of industri‐ al protein reach by-products of oil extraction such as rapeseed meal, cottonseed meal, sun‐ flower meal and others.

products that replaced most of the typical and local productions that previously character‐

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Recently, the need for a more environmental friendly agriculture together with the increas‐ ing request by the consumers of high quality products, stimulated farmers to recapture the market of agricultural products [25]. These farms progressively abandoned standardized and intensive agriculture and dedicated to animal and crop productions following the voca‐

As far as animal production chains are concerned, the reconstitution of local production sys‐ tems led to the valorisation of animal feeding systems based on locally produced raw mate‐ rials, both forages and protein (grain legumes) or energy (cereals) concentrates. This allowed these farms to reintroduce rational crop rotations, that were abandoned due to the diffusion of monocultures, by alternately cultivating those cereals and legumes that could also be in‐ tended for animal feeding. Most of these farms started to commercialize by their own the products of their farms by an action aimed at informing the consumers about all aspects of

This approach led to partly or completely replace soybean in animal diets, to reintroduce forage crops both in field crop rotations and in animal feeding systems, to develop less in‐ tensive animal farming systems, to stimulate creation of local networks among farmers which could represent a further stimulus for local farms to reintroduce grain legumes and forage crops for animal feeding by making agreements with local animal farms that would

**5. Experiences on soybean replacement carried out in the Marche Region**

On the feasibility of local animal production chains a research, funded by the Marche Re‐ gion (Central Italy), has been carried out by our research group since year 2000 to evaluate the technical and economical possibilities of soybean replacement with grain legumes such as *faba* bean and field pea. At the same time research evaluated the possibility of a total or partial replacement of maize with barley or sorghum grain, since both these crops are valua‐ ble energy crops for areas characterized by high temperatures and low rainfall during summer, since barley is a cool season cereal whereas sorghum is a worm season but drought

The first objective of the project was to test the agronomic feasibility of both faba bean and field pea in different areas of the Marche Region [26-27]. Very favourable lands, where these crops have been evaluated in optimal agronomic conditions, and more marginal fields were included in field trials. Moreover, several farms including both conventional and or‐ ganic farms were involved as partners of the project to carry out "on farm" field trials based on large plots that were managed by the farmers themselves. The "on farm" ap‐ proach allowed the evaluation of the real potential of these crops in the areas under exami‐

ized agricultural systems strictly integrated with the areas of production.

their production system, receiving economical and professional satisfactions.

tion of their own local region.

withdraw their legume products.

**(Central Italy)**

resistant cereal crop.

Therefore, the identification of grain legumes such as faba bean, field pea, chickpea and lu‐ pin that could at least partially replace soybean in animal feeding systems and their intro‐ duction of in crop rotation systems targeted at supplying animal production chains with locally produced protein concentrates could have several positive effects on European agri‐ cultural systems. This set of crops could guarantee, together with forage crops and pastures, the development of animal production chains that were fully integrated with local environ‐ mental characteristics. Moreover, the close link between animal farming and field produc‐ tions supported the maintenance of good soil fertility and organic matter content. Finally, new strategies for the commercialization of final animal products must be undertaken for the full valorisation of the whole production chain.

## **4. Consequences of simplified cropping systems**

The negative trend shown by grain legume diffusion was therefore a consequence of agri‐ culture evolution toward highly specialized intensive production systems that determined the progressive gap between animal productions and field agriculture. This trend led to well-known agricultural and economical drawbacks such as lost of soil fertility, dramatic de‐ crease of soil organic matter content, increased need of inputs (fertilizers and pesticides) to reach the highest agronomic performances, search for high productions to counterbalance the lowering prices of raw materials on both global and local markets. Moreover, a clear sep‐ aration between farmers that progressively became simple producers of raw materials and industry that managed commercialization and transformation of agricultural products, over time led farmers to lose any possibility of market control. These aspects determined a deep crisis in the agricultural sectors of countries where agriculture was characterized by small or medium sized farms that lost their ability to compete on the market since they were con‐ fined to the role of simple low value raw material producers. The diffusion of monoculture, the reduction of forage crops due to the intensive feeding systems mainly based of protein and energy concentrates, the trend toward part-time agriculture, the massive use of chemi‐ cals to maximize crop production characterized agriculture for several decades after World War II. As a consequence, market of agricultural products was invaded by standardized products that replaced most of the typical and local productions that previously character‐ ized agricultural systems strictly integrated with the areas of production.

As a consequence, scientific research was directed to increase knowledge on cultivation, plant breeding and utilization as animal feed of these grain legumes [13-23]. Results showed that in ruminant, monogastric and avian animals at least a partial replacement of soybeans is feasible in intensive animal farming systems, whereas in low input or organic farming systems soybean can be completely replaced by grain legumes that can be grown on farm where soybean cultivation is not feasible [24]. However, despite these encouraging results, grain legume cultivation has suffered a clear reduction in Europe. This trend was related to the development of feed industry that, due to the large quantity of raw materials handled, rely mostly on commodities available on the international market and on the use of industri‐ al protein reach by-products of oil extraction such as rapeseed meal, cottonseed meal, sun‐

Therefore, the identification of grain legumes such as faba bean, field pea, chickpea and lu‐ pin that could at least partially replace soybean in animal feeding systems and their intro‐ duction of in crop rotation systems targeted at supplying animal production chains with locally produced protein concentrates could have several positive effects on European agri‐ cultural systems. This set of crops could guarantee, together with forage crops and pastures, the development of animal production chains that were fully integrated with local environ‐ mental characteristics. Moreover, the close link between animal farming and field produc‐ tions supported the maintenance of good soil fertility and organic matter content. Finally, new strategies for the commercialization of final animal products must be undertaken for

The negative trend shown by grain legume diffusion was therefore a consequence of agri‐ culture evolution toward highly specialized intensive production systems that determined the progressive gap between animal productions and field agriculture. This trend led to well-known agricultural and economical drawbacks such as lost of soil fertility, dramatic de‐ crease of soil organic matter content, increased need of inputs (fertilizers and pesticides) to reach the highest agronomic performances, search for high productions to counterbalance the lowering prices of raw materials on both global and local markets. Moreover, a clear sep‐ aration between farmers that progressively became simple producers of raw materials and industry that managed commercialization and transformation of agricultural products, over time led farmers to lose any possibility of market control. These aspects determined a deep crisis in the agricultural sectors of countries where agriculture was characterized by small or medium sized farms that lost their ability to compete on the market since they were con‐ fined to the role of simple low value raw material producers. The diffusion of monoculture, the reduction of forage crops due to the intensive feeding systems mainly based of protein and energy concentrates, the trend toward part-time agriculture, the massive use of chemi‐ cals to maximize crop production characterized agriculture for several decades after World War II. As a consequence, market of agricultural products was invaded by standardized

flower meal and others.

12 Soybean - Bio-Active Compounds

the full valorisation of the whole production chain.

**4. Consequences of simplified cropping systems**

Recently, the need for a more environmental friendly agriculture together with the increas‐ ing request by the consumers of high quality products, stimulated farmers to recapture the market of agricultural products [25]. These farms progressively abandoned standardized and intensive agriculture and dedicated to animal and crop productions following the voca‐ tion of their own local region.

As far as animal production chains are concerned, the reconstitution of local production sys‐ tems led to the valorisation of animal feeding systems based on locally produced raw mate‐ rials, both forages and protein (grain legumes) or energy (cereals) concentrates. This allowed these farms to reintroduce rational crop rotations, that were abandoned due to the diffusion of monocultures, by alternately cultivating those cereals and legumes that could also be in‐ tended for animal feeding. Most of these farms started to commercialize by their own the products of their farms by an action aimed at informing the consumers about all aspects of their production system, receiving economical and professional satisfactions.

This approach led to partly or completely replace soybean in animal diets, to reintroduce forage crops both in field crop rotations and in animal feeding systems, to develop less in‐ tensive animal farming systems, to stimulate creation of local networks among farmers which could represent a further stimulus for local farms to reintroduce grain legumes and forage crops for animal feeding by making agreements with local animal farms that would withdraw their legume products.

## **5. Experiences on soybean replacement carried out in the Marche Region (Central Italy)**

On the feasibility of local animal production chains a research, funded by the Marche Re‐ gion (Central Italy), has been carried out by our research group since year 2000 to evaluate the technical and economical possibilities of soybean replacement with grain legumes such as *faba* bean and field pea. At the same time research evaluated the possibility of a total or partial replacement of maize with barley or sorghum grain, since both these crops are valua‐ ble energy crops for areas characterized by high temperatures and low rainfall during summer, since barley is a cool season cereal whereas sorghum is a worm season but drought resistant cereal crop.

The first objective of the project was to test the agronomic feasibility of both faba bean and field pea in different areas of the Marche Region [26-27]. Very favourable lands, where these crops have been evaluated in optimal agronomic conditions, and more marginal fields were included in field trials. Moreover, several farms including both conventional and or‐ ganic farms were involved as partners of the project to carry out "on farm" field trials based on large plots that were managed by the farmers themselves. The "on farm" ap‐ proach allowed the evaluation of the real potential of these crops in the areas under exami‐ nation, that were mainly located in the inner part of the Marche region were irrigation is not feasible (Figure 12).

Results of the field trials showed that these grain legumes can be effectively produced in in‐ ner areas of the Marche Region where soybean cannot be cultivated and animal farming is traditionally an economic source of income for local farmers. After gathering information on the agronomic feasibility, research has been addressed to verify the possibility of total or partial substitution of soybean with faba beans and/or field pea in beef cattle, dairy cattle and swine feeding systems. Therefore, feeding trials were conducted in one large dairy farm, located in the Province of Ancona, 4 organic farms rearing beef cattle of the Marchigi‐ ana breed and located in the Provinces of Macerata and Fermo, one conventional farm locat‐ ed in the Province of Pesaro-Urbino, and one conventional small familiar swine farm,

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located in the Province of Fermo, rearing pigs using a non-intensive farming system.

Concerning the dairy sector, our experimental trials were carried out while farmers were expe‐ riencing the continuous fluctuation of the prices of raw materials, mainly commodities such as soybean meal and corn grain, coupled with the crisis of the dairy sector across all Europe that determined low milk prices despite the increasing costs of production. Therefore, we were able to evaluate both the potential and the limits of soybean and corn partial replacement in a very critical agricultural sector such as dairy farming. Due to the peculiarities of dairy production, based on the animal physiology and on the nutritional characteristics of the raw materials un‐ der examination, soybean meal cannot be totally replaced by faba bean and field pea. Based on the results of the "on farm" feeding trial carried out at this dairy farm about 50% of soybean meal present in the ration was replaced by faba bean and field pea. To understand the poten‐ tial effect of this partial substitution on the local agricultural system it can be considered that the farm was initially using 3kg/cow of soybean meal. Therefore, 1.5 kg of soybean meal were substituted by about 2 kg/cow of faba bean/field pea mix, considering that also part of the corn grain was also partially replaced by these grain legumes due to their starch content. Having the farm an average of 450 lactating cows per day, daily feeding requested about 9 tons/day of fa‐ ba bean and field pea, which was about 330 tons/year. Based on the results of the field trials, as‐ suming an average field production of 2.5 tons/hectare (2 tons/ha for faba beans and 3 tons/ha for field pea) it can be estimated that this farm could support about 130 hectares cultivated with grain legumes for animal feeding. Assuming a multi-rotation such as wheat-grain legume-bar‐ ley- 3 years of alfalfa we can roughly estimate that partial substitution of soybean in this case could support an overall agricultural system covering about 790 hectares. Moreover, the pres‐ ence of a 3 years forage crop such as alfalfa would reduce drastically the use of pesticides and fertilizers and also would increase nitrogen fixation and soil organic matter content, the intro‐ duction of organic farming practices to manage cereal and grain legume crops would avoid or at least reduce the use of chemicals and in particular of herbicides also in conventional farms, the milk production would be closely linked to the area of production. However, to make this system working it is necessary to make it economically profitable for the farm. When soybean meal prices increased the use of grain legumes produced by local farms helped the dairy farm to compensate for the increased costs of production. Despite these encouraging results the cri‐ sis of the dairy sector is hampering the implementation of this integrated production system that is based on close relationships between the dairy farm and farmers producing raw materi‐ als used as animal feed. The identification of different commercialization systems able to fully

**Figure 12.** Field experimental trial including faba bean and field pea carried out in typical agricultural landscape of inner areas of the Marche Region (Province of Ancona).

Results showed that both faba beans and field pea could be effectively reintroduced in crop rotations with winter cereals such as wheat and barley, reconstituting a correct alternation between nitrogen fixing legumes and cereals. However, the grain productions obviously varied based on the environmental and soil conditions and the seasonal climatic conditions that varied from year to year. This experience allowed the creation of a useful data set indi‐ cating that faba beans showed, on the average, a range between 1.0 and 3.5 tons/hectare of grain, the lowest productions being obtained when very dry growing seasons occurred with extremely low values of rainfall during end of winter and spring. Farmers know very well these characteristics of faba beans since this crop has been traditionally used across the whole region mainly as protein grain for beef cattle of the Marchigiana breed. However, plant breeding efforts are requested to stabilize grain production in the variable environ‐ mental conditions characterizing the inner areas of Central Italy.

Field pea was characterized by a higher average seed production than faba beans, showing a range between 1.5 and 4.5 tons/hectare. For this crop low production can be due to environ‐ mental adverse conditions but also grain loss due to seed shattering during harvesting is a primary cause of production losses mainly when the crop is grown on soils with an irregular surface that makes threshing difficult.

To compare faba bean and field pea with soybean, few farms where irrigation was feasible were asked to try cultivation of soybean. The results showed an average seed production be‐ tween 2.5 and 3 tons/hectare, similar to a good faba bean or field pea harvest, but costs of irrigation made this crop unprofitable. Moreover, chemical weed control was necessary in order to obtain acceptable seed production because watering also favoured the development of weeds. Herbicides are used also to protect faba beans and field pea against weed competi‐ tion. However, our field trials carried out in organic farms showed that multi-year rotations including forage crops such as alfalfa, highly competitive cereals against weeds such as bar‐ ley or wheat, and a higher crop density (number of plants/m2 ) than conventional cultivation can avoid the use of herbicides on crops such as faba beans and field pea.

Results of the field trials showed that these grain legumes can be effectively produced in in‐ ner areas of the Marche Region where soybean cannot be cultivated and animal farming is traditionally an economic source of income for local farmers. After gathering information on the agronomic feasibility, research has been addressed to verify the possibility of total or partial substitution of soybean with faba beans and/or field pea in beef cattle, dairy cattle and swine feeding systems. Therefore, feeding trials were conducted in one large dairy farm, located in the Province of Ancona, 4 organic farms rearing beef cattle of the Marchigi‐ ana breed and located in the Provinces of Macerata and Fermo, one conventional farm locat‐ ed in the Province of Pesaro-Urbino, and one conventional small familiar swine farm, located in the Province of Fermo, rearing pigs using a non-intensive farming system.

nation, that were mainly located in the inner part of the Marche region were irrigation is

**Figure 12.** Field experimental trial including faba bean and field pea carried out in typical agricultural landscape of

Results showed that both faba beans and field pea could be effectively reintroduced in crop rotations with winter cereals such as wheat and barley, reconstituting a correct alternation between nitrogen fixing legumes and cereals. However, the grain productions obviously varied based on the environmental and soil conditions and the seasonal climatic conditions that varied from year to year. This experience allowed the creation of a useful data set indi‐ cating that faba beans showed, on the average, a range between 1.0 and 3.5 tons/hectare of grain, the lowest productions being obtained when very dry growing seasons occurred with extremely low values of rainfall during end of winter and spring. Farmers know very well these characteristics of faba beans since this crop has been traditionally used across the whole region mainly as protein grain for beef cattle of the Marchigiana breed. However, plant breeding efforts are requested to stabilize grain production in the variable environ‐

Field pea was characterized by a higher average seed production than faba beans, showing a range between 1.5 and 4.5 tons/hectare. For this crop low production can be due to environ‐ mental adverse conditions but also grain loss due to seed shattering during harvesting is a primary cause of production losses mainly when the crop is grown on soils with an irregular

To compare faba bean and field pea with soybean, few farms where irrigation was feasible were asked to try cultivation of soybean. The results showed an average seed production be‐ tween 2.5 and 3 tons/hectare, similar to a good faba bean or field pea harvest, but costs of irrigation made this crop unprofitable. Moreover, chemical weed control was necessary in order to obtain acceptable seed production because watering also favoured the development of weeds. Herbicides are used also to protect faba beans and field pea against weed competi‐ tion. However, our field trials carried out in organic farms showed that multi-year rotations including forage crops such as alfalfa, highly competitive cereals against weeds such as bar‐

) than conventional cultivation

not feasible (Figure 12).

14 Soybean - Bio-Active Compounds

inner areas of the Marche Region (Province of Ancona).

surface that makes threshing difficult.

mental conditions characterizing the inner areas of Central Italy.

ley or wheat, and a higher crop density (number of plants/m2

can avoid the use of herbicides on crops such as faba beans and field pea.

Concerning the dairy sector, our experimental trials were carried out while farmers were expe‐ riencing the continuous fluctuation of the prices of raw materials, mainly commodities such as soybean meal and corn grain, coupled with the crisis of the dairy sector across all Europe that determined low milk prices despite the increasing costs of production. Therefore, we were able to evaluate both the potential and the limits of soybean and corn partial replacement in a very critical agricultural sector such as dairy farming. Due to the peculiarities of dairy production, based on the animal physiology and on the nutritional characteristics of the raw materials un‐ der examination, soybean meal cannot be totally replaced by faba bean and field pea. Based on the results of the "on farm" feeding trial carried out at this dairy farm about 50% of soybean meal present in the ration was replaced by faba bean and field pea. To understand the poten‐ tial effect of this partial substitution on the local agricultural system it can be considered that the farm was initially using 3kg/cow of soybean meal. Therefore, 1.5 kg of soybean meal were substituted by about 2 kg/cow of faba bean/field pea mix, considering that also part of the corn grain was also partially replaced by these grain legumes due to their starch content. Having the farm an average of 450 lactating cows per day, daily feeding requested about 9 tons/day of fa‐ ba bean and field pea, which was about 330 tons/year. Based on the results of the field trials, as‐ suming an average field production of 2.5 tons/hectare (2 tons/ha for faba beans and 3 tons/ha for field pea) it can be estimated that this farm could support about 130 hectares cultivated with grain legumes for animal feeding. Assuming a multi-rotation such as wheat-grain legume-bar‐ ley- 3 years of alfalfa we can roughly estimate that partial substitution of soybean in this case could support an overall agricultural system covering about 790 hectares. Moreover, the pres‐ ence of a 3 years forage crop such as alfalfa would reduce drastically the use of pesticides and fertilizers and also would increase nitrogen fixation and soil organic matter content, the intro‐ duction of organic farming practices to manage cereal and grain legume crops would avoid or at least reduce the use of chemicals and in particular of herbicides also in conventional farms, the milk production would be closely linked to the area of production. However, to make this system working it is necessary to make it economically profitable for the farm. When soybean meal prices increased the use of grain legumes produced by local farms helped the dairy farm to compensate for the increased costs of production. Despite these encouraging results the cri‐ sis of the dairy sector is hampering the implementation of this integrated production system that is based on close relationships between the dairy farm and farmers producing raw materi‐ als used as animal feed. The identification of different commercialization systems able to fully valorise the quality of the overall production chains is becoming a fundamental step in order to counteract the continuous decrease of milk price on the national and international market.

ion is imported by American countries, the risk of GM soybean contamination is very high. This is confirmed by the introduction of a threshold of 0.9% technically unavoidable con‐ tamination also in organic feedstuff. Therefore, our results demonstrated that also organic farming systems could avoid GM contamination whenever a strict control of the raw materi‐ al production or origin is made directly from the farmers. Feed composition therefore be‐ comes an index of the raw materials used in the production chains and can be used as further information for the consumers to valorise the value of the final product. For these reason a DNA method has been developed as further result of the project aimed at the iden‐ tification of the presence of faba beans and/or field pea within feed samples [28]. This could be a simple and not expensive approach to certify the use of local raw materials as feedstuff together with the absence of soybean from the ration. Moreover, an attempt to increase con‐ sumers' information about the characteristics of GM *free* organic production has been started as part of dissemination of the project activities and this increased the number of consumers interested in purchasing GM *free* products. On the whole, results on organic beef cattle showed that GM *free* production chains based on feeding systems that rely on locally pro‐ duced raw materials can be an efficient alternative to intensive production chains. This ap‐ proach could also be useful to maintain or increase economically effective agricultural systems in inner areas of Central Italy by reducing the dependence from the international

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced

Feedstuff

17

http://dx.doi.org/10.5772/52476

Encouraging results have also been obtained concerning the swine production chain. The farm where feeding trials were carried out had the possibility of rearing pigs both indoor and outdoor (open air). Therefore a feeding trial was conducted to compare one convention‐ al feed (Control) based on the use of soybean meal and corn with an experimental feed where soybean meal was replaced completely by faba beans and field pea and corn was par‐ tially replaced with barley. Both feeds were formulated respecting the differences requested between the growth and the finishing phases. No differences in animal growth rate (600 g/ day) were detected between the two feeds (Control vs Experimental). At the same time a group of pigs was reared outdoor and fed with the experimental feed. Average daily growth rate was slightly lower than observed in the indoor trial. The same experimental feed was subsequently tested in one organic and one conventional farm and results confirmed that regular growth rates can be obtained when soybean is not included in the feed, with slightly higher average daily gains obtained in the conventional farm (750 g/day). Therefore, non in‐ tensive swine production chains could represent another animal farming system that could stimulate the development of production systems linked to the production area, the net‐ working among local farms concerning the exchange of raw materials for animal feeding, the reintroduction of rational not intensive agricultural systems. Commercialization of the final products is again fundamental to guarantee profitability for all the actors of the pro‐ duction chain and for this purpose direct selling is showing to be an effective marketing

market of commodities.

strategy to reach this objective.

Beef cattle field trials were carried out at four organic farms located in the inner areas of Provinces of Macerata and Fermo. Differently from the experience previously described in the dairy sector, the small size of these farms, the high quality of both final products and production system and a different approach to the product valorisation based on the direct sales of meat by the producers themselves, allowed research results to be transferred to the final step of the production chain: the product marketing and commercialization.

Feeding trials on organic beef cattle showed that for not intensive production systems soy‐ bean can be totally replaced by faba beans and field pea. Moreover, these farms are charac‐ terized by self-producing almost all the forage and a high amount of protein (grain legumes) and energy (mainly barley) needed for animal feeding. It is relatively simple for these farms to make arrangements with neighbouring farmers to secure themselves the supply of the raw materials they are not able to self produce. This production system is extremely interest‐ ing because, as shown in Figure 13, it is based on rational agronomic crop rotations main‐ taining both a high level of crop diversification together with a low if not positive environmental impact due to the organic farming practices. The lower animal daily growth rate characterizing organic or non-intensive animal farming (1-1.2 kg/day), compared to growth rate of intensive systems (higher than 1.6 kg/day), is also an aspect of the production system which is valorised in the final product.

**Figure 13.** Organic animal farm located at Monte San Martino (Province of Macerata) where both experimental field and feeding trials were carried out.

Aim of the research project was also to verify the technical and economic feasibility of GM*free* production chains, that is production systems that do not include genetically modi‐ fied (GM) feed in the feeding system. Among the commodities, soybean show the highest amount of worldwide production obtained from GM varieties, mainly cultivated in USA or South America [12]. Therefore, since almost soybean seed or meal used in the European Un‐ ion is imported by American countries, the risk of GM soybean contamination is very high. This is confirmed by the introduction of a threshold of 0.9% technically unavoidable con‐ tamination also in organic feedstuff. Therefore, our results demonstrated that also organic farming systems could avoid GM contamination whenever a strict control of the raw materi‐ al production or origin is made directly from the farmers. Feed composition therefore be‐ comes an index of the raw materials used in the production chains and can be used as further information for the consumers to valorise the value of the final product. For these reason a DNA method has been developed as further result of the project aimed at the iden‐ tification of the presence of faba beans and/or field pea within feed samples [28]. This could be a simple and not expensive approach to certify the use of local raw materials as feedstuff together with the absence of soybean from the ration. Moreover, an attempt to increase con‐ sumers' information about the characteristics of GM *free* organic production has been started as part of dissemination of the project activities and this increased the number of consumers interested in purchasing GM *free* products. On the whole, results on organic beef cattle showed that GM *free* production chains based on feeding systems that rely on locally pro‐ duced raw materials can be an efficient alternative to intensive production chains. This ap‐ proach could also be useful to maintain or increase economically effective agricultural systems in inner areas of Central Italy by reducing the dependence from the international market of commodities.

valorise the quality of the overall production chains is becoming a fundamental step in order to counteract the continuous decrease of milk price on the national and international market.

Beef cattle field trials were carried out at four organic farms located in the inner areas of Provinces of Macerata and Fermo. Differently from the experience previously described in the dairy sector, the small size of these farms, the high quality of both final products and production system and a different approach to the product valorisation based on the direct sales of meat by the producers themselves, allowed research results to be transferred to the

Feeding trials on organic beef cattle showed that for not intensive production systems soy‐ bean can be totally replaced by faba beans and field pea. Moreover, these farms are charac‐ terized by self-producing almost all the forage and a high amount of protein (grain legumes) and energy (mainly barley) needed for animal feeding. It is relatively simple for these farms to make arrangements with neighbouring farmers to secure themselves the supply of the raw materials they are not able to self produce. This production system is extremely interest‐ ing because, as shown in Figure 13, it is based on rational agronomic crop rotations main‐ taining both a high level of crop diversification together with a low if not positive environmental impact due to the organic farming practices. The lower animal daily growth rate characterizing organic or non-intensive animal farming (1-1.2 kg/day), compared to growth rate of intensive systems (higher than 1.6 kg/day), is also an aspect of the production

**Figure 13.** Organic animal farm located at Monte San Martino (Province of Macerata) where both experimental field

Aim of the research project was also to verify the technical and economic feasibility of GM*free* production chains, that is production systems that do not include genetically modi‐ fied (GM) feed in the feeding system. Among the commodities, soybean show the highest amount of worldwide production obtained from GM varieties, mainly cultivated in USA or South America [12]. Therefore, since almost soybean seed or meal used in the European Un‐

final step of the production chain: the product marketing and commercialization.

system which is valorised in the final product.

16 Soybean - Bio-Active Compounds

and feeding trials were carried out.

Encouraging results have also been obtained concerning the swine production chain. The farm where feeding trials were carried out had the possibility of rearing pigs both indoor and outdoor (open air). Therefore a feeding trial was conducted to compare one convention‐ al feed (Control) based on the use of soybean meal and corn with an experimental feed where soybean meal was replaced completely by faba beans and field pea and corn was par‐ tially replaced with barley. Both feeds were formulated respecting the differences requested between the growth and the finishing phases. No differences in animal growth rate (600 g/ day) were detected between the two feeds (Control vs Experimental). At the same time a group of pigs was reared outdoor and fed with the experimental feed. Average daily growth rate was slightly lower than observed in the indoor trial. The same experimental feed was subsequently tested in one organic and one conventional farm and results confirmed that regular growth rates can be obtained when soybean is not included in the feed, with slightly higher average daily gains obtained in the conventional farm (750 g/day). Therefore, non in‐ tensive swine production chains could represent another animal farming system that could stimulate the development of production systems linked to the production area, the net‐ working among local farms concerning the exchange of raw materials for animal feeding, the reintroduction of rational not intensive agricultural systems. Commercialization of the final products is again fundamental to guarantee profitability for all the actors of the pro‐ duction chain and for this purpose direct selling is showing to be an effective marketing strategy to reach this objective.

### **6. Conclusions**

The main objective of this paper was to stimulate a critical evaluation of soybean impact on agricultural systems where soybean cannot be cultivated. Notwithstanding soybean positive nutritional characteristics, this commodity may not be the only solution for animal produc‐ tion chains for those countries that may suffer from a complete dependence on import of raw materials.

**Author details**

Stefano Tavoletti \*

**References**

Address all correspondence to: s.tavoletti@univpm.it

Marche - Via Brecce Bianche, 60131 ANCONA, Italy

*sociation Review*, 12(4), 143-162.

*and Water Conservation* 50(3) 253-261.

*and Ecology*, 9(1/2) 1-14.

108(50), 20260-20264.

316(5831), 1570-1571.

*N.18.*

*Press Inc*.

Dipartimento di Scienze Agrarie, Alimentari ed Ambientali – Università Politecnica delle

Critical Evaluation of Soybean Role in Animal Production Chains Based on the Valorization of Locally Produced

Feedstuff

19

http://dx.doi.org/10.5772/52476

[1] Masuda, T., & Goldsmith, P. D. (2009). World Soybean Production: Area Harvested, Yield, and Long-Term Projections. *International Food and Agribusiness Management As‐*

[2] Borsari, B. (2011). Agroecology as a Needed Paradigm Shift to Insure Food Security in a Global Market Economy. *International Journal of Agricultural Resources, Governance*

[3] Altieri MA. (1995). Agroecology: the Science of Sustainable Agriculture. *Westview*

[4] Reicosky D.C, Kemper W.D, Langdale C.W, Douglas C.LJ., 1995. Rasmussen PE. Soil organic matter changes resulting from tillage and biomass production. *Journal of Soil*

[5] Tilman, D., Cassman, K. G., Matson, P. A., Naylor, R., & Polasky, S. (2002). Agricul‐ tural sustainability and intensive production practices. *Nature*, 418(6898), 671-677. [6] Montgomery, DR. (2007). Soil erosion and agricultural sustainability. *Proceedings of*

[7] Tilman, D., Balzer, C., Hill, J., & Befort, B. L. (2011). Global food demand and sustain‐ able intensification of agriculture. Proceedings of National Academy of Science; ,

[8] Sleper D.A., Barker T.C., Bramel-Cox P.J. editors. 1991. Plant Breeding and Sustaina‐ ble Agriculture: Considerations for Objectives and Methods. *CSSA Special Publication*

[9] Jackson, D.L., & Jackson, L.L. (2002). The Farm as Natural Habitat. Reconnecting

[10] Jordan, N., Boody, G., Broussard, W., Glover, Keeney. D., Mc Cown, B. H., Mc Isaac, G., Muller, M., Murray, H., Neal, J., Pansing, C., Turner, R. E., Warner, K., & Wyse, D. (2007). Sustainable Development of the Agricultural Bio-Economy. *Science*,

Food Systems with Ecosystems. *Island Press, Washington DC*.

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Field and feeding trials carried "on farm" furnished information on the feasibility of local animal production chains using feeding systems based on locally produced raw materials that could partially or completely replace soybean. This possibility would be a stimulus to recreate networks among local farmers in order to develop local production chains that could restore an economically feasible agricultural systems to farms that are unable to com‐ pete on the global market. This also represents an attempt to reintroduce sustainable agri‐ cultural systems characterized by a reduced use of chemicals and pesticides due to the cultivation of low input cereals and legumes in rationale crop rotations.

Moreover, the interest toward research results on feedstuff that do not include soybean is also related to the risk of GM contamination due to this commodity. GM free animal pro‐ duction chains are strongly exposed to GM contamination when soybean is included as the main protein source in the feed. The decision to include the 0,9% threshold also for organic farming production chains underlines the real risk of GM contamination and the difficulties to create GM free production chains when the feed is based on the use of the same raw ma‐ terials characterizing GM animal products.

Our results however showed that for large animal farms, that carryout intensive production systems, it is more difficult than for small farms, characterized by not intensive production chains, to manage soybean replacement. This aspect confirms the almost complete depend‐ ence from imported commodities that has been reached in the time by agricultural produc‐ tion sectors aimed at the mass production of large amounts of standardized products. The large volume of raw materials requested followed by the low internal availability of feed‐ stuff that can be used as an alternative to soybean exposes these farms to the risks of interna‐ tional market variations both in the availability and in the price of this commodity.

On the other hand, the implementation of animal production chains based on the use of lo‐ cally produced feedstuff is a valid approach for small farms producing high quality prod‐ ucts using not intensive animal farming systems. These farms can in this way gain a market space for products that can be an alternative to standardized products and at the same time activate agricultural systems well integrated with local environmental features, that make less use of intensive production techniques, reduce the use of fertilizers and pesti‐ cides, restore soil fertility. However, new marketing strategies are necessary to make con‐ sumers aware of the importance of the overall characteristics of local production chains in defining the quality of a final product and to ensure at the same time a profitable price for the producers.

## **Author details**

**6. Conclusions**

18 Soybean - Bio-Active Compounds

raw materials.

The main objective of this paper was to stimulate a critical evaluation of soybean impact on agricultural systems where soybean cannot be cultivated. Notwithstanding soybean positive nutritional characteristics, this commodity may not be the only solution for animal produc‐ tion chains for those countries that may suffer from a complete dependence on import of

Field and feeding trials carried "on farm" furnished information on the feasibility of local animal production chains using feeding systems based on locally produced raw materials that could partially or completely replace soybean. This possibility would be a stimulus to recreate networks among local farmers in order to develop local production chains that could restore an economically feasible agricultural systems to farms that are unable to com‐ pete on the global market. This also represents an attempt to reintroduce sustainable agri‐ cultural systems characterized by a reduced use of chemicals and pesticides due to the

Moreover, the interest toward research results on feedstuff that do not include soybean is also related to the risk of GM contamination due to this commodity. GM free animal pro‐ duction chains are strongly exposed to GM contamination when soybean is included as the main protein source in the feed. The decision to include the 0,9% threshold also for organic farming production chains underlines the real risk of GM contamination and the difficulties to create GM free production chains when the feed is based on the use of the same raw ma‐

Our results however showed that for large animal farms, that carryout intensive production systems, it is more difficult than for small farms, characterized by not intensive production chains, to manage soybean replacement. This aspect confirms the almost complete depend‐ ence from imported commodities that has been reached in the time by agricultural produc‐ tion sectors aimed at the mass production of large amounts of standardized products. The large volume of raw materials requested followed by the low internal availability of feed‐ stuff that can be used as an alternative to soybean exposes these farms to the risks of interna‐

On the other hand, the implementation of animal production chains based on the use of lo‐ cally produced feedstuff is a valid approach for small farms producing high quality prod‐ ucts using not intensive animal farming systems. These farms can in this way gain a market space for products that can be an alternative to standardized products and at the same time activate agricultural systems well integrated with local environmental features, that make less use of intensive production techniques, reduce the use of fertilizers and pesti‐ cides, restore soil fertility. However, new marketing strategies are necessary to make con‐ sumers aware of the importance of the overall characteristics of local production chains in defining the quality of a final product and to ensure at the same time a profitable price

tional market variations both in the availability and in the price of this commodity.

cultivation of low input cereals and legumes in rationale crop rotations.

terials characterizing GM animal products.

for the producers.

Stefano Tavoletti \*

Address all correspondence to: s.tavoletti@univpm.it

Dipartimento di Scienze Agrarie, Alimentari ed Ambientali – Università Politecnica delle Marche - Via Brecce Bianche, 60131 ANCONA, Italy

## **References**


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[26] Tavoletti, S., Mattii, S., Pasquini, M., & Trombetta, M. F. (2004). La reintroduzione delle colture proteiche nelle filiere agrozootecniche marchigiane. *L'Informatore Agrar‐*

[27] Migliorini, P., Tavoletti, S., Moschini, V., & Iommarini, L. (2008). Performance of grain legume crops in organic farms of central Italy. *In: Neuhoff D, Halberg N, Alföldi T, Lockeretz W, Thommen A, Rasmussen IA, Hermansen J, Vaarst M, Lueck L, Caporali F, Jensen HH, Migliorini P, Willer H (eds.) Cultivating the future based on science. Proc. Of*

[28] Tavoletti, S., Iommarini, L., & Pasquini, M. (2009). A DNA method for qualitative identiWcationof plant raw materials in feedstuff. *European Food Research Technology*,

*16th IFOAM Organic World Congress*, 16-20, 978-3-03736-023-1.


[24] Blair, R. (2011). Nutrition and Feeding of organic cattle. *CABI, UK*, 13978184593758.

[11] Index Mundi.

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[13] Yu, P., Goelema, J. O., Leury, B. J., Tamminga, S., & Egan, A. R. (2002). An analysis of the nutritive value of heat processed legume seeds for animal production using the

[14] Froidmont, E., & Bartiaux-Thill, N. (2004). Suitability of lupin and pea seeds as a sub‐ stitute for soybean meal in high-producing dairy cow feed. *Animal Research*, 53(6),

[15] Prandini, A., Morlacchini, M., Moschini, M., Fusconi, G., Masoero, F., & Piva, G. (2005). Raw and extruded pea (Pisum sativum) and lupin (Lupinus albus var. Multi‐ talia) seeds as protein sources in weaned piglets' diets: effect on growth rate and

[16] Stein, H. H., Everts, A. K. R., Sweeter, K. K., Peters, D. N., Maddock, R. J., Wulf, D. M., & Pedersen, C. (2006). The influence of dietary field peas (Pisum sativum L.) on pig performance, carcass quality, and the palatability of pork. *Journal of Animal Sci‐*

[17] Gilbery T.C., Lardy G.P., Soto-Navarro S.A., Bauer M.L. and Anderson V.L. 2007 Ef‐ fect of field peas, chickpeas, and lentils on rumen fermentation, digestion, microbial protein synthesis and feedlot performance in receiving diets for beef cattle. *J Anim Sci*

[18] Perella, F., Mugnai, C., Dal, Bosco. A., Sirri, F., Cestola, E., & Castellini, C. (2009). Fa‐ ba bean (Vicia faba var. minor) as a protein source for organic chickens: performance

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DVE/OEB model: a review. *Animal Feed Science and Technology*, 99(1), 141-176.


**Chapter 2**

**Advanced Techniques in Soybean Biodiesel**

The planet where we inhabit, live, has experienced a great transformation period in the most different fields. The evolution followed by a great technological development, on the other side has caused an imbalance both in society itself, as in the material environment in which we live. The planet earth has visibly demonstrated how has been affected by this imbalance and how it has naturally reacted. In recent years, all this has being reported through the lit‐ erature, studied by different scientific research groups, as also observed by what is reported in the media in general, even in the form of documentaries and films, as the documentary performed by former USA vice president Al Gore, an inconvenient truth (an inconvenient truth, 2006). All learning takes a certain time to begin to be assimilated and been put in prac‐ tice effectively, so humanity has learned, been advised by the latest natural disasters of this century, as in the case of Japan's earthquake, tsunami, the strong hurricanes that plague the northern hemisphere summer, as in fact glaciers melting that were called eternal, the poles of this planet, strong climate change experienced over the past years and the major pollution in large urban cities where population are forced to live in many different fields, has signal‐ ing how much real acts, changes are necessary to continue to be possible living an inhabited planet.In this century, XXI, the world main problems, which it has experienced, are related to the scarcity of natural resources such as water, which had been mismanaged, contaminat‐ ed by urban and industrial solid waste disposal, and in relation to generation and use of en‐

These energy sources can be broadly classified into three categories: fossil fuels (coal, oil and natural gas), renewable (hydroelectric, wind, solar and biomass) and nuclear sources. Among those can be highlighted Biomass, where all organic matter that is produced by

> © 2013 Fonseca 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 Fonseca 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.

Mauricio G. Fonseca, Luciano N. Batista,

Monique R. Jesus and Georgiana F. Cruz

http://dx.doi.org/10.5772/52990

ergy the most diverse shapes.

**1. Introduction**

Additional information is available at the end of the chapter

Viviane F. Silva, Erica C. G. Pissurno, Thais C. Soares,

## **Advanced Techniques in Soybean Biodiesel**

Mauricio G. Fonseca, Luciano N. Batista, Viviane F. Silva, Erica C. G. Pissurno, Thais C. Soares, Monique R. Jesus and Georgiana F. Cruz

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52990

## **1. Introduction**

The planet where we inhabit, live, has experienced a great transformation period in the most different fields. The evolution followed by a great technological development, on the other side has caused an imbalance both in society itself, as in the material environment in which we live. The planet earth has visibly demonstrated how has been affected by this imbalance and how it has naturally reacted. In recent years, all this has being reported through the lit‐ erature, studied by different scientific research groups, as also observed by what is reported in the media in general, even in the form of documentaries and films, as the documentary performed by former USA vice president Al Gore, an inconvenient truth (an inconvenient truth, 2006). All learning takes a certain time to begin to be assimilated and been put in prac‐ tice effectively, so humanity has learned, been advised by the latest natural disasters of this century, as in the case of Japan's earthquake, tsunami, the strong hurricanes that plague the northern hemisphere summer, as in fact glaciers melting that were called eternal, the poles of this planet, strong climate change experienced over the past years and the major pollution in large urban cities where population are forced to live in many different fields, has signal‐ ing how much real acts, changes are necessary to continue to be possible living an inhabited planet.In this century, XXI, the world main problems, which it has experienced, are related to the scarcity of natural resources such as water, which had been mismanaged, contaminat‐ ed by urban and industrial solid waste disposal, and in relation to generation and use of en‐ ergy the most diverse shapes.

