Soybeans : cultivation, uses and nutrition

AGRICULTURE ISSUES AND POLICIES

SOYBEANS: CULTIVATION, USES AND NUTRITION No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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AGRICULTURE ISSUES AND POLICIES

SOYBEANS: CULTIVATION, USES AND NUTRITION

JASON E. MAXWELL EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Soybeans : cultivation, uses and nutrition / editor: Jason E. Maxwell. p. cm. Includes index. ISBN 978-1-61122-092-6 (eBook) 1. Soybean. 2. Soybean--Utilization. 3. Soybean--Nutrition. I. Maxwell, Jason E. SB205.S7S5543 2011 633.3'4--dc22 2010031753

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface Chapter 1

ix Soybean Seed Composition and Quality: Interactions of Environment, Genotype, and Management Practices Nacer Bellaloui, Krishna N. Reddy, H. Arnold Bruns, Anne M. Gillen, Alemu Mengistu, Luiz H. S. Zobiole, Daniel K. Fisher, Hamed K. Abbas, Robert M. Zablotowicz and Robert J. Kremer

Chapter 2

Soy Food in Demand: A Present and Future Perspective M.K.Tripathi and S.Mangaraj

Chapter 3

Processed Foods from Soybean Seeds in Japan and the Characteristics Toshikazu Nishiwaki, Keiko Morohashi, Satoshi Watanabe, Sayaka Shimojo and Takuji Ohyama

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79

Chapter 4

Soybean Meal in Diets for Cultured Fishes Anne Marie Bakke

125

Chapter 5

Soybean, a Peroxidase Source for the Biotreatment of Effluents J.L. Gómez, M. Gómez and M.D. Murcia

155

Chapter 6

Soybean Peroxidase Applications in Wastewater Treatment Mohammad Mousa Al-Ansari, Beeta Saha, Samar Mazloum, K. E. Taylor, J. K. Bewtra and N. Biswas

189

Chapter 7

Soybean Germination and Cancer Disease María del Carmen Robles Ramírez, Eva Ramón Gallegos and Rosalva Mora Escobedo

223

Chapter 8

Glyphosate Interactions with Physiological, Microbiological, and Nutritional Parameters in Glyphosate-Resistant Soybeans Luiz Henrique Saes Zobiole, Robert John Kremer and Rubem Silvério de Oliveira Jr.

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Chapter 9

Oxidative Stress in Soybean: Role of Iron Andrea Galatro and Susana Puntarulo

Chapter 10

Using Transgenic Strategies to Obtain Soybean Plants Expressing Resistance to Insects Milena Schenkel Homrich, Maria Helena Bodanese-Zanettini and Luciane M. P. Passaglia

Chapter 11

Chapter 12

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Soybean Oil Deodorizer Distillate: An Integrated Isolation and Analyses System of Its Bioactive Compounds Setiyo Gunawan, Novy S. Kasim and Yi-Hsu Ju

309

Biofilm Formed on the Soybean Curd Residue Is Associated with Efficient Production of a Lipopeptide by Bacillus Subtilis Makoto Shoda and Shinji Mizumoto

333

Chapter 13

Extraction and Characterization of Soybean Proteins Savithiry S. Natarajan and Devanand L. Luthria

353

Chapter 14

Soybean Oil in Health and Disease Cristina M. Sena and Raquel M. Seiça

371

Chapter 15

The Use of Soybean Peptone in Bacterial Cultivations for Vaccine Production M.W. Wilwert, J.C. Parizoto, M.R. Silva, S.M.F. Albani, M.M. Machado, G.S. Azarias, A.J. Silva, A.C.L. Horta, J. Cabrera-Crespo, T.C. Zangirolami, and M. Takagi