These energy sources can be broadly classified into three categories: fossil fuels (coal, oil and natural gas), renewable (hydroelectric, wind, solar and biomass) and nuclear sources. Among those can be highlighted Biomass, where all organic matter that is produced by

© 2013 Fonseca 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 Fonseca 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.

this process is called biomass. This has a great advantage over fossil fuels, it's less pollut‐ ing, because its processes do not add carbon dioxide to the atmosphere, the environment. The biomass process reduces the carbon dioxide amount in atmosphere through the pho‐ tosynthesis, performed by increasing the planted green areas, to cultivate the seeds crops. Research and development departments have been engaged in fuels discovery that do not cause much environment damage and that can replace fossil fuels, reducing the toxic emissions level, replacing the rare fossil fuel used to date. In the midst of these researches has been observed that the use of vegetable oils has shown great ability to make this one a possible alternative renewable energy (Agarwal & Das, 2000).A related problem in the replacement of diesel for oil plant was related to physical and chemical factors such as high viscosity, low volatility which results in incomplete combustion, leading to forma‐ tion of carbon deposits in the engine and a high unsaturations degree (Meher et al, 2008), factor that reduces the power of the fuel at the lowest level of cetane,but also favors oxi‐ dation. Studies have shown that vegetable oils characteristics can be modified through four ways (Shrivastava & Presad, 2002): By pyrolysis, microemulsification, dilution and transesterification process. The latter originates the alkyl esters that constitute what is called biodiesel.

This parameter is limited by the solvent, reactant boiling point. The second factor to im‐ prove reaction yield, vigorous mixing, possibilities a higher collision rate between the re‐ actants, been obtained a reaction mixture plus homogenized, yielding a higher rate of methyl esters obtained. In general alcohols and triglyceride sources are immiscible, vigo‐ rous mixing possibilities the obtaining of alcohol dispersed as fine droplets, increasing the contact surface between the two immiscible reactants (Stamenkovic et al, 2008). The use of a secondary solvent, a co-solvent as THF, possibilities a higher miscibility of the alcohol in the triglyceride phase, obtaining a better mixing of the two phases and hence a more reactions to take place, improving the biodiesel yield. The following Figure 1 illustrates a

In general terms, these reactions take place under homogeneous catalysts, acid or base catalysts, enzyme or through the use of heterogeneous catalysts. The selection of appro‐ priate catalyst depends on the amount of free fatty acids in the oil. Heterogeneous cata‐ lyst provides high activity; high selectivity, high water tolerance properties and these properties depend on the amount and strengths of active acid or basic sites. Basic catalyst can be subdivided based on the type of metal oxides and their derivatives. Similarly, acid‐ ic catalyst can be subdivided depending upon their active acidic sites (Singh & Sarma, 2011). Generally, a basic catalyst gives better yields than the acids catalysts in both homo‐ geneous and heterogeneous catalysts. The better results of homogeneous catalysts are re‐ lated to the fact that base catalysts are kinetically much faster than heterogeneously catalyzed transesterification and are economically viable. There are many factors which govern the path of transesterification reactions, between these can be stand out the fol‐ lowing parameters: the nature o raw material, the optimum experimental conditions, as the ratio oil/methanol, the temperature and the catalyst concentration, for example. Com‐ paring heterogeneous catalyst with homogeneous catalysts can be observed that the use of solid heterogeneous use more extreme reaction conditions, higher pressure and temper‐ ature due the fact of the difficulty in the limited mass transfer between the three phase system solid-liquid-liquid immiscible (catalyst, oil, methanol). The main advantages in the use of solid catalysts are related to the easy work up when compared with homogeneous catalysts. Solid catalysts are separated just by filtration and centrifugation and are envi‐ ronmentally friendly, because they are reusable and reduce the amount of wasted, treated water used. Among the heterogeneous catalysts, we can highlight the use of zeolites

HO

OH

OH

+ H3<sup>C</sup> OCOR

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 25

biodiesel type of reaction.

OCOR

OCOR

+

**Figure 1.** General transesterification reaction to produce biodiesel

(Suppes et al, 2004), clays, ion exchange resins and oxides.

H3C OH

OCOR

### **2. Biodiesel**

The use of vegetable oils as an alternative fuel for diesel engine was discovered more than100 years ago, in the Paris world exhibition in 1900, when Rudolph Diesel used pea‐ nut oil in an engine ignition (Shay, 1993).This predicted saying, "The use of vegetable oils as fuel engine may be negligible in the present moment, but in the future may become so important as oil and coal as energy sources. The biodiesel term is a subject still under dis‐ cussion. Some definitions consider biodiesel as a mixture of any vegetable oils with fuel, diesel and fossil derivative others consider the alkyl esters mixture from vegetable oils or animal fats with fossil fuels. Under the chemical aspect biodiesel, an alternative fuel can be defined as alkyl esters derived from fatty acids obtained from oils, vegetables or ani‐ mal fats, which suffering a chemical reaction, transesterification with short chain alcohols such as methanol and ethanol (Pinto et al, 2005). Transesterification: Chemical reaction be‐ tween an ester (RCOOR ') and an alcohol (R''COH) resulting in a new ester (R''COOR') and an alcohol (RCOH).

This reaction type, used in biodiesel production is the reaction between the triglycerides, main components of vegetable oils and fats that react with short chain alcohols, methanol and ethanol, resulting in two products, methyl esters derived from fatty acids, and the second product glycerol formation. Transesterification reaction rates can be affected by some aspects: The catalyst type (acid or alkaline), purity of reactants (mainly water con‐ tent), free fatty acid content and alcohol/vegetable oil molar ratio (Helwani et al, 2009). The biodiesel reaction can be optimized specially by three factors: The first is an increase in the temperature. An increase in temperature increases reaction rate in exponential, al‐ lowing the reactants to be more miscible, obtaining a higher reaction rate to take place. This parameter is limited by the solvent, reactant boiling point. The second factor to im‐ prove reaction yield, vigorous mixing, possibilities a higher collision rate between the re‐ actants, been obtained a reaction mixture plus homogenized, yielding a higher rate of methyl esters obtained. In general alcohols and triglyceride sources are immiscible, vigo‐ rous mixing possibilities the obtaining of alcohol dispersed as fine droplets, increasing the contact surface between the two immiscible reactants (Stamenkovic et al, 2008). The use of a secondary solvent, a co-solvent as THF, possibilities a higher miscibility of the alcohol in the triglyceride phase, obtaining a better mixing of the two phases and hence a more reactions to take place, improving the biodiesel yield. The following Figure 1 illustrates a biodiesel type of reaction.

**Figure 1.** General transesterification reaction to produce biodiesel

this process is called biomass. This has a great advantage over fossil fuels, it's less pollut‐ ing, because its processes do not add carbon dioxide to the atmosphere, the environment. The biomass process reduces the carbon dioxide amount in atmosphere through the pho‐ tosynthesis, performed by increasing the planted green areas, to cultivate the seeds crops. Research and development departments have been engaged in fuels discovery that do not cause much environment damage and that can replace fossil fuels, reducing the toxic emissions level, replacing the rare fossil fuel used to date. In the midst of these researches has been observed that the use of vegetable oils has shown great ability to make this one a possible alternative renewable energy (Agarwal & Das, 2000).A related problem in the replacement of diesel for oil plant was related to physical and chemical factors such as high viscosity, low volatility which results in incomplete combustion, leading to forma‐ tion of carbon deposits in the engine and a high unsaturations degree (Meher et al, 2008), factor that reduces the power of the fuel at the lowest level of cetane,but also favors oxi‐ dation. Studies have shown that vegetable oils characteristics can be modified through four ways (Shrivastava & Presad, 2002): By pyrolysis, microemulsification, dilution and transesterification process. The latter originates the alkyl esters that constitute what is

The use of vegetable oils as an alternative fuel for diesel engine was discovered more than100 years ago, in the Paris world exhibition in 1900, when Rudolph Diesel used pea‐ nut oil in an engine ignition (Shay, 1993).This predicted saying, "The use of vegetable oils as fuel engine may be negligible in the present moment, but in the future may become so important as oil and coal as energy sources. The biodiesel term is a subject still under dis‐ cussion. Some definitions consider biodiesel as a mixture of any vegetable oils with fuel, diesel and fossil derivative others consider the alkyl esters mixture from vegetable oils or animal fats with fossil fuels. Under the chemical aspect biodiesel, an alternative fuel can be defined as alkyl esters derived from fatty acids obtained from oils, vegetables or ani‐ mal fats, which suffering a chemical reaction, transesterification with short chain alcohols such as methanol and ethanol (Pinto et al, 2005). Transesterification: Chemical reaction be‐ tween an ester (RCOOR ') and an alcohol (R''COH) resulting in a new ester (R''COOR')

This reaction type, used in biodiesel production is the reaction between the triglycerides, main components of vegetable oils and fats that react with short chain alcohols, methanol and ethanol, resulting in two products, methyl esters derived from fatty acids, and the second product glycerol formation. Transesterification reaction rates can be affected by some aspects: The catalyst type (acid or alkaline), purity of reactants (mainly water con‐ tent), free fatty acid content and alcohol/vegetable oil molar ratio (Helwani et al, 2009). The biodiesel reaction can be optimized specially by three factors: The first is an increase in the temperature. An increase in temperature increases reaction rate in exponential, al‐ lowing the reactants to be more miscible, obtaining a higher reaction rate to take place.

called biodiesel.

24 Soybean - Bio-Active Compounds

**2. Biodiesel**

and an alcohol (RCOH).

In general terms, these reactions take place under homogeneous catalysts, acid or base catalysts, enzyme or through the use of heterogeneous catalysts. The selection of appro‐ priate catalyst depends on the amount of free fatty acids in the oil. Heterogeneous cata‐ lyst provides high activity; high selectivity, high water tolerance properties and these properties depend on the amount and strengths of active acid or basic sites. Basic catalyst can be subdivided based on the type of metal oxides and their derivatives. Similarly, acid‐ ic catalyst can be subdivided depending upon their active acidic sites (Singh & Sarma, 2011). Generally, a basic catalyst gives better yields than the acids catalysts in both homo‐ geneous and heterogeneous catalysts. The better results of homogeneous catalysts are re‐ lated to the fact that base catalysts are kinetically much faster than heterogeneously catalyzed transesterification and are economically viable. There are many factors which govern the path of transesterification reactions, between these can be stand out the fol‐ lowing parameters: the nature o raw material, the optimum experimental conditions, as the ratio oil/methanol, the temperature and the catalyst concentration, for example. Com‐ paring heterogeneous catalyst with homogeneous catalysts can be observed that the use of solid heterogeneous use more extreme reaction conditions, higher pressure and temper‐ ature due the fact of the difficulty in the limited mass transfer between the three phase system solid-liquid-liquid immiscible (catalyst, oil, methanol). The main advantages in the use of solid catalysts are related to the easy work up when compared with homogeneous catalysts. Solid catalysts are separated just by filtration and centrifugation and are envi‐ ronmentally friendly, because they are reusable and reduce the amount of wasted, treated water used. Among the heterogeneous catalysts, we can highlight the use of zeolites (Suppes et al, 2004), clays, ion exchange resins and oxides.

## **3. Catalysts**

#### **3.1. Heterogeneous catalysts**

The useof heterogeneous catalysts (Wang & Yang, 2007 and Leclercq et al, 2001) has as major advantage the reaction work-up, i.e., post-treatment reaction, separation and purification steps, since these can be easily removed and can be reused. Another interesting factor is the fact that this type of catalysis, there is no formation of by products, such as saponification (Suppes et al, 2001; Tomasevic et al, 2003 and Gryglewicz, 1999). The greatest difficulty en‐ countered in using this reaction type is directly related to problems in relation between the diffusion systems, oil /catalyst /methanol.

**Vegetable oil Catalysts Ratio**

Mesoporous silica loaded with MgO

ate results in the formation of biodiesel and glycerol.

dium, the rinse water should be neutralized.

**Table 1.** Different heterogeneous catalysts used for transesterification of vegetable oils.

In the following figure 2, is exemplified the mechanism of base catalyzed transesterification. The mechanism can be resumed in the following way. In the first step the methoxide anion attaches to the carbonyl carbon atom of the triglyceride. In the second step, the oxygen picks up an acid H+ from the alcohol. In the last step a rearrangement of the tetrahedral intermedi‐

Sulfur and chlorides compounds are the most commonly used acid catalysts. This type of catalysis[(Mohamad & Ali, 2002) has as main advantages the absence of products derived from saponification reactions, higher yields but has some disadvantages such as the fact that the reactions are performed in a highly corrosive and reactive post-treatment, where the me‐

Enzimes are a fourth class of compounds used to produce biodiesel (Fukuda et al, 2001). In general its use is complicated by the fact that the enzyme generally are a specific material,

**Blended vegetable**

*3.2.2. Acid catalysts*

**4. Enzymes**

**MeOH/Oil**

**Soybean** Calcined LDH (Li-Al) 15 1-6 65 71.9 Li **Soybean** La/zeolite beta 14.5 4 160 48.9 Furata **Soybean** MgOMgAl2O4 3 10 65 57 Schumaker **Soybean** MgO, ZnO, Al2O3 55 7 70-130 82 Trakarnpruk **Soybean** Cu and Co 5 3 70 Shu **Soybean** CaO, SrO 12 0.5-3 65 95 Wang **Soybean** ETS-10 6 24 120 94.6 Arzamendi **Cotton seed** Mg-Al-CO3 HT 6 12 180-210 87 Wang **Jatropha Curcas** CaO 9 2.5 70 93 Albuquerque **Palm** Mg-Al-CO3 (hydrotalcite) 30 6 100 86.6 Huaping **Rape** Mg-Al HT 6 4 65 90.5 Zeng **Sunflower** NaOH/Alumina 6-48 1 50 99 Liu **Sunflower** CaO/SBA-14 12 5 160 95 Suppes

**Reaction time (h)**

**Temperature (ºC)**

8 5 220 96 Barakos

**Conversion (%)**

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990

**References**

27

#### **3.2. Homogeneous catalysts**

#### *3.2.1. Basic catalysts*

Basic Catalysis (Zhou et al, 2003) are procedures that use in general alkoxides of sodium and potassium, carbonates and hydroxides of these elements. Among these three groups it is found that alkoxides catalysts are financially unfavorable because they are more expensive but also difficult to handle because they are hygroscopic, and facilitate the achievement of side products such as derivatives of saponification, but have the advantage of carrying out the reactions in milder temperatures, produces high levels of esters derived from fatty acids and do not have corrosive properties as acid catalysts. A solution used to minimize the soap formation when biodiesel has a high free fatty acid content or water is the use of 2 or 3% mol of K2CO3 that will form the corresponding bicarbonate salt instead of water.

In the following, table 1, it's possible to find diverse types of heterogeneous catalysts used to obtain biodiesel of soybean, cotton seed, *Jatropha curcas*, palm, rape oil and sunflower.

Among the studies using soybean oil to obtain biodiesel, can be stand out the work devel‐ oped by Wang et al, using CaO, SrO as a solid catalyst used in a heterogeneous process to obtain biodiesel. Cao, is a typical basic solid catalyst used in the most different ways. This compound has many advantages as a reusable due to its long catalyst lifetime, high‐ er activity and requirement of only mild reaction conditions. At the example of table 1, is observed that in the best conditions to obtain biodiesel in yield of 95% is necessary a tem‐ perature of 65ºC, a molar ratio of MeOH/Oil of 5 and even a little reaction time from 0.5 to 3 hours. Even with all these specific positive factors, solid acid catalysts have been very useful at many industrial processes. Acid catalysts contain a large variety of acid sites with different strength of Bronsted, Lewis acidity, which is considered a good advantage at the transesterification process. These catalysts are even very useful, when is necessary to obtain biodiesel from oils rich in FFA, free fatty acids, because they convert the FFA in‐ to FAME prior to the biodiesel production, avoiding by this way the problem encoun‐ tered at base catalysts, the soap formation.


**Table 1.** Different heterogeneous catalysts used for transesterification of vegetable oils.

In the following figure 2, is exemplified the mechanism of base catalyzed transesterification. The mechanism can be resumed in the following way. In the first step the methoxide anion attaches to the carbonyl carbon atom of the triglyceride. In the second step, the oxygen picks up an acid H+ from the alcohol. In the last step a rearrangement of the tetrahedral intermedi‐ ate results in the formation of biodiesel and glycerol.

#### *3.2.2. Acid catalysts*

**3. Catalysts**

26 Soybean - Bio-Active Compounds

**3.1. Heterogeneous catalysts**

**3.2. Homogeneous catalysts**

*3.2.1. Basic catalysts*

diffusion systems, oil /catalyst /methanol.

tered at base catalysts, the soap formation.

The useof heterogeneous catalysts (Wang & Yang, 2007 and Leclercq et al, 2001) has as major advantage the reaction work-up, i.e., post-treatment reaction, separation and purification steps, since these can be easily removed and can be reused. Another interesting factor is the fact that this type of catalysis, there is no formation of by products, such as saponification (Suppes et al, 2001; Tomasevic et al, 2003 and Gryglewicz, 1999). The greatest difficulty en‐ countered in using this reaction type is directly related to problems in relation between the

Basic Catalysis (Zhou et al, 2003) are procedures that use in general alkoxides of sodium and potassium, carbonates and hydroxides of these elements. Among these three groups it is found that alkoxides catalysts are financially unfavorable because they are more expensive but also difficult to handle because they are hygroscopic, and facilitate the achievement of side products such as derivatives of saponification, but have the advantage of carrying out the reactions in milder temperatures, produces high levels of esters derived from fatty acids and do not have corrosive properties as acid catalysts. A solution used to minimize the soap formation when biodiesel has a high free fatty acid content or water is the use of 2 or 3% mol

In the following, table 1, it's possible to find diverse types of heterogeneous catalysts used to obtain biodiesel of soybean, cotton seed, *Jatropha curcas*, palm, rape oil and sunflower.

Among the studies using soybean oil to obtain biodiesel, can be stand out the work devel‐ oped by Wang et al, using CaO, SrO as a solid catalyst used in a heterogeneous process to obtain biodiesel. Cao, is a typical basic solid catalyst used in the most different ways. This compound has many advantages as a reusable due to its long catalyst lifetime, high‐ er activity and requirement of only mild reaction conditions. At the example of table 1, is observed that in the best conditions to obtain biodiesel in yield of 95% is necessary a tem‐ perature of 65ºC, a molar ratio of MeOH/Oil of 5 and even a little reaction time from 0.5 to 3 hours. Even with all these specific positive factors, solid acid catalysts have been very useful at many industrial processes. Acid catalysts contain a large variety of acid sites with different strength of Bronsted, Lewis acidity, which is considered a good advantage at the transesterification process. These catalysts are even very useful, when is necessary to obtain biodiesel from oils rich in FFA, free fatty acids, because they convert the FFA in‐ to FAME prior to the biodiesel production, avoiding by this way the problem encoun‐

of K2CO3 that will form the corresponding bicarbonate salt instead of water.

Sulfur and chlorides compounds are the most commonly used acid catalysts. This type of catalysis[(Mohamad & Ali, 2002) has as main advantages the absence of products derived from saponification reactions, higher yields but has some disadvantages such as the fact that the reactions are performed in a highly corrosive and reactive post-treatment, where the me‐ dium, the rinse water should be neutralized.

#### **4. Enzymes**

Enzimes are a fourth class of compounds used to produce biodiesel (Fukuda et al, 2001). In general its use is complicated by the fact that the enzyme generally are a specific material,

**Figure 2.** Mechanism of base catalyzed transesterification.

and extremely expensive in relation to this type of reaction, are sensitive to the presence of methanol and ethanol, which causes deactivation of the same (Salis et al, 2008). Literature (Modi et al, 2007) shows that this problem can be circumvented by the water (Kaieda et al, 2001 & Kaieda et al, 1998) use and organic (Raganathan, 2008 & Harding et al, 2008) solvents such as dioxanes and petroleum ether, for example.

**5. Non catalytic fatty acid alkyl ester production**

ing biodiesel by supercritical method, figure 4.

**Figure 3.** Mechanism of acid catalyzed transesterification

R1 OR2

OH

R1 OR2 OH

O+ R H

OR2

+

H +

> H O R

> > - H<sup>+</sup>

R1

O+ H

OR2

R1 C +

R1 OR2 OH O+ R H

R

R1 O

O

R1

O

R1

R = Alkyl group of the alcohol R1 = Carbon chain of fatty acid

O

O

O

<sup>R</sup> <sup>O</sup> <sup>2</sup><sup>=</sup>

R1

O

OH

R2 O H

OR2

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 29

O

R1 C +

The use of supercritical methanol process (Marulanda, 2012) to obtain biodiesel has been a useful method when the feed stock oil contains high amount of free fatty acids. This meth‐ odology has solved the problem encountered at the use of solid catalysts, low reaction rates due to low level of mass transfer, limitation between liquid and solid phase of catalysts and reactants. By this process, the dielectric constant of liquid methanol which tends to decrease in the supercritical state, increase the oil in to methanol solubility, resulting in a single phase oil/methanol system (Lee et al, 2012). In the next page, is exemplified the process of obtain‐

**Figure 3.** Mechanism of acid catalyzed transesterification

and extremely expensive in relation to this type of reaction, are sensitive to the presence of methanol and ethanol, which causes deactivation of the same (Salis et al, 2008). Literature (Modi et al, 2007) shows that this problem can be circumvented by the water (Kaieda et al, 2001 & Kaieda et al, 1998) use and organic (Raganathan, 2008 & Harding et al, 2008) solvents

O

O

R1 O

R1

R1 = Carbon chain of fatty acid

R = alkyl group pf alcohol

O

such as dioxanes and petroleum ether, for example.

**Figure 2.** Mechanism of base catalyzed transesterification.

Pre-step OH-

R 1

28 Soybean - Bio-Active Compounds

R 1

R 1

O

O-

O

O

O - O

O R 2

R

O+ R 2 H

R

Where R

<sup>2</sup> <sup>=</sup>

R 2

Or NaOR RO-

+ RO-

+ ROH RO-

+ ROH R

R 2 O

O

R1 O

+ Na<sup>+</sup>

+ H2O

R 1

1

O

<sup>H</sup> <sup>+</sup> <sup>R</sup>

O-

O

O+ R 2

R

1

O

O R

O R 2

R

H

<sup>+</sup> RO-

O -

### **5. Non catalytic fatty acid alkyl ester production**

The use of supercritical methanol process (Marulanda, 2012) to obtain biodiesel has been a useful method when the feed stock oil contains high amount of free fatty acids. This meth‐ odology has solved the problem encountered at the use of solid catalysts, low reaction rates due to low level of mass transfer, limitation between liquid and solid phase of catalysts and reactants. By this process, the dielectric constant of liquid methanol which tends to decrease in the supercritical state, increase the oil in to methanol solubility, resulting in a single phase oil/methanol system (Lee et al, 2012). In the next page, is exemplified the process of obtain‐ ing biodiesel by supercritical method, figure 4.

(Usta, 2005), *JatropaCurcas* (Berchmans & Hirata, 2008), Karanja (*Pongamiaglabra*) (Meher et al, 2006), salmon (El-Mashad et al, 2008), cooking oils, among others. All biodiesel sources are chosen according to the chemical composition of their fatty acids in relation to the size of their chains, unsaturation degree and the presence of other chemical functions, as these fac‐

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 31

In recent years soybean biodiesel has achieved a high level of advanced techniques to im‐ prove its production. Has been developed a methodology using microwave assistance to im‐ prove the esters conversion rates,, using heterogeneous catalysts, nano Cao, for example which facilitates the interaction between the molecules (Hsiao et al, 2011). Dr Hsiao and his research group has proved that by this methodology, is possible to obtain a higher biodiesel yield in less time. There are two factors that influence this reaction type. The use of nano‐ compounds facilitates the interaction between the molecules, nanocompounds possibility a high contact surface between the molecules. The microwave methodology reduces even the reaction time due to changing the electrical field activates the smallest variance degree of ions and molecules leading to molecular friction, enabling the initiation of molecule, chemi‐ cal reaction. This methodology also provides an easier access to susceptible bonds, so, in‐ creases the chemical interaction, been obtained products in less time and higher yields. Microwave methodologies has proved to be part of desirable green chemistry, cause it is a safe, comfortable and clean way of working with chemical reactions. Microwave flow sys‐ tem assistance through homogeneous catalysis is another example which has improved the biodiesel production in less time, depending of some factors as reaction residence time, cata‐ lyst amount and temperature at the exit point (Encinar et al, 2012). In attempt to improve the microwave assisted methodology to obtain biodiesel another technique was added, used, the ultrasound. Microwave and ultrasound developed methodology has proved to be very efficient when used together. In this process, the first step used is ultrasound, cause ultra‐ sonic field induced an effective emulsification and mass transfer that increases the rate of ester formation due to ultrasonic mixing causes cavitations of bubble near the phase boun‐ dary between the methanol and seeds oil, facilitating the thoroughly mixing, interaction be‐ tween the oil and the reactant, methanol (Hsiao et al, 2010).Another technique has been also developed using ultrasonic irradiation with vibration ultrasonic. Instead of using heteroge‐ neous or homogeneous transesterification catalyst, was used the enzyme methodology, through the application of Novozym 435. This methodology has proved to be efficient in en‐ zymatic reaction to obtain biodiesel. Was observed that the use of ultrasonic added to vibra‐ tion is a further factor to obtain higher values of biodiesel, cause the movement increase might facilitate the interaction between the substrate and the active site (Yu et al, 2010).Transesterification of soybean oil was achieved using ultrasonic water bath and two different commercial lipases in organic solvents (*n*-hexane) for example (Batistella et al, 2012). Dimethyl carbonate is a useful alternative to obtain biodiesel, this one is nontoxic, cheapness product and the reaction obtained product, glycerol carbonate is a value added

tors influence the biodiesel quality.

**7. New advanced techniques to obtain biodiesel**

**Figure 4.** Schematic process of biodiesel production by supercritical method.

The methodology using supercritical methanol to obtain biodiesel by transesterification has reached the possibility to realize the reactions under mild, relatively moderate reactions to avoid the thermal degradation of fatty acid methyl esters (FAME). The reactions has been investigated in a wide range of reaction conditions (T = 200 – 425 ºC, time = 2 – 40 min. and P = 9.6 – 43 MPa). Thermal stability studies of methyl esters has showed that the best reaction conditions by supercritical methanol methodology to obtain biodiesel, consists in the tem‐ perature of 270ºC and reaction pressure of 8.09 MPa. The use of a co-solvent, as propane, CO2 and heptane, diminishes, decrease the reaction temperature and the pressure needed to achieve a high yield of biodiesel obtained. The following table 2 exemplifies the investigated reactions conditions to obtain biodiesel.


**Table 2.** Examples of biodiesel production, experiment data using supercritical methanol.

#### **6. Sources to obtain biodiesel**

The sources for biodiesel production are chosen according to availability of the same in each country, region, taking into account the relative low cost of production and favorable econo‐ mies of scale. For example, the use of refined oil would not be favorable due to high produc‐ tion costs and low production scale, on the other hand the use of seeds, algae and fat have a low production cost and greater availability than refined oils or recycled, which is a favora‐ ble factor for the production of biodiesel from these elements. When choosing a source of biodiesel production plants, a relationship is taken into account is how much they produce and the yield of oil per hectare. Following some examples of studied seeds: soybean, Babas‐ su(*Orbiginiasp*.), castor oil, fish oil, microalgae(*Chorellavulgaris*) (Miao & Wu, 2006), tobacco (Usta, 2005), *JatropaCurcas* (Berchmans & Hirata, 2008), Karanja (*Pongamiaglabra*) (Meher et al, 2006), salmon (El-Mashad et al, 2008), cooking oils, among others. All biodiesel sources are chosen according to the chemical composition of their fatty acids in relation to the size of their chains, unsaturation degree and the presence of other chemical functions, as these fac‐ tors influence the biodiesel quality.

## **7. New advanced techniques to obtain biodiesel**

**Figure 4.** Schematic process of biodiesel production by supercritical method.

**Oil (co-solvent) T (ºC) P (MPa) MeOH/Oil**

**Table 2.** Examples of biodiesel production, experiment data using supercritical methanol.

reactions conditions to obtain biodiesel.

30 Soybean - Bio-Active Compounds

**6. Sources to obtain biodiesel**

The methodology using supercritical methanol to obtain biodiesel by transesterification has reached the possibility to realize the reactions under mild, relatively moderate reactions to avoid the thermal degradation of fatty acid methyl esters (FAME). The reactions has been investigated in a wide range of reaction conditions (T = 200 – 425 ºC, time = 2 – 40 min. and P = 9.6 – 43 MPa). Thermal stability studies of methyl esters has showed that the best reaction conditions by supercritical methanol methodology to obtain biodiesel, consists in the tem‐ perature of 270ºC and reaction pressure of 8.09 MPa. The use of a co-solvent, as propane, CO2 and heptane, diminishes, decrease the reaction temperature and the pressure needed to achieve a high yield of biodiesel obtained. The following table 2 exemplifies the investigated

**ratio**

**Soybean** 100 -320 32 40 25 C 96 He H. et al **Soybean (**CH3H8/MeOH = 0.05) 280 12.8 24 10 B 98 Cao W. et al **Soybean (**CH3H8/MeOH = 0.05) 288 9.6 65.8 10 B 99 Hegel P. et al **Soybean** (CO2/MeOH = 0.1) 280 14.3 24 10 B 98 Han H. et al **Soybean** (CO2/MeOH = 0.1) 350-425 10 - 25 3-6 2 - 3 C 100 Anitescu G. et al

The sources for biodiesel production are chosen according to availability of the same in each country, region, taking into account the relative low cost of production and favorable econo‐ mies of scale. For example, the use of refined oil would not be favorable due to high produc‐ tion costs and low production scale, on the other hand the use of seeds, algae and fat have a low production cost and greater availability than refined oils or recycled, which is a favora‐ ble factor for the production of biodiesel from these elements. When choosing a source of biodiesel production plants, a relationship is taken into account is how much they produce and the yield of oil per hectare. Following some examples of studied seeds: soybean, Babas‐ su(*Orbiginiasp*.), castor oil, fish oil, microalgae(*Chorellavulgaris*) (Miao & Wu, 2006), tobacco

**Time (min)** **B/C Yield (%)**

**Refs.**

In recent years soybean biodiesel has achieved a high level of advanced techniques to im‐ prove its production. Has been developed a methodology using microwave assistance to im‐ prove the esters conversion rates,, using heterogeneous catalysts, nano Cao, for example which facilitates the interaction between the molecules (Hsiao et al, 2011). Dr Hsiao and his research group has proved that by this methodology, is possible to obtain a higher biodiesel yield in less time. There are two factors that influence this reaction type. The use of nano‐ compounds facilitates the interaction between the molecules, nanocompounds possibility a high contact surface between the molecules. The microwave methodology reduces even the reaction time due to changing the electrical field activates the smallest variance degree of ions and molecules leading to molecular friction, enabling the initiation of molecule, chemi‐ cal reaction. This methodology also provides an easier access to susceptible bonds, so, in‐ creases the chemical interaction, been obtained products in less time and higher yields. Microwave methodologies has proved to be part of desirable green chemistry, cause it is a safe, comfortable and clean way of working with chemical reactions. Microwave flow sys‐ tem assistance through homogeneous catalysis is another example which has improved the biodiesel production in less time, depending of some factors as reaction residence time, cata‐ lyst amount and temperature at the exit point (Encinar et al, 2012). In attempt to improve the microwave assisted methodology to obtain biodiesel another technique was added, used, the ultrasound. Microwave and ultrasound developed methodology has proved to be very efficient when used together. In this process, the first step used is ultrasound, cause ultra‐ sonic field induced an effective emulsification and mass transfer that increases the rate of ester formation due to ultrasonic mixing causes cavitations of bubble near the phase boun‐ dary between the methanol and seeds oil, facilitating the thoroughly mixing, interaction be‐ tween the oil and the reactant, methanol (Hsiao et al, 2010).Another technique has been also developed using ultrasonic irradiation with vibration ultrasonic. Instead of using heteroge‐ neous or homogeneous transesterification catalyst, was used the enzyme methodology, through the application of Novozym 435. This methodology has proved to be efficient in en‐ zymatic reaction to obtain biodiesel. Was observed that the use of ultrasonic added to vibra‐ tion is a further factor to obtain higher values of biodiesel, cause the movement increase might facilitate the interaction between the substrate and the active site (Yu et al, 2010).Transesterification of soybean oil was achieved using ultrasonic water bath and two different commercial lipases in organic solvents (*n*-hexane) for example (Batistella et al, 2012). Dimethyl carbonate is a useful alternative to obtain biodiesel, this one is nontoxic, cheapness product and the reaction obtained product, glycerol carbonate is a value added substance with various useful applications. This can be obtained through enzymatic transes‐ terification of soybean oil in organic solvents in mild conditions (Seong et al, 2011).The products, biodiesel are obtained in more time, but by other side this methodology has many advantages: It is an easy to use methodology, the enzyme can be reusable and the reaction work up is chemically friendly, cause it's not necessary the treatment of water used to purify biodiesel by the traditional, usual transesterification by homogeneous basic catalyst and is obtained an added value product, the glycerol carbonates. Heterogeneous catalysis using subcritical methanol is an advance in the soybean biodiesel obtaining methodology. By this technique is possible to use less amount of catalyst and have as main advantages the catalyst reusable and the separation, obtaining from reaction medium through centrifugation. The use of small amount of a catalyst, K3PO4, 0.1%wt, insoluble in methanol has transformed the reaction in subcritical methanol more available, cause has reduced the temperatures from 350ºC to 160ºC and the methanol molar ratio from 42 to 24, for example. The catalyst can be reusable at least three times. (Yin et al, 2012). KF Modified calcium magnesium oxide cata‐ lyst is an example of heterogeneous catalysis to obtain soybean biodiesel and even to recycle the catalysts due to be easily removed from the reaction through centrifugation and the use of a reaction under atmosphere pressure and 65ºC of temperature. This new catalyst has even improved the ester methyl yield from 63.6% (CaO-MgO catalyst) to 97.9% (KF-MgO-CaO) (Fan et al, 2012). Response surface methodology is an applicable technique to improve the results in obtaining soybean biodiesel. This methodology verifies the main parameters to optimize the biodiesel production process (Silva et al, 2011).The use of a process entirely in‐ dependent from petroleum has been reached by the use of ethanol, obtained from a renewa‐ ble source, sugar cane and seed oil. Gomes and his research group has developed a methodology to obtain ethyl biodiesel and even has developed a methodology to simplify the work-up process. In order to optimize the separation step of glycerol from biodiesel, many techniques have been studied. The microfiltration through ceramic membrane has demonstrated to be a useful technique to obtain biodiesel. This methodology is environmen‐ tally friendly cause reduces the amount of used water to purify the biodiesel. This technique simplify the entire purification process, the biodiesel is obtained by transesterification, after the end of reaction is added acidified water, this process facilitates the separation in two phases, the organic one, rich in oil and the aqueous, which posses the soaps converted in water soluble salts, catalysts, glycerol and other water soluble substances (Gomes et al, 2011). In water, glycerol forms greater droplets that are retained during the microfiltration step, been the biodiesel obtained by this way with glycerol content lower than 0.02% wt, the limit of free glycerol specified by Brazilian regulation. Mesoporous silica catalyst was used in a heterogeneous catalysis of soybean transesterification with methanol. La50SBA-15 is used to obtain an ethyl biodiesel in mild conditions after 6 hours. The main advantage of this technique is the use of a heterogeneous catalysis to obtain ethyl biodiesel in mild condi‐ tions, don't use the usual high temperatures 473K and lower amounts of the ratio Oil/ Alco‐ hol, from 36 to 20. This lower amount of alcohol facilitates the phase separation organic/ water, less amount of alcohol causes an easier purification process, because diminishes the possibility of emulsion formation (Quintela et al, 2012). The heterogeneous catalysis of bio‐ diesel has reached an advance with the development of methodologies using membranes.

These membranes can be prepared in a simple way by the use of clays as hydrotalcite and poly (vinyl alcohol). The biodiesel is prepared by transesterification and can be obtained in mild conditions, 60ºC in a volume ratio oil/methanol of 5:60. The methyl biodiesel by this methodology can be obtained in 90%. The catalyst can be reused at least by 7 times (Guer‐ reiro et al, 2010). An alternative method to obtain biodiesel is the enzymatic-catalytic way. In general this methodology has as main advantage the enzymatic selectivity, is a reusable cat‐ alytic and facilitates the separation, purification process. Methodology using a immobilized lipase onto a nanostructure has been developed due to the good transesterification activity of lipase and the use of electrospinning method to obtain nanofibrous membranes, which have larger surface area and porous structure that can lower the substrate resistance and fa‐ cilitate enzyme immobilization, generating a reusable catalyst for biodiesel synthesis. The use of this methodology simplifies the separation process, where after the reaction, glycerol can be removed by centrifugation and the biodiesel obtained in the 90% range (Li et al,

**Variable Base Catalyst Acid Catalyst Lipase Catalyst Supercritical**

Interfere with reaction

Cheap Cheap Relatively

**8. Non usual methods of soybean biodiesel analysis of cold properties**

Several methods have been used to characterize biodiesel, and each methodology analyses some aspects of biodiesel as cold properties and oxidation process. Most legislation assumes a small group of tests to determination of biodiesel quality. Eighteen percents of Brazilian biodiesel production uses soybean as oil sources. Several nations have been establishing

**Alcohol**

No influence Non sensitive

Medium Potentially cheaper

Esters Methyl esters Esters Non sensitive

60 - 70 55 - 80 30 - 40 239 - 385 180 - 220

Normal Normal Higher Good Normal

Difficult Difficult Easy Easy

expensive

Repeated washing None Easy

**Heterogeneous Catalyst**

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 33

2010).

Reaction temperature (ºC)

Free fatty acid in raw material

> Water in raw materials

Yields of methyl esters

> Recovery of glycerol

Purification of methyl esters

Production cost of catalyst

**and oxidation state**

Saponified products

Interfere with reaction

> Repeated washing

**Table 3.** Example of different technologies to produce biodiesel.

These membranes can be prepared in a simple way by the use of clays as hydrotalcite and poly (vinyl alcohol). The biodiesel is prepared by transesterification and can be obtained in mild conditions, 60ºC in a volume ratio oil/methanol of 5:60. The methyl biodiesel by this methodology can be obtained in 90%. The catalyst can be reused at least by 7 times (Guer‐ reiro et al, 2010). An alternative method to obtain biodiesel is the enzymatic-catalytic way. In general this methodology has as main advantage the enzymatic selectivity, is a reusable cat‐ alytic and facilitates the separation, purification process. Methodology using a immobilized lipase onto a nanostructure has been developed due to the good transesterification activity of lipase and the use of electrospinning method to obtain nanofibrous membranes, which have larger surface area and porous structure that can lower the substrate resistance and fa‐ cilitate enzyme immobilization, generating a reusable catalyst for biodiesel synthesis. The use of this methodology simplifies the separation process, where after the reaction, glycerol can be removed by centrifugation and the biodiesel obtained in the 90% range (Li et al, 2010).


**Table 3.** Example of different technologies to produce biodiesel.

substance with various useful applications. This can be obtained through enzymatic transes‐ terification of soybean oil in organic solvents in mild conditions (Seong et al, 2011).The products, biodiesel are obtained in more time, but by other side this methodology has many advantages: It is an easy to use methodology, the enzyme can be reusable and the reaction work up is chemically friendly, cause it's not necessary the treatment of water used to purify biodiesel by the traditional, usual transesterification by homogeneous basic catalyst and is obtained an added value product, the glycerol carbonates. Heterogeneous catalysis using subcritical methanol is an advance in the soybean biodiesel obtaining methodology. By this technique is possible to use less amount of catalyst and have as main advantages the catalyst reusable and the separation, obtaining from reaction medium through centrifugation. The use of small amount of a catalyst, K3PO4, 0.1%wt, insoluble in methanol has transformed the reaction in subcritical methanol more available, cause has reduced the temperatures from 350ºC to 160ºC and the methanol molar ratio from 42 to 24, for example. The catalyst can be reusable at least three times. (Yin et al, 2012). KF Modified calcium magnesium oxide cata‐ lyst is an example of heterogeneous catalysis to obtain soybean biodiesel and even to recycle the catalysts due to be easily removed from the reaction through centrifugation and the use of a reaction under atmosphere pressure and 65ºC of temperature. This new catalyst has even improved the ester methyl yield from 63.6% (CaO-MgO catalyst) to 97.9% (KF-MgO-CaO) (Fan et al, 2012). Response surface methodology is an applicable technique to improve the results in obtaining soybean biodiesel. This methodology verifies the main parameters to optimize the biodiesel production process (Silva et al, 2011).The use of a process entirely in‐ dependent from petroleum has been reached by the use of ethanol, obtained from a renewa‐ ble source, sugar cane and seed oil. Gomes and his research group has developed a methodology to obtain ethyl biodiesel and even has developed a methodology to simplify the work-up process. In order to optimize the separation step of glycerol from biodiesel, many techniques have been studied. The microfiltration through ceramic membrane has demonstrated to be a useful technique to obtain biodiesel. This methodology is environmen‐ tally friendly cause reduces the amount of used water to purify the biodiesel. This technique simplify the entire purification process, the biodiesel is obtained by transesterification, after the end of reaction is added acidified water, this process facilitates the separation in two phases, the organic one, rich in oil and the aqueous, which posses the soaps converted in water soluble salts, catalysts, glycerol and other water soluble substances (Gomes et al, 2011). In water, glycerol forms greater droplets that are retained during the microfiltration step, been the biodiesel obtained by this way with glycerol content lower than 0.02% wt, the limit of free glycerol specified by Brazilian regulation. Mesoporous silica catalyst was used in a heterogeneous catalysis of soybean transesterification with methanol. La50SBA-15 is used to obtain an ethyl biodiesel in mild conditions after 6 hours. The main advantage of this technique is the use of a heterogeneous catalysis to obtain ethyl biodiesel in mild condi‐ tions, don't use the usual high temperatures 473K and lower amounts of the ratio Oil/ Alco‐ hol, from 36 to 20. This lower amount of alcohol facilitates the phase separation organic/ water, less amount of alcohol causes an easier purification process, because diminishes the possibility of emulsion formation (Quintela et al, 2012). The heterogeneous catalysis of bio‐ diesel has reached an advance with the development of methodologies using membranes.

32 Soybean - Bio-Active Compounds

## **8. Non usual methods of soybean biodiesel analysis of cold properties and oxidation state**

Several methods have been used to characterize biodiesel, and each methodology analyses some aspects of biodiesel as cold properties and oxidation process. Most legislation assumes a small group of tests to determination of biodiesel quality. Eighteen percents of Brazilian biodiesel production uses soybean as oil sources. Several nations have been establishing standards and legislation about biodiesel. Mainly determinations include iodine value, acid content, specific mass, esters content among others. The aim of this work are the availability of methodologies that wasn`t includes on official methodologies.

Small signal at temperature of -1.1ºC is due a crystallization of saturated ester notably pal‐ mitic and stearic methyl esters, shaped signal of -60ºC is caused by crystallization of unsatu‐ rated esters ( oleic, linoleic and linolenic methyl esters). These temperatures can change by

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 35

DSC trying to be associates with pour point results, because, a priori, both methods analyze a formation of firsts crystals, Formation of crystals initiate with nucleation of crystals that precedes crystallization is dependent on the formation and growth of aggregates or clusters of molecules. These aggregates must overcome a critical size in order to keep a steady growth and become a crystal of detectable dimensions [Avrami, 1940; Avrami, 1941]. At the stage of crystal growth, molecules of solute adsorb on the crystal surface and the process de‐ pends on the diffusion of material from the liquid phase to the solid phase which is being formed. Any of these stages can control crystal growth. The added substance must be capa‐ ble to interfere with one of these stages: either avoiding or delaying the growing of aggre‐

In this point it's crucial to understand the difference of results obtained by pour point analy‐ ses and DSC. Calorimetric method is very sensible and detect energy associate of crystalliza‐ tion phenomena under of critical crystal size. While pour point detection occur only when

**Figure 6.** Impact of additives into onset temperature of biodiesel crystallization based on Soares et al, 2009

cooling rate, so is important promoting standardization for independent analysis.

gates to a critical size or reducing crystal growth rate [Mullin,2001].

crystal reached a minimum size.

#### **8.1. Cold properties**

Official methodologies of biodiesel are: Cold Filter Plugging Point (CFPP), Cloud Point and Pour Point. Pour point indicates a moment of initial crystallization, but this methodology has low accuracy. For studies with more complexity other methodologies has better performance.

#### **8.2. Differential scanning calorimetry**

One of most usual and versatility methodology is a differential scanning calorimetry, these methodology are based on monitoring the difference in energy provided/released to/ by the sample (reagent system) in relation to a reference system (inert) as a function of temperature when both systems are subjected to a controlled temperature program. The changes in the temperature of the sample are caused by phase rearrangements, dehydration reaction, disso‐ ciation or decomposition reactions, oxidation or reduction reaction, gelatinization and other chemical reactions. DSC evaluates absorption or energy liberation to determine the initial of the reaction. A typical curve of biodiesel is presented at figure 1:

**Figure 5.** Differential Scanning Calorimetry of biodiesel.

Small signal at temperature of -1.1ºC is due a crystallization of saturated ester notably pal‐ mitic and stearic methyl esters, shaped signal of -60ºC is caused by crystallization of unsatu‐ rated esters ( oleic, linoleic and linolenic methyl esters). These temperatures can change by cooling rate, so is important promoting standardization for independent analysis.

standards and legislation about biodiesel. Mainly determinations include iodine value, acid content, specific mass, esters content among others. The aim of this work are the availability

Official methodologies of biodiesel are: Cold Filter Plugging Point (CFPP), Cloud Point and Pour Point. Pour point indicates a moment of initial crystallization, but this methodology has low accuracy. For studies with more complexity other methodologies has better performance.

One of most usual and versatility methodology is a differential scanning calorimetry, these methodology are based on monitoring the difference in energy provided/released to/ by the sample (reagent system) in relation to a reference system (inert) as a function of temperature when both systems are subjected to a controlled temperature program. The changes in the temperature of the sample are caused by phase rearrangements, dehydration reaction, disso‐ ciation or decomposition reactions, oxidation or reduction reaction, gelatinization and other chemical reactions. DSC evaluates absorption or energy liberation to determine the initial of

Method: Biodiesel Lamoc DSC File: C:...\Biodiesel de girassol metílico.001


Exo Up Universal V4.5A TA Instruments

1.11°C -0.003926W/g

Operator: Gabriela / Viviane Run Date: 01-Oct-2010 14:08 Instrument: DSC Q2000 V24.4 Build 116

of methodologies that wasn`t includes on official methodologies.

the reaction. A typical curve of biodiesel is presented at figure 1:

**8.1. Cold properties**

34 Soybean - Bio-Active Compounds

**8.2. Differential scanning calorimetry**


**Figure 5.** Differential Scanning Calorimetry of biodiesel.



Heat Flow (W/g)

0.0

0.1

0.2

Size: 2.8690 mg

Sample: Biodiesel de girassol metílico

DSC trying to be associates with pour point results, because, a priori, both methods analyze a formation of firsts crystals, Formation of crystals initiate with nucleation of crystals that precedes crystallization is dependent on the formation and growth of aggregates or clusters of molecules. These aggregates must overcome a critical size in order to keep a steady growth and become a crystal of detectable dimensions [Avrami, 1940; Avrami, 1941]. At the stage of crystal growth, molecules of solute adsorb on the crystal surface and the process de‐ pends on the diffusion of material from the liquid phase to the solid phase which is being formed. Any of these stages can control crystal growth. The added substance must be capa‐ ble to interfere with one of these stages: either avoiding or delaying the growing of aggre‐ gates to a critical size or reducing crystal growth rate [Mullin,2001].

In this point it's crucial to understand the difference of results obtained by pour point analy‐ ses and DSC. Calorimetric method is very sensible and detect energy associate of crystalliza‐ tion phenomena under of critical crystal size. While pour point detection occur only when crystal reached a minimum size.

**Figure 6.** Impact of additives into onset temperature of biodiesel crystallization based on Soares et al, 2009

Soares et al (2009) present a work in which several esters derived from branched chain, cy‐ clic monohydroxylated alcohols or polyhydroxylated alcohols were added to methyl trans‐ esterified soybean oil ( biodiesel like) to investigate their effect on the transesterified soybean oil crystallization. In this work were added kinetic studies, were conducted in order to detect differences in crystallization mechanisms due to differences in additive structures.

to be transformed and that these aggregates must grow to a critical size to start a steady growth. By simplifying his statistical treatment presented in the calculation of transformed

Where α is the volume fraction transformed (crystallized mass); k is dependent on a shape factor, on nucleation probability, on nucleation and growth rates and on the dimensionality of crystal growth while n reflects the mechanism of nucleation and growth and the crystal morphology [Avrami, 1940; Avrami, 1941]. Several works has been using DSC as assessment

Oxidative properties of biodiesel commonly assessment by EN 14112 called rancimat and Io‐ dine value, but several methods have been used by analysis of oxidation state of oil and bio‐ diesel. Oxidation products from these compounds as Petrooxy, differential scanning calorimetry (DSC), Pressure Differential Scanning Calorimetry (PDSC) (Dufaure et al, 1999) iodine value (IV) and mainly Rancimat Method. Each method is based at one step, inter‐

At the PetroOxy, the sample is inducted to oxidation through an intense oxygen flow, ma‐ nipulating by this way the stability conditions through a specific apparatus. The analysis time is recorded as the required time to the sample absorbs 10% of oxygen pressure. Analy‐

The differential scanning calorimetry (DSC) monitors the difference in energy provided/ released between the sample (reagent system) and the reference system (inert) as a function of temperature when both, the system are subjected to a controlled temperature program. Changes in temperature sample are caused by rearrangements of induced phase changes, dehydration reaction, dissociation or decomposition reactions, oxidation or reduction reac‐ tion, gelatinization and other chemical reactions. DSC evaluated absorption or energy libera‐ tion for determining initial reaction. This process can present some problems because of formation of lipid alkyl radical is an endotermic process and others reactions are exotermic (Santos et al, 2011). The time for secondary product formation from the primary oxidation product, hydroperoxide, varies with different oils. Secondary oxidation products are formed immediately after hydroperoxide formation in olive and rapeseed oils. However, in sun‐ flower and safflower oils, secondary oxidation products are formed when the concentration

At the Rancimat technique, oxidative stability is based at the electric conductivity increase (Hadorn & Zurcher, 1974.). The biodiesel is prematurely aged by the thermal decomposi‐ tion. The formed products by the decomposition are blown by an air flow (10L/ 110 ºC) into a measuring cell that contains bi-distilled, ionized water. The induction time is determined by the conductivity measure and this can be totally automatized. Rancimat is the most used

α=1-exp (-ktn) (1)

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 37

matter, he came to the generalized expression:

cold properties (ref), with better accuracy and precision.

mediate compounds or reactants of biodiesel oxidation.

of hydroperoxides is appreciable (Guillen and Cabo 2002).

sis is based on oxygen consumption (reactant) but not detect products.

**8.3. Oxidative properties**

They obtained a depression of onset crystallization point about 2ºC when used an additive ( 0,08mol/100g of transesterifed soybean oil)

Still about this work were determined induction times of crystallization. Induction time in‐ dicates how many time initiate crystallization at determined temperature. This time tends to reduce while temperature decreases. This information is important because permitted asso‐ ciation with limit time to storage before crystallization.

**Figure 7.** Induction time of crystallization based on Soares et al ( 2009)

These kinetic models describe how the extension of the phase transformation of a given ma‐ terial occurs as a function of time and temperature. Their equation is based on the supposi‐ tions of isothermal condition of crystallization, aleatory homogeneous or heterogeneous nucleation and that the new phase growth rate is temperature dependent. Avrami admitted that a number of tiny nuclei (aggregates of subcritical size) are already present in the phase to be transformed and that these aggregates must grow to a critical size to start a steady growth. By simplifying his statistical treatment presented in the calculation of transformed matter, he came to the generalized expression:

$$\alpha \text{=1-exp (-kt^{\circ})}\tag{1}$$

Where α is the volume fraction transformed (crystallized mass); k is dependent on a shape factor, on nucleation probability, on nucleation and growth rates and on the dimensionality of crystal growth while n reflects the mechanism of nucleation and growth and the crystal morphology [Avrami, 1940; Avrami, 1941]. Several works has been using DSC as assessment cold properties (ref), with better accuracy and precision.

### **8.3. Oxidative properties**

Soares et al (2009) present a work in which several esters derived from branched chain, cy‐ clic monohydroxylated alcohols or polyhydroxylated alcohols were added to methyl trans‐ esterified soybean oil ( biodiesel like) to investigate their effect on the transesterified soybean oil crystallization. In this work were added kinetic studies, were conducted in order to detect differences in crystallization mechanisms due to differences in additive structures. They obtained a depression of onset crystallization point about 2ºC when used an additive

Still about this work were determined induction times of crystallization. Induction time in‐ dicates how many time initiate crystallization at determined temperature. This time tends to reduce while temperature decreases. This information is important because permitted asso‐

These kinetic models describe how the extension of the phase transformation of a given ma‐ terial occurs as a function of time and temperature. Their equation is based on the supposi‐ tions of isothermal condition of crystallization, aleatory homogeneous or heterogeneous nucleation and that the new phase growth rate is temperature dependent. Avrami admitted that a number of tiny nuclei (aggregates of subcritical size) are already present in the phase

( 0,08mol/100g of transesterifed soybean oil)

36 Soybean - Bio-Active Compounds

ciation with limit time to storage before crystallization.

**Figure 7.** Induction time of crystallization based on Soares et al ( 2009)

Oxidative properties of biodiesel commonly assessment by EN 14112 called rancimat and Io‐ dine value, but several methods have been used by analysis of oxidation state of oil and bio‐ diesel. Oxidation products from these compounds as Petrooxy, differential scanning calorimetry (DSC), Pressure Differential Scanning Calorimetry (PDSC) (Dufaure et al, 1999) iodine value (IV) and mainly Rancimat Method. Each method is based at one step, inter‐ mediate compounds or reactants of biodiesel oxidation.

At the PetroOxy, the sample is inducted to oxidation through an intense oxygen flow, ma‐ nipulating by this way the stability conditions through a specific apparatus. The analysis time is recorded as the required time to the sample absorbs 10% of oxygen pressure. Analy‐ sis is based on oxygen consumption (reactant) but not detect products.

The differential scanning calorimetry (DSC) monitors the difference in energy provided/ released between the sample (reagent system) and the reference system (inert) as a function of temperature when both, the system are subjected to a controlled temperature program. Changes in temperature sample are caused by rearrangements of induced phase changes, dehydration reaction, dissociation or decomposition reactions, oxidation or reduction reac‐ tion, gelatinization and other chemical reactions. DSC evaluated absorption or energy libera‐ tion for determining initial reaction. This process can present some problems because of formation of lipid alkyl radical is an endotermic process and others reactions are exotermic (Santos et al, 2011). The time for secondary product formation from the primary oxidation product, hydroperoxide, varies with different oils. Secondary oxidation products are formed immediately after hydroperoxide formation in olive and rapeseed oils. However, in sun‐ flower and safflower oils, secondary oxidation products are formed when the concentration of hydroperoxides is appreciable (Guillen and Cabo 2002).

At the Rancimat technique, oxidative stability is based at the electric conductivity increase (Hadorn & Zurcher, 1974.). The biodiesel is prematurely aged by the thermal decomposi‐ tion. The formed products by the decomposition are blown by an air flow (10L/ 110 ºC) into a measuring cell that contains bi-distilled, ionized water. The induction time is determined by the conductivity measure and this can be totally automatized. Rancimat is the most used technique to determine finalized biodiesel stability, under oxidative accelerated conditions, according to standard EN14112. This technique evaluated final products of thermal decom‐ position.

The differential scanning calorimetry (DSC) monitors the difference in energy provided/ released between the sample (reagent system) and the reference system (inert) as a function of temperature when both in the system are subjected to a controlled temperature program. Changes in temperature sample are caused by rearrangements of induced phase changes, dehydration reaction, dissociation or decomposition reactions, oxidation or reduction reac‐ tion, gelatinization and other chemical reactions. DSC evaluated consumption or energy lib‐ eration for determining initial reaction.

The Pressure Differential Scanning Calorimetry (P-DSC) is a thermo analytical technique that measures the oxidative stability using a differential heat flow between sample and ref‐ erence thermocouple under variations of temperatures and pressure. This technique differs from the Rancimat for being a fast method and presents one more variable - the pressure, allowing to work at low temperatures and using a small amount of sample (Candeia, 2009). All of these methods evaluated one aspects of oxidation process. But to predict behavior or design an adequate biodiesel its necessary to associate a oxidation process with structural properties or composition.

Iodine Value (IV) has been used for a long time to quantify unsaturated bonds on vegetable oil and, actually, biodiesel. Iodine Value is considering mainly structural method to assess‐ ment oxidation stability. Although currently some authors agree that this method is not nec‐ essarily the better method to evaluate stability.

Agreement with oxidation mechanism of fatty acids its very common associated presence of unsaturated with tendency of low stability, but Jain and Sharma (2010) presents weak rela‐ tionship (R2 = 0,4374) between unsaturated esters content and induction period for several bi‐ odiesels, its associate this discrepancy with differ technology productions or presence of impurities.

**Figure 8.** Formation of high molecular level species tends to increase oil viscosity

stage.

**9. Conclusion**

In presence of oxygen molecule polymer are forming by C-O-C linkages (Jonhson et al,1957; Wexler, 1964; Formo et al, 1979) and C-C linkages, while under an inert atmosphere only polymers with C-C linkages primordially are founded. High contents of polyunsaturated fatty acid chains enhance oxidative polymerization in fatty oils (Korus et al, 1983).Trimers and other fatty acid polymers presents higher thermal stability enhanced Termogravimetric curves of biodiesel, therefore this methodology can be used to determine biodiesel oxidation

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 39

Many advanced techniques are obtained throughout the world to obtain and characterize bi‐ odiesel. To follow all these methodologies is necessary a constant research in many different science fields as chemistry, theoretical chemistry and even physics methodologies. This

Knothe (2007) bring an important discussion about relevance of Iodine value, its associate iodine value with some others structural index as APE and BAPE and associate to several properties of biodiesel in his work; he gives examples of how different mixtures of methyl esters of the three most common unsaturated FA—oleic, linoleic, and linolenic—can achieve nearly identical IV just slightly below the value of 115. This is an important fact because if oxidative stability depends of how many times one biodiesel can resist to be oxidized, and Linolenic acid reacts more fast then linoleic and oleic acid, this mixtures should present a differing oxidation behavior independently to have same iodine Value.

One of innovative uses of Thermogravimetric analysis is based on gum formation during bi‐ odiesel storage. Figure 4 shows a impact of storage at high temperature storage (85°C) of bi‐ odiesel. High resistance of biodiesel after four weeks is probably due formation of polymeric species as trimmers or tetramers of unsaturation esters.

**Figure 8.** Formation of high molecular level species tends to increase oil viscosity

In presence of oxygen molecule polymer are forming by C-O-C linkages (Jonhson et al,1957; Wexler, 1964; Formo et al, 1979) and C-C linkages, while under an inert atmosphere only polymers with C-C linkages primordially are founded. High contents of polyunsaturated fatty acid chains enhance oxidative polymerization in fatty oils (Korus et al, 1983).Trimers and other fatty acid polymers presents higher thermal stability enhanced Termogravimetric curves of biodiesel, therefore this methodology can be used to determine biodiesel oxidation stage.

### **9. Conclusion**

technique to determine finalized biodiesel stability, under oxidative accelerated conditions, according to standard EN14112. This technique evaluated final products of thermal decom‐

The differential scanning calorimetry (DSC) monitors the difference in energy provided/ released between the sample (reagent system) and the reference system (inert) as a function of temperature when both in the system are subjected to a controlled temperature program. Changes in temperature sample are caused by rearrangements of induced phase changes, dehydration reaction, dissociation or decomposition reactions, oxidation or reduction reac‐ tion, gelatinization and other chemical reactions. DSC evaluated consumption or energy lib‐

The Pressure Differential Scanning Calorimetry (P-DSC) is a thermo analytical technique that measures the oxidative stability using a differential heat flow between sample and ref‐ erence thermocouple under variations of temperatures and pressure. This technique differs from the Rancimat for being a fast method and presents one more variable - the pressure, allowing to work at low temperatures and using a small amount of sample (Candeia, 2009). All of these methods evaluated one aspects of oxidation process. But to predict behavior or design an adequate biodiesel its necessary to associate a oxidation process with structural

Iodine Value (IV) has been used for a long time to quantify unsaturated bonds on vegetable oil and, actually, biodiesel. Iodine Value is considering mainly structural method to assess‐ ment oxidation stability. Although currently some authors agree that this method is not nec‐

Agreement with oxidation mechanism of fatty acids its very common associated presence of unsaturated with tendency of low stability, but Jain and Sharma (2010) presents weak rela‐

odiesels, its associate this discrepancy with differ technology productions or presence of

Knothe (2007) bring an important discussion about relevance of Iodine value, its associate iodine value with some others structural index as APE and BAPE and associate to several properties of biodiesel in his work; he gives examples of how different mixtures of methyl esters of the three most common unsaturated FA—oleic, linoleic, and linolenic—can achieve nearly identical IV just slightly below the value of 115. This is an important fact because if oxidative stability depends of how many times one biodiesel can resist to be oxidized, and Linolenic acid reacts more fast then linoleic and oleic acid, this mixtures should present a

One of innovative uses of Thermogravimetric analysis is based on gum formation during bi‐ odiesel storage. Figure 4 shows a impact of storage at high temperature storage (85°C) of bi‐ odiesel. High resistance of biodiesel after four weeks is probably due formation of polymeric

differing oxidation behavior independently to have same iodine Value.

species as trimmers or tetramers of unsaturation esters.

= 0,4374) between unsaturated esters content and induction period for several bi‐

position.

38 Soybean - Bio-Active Compounds

eration for determining initial reaction.

essarily the better method to evaluate stability.

properties or composition.

tionship (R2

impurities.

Many advanced techniques are obtained throughout the world to obtain and characterize bi‐ odiesel. To follow all these methodologies is necessary a constant research in many different science fields as chemistry, theoretical chemistry and even physics methodologies. This chapter contains only a simple updated tool to help to verify all the nowadays latest news, but never forget the most important tool to use is your brain, the determination and interest in the studied subject to unravel the science frontiers.

[8] Barakos, N., Pasias, S., & Papayannakos, N. (2008). Transesterification of triglycerides in high and low quality oil feeds over an HT2 hydrotalcite catalyst. *Bioresource Tech‐*

Advanced Techniques in Soybean Biodiesel http://dx.doi.org/10.5772/52990 41

[9] Batistella, L., Lerin, L. A., Brugnerotto, P., Danielli, A. J., Trentin, C. M., Popiolski, A., Treichel, H., Oliveira, J. V., & Oliveira, D. (2012). Ultrasound-assisted lipase-cata‐ lyzed transesterification of soybean oil in organic solvent system. *UltrasonicsSono‐*

[10] Candeia, R. A., Silva, M. C. D., Carvalho, Filho. J. R., Brasilino, M., Bicudo, T. C., San‐ tos, I. M. G., & Souza, A. G. (2009). Influence of soybean biodiesel content on basic

[11] Dufaure, C., Thamrin, U., & Moulongui, Z. (1999). Comparison of the thermal behav‐ iour of some fatty esters and related ethers by TGA-DTA analysis. *Thermochim. Acta,*,

[12] Berchmans, H. J., & Hirata, S. (2008). Biodiesel production from crude Jathropa Cur‐ cas L. Seed oil with a high content of free fatty acids. *Bioresour. Technol.,*, 99,

[13] Cao, W., Han, H., & Zhang, J. (2005). Preparation of biodiesel from soybean oil using

[14] El -Mashad, H. M., Zhang, R., & Avena-Bustillos, R. J. (2008). A two steps process for

[15] Encinar, J. M., González, J. F., Martínez, G., Sánchez, N., & Pardal, A. (2012). Soybean oil transesterification by the use of a microwave flow system. *Fuel*, 95, 385-393.

[16] Fan, M., Zhang, P., & , Q. (2012). Enhancement of biodiesel synthesis from soybean oil by potassium fluoride modification of a calcium magnesium oxides catalyst. *Biore‐*

[17] Formo, M. W., Jungermann, E., Noris, F., & Sonntag, N. O. (1979). *Bailey's Industrial Oil and Fat Products,*, 1(4), Daniel Swern, Editor: John Wiley and Son,, 698-711.

[18] Fukuda, H., Konda, A., & Noda, H. (2001). Bioidesel fuel production by transesterifi‐

[19] Furata, S., Matsuhasbi, H., & Arata, K. (2004). Biodiesel fuel production with solid superacid catalysis in fixed bed reactor under atmospheric pressure. *Catal. Commun*,

[20] Gomes, M. C. S., Arroyo, P. A., & Pereira, N. C. (1999). Biodiesel production from de‐ gummed soybean oil and glycerol removal using ceramic membrane. *Journal of Mem‐*

[21] Gryglewicz, S. (1999). Rapeseed oil methyl esters preparation using heterogeneous

properties of biodiesel diesel blends. *Fuel*, 88, 738-743.

supercritical methanol and co-solvent. *Fuel*, 84, 347-351.

biodiesel production from salmon oil. *Bisosys. Engin.,*, 99, 220-227.

*nol*, 99, 5037-5042.

*chemistry*, 19, 452-458.

368, 77-83.

1716-1721.

5, 721-723.

*source technology*, 104, 447-450.

*brane Science*, 378, 453-461.

cation of oils. *J. Biosc. Bioeng.,*, 92, 405-416.

catalysts. *BioresourTechnol*, 70, 249-253.

## **Author details**

Mauricio G. Fonseca1\*, Luciano N. Batista1 , Viviane F. Silva1 , Erica C. G. Pissurno1 , Thais C. Soares1 , Monique R. Jesus1 and Georgiana F. Cruz2

1 INMETRO – National Institute of Metrology, Quality and Technology, Metrological Chemistry Division Xerém, Duque de Caxias, Rio de Janeiro, Brasil

2 UENF – North Fluminense University, Engineering and exploitation of petroleum labora‐ tory, Macaé, Rio de Janeiro, Brasil

## **References**


[8] Barakos, N., Pasias, S., & Papayannakos, N. (2008). Transesterification of triglycerides in high and low quality oil feeds over an HT2 hydrotalcite catalyst. *Bioresource Tech‐ nol*, 99, 5037-5042.

chapter contains only a simple updated tool to help to verify all the nowadays latest news, but never forget the most important tool to use is your brain, the determination and interest

, Viviane F. Silva1

and Georgiana F. Cruz2

1 INMETRO – National Institute of Metrology, Quality and Technology, Metrological

2 UENF – North Fluminense University, Engineering and exploitation of petroleum labora‐

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Chemistry Division Xerém, Duque de Caxias, Rio de Janeiro, Brasil

**Author details**

40 Soybean - Bio-Active Compounds

Thais C. Soares1

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**Chapter 3**

**Facilities for Obtaining Soybean Oil in Small Plants**

In this chapter we will study the processes, procedures and types of equipment used in in‐ dustrial processing of soybeans for obtaining vegetable oil and its adequatestorage in small

Soybeans, despite its low oil content (18% to 22%) is the second most important oilseed crop of the world, after palm oil. In 2010, it represented 27.3% of vegetable oil total produced worldwide, compared with 33.7% palm oil (pulp and almond), 15.6% rapeseed oil and 8.7% sunflower oil, together are account for 85.3% of vegetable oil total produced globally [12]. The high protein content (37% to 40%) of soybean is the main feedstock in the manufacture of feed for domestic animals. Almost 70% of meal protein that makes up the animal feed

The demand for vegetable oils will grow, mainly by increased consumption / capita in emerging countries. The average annual consumption of edible oil of a citizen of a devel‐ oped country is about 50 liters, while the world average is about 20 liters / head / year. An‐ other factor that will contribute to this increase is the use as biofuel (biodiesel and H-Bio),

The soybean oil currently holds the 2nd position in the world supply of oils and fats, accord‐ ing to Oilworld. In 1990, production of oil stood at around 16.1 million tonnes, followed by palm oil with 10.8 million tons. Other vegetable oils were the significant world production of rapeseed and sunflower, both with approximately 8 million tons, and cotton and peanuts, with

> © 2013 Tavares de Andrade 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 Tavares de Andrade 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.

Ednilton Tavares de Andrade,

Roberto Guimarães Pereira,

http://dx.doi.org/10.5772/52477

**1. Introduction**

comes from soybean [7].

the new lever to consumption of vegetable oil.

plants.

Oscar Edwin Piamba Tulcan and Danielle Oliveira de Andrade

Luciana Pinto Teixeira, Ivênio Moreira da Silva,

Additional information is available at the end of the chapter

## **Facilities for Obtaining Soybean Oil in Small Plants**

Ednilton Tavares de Andrade, Luciana Pinto Teixeira, Ivênio Moreira da Silva, Roberto Guimarães Pereira, Oscar Edwin Piamba Tulcan and Danielle Oliveira de Andrade

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52477

## **1. Introduction**

In this chapter we will study the processes, procedures and types of equipment used in in‐ dustrial processing of soybeans for obtaining vegetable oil and its adequatestorage in small plants.

Soybeans, despite its low oil content (18% to 22%) is the second most important oilseed crop of the world, after palm oil. In 2010, it represented 27.3% of vegetable oil total produced worldwide, compared with 33.7% palm oil (pulp and almond), 15.6% rapeseed oil and 8.7% sunflower oil, together are account for 85.3% of vegetable oil total produced globally [12]. The high protein content (37% to 40%) of soybean is the main feedstock in the manufacture of feed for domestic animals. Almost 70% of meal protein that makes up the animal feed comes from soybean [7].

The demand for vegetable oils will grow, mainly by increased consumption / capita in emerging countries. The average annual consumption of edible oil of a citizen of a devel‐ oped country is about 50 liters, while the world average is about 20 liters / head / year. An‐ other factor that will contribute to this increase is the use as biofuel (biodiesel and H-Bio), the new lever to consumption of vegetable oil.

The soybean oil currently holds the 2nd position in the world supply of oils and fats, accord‐ ing to Oilworld. In 1990, production of oil stood at around 16.1 million tonnes, followed by palm oil with 10.8 million tons. Other vegetable oils were the significant world production of rapeseed and sunflower, both with approximately 8 million tons, and cotton and peanuts, with

approximately 4 million tons each. Although interchangeable, each one of these oils has specif‐ ic characteristics that makes it more or less appropriate depending on its final use [12].

in hygiene and microbiological control. There is no waste in the process, which may cause

Facilities for Obtaining Soybean Oil in Small Plants

http://dx.doi.org/10.5772/52477

49

Compared with the traditional processes, the main disadvantage is the high content of resid‐ ual oil in the semi-defatted meal (about 8%). Soybean oil is very unsaturated and may lead to rancidity bran under these conditions. In a small scale production, it is expected that the soy meal is consumed as it is produced, ie the time of storage would be small. Furthermore, the oil in the meal will replace the oil which is usually added in the preparation of feeds.

The second biggest problem is the allocation of oil produced. Because crude soybean oil, cannot be consumed as food without the refining process - the taste is very bad. Although oil extracted by extraction with low concentrations of phosphatides, which is equivalent to the degummed oil, its storage is not recommended for long periods. Hardly the big oil refin‐

In the case of commodities, the market promotes an intense coordination of the system, con‐ trolling the prices of commodities. As soybean production-level family does not benefit from economies of scale, the tendency is to seek new ways to add value to the product, incorpo‐ rating new features into grains. The differentiation of the grains can open prospects for more efficient production, processing or use, making common grains, marketed as mere commod‐

There are two production processes oils and fats. For materials with high oil contents (over 30%), it uses the pressing process. For raw materials with lower levels of oil, it uses the sol‐ vent extraction. In the extraction by pressing the residual oil content of the raw material is

In industrial processes, typically raw materials rich in oil is pressed up to a residual oil con‐ tent of about 20% and the remaining oil is extracted by solvent. Thus, the soybean oil (20% oil) is usually extracted only solvent as sunflower oil (45% oil) is partially removed by press‐

The oil obtained in these processes, known as crude oil, generally undergoes a purification process (refining) before being consumed as food. The only exception is the commercialscale oil (olive oil) olive oil that is consumed without refining (oil "virgin"), although other oils such as sesame, sunflower, peanut oils can be consumed raw. The soybean oil, cotton

The residues of extraction, pie, if the pressing, bran, in the case of solvent extraction, less than 20% are used for human consumption. They are generally used for the preparation of

The oil extraction process can be divided into three phases. The first involves the pre-cleani‐ ness, drying and storage of product to be processed. The second step concerns the prepara‐

around 10%, while in this extraction solvent content can be reduced to less than 1%.

ities, specializing in products with high added value and commercial [11, 10].

environmental problems.

ers will buy this product.

**3. Production Process**

ing and the remaining solvent.

animal feed.

and canola are consumed only after refined.

While the supply of vegetable oils is large, each of these oils have specific characteristics that make them more or less suitable for use as a biofuel [12]. The restriction on the use of soy for biodiesel is compared to the low oil content in their grains. The oil yield per hectare of soy‐ bean, considering an average oil content of 20% and within the grain yield per area of 400 to 800 kg in a crop that produces 2000 to 4000 kg / ha, respectively [21]. The yield of soybean is around 2.8 to 2.95 t / ha.

According to the USDA (U.S. Department of Agriculture United States) for the 2010/2011 harvest, the estimate of global soybean production was of 256.1 million tons (Table 1), down 1.46% compared to 259.89 million tons produced in 2009/2010. Likewise, the global con‐ sumption of 2010/2011 was estimated at 255.284.000 tons, an increase of 7.5% compared to 237.430.000 tons achieved in the previous crop. Still, world ending stocks of the product in 2010/2011 will be at 58.21 million tons, 3.26% below the world ending stocks of previous crop (2009/2010) of 60.17 million tons.


**Table 1.** Estimates of global soybean production for 2011/2012 harvest [30]

### **2. The Product: The Meal and Soybean Oil**

The products of the processes are simplified form the crude oil and meal (cake) semi-defat‐ ted, which can be used for the preparation of animal feed. It is planned to be used for hu‐ man consumption, since such use would imply the need for greater care and sophistication in hygiene and microbiological control. There is no waste in the process, which may cause environmental problems.

Compared with the traditional processes, the main disadvantage is the high content of resid‐ ual oil in the semi-defatted meal (about 8%). Soybean oil is very unsaturated and may lead to rancidity bran under these conditions. In a small scale production, it is expected that the soy meal is consumed as it is produced, ie the time of storage would be small. Furthermore, the oil in the meal will replace the oil which is usually added in the preparation of feeds.

The second biggest problem is the allocation of oil produced. Because crude soybean oil, cannot be consumed as food without the refining process - the taste is very bad. Although oil extracted by extraction with low concentrations of phosphatides, which is equivalent to the degummed oil, its storage is not recommended for long periods. Hardly the big oil refin‐ ers will buy this product.

In the case of commodities, the market promotes an intense coordination of the system, con‐ trolling the prices of commodities. As soybean production-level family does not benefit from economies of scale, the tendency is to seek new ways to add value to the product, incorpo‐ rating new features into grains. The differentiation of the grains can open prospects for more efficient production, processing or use, making common grains, marketed as mere commod‐ ities, specializing in products with high added value and commercial [11, 10].

## **3. Production Process**

approximately 4 million tons each. Although interchangeable, each one of these oils has specif‐

While the supply of vegetable oils is large, each of these oils have specific characteristics that make them more or less suitable for use as a biofuel [12]. The restriction on the use of soy for biodiesel is compared to the low oil content in their grains. The oil yield per hectare of soy‐ bean, considering an average oil content of 20% and within the grain yield per area of 400 to 800 kg in a crop that produces 2000 to 4000 kg / ha, respectively [21]. The yield of soybean is

According to the USDA (U.S. Department of Agriculture United States) for the 2010/2011 harvest, the estimate of global soybean production was of 256.1 million tons (Table 1), down 1.46% compared to 259.89 million tons produced in 2009/2010. Likewise, the global con‐ sumption of 2010/2011 was estimated at 255.284.000 tons, an increase of 7.5% compared to 237.430.000 tons achieved in the previous crop. Still, world ending stocks of the product in 2010/2011 will be at 58.21 million tons, 3.26% below the world ending stocks of previous

**Country Harvested Area (Million Hectares) Production (1000 MT)** United States 29.800 83.172 Brazil 25.000 68.500 Argentina 18.600 46.500 China 7.650 13.500 India 10.270 11.000 Paraguay 2.600 5.000 Canada 1.542 4.246 Russia 1.180 1.749 Ukraine 1.100 2.200 Uruguay 1.000 1.700 Bolivia 900 1.580 World Total 103.094 245.065

The products of the processes are simplified form the crude oil and meal (cake) semi-defat‐ ted, which can be used for the preparation of animal feed. It is planned to be used for hu‐ man consumption, since such use would imply the need for greater care and sophistication

ic characteristics that makes it more or less appropriate depending on its final use [12].

around 2.8 to 2.95 t / ha.

48 Soybean - Bio-Active Compounds

crop (2009/2010) of 60.17 million tons.

**Table 1.** Estimates of global soybean production for 2011/2012 harvest [30]

**2. The Product: The Meal and Soybean Oil**

There are two production processes oils and fats. For materials with high oil contents (over 30%), it uses the pressing process. For raw materials with lower levels of oil, it uses the sol‐ vent extraction. In the extraction by pressing the residual oil content of the raw material is around 10%, while in this extraction solvent content can be reduced to less than 1%.

In industrial processes, typically raw materials rich in oil is pressed up to a residual oil con‐ tent of about 20% and the remaining oil is extracted by solvent. Thus, the soybean oil (20% oil) is usually extracted only solvent as sunflower oil (45% oil) is partially removed by press‐ ing and the remaining solvent.

The oil obtained in these processes, known as crude oil, generally undergoes a purification process (refining) before being consumed as food. The only exception is the commercialscale oil (olive oil) olive oil that is consumed without refining (oil "virgin"), although other oils such as sesame, sunflower, peanut oils can be consumed raw. The soybean oil, cotton and canola are consumed only after refined.

The residues of extraction, pie, if the pressing, bran, in the case of solvent extraction, less than 20% are used for human consumption. They are generally used for the preparation of animal feed.

The oil extraction process can be divided into three phases. The first involves the pre-cleani‐ ness, drying and storage of product to be processed. The second step concerns the prepara‐ tion of the grains for the oil extraction, by facilitating the extraction processes, such as the loss of grain, conditioning or heating, lamination, and expander. Finally, the third stage in‐ volves the extraction itself, which may develop by pressing or solvent. Figure 1 details the phases and steps of the extraction process.

**3.1. Storage of raw material**

terioration processes during the storage.

**3.2. Preparation of the raw material**

the two forms of extraction.

of oxidative processes in the interior of the grains.