Chapter 16

Chapter 17

Chapter 18

Chapter 19

In Vitro Inhibitory Activity on Angiotensin-Converting Enzyme of Okara Protein Hydrolysates Produced with Pepsin Antonio Jiménez-Escrig, Manuel Alaiz, Javier Vioque and Pilar Rupérez Use of Soy Peptones for Streptococcus Pneumoniae Cultivation for Vaccine Production C. Liberman, D. Kolling, R. S. Sari, M. Takagi, J. Cabrera Crespo, M. E. Sbrogio Almeida, J. G. C. Pradella, L. C. C. Leite and V. M. Gonçalves Production and Consumption of Green Vegetable Soybeans ―Edamame‖ Yoshihiko Takahashi and Takuji Ohyama Soybean as a Model to Study Defensive Responses Enhanced by Fungal Elicitors, Nitric Oxide and Elevated CO2 Marcia Regina Braga, Kelly Simões, Sonia Machado de Campos Dietrich, Luzia Valentina Modolo and Ana Paula de Faria

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Chapter 20

Applications of Soybean Peroxidase in Analytical Science Shuang Han, Lihong Shi and Guobao Xu

455

Chapter 21

Soybean Nutrition: Protein and Isoflavones Savithiry S. Natarajan, Devanand L. Luthria and Ronita Ghatak

465

Chapter 22

Nutritional Enhancement of Soybean Meal and Hull via Enzymatic and Microbial Bioconversion Liyan Chen, Yixing Zhang, Ronald L. Madl and Praveen V. Vadlani

Index

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PREFACE Soybean is one of the major world crops. In 2008, the world consumption of soybean was over 221 million metric tons of which approximately 50% came from the U.S. Soybean seed is a major source of protein, oil, carbohydrates, isoflavones, and minerals for humans and animals. This book presents current research data from across the globe in the study of soybean cultivation, its uses and nutrition. Some topics discussed herein include soybean seed composition and quality; soybean peroxidase applications in wastewater treatment; soybean germination and cancer disease; soybean oil deodorizer distillate; soybean oil in health and disease; and the use of soybean peptone in bacterial cultivations for vaccine production. Chapter 1 – Soybean [Glycine max (L) Merr.] seed is a major source of protein, oil, carbohydrates, isoflavones, and minerals for human and animal nutrition. About one-third of the world's edible oils and two-thirds of protein meal are derived from soybean seed. Thus, improving soybean seed composition and quality is key to improving human and animal nutrition. Seed composition refers to major constituents found within the seed including protein, oil, fatty acids, carbohydrates, isoflavones, and mineral content which determine seed nutritional value. Soybean seed quality refers to viability, germination, and seedling vigor which directly impact yield. Seed composition and quality are known to be genetically controlled, and significant variability in seed quality and composition exist due to differences in the gene pool. The physiological and biochemical mechanisms by which this variability is expressed are still not completely understood, but are known to be significantly influenced by genotype (G), environment (E), management practices (MP), and their interactions. Understanding the interaction of these factors and how they impact seed composition and quality is crucial for maintaining high yield and quality. This review highlights the current research on seed composition and quality as influenced by G, E, MP, plant diseases, and their interactions from physiological, biochemical, and genetic perspective. This review also highlights major challenges in soybean seed composition and quality improvement, and examines how current research techniques have been used for improving seed nutritional value for conventional uses such as food and feed or for specialty uses such as tofu, soymilk, and other soy products. This information is beneficial as a source in soybean breeding, physiology, and biochemistry research. Chapter 2 – The soybean is widely believed to have originated 4000-5000 years ago in the north and central regions of China. Historically, soybeans have played an important part in Asian culture, both as a food and as a medicine. Soybeans have been consumed throughout