Considering itself it importance of all previous the productive stages the harvest, since the election of the seeds to the cultural treatments, to one adjusted storage, the grains must pass for two important stages: the first one is the daily pay-cleanness, in which all the impurities must be removed, therefore it intervenes directly with the income of the process of oil ex‐ traction, useful life of the involved machines, beyond serving as inoculate of plagues and harmful microorganisms. The second stage is the process of drying that has as objective to guarantee the reduction of the moisture content of the product, of form to minimize the de‐

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The stage of storage is essential for the maintenance of the quality of the raw material to be processed, influencing directly in the final product quality [23]. During the storage of the grains, that must be carried through in silos or specifically projected warehouses, the charac‐ teristics of temperature and moisture content of the product they must be monitored. Such characteristics are determinative in the minimization of the losses for deteriorations caused for microorganisms and attack of plagues, beyond being essential to prevent the occurrence

In what it says respect the quality of the oil during the storage, the variations in the moisture content of the product, as well as of the temperature can provide to enzymatic and oxidative reactions of the present oil in the interior of the grain, providing alterations in the character‐ istics and disposal of acid the fatty gifts. For the case of the soy grains, the oil if finds depos‐ ited in lipid bodies (the Spherosomes) distributed throughout its endosperm. Therefore, the control of such characteristics throughout all the previous stage the extraction of the oil is of basic importance for the guarantee of a quality by-product, as much in artisan scale as in industrial scale. In accordance with [23], low the quality of the crude oil influences in the

The extraction of the oil presents as by-products, beyond the involved lipid fraction with the rude oil, also proteins and carbohydrates that constitute the pie or bran, much used in the food industry, as much for animals as for human beings. Therefore, for the guarantee of the quality and not contamination of by-products, the value aggregation, and the integral ex‐ ploitation of the processed product, must be carried through the preparation of the grain the

In what the oil production says respect proceeding from the soy grain, that presents about 20% of oil, two methods extraction can be used: the solvent extraction for and the extraction for pressing. Although some authors to characterize the extraction for pressing of the soy as not being economically interesting in reason of its low oil content, indicating the extraction for solvent, in this book will be presented the preparations of the necessary raw material for

The purpose of the stage of preparation of the grain for the extraction of the oil involves to provide the increase of the susceptibility of disruption of lipid organelles, contained in en‐

increase of the losses and expenses with refining, providing a lesser income.

form to guarantee a maximum separation between the oil and the bran.

**Figure 1.** Flowchart for the production of soybean oil

#### **3.1. Storage of raw material**

tion of the grains for the oil extraction, by facilitating the extraction processes, such as the loss of grain, conditioning or heating, lamination, and expander. Finally, the third stage in‐ volves the extraction itself, which may develop by pressing or solvent. Figure 1 details the

**Reception**

**Pay-cleanness**

**Drying**

**Storage**

**Cleaning**

**Loss of grain**

**Heating**

**Lamination** 

**Expander** 

**Extraction by prenssing Solvent extraction**

**Immersion Percolation**

**Refining**

**Refined oil**

**Mechanical press**

**Filtration**

**Crude oil reservoir**

**Figure 1.** Flowchart for the production of soybean oil

phases and steps of the extraction process.

50 Soybean - Bio-Active Compounds

Considering itself it importance of all previous the productive stages the harvest, since the election of the seeds to the cultural treatments, to one adjusted storage, the grains must pass for two important stages: the first one is the daily pay-cleanness, in which all the impurities must be removed, therefore it intervenes directly with the income of the process of oil ex‐ traction, useful life of the involved machines, beyond serving as inoculate of plagues and harmful microorganisms. The second stage is the process of drying that has as objective to guarantee the reduction of the moisture content of the product, of form to minimize the de‐ terioration processes during the storage.

The stage of storage is essential for the maintenance of the quality of the raw material to be processed, influencing directly in the final product quality [23]. During the storage of the grains, that must be carried through in silos or specifically projected warehouses, the charac‐ teristics of temperature and moisture content of the product they must be monitored. Such characteristics are determinative in the minimization of the losses for deteriorations caused for microorganisms and attack of plagues, beyond being essential to prevent the occurrence of oxidative processes in the interior of the grains.

In what it says respect the quality of the oil during the storage, the variations in the moisture content of the product, as well as of the temperature can provide to enzymatic and oxidative reactions of the present oil in the interior of the grain, providing alterations in the character‐ istics and disposal of acid the fatty gifts. For the case of the soy grains, the oil if finds depos‐ ited in lipid bodies (the Spherosomes) distributed throughout its endosperm. Therefore, the control of such characteristics throughout all the previous stage the extraction of the oil is of basic importance for the guarantee of a quality by-product, as much in artisan scale as in industrial scale. In accordance with [23], low the quality of the crude oil influences in the increase of the losses and expenses with refining, providing a lesser income.

#### **3.2. Preparation of the raw material**

The extraction of the oil presents as by-products, beyond the involved lipid fraction with the rude oil, also proteins and carbohydrates that constitute the pie or bran, much used in the food industry, as much for animals as for human beings. Therefore, for the guarantee of the quality and not contamination of by-products, the value aggregation, and the integral ex‐ ploitation of the processed product, must be carried through the preparation of the grain the form to guarantee a maximum separation between the oil and the bran.

In what the oil production says respect proceeding from the soy grain, that presents about 20% of oil, two methods extraction can be used: the solvent extraction for and the extraction for pressing. Although some authors to characterize the extraction for pressing of the soy as not being economically interesting in reason of its low oil content, indicating the extraction for solvent, in this book will be presented the preparations of the necessary raw material for the two forms of extraction.

The purpose of the stage of preparation of the grain for the extraction of the oil involves to provide the increase of the susceptibility of disruption of lipid organelles, contained in en‐ dosperm, through thermal and mechanical treatments. For this, after the withdrawal of the mass of grains of the storing unit, with moisture content of approximately 10%, in wet base (b.w.), is indicated, again the separation of the impurities that still can be contained in the mass of grains.

The following stage is called of expansion. In this stage, the flakes are, again, humidified with warm water vapor and for attrition throughout screws without end that lead the mate‐ rial until a perforated plate. In reason of the difference of pressure before and after the ticket for this plate, the warm and humidified flake suffers the process from expansion. In accord‐

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The ticket of the mass of grains for the expander, or expander - extruder, propitiates greater porosity to the mass and permeability to solvent, favoring the contact between solvent and the expanded cell, guaranteeing a more efficient percolating between the oil the solvent. Ac‐ cording [23], the use of the expander implies in the increase of about 40% in the capacity of extraction for solvent. After the expansion, the material must be cooled until the tempera‐ ture of extraction of the oil, and depending on the conditions of the processing, the mass must pass for the form drying to guarantee a bigger efficiency in the extraction process.

The extraction of the oil for being carried through two different methods: the extraction me‐ chanics through the pressing, or by means of the chemical extraction for solvent. In situa‐ tions special, of form if to get the maximum efficiency of extraction it can be used the two methods sequentially. To follow, the two forms of oil extraction will be presented proceed‐

The method of extraction for pressing consists of the withdrawal of the oil by means of the application of a external pressure on the mass of grains, through the pressing mechanics. As main involved advantages with the use of the press mechanics for the oil extraction low cost of installation is its, not the use of solvent, and not the necessity of posterior refining of the oil, what it implies in the reduction of the processing cost and, consequently, of the gotten

Currently, the press more common mechanics is the continuous press of screw, also call of expeller, that hopper of feeding is composed for one, that leads the material to be pressed by means of a screw without end of step interrupted for steel rings, made use parallel; to the end of the set, they find if a cone tip that regulates the speed of exit of the pie and the cham‐

The pressing process if develops from the introduction of the mass of grains in hopper that it feeds the screw without end, compressing it against steel rings, providing the elimination of the oil for the orifices. The extraction speed depends directly on the imposed pressure, that initially must understand of 300 the 400 kg cm-2, but throughout the process due to the gradual accumulation of mass in the interior of the press, the pressure can be superior the

During the process, the mass of grains equally is pressed, preventing the resorption of the oil for other parcels of the mass. After the ticket for the press, the rude oil must be filtered

with the purpose to separate the solid residues proceeding from the remaining pie.

ance with [23], this expansion occurs in reason of the starch presence in the grain.

**3.3. The extraction**

*3.3.1. Extraction for pressing*

ber pressure on the mass.

1,000 kg cm-2 [23].

ing from oil seeds, as it is the case of the soy.

oil, favoring the use of the same one for small producers.

After this stage, must be carried through the form grain in addition to lead the unfastening and the separation of the rinds, that are abrasive, and to favor the uniformity of the size of particles to be processed. The grain in addition can be carried through by means of a breaker called equipment of coil that consists of the disposal of two corrugated cylinders, parallel made use that turns with different speeds, while the mass of grains is lead between them. During this process, for the withdrawal of the remaining rind a pneumatic aspiration is car‐ ried through, jointly with bolters splitting.

In this process of in addition the grains the milling of the grain is not indicated, since it neg‐ ative intervenes with the separation between the rind and the remaining structure of the grain, the cotyledon; beyond, for the case of the extraction for solvent, to make it difficult the separation of the solvent and the oil of the bran. Another important determinative character‐ istic in the success of this stage is the maintenance of the grains with moisture content of, approximately, 10%, in b.u., of form to prevent the embouchement of the machine for higher the moisture content cases, or the dust production for very low moisture contents.

After the grain in addition, must be carried through the baking of the processed mass of grains. This stage has as objective to provide to a fast increase of the moisture content of the mass of grains, producing a bigger plasticity to the mass, and minimizing the dust produc‐ tion. This process is presented as a facilitator to rupture of the Spherosomes cell walls, in order to facilitate the leakage oil.

In accordance with [18], the increasing of the moisture of the flakes, the cell wall disruption and subsequent increase in the permeability of cell membranes, facilitates the exit of the oil, reducing its viscosity and its surface tension, which allow agglomeration of the oil droplets and its subsequent extraction. The baking can be carried through by warm vertical cookers through chambers with warm vapor, or horizontal rotating drums for a warm tubular beam the vapor.

After this preparation of the raw material, mainly for the case of the extraction with solvent, is indicated the accomplishment of plus others two stages: the lamination and the expan‐ sion. These two stages have the objective in the distance to provide the minimization of be‐ tween solvent and the oil, favoring the extraction process.

The lamination if bases on the flake attainment from pieces of grains, with the objective to minimize the internal resistance in favor of the extraction of the oil. For the confection of flakes, the grains broken and with raised temperature more are lead, in the rolling mill, en‐ ter a pair of made use smooth cylinders horizontally that 0,3 mm jam pieces of the grains of soy in blades of 0,2, forming flakes [23]. In this process, the flake production with homoge‐ neous thickness directly is related with the efficiency of extraction of the oil and, mainly, with the quality and pureness of the produced bran, since this characteristic influences in the interaction between the oil and the solvent during the extraction.

The following stage is called of expansion. In this stage, the flakes are, again, humidified with warm water vapor and for attrition throughout screws without end that lead the mate‐ rial until a perforated plate. In reason of the difference of pressure before and after the ticket for this plate, the warm and humidified flake suffers the process from expansion. In accord‐ ance with [23], this expansion occurs in reason of the starch presence in the grain.

The ticket of the mass of grains for the expander, or expander - extruder, propitiates greater porosity to the mass and permeability to solvent, favoring the contact between solvent and the expanded cell, guaranteeing a more efficient percolating between the oil the solvent. Ac‐ cording [23], the use of the expander implies in the increase of about 40% in the capacity of extraction for solvent. After the expansion, the material must be cooled until the tempera‐ ture of extraction of the oil, and depending on the conditions of the processing, the mass must pass for the form drying to guarantee a bigger efficiency in the extraction process.

#### **3.3. The extraction**

dosperm, through thermal and mechanical treatments. For this, after the withdrawal of the mass of grains of the storing unit, with moisture content of approximately 10%, in wet base (b.w.), is indicated, again the separation of the impurities that still can be contained in the

After this stage, must be carried through the form grain in addition to lead the unfastening and the separation of the rinds, that are abrasive, and to favor the uniformity of the size of particles to be processed. The grain in addition can be carried through by means of a breaker called equipment of coil that consists of the disposal of two corrugated cylinders, parallel made use that turns with different speeds, while the mass of grains is lead between them. During this process, for the withdrawal of the remaining rind a pneumatic aspiration is car‐

In this process of in addition the grains the milling of the grain is not indicated, since it neg‐ ative intervenes with the separation between the rind and the remaining structure of the grain, the cotyledon; beyond, for the case of the extraction for solvent, to make it difficult the separation of the solvent and the oil of the bran. Another important determinative character‐ istic in the success of this stage is the maintenance of the grains with moisture content of, approximately, 10%, in b.u., of form to prevent the embouchement of the machine for higher

After the grain in addition, must be carried through the baking of the processed mass of grains. This stage has as objective to provide to a fast increase of the moisture content of the mass of grains, producing a bigger plasticity to the mass, and minimizing the dust produc‐ tion. This process is presented as a facilitator to rupture of the Spherosomes cell walls, in

In accordance with [18], the increasing of the moisture of the flakes, the cell wall disruption and subsequent increase in the permeability of cell membranes, facilitates the exit of the oil, reducing its viscosity and its surface tension, which allow agglomeration of the oil droplets and its subsequent extraction. The baking can be carried through by warm vertical cookers through chambers with warm vapor, or horizontal rotating drums for a warm tubular beam

After this preparation of the raw material, mainly for the case of the extraction with solvent, is indicated the accomplishment of plus others two stages: the lamination and the expan‐ sion. These two stages have the objective in the distance to provide the minimization of be‐

The lamination if bases on the flake attainment from pieces of grains, with the objective to minimize the internal resistance in favor of the extraction of the oil. For the confection of flakes, the grains broken and with raised temperature more are lead, in the rolling mill, en‐ ter a pair of made use smooth cylinders horizontally that 0,3 mm jam pieces of the grains of soy in blades of 0,2, forming flakes [23]. In this process, the flake production with homoge‐ neous thickness directly is related with the efficiency of extraction of the oil and, mainly, with the quality and pureness of the produced bran, since this characteristic influences in

the moisture content cases, or the dust production for very low moisture contents.

mass of grains.

52 Soybean - Bio-Active Compounds

ried through, jointly with bolters splitting.

order to facilitate the leakage oil.

tween solvent and the oil, favoring the extraction process.

the interaction between the oil and the solvent during the extraction.

the vapor.

The extraction of the oil for being carried through two different methods: the extraction me‐ chanics through the pressing, or by means of the chemical extraction for solvent. In situa‐ tions special, of form if to get the maximum efficiency of extraction it can be used the two methods sequentially. To follow, the two forms of oil extraction will be presented proceed‐ ing from oil seeds, as it is the case of the soy.

#### *3.3.1. Extraction for pressing*

The method of extraction for pressing consists of the withdrawal of the oil by means of the application of a external pressure on the mass of grains, through the pressing mechanics. As main involved advantages with the use of the press mechanics for the oil extraction low cost of installation is its, not the use of solvent, and not the necessity of posterior refining of the oil, what it implies in the reduction of the processing cost and, consequently, of the gotten oil, favoring the use of the same one for small producers.

Currently, the press more common mechanics is the continuous press of screw, also call of expeller, that hopper of feeding is composed for one, that leads the material to be pressed by means of a screw without end of step interrupted for steel rings, made use parallel; to the end of the set, they find if a cone tip that regulates the speed of exit of the pie and the cham‐ ber pressure on the mass.

The pressing process if develops from the introduction of the mass of grains in hopper that it feeds the screw without end, compressing it against steel rings, providing the elimination of the oil for the orifices. The extraction speed depends directly on the imposed pressure, that initially must understand of 300 the 400 kg cm-2, but throughout the process due to the gradual accumulation of mass in the interior of the press, the pressure can be superior the 1,000 kg cm-2 [23].

During the process, the mass of grains equally is pressed, preventing the resorption of the oil for other parcels of the mass. After the ticket for the press, the rude oil must be filtered with the purpose to separate the solid residues proceeding from the remaining pie.

#### *3.3.2. Extraction for solvent*

The oil extraction for solvent can be used as only method of extraction, or same as comple‐ ment to the extraction mechanics, in the daily pay-extraction form. This method of extrac‐ tion if bases on the absorption of the solvent for the lipid cells, where in its interior it has the dissolution of the oil, that, later, for leaching, he is loaded for the exterior of the cell. This process, in adjusted conditions, approximately removes 99% of the oil contained in the mass of grains [23].

curity in the industrial plant and with its manuscript, seen its high inflammability, explosion

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The refining if characterizes as the set of operations carried through after the process of ex‐ traction for the removal of residues gifts in the crude oil that can affect the color, stability, aroma, and flavor, beyond its physical characteristics. These residues are preceding from drag mechanics, and/or solubilization of other substances in the oil or solvent the occurrenc‐

The refining takes place in two stages: the first step is the physical removal of substances, while the second involves the refining processes through neutralization, clarification, and deodorization. In accordance with [31], initially the oil passes for a stage of physical separa‐ tion, where, in a tank, the separation for gravity of insoluble substances is carried through. After this stage, the oil passes for the degumming that consists of the removal of phospholi‐

In accordance with [23], the presence of phospholipids in the soy oil favors the occurrence of losses, in reason of the formation of depositions with presence of about 35% of oil in the deep one the tanks of deposition, with aspect similar to a gum. In this in case that, the phos‐ pholipids can be presented of two forms: hydratable, being liable of withdrawal with addi‐ tion of water and centrifugation, and not hydratable that needs the addition of acid citric

Still of according to [31], after the degumming process, must be proceeded with the neutrali‐ zation, that the withdrawal of fatty acid involves, pigments, remaining phospholipids of the degumming, and soluble sulfur composites in water. In the neutralization process it has the caustic soda water addition, with fatty acid the purpose to reduce the text of free, to clot the phosphatides and the gums, to degrade part of the dyes gifts, and to load the insoluble sub‐ stance for the clotted material. Of this process it has the production of you leave organic so‐

The laundering is a process that results in the withdrawal of the soap produced during the neutralization. It is based on the *distilled* water addition the temperature of 85-95°C (about 10-20% of the volume of oil) for the elimination of the soda water and the foam of the oil, however case the separation between the oil and the water is difficult indicates it dilution in the aluminum sulfate water. The laundering process can more than proceed a time until the total exemption from soap from the oil. To finish the process laundering, in reason of the

After the drying, the oil is directed to the *clarification* (*bleaching)* that objective the pigment elimination, residual products of the oxidation, metals weighed, and soaps, of form to guar‐ antee the improvement of the flavor, odor and oxidative stability of the oil. Bleaching occurs from the adsorption of specific adsorbents substances for the elimination of substances that

dium or soaps, what it results in the necessity of one another stage, the *laundering*.

pids, sugars, resins, and breaks up of soluble proteins in water.

phosphoric or for becomes it hydratable for its posterior withdrawal.

water addition, the drying must be proceeded from the oil.

and toxicity [23].

**4. Refining**

es during the extraction process.

However, for the guarantee of the efficiency of the process, it is essential the adjusted prepa‐ ration of the grain and the choice of to be used extractor, of form to guarantee the maximum contact of the solvent with the cellular wall. Of this form, how much bigger it is the amount of cells breached throughout the preparation of the mass of grains, faster it is the extraction process, since the solvent will only go to dissolve the free oil, not needing to carry for diffu‐ sion the dissolved oil to the external region to the cell.

In accordance with [23], the transport of lipid throughout the cellular membrane occurs in function of the variation of its permeability (initially impermeable to the lipid) in function of the difference of the internal and external osmotic pressures to the cells. The increase of in‐ tracellular pressure in virtue of the action of the solvent it provides the expansion of the membrane and, consequently, the dilatation of the pores of the cellular membrane, allowing the ticket of the solvent oil solution and for the extracellular region, which had to the gradi‐ ent of existing concentration [23, 26].

The extraction process occurs in higher temperatures, seen its influence in the viscosity of the solvent oil mixture and, and in the solubilization of the oil in the solvent. The extraction speeds from beginning to end of the process of solvent extraction progresses differently. Ini‐ tially, when the oil of better quality is extracted, the process if develops of fast form, due to the biggest gradient of concentration, however, throughout the process this speed diminish‐ es, and the extracted oil to the end presents minor quality, in reason, mainly, of the presence of other cellular composites that provide losses throughout the refining.

From this process, the oil extraction for solvent can be developed of two forms, for immer‐ sion, where the mass of grains is kept immersed in the solvent for a definitive period of time, or by percolating, which the mass of grains is made use in layers to guarantee opti‐ mum contact of the solvent that it passes freely between the grains and it is renewed when it has saturation. To the end of the extraction, as much the solvent mixed to the oil, as the sol‐ vent gift next to the remaining bran can be recouped and be reintroduced in the process. For the grains to solvent extraction with low concentrations of oil is indicated by immersing the system [2].

For the extraction of the oil, frequent the commercial hexane is used as solvent. Proceeding from the refining of the oil, the hexane presents determinative characteristics for its good performance as solvent, as: it is a composition to apolar total being miscible in oil at the same time that it is immiscible in water, presents low latent heat of boiling, and it does not react with the constituent material of the equipment used for the extraction. Although its fa‐ vorable characteristics physicist-chemistries, this solvent requires special care with the se‐ curity in the industrial plant and with its manuscript, seen its high inflammability, explosion and toxicity [23].

## **4. Refining**

*3.3.2. Extraction for solvent*

54 Soybean - Bio-Active Compounds

sion the dissolved oil to the external region to the cell.

ent of existing concentration [23, 26].

of grains [23].

system [2].

The oil extraction for solvent can be used as only method of extraction, or same as comple‐ ment to the extraction mechanics, in the daily pay-extraction form. This method of extrac‐ tion if bases on the absorption of the solvent for the lipid cells, where in its interior it has the dissolution of the oil, that, later, for leaching, he is loaded for the exterior of the cell. This process, in adjusted conditions, approximately removes 99% of the oil contained in the mass

However, for the guarantee of the efficiency of the process, it is essential the adjusted prepa‐ ration of the grain and the choice of to be used extractor, of form to guarantee the maximum contact of the solvent with the cellular wall. Of this form, how much bigger it is the amount of cells breached throughout the preparation of the mass of grains, faster it is the extraction process, since the solvent will only go to dissolve the free oil, not needing to carry for diffu‐

In accordance with [23], the transport of lipid throughout the cellular membrane occurs in function of the variation of its permeability (initially impermeable to the lipid) in function of the difference of the internal and external osmotic pressures to the cells. The increase of in‐ tracellular pressure in virtue of the action of the solvent it provides the expansion of the membrane and, consequently, the dilatation of the pores of the cellular membrane, allowing the ticket of the solvent oil solution and for the extracellular region, which had to the gradi‐

The extraction process occurs in higher temperatures, seen its influence in the viscosity of the solvent oil mixture and, and in the solubilization of the oil in the solvent. The extraction speeds from beginning to end of the process of solvent extraction progresses differently. Ini‐ tially, when the oil of better quality is extracted, the process if develops of fast form, due to the biggest gradient of concentration, however, throughout the process this speed diminish‐ es, and the extracted oil to the end presents minor quality, in reason, mainly, of the presence

From this process, the oil extraction for solvent can be developed of two forms, for immer‐ sion, where the mass of grains is kept immersed in the solvent for a definitive period of time, or by percolating, which the mass of grains is made use in layers to guarantee opti‐ mum contact of the solvent that it passes freely between the grains and it is renewed when it has saturation. To the end of the extraction, as much the solvent mixed to the oil, as the sol‐ vent gift next to the remaining bran can be recouped and be reintroduced in the process. For the grains to solvent extraction with low concentrations of oil is indicated by immersing the

For the extraction of the oil, frequent the commercial hexane is used as solvent. Proceeding from the refining of the oil, the hexane presents determinative characteristics for its good performance as solvent, as: it is a composition to apolar total being miscible in oil at the same time that it is immiscible in water, presents low latent heat of boiling, and it does not react with the constituent material of the equipment used for the extraction. Although its fa‐ vorable characteristics physicist-chemistries, this solvent requires special care with the se‐

of other cellular composites that provide losses throughout the refining.

The refining if characterizes as the set of operations carried through after the process of ex‐ traction for the removal of residues gifts in the crude oil that can affect the color, stability, aroma, and flavor, beyond its physical characteristics. These residues are preceding from drag mechanics, and/or solubilization of other substances in the oil or solvent the occurrenc‐ es during the extraction process.

The refining takes place in two stages: the first step is the physical removal of substances, while the second involves the refining processes through neutralization, clarification, and deodorization. In accordance with [31], initially the oil passes for a stage of physical separa‐ tion, where, in a tank, the separation for gravity of insoluble substances is carried through. After this stage, the oil passes for the degumming that consists of the removal of phospholi‐ pids, sugars, resins, and breaks up of soluble proteins in water.

In accordance with [23], the presence of phospholipids in the soy oil favors the occurrence of losses, in reason of the formation of depositions with presence of about 35% of oil in the deep one the tanks of deposition, with aspect similar to a gum. In this in case that, the phos‐ pholipids can be presented of two forms: hydratable, being liable of withdrawal with addi‐ tion of water and centrifugation, and not hydratable that needs the addition of acid citric phosphoric or for becomes it hydratable for its posterior withdrawal.

Still of according to [31], after the degumming process, must be proceeded with the neutrali‐ zation, that the withdrawal of fatty acid involves, pigments, remaining phospholipids of the degumming, and soluble sulfur composites in water. In the neutralization process it has the caustic soda water addition, with fatty acid the purpose to reduce the text of free, to clot the phosphatides and the gums, to degrade part of the dyes gifts, and to load the insoluble sub‐ stance for the clotted material. Of this process it has the production of you leave organic so‐ dium or soaps, what it results in the necessity of one another stage, the *laundering*.

The laundering is a process that results in the withdrawal of the soap produced during the neutralization. It is based on the *distilled* water addition the temperature of 85-95°C (about 10-20% of the volume of oil) for the elimination of the soda water and the foam of the oil, however case the separation between the oil and the water is difficult indicates it dilution in the aluminum sulfate water. The laundering process can more than proceed a time until the total exemption from soap from the oil. To finish the process laundering, in reason of the water addition, the drying must be proceeded from the oil.

After the drying, the oil is directed to the *clarification* (*bleaching)* that objective the pigment elimination, residual products of the oxidation, metals weighed, and soaps, of form to guar‐ antee the improvement of the flavor, odor and oxidative stability of the oil. Bleaching occurs from the adsorption of specific adsorbents substances for the elimination of substances that confer coloration, sulfur, soaps and metals to the soy oil. The adsorbents substances can be some types of silicates, diatoms lands, clays acid activated, silica and active coal [23].

storage and processing of the beans, as has been seen above, as well as the characteristics,

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Overall, the changes and loss of quality of the agricultural products are related with the growth and activity of microorganisms, the action of enzymes, chemical reactions, attack by insects and rodents, and physical changes caused by mechanical agents. According [17], the major causes of food deterioration are, respectively, the attack of microorganisms and oxida‐

The vegetable oils have fewer characteristics in your reactive molecule, unlike proteins and carbohydrates presents in the grains. The reactivity of the oil is concentrated, mostly, in the hydrolysis that produces free fatty acids according to the moisture content present, lipolytic enzymes and temperature, and the oxidation reaction of lipid compound that is a function

The hydrolysis process provides the breakdown of triglycerides and therefore the increase of free fatty acids, that influences the acidity of the vegetable oil. High levels of acidity in vegetable oils leads to high losses during the refining stage [23]. However, in general, for to monitor the deterioration of the vegetable oils is accomplished due to the oxidative reactions

12:00 14:00 16:00 18:00 18:01 18:02 18:03 22:01 IV

Canola 0 0 4.5 1.5 58.5 25 9 1.5 118 Corn 0 0 11 2 36 50 1 121 Sunflower 0 6 4.5 32 57.5 126 Soybean 0 8 4 28 53 7 130

According [24], all vegetable oils consist primarily of triglycerides. The triglycerides have a three-carbon backbone with a long hydrocarbon chain attached to each of the carbons. These chains are attached through an oxygen atom and a carbonyl carbon, which is a carbon atom that is double-bonded to second oxygen. The differences between oils from different sources relate to the length of the fatty acid chains attached to the backbone and the number of car‐ bon–carbon double bonds on the chain. Most fatty acid chains from plant based oils are 18 carbons long with between zero and three double bonds. Fatty acid chains without double bonds are said to be saturated and those with double bonds are unsaturated (Misra and

In general, vegetable oils are made especially for fatty acids with chains between 12 and 24 carbons: Lauric (C12:0), Myristic (C14:0), Palmitic (C16:0); Palmitoleic (C16:1) Stearic (C18:0), Oleic (C18:1), Linoleic (C18:2); Linolenic (C18:3); Arachidic (C20:0); Gadoleic (C20:1); Behen‐ ic (C22:0), Erucic (C22:1); Lignoceric (C24:0). The proportions of the fatty acid composition

extraction and treating the produced oil, and form of storage and shipping.

Oils **Fatty acids composition (Wt.-%)**

tive processes.

of the concentration of oxygen in the medium.

occurred in the triglyceride molecule.

**Table 2.** Vegetable oils fatty acid composition

Murthy, 2010).

In accordance with [14], throughout the refining, the clarification produces an oil with big‐ ger susceptibility to the oxidation, being indicated for the storage the use of absent nitrogen stream bed of oxygen.

After the clarification, the oil follows for its last stage of refining, the deodorization. The de‐ odorization of characterizes for the substance elimination formed during the storage and processing of the grain and other natural substances of the oil that can provide to awkward flavor and odor. For the withdrawal of these substances to use it distillation with chain of vapor to the vacuum, of form to guarantee the protection of the oil to the effect of the atmos‐ pheric oxidation, prevention of hydrolysis for the vapor, and the reduction of the necessary amount of vapor for the process.

For some types of oils, as it is the case of the palm oil and peanut, the physical refining is pro‐ ceeded after the accomplishment from the deodorization. The physical refining consists of the elimination for evaporation of fatty acid the free gifts in the oil, providing bigger income of the fine oil. For the case of the soy oil, the physical refining is not indicated, in reason of its low acidity after to the end of the process and the difficulty in the degumming process.

## **5. Quality**

Every product has a number of characteristic attributes. It's called quality, whose existence will define the success or failure in their marketing. This quality is mainly observed by two fundamental aspects: the first relates to the consumer who seeks desirable characteristics, whether from an economic standpoint, nutritional, aesthetic, etc. The second aspect refers to the legality, where the product goes through a series laboratory analysis and is classified in‐ to pre-established standards and its final quality is attested.

According [13], quality can be defined as a set of characteristics that will directly impact on the acceptability of the product. In this context, the subjectivity of the sensory analysis of each person are determinants for to preset of the quality, that can involve the appearance, texture, flavor and aroma.

The color, size, shape, integrity and consistency are factors directly involved in the appear‐ ance of the product, already the relateds with the physical senses of touch, and mouth are determined on the according to texture; the flavor factors involve the taste, the aroma, and correlate with the olfactory sensitivity. Besides these, you can also to relate the after taste that occurs according to a secondary analysis of the product [13].

Associated with these organoleptics factors for the acceptance of the product are also the nu‐ tritional value, the presence of toxic substances and the final price. All these characteristics are to related to the type of raw material and production method. In the case of vegetable oils, with is the case of the soybean oil, these factors are directly related to the conditions of storage and processing of the beans, as has been seen above, as well as the characteristics, extraction and treating the produced oil, and form of storage and shipping.

Overall, the changes and loss of quality of the agricultural products are related with the growth and activity of microorganisms, the action of enzymes, chemical reactions, attack by insects and rodents, and physical changes caused by mechanical agents. According [17], the major causes of food deterioration are, respectively, the attack of microorganisms and oxida‐ tive processes.

The vegetable oils have fewer characteristics in your reactive molecule, unlike proteins and carbohydrates presents in the grains. The reactivity of the oil is concentrated, mostly, in the hydrolysis that produces free fatty acids according to the moisture content present, lipolytic enzymes and temperature, and the oxidation reaction of lipid compound that is a function of the concentration of oxygen in the medium.

The hydrolysis process provides the breakdown of triglycerides and therefore the increase of free fatty acids, that influences the acidity of the vegetable oil. High levels of acidity in vegetable oils leads to high losses during the refining stage [23]. However, in general, for to monitor the deterioration of the vegetable oils is accomplished due to the oxidative reactions occurred in the triglyceride molecule.


**Table 2.** Vegetable oils fatty acid composition

confer coloration, sulfur, soaps and metals to the soy oil. The adsorbents substances can be

In accordance with [14], throughout the refining, the clarification produces an oil with big‐ ger susceptibility to the oxidation, being indicated for the storage the use of absent nitrogen

After the clarification, the oil follows for its last stage of refining, the deodorization. The de‐ odorization of characterizes for the substance elimination formed during the storage and processing of the grain and other natural substances of the oil that can provide to awkward flavor and odor. For the withdrawal of these substances to use it distillation with chain of vapor to the vacuum, of form to guarantee the protection of the oil to the effect of the atmos‐ pheric oxidation, prevention of hydrolysis for the vapor, and the reduction of the necessary

For some types of oils, as it is the case of the palm oil and peanut, the physical refining is pro‐ ceeded after the accomplishment from the deodorization. The physical refining consists of the elimination for evaporation of fatty acid the free gifts in the oil, providing bigger income of the fine oil. For the case of the soy oil, the physical refining is not indicated, in reason of its low

Every product has a number of characteristic attributes. It's called quality, whose existence will define the success or failure in their marketing. This quality is mainly observed by two fundamental aspects: the first relates to the consumer who seeks desirable characteristics, whether from an economic standpoint, nutritional, aesthetic, etc. The second aspect refers to the legality, where the product goes through a series laboratory analysis and is classified in‐

According [13], quality can be defined as a set of characteristics that will directly impact on the acceptability of the product. In this context, the subjectivity of the sensory analysis of each person are determinants for to preset of the quality, that can involve the appearance,

The color, size, shape, integrity and consistency are factors directly involved in the appear‐ ance of the product, already the relateds with the physical senses of touch, and mouth are determined on the according to texture; the flavor factors involve the taste, the aroma, and correlate with the olfactory sensitivity. Besides these, you can also to relate the after taste

Associated with these organoleptics factors for the acceptance of the product are also the nu‐ tritional value, the presence of toxic substances and the final price. All these characteristics are to related to the type of raw material and production method. In the case of vegetable oils, with is the case of the soybean oil, these factors are directly related to the conditions of

acidity after to the end of the process and the difficulty in the degumming process.

to pre-established standards and its final quality is attested.

that occurs according to a secondary analysis of the product [13].

some types of silicates, diatoms lands, clays acid activated, silica and active coal [23].

stream bed of oxygen.

56 Soybean - Bio-Active Compounds

**5. Quality**

texture, flavor and aroma.

amount of vapor for the process.

According [24], all vegetable oils consist primarily of triglycerides. The triglycerides have a three-carbon backbone with a long hydrocarbon chain attached to each of the carbons. These chains are attached through an oxygen atom and a carbonyl carbon, which is a carbon atom that is double-bonded to second oxygen. The differences between oils from different sources relate to the length of the fatty acid chains attached to the backbone and the number of car‐ bon–carbon double bonds on the chain. Most fatty acid chains from plant based oils are 18 carbons long with between zero and three double bonds. Fatty acid chains without double bonds are said to be saturated and those with double bonds are unsaturated (Misra and Murthy, 2010).

In general, vegetable oils are made especially for fatty acids with chains between 12 and 24 carbons: Lauric (C12:0), Myristic (C14:0), Palmitic (C16:0); Palmitoleic (C16:1) Stearic (C18:0), Oleic (C18:1), Linoleic (C18:2); Linolenic (C18:3); Arachidic (C20:0); Gadoleic (C20:1); Behen‐ ic (C22:0), Erucic (C22:1); Lignoceric (C24:0). The proportions of the fatty acid composition can be determined by gas chromatography method. The weight composition of fatty acids found in soybean oil was: C16:0=11.6%, C16:1=0.1%, C18:0=32%, C18:1=20.4%; C18:2=57.1% and C18:3=5% [24].

Average values of the chemical composition of vegetable oils and values of iodine index are presented in the Table 2 [15] and physical properties of soybean oil in Table 3. The iodine In‐ dex or Number is one characteristic of the oil that measures its index of unsaturation, when evaluating the amount of iodine necessary to saturate the double links of the molecule.


**Figure 2.** Density in temperatures of 15 and 20 ºC

**Table 4.** Corrosivity of vegetable oils at temperature of 100 ºC

diesel has a density of around 0.865 g.cm-3 (Figure 2).

**Table 5.** Kinematic viscosity values of vegetable oils obtained experimentally

shown.

**Vegetable Oil Corrosivity** Canola 1a Sunflower 1a Corn 1a Soybean 1b

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Looking at Table 5, 6 and 7, canola oil has the lowest density of 0.917 g.cm-3 at a temperature of 15 °C and 0.916 g.cm-3 at a temperature of 20 °C and soybean oil has the highest density, 0.922 g.cm-3 at a temperature of 15 °C and sunflower and corn oil showed a higher density at a temperature of 20 °C, 0.919 g.cm-3. According to the specifications of the ABNT standard,

In Table 3 the mean values and standard deviation of the viscosity of vegetable oils are

**Vegetable Oil Kinematic Viscosity (mm2.s-1) Standard deviation**

Canola 35.5278 0.0081

Sunflower 31.7275 0.0726

Corn 33.7713 0.0409

Soybean 31.6107 0.0093

The test results of kinematic viscosity of vegetable oils are shown in Figure 3 below:

#### **Table 3.** Physical properties of soybean oil [24]

Both the hydrolysis reaction as the oxidation reaction can also be influenced by temperature, light, presence of unsaturated fatty acids, moisture content, and product type. As a result of these reactions is the development of different flavors and odors that compromise the quali‐ ty and acceptability of the product. For these changes organoleptics give the name of rancid‐ ity. Addition, for the production of biodiesel, the presence of free fatty acids in vegetable oils affect the process of separation of soaps, and influence on the reactions of esterification, compromising the efficiency of the process.

Thus, in that it involves, specifically, the oxidation of lipids, the main causative factors are the composition of fats, the presence of oxygen, temperature, and luminosity. The composi‐ tion of fats influence on the presence of unsaturation in the molecule, as the unsaturation increases the susceptibility of oxygen absorption, that the more present in the environment, more available for the reactions is presented. Both the temperature and luminosity influ‐ ence, proportionally, in the rates of reactions. For the specific case of light, its presence influ‐ ences on accelerating the development of rancidity in fats.

According [8] and [27], the quality of the oil can be followed depending on the index of acid determined by the presence of free fatty acids; index of saponification which demonstrates the presence of oils or fats high proportion fatty acids, color; foaming, viscosity, density, and index of peroxide that is determined by the presence of iodine. According to [24], the Table 3 shows the properties of the soybean oil.

It is shown in Table 4 that vegetable oils present corrosion within the pattern established for diesel oil (Standard corrosion = 1) according to ANP.

**Figure 2.** Density in temperatures of 15 and 20 ºC

can be determined by gas chromatography method. The weight composition of fatty acids found in soybean oil was: C16:0=11.6%, C16:1=0.1%, C18:0=32%, C18:1=20.4%; C18:2=57.1%

Average values of the chemical composition of vegetable oils and values of iodine index are presented in the Table 2 [15] and physical properties of soybean oil in Table 3. The iodine In‐ dex or Number is one characteristic of the oil that measures its index of unsaturation, when

Both the hydrolysis reaction as the oxidation reaction can also be influenced by temperature, light, presence of unsaturated fatty acids, moisture content, and product type. As a result of these reactions is the development of different flavors and odors that compromise the quali‐ ty and acceptability of the product. For these changes organoleptics give the name of rancid‐ ity. Addition, for the production of biodiesel, the presence of free fatty acids in vegetable oils affect the process of separation of soaps, and influence on the reactions of esterification,

Thus, in that it involves, specifically, the oxidation of lipids, the main causative factors are the composition of fats, the presence of oxygen, temperature, and luminosity. The composi‐ tion of fats influence on the presence of unsaturation in the molecule, as the unsaturation increases the susceptibility of oxygen absorption, that the more present in the environment, more available for the reactions is presented. Both the temperature and luminosity influ‐ ence, proportionally, in the rates of reactions. For the specific case of light, its presence influ‐

According [8] and [27], the quality of the oil can be followed depending on the index of acid determined by the presence of free fatty acids; index of saponification which demonstrates the presence of oils or fats high proportion fatty acids, color; foaming, viscosity, density, and index of peroxide that is determined by the presence of iodine. According to [24], the Table 3

It is shown in Table 4 that vegetable oils present corrosion within the pattern established for

**PROPERTY SOYBEAN OIL**

evaluating the amount of iodine necessary to saturate the double links of the molecule.

Density kg/L (20 °C) 0.92037 Viscosity mm2/s (40 °C) 30.787 Flash Point °C 332 Cloud Point °C -2 Pour Point °C -14 Copper strip corrosion 1b

and C18:3=5% [24].

58 Soybean - Bio-Active Compounds

**Table 3.** Physical properties of soybean oil [24]

compromising the efficiency of the process.

shows the properties of the soybean oil.

ences on accelerating the development of rancidity in fats.

diesel oil (Standard corrosion = 1) according to ANP.