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Asia for more than 1000 years in a variety of traditional soy food products. Asian countries still utilize soybeans largely for traditional soy food production. In 1999, Asian countries accounted for nearly half of the 23 million tons of soybeans exported by the U.S. traditional soy foods, those foods prepared from whole soybeans, are typically divided into two categories: nonfermented and fermented. Traditional nonfermented soy foods include fresh green soybeans, whole dry soybeans, soy nuts, soy sprouts, whole-fat soy flour, soymilk, tofu, okara, and yuba. Fresh green soybeans and whole dried soybeans are prepared and consumed in much the same fashion as Western bean dishes, eaten plainly or serving as additions to soups and stews. In addition to the drying of soybeans to create whole dried varieties, dry roasting may also be employed to create a crunchier product known as a soy nut. Soy nuts can be eaten whole as a snack food, or used in food applications similar to dry roasted peanuts. Soy sprouts, prepared by the soaking, washing, and sprouting of soybeans, are consumed as a vegetable throughout the year in many Asian countries, and are used in soups, salads, and side dishes. Whole-fat soy flour, prepared from the grinding of whole dried soybeans, is used in bakery applications in the place of milk powder applications or as a substitute for wholewheat flour. Preparations of soymilk, tofu, okara, and yuba begin with the soaking of whole soybeans, followed by rinsing, grinding, and filtering. The insoluble residue at this point is called okara, it can be used in the making of a dish, salted as a pickle, or fermented in the production of tempeh. Further processing of the filtered soybean liquid includes cooking to yield soymilk. Tofu is made from the further processing of soymilk in which different coagulants are added to precipitate the protein from the soymilk. Traditional fermented soy foods include tempeh, miso, soy sauces, natto, and fermented tofu (sufu) and soymilk products. Tempeh is a product produced from the fermentation of dehulled, boiled soybeans by Rhizopus oligosporus. The process of fermentation yields a cake like product with a clean yeasty odor. Tempeh is usually served as meat substitute that, when sliced and deep-fried, has a nutty flavor, pleasant aroma, and crunchy texture. Miso is the Japanese term used for bean paste and refers to fermented paste like products. Miso production begins with making a starter culture, or koji, which consists of a cereal (rice, barley, or soybeans) which, is soaked, cooked, cooled, and inoculated with a mixture of strains of Aspergillus orzae and Aspergillus soyae. Soy sauce is a salty, sharp tasting, dark-brown liquid extracted from fermented mixture of soybeans and wheat. Natto is a sweet, aromatic product made from the fermentation whole soybeans with Bacillus natto. A number of traditional fermented soy food products also exist that are made from the fermentation of tofu and soymilk. Sufu, or Chinese cheese, is produced by the fermentation of fresh tofu by fungi, such as Mucor hiemalis or Actinomucor elegans. Soymilk is used to produce soybean yogurt, which is similar to western dairy yogurt, and is basically a mixture of soymilk, whey, and sucrose that has been cooked and cooled and inoculated with Lactcoccus acidophilus. Although soybeans were introduced to Europe in the 1700s, little interest developed until the early 1900s, primarily because of the plants inferior flavor quality compared to the native oil and meal products. Soybeans were introduced to the Eastern United States (U.S.) in the late 1800‘s with production spreading to the Midwest by 1920. Through the early 1930s, soybeans were grown primarily as a pasture and forage crop. However, by 1947, 85% of the crop was harvested for seed processing in the production of oil. As demand increased, markets developed for soybean oil and later for the high-quality soybean meal used as a protein source for animal feeds. Soybeans contain about 40 % fat and oil from soybeans is the world‘s leading vegetable oil and accounts for well over half the fats and oils going into food products in the U.S. The bulk of the soybean oil produced is