**Table 4.** Corrosivity of vegetable oils at temperature of 100 ºC

Looking at Table 5, 6 and 7, canola oil has the lowest density of 0.917 g.cm-3 at a temperature of 15 °C and 0.916 g.cm-3 at a temperature of 20 °C and soybean oil has the highest density, 0.922 g.cm-3 at a temperature of 15 °C and sunflower and corn oil showed a higher density at a temperature of 20 °C, 0.919 g.cm-3. According to the specifications of the ABNT standard, diesel has a density of around 0.865 g.cm-3 (Figure 2).

In Table 3 the mean values and standard deviation of the viscosity of vegetable oils are shown.


**Table 5.** Kinematic viscosity values of vegetable oils obtained experimentally

The test results of kinematic viscosity of vegetable oils are shown in Figure 3 below:

Generally the oil extracted is sent to the food industry, or the production of biofuels, and in some cases is used as feedstock in other industrial processes. Soybean oil has its applications in the first two cases cited. [16] agribusiness of the soybean oil has a high processing capaci‐

Canola 0.917 0.0005 0.916 0.0005 1a 35.5278 0.0081 -1 ºC -20 ºC Sunflower 0.921 0.0010 0.919 0.0005 1a 31.7275 0.0726 1 ºC -18 ºC Corn 0.919 0.0005 0.919 0.0005 1a 33.7713 0.0409 3 ºC -12.5 ºC Soybean 0.922 0.0000 0.920 0.0006 1b 31.6107 0.0093 -2 ºC -18.5 ºC

It is known specific attributes which must be met after defining the destination of oil extract‐ ed, not all characteristics of a product can be easily evaluated. In the case of agri-food prod‐ ucts there is the existence of personal or subjective parameters that hinder your judgment. Thus, the oil-producing units paying attention to product quality, this will help to stay in

**a.** Quality of product design: that is the result of the activities that translate the knowledge of market needs and opportunities in information technology for the production. **b.** Quality of design process: is results from the efficient translation of the design specifica‐ tions of the product in process at various levels, such as process flow diagram, layout,

**d.** Quality of services associated with the product: it is the result of the quality level of the

[16] reported that the limit of the concept of quality has advanced in segments related to the management of the productive sector. It is clear that the companies' survival now depends on the quality of products or services it offered. Several agents have sought to monitor qual‐ ity through product recalls, allowing verification of consignments dispatched by identifying the weaknesses of the production complex, such as inefficiency in logistics and handling

The production of vegetable oil in small units can be designed more efficiently by under‐ standing the concept of agro-industrial chain, because according to [20] in recent years the so-called industrialization of agriculture has led to increasing dependence on agriculture to industry. Thus small farmers can participate in a competitive market where strategic alli‐

**c.** Quality of conformation: is the result of the efficiency level of actual production.

stages of distribution and marketing, beyond quality of after-sales services.

[3] reported that the quality of a product is resulting from the joining of items:

**Corrosion**

**Kinematic Viscosity (mm2.s-1)**

**Standard**

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**deviation Cloud point**

**Flow point** 61

**Standard deviation**

ty, with oil production exceeding 50 million liters per year.

**Density 20°C**

**Standard deviation**

**Table 7.** Physical characterization of vegetable oils [29, 1]

the market and seeing new opportunities.

conditions and storage of the product.

design tools and equipment, project work etc..

**Vegetable oil Density**

**15°C**

**Figure 3.** Kinematic viscosity of vegetable oils

Table 6 show the temperatures measured in the tests of cloud.


**Table 6.** Temperatures of cloud points and flow points of vegetable oils

Vegetable oils have a density slightly larger than that of diesel fuel and below water, they are products of easy handling and processing. Their viscosities are far from being similar to diesel fuel, but this allows their use in cases of thermo-chemical conversion without major problems, and are easy to carry through pipelines without large deformation of tension and energy.

**Figure 4.** Results obtained experimentally for cloud point and flow point of vegetable oils

### **6. Control the Production Management of Soybean Oil**

The acquisition process of vegetable oils occurs through of relatively simple procedures, starting with the attainment of oilseeds, proceeding to mechanical extraction, filtering and marketing of oil. This understanding contributes to the study of the entire production chain. Generally the oil extracted is sent to the food industry, or the production of biofuels, and in some cases is used as feedstock in other industrial processes. Soybean oil has its applications in the first two cases cited. [16] agribusiness of the soybean oil has a high processing capaci‐ ty, with oil production exceeding 50 million liters per year.


**Table 7.** Physical characterization of vegetable oils [29, 1]

**Figure 3.** Kinematic viscosity of vegetable oils

60 Soybean - Bio-Active Compounds

Table 6 show the temperatures measured in the tests of cloud.

**Table 6.** Temperatures of cloud points and flow points of vegetable oils

**Figure 4.** Results obtained experimentally for cloud point and flow point of vegetable oils

**6. Control the Production Management of Soybean Oil**

**Vegetable Oil Temperatures of Cloud Point Temperatures of Flow Point**

Canola -1 ºC -20 ºC Sunflower 1 ºC -18 ºC Corn 3 ºC -12.5 ºC Soybean -2 ºC -18.5 ºC

Vegetable oils have a density slightly larger than that of diesel fuel and below water, they are products of easy handling and processing. Their viscosities are far from being similar to diesel fuel, but this allows their use in cases of thermo-chemical conversion without major problems, and are easy to carry through pipelines without large deformation of tension and energy.

The acquisition process of vegetable oils occurs through of relatively simple procedures, starting with the attainment of oilseeds, proceeding to mechanical extraction, filtering and marketing of oil. This understanding contributes to the study of the entire production chain.

It is known specific attributes which must be met after defining the destination of oil extract‐ ed, not all characteristics of a product can be easily evaluated. In the case of agri-food prod‐ ucts there is the existence of personal or subjective parameters that hinder your judgment. Thus, the oil-producing units paying attention to product quality, this will help to stay in the market and seeing new opportunities.

[3] reported that the quality of a product is resulting from the joining of items:


[16] reported that the limit of the concept of quality has advanced in segments related to the management of the productive sector. It is clear that the companies' survival now depends on the quality of products or services it offered. Several agents have sought to monitor qual‐ ity through product recalls, allowing verification of consignments dispatched by identifying the weaknesses of the production complex, such as inefficiency in logistics and handling conditions and storage of the product.

The production of vegetable oil in small units can be designed more efficiently by under‐ standing the concept of agro-industrial chain, because according to [20] in recent years the so-called industrialization of agriculture has led to increasing dependence on agriculture to industry. Thus small farmers can participate in a competitive market where strategic alli‐ ances allow to produce and market their products. Given this perception, competitiveness is to migrate the level of individual firms to competition between agribusiness chains. Thus, because it is an integrated system there is a need to coordinate the supply chain, and the search for quality is now undertaking the various agents.

**6.1. Methodologies applied to quality management**

**•** Do: It means the implementation of procedures;

ed searching to standardize the rules for products and industries.

in accordance with the requirements and policies of the organization;

the policies, objectives and product requirements and report the results; **•** Action: It means to take actions to continually improve process performance.

The most common steps for the implementation of ISO 9001 are:

tion of vegetable oils in small units.

*6.1.1. Application of ISO 9000*

Guidelines for SGQs auditing.

**•** Conviction of top management

**•** Assessment of current situation

**•** Establishment of working groups

**•** Training of internal auditors **•** Performing internal audits

**•** Development / deployment documentation

**•** Preparation of schedule

**•** Training leveling

**•** Specific training **•** Process Mapping

**•** Corrective Actions

**•** Training end

**•** Choice of coordinating implementation

ing [19], in which:

Methodologies will be presented that can be applied to quality management in the produc‐

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63

The International Organization for Standadization is responsible for standardization in glob‐ al character, is the ISO 9000 group linked the quality management systems, which was creat‐

In addition to ISO 9000, regarding the standards focused on vocabulary and fundamentals, there is the importance of ISO 9001 that dealing with Quality Management Systems and

A process model of ISO 9001 may be seen as an implementation of the PDCA cycle, accord‐

**•** PLan: It means setting objectives and processes necessary to deliver products or services

**•** Check: It means to monitor and measure processes and products by comparing them to

According [3] the coordination of quality is a set of activities planned and controlled by a coordinator agent, in order to improve quality management in the chain and ensure product quality through a process of transaction information, helping to improve the matching of customers and to reduce costs and losses at all stages of the chain.

This concept of quality coordination, "to plan", "controlling" and "improve" the quality man‐ agement, "the process of information transaction" and "coordinating agent" are:


The coordinator agent can be a company, a group of companies, an association of represen‐ tatives of the segments of the agribusiness chain or a third party contracted to perform the tasks for the agent coordinated.

The coordination process quality in agro-industrial chain of soybean oil, produced in small units, can be strengthened by adopting measures to encourage the involvement of its agents. Usually the resources of small producers to cover production are limited, requiring there‐ fore of a reliable outlook for the sale of its production. At this point, there is the need of pro‐ ductive sector strategies are aligned with unusual strategies for the development of the productive chain.

The literature dedicated to the study of agribusiness systems suggests practices to be im‐ plemented from the producer to the customer. Measures such as discounted prices, flexi‐ bility in time and remuneration for services, contribute to the strengthening of alliances among its members.

The strategies that comprise the agribusiness chain agents, shall consider agribusiness prod‐ ucts are subject to special features such as seasonality of production, the need for special conditions for transport and storage and other care as they are perishable. The following paragraphs deal with aspects related to the quality of agro-industrial products, particularly vegetable oils.

#### **6.1. Methodologies applied to quality management**

Methodologies will be presented that can be applied to quality management in the produc‐ tion of vegetable oils in small units.

#### *6.1.1. Application of ISO 9000*

ances allow to produce and market their products. Given this perception, competitiveness is to migrate the level of individual firms to competition between agribusiness chains. Thus, because it is an integrated system there is a need to coordinate the supply chain, and the

According [3] the coordination of quality is a set of activities planned and controlled by a coordinator agent, in order to improve quality management in the chain and ensure product quality through a process of transaction information, helping to improve the matching of

This concept of quality coordination, "to plan", "controlling" and "improve" the quality man‐

**•** Planning: Planning activities in order to create a process capable of producing products

**•** Control: Control processes and activities with the objective of evaluating the performance

**•** Transition process of information: the acquisition, management and distribution of infor‐

**•** Coordinating agent: a key to the coordination of quality aims to make information related to product quality and quality management are identified, communicated and controlled

The coordinator agent can be a company, a group of companies, an association of represen‐ tatives of the segments of the agribusiness chain or a third party contracted to perform the

The coordination process quality in agro-industrial chain of soybean oil, produced in small units, can be strengthened by adopting measures to encourage the involvement of its agents. Usually the resources of small producers to cover production are limited, requiring there‐ fore of a reliable outlook for the sale of its production. At this point, there is the need of pro‐ ductive sector strategies are aligned with unusual strategies for the development of the

The literature dedicated to the study of agribusiness systems suggests practices to be im‐ plemented from the producer to the customer. Measures such as discounted prices, flexi‐ bility in time and remuneration for services, contribute to the strengthening of alliances

The strategies that comprise the agribusiness chain agents, shall consider agribusiness prod‐ ucts are subject to special features such as seasonality of production, the need for special conditions for transport and storage and other care as they are perishable. The following paragraphs deal with aspects related to the quality of agro-industrial products, particularly

**•** Improve: set of activities that aims to improve the quality of processes and products;

agement, "the process of information transaction" and "coordinating agent" are:

search for quality is now undertaking the various agents.

of real quality and act if there is a diversion;

mation throughout the production chain;

that meet consumers;

62 Soybean - Bio-Active Compounds

throughout the chain.

productive chain.

among its members.

vegetable oils.

tasks for the agent coordinated.

customers and to reduce costs and losses at all stages of the chain.

The International Organization for Standadization is responsible for standardization in glob‐ al character, is the ISO 9000 group linked the quality management systems, which was creat‐ ed searching to standardize the rules for products and industries.

In addition to ISO 9000, regarding the standards focused on vocabulary and fundamentals, there is the importance of ISO 9001 that dealing with Quality Management Systems and Guidelines for SGQs auditing.

A process model of ISO 9001 may be seen as an implementation of the PDCA cycle, accord‐ ing [19], in which:


The most common steps for the implementation of ISO 9001 are:


### **6.2. Hazard Analysis and Critical Control Points (HACCP)**

According [4] the HACCP is based in the analysis method of the mode and effects and fault causes or failure, mode and effect analysis (FMEA), developed by Kaoru Ishikawa in the Japanese industry. The HACCP allows to identify and assess hazards associated with differ‐ ent stages of the food chain, and define the necessary means for its control. The HACCP should be considered as a quality system, a rational practice, organized and systematic, indi‐ cated to provide the necessary confidence that a agri-food product will meet the health and safety requirements expected by the consumer.

**6.4. Good Manufacturing Practices**

**•** Ordinance no. MS 1.428/1993

**6.5. Cleaner Production: CP**

**•** Ordinance no. 368/1997 of the MAP

material and generate less pollution.

Environmental Education.

**•** Ordinance no. 326/1997 of the MS / SVS **•** Resolution RDC / ANVISA no. 275/2002

Such standards can be applied in the production of oil intended for human consumption. Procedures are designed to get quality products. The Good Manufacturing Practices (GMP), according to Tomich et al. (2005), are a set of standards used in products, processes, services

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According to [6] requires that each establishment has its Manual of Good Manufacturing Practices, which details the conditions of hygiene and sanitary food handling procedures, cleaning equipment, utensils, facilities and buildings of establishments, in addition to estab‐ lishment of minimum health requirements of buildings, facilities, equipment and tools, con‐ trol of water supply, health and hygiene of food handlers, the integrated control of pests and

The following legislation provides for the establishment of Good Manufacturing Practices (GMP), in Brazil, whereas other related items: Standards of Identity and Quality, sanitary in‐

The methodology of CP is the application of an economic strategy, environmental and tech‐ nical, integrated processes and products in order to increase the efficiency of use of raw ma‐ terials, water and energy, not the generation, waste minimization and recycling with

To have success in the development of cleaner production, are required to exercise responsi‐ ble environmental management and technology assessment, the initial effort is fundamental

All factors imply aggregate values and services, with the primary concern of consuming less

[25] studied the management of agro-industrial residues. As a result it was proposed a road‐ map for Environmental Management System (EMS). Operational actions were suggested as: Mass Balance identifying and quantifying infrastructure resources; Anticipation and moni‐ toring the adoption of measures to prevent accidents or damage to the ecological nature of the life cycle analysis of products, Cleaner Production (CP) and Reverse Logistics, which is to collect, package, transport and dispose of waste that were generated in the activities of obtaining Raw Material and managerial actions suggesting adoption of sustainable strat‐ egies and policies, Environmental Management System as a mechanism to monitor the ad‐ ministrative and managerial performance the organization, Environmental Audit and

and buildings, for the promotion and certification of quality and food safety.

vectors, and control and quality assurance of final products.

spection, Standard Operating Procedures and Checklist of GMP.

environmental and economic benefits to the productive processes.

in changing attitudes, including a subjective evaluation of the entire process.

Studies based on the concept of prevention, developed by [5], concluded that the HACCP represents an advance in food safety, when the adoption of preventive measures promoted the effective design of food safety and the processes in which, a priori, was analyzed quality (microbiological, physico-chemical and sensory) of the products already processed.

#### **6.3. Traceability**

At first glance, it is believed to be unnecessary to use tools that require high technological standard in small units of oil extraction. However, products offered in different ways may present a promising alternative for small farmers. Traceability aims to identify the origin of the product from the farm to the consumer. This principle is indifferent to processes in which the product has undergone. Thus, it is expected to reach the final with a quality prod‐ uct, and with a known source.

To [9] an efficient screening process should consist of the following elements:

Standards and / or the quality that aim to protect / secure;

Procedures allowed, prohibited, tolerated and required;

Grace periods or transition established as provided in the rules;

Requirement that producers are provided with proof of purchase, sales, everything that al‐ lows inspectors to check compliance of standards by the operator (owner of the process);

Periodic visits to the default setting;

Visits "surprise" the establishment.

According to [19] in soybean production, traceability is an essential tool for identification and separation of genetically modified organisms that are restricted in certain countries or markets. Thus, traceability systems are necessary for the management and development of the agribusiness chain, ensuring quality food offered.

#### **6.4. Good Manufacturing Practices**

**•** Pre-certification audit, and

64 Soybean - Bio-Active Compounds

**6.3. Traceability**

uct, and with a known source.

Periodic visits to the default setting;

Visits "surprise" the establishment.

**•** Setting and maintaining the quality management system ISO 9001

According [4] the HACCP is based in the analysis method of the mode and effects and fault causes or failure, mode and effect analysis (FMEA), developed by Kaoru Ishikawa in the Japanese industry. The HACCP allows to identify and assess hazards associated with differ‐ ent stages of the food chain, and define the necessary means for its control. The HACCP should be considered as a quality system, a rational practice, organized and systematic, indi‐ cated to provide the necessary confidence that a agri-food product will meet the health and

Studies based on the concept of prevention, developed by [5], concluded that the HACCP represents an advance in food safety, when the adoption of preventive measures promoted the effective design of food safety and the processes in which, a priori, was analyzed quality

At first glance, it is believed to be unnecessary to use tools that require high technological standard in small units of oil extraction. However, products offered in different ways may present a promising alternative for small farmers. Traceability aims to identify the origin of the product from the farm to the consumer. This principle is indifferent to processes in which the product has undergone. Thus, it is expected to reach the final with a quality prod‐

Requirement that producers are provided with proof of purchase, sales, everything that al‐ lows inspectors to check compliance of standards by the operator (owner of the process);

According to [19] in soybean production, traceability is an essential tool for identification and separation of genetically modified organisms that are restricted in certain countries or markets. Thus, traceability systems are necessary for the management and development of

(microbiological, physico-chemical and sensory) of the products already processed.

To [9] an efficient screening process should consist of the following elements:

Standards and / or the quality that aim to protect / secure;

Procedures allowed, prohibited, tolerated and required;

the agribusiness chain, ensuring quality food offered.

Grace periods or transition established as provided in the rules;

**6.2. Hazard Analysis and Critical Control Points (HACCP)**

safety requirements expected by the consumer.

Such standards can be applied in the production of oil intended for human consumption. Procedures are designed to get quality products. The Good Manufacturing Practices (GMP), according to Tomich et al. (2005), are a set of standards used in products, processes, services and buildings, for the promotion and certification of quality and food safety.

According to [6] requires that each establishment has its Manual of Good Manufacturing Practices, which details the conditions of hygiene and sanitary food handling procedures, cleaning equipment, utensils, facilities and buildings of establishments, in addition to estab‐ lishment of minimum health requirements of buildings, facilities, equipment and tools, con‐ trol of water supply, health and hygiene of food handlers, the integrated control of pests and vectors, and control and quality assurance of final products.

The following legislation provides for the establishment of Good Manufacturing Practices (GMP), in Brazil, whereas other related items: Standards of Identity and Quality, sanitary in‐ spection, Standard Operating Procedures and Checklist of GMP.


#### **6.5. Cleaner Production: CP**

The methodology of CP is the application of an economic strategy, environmental and tech‐ nical, integrated processes and products in order to increase the efficiency of use of raw ma‐ terials, water and energy, not the generation, waste minimization and recycling with environmental and economic benefits to the productive processes.

To have success in the development of cleaner production, are required to exercise responsi‐ ble environmental management and technology assessment, the initial effort is fundamental in changing attitudes, including a subjective evaluation of the entire process.

All factors imply aggregate values and services, with the primary concern of consuming less material and generate less pollution.

[25] studied the management of agro-industrial residues. As a result it was proposed a road‐ map for Environmental Management System (EMS). Operational actions were suggested as: Mass Balance identifying and quantifying infrastructure resources; Anticipation and moni‐ toring the adoption of measures to prevent accidents or damage to the ecological nature of the life cycle analysis of products, Cleaner Production (CP) and Reverse Logistics, which is to collect, package, transport and dispose of waste that were generated in the activities of obtaining Raw Material and managerial actions suggesting adoption of sustainable strat‐ egies and policies, Environmental Management System as a mechanism to monitor the ad‐ ministrative and managerial performance the organization, Environmental Audit and Environmental Education.

### **7. From Control to Management**

Currently, the concern with quality extends beyond the aspects mentioned above, no longer a mere bureaucratic requirement of regulatory agencies and inspection, but a fundamental strategy and essential to ensure competitiveness. The quality is replaced by a much broader approach, involving all levels of the organization and process.

**10. Installations and Equipments**

ing a site to be deployed to agribusiness:

sion.

eration until the standard is met.

**Figure 5.** Seed cooker

The following relate to the main points that should be taken into consideration when choos‐

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67

**•** the potential for obtaining the raw material in the region should be higher than the pro‐

**•** availability of sufficient area to implement the agricultural industry and its future expan‐

All new property before it began, it must seek approval of their facilities by the regional body of the Labor Ministry or agency responsible, and this after doing a preview, issue a certificate of approval for facilities. This procedure is adopted in order to ensure that the new establishment activities free of accident hazards and / or occupational diseases, which is why the establishment does not meet regulations will be subject to the impediment of its op‐

jected demand of the plant and enable future expansion in production;

**•** water supply reliable and good quality (drinking water);

**•** providing sufficient electrical power without interruption;

**•** availability of skilled labor, including technical personnel;

**•** no contaminants of any kind on the outskirts of agribusiness;

**•** road infrastructure in working condition and easily accessible;

## **8. Quality attributes**

The main attributes that describe the desirable characteristics in grain for the quality of the bran are high in protein concentration, profile and level of amino acids, especially lysine, and high energy. To control the meal must consider factors such as moisture content, oil content, protein content, the urea activity and the rate of protein digestibility. And in the case of crude oil, emphasizes the determination of fatty acids and their state of rancidity.

These attributes directly influence the quality and safety of the product and the forms of controls are given in the text. It should be laid down the acceptable quality level (EQS) according to the intended use of the product. Obviously, the level of residual bran oil obtained by pressing has maximum and minimum other than the one extract‐ ed by solvent. The determination of the EQS should consider the characteristics of the process to optimize your control.

This item will be established quality standards obtained for soybean oil for immediate and latent analysis.

#### **9. Process Control**

As animal feed, the control of raw materials and products may be carried out with simpler and less frequently. The analyzes have suggested the purpose of controlling the quality of raw materials, products and the process yield. The quality and characteristics of the raw ma‐ terial has great influence on product quality and yield. Soybeans should be free of mold, broken grains, greens and other defects.

During the extrusion process, there is an internal friction of grain against the internal ele‐ ments of the extruder rising to temperature and pressure with the complete inactivation of the activities: ureatics, antitrypsin and hemoglutinant (anti-nutritional factors). Exposure of the grain at high temperature and pressure for an extremely short time (20-40 seconds) fa‐ vors the obtaining a quality product without compromising the nutritional quality of soy‐ bean. Therefore, factors such as temperature and pressure during the process should be monitored and recorded continuously. In annex presents tables to assist in process control. Below we suggest some analysis should be performed periodically.

## **10. Installations and Equipments**

**7. From Control to Management**

**8. Quality attributes**

66 Soybean - Bio-Active Compounds

process to optimize your control.

broken grains, greens and other defects.

latent analysis.

**9. Process Control**

approach, involving all levels of the organization and process.

Currently, the concern with quality extends beyond the aspects mentioned above, no longer a mere bureaucratic requirement of regulatory agencies and inspection, but a fundamental strategy and essential to ensure competitiveness. The quality is replaced by a much broader

The main attributes that describe the desirable characteristics in grain for the quality of the bran are high in protein concentration, profile and level of amino acids, especially lysine, and high energy. To control the meal must consider factors such as moisture content, oil content, protein content, the urea activity and the rate of protein digestibility. And in the case of crude oil, emphasizes the determination of fatty acids and their state of rancidity.

These attributes directly influence the quality and safety of the product and the forms of controls are given in the text. It should be laid down the acceptable quality level (EQS) according to the intended use of the product. Obviously, the level of residual bran oil obtained by pressing has maximum and minimum other than the one extract‐ ed by solvent. The determination of the EQS should consider the characteristics of the

This item will be established quality standards obtained for soybean oil for immediate and

As animal feed, the control of raw materials and products may be carried out with simpler and less frequently. The analyzes have suggested the purpose of controlling the quality of raw materials, products and the process yield. The quality and characteristics of the raw ma‐ terial has great influence on product quality and yield. Soybeans should be free of mold,

During the extrusion process, there is an internal friction of grain against the internal ele‐ ments of the extruder rising to temperature and pressure with the complete inactivation of the activities: ureatics, antitrypsin and hemoglutinant (anti-nutritional factors). Exposure of the grain at high temperature and pressure for an extremely short time (20-40 seconds) fa‐ vors the obtaining a quality product without compromising the nutritional quality of soy‐ bean. Therefore, factors such as temperature and pressure during the process should be monitored and recorded continuously. In annex presents tables to assist in process control.

Below we suggest some analysis should be performed periodically.

The following relate to the main points that should be taken into consideration when choos‐ ing a site to be deployed to agribusiness:


All new property before it began, it must seek approval of their facilities by the regional body of the Labor Ministry or agency responsible, and this after doing a preview, issue a certificate of approval for facilities. This procedure is adopted in order to ensure that the new establishment activities free of accident hazards and / or occupational diseases, which is why the establishment does not meet regulations will be subject to the impediment of its op‐ eration until the standard is met.

**Figure 5.** Seed cooker

**Figure 6.** Crusher seeds

**Figure 9.** Small plant for the production of biodiesel

**Figure 10.** Settling tank to the glycerin

A unit of oil extraction on the farm can be mounted in a simplified form with the elements described below. Figure 5 shows a seed cooker being installed, its use may be waived in work with seeds whose oil extraction is done cold, as in the case of sunflower seeds. The crusher shown in Figure 6 can be used to prepare larger seed. Equipment such as the press

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**Figure 7.** Press small

**Figure 8.** Filter with collect the oil bucket

**Figure 9.** Small plant for the production of biodiesel

**Figure 6.** Crusher seeds

68 Soybean - Bio-Active Compounds

**Figure 7.** Press small

**Figure 8.** Filter with collect the oil bucket

A unit of oil extraction on the farm can be mounted in a simplified form with the elements described below. Figure 5 shows a seed cooker being installed, its use may be waived in work with seeds whose oil extraction is done cold, as in the case of sunflower seeds. The crusher shown in Figure 6 can be used to prepare larger seed. Equipment such as the press shown in Figure 7 can perform pressing in an average yield of 40 kg per hour and is suitable for small applications. The filtration must be done efficiently preventing the oil is lost, thus a device for filtering, with collect bucket oil is of great importance in the process (Figure 8). A small plant, for production of oil and biodiesel, you can compose the oil processing unit (Figure 9). Glycerin resulting product from the transesterification of vegetable oils may be separated by centrifugation or decantation. In the second case, the composition of glycerin biodiesel more tanks can be conducted as shown in Figure 10, which should preferably have conical structure inside facilitating the decanting of the glycerine.

3 Federal Fluminense University/TER/PGMEC, Brazil

4 National University of Colombia, Bogota, Colombia

em Indústria de Laticínios de Curitiba- PR.

op\_page=27&cod\_pai=31, Accessed: 26 March.

op\_page=25&cod\_pai=29, Accessed: 26 March.

faostat.fao.org/site/339/default.aspx, Accessed: 26 March.

*Natureza(ISPN).Brasília- DF*, 43.

*cultural Engineering*, 4.

*Saraiva*, 464, cap. 2.

*aiva*, 464, cap. 7.

[1] Andrade, D. O., Tulcan, O. E. P., Andrade, E. T., & Pereira, R. G. (2008). Determina‐ tion of the physical characterization of vegetable oil. *International Conference of Agri‐*

Facilities for Obtaining Soybean Oil in Small Plants

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71

[3] Borrás, Miguel. Ângelo, & Toledo, José. Carlos. (2006). A Coordenação de Cadeias Agroindustriais: Garantindo a Qualidade e Competitividade no Agronegócio. *Zuin, Luiz Fernando Soares; Queiroz, Timóteo Ramos. Agronegócios: Gestão e Inovação. São Paulo:*

[4] Borrás, Miguel. Ângelo, & Toledo, José. Carlos. (2006). Qualidade dos Produtos Agroalimentares: A Importância da Gestão da Qualidade no Agronegócio. *Zuin, Luiz Fernando Soares; Queiroz, Timóteo Ramos. Agronegócios: Gestão e Inovação. São Paulo: Sar‐*

[5] Brum, Jaime. Victor Ferreira. (2004). Análise de Perigos e Pontos Críticos de Controle

[6] Carrazza, Luis. Roberto, Maciel, Luis. Gustavo, Borges, Moacir. Chaves, Cordeiro, Augusto. César Rodrigues., & Ávila, João. Carlos Cruz. (2011). Caderno de Normas Fiscais, Sanitárias e Ambientais Regularização de Agroindústrias Comunitárias de Produtos de Uso Sustentável da Biodiversidade. *SPN- Instituto Sociedade, População e*

[7] Dall´, Agnol. A., Lazarotto, J. J., & Hirakuri, M. H. (2010). Desenvolvimento, Mercado e Rentabilidade da Soja Brasileira. *Circular Técnico 74. Embrapa Soja*, April, 18.

[8] Dobarganes, M. C. (2000). Frying fats: quality control. In: IUPAC workshop on fats, oils and oilseed analysis. *Rio de Janeiro. EMBRAPA. Book of conferences*, 29-45.

[9] Dulley, Richard. Domingues, & Toledo, Alessandra. A. Gayoso Franco. de. (2003). Rastreabilidade dos Produtos Agrícolas. *Informações econômicas, SP*, 33(3), mar. [10] Embrapa Soja. (2012b). Uso industrial. http://www.cnpso.embrapa.br/index.php?

[11] Embrapa Soja. (2012a). Uso. http://www.cnpso.embrapa.br/index.php?

[12] FAO (Food and Agricultural Organization of the United Nations). (2012). http://

[2] Bockisch, M. (1993). Fats and oils handbook. *Champaign, AOCS*, 345-718.

**References**

## **11. Conclusion**

In conclusion we have with the expansion of soy agribusiness, there is a need to add value to the product. And in case of small farmers or family farmers in the production of oil will allow your own farm profit by adding value, besides the possibility of using this product on the farm.

The values of cloud points, close to zero degrees Celsius, and flow, near minus fifteen de‐ grees, makes manipulation of these oils possible in tropical climates, where temperatures barely reach those levels, do not show stoppage problems, flow and clogging the lines of the process involved with the use of oil.

Tests for corrosion highlights low levels of attack in copper sheets, and oil is a product that does not generate large losses of material in the areas of the process which they have contact with, on the other hand the formation of clogging points and false surfaces as a result of pol‐ ymerization of oil when working at higher temperatures can be more dangerous.

The physical and chemical characteristics evaluated in this work present a great relation with the composition of the tested oils, where there is a relation between the density, viscos‐ ity and corrosion index with the iodine index of the fluid. On the other hand, the cloud and flow points are related with the presence of saturated and unsaturated fatty acids. In respect to the saturation of the oil, it can be favorable in problems such as corrosion, of low temper‐ atures or in the case where lower viscosity values are needed.

## **Author details**

Ednilton Tavares de Andrade1 , Luciana Pinto Teixeira3 , Ivênio Moreira da Silva3 , Roberto Guimarães Pereira2 , Oscar Edwin Piamba Tulcan4 and Danielle Oliveira de Andrade3

1 Federal Fluminense University/TER/PGEB/PGMEC, Brazil

2 Federal Fluminense University/TEM/PGMEC/PGEB/MSG, Brazil

#### 3 Federal Fluminense University/TER/PGMEC, Brazil

#### 4 National University of Colombia, Bogota, Colombia

### **References**

shown in Figure 7 can perform pressing in an average yield of 40 kg per hour and is suitable for small applications. The filtration must be done efficiently preventing the oil is lost, thus a device for filtering, with collect bucket oil is of great importance in the process (Figure 8). A small plant, for production of oil and biodiesel, you can compose the oil processing unit (Figure 9). Glycerin resulting product from the transesterification of vegetable oils may be separated by centrifugation or decantation. In the second case, the composition of glycerin biodiesel more tanks can be conducted as shown in Figure 10, which should preferably have

In conclusion we have with the expansion of soy agribusiness, there is a need to add value to the product. And in case of small farmers or family farmers in the production of oil will allow your own farm profit by adding value, besides the possibility of using this product on

The values of cloud points, close to zero degrees Celsius, and flow, near minus fifteen de‐ grees, makes manipulation of these oils possible in tropical climates, where temperatures barely reach those levels, do not show stoppage problems, flow and clogging the lines of the

Tests for corrosion highlights low levels of attack in copper sheets, and oil is a product that does not generate large losses of material in the areas of the process which they have contact with, on the other hand the formation of clogging points and false surfaces as a result of pol‐

The physical and chemical characteristics evaluated in this work present a great relation with the composition of the tested oils, where there is a relation between the density, viscos‐ ity and corrosion index with the iodine index of the fluid. On the other hand, the cloud and flow points are related with the presence of saturated and unsaturated fatty acids. In respect to the saturation of the oil, it can be favorable in problems such as corrosion, of low temper‐

ymerization of oil when working at higher temperatures can be more dangerous.

, Luciana Pinto Teixeira3

, Oscar Edwin Piamba Tulcan4

, Ivênio Moreira da Silva3

and

,

atures or in the case where lower viscosity values are needed.

1 Federal Fluminense University/TER/PGEB/PGMEC, Brazil

2 Federal Fluminense University/TEM/PGMEC/PGEB/MSG, Brazil

conical structure inside facilitating the decanting of the glycerine.

**11. Conclusion**

70 Soybean - Bio-Active Compounds

**Author details**

Ednilton Tavares de Andrade1

Danielle Oliveira de Andrade3

Roberto Guimarães Pereira2

process involved with the use of oil.

the farm.


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[28] Shahidi, F., & Wanasundara, U. N. (1998). Omega-3 fatty acid concentrates: Nutri‐

[29] Tulcan, O. E. P., Andrade, D. O., Andrade, E. T., & Pereira, R. G. (2008). Analisys of physical characteristics of vegetable oil. *International Conference of Agricultural Engi‐*

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*neering*, 4.


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[14] Hui, Y. H. (1996). Baley's industrial oil and fats products. *5. Ed. Nova York, Wiley*, 4.

[15] Knothe, G., Dunn, R., & Bagby, M. O. (2003). Biodiesel: The Use of Vegetable Oils and Their Derivatives as Alternative Diesel Fuels. *Fuels and Chemicals from Biomass.*

[16] Lima, Suzana. Maria Valle., & Castro, Antonio. Maria Gomes. de. (2010). A Agroin‐ dústria de Óleo Vegetal para a Produção de Biodiesel. *Castro, Antonio Maria Gomes de; LIMA, Suzana Maria Valle; SILVA, João Flávio Veloso. Complexo Agroindutrial de Biodiesel no Brasil: Competitividade das Cadeias Produtivas de Matérias-Primas. Brasília, DF: Embra‐*

[17] Lindley, M. G. (1998). The impact of food processing on antioxidants in vegetable oil,

[18] Mandarino, José. Marcos Gontijo., & Roessing, Antônio. Carlos. (2001). Tecnologia para produção do óleo de soja: descrição das etapas, equipamentos, produtos e sub‐

[19] Martins, Roberto. Antônio. (2009). Gestão da Qualidade Agroindustrial. *BATALHA,*

[20] Mendes, Judas. Tadeu Grassi., & Padilha, Junior. João Batista. (2007). Agronegócio:

[21] Moraes, R. M. A. (2007). Potencial da soja na produção de biodiesel. http://www.ciso‐

[22] Moraes, R. M. A. (2007). Potencial da soja na produção de biodiesel.. http:// www.cisoja.com.br/index.php?p=artigo&idA=1, Accessed: 26 March 2012.

[23] Oetterer, M., Regitano-D'Arce, M. A. B., & Spoto, M. H. F. (2006). Fundamentos de

[24] Pereira, R. G. ., Tulcan, O. P. ., Lameira, V. J. ., Espirito, Santo., Filho, D. M. ., & An‐ drade, E. T. (2011). Use of Soybean Oil in Energy Generation. *Dora Krezhova. (Org.). Recent Trends for Enhancing the Diversity and Quality of Soybean Products. Recent Trends for Enhancing the Diversity and Quality of Soybean Products. Rijeka: InTech*, 01, 301-320.

[25] Schenini, Pedro. Carlos. (2011). Gerenciamento de Resíduos da Agroindustria. *II Sim‐ pósio Internacional sobre Gerenciamento de Resíduos Agropecuários e Agroindustriais- II SI‐*

[26] Schneider, F. H. (1980). Zur extraktiven lipid-freisetzung aus pflanzlichen zellen.

*Mário Otávio. Gestão Agroindustrial. GEPAI. 3ª Ed. São Paulo: Atlas*, cap. 8.

fruits and vegetables. *Trend in Food Science and Technology*, 9.

Uma abordagem Econômica. *São Paulo: Pearson Prentice Hall*.

Ciência e Tecnologia de Alimentos. *Barueri, SP: Manole*, 612.

*GERA.15 a 17 de março de- Foz do Iguaçu, PR, Volume I- Palestras*.

*Fette Seifen Antstrichmittel, Hamburgo*, 82(1), 16-23.

ja.com.br/index.php?p=artigo&idA=1, Accessed: 26 March. 2012.

e aplicações. *São Paulo: Nobel*.

72 Soybean - Bio-Active Compounds

*pa Agroenergia*, cap. 6.

*Washington, D.C.: American Chemical Society*.

produtos. *Londrina: Embrapa Soja*, 40.


**Chapter 4**

**Phytoestrogens and Colon Cancer**

B. Pampaloni, C. Mavilia, E. Bartolini, F. Tonelli,

Additional information is available at the end of the chapter

Colorectal carcinoma (CRC) represents the most frequent malignancy of the gastrointestinal tract in the Western world in both genders. There is a wide variation of incidence rate for both colonic and rectal cancer among the populations of different countries: up to a 30-40 fold difference is seen between North America (Canada, Los Angeles, San Francisco), New-Zealand (non–Maori), Northern Italy (Trieste), Northern France (Haut- and Bas-Rhin) in which the rate of CRC is around 50/100,000 inhabitants, and India (Madras, Bangalore, Tri‐ vadrum, Barshi, Paranda, Bhum, Karunagappally), Algeria (Setif), and Mali (Bamako) in which the rate is around 3/100,000 [1]. It is estimated that approximately 6% of the United States population will eventually develop a CRC, and that 6 million of American citizens

The geographic differences in CRC incidence are due more to environment, life-style, and diet than to racial or ethnic factors. Demonstration of this fact is that migrants from low to high incidence areas have the same incidence as the host country within one generation,

Colonoscopy to screen asymptomatic adults older than 50 years allows an estimation of the prevalence of adenomatous polyps or CRC: in North America CRC is found in 2%, and ad‐

Population-based studies have investigated several environmental factors as contributors to the initiation of sporadic colorectal carcinogenesis. High-calorie diet, high red meat con‐ sumption, overcooked red meat consumption, high saturated fat consumption, excess alco‐ hol consumption, cigarette smoking, sedentary lifestyle, and obesity are considered to increase the incidence of CRC, while consumption of fiber, fresh fruit and vegetables, and a high-calcium diet could have a protective effect [5]. A recent review [6] provided an over‐

> © 2013 Pampaloni 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 Pampaloni 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.

M.L. Brandi and Federica D'Asta

http://dx.doi.org/10.5772/54065

who are living will die of CRC [2].

having assimilate western lifestyle and diet [3].

vanced adenoma (more than 1 cm in diameter) in 10% [4[.

**1. Introduction**

## **Chapter 4**

## **Phytoestrogens and Colon Cancer**

B. Pampaloni, C. Mavilia, E. Bartolini, F. Tonelli, M.L. Brandi and Federica D'Asta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54065

## **1. Introduction**

Colorectal carcinoma (CRC) represents the most frequent malignancy of the gastrointestinal tract in the Western world in both genders. There is a wide variation of incidence rate for both colonic and rectal cancer among the populations of different countries: up to a 30-40 fold difference is seen between North America (Canada, Los Angeles, San Francisco), New-Zealand (non–Maori), Northern Italy (Trieste), Northern France (Haut- and Bas-Rhin) in which the rate of CRC is around 50/100,000 inhabitants, and India (Madras, Bangalore, Tri‐ vadrum, Barshi, Paranda, Bhum, Karunagappally), Algeria (Setif), and Mali (Bamako) in which the rate is around 3/100,000 [1]. It is estimated that approximately 6% of the United States population will eventually develop a CRC, and that 6 million of American citizens who are living will die of CRC [2].