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consumed as salad oil. Industrial applications for soybean oil also exist and include soap manufacture, paints, resins, and drying oil products. The major uses domestically are cooking and salad oils, shortening, and margarine. Chapter 3 – The average life span in Japan was 83 years old (women 86.08, men 79.29) in 2009, and that of women was the longest in the world. This may be partly due to low fat Asian dishes with rice, soybean products, fish, and vegetables. Soybeans originated from East Asia, and Japanese people eat many traditional foods made from soybeans, such as Shoyu, Miso, Tofu, Natto etc. Soybean seed is one of the most important protein sources for humans and livestock all over the world. The amino acid composition of soybean is relatively well balanced, although soybean seeds contain a relatively low amount of sulfur amino acids, methionine, and cysteine. Soybean seeds contain a large amount of lipids, minerals, and vitamins. Soybean seeds also contain isoflavonoids, daidzein and genestein, and other components to keep human health well. The isoflavonoids are expected to play a role like a female hormone or to decrease fat in blood. In this chapter, the authors would like to introduce the nutritional value of soybean seeds, and the production, cooking and nutrition of some traditional Japanese soy foods, such as Shoyu (soy sauce), Miso (fermented soy paste), Tofu (soy curd), and Natto (fermented soybean with bacteria). In addition, recent advances in the physiological effects of soy protein and processed foods in Japan are introduced. Chapter 4 – According to the Food and Agriculture Organization of the United Nations (FAO), aquaculture‘s contribution to total global fisheries is increasing faster than capture fisheries, and aquaculture is now supplying about half the fish consumed by the human population worldwide. Capture fisheries of wild fish stocks, on the other hand, has shown zero or negative growth in recent years. Capture fisheries have been the main sources of fishmeal and fish oil, traditionally used as the main protein and lipid sources in formulated diets for many cultured fish species, especially carnivorous fish. However, the limited global supply of fishmeal and fish oil has led to the increasing use of alternative protein and lipidrich feed ingredients in aquafeeds, mostly from plant crops. Soybean meal is used for partial replacement of fishmeal in formulated aquafeeds for many species, due to the ample market supply, lower market price, high protein concentration and favorable amino acid composition. Among alternative feed ingredients, by far the most research on effects on growth performance as well as digestive and immune function in teleost fishes has been carried out with soybean products. This may partly be due to early reports of the negative impact that de-hulled, full-fat or de-fatted (hexane extracted) soybean meal (SBM)-containing diets have on some production animals, including salmonid fishes such as Atlantic salmon and rainbow trout. In salmonids, dietary inclusion of SBM can cause dramatic reductions in growth performance, nutrient digestibilities, and diarrhea. These can at least partially be explained by an inflammatory response in the distal intestine (hind gut), termed soybean meal-induced enteropathy. It is characterized by a high level of Tlymphocyte infiltration into the intestinal mucosa. However, the decreased nutrient digestibility and utilization of SBM in salmonid diets also appears to be due to decreased enzymatic digestion and assimilation of nutrients, and increased production and loss of endogenous secretions/enzymes in the more proximal regions of the intestine. The metabolic cost of the inflammatory response in the distal intestine may also lead to reduced growth performance. In salmonids, the inflammatory reaction in the distal intestine has been observed in all individuals exposed to dietary SBM at various stages of development. As for other fish species, SBM in diets for gilthead sea bream and common carp have also been shown to

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cause an inflammatory response in the intestine. But salmonids appear to be the most sensitive species. There are indications that solvent-extracted SBM in diets can have negative consequences on the salmon‘s and also channel catfish‘s susceptibility to infectious diseases. The contribution of the intestinal microbiota and any changes in its species composition caused by SBM has not been clearly established. The long-term consequences to the health of the fish, as well as their response to orally or intestinally administered vaccines, medicines and other compounds of biological significance, may be compromised and merit further investigation. Although the causative agent(s) of the pathophysiological responses in salmonids has not been conclusively identified, some recent findings suggest that soy saponins may be involved, most likely in combination with other, as yet unidentified components. The alcohol extraction of soybean meal, a processing step used in the production of soy protein concentrate, appears to remove the causatory agents in most cases. This highly refined soybean product has generally been found to be a safe, high quality alternative protein source in salmonid feeds. But it is costly. Fermentation also appears to improve the acceptability of SBM for the fish. Further research on cost-efficient processing methods of SBM to remove antinutritional factors or development of soy strains with low levels of antinutritional factors can aid in reducing the negative impact on the health and welfare of cultured fish fed soybean products. Such efforts can potentially increase the use of soybean products in aquafeeds and thus contribute to developing a more sustainable aquaculture industry. Chapter 5 – Nowadays the massive industrialization of society has to deal with the efficient utilization and recycling of natural resources to achieve modern industrial manufacturing processes. Many industrial wastewater streams, mainly from producing polymers, resins, coatings, paints, dyes, agrochemicals, pharmaceuticals and oil refining are being discharged into the environment. Because of their toxicity level, higher than permitted, extensive research in economic and efficient technological treatments has been done recently without reaching a definitive solution. Among the numerous applications of soybean, its use as a source of soybean peroxidase is of particular interest in the area of bioremediation. Peroxidases are oxidoreductase enzymes with a wide variety of substrate specificity, that catalyze, in the presence of hydrogen peroxide, the oxidative polymerization of different pollutants widely found in industrial effluents, such as phenolic compounds, polycyclic aromatic hydrocarbons (PAHs), aromatic amines, non-phenolic aromatics and dyes. In general, these pollutants are highly toxic for both human beings and the environment, and they must be removed from wastewater before they are discharged into the environment. The use of enzymes is presented as an alternative method for the industrial wastewater treatment when conventional physical, chemical and biological methods such as adsorption in active carbon, chemical oxidation and microorganism treatment may be ineffective. Enzymes present several advantages: mainly they are easy to handle and store, operate within a wide range of conditions, present high specificity towards the targeted compounds and minimum environmental impact. In particular, in the presence of peroxidases, the previously mentioned pollutants are oxidised forming free radicals that subsequently polymerize until the formation of high molecular weight products, with the final products being insoluble polymers that can be easily precipitated from the wastewater without causing environmental problems. Soybean has the additional advantage of being a cheap and abundant source of peroxidase, easily extracted from soybean seed hulls that are a by-product

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in the food industry, and capable of maintaining its enzymatic activity over a wide range of temperature and pH, which explains its increasing use over the last years in the biotreatment of effluents. In order to decrease the systems‘ costs, one important area for future research will be the study of the direct use of soybean seed hulls without any extraction of the enzyme. Chapter 6 – Soybean peroxidase (SBP) is an oxidoreductase-class enzyme extracted from soybean seed coats (hulls). SBP has been extensively studied in recent years for its role in various biotechnological applications. It has many attractive properties due to its structure, conformational flexibility, activity and stability under various environmental conditions. This review will focus on the application of SBP in industrial waste- and process-water treatment. SBP catalyzes oxidative polymerization of a wide range of hazardous aqueous aromatic pollutants which are present in wastewater streams of various industries such as petroleum refining, coal conversion, wood products and preservation, metal casting, pulp and paper, dyes, adhesives, resins, plastics and textile manufacturing. These pollutants can have adverse health effects on human, animal and aquatic life upon exposure by direct absorption through the skin, ingestion and/or inhalation. Hence, their discharge is regulated. The review will cover pollutants investigated with SBP, advantages of SBP over other enzymes, pretreatment methods to broaden the scope of SBP polymerization, use of additives/surfactants to enhance SBP activity, application of SBP in real industrial wastewater, immobilized SBP and its limitations. Chapter 7 – Cancer is one of the chronic diseases that has the highest incidence in the world. Extensive epidemiological, in vitro, and animal data suggest that soybean consumption reduces the risk of developing several types of cancer. To date, a number of nutrients and micronutrients with anticancer properties have been identified in soybean, including isoflavones, saponins, inositol hexaphosphate, and biologically active proteins and peptides such as protease inhibitors, lectins, low molecular weight peptides and the most recently discovered peptide lunasin. The anticancer properties of soybean may also be due to its amino acid balance since it has low methionine and high arginine content; both conditions are known to inhibit tumor development. The ability to block DNA damage caused by reactive oxygen species and/or carcinogens is the most direct strategy for preventing the initiation of cancer and for slowing down disease progression; studies have demonstrated the antioxidant capacity of soybeans due to their content of compounds such as isoflavones and other phenolic compounds, phytates, tocopherols, carotenoids, and peptides derived from its hydrolysis. Furthermore, soy may protect against cancer through other different mechanisms including increased cell differentiation, decreased activation of procarcinogens to carcinogens, and regulation of genes involved in signal transduction pathways critical in tumor initiation, promotion and /or progression. Germination is a simple, low-cost process that can improve the nutraceutical properties of plants by modifying metabolite content and generating peptides and amino acids with possible biological activity. A previous study from the authors laboratory showed that germination could improve the antiproliferative effect of soybean protein on HeLa and C-33 cervical cancer cells. In this chapter the authors will review the different components of soy with anticarcinogenic properties and how they are affected by germination. In addition, the authors will explore the possibility of using germination as a means to improve these properties. Chapter 8 – The crop area planted to conventional soybeans has decreased annually while that planted to glyphosate-resistant (GR) soybean has drastically increased, mainly due to the