The geographic differences in CRC incidence are due more to environment, life-style, and diet than to racial or ethnic factors. Demonstration of this fact is that migrants from low to high incidence areas have the same incidence as the host country within one generation, having assimilate western lifestyle and diet [3].

Colonoscopy to screen asymptomatic adults older than 50 years allows an estimation of the prevalence of adenomatous polyps or CRC: in North America CRC is found in 2%, and ad‐ vanced adenoma (more than 1 cm in diameter) in 10% [4[.

Population-based studies have investigated several environmental factors as contributors to the initiation of sporadic colorectal carcinogenesis. High-calorie diet, high red meat con‐ sumption, overcooked red meat consumption, high saturated fat consumption, excess alco‐ hol consumption, cigarette smoking, sedentary lifestyle, and obesity are considered to increase the incidence of CRC, while consumption of fiber, fresh fruit and vegetables, and a high-calcium diet could have a protective effect [5]. A recent review [6] provided an over‐

view of the epidemiological evidence supporting the roles of diet, lifestyle, and medication in reducing the risk of colorectal cancer. Similarly, many studies that implicate effects of di‐ etary agents in various types of cancers are available and suggest that much of the suffering and death from cancer could be prevented by consuming a healthy diet, reducing tobacco use, performing regular physical activity, and maintaining an optimal body weight [7]. Even if several epidemiological and experimental studies support the role of these factors in the genesis of CRC, other well-designed prospective and randomized clinical trials conducted in recent years report conflicting evidence, in particular on the role of the diet component in the etiology of CRC [8, 9].

crypt foci, and followed by the formation of an adenomatous polyp. These pathological features are considered the precursor of the carcinoma in a temporal sequence that also can be completed in several years. However, CRC is not a homogenous disease: several histological types can be distinguished such as tubular or villous, mucinous, serrated, medullary, signet-ring, squamous cell, adenosquamous, small cell, and undifferentiated, and different molecular basis can also be recognized in histologically similar tumors. In recent years, the identification of the genetic mutations of hereditary forms of CRC has clarified two fundamentals types of carcinogenesis. The first is similar to that described for the development of the FAP, and is characterized by a progressive accumulation of genetic changes starting from a biallelic inactivation of *APC*. Additional mutations either of oncogenes *KRAS* and *p53* or of oncosuppressor genes (*DCC* and *DPC4*) are necessary for the neoplastic progression and invasivity [12]. The genetic alterations are responsible for an increased mucosal proliferation and a reduced apoptosis, causing a clonal cellular expansion. The second, similar to the CRC arising in the HNPCC, is due to inactivation of *MLH1* or of other *MMR* genes. Repetitive sequences of DNA, sited in non-encoding microsatellite regions throughout the genome, are specifically found in this type of CRC, hence, the definition of micro satellite instability (MSI). The mechanism responsible for the carcinogenesis is epigenetic due to an extensive DNA methylation. Rarely in this type of CRC both proto-oncogenes (*KRAS, p53*) and oncosuppressor genes (*APC, TGFBRII, IGF2R, BAX*) are mutated or inactivated [13]. The former genetic mechanism explains the most frequent form of sporadic CRC characterized by the sequence adeno‐ ma-carcinoma and a long period for the formation of cancer; vice versa, the last mecha‐ nism is only present in 15% of sporadic CRC, and can have the character of an

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 77

Improved knowledge of the molecular mechanisms of colorectal carcinogenesis allows a rationale chemopreventive use in individuals who have an increased risk of developing colorectal adenomas or cancer. Both natural or synthetic agents have been employed to prevent or suppress the colorectal tumorigenesis. In particular, in experimental animals, cohort and clinical case-control studies have shown inverse association between the use of either anti-inflammatory non steroidal drugs (NSAIDs), estrogens or phytoestrogens, and incidence of both colonic adenomas and CRC. NSAID use appears to prevent the oc‐ currence of carcinogen-induced animal colonic tumors [14] and to decrease the number and size of colo-rectal polyps in FAP (Familial Adenomatous Polyposis) patients [15]. Randomized placebo controller trials showed that aspirin reduced the risk of colorectal adenomas in populations with an intermediate risk of developing adenomas [16]. Fur‐ thermore, NSAIDs or selective COX-2 inhibitors reduce the in vitro growth of human co‐ lon cancer cell [17]. The effect of NSAIDs is mediated by cell cycle arrest due to inhibition of the Wnt-signaling pathway that favors the phosphorilation of beta-catenin

The fact that estrogens have an effect in decreasing the risk of colo-rectal cancer is shown by

accelerated carcinogenesis.

and by induction of apoptosis [18, 19].

the following data:

Meanwhile, the great majority of CRC are sporadic, with 2 to 6% of them related to a heredi‐ tary disease due to mutations of highly penetrant autosomic dominant genes. Mutations of *APC* tumor suppressor gene is responsible for familial adenomatous polyposis (FAP), and mutations of the mismatch repair (*MMR*) genes are related to hereditary non polypoid col‐ orectal cancer (HNPCC or Lynch's syndrome). Mutations of *MLH1* and *MSH2* are responsi‐ ble for more than 90% of the family affected by HNPCC. In these familial events, the onset of CRC is greatly anticipated in comparison to the sporadic counterpart which is usually di‐ agnosed after 50 years of age. However, an increasing incidence rate of CRC not clearly re‐ lated to the presence of inheritable or predisposing colonic diseases was observed in individuals less than 40 years of age in recent decades [10]. Furthermore, an enhanced risk for CRC and colonic adenomas is present in individuals whose first-degree relatives are af‐ fected by CRC, especially if the tumor occurs before the age of 60 [8]. Possible factors of this inherited susceptibility to CRC are polymorphisms of genes deputed to glutathione synthe‐ sis such as *GSTP1*, *GSTM1* and *GSTT1* genes [11].

Prognosis of CRC is in relationship to local and distant tumor progression. Deep penetration of carcinogenic cells in the colonic wall, invasion of adjacent organs, diffusion in lymph no‐ des or peritoneum, and distant metastases must be evaluated for staging of the disease and correct therapeutic planning. One third of all colorectal tumors are located in the rectum: prognosis of distally sited rectal cancer is worse than that of proximally sited rectal cancer or of colonic cancer. Despite great advances in population screening, early diagnosis, surgical interventions, and complementary therapies, long-term survival for CRC remains in the range of 50-60%.

Tumor formation in humans is a multistage process involving a series of events, and gener‐ ally occurs over an extended period. During this process, several genetic and epigenetic al‐ terations lead to the progressive transformation of a normal cell into a cancer cell. These cells acquire various abilities that transform them into malignant cells: they become resistant to growth inhibition, proliferate without dependence on growth factors, replicate without limit, evade apoptosis, and invade, metastasize, and support angiogenesis. Mechanisms by which cancer cells acquire these capabilities can vary considerably, but most of the physio‐ logical changes associated with these mechanisms involve alteration of signal transduction pathways [7].

It is commonly agreed that the first step of colorectal tumorigenesis is the shift of the proliferative zone in the glandular crypts, accompanied by the development of aberrant crypt foci, and followed by the formation of an adenomatous polyp. These pathological features are considered the precursor of the carcinoma in a temporal sequence that also can be completed in several years. However, CRC is not a homogenous disease: several histological types can be distinguished such as tubular or villous, mucinous, serrated, medullary, signet-ring, squamous cell, adenosquamous, small cell, and undifferentiated, and different molecular basis can also be recognized in histologically similar tumors. In recent years, the identification of the genetic mutations of hereditary forms of CRC has clarified two fundamentals types of carcinogenesis. The first is similar to that described for the development of the FAP, and is characterized by a progressive accumulation of genetic changes starting from a biallelic inactivation of *APC*. Additional mutations either of oncogenes *KRAS* and *p53* or of oncosuppressor genes (*DCC* and *DPC4*) are necessary for the neoplastic progression and invasivity [12]. The genetic alterations are responsible for an increased mucosal proliferation and a reduced apoptosis, causing a clonal cellular expansion. The second, similar to the CRC arising in the HNPCC, is due to inactivation of *MLH1* or of other *MMR* genes. Repetitive sequences of DNA, sited in non-encoding microsatellite regions throughout the genome, are specifically found in this type of CRC, hence, the definition of micro satellite instability (MSI). The mechanism responsible for the carcinogenesis is epigenetic due to an extensive DNA methylation. Rarely in this type of CRC both proto-oncogenes (*KRAS, p53*) and oncosuppressor genes (*APC, TGFBRII, IGF2R, BAX*) are mutated or inactivated [13]. The former genetic mechanism explains the most frequent form of sporadic CRC characterized by the sequence adeno‐ ma-carcinoma and a long period for the formation of cancer; vice versa, the last mecha‐ nism is only present in 15% of sporadic CRC, and can have the character of an accelerated carcinogenesis.

view of the epidemiological evidence supporting the roles of diet, lifestyle, and medication in reducing the risk of colorectal cancer. Similarly, many studies that implicate effects of di‐ etary agents in various types of cancers are available and suggest that much of the suffering and death from cancer could be prevented by consuming a healthy diet, reducing tobacco use, performing regular physical activity, and maintaining an optimal body weight [7]. Even if several epidemiological and experimental studies support the role of these factors in the genesis of CRC, other well-designed prospective and randomized clinical trials conducted in recent years report conflicting evidence, in particular on the role of the diet component in

Meanwhile, the great majority of CRC are sporadic, with 2 to 6% of them related to a heredi‐ tary disease due to mutations of highly penetrant autosomic dominant genes. Mutations of *APC* tumor suppressor gene is responsible for familial adenomatous polyposis (FAP), and mutations of the mismatch repair (*MMR*) genes are related to hereditary non polypoid col‐ orectal cancer (HNPCC or Lynch's syndrome). Mutations of *MLH1* and *MSH2* are responsi‐ ble for more than 90% of the family affected by HNPCC. In these familial events, the onset of CRC is greatly anticipated in comparison to the sporadic counterpart which is usually di‐ agnosed after 50 years of age. However, an increasing incidence rate of CRC not clearly re‐ lated to the presence of inheritable or predisposing colonic diseases was observed in individuals less than 40 years of age in recent decades [10]. Furthermore, an enhanced risk for CRC and colonic adenomas is present in individuals whose first-degree relatives are af‐ fected by CRC, especially if the tumor occurs before the age of 60 [8]. Possible factors of this inherited susceptibility to CRC are polymorphisms of genes deputed to glutathione synthe‐

Prognosis of CRC is in relationship to local and distant tumor progression. Deep penetration of carcinogenic cells in the colonic wall, invasion of adjacent organs, diffusion in lymph no‐ des or peritoneum, and distant metastases must be evaluated for staging of the disease and correct therapeutic planning. One third of all colorectal tumors are located in the rectum: prognosis of distally sited rectal cancer is worse than that of proximally sited rectal cancer or of colonic cancer. Despite great advances in population screening, early diagnosis, surgical interventions, and complementary therapies, long-term survival for CRC remains in the

Tumor formation in humans is a multistage process involving a series of events, and gener‐ ally occurs over an extended period. During this process, several genetic and epigenetic al‐ terations lead to the progressive transformation of a normal cell into a cancer cell. These cells acquire various abilities that transform them into malignant cells: they become resistant to growth inhibition, proliferate without dependence on growth factors, replicate without limit, evade apoptosis, and invade, metastasize, and support angiogenesis. Mechanisms by which cancer cells acquire these capabilities can vary considerably, but most of the physio‐ logical changes associated with these mechanisms involve alteration of signal transduction

It is commonly agreed that the first step of colorectal tumorigenesis is the shift of the proliferative zone in the glandular crypts, accompanied by the development of aberrant

the etiology of CRC [8, 9].

76 Soybean - Bio-Active Compounds

range of 50-60%.

pathways [7].

sis such as *GSTP1*, *GSTM1* and *GSTT1* genes [11].

Improved knowledge of the molecular mechanisms of colorectal carcinogenesis allows a rationale chemopreventive use in individuals who have an increased risk of developing colorectal adenomas or cancer. Both natural or synthetic agents have been employed to prevent or suppress the colorectal tumorigenesis. In particular, in experimental animals, cohort and clinical case-control studies have shown inverse association between the use of either anti-inflammatory non steroidal drugs (NSAIDs), estrogens or phytoestrogens, and incidence of both colonic adenomas and CRC. NSAID use appears to prevent the oc‐ currence of carcinogen-induced animal colonic tumors [14] and to decrease the number and size of colo-rectal polyps in FAP (Familial Adenomatous Polyposis) patients [15]. Randomized placebo controller trials showed that aspirin reduced the risk of colorectal adenomas in populations with an intermediate risk of developing adenomas [16]. Fur‐ thermore, NSAIDs or selective COX-2 inhibitors reduce the in vitro growth of human co‐ lon cancer cell [17]. The effect of NSAIDs is mediated by cell cycle arrest due to inhibition of the Wnt-signaling pathway that favors the phosphorilation of beta-catenin and by induction of apoptosis [18, 19].

The fact that estrogens have an effect in decreasing the risk of colo-rectal cancer is shown by the following data:

**1.** Several epidemiologic studies show a smaller incidence of sporadic CRC in the female gender. Also the occurrence of CRC in HNPCC is lower in females than in males;

bly fruits, and lower risk of colon cancer [28]. A meta-analysis of six case-control studies found that a high intake of vegetables or fiber was associated with an approximate 40%–50% reduction in risk for colon cancer [29]. Similarly, a pooled analysis of 13 case-control studies reported an approximately 50% lower risk of colon cancer associated with higher intake of

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 79

Increasing intake of fruits, vegetables, or fiber is unlikely to prevent a large proportion of colorectal cancers, particularly among the US population, which has a food supply already fortified with folate and other dietary factors that might protect against colorectal neoplasia. There is also little evidence that concentrated sources of one type of fiber are efficacious, al‐ though fiber-rich diets have health benefits for other gastrointestinal conditions, such as di‐

All evidence supporting the decreased risk include results from a few studies of ade‐ nomatous polyps (which may progress to colorectal carcinomas). Fruit and grain intake also appears to be inversely related to risk of colorectal cancer and polyps, although less consistently than vegetables. These potentially protective associations may result from the high levels of dietary fibres, antioxidants (e.g., beta-carotene, vitamin C), or other an‐ ticarcinogenic constituents (e.g., protease inhibitors, phytoestrogens) in these vegetables, fruits, and grains. However, the association of adenomatous polyps of the large bowel with intake of vegetables, fruits, and grains has not been studied to any great degree, and existing data on these associations are not entirely consistent. Because adenomatous polyps are precursors to colorectal cancer, studying polyps instead of cancer might allow one to measure the diet of relatively asymptomatic subjects closer to the time of the ini‐

A recent meta analysis and data review, conducted by Magalhães B. [32], substantiates that the risk of colon cancer was increased with patterns characterized by high intake of red and

There are many plausible mechanisms by which intake of vegetables, fruits and "healthy

Plant foods contain a wide variety of anticancer phytochemicals with many potential bioac‐

Many epidemiologic studies evidence a lower rate of hormone-related cancers among Asian populations which are characterized by regular consumption of soy based foods. Soy is a major plant source of dietary protein for humans. A review of epidemiologic studies (most of which were case-control studies published before 2000) suggested an inverse association between high soy intake and colon cancer risk in humans [35]. Moreover, migration studies show that Japanese immigrants in the United Status have incidence rates of colorectal can‐

processed meat, and decreased with those labelled as 'healthy.'

tivities that may reduce cancer susceptibility [7,33, 34].

verticular disease and constipation, and possibly other chronic diseases [6].

fiber [30].

tial neoplastic process. [31].

foods" may prevent carcinogenesis.

**3. Soyfoods and colon cancer**


## **2. Nutrition and colon cancer**

It is now believed that 90–95% of all cancers are attributed to lifestyle, with the remaining 5– 10% attributed to faulty genes [7]. Almost 30 years ago epidemiological research suggested that appropriate nutrition could prevent approximately 35% of cancer deaths, and up to 90% of certain cancers could be avoided by dietary enhancement [23, 24].

Colon cancer is a multifactorial disease that results from the interaction of different factors such as aging, family history, and dietary style. Identifying modifiable factors associated with colorectal cancer is of importance, the ultimate goal being primary prevention, and particularly the role of diet in the aetiology, initiation, and progression of colorectal cancer remains an area of important research. Moreover, several components of food can exert a potent activity also in the later stages of cancer. Several studies have indicated that inhibi‐ tion of metastasis by genistein, one of the most important constituents of soy foods, repre‐ sents an important mechanism by which it is possible to reduce mortality associated with solid organ cancer.

Many plant-derived dietary agents have multitargeting properties and are therefore called nutraceuticals. A nutraceutical (a term formed by combining the words "nutrition" and "pharmaceutical") is simply any substance considered to be a food or part of a food that provides medical and health benefits. During the past decade, a number of nutraceuticals have been identified from natural sources. Nutraceuticals are chemically diverse and target various steps in tumor cell development [7].

Several epidemiological studies have consistently shown an inverse association between consumption of vegetables and fruits and the risk of human cancers at many sites. Wickia & Hagmannc (2011) recently reported that many case-control and cohort studies are dealing with the effect of fruits and vegetables on cancer incidence [25]. Early data indicated a bene‐ ficial effect [26] and, as recently as 2008, Freedman et al. found a reduced occurrence of head and neck cancers with increased fruit and vegetable consumption [27].

The concept that a diet that is high in fiber, especially from fruits and vegetables, lowers risk of colorectal cancer has been in existence for more than 4 decades. The majority of case-con‐ trol studies have shown an association between higher intake of fiber, vegetables, and possi‐ bly fruits, and lower risk of colon cancer [28]. A meta-analysis of six case-control studies found that a high intake of vegetables or fiber was associated with an approximate 40%–50% reduction in risk for colon cancer [29]. Similarly, a pooled analysis of 13 case-control studies reported an approximately 50% lower risk of colon cancer associated with higher intake of fiber [30].

Increasing intake of fruits, vegetables, or fiber is unlikely to prevent a large proportion of colorectal cancers, particularly among the US population, which has a food supply already fortified with folate and other dietary factors that might protect against colorectal neoplasia. There is also little evidence that concentrated sources of one type of fiber are efficacious, al‐ though fiber-rich diets have health benefits for other gastrointestinal conditions, such as di‐ verticular disease and constipation, and possibly other chronic diseases [6].

All evidence supporting the decreased risk include results from a few studies of ade‐ nomatous polyps (which may progress to colorectal carcinomas). Fruit and grain intake also appears to be inversely related to risk of colorectal cancer and polyps, although less consistently than vegetables. These potentially protective associations may result from the high levels of dietary fibres, antioxidants (e.g., beta-carotene, vitamin C), or other an‐ ticarcinogenic constituents (e.g., protease inhibitors, phytoestrogens) in these vegetables, fruits, and grains. However, the association of adenomatous polyps of the large bowel with intake of vegetables, fruits, and grains has not been studied to any great degree, and existing data on these associations are not entirely consistent. Because adenomatous polyps are precursors to colorectal cancer, studying polyps instead of cancer might allow one to measure the diet of relatively asymptomatic subjects closer to the time of the ini‐ tial neoplastic process. [31].

A recent meta analysis and data review, conducted by Magalhães B. [32], substantiates that the risk of colon cancer was increased with patterns characterized by high intake of red and processed meat, and decreased with those labelled as 'healthy.'

There are many plausible mechanisms by which intake of vegetables, fruits and "healthy foods" may prevent carcinogenesis.

Plant foods contain a wide variety of anticancer phytochemicals with many potential bioac‐ tivities that may reduce cancer susceptibility [7,33, 34].

## **3. Soyfoods and colon cancer**

**1.** Several epidemiologic studies show a smaller incidence of sporadic CRC in the female gender. Also the occurrence of CRC in HNPCC is lower in females than in males;

**3.** epidemiologic studies of postmenopausal women show that users of HRT have a signif‐ icant reduction of CRC development in respect to women who had never used HRT.

**4.** use of non-contraceptive hormones for more than 5 years reduces by (OR = 0.47, 95 per‐

It is now believed that 90–95% of all cancers are attributed to lifestyle, with the remaining 5– 10% attributed to faulty genes [7]. Almost 30 years ago epidemiological research suggested that appropriate nutrition could prevent approximately 35% of cancer deaths, and up to 90%

Colon cancer is a multifactorial disease that results from the interaction of different factors such as aging, family history, and dietary style. Identifying modifiable factors associated with colorectal cancer is of importance, the ultimate goal being primary prevention, and particularly the role of diet in the aetiology, initiation, and progression of colorectal cancer remains an area of important research. Moreover, several components of food can exert a potent activity also in the later stages of cancer. Several studies have indicated that inhibi‐ tion of metastasis by genistein, one of the most important constituents of soy foods, repre‐ sents an important mechanism by which it is possible to reduce mortality associated with

Many plant-derived dietary agents have multitargeting properties and are therefore called nutraceuticals. A nutraceutical (a term formed by combining the words "nutrition" and "pharmaceutical") is simply any substance considered to be a food or part of a food that provides medical and health benefits. During the past decade, a number of nutraceuticals have been identified from natural sources. Nutraceuticals are chemically diverse and target

Several epidemiological studies have consistently shown an inverse association between consumption of vegetables and fruits and the risk of human cancers at many sites. Wickia & Hagmannc (2011) recently reported that many case-control and cohort studies are dealing with the effect of fruits and vegetables on cancer incidence [25]. Early data indicated a bene‐ ficial effect [26] and, as recently as 2008, Freedman et al. found a reduced occurrence of head

The concept that a diet that is high in fiber, especially from fruits and vegetables, lowers risk of colorectal cancer has been in existence for more than 4 decades. The majority of case-con‐ trol studies have shown an association between higher intake of fiber, vegetables, and possi‐

and neck cancers with increased fruit and vegetable consumption [27].

**2.** women who are multipare are a reduced risk of CRC in confront to nullipare;

The risk appears to be halved with 5-10 years of HRT use [20, 21];

of certain cancers could be avoided by dietary enhancement [23, 24].

cent CI = 0.24-0.91) the risk of colon cancer [22].

**2. Nutrition and colon cancer**

78 Soybean - Bio-Active Compounds

solid organ cancer.

various steps in tumor cell development [7].

Many epidemiologic studies evidence a lower rate of hormone-related cancers among Asian populations which are characterized by regular consumption of soy based foods. Soy is a major plant source of dietary protein for humans. A review of epidemiologic studies (most of which were case-control studies published before 2000) suggested an inverse association between high soy intake and colon cancer risk in humans [35]. Moreover, migration studies show that Japanese immigrants in the United Status have incidence rates of colorectal can‐ cers very near to the rates among the whites in the country [6]. Thus the protective effect of soy foods and isoflavones is a matter of interest in the etiology of colorectal cancer.

The presence of hydroxylated and methylated genistein metabolites correlated positively with inhibition of cancer cell proliferation, but genistein sulfates were not associated with antiproliferative effects of genistein, suggesting that some types of metabolism of the isofla‐

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 81

Witte, et al, showed that higher consumption of tofu (or soybeans) was inversely associated with polyps. Tofu (or soybeans) contain a number of potentially anticarcinogenic constitu‐ ents, including isoflavones, saponins, genistein, and phytosterols. They were able to look at tofu (or soybeans) as a single food item (i.e., separate from legumes) because almost 15 per‐ cent of our multiethnic study population reported consuming tofu (or soybeans) at least once a week. The strongest association observed was for vegetables—including those high in carotenoids, cruciferae, and broccoli—as well as garlic and tofu (or soybeans), and these associations were found even after adjusting for dietary fiber, folate, beta-carotene, vitamin

Men tend to have a slightly higher incidence of colorectal cancer than women of similar age (American Cancer Society, 2007), and oestrogen seems to be implicated for this decreased risk in women. Epidemiological studies and results of a Women's Health Initiative (WHI) clinical trial provide strong evidence that colorectal cancer is hormone sensitive because the

In effect, many epidemiological and experimental studies suggest a protective role of estro‐ gens against colorectal cancer. The decrease in the number of deaths from large bowel carci‐ noma observed in the United States in the last 40 years was significantly higher in women (30%) as compared to men (7%). A link was observed between oral contraceptive use and a reduction of colorectal cancer, whereas there was a higher than expected frequency of color‐

Interestingly, as reported by Barone et al., although several experimental studies have con‐ firmed a protective role of estrogens for CRC, few studies have been conducted, and with conflicting results, on the possible protective effect of estrogens against the development of adenomatous polyps in the colon, although it is well known that the development of adeno‐

Gender differences in the incidence and behavior of colorectal cancer (CRC), as well as epi‐ demiologic data indicating a protective effect of hormone replacement therapy in women, have further supported the concept of hormonal influence on the development of CRC. It has been suggested that the protective effect of estrogens (or phytoestrogens) may be medi‐ ated through activation of ERβ, which has been shown to be the predominant subtype of ER

ERs are nuclear receptors belonging to the steroid hormone receptor superfamily which have the characteristic of being activated upon binding of the ligand. If the ligand is not present, ERs bind to a shock protein. Otherwise, when the ligand is present, the ERs make a stable dimer and initiate the specific estrogenic response, with transcription of the target genes. Two main types of ER have been identified: alfa (ERα) and beta (ERβ). They are the so-called ligand-activated transcriptional factors through which estrogens

vones may be crucial for their action [44].

C, and other commonly measured antioxidants [31].

ectal tumors among non users [45].

in the gastrointestinal tract [47].

carcinoma mostly involves polyp formation [46].

cancer risk is reduced by post-menopause hormone therapy [35]

Soy and soy foods contain a wide variety of chemical compounds, biologically active, that may contribute, individually or synergistically, to the health benefits of this plant; in partic‐ ular, polyphenols are considered to possess chemopreventive and therapeutic properties against cancer.

Among these compounds, certainly, there are isoflavones, the most important and abundant of which is genistein, which also have estrogenic properties. In fact, in recent decades, there have been several studies showing that isoflavones are promising candidates for cancer pre‐ vention [36, 37, 38, 39].

Data associating soybean consumption with reduced cancer rates have been used as evi‐ dence for a role of isoflavones in cancer prevention. However, soybeans are also a rich source of trypsin inhibitor, other proteins with health benefits, phosphatidyl inositol, sapo‐ nins, and sphingolipids, all of which have potential health benefits. All of these soybean constituents demonstrate tumor preventive properties in animal models. Research by Birt et al. demonstrated that 20% by weight of dietary soy protein significantly reduced rat intesti‐ nal mucosa levels of polyamine, a biomarker of cellular proliferation for colorectal cancer risk [39].

Surely, soy foods are complex foods, and it is difficult to assume that associations which suggest protective properties of soy foods are due only to a single constituent. Because of the association between diets in Japan and China and lower rates of cancers, such as those of the breast, prostate, and colon, than in Europe and the United States, many investigators have assumed that this is due to soy food consumption in Japan and China.

Other factors in the Asian diet may be responsible, and it's important to evaluate the possi‐ ble confounding dietetic factors in the studies.

Several studies suggest that soy foods, the predominant source of isoflavones, are associated with reductions in cancer rate, but they do not consistently appear to be the primary protec‐ tive component of the Asian diet.

Wu et al. noted the difficulties in assessing the relationship between the level of intake and protection. Case control and prospective epidemiological investigations that have provided a suggestion of protection against cancer by soy foods have not provided adequate informa‐ tion on the bioactive constituents in the soy foods, the portion size, or other components that may be protective in the diets of people who eat soy foods [40].

Isoflavones and flavonoids may be rapidly and predominately glucuronidated in the GI mu‐ cosa, if genistein can be considered a model for all of these phenolic compounds [41]. Fur‐ ther, glucuronidation occurs in the liver. Genistein undergoes biliary excretion, with more than 70% of a dose recovered in bile within 4 hr after dosing in rats. Although genistein may be absorbed well initially, a maximum of 25% of an oral genistein dose would be eliminated in rat urine. About 20–25% of an oral dose of genistein (predominantly as its glucoside from soy foods) is recovered in human urine [42, 43].

The presence of hydroxylated and methylated genistein metabolites correlated positively with inhibition of cancer cell proliferation, but genistein sulfates were not associated with antiproliferative effects of genistein, suggesting that some types of metabolism of the isofla‐ vones may be crucial for their action [44].

cers very near to the rates among the whites in the country [6]. Thus the protective effect of

Soy and soy foods contain a wide variety of chemical compounds, biologically active, that may contribute, individually or synergistically, to the health benefits of this plant; in partic‐ ular, polyphenols are considered to possess chemopreventive and therapeutic properties

Among these compounds, certainly, there are isoflavones, the most important and abundant of which is genistein, which also have estrogenic properties. In fact, in recent decades, there have been several studies showing that isoflavones are promising candidates for cancer pre‐

Data associating soybean consumption with reduced cancer rates have been used as evi‐ dence for a role of isoflavones in cancer prevention. However, soybeans are also a rich source of trypsin inhibitor, other proteins with health benefits, phosphatidyl inositol, sapo‐ nins, and sphingolipids, all of which have potential health benefits. All of these soybean constituents demonstrate tumor preventive properties in animal models. Research by Birt et al. demonstrated that 20% by weight of dietary soy protein significantly reduced rat intesti‐ nal mucosa levels of polyamine, a biomarker of cellular proliferation for colorectal cancer

Surely, soy foods are complex foods, and it is difficult to assume that associations which suggest protective properties of soy foods are due only to a single constituent. Because of the association between diets in Japan and China and lower rates of cancers, such as those of the breast, prostate, and colon, than in Europe and the United States, many investigators

Other factors in the Asian diet may be responsible, and it's important to evaluate the possi‐

Several studies suggest that soy foods, the predominant source of isoflavones, are associated with reductions in cancer rate, but they do not consistently appear to be the primary protec‐

Wu et al. noted the difficulties in assessing the relationship between the level of intake and protection. Case control and prospective epidemiological investigations that have provided a suggestion of protection against cancer by soy foods have not provided adequate informa‐ tion on the bioactive constituents in the soy foods, the portion size, or other components that

Isoflavones and flavonoids may be rapidly and predominately glucuronidated in the GI mu‐ cosa, if genistein can be considered a model for all of these phenolic compounds [41]. Fur‐ ther, glucuronidation occurs in the liver. Genistein undergoes biliary excretion, with more than 70% of a dose recovered in bile within 4 hr after dosing in rats. Although genistein may be absorbed well initially, a maximum of 25% of an oral genistein dose would be eliminated in rat urine. About 20–25% of an oral dose of genistein (predominantly as its glucoside from

have assumed that this is due to soy food consumption in Japan and China.

may be protective in the diets of people who eat soy foods [40].

ble confounding dietetic factors in the studies.

soy foods) is recovered in human urine [42, 43].

tive component of the Asian diet.

soy foods and isoflavones is a matter of interest in the etiology of colorectal cancer.

against cancer.

risk [39].

vention [36, 37, 38, 39].

80 Soybean - Bio-Active Compounds

Witte, et al, showed that higher consumption of tofu (or soybeans) was inversely associated with polyps. Tofu (or soybeans) contain a number of potentially anticarcinogenic constitu‐ ents, including isoflavones, saponins, genistein, and phytosterols. They were able to look at tofu (or soybeans) as a single food item (i.e., separate from legumes) because almost 15 per‐ cent of our multiethnic study population reported consuming tofu (or soybeans) at least once a week. The strongest association observed was for vegetables—including those high in carotenoids, cruciferae, and broccoli—as well as garlic and tofu (or soybeans), and these associations were found even after adjusting for dietary fiber, folate, beta-carotene, vitamin C, and other commonly measured antioxidants [31].

Men tend to have a slightly higher incidence of colorectal cancer than women of similar age (American Cancer Society, 2007), and oestrogen seems to be implicated for this decreased risk in women. Epidemiological studies and results of a Women's Health Initiative (WHI) clinical trial provide strong evidence that colorectal cancer is hormone sensitive because the cancer risk is reduced by post-menopause hormone therapy [35]

In effect, many epidemiological and experimental studies suggest a protective role of estro‐ gens against colorectal cancer. The decrease in the number of deaths from large bowel carci‐ noma observed in the United States in the last 40 years was significantly higher in women (30%) as compared to men (7%). A link was observed between oral contraceptive use and a reduction of colorectal cancer, whereas there was a higher than expected frequency of color‐ ectal tumors among non users [45].

Interestingly, as reported by Barone et al., although several experimental studies have con‐ firmed a protective role of estrogens for CRC, few studies have been conducted, and with conflicting results, on the possible protective effect of estrogens against the development of adenomatous polyps in the colon, although it is well known that the development of adeno‐ carcinoma mostly involves polyp formation [46].

Gender differences in the incidence and behavior of colorectal cancer (CRC), as well as epi‐ demiologic data indicating a protective effect of hormone replacement therapy in women, have further supported the concept of hormonal influence on the development of CRC. It has been suggested that the protective effect of estrogens (or phytoestrogens) may be medi‐ ated through activation of ERβ, which has been shown to be the predominant subtype of ER in the gastrointestinal tract [47].

ERs are nuclear receptors belonging to the steroid hormone receptor superfamily which have the characteristic of being activated upon binding of the ligand. If the ligand is not present, ERs bind to a shock protein. Otherwise, when the ligand is present, the ERs make a stable dimer and initiate the specific estrogenic response, with transcription of the target genes. Two main types of ER have been identified: alfa (ERα) and beta (ERβ). They are the so-called ligand-activated transcriptional factors through which estrogens exert their effects on various tissues and have a different tissue distribution. ERα is mainly present in the mammary glands and in the utterus; ERβ is mainly present in en‐ dothelial cells, the urogenital tract, the central nervous system, and the colonic mucosa. Experimental data have demonstrated that CRC express an elevated number of estrogen receptors (ERs), but while ERα is detected in very low levels either in normal or patho‐ logical colonic mucosa (adenoma and carcinoma), ERβ expression is high in the normal colonic mucosa, and progressively decreased in the pathological mucosa in relationship to the cellular differentiation and CRC stage.

As proposed for estrogens, genomic and non-genomic mechanisms have also been suggest‐

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 83

As reported by several authors in the past, genomic pathways are mediated through the ability of phytoestrogens to interact with enzymes and receptors, and cross the plasma membrane. In this way, they bind ERs and induce the transcription of estrogen-responsive genes, stimulate cell growth in the breast, and modify ER transcription itself. However, some of their effects are not due to interaction with ERs, and are therefore denominated non-genomic effects. For example: inhibition of tyrosine kinase and DNA topoisomerase,

The bioavailability of phytoestrogens (determined by: absorption, distribution, metabo‐ lism (bioconversion in the gut and biotransformation in the liver) and escretion) and their activity is highly variable and changes with respect to several factors, such as ad‐ ministration rules, dosage, metabolism and interaction with other pharmacological sub‐ stances. Moreover, their biological effect is influenced by the type of target tissue, the number and type of ERs expressed in the tissue, their serum concentration, and sex ste‐

Phytoestrogens, present in soy and soy-based food, may act through hormonal mechanisms to reduce cancer risk by binding to estrogen receptors (ER) or interacting with enzymes in‐

Although cancer incidence in women is much lower than in men in both countries, there is also a difference when the 2 countries are compared. Japanese men as well as women have a lower colorectal cancer incidence than their American counterparts, although mortality is quite similar when related to specific incidence data. In hormone-dependent cancers such as those of the breast and prostate, incidence is exceedingly low in Japan (and was even lower in earlier decades) compared with that in the United States. Mortality, again in proportion to incidence, is rather similar. Numerous reports have suggested that this difference in tumor incidence is probably due to consumption of soy as a staple food in Asian countries in con‐ trast to Western industrialized countries. These substances, through their potential to act as selective estrogen receptor modulators, may affect vitamin D–related inhibition of tumor growth by upregulating extrarenal synthesis of 1,25- D3. Genistein, the most prominent phytoestrogen in soy, is known to regulate other P450 enzymes, such as 5-reductase and 17 hydroxysteroid dehydrogenase, which are essential for metabolism of sex hormones [54].

In vitro studies of DLD1 colon adenocarcinoma cells have linked the effects of soy with es‐ trogen receptor beta. Experiments conducted on this cell line, with or without ER-β gene si‐ lencing by RNA interference (RNAi), have shown that soy isoflavones decreased the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated kin‐ ase (ERK)-1/2, AKT, and nuclear factor (NF)-κB. Soy isoflavones dose-dependently caused G2/M cell cycle arrest and downregulated the expression of cyclin A. This was associated with inhibition of cyclin dependent kinase (CDK)-4 and upregulation of its inhibitor p21 ex‐ pressions. ER-β gene silencing lowered soy isoflavone-mediated suppression of cell viability

ed for phytoestrogens to explain their biological activities

suppression of angiogenesis, and antioxidant effects [33, 36, 46].

roid hormone concentration [51, 52].

volved in sex steroid biosynthesis and metabolism [53].

The observation that the level of ERβ protein is lower in malignant tumors than in normal tissue of the same organ has fostered the hypothesis that ERβ may function as a tumor sup‐ pressor, protecting cells against malignant transformation and uncontrolled proliferation.

ERβ is present in various isoforms: studying different types of colonic tumoral cells, isoform 1 of ERβ is found in the Lo-Vo, HCT8, HCT116, DLD-1 and isoform 2,3,4 and 5 only in the HCT8 and HCT116. It has not been well investigated whether the function of the various isoforms of ERβ, but loss of the expression of isoform 1 of ERβ, is accompanied by undiffer‐ entiated proliferation, mucinous histological type, and tumor progression [48]. It is accepted that the binding of estrogens to the ERβ blocks the activity of AP-1 on the genes involved in the cellular proliferation and provokes an activation of p53. Conversely, SERM, such as ta‐ moxifene and raloxifene, induce an antiproliferative effect in human colorectal cell lines by a citostatic or cytotoxic effect [49]. Several observations on the CRC cellular cultures and on the experimental mouse with germinal mutation of APC have clarified the role of the ER and estrogenes for colorectal cancerogenesis: 17β estadiol decreases the proliferation in vitro of the HCT116, Lo-Vo and DLD1 cells, but increases the proliferation of the HCT8 cells. However, the effect on the last type of cells is completely changed by increasing the level of RRb by transfection with ERβ. The overexpression of ERβ can have an inhibitory effect on the proliferation. In the transfected HCT8 cells the levels of CD4 and CP21, which are onco‐ suppressor genes, are significantly increased, and the level of cyclinE, which have oncogenic activity, significantly decreased, in respect to normal HCT8 [50].

ERβ is lower in the adenomatous polyps of FAP patients and in the intestinal adenomas which develop in APC Min+/- mouse than in the colonic normal mucosa. The restoration of normal levels of ERβ obtained with dietary phytoestrogenes is accompained by regression or disappearance of the polyps in the experimental animal. Patients with sporadic adenomas in the colon show an increase of apoptoic activity, and ERβ expression of the colonic muco‐ sa, if their diet is supplemented by phytoestrogenes [45]. These data strongly support a piv‐ otal role of ERβ in a protective action against the initiation and progression of colorectal carcinogenesis.

Many epidemiologic studies evidence a lower rate of hormone-related cancers among Asian populations which are characterized by regular consumption of soy based foods. Soy is a major plant source of dietary protein for humans. Among other components, soy contains large amounts of phytoestrogens.

As proposed for estrogens, genomic and non-genomic mechanisms have also been suggest‐ ed for phytoestrogens to explain their biological activities

exert their effects on various tissues and have a different tissue distribution. ERα is mainly present in the mammary glands and in the utterus; ERβ is mainly present in en‐ dothelial cells, the urogenital tract, the central nervous system, and the colonic mucosa. Experimental data have demonstrated that CRC express an elevated number of estrogen receptors (ERs), but while ERα is detected in very low levels either in normal or patho‐ logical colonic mucosa (adenoma and carcinoma), ERβ expression is high in the normal colonic mucosa, and progressively decreased in the pathological mucosa in relationship

The observation that the level of ERβ protein is lower in malignant tumors than in normal tissue of the same organ has fostered the hypothesis that ERβ may function as a tumor sup‐ pressor, protecting cells against malignant transformation and uncontrolled proliferation.

ERβ is present in various isoforms: studying different types of colonic tumoral cells, isoform 1 of ERβ is found in the Lo-Vo, HCT8, HCT116, DLD-1 and isoform 2,3,4 and 5 only in the HCT8 and HCT116. It has not been well investigated whether the function of the various isoforms of ERβ, but loss of the expression of isoform 1 of ERβ, is accompanied by undiffer‐ entiated proliferation, mucinous histological type, and tumor progression [48]. It is accepted that the binding of estrogens to the ERβ blocks the activity of AP-1 on the genes involved in the cellular proliferation and provokes an activation of p53. Conversely, SERM, such as ta‐ moxifene and raloxifene, induce an antiproliferative effect in human colorectal cell lines by a citostatic or cytotoxic effect [49]. Several observations on the CRC cellular cultures and on the experimental mouse with germinal mutation of APC have clarified the role of the ER and estrogenes for colorectal cancerogenesis: 17β estadiol decreases the proliferation in vitro of the HCT116, Lo-Vo and DLD1 cells, but increases the proliferation of the HCT8 cells. However, the effect on the last type of cells is completely changed by increasing the level of RRb by transfection with ERβ. The overexpression of ERβ can have an inhibitory effect on the proliferation. In the transfected HCT8 cells the levels of CD4 and CP21, which are onco‐ suppressor genes, are significantly increased, and the level of cyclinE, which have oncogenic

ERβ is lower in the adenomatous polyps of FAP patients and in the intestinal adenomas which develop in APC Min+/- mouse than in the colonic normal mucosa. The restoration of normal levels of ERβ obtained with dietary phytoestrogenes is accompained by regression or disappearance of the polyps in the experimental animal. Patients with sporadic adenomas in the colon show an increase of apoptoic activity, and ERβ expression of the colonic muco‐ sa, if their diet is supplemented by phytoestrogenes [45]. These data strongly support a piv‐ otal role of ERβ in a protective action against the initiation and progression of colorectal

Many epidemiologic studies evidence a lower rate of hormone-related cancers among Asian populations which are characterized by regular consumption of soy based foods. Soy is a major plant source of dietary protein for humans. Among other components, soy contains

to the cellular differentiation and CRC stage.

82 Soybean - Bio-Active Compounds

activity, significantly decreased, in respect to normal HCT8 [50].

carcinogenesis.

large amounts of phytoestrogens.

As reported by several authors in the past, genomic pathways are mediated through the ability of phytoestrogens to interact with enzymes and receptors, and cross the plasma membrane. In this way, they bind ERs and induce the transcription of estrogen-responsive genes, stimulate cell growth in the breast, and modify ER transcription itself. However, some of their effects are not due to interaction with ERs, and are therefore denominated non-genomic effects. For example: inhibition of tyrosine kinase and DNA topoisomerase, suppression of angiogenesis, and antioxidant effects [33, 36, 46].

The bioavailability of phytoestrogens (determined by: absorption, distribution, metabo‐ lism (bioconversion in the gut and biotransformation in the liver) and escretion) and their activity is highly variable and changes with respect to several factors, such as ad‐ ministration rules, dosage, metabolism and interaction with other pharmacological sub‐ stances. Moreover, their biological effect is influenced by the type of target tissue, the number and type of ERs expressed in the tissue, their serum concentration, and sex ste‐ roid hormone concentration [51, 52].

Phytoestrogens, present in soy and soy-based food, may act through hormonal mechanisms to reduce cancer risk by binding to estrogen receptors (ER) or interacting with enzymes in‐ volved in sex steroid biosynthesis and metabolism [53].

Although cancer incidence in women is much lower than in men in both countries, there is also a difference when the 2 countries are compared. Japanese men as well as women have a lower colorectal cancer incidence than their American counterparts, although mortality is quite similar when related to specific incidence data. In hormone-dependent cancers such as those of the breast and prostate, incidence is exceedingly low in Japan (and was even lower in earlier decades) compared with that in the United States. Mortality, again in proportion to incidence, is rather similar. Numerous reports have suggested that this difference in tumor incidence is probably due to consumption of soy as a staple food in Asian countries in con‐ trast to Western industrialized countries. These substances, through their potential to act as selective estrogen receptor modulators, may affect vitamin D–related inhibition of tumor growth by upregulating extrarenal synthesis of 1,25- D3. Genistein, the most prominent phytoestrogen in soy, is known to regulate other P450 enzymes, such as 5-reductase and 17 hydroxysteroid dehydrogenase, which are essential for metabolism of sex hormones [54].

In vitro studies of DLD1 colon adenocarcinoma cells have linked the effects of soy with es‐ trogen receptor beta. Experiments conducted on this cell line, with or without ER-β gene si‐ lencing by RNA interference (RNAi), have shown that soy isoflavones decreased the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated kin‐ ase (ERK)-1/2, AKT, and nuclear factor (NF)-κB. Soy isoflavones dose-dependently caused G2/M cell cycle arrest and downregulated the expression of cyclin A. This was associated with inhibition of cyclin dependent kinase (CDK)-4 and upregulation of its inhibitor p21 ex‐ pressions. ER-β gene silencing lowered soy isoflavone-mediated suppression of cell viability and proliferation. ERK-1/2 and AKT expressions were unaltered and NF-κB was modestly upregulated by soy isoflavones after transient knockdown of ER-β expression.

therapy of breast cancer. However, effects independent of this activity have also been dem‐

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 85

Soy isoflavone supplemented diets also prevented the development of adenocarcinomas in

Phytoestrogens, present in soy based food, may act through hormonal mechanisms to reduce cancer risk by binding to estrogen receptors (ER) or interacting with enzymes in‐ volved in sex steroid biosynthesis and metabolism [53]. Moreover, genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, and the mecha‐ nisms by which genistein exerts its anti-tumor effects have been the subject of consider‐

Studies demonstrate that ERβ is highly expressed in superficial and crypt epithelium of the normal colon in both genders. ERβ expression was highly correlated among all cell types in both genders, and the strongest correlation was observed between surface and crypt ERβ ex‐ pression. This finding suggests that there may be an intersubject difference in ERβ expres‐ sion that is manifested in all cell types. ERβ expression was significantly lower in colon cancer cells compared with normal colonic epithelium, and there was a progressive decline in ERβ expression that paralleled the loss of cancer cell differentiation. The present findings are consonant with previous results reported by Foley and colleagues [69], who also detect‐ ed a loss of ERβ protein expression in malignant colon tissue by western immunoblotting. Another immunohistochemical study of ERβ in 55 patients with colorectal adenocarcinomas showed that 32% of all tumors in both genders were ERβ-negative; the 10% cut-off threshold

Studies conducted with ER subtype-specific ligands and those performed with estrogen re‐ ceptor b-knockout mice (ERβKOs) have illustrated the involvement of ERβ in cellular antiinflammatory pathways and tissue homeostasis in the colon. These results suggest that ERβspecific ligands may be promising targets in the pharmaceutical and therapeutical treatment of inflammatory bowel disease and the prevention of CRC. ERβKOs suggest that ERβ-spe‐ cific agonists and ERβ-selective phytoestrogens like genistein (GEN) and coumestrol may

Schleipen et al. investigate the influence of ERα and ERβ-specific agonists, and of genestein on cell proliferation and apoptosis of the small intestine and the colon. Recent data indicate that ERβ-specific agonists and GEN inhibit epithelial proliferation of the prostate and mam‐ mary gland, and can even impede prostate cancer development [74, 76, 75]. It can therefore be assumed that ERβ-specific agonists may also inhibit the proliferation of the intestinal epi‐ thelium. To prove this hypothesis in the study, ovariectomized rats were treated with 17β-Estradiol (E2), the phytoestrogen GEN and ER subtype-specific agonists for ERα and ERβ

Genistein has been shown to induce epigenetic changes in several cancer cell lines and in *in vivo* animal models [64]. Recent studies show that genistein may affect DNA methylation, serves as a natural demethylation agent, and is specifically effective on colon cancer cells

the prostate and seminal vesicles in a rat carcinogenesis model [68].

was used to distinguish ERβ-positive from negative tumors [70].

serve as potential regulators of intestinal tissue homeostasis [71, 72, 73].

onstrated [66, 67].

able interest [61, 62, 63].

for 3 weeks.

from early-stage colon cancer [58].

Soy isoflavone-mediated arrest of cells at G2/M phase and upregulation of p21 expression were not observed when ER-β gene was silenced. These findings suggest that maintaining the expression of ER-β is crucial in mediating the growth-suppressive effects of soy isofla‐ vones against colon tumors. Thus, upregulation of ER-β status by specific foodborne ER-li‐ gands such as soy isoflavones could potentially be a dietary prevention or therapeutic strategy for colon cancer [55].

## **4. Genistein and isoflavones: Other mechanisms of action**

In addition to estrogenic/antiestrogenic activity, some mechanisms of action have been iden‐ tified for isoflavone/flavone prevention of cancer: antiproliferation, induction of cell cycle arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regula‐ tion of host immune system, and changes in cellular signaling [39, 56, 57]. It is expected that also combinations of these mechanisms may contribute to cancer prevention.

Gene silencing due to the promoter methylation provides an opportunity for clinical inter‐ vention, as gene-re-expression can be induced by a variety of DNA demethylating agents.

Recent studies show that genistein may affect DNA methylation, serves as a natural deme‐ thylation agent, and that it is specifically effective on colon cancer cells from early-stage co‐ lon cancer [58]. WNT family members are highly conserved, secreted signaling molecules that play important roles in both tumorigenesis and normal development and differentia‐ tion. Study of Hibi *et al.* evidences that genistein treatment affected the DNA methylation of *WNT5a,* and that *WNT5a* downregulation is correlated with hypermethylation of its promot‐ er in human colon cancer patients [60, 59].

Moreover, genistein may inhibit cancer progression by inducing apoptosis or inhibiting pro‐ liferation, and the mechanisms by which genistein exerts its anti-tumor effects has been the subject of considerable interest [61, 62, 63].

Genistein has been shown to induce epigenetic changes in several cancer cell lines and in the in vivo animal models. [64].

The presence of hydroxylated and methylated genistein metabolites correlated positively with inhibition of cancer cell proliferation, but genistein sulfates were not associated with antiproliferative effects of genistein, suggesting that some types of metabolism of the isofla‐ vones may be crucial for their action.

Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting PTK-mediated signaling mechanisms [65]. Sakla et al. (2007) recently reported that genistein inhibits the protooncogene HER-2 protein tyrosine phosphorylation in breast cancer cells as well as delaying tumor onset in transgenic mice that overexpress the HER-2 gene. These data support its potential anti-cancer role in chemo‐ therapy of breast cancer. However, effects independent of this activity have also been dem‐ onstrated [66, 67].

and proliferation. ERK-1/2 and AKT expressions were unaltered and NF-κB was modestly

Soy isoflavone-mediated arrest of cells at G2/M phase and upregulation of p21 expression were not observed when ER-β gene was silenced. These findings suggest that maintaining the expression of ER-β is crucial in mediating the growth-suppressive effects of soy isofla‐ vones against colon tumors. Thus, upregulation of ER-β status by specific foodborne ER-li‐ gands such as soy isoflavones could potentially be a dietary prevention or therapeutic

In addition to estrogenic/antiestrogenic activity, some mechanisms of action have been iden‐ tified for isoflavone/flavone prevention of cancer: antiproliferation, induction of cell cycle arrest and apoptosis, prevention of oxidation, induction of detoxification enzymes, regula‐ tion of host immune system, and changes in cellular signaling [39, 56, 57]. It is expected that

Gene silencing due to the promoter methylation provides an opportunity for clinical inter‐ vention, as gene-re-expression can be induced by a variety of DNA demethylating agents.

Recent studies show that genistein may affect DNA methylation, serves as a natural deme‐ thylation agent, and that it is specifically effective on colon cancer cells from early-stage co‐ lon cancer [58]. WNT family members are highly conserved, secreted signaling molecules that play important roles in both tumorigenesis and normal development and differentia‐ tion. Study of Hibi *et al.* evidences that genistein treatment affected the DNA methylation of *WNT5a,* and that *WNT5a* downregulation is correlated with hypermethylation of its promot‐

Moreover, genistein may inhibit cancer progression by inducing apoptosis or inhibiting pro‐ liferation, and the mechanisms by which genistein exerts its anti-tumor effects has been the

Genistein has been shown to induce epigenetic changes in several cancer cell lines and in the

The presence of hydroxylated and methylated genistein metabolites correlated positively with inhibition of cancer cell proliferation, but genistein sulfates were not associated with antiproliferative effects of genistein, suggesting that some types of metabolism of the isofla‐

Genistein is a known inhibitor of protein-tyrosine kinase (PTK), which may attenuate the growth of cancer cells by inhibiting PTK-mediated signaling mechanisms [65]. Sakla et al. (2007) recently reported that genistein inhibits the protooncogene HER-2 protein tyrosine phosphorylation in breast cancer cells as well as delaying tumor onset in transgenic mice that overexpress the HER-2 gene. These data support its potential anti-cancer role in chemo‐

upregulated by soy isoflavones after transient knockdown of ER-β expression.

**4. Genistein and isoflavones: Other mechanisms of action**

also combinations of these mechanisms may contribute to cancer prevention.

strategy for colon cancer [55].

84 Soybean - Bio-Active Compounds

er in human colon cancer patients [60, 59].

subject of considerable interest [61, 62, 63].

vones may be crucial for their action.

in vivo animal models. [64].

Soy isoflavone supplemented diets also prevented the development of adenocarcinomas in the prostate and seminal vesicles in a rat carcinogenesis model [68].

Phytoestrogens, present in soy based food, may act through hormonal mechanisms to reduce cancer risk by binding to estrogen receptors (ER) or interacting with enzymes in‐ volved in sex steroid biosynthesis and metabolism [53]. Moreover, genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, and the mecha‐ nisms by which genistein exerts its anti-tumor effects have been the subject of consider‐ able interest [61, 62, 63].

Studies demonstrate that ERβ is highly expressed in superficial and crypt epithelium of the normal colon in both genders. ERβ expression was highly correlated among all cell types in both genders, and the strongest correlation was observed between surface and crypt ERβ ex‐ pression. This finding suggests that there may be an intersubject difference in ERβ expres‐ sion that is manifested in all cell types. ERβ expression was significantly lower in colon cancer cells compared with normal colonic epithelium, and there was a progressive decline in ERβ expression that paralleled the loss of cancer cell differentiation. The present findings are consonant with previous results reported by Foley and colleagues [69], who also detect‐ ed a loss of ERβ protein expression in malignant colon tissue by western immunoblotting. Another immunohistochemical study of ERβ in 55 patients with colorectal adenocarcinomas showed that 32% of all tumors in both genders were ERβ-negative; the 10% cut-off threshold was used to distinguish ERβ-positive from negative tumors [70].

Studies conducted with ER subtype-specific ligands and those performed with estrogen re‐ ceptor b-knockout mice (ERβKOs) have illustrated the involvement of ERβ in cellular antiinflammatory pathways and tissue homeostasis in the colon. These results suggest that ERβspecific ligands may be promising targets in the pharmaceutical and therapeutical treatment of inflammatory bowel disease and the prevention of CRC. ERβKOs suggest that ERβ-spe‐ cific agonists and ERβ-selective phytoestrogens like genistein (GEN) and coumestrol may serve as potential regulators of intestinal tissue homeostasis [71, 72, 73].

Schleipen et al. investigate the influence of ERα and ERβ-specific agonists, and of genestein on cell proliferation and apoptosis of the small intestine and the colon. Recent data indicate that ERβ-specific agonists and GEN inhibit epithelial proliferation of the prostate and mam‐ mary gland, and can even impede prostate cancer development [74, 76, 75]. It can therefore be assumed that ERβ-specific agonists may also inhibit the proliferation of the intestinal epi‐ thelium. To prove this hypothesis in the study, ovariectomized rats were treated with 17β-Estradiol (E2), the phytoestrogen GEN and ER subtype-specific agonists for ERα and ERβ for 3 weeks.

Genistein has been shown to induce epigenetic changes in several cancer cell lines and in *in vivo* animal models [64]. Recent studies show that genistein may affect DNA methylation, serves as a natural demethylation agent, and is specifically effective on colon cancer cells from early-stage colon cancer [58].

WNT family members are highly conserved, secreted signaling molecules that play impor‐ tant roles in both tumorigenesis and normal development and differentiation. Study of Hibi *et al.* evidence that genistein treatment affected the DNA methylation of *WNT5a,* and that *WNT5a* down-regulation is correlated with hypermethylation of its promoter in human co‐ lon cancer patients [59, 60]. Aberrant WNT signaling is considered one of the most correlat‐ ed factors in over 90% of both benign and malignant colorectal tumors [77].

Genistein has been shown to inhibit cancer metastasis through its ability to regulate nearly every step of the metastatic cascade, including cell adhesion, migration invasion, and angiogenesis. The effect of genistein on the metastaic cascade involves many meta‐ stasis suppressor or related signaling pathways, such as NFKappaB. Genistein can affect both of these processes, as well as modulate key regulatory protein such as Akt and nuclear factor κB (NF-κB). In general, low-to-mid micro molar concentrations of genis‐ tein are required for these effects in cell-culture-based models, although, interestingly, effects in animal models have been observed at lower concentrations. Genistein inhibits critical pathways in cancer invasion and can specifically target MEK4. This inhibition re‐ sults in inactivation of the MEK4 pathway, decreased MMP-2 production, and de‐ creased cell invasion. Genistein also activates Smad1, which is activated by the endoglin signaling pathway, and causes decreased cell invasion. Additionally, genistein inhibits FAK activation, resulting in increased cell adhesion. At this time, it is unclear whether the activation of Smad1 and FAK are due to genistein's inhibition of MEK4 or via a

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 87

Several reports have demonstrated that genistein can induce cell cycle arrest and that it can therapeutically modulate key regulator cell cycle proteins at concentrations ranging from 5 to 200 μM [84]. It is important to note that these concentrations are greater than the blood levels that are observed with dietary consumption, indicating that this is likely not the pri‐ mary mechanism by which genistein inhibits metastasis. However, it is theoretically possi‐ ble to achieve these levels in humans, and various animal studies have also demonstrated

Studies by Wentao et al. show that genistein inhibits EGF induced loss of FOXO3 activ‐ ity by targeting the PI3K/ Akt pathway. Downstream, genistein inhibits EGF induced FOXO3 disassociation from p53(mut), which further promotes FOXO3 activity and leads to increased expression of the p27kip1 cell cycle inhibitor, which inhibits proliferation in colon cancer cells. The author demonstrated that one of the anti-proliferative mecha‐ nisms of genistein in colon cancer cells is to promote FOXO3 activity by inhibiting EGFinduced FOXO3 phosphorylation (inactivation) via the PI3K/Akt pathway. Active FOXO3 negatively regulates proliferation of colon cancer cells and shows that its inacti‐

Several studies shown that consumption of fiber, fresh fruit and vegetables, a high-calcium diet could have a protective effect on the increased risk of colorectal cancer, and suggest that much of the suffering and death from cancer could be prevented by consuming a healthy diet, reducing tobacco use, performing regular physical activity, and maintaining an optimal

that genistein can reduce the primary tumor size in certain contexts.

vation is an essential step in EGF-mediated proliferation [85, 86].

different signaling mechanism [83].

**5. Conclusion**

body weight [5].

Many epigenetic silencing and activating events have been discovered in the WNT pathway that are also related to aberrant WNT signaling, including aberrant expression of *sFRP1, DKK1*, and *APC* [78, 79]. Therefore, Wang and Chen investigate the effect of genistein on WNT pathway regulation in colon cancer development [58]. This study showed that: genis‐ tein treatment selectively induced *WNT5a* expression in specific colon cancer cell lines; *WNT5a* showed the lowest expression compared to other more advanced tumor cell lines; and the novel finding that *WNT5a* mRNA expression was upregulated by genistein in this early-stage colon cancer cell line.

These results support the notion that genistein serves as a natural demethylation agent and that it is specifically effective on colon cancer cells from early-stage colon cancer. Genistein treatment affected the DNA methylation of *WNT5a*. It has been shown that *WNT5a* downre‐ gulation is correlated with hypermethylation of its promoter in human colon cancer patients [59, 60].

Wang and Chen studies showed that the time dependent induction of WNT5a by genistein in colon cancer cell line SW 1116 was correlated with decreased methylation of a CpG island within its promoter, as determined by bisulfate sequencing [58].

Demethylation of CpGs inhibition of Dnmt and MBD2 activity, and activation of the histo‐ nes by acetylation and demethylation at the BTG3 promoter followed by genistein treat‐ ment, were observed in renal cancer cells [80]. Using the mouse differential methylation hybridization array, alteration of DNA methylation in specific genes in mice was observed following feeding of a diet containing genistein compared to that in mice fed a control ca‐ sein diet [81].

Other direct evidence that genistein affected DNA methylation was that maternal exposure to dietary genistein altered the epigenome of offspring in viable yellow agouti (Avy/a) mice. Overall, the potential of genistein as an effective epigenome modifier, which may greatly impact CRC metastasis, highlights the potential ability of dietary genistein to improve CRC prognosis [82].

Downregulation by promoter hypermethylation occurs in cell lines from earlier stages of co‐ lon cancer but not in cell lines from later stages.

These findings suggest that maintaining the expression of ER-β is crucial in mediating the growth-suppressive effects of soy isoflavones against colon tumors. Upregulation of ER-β by specific foodborne ER-ligands, such as soy isoflavones, could potentially be a dietary pre‐ vention strategy for colon cancer. [55].

Genistein has been shown to inhibit cancer metastasis through its ability to regulate nearly every step of the metastatic cascade, including cell adhesion, migration invasion, and angiogenesis. The effect of genistein on the metastaic cascade involves many meta‐ stasis suppressor or related signaling pathways, such as NFKappaB. Genistein can affect both of these processes, as well as modulate key regulatory protein such as Akt and nuclear factor κB (NF-κB). In general, low-to-mid micro molar concentrations of genis‐ tein are required for these effects in cell-culture-based models, although, interestingly, effects in animal models have been observed at lower concentrations. Genistein inhibits critical pathways in cancer invasion and can specifically target MEK4. This inhibition re‐ sults in inactivation of the MEK4 pathway, decreased MMP-2 production, and de‐ creased cell invasion. Genistein also activates Smad1, which is activated by the endoglin signaling pathway, and causes decreased cell invasion. Additionally, genistein inhibits FAK activation, resulting in increased cell adhesion. At this time, it is unclear whether the activation of Smad1 and FAK are due to genistein's inhibition of MEK4 or via a different signaling mechanism [83].

Several reports have demonstrated that genistein can induce cell cycle arrest and that it can therapeutically modulate key regulator cell cycle proteins at concentrations ranging from 5 to 200 μM [84]. It is important to note that these concentrations are greater than the blood levels that are observed with dietary consumption, indicating that this is likely not the pri‐ mary mechanism by which genistein inhibits metastasis. However, it is theoretically possi‐ ble to achieve these levels in humans, and various animal studies have also demonstrated that genistein can reduce the primary tumor size in certain contexts.

Studies by Wentao et al. show that genistein inhibits EGF induced loss of FOXO3 activ‐ ity by targeting the PI3K/ Akt pathway. Downstream, genistein inhibits EGF induced FOXO3 disassociation from p53(mut), which further promotes FOXO3 activity and leads to increased expression of the p27kip1 cell cycle inhibitor, which inhibits proliferation in colon cancer cells. The author demonstrated that one of the anti-proliferative mecha‐ nisms of genistein in colon cancer cells is to promote FOXO3 activity by inhibiting EGFinduced FOXO3 phosphorylation (inactivation) via the PI3K/Akt pathway. Active FOXO3 negatively regulates proliferation of colon cancer cells and shows that its inacti‐ vation is an essential step in EGF-mediated proliferation [85, 86].

## **5. Conclusion**

WNT family members are highly conserved, secreted signaling molecules that play impor‐ tant roles in both tumorigenesis and normal development and differentiation. Study of Hibi *et al.* evidence that genistein treatment affected the DNA methylation of *WNT5a,* and that *WNT5a* down-regulation is correlated with hypermethylation of its promoter in human co‐ lon cancer patients [59, 60]. Aberrant WNT signaling is considered one of the most correlat‐

Many epigenetic silencing and activating events have been discovered in the WNT pathway that are also related to aberrant WNT signaling, including aberrant expression of *sFRP1, DKK1*, and *APC* [78, 79]. Therefore, Wang and Chen investigate the effect of genistein on WNT pathway regulation in colon cancer development [58]. This study showed that: genis‐ tein treatment selectively induced *WNT5a* expression in specific colon cancer cell lines; *WNT5a* showed the lowest expression compared to other more advanced tumor cell lines; and the novel finding that *WNT5a* mRNA expression was upregulated by genistein in this

These results support the notion that genistein serves as a natural demethylation agent and that it is specifically effective on colon cancer cells from early-stage colon cancer. Genistein treatment affected the DNA methylation of *WNT5a*. It has been shown that *WNT5a* downre‐ gulation is correlated with hypermethylation of its promoter in human colon cancer patients

Wang and Chen studies showed that the time dependent induction of WNT5a by genistein in colon cancer cell line SW 1116 was correlated with decreased methylation of a CpG island

Demethylation of CpGs inhibition of Dnmt and MBD2 activity, and activation of the histo‐ nes by acetylation and demethylation at the BTG3 promoter followed by genistein treat‐ ment, were observed in renal cancer cells [80]. Using the mouse differential methylation hybridization array, alteration of DNA methylation in specific genes in mice was observed following feeding of a diet containing genistein compared to that in mice fed a control ca‐

Other direct evidence that genistein affected DNA methylation was that maternal exposure to dietary genistein altered the epigenome of offspring in viable yellow agouti (Avy/a) mice. Overall, the potential of genistein as an effective epigenome modifier, which may greatly impact CRC metastasis, highlights the potential ability of dietary genistein to improve CRC

Downregulation by promoter hypermethylation occurs in cell lines from earlier stages of co‐

These findings suggest that maintaining the expression of ER-β is crucial in mediating the growth-suppressive effects of soy isoflavones against colon tumors. Upregulation of ER-β by specific foodborne ER-ligands, such as soy isoflavones, could potentially be a dietary pre‐

within its promoter, as determined by bisulfate sequencing [58].

lon cancer but not in cell lines from later stages.

vention strategy for colon cancer. [55].

ed factors in over 90% of both benign and malignant colorectal tumors [77].

early-stage colon cancer cell line.

86 Soybean - Bio-Active Compounds

[59, 60].

sein diet [81].

prognosis [82].

Several studies shown that consumption of fiber, fresh fruit and vegetables, a high-calcium diet could have a protective effect on the increased risk of colorectal cancer, and suggest that much of the suffering and death from cancer could be prevented by consuming a healthy diet, reducing tobacco use, performing regular physical activity, and maintaining an optimal body weight [5].

Soy is one of the most consumed foods worldwide. Soy foods contain larger amounts of phytoestrogens of which the isoflavon genistein is surely the biologically most important.

This compound, in recent years, has received much attention in the field of oncology re‐ search, as it exerts a wide range of biological effects of direct relevance to cancer.

Phytoestrogens and in particular genistein, have shown to be an important tool for the in‐ hibition of cancer metastasis, exerting effects on both the initial steps of primary tumor growth as well as the later steps of the metastatic cascade.

The international literature suggests that phytoestrogens have potentially a high clinical im‐ pact and the expansion of knowledge on soy, soy foods, and soy products will lead to novel future developments in the field of cancer treatment.

**Figure 1.** Chemical structures of soy phytoestrogens are similar to17 beta estadiol

, E. Bartolini1

IARC Scientific Publications No.143. Lyon: IARC,1997

pincott &W, Philadelphia, 2005, pag 1062

, F. Tonelli2

1 Department of Internal Medicine, School of Medicine, University of Florence, Florence, Italy

2 Department of Clinical Physiopathology, School of Medicine, University of Florence,

3 Department of Neurosciences, Psychology, DrugArea and Child Health, School of Medi‐

[1] Parkin DM, Whelan SL, Ferlay L et al Cancer incidence in five continents, vol VII.

[2] Jemal A, Siegel R, Ward E et al: Cancer statistics, 2006. CA Cancer J Clin 2006;56:107 [3] Whittemore As, Wu-Williams AH, Lee M et al. Diet, physical activity, and colorectal cancer among Chinese in North America and China. J Natl Cancer Inst 1990, 82:915 [4] Lieberman DA, Weiss DG, Bond JH et al. Use of colonoscopy to screen asymptomatic adults for colorectal cancer. Veterans Affairs Cooperative Study Group 380. N Engl J

[5] Libutti SK, Saltz LB, Rustgi AK, Tepper JE. Cancer of the colon in DeVita VT, Hell‐ man S, Rosenberg SA eds Cancer: Principles & Practis of Oncology. 7th Edition, Lip‐

, M.L. Brandi1

and Federica D'Asta3

Phytoestrogens and Colon Cancer http://dx.doi.org/10.5772/54065 89

**Author details**

, C. Mavilia1

cine, University of Florence, Florence, Italy

Med 2000;343:162

B. Pampaloni1

Florence, Italy

**References**


**Table 1.** Isoflavone Content of Selected Soy Foods (USDA Database 2008)

**Figure 1.** Chemical structures of soy phytoestrogens are similar to17 beta estadiol

## **Author details**

Soy is one of the most consumed foods worldwide. Soy foods contain larger amounts of phytoestrogens of which the isoflavon genistein is surely the biologically most important.

This compound, in recent years, has received much attention in the field of oncology re‐

Phytoestrogens and in particular genistein, have shown to be an important tool for the in‐ hibition of cancer metastasis, exerting effects on both the initial steps of primary tumor

The international literature suggests that phytoestrogens have potentially a high clinical im‐ pact and the expansion of knowledge on soy, soy foods, and soy products will lead to novel

**Phytoestrogens in soy foods**

Miso 41.45 Natto 82.29 Roasted soybeans 148.50 Soy beans 154.53 Soy cheese american 17.95 Soy flour (textured) 172.55 Soy milk 10-200 Soy milk curd, dried 83.30 Soy milk fortified or unfortified 10.73 Soy milk skin or film (Foo jook or yuba), cooked 44.67 Soy milk skin or film (Foo jook or yuba), raw 196.05 Soy protein concentrate 94.65 Soy protein drink 81.65 Soy protein isolate 91.05 Soy yogurth 33.17 Tempeh 60.61 Tofu (dried frozen) 83.20 Tofu raw regular with calcium and sulphate 22.73 Tofu yogurt 16.30

**Foods Total isoflavons (mg/100 g)**

search, as it exerts a wide range of biological effects of direct relevance to cancer.

growth as well as the later steps of the metastatic cascade.

**Table 1.** Isoflavone Content of Selected Soy Foods (USDA Database 2008)

future developments in the field of cancer treatment.

88 Soybean - Bio-Active Compounds

B. Pampaloni1 , C. Mavilia1 , E. Bartolini1 , F. Tonelli2 , M.L. Brandi1 and Federica D'Asta3

1 Department of Internal Medicine, School of Medicine, University of Florence, Florence, Italy

2 Department of Clinical Physiopathology, School of Medicine, University of Florence, Florence, Italy

3 Department of Neurosciences, Psychology, DrugArea and Child Health, School of Medi‐ cine, University of Florence, Florence, Italy

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[78] Aguilera O, Fraga MF, Ballestar E, Paz MF, Herranz M, Espada J et al: Epigenetic in‐ activation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal

[79] Suzuki H, Watkins DN, Jair KW, Schuebel KE, Markowitz SD, Chen WD et al: Epige‐ netic inactivation of SFRP genes allows constitutive WNT signaling in colorectal can‐

[80] Shahana Majid, Altaf A.Dar1, Ardalan E.Ahmad, Hiroshi Hirata, Kazumori Kawaka‐ mi, Varahram Shahryari, Sharanjot Saini, Yuichiro Tanaka, Angela V.Dahiya, Gaurav Khatri and Rajvir Dahiya BTG3 tumor suppressor gene promoter demethylation, his‐ tone modification and cell cycle arrest by genistein in renal cancer. Carcinogenesis

[81] Kevin J. Day et al Genistein Alters Methylation Patterns in Mice 2002 American Soci‐

[82] Qian Li and Hong Chen "Epigenetic modifications of metastasis suppressor genes in

[83] Pavese Janet M. & Rebecca L. Farmer & Raymond C. Bergan: Inhibition of cancer cell invasion and metastasis by genistein. Cancer Metastasis Rev (2010) 29:465–482

[84] Ramos, S. (2007). Effects of dietary flavonoids on apoptotic pathways related to can‐ cer chemoprevention. The Journal of Nutritional Biochemistry, 18(7), 427–442

[85] Wentao Qi1, Christopher R Weber2\*, Kaarin Wasland1 and Suzana D Savkovic1: Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity. BMC Cancer 2011,

[86] Dijkers PF, Medema RH, Pals C, Banerji L, Thomas NS, Lam EW, Burgering BM, Raaijmakers JA, Lammers JW, Koenderman L, Coffer PJ: Forkhead transcription fac‐ tor FKHR-L1 modulates cytokine-dependent transcriptional regulation of p27(KIP1).

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**Chapter 5**

**Bowman-Birk Protease Inhibitor as a**

Farinaz Safavi and Abdolmohamad Rostami

Additional information is available at the end of the chapter

**1.1. The Bowman-Birk Protease Inhibitor (BBI)**

cellent targets for treatment of many disorders.

tors that has been widely studied for the past 60 years [3, 4].

date for use as an oral agent for therapeutic purposes.

http://dx.doi.org/10.5772/54066

**1. Introduction**

**Potential Oral Therapy for Multiple Sclerosis**

Legume seeds contain different kinds of proteins and protease inhibitors. Serine proteases are a large sub-group of the protease family [1] and they play a role in various pathological conditions such as cancer and thrombotic and inflammatory diseases [2]. Thus they are ex‐

Various plant species and, in particular, legumes contain a great number of serine protease inhibitors. The Bowman-Birk protease inhibitor belongs to a family of serine protease inhibi‐

The soybean-derived Bowman-Birk protease inhibitor (BBI) is a small protein consisting of 71 amino acids and 7 disulfide bonds [4]. BBI is a double-headed serine protease inhibitor, with two functional active sites at opposite sides of the molecule, which inhibits both trypsin and chymotrypsin-like proteases [1,3] (Figure 1). It is a water-soluble protein that is resistant to acidic conditions and proteolytic enzymes [3]. These characteristics make it a good candi‐

Crude soybean contains a small amount of BBI and may have components that counter some of the beneficial effects of BBI. Bowman-Birk Inhibitor Concentrate (BBIC) is a soybean extract enriched in BBI [5]. Researchers prefer to use BBIC in their studies because a smaller

In rodents, BBI is detectable in the blood, tissue and urine after ingestion [6]. Interestingly, BBI can be detected in the central nervous system (CNS) of animals even when the blood-

> © 2013 Safavi and Rostami; 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.

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 Safavi and Rostami; licensee InTech. This is a paper distributed under the terms of the Creative Commons

amount of BBIC contains the proposed dose of BBI compared to crude soybean.

**Chapter 5**

## **Bowman-Birk Protease Inhibitor as a Potential Oral Therapy for Multiple Sclerosis**

Farinaz Safavi and Abdolmohamad Rostami

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54066

**1. Introduction**

### **1.1. The Bowman-Birk Protease Inhibitor (BBI)**

Legume seeds contain different kinds of proteins and protease inhibitors. Serine proteases are a large sub-group of the protease family [1] and they play a role in various pathological conditions such as cancer and thrombotic and inflammatory diseases [2]. Thus they are ex‐ cellent targets for treatment of many disorders.

Various plant species and, in particular, legumes contain a great number of serine protease inhibitors. The Bowman-Birk protease inhibitor belongs to a family of serine protease inhibi‐ tors that has been widely studied for the past 60 years [3, 4].

The soybean-derived Bowman-Birk protease inhibitor (BBI) is a small protein consisting of 71 amino acids and 7 disulfide bonds [4]. BBI is a double-headed serine protease inhibitor, with two functional active sites at opposite sides of the molecule, which inhibits both trypsin and chymotrypsin-like proteases [1,3] (Figure 1). It is a water-soluble protein that is resistant to acidic conditions and proteolytic enzymes [3]. These characteristics make it a good candi‐ date for use as an oral agent for therapeutic purposes.

Crude soybean contains a small amount of BBI and may have components that counter some of the beneficial effects of BBI. Bowman-Birk Inhibitor Concentrate (BBIC) is a soybean extract enriched in BBI [5]. Researchers prefer to use BBIC in their studies because a smaller amount of BBIC contains the proposed dose of BBI compared to crude soybean.

In rodents, BBI is detectable in the blood, tissue and urine after ingestion [6]. Interestingly, BBI can be detected in the central nervous system (CNS) of animals even when the blood-

brain barrier is intact. In human studies, although the BBI level could not be detected in blood after oral BBIC dosing, it could be measured in urine [6].

Activated antigen presenting cells (APCs) and auto-reactive T cells produce pro-inflamma‐ tory cytokines, including IL-23, IFN-γ, TNF-α, IL-17, that enhance cell-mediated immunity in the CNS [15-17]. Conversely, other cytokines, such as IL-10, IL-27, IL-4 and TGF-β, play

Bowman-Birk Protease Inhibitor as a Potential Oral Therapy for Multiple Sclerosis

http://dx.doi.org/10.5772/54066

99

Despite extensive research, only a few pharmacotherapeutic agents (e.g., IFN-β, glatiramer acetate, and mitoxantrone) are available, all of which are administered by injection, demon‐ strate mild to moderate efficacy and have potential side effects [21,22]. A new oral therapeu‐ tic agent (Fingolimod) was approved by the FDA and shows potential benefits in MS

Recently, in two phase three clinical trials, BG-12 (dimethyl fumarate), a newly proposed or‐ al drug for the treatment of multiple sclerosis, showed a significant reduction in relapse rate and number of MRI lesions in treated patients compared to the placebo group [68, 69].

Development of a new, effective and oral therapy for MS, with fewer side effects, is there‐

Experimental Autoimmune Encephalomyelitis (EAE) is an autoimmune animal model of MS. Immunization with myelin peptides in different strains of mice induces chronic or re‐ lapsing types of the disease, which makes EAE a good tool for studying disease mechanisms and testing therapeutic agents [24] To date, three of the four therapies currently approved

After immunization with myelin protein, APCs present myelin on the surface of MHC II and produce pro-inflammatory cytokines. Dendritic cell-derived IL-12 and IL-23 lead to de‐ velopment of myelin-specific Th1 and Th17 cells, respectively. Th1 and Th17 cells are the two main culprits in pathogenesis of EAE and MS [25]. Auto-reactive T cells enter the CNS and facilitate recruitment of other immune cells such as monocytes and neutrophils. Accu‐ mulation of inflammatory cells within the CNS promotes myelin damage, axonal loss and

Recently it has been shown that dendritic cells are also able to produce another cytokine from the IL-12 family called IL-27. Compared with IL-12 and IL-23, IL-27 elicits different im‐ munoregulatory effects. IL-27 inhibits encephalitogenicity of T cells and suppresses EAE disease [26]. In addition, it stimulates IL-10 production in T cells and induces Tr1 cells [17]. IL-10 is a widely studied immunoregulatory cytokine, which virtually all immune cells are able, in different conditions, to release and which suppresses inflammatory response [27].

In general, if a therapeutic agent is able to stimulate IL-10 production and Tr1 cells, it could

an immunoregulatory role and may be protective in MS [17-20].

**3. Experimental Autoimmune Encephalomyelitis (EAE)**

for MS were first tested in this animal model [24].

clinical manifestations in affected animals [24].

be an excellent candidate for MS therapy.

IL-10 also plays a significant role in suppression of EAE [28-30].

patients [23].

fore desirable.

**Figure 1.** Crystal structure of soybean-derived Bowman-Birk protease inhibitor

The ability of certain serine protease inhibitors to prevent the malignant transformation of cells was shown two decades ago [7, 8]. BBI prevents/suppresses carcinogenesis in a variety of in vitro and in vivo systems [8, 9].

Several human clinical trials to evaluate the effect of BBIC have been completed or are in progress [10-12]. To date, in completed clinical trials, neither toxicity nor neutralizing anti‐ bodies against BBIC have been reported in patients receiving BBIC [9].

## **2. Multiple Sclerosis (MS)**

MS is the second cause of disability in young adults and is considered to be a demyelinating disease of the central nervous system (CNS) along with chronic inflammation, demyelina‐ tion and gliosis [13]. Lesions are characterized by periventricular cuffing and infiltration consisting mainly of T lymphocytes and macrophages, leading to myelin destruction. Re‐ cently neuronal degeneration and axonal involvement have also been shown in MS lesions [14]. Current findings therefore raise some doubts about the original assumption that MS is exclusively a white matter disease.

Based on the MS inflammatory phenotype, it has been considered an autoimmune disorder in which peripherally activated myelin-reactive T cells enter the CNS and begin an immuno‐ logic cascade that subsequently causes myelin damage.

Activated antigen presenting cells (APCs) and auto-reactive T cells produce pro-inflamma‐ tory cytokines, including IL-23, IFN-γ, TNF-α, IL-17, that enhance cell-mediated immunity in the CNS [15-17]. Conversely, other cytokines, such as IL-10, IL-27, IL-4 and TGF-β, play an immunoregulatory role and may be protective in MS [17-20].

Despite extensive research, only a few pharmacotherapeutic agents (e.g., IFN-β, glatiramer acetate, and mitoxantrone) are available, all of which are administered by injection, demon‐ strate mild to moderate efficacy and have potential side effects [21,22]. A new oral therapeu‐ tic agent (Fingolimod) was approved by the FDA and shows potential benefits in MS patients [23].

Recently, in two phase three clinical trials, BG-12 (dimethyl fumarate), a newly proposed or‐ al drug for the treatment of multiple sclerosis, showed a significant reduction in relapse rate and number of MRI lesions in treated patients compared to the placebo group [68, 69].

Development of a new, effective and oral therapy for MS, with fewer side effects, is there‐ fore desirable.

## **3. Experimental Autoimmune Encephalomyelitis (EAE)**

brain barrier is intact. In human studies, although the BBI level could not be detected in

The ability of certain serine protease inhibitors to prevent the malignant transformation of cells was shown two decades ago [7, 8]. BBI prevents/suppresses carcinogenesis in a variety

Several human clinical trials to evaluate the effect of BBIC have been completed or are in progress [10-12]. To date, in completed clinical trials, neither toxicity nor neutralizing anti‐

MS is the second cause of disability in young adults and is considered to be a demyelinating disease of the central nervous system (CNS) along with chronic inflammation, demyelina‐ tion and gliosis [13]. Lesions are characterized by periventricular cuffing and infiltration consisting mainly of T lymphocytes and macrophages, leading to myelin destruction. Re‐ cently neuronal degeneration and axonal involvement have also been shown in MS lesions [14]. Current findings therefore raise some doubts about the original assumption that MS is

Based on the MS inflammatory phenotype, it has been considered an autoimmune disorder in which peripherally activated myelin-reactive T cells enter the CNS and begin an immuno‐

blood after oral BBIC dosing, it could be measured in urine [6].

98 Soybean - Bio-Active Compounds

**Figure 1.** Crystal structure of soybean-derived Bowman-Birk protease inhibitor

bodies against BBIC have been reported in patients receiving BBIC [9].

of in vitro and in vivo systems [8, 9].

**2. Multiple Sclerosis (MS)**

exclusively a white matter disease.

logic cascade that subsequently causes myelin damage.

Experimental Autoimmune Encephalomyelitis (EAE) is an autoimmune animal model of MS. Immunization with myelin peptides in different strains of mice induces chronic or re‐ lapsing types of the disease, which makes EAE a good tool for studying disease mechanisms and testing therapeutic agents [24] To date, three of the four therapies currently approved for MS were first tested in this animal model [24].

After immunization with myelin protein, APCs present myelin on the surface of MHC II and produce pro-inflammatory cytokines. Dendritic cell-derived IL-12 and IL-23 lead to de‐ velopment of myelin-specific Th1 and Th17 cells, respectively. Th1 and Th17 cells are the two main culprits in pathogenesis of EAE and MS [25]. Auto-reactive T cells enter the CNS and facilitate recruitment of other immune cells such as monocytes and neutrophils. Accu‐ mulation of inflammatory cells within the CNS promotes myelin damage, axonal loss and clinical manifestations in affected animals [24].

Recently it has been shown that dendritic cells are also able to produce another cytokine from the IL-12 family called IL-27. Compared with IL-12 and IL-23, IL-27 elicits different im‐ munoregulatory effects. IL-27 inhibits encephalitogenicity of T cells and suppresses EAE disease [26]. In addition, it stimulates IL-10 production in T cells and induces Tr1 cells [17]. IL-10 is a widely studied immunoregulatory cytokine, which virtually all immune cells are able, in different conditions, to release and which suppresses inflammatory response [27]. IL-10 also plays a significant role in suppression of EAE [28-30].

In general, if a therapeutic agent is able to stimulate IL-10 production and Tr1 cells, it could be an excellent candidate for MS therapy.

## **4. Proteases in inflammation**

Several proteases are associated with the pathogenesis of inflammatory disorders [24, 31]. Proteolytic enzymes are involved in activation and migration of immune cells, cytokine and chemokine activation/inactivation and complement function [32].

lates migration of lymphocytes and purified T cells, and BBI inhibits this enzyme quite efficiently [49]. In addition, BBI significantly suppresses the chemotactic activity of chymase,

Bowman-Birk Protease Inhibitor as a Potential Oral Therapy for Multiple Sclerosis

http://dx.doi.org/10.5772/54066

101

Stimulated human polymorphonuclear leukocytes produce reactive oxygen species (super‐ oxide and hydrogen peroxide) that may damage cell membranes by reacting with phoso‐ pholipids to form peroxides [52]. BBI is able to suppress the production of reactive oxygen species and inhibits their destructive effects [53]. Macrophage-derived proteases and free radicals are also associated with inflammation. BBI down-regulates NO and PGE2 inflam‐ matory pathways in LPS-activated macrophages [54]. Activated macrophages also induce neurotoxicity in the CNS. Anti-inflammatory effects of BBI prevent macrophage-induced

Serine protease inhibitors can prevent conversion of pro-MMPs to enzymatically active forms [56, 57]. BBI inhibits generation of active MMP-1 and MMP-9 in vitro, and BBIC re‐

The aforementioned mechanisms may be particularly relevant in the context of the patho‐

BBI may have significant immunomodulatory effects and can be an excellent potential can‐

**7. BBI and other protease inhibitors in treatment of inflammatory disease**

The role of proteases in inflammation has been reviewed in previous sections. Based on the fact that proteases are actively involved in inflammation, they can be a good therapeutic tar‐ get in suppression of inflammatory response and treatment of inflammatory diseases.

RWJ-355871 is a synthetic protease inhibitor that effectively suppresses allergic inflammato‐ ry diseases of the respiratory system [59]. 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) is another protease inhibitor that attenuates ovalbumin-induced allergic airway in‐

Several studies have reported that protease inhibitors diminish inflammatory response in in‐ flammatory bowel diseases [1]. Nafamostat is a serine protease inhibitor that suppresses dextran sulfate sodium-induced colitis and diminishes inflammatory infiltration in the colon [61]. BBI is able to suppress gland inflammation in the gastrointestinal tract and shows a strong anti-inflammatory effect in the acute colitis model [62]. In addition, in a completed clinical trial [12], BBI demonstrates anti-inflammatory effects and a degree of amelioration of clinical disease and remission rate in patients with ulcerative colitis. We have also shown that administration of oral BBIC significantly inhibits experimental autoimmune neuritis

All of the above findings show the potential immunomodulatory and therapeutic effect of

duces MMP-2 and -9 activity in supernatants of spleen cells [58].

genesis of multiple sclerosis and myelin destruction in the CNS.

didate for treatment of inflammatory and autoimmune diseases.

flammation in its animal model [60].

(EAN) in rats [63, 64].

BBI in autoimmune diseases.

thus suppressing lymphocyte migration [51].

neurotoxicity [55].

Various studies demonstrate that neutrophil serine proteases induce proinflammatory activ‐ ity of both IL-32 and IL-33 cytokines [33, 34]. They are also able to convert inactive forms of IL-1 and IL-18 to the active form of these cytokines [35]. Cytotoxic T cell-derived proteases called granzymes are also involved in inflammation. Granzymes promote T cell entry into the site of inflammation. In addition, they stimulate B cell proliferation [36].

The complement cascade contains different enzymes that activate each other and proteases that play a role in initiation of the cascade, which results in formation of the membrane at‐ tack complex [37].

In general, proteases are involved in all aspects of the immune response and play a signifi‐ cant role in inflammation.

## **5. Proteases in pathogenesis of EAE and MS**

Modulators of neuronal and endogenous proteolysis show a different pattern in spinal cords of EAE rats compared to control animals. This finding indicates higher activity of some pro‐ teases in EAE than in control groups, which makes specific proteases good potential bio‐ markers for disease activity or therapeutic targets in the EAE model and MS [38]. Various types of proteases, including lysosomal proteases and matrix metalloproteinases (MMPs), are highly expressed in MS lesions [24, 39-42]. Serine proteases such as plasmin, cathepsin G, chymase and trypsin activate inert MMP proenzymes to their active forms [24, 41, 42].

GelatinaseB (MMP-9] increases the number of leukocytes entering the site of inflammation and promotes myelin breakdown [39, 43]. Plasmin is a serine protease that mainly partici‐ pates in the coagulation cascade. It has been demonstrated that plasmin directly induces myelin destruction and demyelination [44].

Levels of gelatinase and tissue plasminogen activator (t-PA) are also increased in MS lesions and in the cerebrospinal fluid (CSF) of active MS patients [46, 47]. Reactive astrocytes and infiltrating lymphocytes, macrophages and microglia express MMP-2, MMP-9 and t-PA in early active MS plaques [24, 41, 45, 47].

## **6. Anti-inflammatory effects of BBI**

BBI suppresses the function of several proteases such as leukocyte elastase, trypsin and hu‐ man cathepsin G released from human inflammatory cells. [48-50]. Mast cell chymase stimu‐

lates migration of lymphocytes and purified T cells, and BBI inhibits this enzyme quite efficiently [49]. In addition, BBI significantly suppresses the chemotactic activity of chymase, thus suppressing lymphocyte migration [51].

**4. Proteases in inflammation**

100 Soybean - Bio-Active Compounds

tack complex [37].

cant role in inflammation.

Several proteases are associated with the pathogenesis of inflammatory disorders [24, 31]. Proteolytic enzymes are involved in activation and migration of immune cells, cytokine and

Various studies demonstrate that neutrophil serine proteases induce proinflammatory activ‐ ity of both IL-32 and IL-33 cytokines [33, 34]. They are also able to convert inactive forms of IL-1 and IL-18 to the active form of these cytokines [35]. Cytotoxic T cell-derived proteases called granzymes are also involved in inflammation. Granzymes promote T cell entry into

The complement cascade contains different enzymes that activate each other and proteases that play a role in initiation of the cascade, which results in formation of the membrane at‐

In general, proteases are involved in all aspects of the immune response and play a signifi‐

Modulators of neuronal and endogenous proteolysis show a different pattern in spinal cords of EAE rats compared to control animals. This finding indicates higher activity of some pro‐ teases in EAE than in control groups, which makes specific proteases good potential bio‐ markers for disease activity or therapeutic targets in the EAE model and MS [38]. Various types of proteases, including lysosomal proteases and matrix metalloproteinases (MMPs), are highly expressed in MS lesions [24, 39-42]. Serine proteases such as plasmin, cathepsin G, chymase and trypsin activate inert MMP proenzymes to their active forms [24, 41, 42].

GelatinaseB (MMP-9] increases the number of leukocytes entering the site of inflammation and promotes myelin breakdown [39, 43]. Plasmin is a serine protease that mainly partici‐ pates in the coagulation cascade. It has been demonstrated that plasmin directly induces

Levels of gelatinase and tissue plasminogen activator (t-PA) are also increased in MS lesions and in the cerebrospinal fluid (CSF) of active MS patients [46, 47]. Reactive astrocytes and infiltrating lymphocytes, macrophages and microglia express MMP-2, MMP-9 and t-PA in

BBI suppresses the function of several proteases such as leukocyte elastase, trypsin and hu‐ man cathepsin G released from human inflammatory cells. [48-50]. Mast cell chymase stimu‐

chemokine activation/inactivation and complement function [32].

**5. Proteases in pathogenesis of EAE and MS**

myelin destruction and demyelination [44].

early active MS plaques [24, 41, 45, 47].

**6. Anti-inflammatory effects of BBI**

the site of inflammation. In addition, they stimulate B cell proliferation [36].

Stimulated human polymorphonuclear leukocytes produce reactive oxygen species (super‐ oxide and hydrogen peroxide) that may damage cell membranes by reacting with phoso‐ pholipids to form peroxides [52]. BBI is able to suppress the production of reactive oxygen species and inhibits their destructive effects [53]. Macrophage-derived proteases and free radicals are also associated with inflammation. BBI down-regulates NO and PGE2 inflam‐ matory pathways in LPS-activated macrophages [54]. Activated macrophages also induce neurotoxicity in the CNS. Anti-inflammatory effects of BBI prevent macrophage-induced neurotoxicity [55].

Serine protease inhibitors can prevent conversion of pro-MMPs to enzymatically active forms [56, 57]. BBI inhibits generation of active MMP-1 and MMP-9 in vitro, and BBIC re‐ duces MMP-2 and -9 activity in supernatants of spleen cells [58].

The aforementioned mechanisms may be particularly relevant in the context of the patho‐ genesis of multiple sclerosis and myelin destruction in the CNS.

BBI may have significant immunomodulatory effects and can be an excellent potential can‐ didate for treatment of inflammatory and autoimmune diseases.

## **7. BBI and other protease inhibitors in treatment of inflammatory disease**

The role of proteases in inflammation has been reviewed in previous sections. Based on the fact that proteases are actively involved in inflammation, they can be a good therapeutic tar‐ get in suppression of inflammatory response and treatment of inflammatory diseases.

RWJ-355871 is a synthetic protease inhibitor that effectively suppresses allergic inflammato‐ ry diseases of the respiratory system [59]. 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF) is another protease inhibitor that attenuates ovalbumin-induced allergic airway in‐ flammation in its animal model [60].

Several studies have reported that protease inhibitors diminish inflammatory response in in‐ flammatory bowel diseases [1]. Nafamostat is a serine protease inhibitor that suppresses dextran sulfate sodium-induced colitis and diminishes inflammatory infiltration in the colon [61]. BBI is able to suppress gland inflammation in the gastrointestinal tract and shows a strong anti-inflammatory effect in the acute colitis model [62]. In addition, in a completed clinical trial [12], BBI demonstrates anti-inflammatory effects and a degree of amelioration of clinical disease and remission rate in patients with ulcerative colitis. We have also shown that administration of oral BBIC significantly inhibits experimental autoimmune neuritis (EAN) in rats [63, 64].

All of the above findings show the potential immunomodulatory and therapeutic effect of BBI in autoimmune diseases.

## **8. Immunoregulatory effect of BBI in the EAE model**

We have shown that oral treatment of BBIC in MBP-induced EAE in rats, reduces disease severity from clinical score 3 (complete hind limb paralysis) to less than 1 (flaccid tail) com‐ pared to control animals. In addition, BBIC treatment significantly diminished demyelina‐ tion in the peripheral nerve tissue of treated animals [58]. We have also shown that both BBI and BBIC suppress clinical and pathologic manifestations of chronic and relapsing EAE in B6 and SJL mice. In addition, the therapeutic effect of oral BBI is dose-dependent, and oral administration of higher amounts of BBI inhibits EAE more efficiently [65] (Figure 1).

BBI in EAE in WT and IL-10 KO mice. Although BBI-treated WT mice showed less severe disease, BBI treatment did not affect clinical disease in BBI-treated IL-10 KO mice compared

Bowman-Birk Protease Inhibitor as a Potential Oral Therapy for Multiple Sclerosis

http://dx.doi.org/10.5772/54066

103

**Figure 3.** Effect of oral BBI compared to PBS treatment in EAE in WT and IL-10 KO mice. Although BBI-treated WT mice showed significantly less severe disease compared to the control group, there was no significant difference in treated and control IL-10 KO mice. Figure reproduced from Dai et al., 2012 with the kind permission of Elsevier Publications Ltd.

Different types of immune cells can release IL-10 cytokine [27]. However, BBI treatment in‐

lished data), demonstrating that BBI has a strong ability to activate IL-10 producing pathways in T cells. Exploring these underlying mechanisms will be a major focus of our fu‐

**Figure 4.** Higher expression of Foxp3+ Treg cells in CNS infiltrating cells after oral treatment with BBI, Figure repro‐

Treg cells are a subgroup of CD4+ T cells that expresses the Foxp3+ transcription factor. They produce IL-10 in the CNS and can suppress EAE disease [67]. Oral administration of BBI al‐

duced from Dai et al., 2012 with the kind permission of Elsevier Publications Ltd.

T cells [66]; it increases IL-10 production in CD8+

T cells (unpub‐

to the control group [66] (Figure 3).

duces IL-10 mainly in CD4+

ture studies.

BBI treatment also decreased pathogenicity of myelin-reactive T cells and induced milder disease in the adoptively transferred EAE model (unpublished data).

**Figure 2.** Effect of oral BBI compared to PBS treatment in EAE. Mice that received BBI showed significantly less severe disease compared to control group. The therapeutic effect of BBI is dose-dependent. Elsevier Publications Ltd. has kindly granted us permission to reproduce this figure from Touil et al., 2008.

BBI inhibits invasion of immune cells through the blood-brain barrier (BBB). BBI-treated mice showed dramatically lower numbers of CNS-infiltrating MNCs than control animals [58, 65, 66]. In addition, BBI suppresses generation of active MMP-1 and MMP-9 in vitro, and BBIC reduces MMP-2 and -9 activity in supernatants of spleen cells [58]. Consistent with other findings, BBI decreased migration of splenocytes in Boyden's chamber assay [65].

However, BBI may inhibit release of active MMP-2 and MMP-9 at the blood-brain barrier and prevent immune cell infiltration into the CNS; it might decrease expression of adhesion molecules on immune cells or invasiveness of immune cells, resulting in an altered cytokine pattern of inflammatory cells that hinders their migration from peripheral immune organs to the site of inflammation.

In order to clarify immunoregulatory mechanisms of BBI, the direct effect of BBI on immune cells was evaluated, and it was shown that splenocytes produce a higher amount of IL-10 following BBI treatment [65, 66]. Several reports have demonstrated the immunoregulatory effect of IL-10 in the EAE model of multiple sclerosis. To determine whether the immuno‐ modulatory effect of BBI depends on IL-10, we have compared the therapeutic effect of oral BBI in EAE in WT and IL-10 KO mice. Although BBI-treated WT mice showed less severe disease, BBI treatment did not affect clinical disease in BBI-treated IL-10 KO mice compared to the control group [66] (Figure 3).

**8. Immunoregulatory effect of BBI in the EAE model**

102 Soybean - Bio-Active Compounds

disease in the adoptively transferred EAE model (unpublished data).

kindly granted us permission to reproduce this figure from Touil et al., 2008.

to the site of inflammation.

We have shown that oral treatment of BBIC in MBP-induced EAE in rats, reduces disease severity from clinical score 3 (complete hind limb paralysis) to less than 1 (flaccid tail) com‐ pared to control animals. In addition, BBIC treatment significantly diminished demyelina‐ tion in the peripheral nerve tissue of treated animals [58]. We have also shown that both BBI and BBIC suppress clinical and pathologic manifestations of chronic and relapsing EAE in B6 and SJL mice. In addition, the therapeutic effect of oral BBI is dose-dependent, and oral

BBI treatment also decreased pathogenicity of myelin-reactive T cells and induced milder

**Figure 2.** Effect of oral BBI compared to PBS treatment in EAE. Mice that received BBI showed significantly less severe disease compared to control group. The therapeutic effect of BBI is dose-dependent. Elsevier Publications Ltd. has

BBI inhibits invasion of immune cells through the blood-brain barrier (BBB). BBI-treated mice showed dramatically lower numbers of CNS-infiltrating MNCs than control animals [58, 65, 66]. In addition, BBI suppresses generation of active MMP-1 and MMP-9 in vitro, and BBIC reduces MMP-2 and -9 activity in supernatants of spleen cells [58]. Consistent with other findings, BBI decreased migration of splenocytes in Boyden's chamber assay [65]. However, BBI may inhibit release of active MMP-2 and MMP-9 at the blood-brain barrier and prevent immune cell infiltration into the CNS; it might decrease expression of adhesion molecules on immune cells or invasiveness of immune cells, resulting in an altered cytokine pattern of inflammatory cells that hinders their migration from peripheral immune organs

In order to clarify immunoregulatory mechanisms of BBI, the direct effect of BBI on immune cells was evaluated, and it was shown that splenocytes produce a higher amount of IL-10 following BBI treatment [65, 66]. Several reports have demonstrated the immunoregulatory effect of IL-10 in the EAE model of multiple sclerosis. To determine whether the immuno‐ modulatory effect of BBI depends on IL-10, we have compared the therapeutic effect of oral

administration of higher amounts of BBI inhibits EAE more efficiently [65] (Figure 1).

**Figure 3.** Effect of oral BBI compared to PBS treatment in EAE in WT and IL-10 KO mice. Although BBI-treated WT mice showed significantly less severe disease compared to the control group, there was no significant difference in treated and control IL-10 KO mice. Figure reproduced from Dai et al., 2012 with the kind permission of Elsevier Publications Ltd.

Different types of immune cells can release IL-10 cytokine [27]. However, BBI treatment in‐ duces IL-10 mainly in CD4+ T cells [66]; it increases IL-10 production in CD8+ T cells (unpub‐ lished data), demonstrating that BBI has a strong ability to activate IL-10 producing pathways in T cells. Exploring these underlying mechanisms will be a major focus of our fu‐ ture studies.

**Figure 4.** Higher expression of Foxp3+ Treg cells in CNS infiltrating cells after oral treatment with BBI, Figure repro‐ duced from Dai et al., 2012 with the kind permission of Elsevier Publications Ltd.

Treg cells are a subgroup of CD4+ T cells that expresses the Foxp3+ transcription factor. They produce IL-10 in the CNS and can suppress EAE disease [67]. Oral administration of BBI al‐ so induces Treg cells in the CNS, which might be one of the underlying mechanisms of the therapeutic effect of BBI in EAE [66] (Figure 4)

[4] Odani S, Ikenaka T. Studies on soybean trypsin inhibitors. 8. Disulfide bridges in

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[11] Armstrong WB, Kennedy AR, Wan XS, Taylor TH, Nguyen QA, Jensen J, et al. Clini‐ cal modulation of oral leukoplakia and protease activity by Bowman-Birk inhibitor concentrate in a phase IIa chemoprevention trial. Clin Cancer Res. 2000;6:4684-4691.

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soybean Bowman-Birk proteinase inhibitor. J Biochem. 1973;74:697-715.

Plenum Press, New York. 9-64.

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BBI also induces IL-10 in other types of effector T cells, and the immunomodulatory effect of BBI might be related to an increase in Tr1 cells. Should this be the case, BBI can be used to in‐ duce regulatory T cells and for treatment of autoimmune diseases such as multiple sclerosis.

## **9. Conclusion**

BBI is a soybean-derived serine protease inhibitor. It can be administered orally with several immunomodulatory characteristics and no major side effects. Our observations have shown that BBI dramatically decreases severity of EAE and that its therapeutic effect is mediated through IL-10. In addition, BBI decreases infiltration of inflammatory cells across the BBB and inflammation in the CNS. BBI has potential as a safe and effective oral therapy for mul‐ tiple sclerosis and other autoimmune diseases.

## **Acknowledgments**

This study was supported by a grant from the National Institutes of Health (NIH 1R01AT005322). We are very grateful to Katherine Regan for editing the chapter.

## **Author details**

Farinaz Safavi and Abdolmohamad Rostami

Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA

## **References**


[4] Odani S, Ikenaka T. Studies on soybean trypsin inhibitors. 8. Disulfide bridges in soybean Bowman-Birk proteinase inhibitor. J Biochem. 1973;74:697-715.

so induces Treg cells in the CNS, which might be one of the underlying mechanisms of the

BBI also induces IL-10 in other types of effector T cells, and the immunomodulatory effect of BBI might be related to an increase in Tr1 cells. Should this be the case, BBI can be used to in‐ duce regulatory T cells and for treatment of autoimmune diseases such as multiple sclerosis.

BBI is a soybean-derived serine protease inhibitor. It can be administered orally with several immunomodulatory characteristics and no major side effects. Our observations have shown that BBI dramatically decreases severity of EAE and that its therapeutic effect is mediated through IL-10. In addition, BBI decreases infiltration of inflammatory cells across the BBB and inflammation in the CNS. BBI has potential as a safe and effective oral therapy for mul‐

This study was supported by a grant from the National Institutes of Health (NIH

[1] Clemente A, Sonnante G, Domoney C. Bowman-Birk inhibitors from legumes and human gastrointestinal health: current status and perspectives. Curr Protein Pept Sci.

[2] Losso JN. The biochemical and functional food properties of the bowman-birk inhibi‐ tor. Crit Rev Food Sci Nutr. 2008;48:94-118. doi: 10.1080/10408390601177589.

[3] Birk Y. The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soy‐

1R01AT005322). We are very grateful to Katherine Regan for editing the chapter.

Department of Neurology, Thomas Jefferson University, Philadelphia, PA, USA

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Farinaz Safavi and Abdolmohamad Rostami

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**9. Conclusion**

104 Soybean - Bio-Active Compounds

**Acknowledgments**

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controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N Engl J Med. 2012 Sep 20; 367[12]:1098-107.

**Chapter 6**

bbls [4] -re‐

**Soybean Oil Derivatives**

Joanna McFarlane

**1. Introduction**

http://dx.doi.org/10.5772/54067

last few years, but was 241x106

bean derived products.

**for Fuel and Chemical Feedstocks**

Additional information is available at the end of the chapter

Plant-based sources of hydrocarbons are being considered as alternatives to petrochemicals because of the need to conserve petroleum resources for reasons of national security and cli‐ mate change [1]. Changes in fuel formulations to include ethanol from corn sugar and meth‐ yl esters from agricultural products are examples of this policy in the United States and elsewhere as biofuels from efficiently grown and processed biomass are claimed to be car‐ bon neutral. In the United States, the mandate to include biofuels has been implemented as the Renewable Fuels Standards (RFS1 and RFS2) [2] with biobased diesel fuel as one of the categories. The production of biodiesel in the United States has varied considerably over the

that still only represents 2% of the total volume of diesel fuel produced for heating and vehi‐ cles [3]. Most of the biodiesel comes from soybean oil, more than double the contribution of

Replacements for commodity chemicals are also being considered, as this value stream represents much of the profit for the oil industry and one that would be affected by short‐ ages in oil or other fossil fuels. While the discovery of large amounts of natural gas asso‐ ciated with oil shale deposits have reduced this as an immediate concern for instance the

search into bio-based feedstock materials continues for the expected long-term benefit. In particular, this chapter reviews a literature on the conversion of bio-based extracts to hy‐ drocarbons for fuels and for building block commodity chemicals, with a focus on soy‐

> © 2013 McFarlane; 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,

© 2013 McFarlane; 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.

distribution, and reproduction in any medium, provided the original work is properly cited.

the other major feedstocks combined: canola oil, yellow grease, and tallow.

estimated recoverable reserves in the Western US have now reached 800x109

gallons in the first quarter of 2012, a high number but one

[69] Fox RJ, Miller DH, Phillips JT, Hutchinson M, Havrdova E, Kita M, Yang M, Raghu‐ pathi K, Novas M, Sweetser MT, Viglietta V, Dawson KT; CONFIRM Study Investi‐ gators.Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple sclerosis. N Engl J Med. 2012 Sep 20;367[12]:1087-97.

## **Chapter 6**

## **Soybean Oil Derivatives for Fuel and Chemical Feedstocks**

## Joanna McFarlane

controlled phase 3 study of oral BG-12 for relapsing multiple sclerosis. N Engl J Med.

[69] Fox RJ, Miller DH, Phillips JT, Hutchinson M, Havrdova E, Kita M, Yang M, Raghu‐ pathi K, Novas M, Sweetser MT, Viglietta V, Dawson KT; CONFIRM Study Investi‐ gators.Placebo-controlled phase 3 study of oral BG-12 or glatiramer in multiple

2012 Sep 20; 367[12]:1098-107.

110 Soybean - Bio-Active Compounds

sclerosis. N Engl J Med. 2012 Sep 20;367[12]:1087-97.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54067

## **1. Introduction**

Plant-based sources of hydrocarbons are being considered as alternatives to petrochemicals because of the need to conserve petroleum resources for reasons of national security and cli‐ mate change [1]. Changes in fuel formulations to include ethanol from corn sugar and meth‐ yl esters from agricultural products are examples of this policy in the United States and elsewhere as biofuels from efficiently grown and processed biomass are claimed to be car‐ bon neutral. In the United States, the mandate to include biofuels has been implemented as the Renewable Fuels Standards (RFS1 and RFS2) [2] with biobased diesel fuel as one of the categories. The production of biodiesel in the United States has varied considerably over the last few years, but was 241x106 gallons in the first quarter of 2012, a high number but one that still only represents 2% of the total volume of diesel fuel produced for heating and vehi‐ cles [3]. Most of the biodiesel comes from soybean oil, more than double the contribution of the other major feedstocks combined: canola oil, yellow grease, and tallow.

Replacements for commodity chemicals are also being considered, as this value stream represents much of the profit for the oil industry and one that would be affected by short‐ ages in oil or other fossil fuels. While the discovery of large amounts of natural gas asso‐ ciated with oil shale deposits have reduced this as an immediate concern for instance the estimated recoverable reserves in the Western US have now reached 800x109 bbls [4] -re‐ search into bio-based feedstock materials continues for the expected long-term benefit. In particular, this chapter reviews a literature on the conversion of bio-based extracts to hy‐ drocarbons for fuels and for building block commodity chemicals, with a focus on soy‐ bean derived products.

© 2013 McFarlane; 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 McFarlane; 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.

### **2. Fuels**

Although commercially produced, more economical conversion of methyl esters from soy‐ bean triglycerides is an active area of research to make the product more cost competitive in comparison with standard petrochemical diesel [5]. The processes of esterification and trans‐ esterification to produce methyl esters that can be burned directly in compression -gnition engines has been reviewed elsewhere [6, 7].The fatty acid chains on the lipid molecule that constitutes soybean oil, also called a triacylglycerol or TAG, are split from the glycerol back‐ bone and esterified with an alcohol, generally methanol, in the presence of a homogeneous base or acid catalyst, Reaction 1.

0.0 0.2 0.4 0.6 0.8 1.0

**Fractional Yield of Methyl Ester**

of 60% [9].

( )( )

0 2 4 6 8 10

**Fractional Yield of Methyl Ester**

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

**Fractional Yield of Methyl Ester**

0 0.2 0.4 0.6 0.8 1

**c) Reaction Volume Fraction**

**Figure 1.** Results of parametric studies on methyl ester production in a continuous reactor showing (a) asymptotic ap‐ proach to a maximum yield with methanol-to-oil molar ratio and (b) with reactor temperature. The dependence of yield on volume fraction simulated the effect of mixing in the reactor. These calculations were programmed in Mat‐ Lab® [12], for nominal reaction conditions of 50°C, 7:1 methanol-to-oil molar ratio, and an effective reaction volume

123 1 2 3

( )( )( ) ( )( )( )

The chemical conversion to FAME produces a low viscosity, high-cetane number fuel that can be mixed directly with conventional diesel. The physical properties of diesel and biodiesel, or FAME, are compared in Table 1 [14]. The properties in the table are given for the liquid phase at 25°C and for vapor phase at 527°C, corresponding to pre-ignition conditions in a compres‐ sion ignition engine. Although similar in carbon chain length and cetane number, biodiesel differs from diesel significantly in its vapor pressure, liquid viscosity, and vapor diffusion co‐ efficient. The properties of the biodiesel depend on the length and unsaturation of the fatty acid chains, Table 1. Where the data are lacking for the soybean-derived methyl esters, the va‐

+® +

35 2 2 2 3 32 3 5 2 2 2 3 2 3

*C H CO R CO R CO R CH OH CH O R C H OH CO R CO R*

( )( )( ) ( )( )

por phase properties for biodiesel have been replaced by those of methyl oleate.

+® +

35 2 2 3 32 3 5 2 2

*C H OH CO R CO R CH OH CH O R C H OH CO R*

3 3 35 2 2 3 32 3 83

+® +

*C H OH CO R CH OH CH O R C H O*

Triglyceride + 3 Methanoln3 Methyl Esters + Glycerine

0 20 40 60 80 100

Soybean Oil Derivatives for Fuel and Chemical Feedstocks

http://dx.doi.org/10.5772/54067

113

(1)

**b) Temperature (°C)**

**a) Methanol-to-Oil Ratio**

Commercial processing of biodiesel through homogeneous catalysis suffers from high feed‐ stock costs and batch processing that requires long residence times to achieve good conver‐ sion. Hence, ongoing research continues to explore methods how to best use low-quality feedstocks, and to reduce the reagent requirements, energy usage, processing time, and com‐ plexity [8]. Figure 1 shows results from simulation of a continuous process to make biodiesel, varying temperature (a) and methanol content (b) to determine conditions for the optimal pro‐ duction of high quality grade biodiesel. As the process is limited by kinetics and mass trans‐ fer, the effect of mixing has also been investigated by considering the available volume fraction of reagents (c) [9], defined as the molar ratio of reagents in the reaction zone versus the overall reagent volume in the vessel. The available volume can be changed by increasing the contact zone between the immiscible reagents where the reactions take place. The interfacial surface area is dependent on the intensity of mixing in the multiphase system. Interfacial area can also be increased by reducing the size of the dispersed phase droplets, such as by bub‐ bling reagent methanol into the oil through a frit. Novel approaches to biodiesel production continue to be explored, particularly for lower grade and waste feedstocks, such as the direct extraction of fatty acid chains through use of a solvent such as an ionic liquid to pretreat esteri‐ fication to the methyl ester [10]. Other work has examined methanol-based transesterification of waste cooking oil under quite mild conditions (110°C in 2 h) in contact with tungsten oxide solid acid catalysts, giving yields of fatty acid methyl esters (FAME) that are close to the Amer‐ ican Society of Testing and Materials (ASTM) standard for biodiesel [11]. The authors of that study, Komintarachat and Chuepeng, reported several advantages of working with a WOx/ Al2O3 catalyst. Prior separation of free fatty acids, in their sample of waste cooking oil report‐ ed as 15%, was not necessary to achieve high yields in a one step process. In addition, they found the catalyst has desirable properties for scale-up, being low cost, reusable, and less reac‐ tive than traditional homogeneous catalysts.

The choice of acid or base homogeneous catalysis depends on the concentration of free fatty acids (FFA) in the triglyceride feedstock. Virgin soybean oil has a low FFA content, <4%, and so can be converted to biodiesel by transesterification without an acid-catalyzed esterifica‐ tion pretreatment. However, oil that has been degraded by heat, such as waste oil, requires a two-step conversion. FFA produced during heating have to be esterified, otherwise they be‐ come saponified during transesterification. New processes are being developed to simplify the conversion of waste oil, such as the use of a supported heteropolyacid catalyst that si‐ multaneously promotes both the esterification and transesterification processes [13].

**Figure 1.** Results of parametric studies on methyl ester production in a continuous reactor showing (a) asymptotic ap‐ proach to a maximum yield with methanol-to-oil molar ratio and (b) with reactor temperature. The dependence of yield on volume fraction simulated the effect of mixing in the reactor. These calculations were programmed in Mat‐ Lab® [12], for nominal reaction conditions of 50°C, 7:1 methanol-to-oil molar ratio, and an effective reaction volume of 60% [9].

$$\begin{aligned} \text{C}\_3\text{H}\_5\text{(CO}\_2\text{R}^1\text{)(CO}\_2\text{R}^2)\text{(CO}\_2\text{R}^3) + \text{CH}\_3\text{OH} &\rightarrow \text{CH}\_3\text{O}\_2\text{R}^1 + \text{C}\_3\text{H}\_5\text{(OH)(CO}\_2\text{R}^2)\text{(CO}\_2\text{R}^3) \\ \text{C}\_3\text{H}\_5\text{(OH)(CO}\_2\text{R}^2)\text{(CO}\_2\text{R}^3) + \text{CH}\_3\text{OH} &\rightarrow \text{CH}\_3\text{O}\_2\text{R}^2 + \text{C}\_3\text{H}\_5\text{(OH)}\_2\text{(CO}\_2\text{R}^3) \\ \text{C}\_3\text{H}\_5\text{(OH)}\_2\text{(CO}\_2\text{R}^3) + \text{CH}\_3\text{OH} &\rightarrow \text{CH}\_3\text{O}\_2\text{R}^3 + \text{C}\_3\text{H}\_8\text{O}\_3 \end{aligned} \tag{1}$$

Triglyceride + 3 Methanoln3 Methyl Esters + Glycerine

**2. Fuels**

112 Soybean - Bio-Active Compounds

base or acid catalyst, Reaction 1.

tive than traditional homogeneous catalysts.

Although commercially produced, more economical conversion of methyl esters from soy‐ bean triglycerides is an active area of research to make the product more cost competitive in comparison with standard petrochemical diesel [5]. The processes of esterification and trans‐ esterification to produce methyl esters that can be burned directly in compression -gnition engines has been reviewed elsewhere [6, 7].The fatty acid chains on the lipid molecule that constitutes soybean oil, also called a triacylglycerol or TAG, are split from the glycerol back‐ bone and esterified with an alcohol, generally methanol, in the presence of a homogeneous

Commercial processing of biodiesel through homogeneous catalysis suffers from high feed‐ stock costs and batch processing that requires long residence times to achieve good conver‐ sion. Hence, ongoing research continues to explore methods how to best use low-quality feedstocks, and to reduce the reagent requirements, energy usage, processing time, and com‐ plexity [8]. Figure 1 shows results from simulation of a continuous process to make biodiesel, varying temperature (a) and methanol content (b) to determine conditions for the optimal pro‐ duction of high quality grade biodiesel. As the process is limited by kinetics and mass trans‐ fer, the effect of mixing has also been investigated by considering the available volume fraction of reagents (c) [9], defined as the molar ratio of reagents in the reaction zone versus the overall reagent volume in the vessel. The available volume can be changed by increasing the contact zone between the immiscible reagents where the reactions take place. The interfacial surface area is dependent on the intensity of mixing in the multiphase system. Interfacial area can also be increased by reducing the size of the dispersed phase droplets, such as by bub‐ bling reagent methanol into the oil through a frit. Novel approaches to biodiesel production continue to be explored, particularly for lower grade and waste feedstocks, such as the direct extraction of fatty acid chains through use of a solvent such as an ionic liquid to pretreat esteri‐ fication to the methyl ester [10]. Other work has examined methanol-based transesterification of waste cooking oil under quite mild conditions (110°C in 2 h) in contact with tungsten oxide solid acid catalysts, giving yields of fatty acid methyl esters (FAME) that are close to the Amer‐ ican Society of Testing and Materials (ASTM) standard for biodiesel [11]. The authors of that study, Komintarachat and Chuepeng, reported several advantages of working with a WOx/ Al2O3 catalyst. Prior separation of free fatty acids, in their sample of waste cooking oil report‐ ed as 15%, was not necessary to achieve high yields in a one step process. In addition, they found the catalyst has desirable properties for scale-up, being low cost, reusable, and less reac‐

The choice of acid or base homogeneous catalysis depends on the concentration of free fatty acids (FFA) in the triglyceride feedstock. Virgin soybean oil has a low FFA content, <4%, and so can be converted to biodiesel by transesterification without an acid-catalyzed esterifica‐ tion pretreatment. However, oil that has been degraded by heat, such as waste oil, requires a two-step conversion. FFA produced during heating have to be esterified, otherwise they be‐ come saponified during transesterification. New processes are being developed to simplify the conversion of waste oil, such as the use of a supported heteropolyacid catalyst that si‐

multaneously promotes both the esterification and transesterification processes [13].

The chemical conversion to FAME produces a low viscosity, high-cetane number fuel that can be mixed directly with conventional diesel. The physical properties of diesel and biodiesel, or FAME, are compared in Table 1 [14]. The properties in the table are given for the liquid phase at 25°C and for vapor phase at 527°C, corresponding to pre-ignition conditions in a compres‐ sion ignition engine. Although similar in carbon chain length and cetane number, biodiesel differs from diesel significantly in its vapor pressure, liquid viscosity, and vapor diffusion co‐ efficient. The properties of the biodiesel depend on the length and unsaturation of the fatty acid chains, Table 1. Where the data are lacking for the soybean-derived methyl esters, the va‐ por phase properties for biodiesel have been replaced by those of methyl oleate.