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wide adoption of glyphosate in current weed management systems. With the extensive use of glyphosate, many farmers have noted visual plant injury in GR soybean varieties after glyphosate application. In fact, glyphosate has intensified deficiencies of numerous essential micronutrients and some macronutrients. The typical symptom is known as "yellow flashing" or yellowing of the upper leaves and, in some cases, has been attributed to the accumulation of the primary phytotoxic metabolite aminomethylphosphonic acid (AMPA); other reports suggest the symptom results from direct damage to chlorophyll, since glyphosate is a strong metal chelator which forms insoluble glyphosate-metal complexes, may immobilize essential micronutrients required as components, co-factors or regulators of physiological functions. Although glyphosate affected photosynthesis of some GR soybean cultivars, nutrient uptake from soil is also reduced indirectly through its toxicity to many soil microorganisms responsible for increasing plant availability of nutrients through mineralization, reduction, and symbiosis associations. Several reports show that glyphosate can directly affect the nitrogen fixing symbiont Bradyrhizobium japonicum. Since this microorganism possesses a glyphosate-sensitive EPSP synthase, some intermediate metabolites including shikimic, hydroxybenzoic and protocatechuic acids accumulate, which inhibit growth and induces death at high concentrations, due to inability of the organism to synthesize essential aromatic amino acids. The loss of energy and fixed N2 provided by B. japonicum may be significant factors responsible for reduced growth and yield in GR soybean. Glyphosate and other herbicides can influence nitrogen metabolism through direct effects on the rhizobial symbiont or through indirect effects on the physiology of the host plant. Glyphosate and root exudates released into the rhizosphere from GR soybean also influence microbial populations and/or activity in the rhizosphere. Previous findings that glyphosate and high concentrations of soluble carbohydrates and amino acids exuded from roots of glyphosate-treated GR soybean, suggest that promotion of rhizosphere and root colonization of GR soybean by specific microbial groups (i.e. Fusarium sp.) may be a combination of stimulation by glyphosate released through root exudation and altered physiology leading to exudation into the rhizosphere of high levels of carbohydrates and amino acids Limited data are available regarding effects of glyphosate on GR soybean physiology, especially those related to photosynthesis. This chapter aims to show that some GR soybeans can be extremely affected by glyphosate, not only by reduced photosynthesis but also decreased nutrient uptake, and reduced shoot and root biomass production, as well as by modified interactions with specific microorganisms, which may negatively impact soybean growth and production. Herbicides are advertised heavily in popular magazines, whereas relatively few publications inform farmers about weed management through the integrated use of cover crops, crop rotation, cultivation and other ecologically based tactics. Thus, with increased problems related to glyphosate use in GR soybean, an approach to minimize glyphosate effects based on increased efficiency of glyphosate by treating weed populations only where and when their densities warrant application, could greatly reduce glyphosate use with a likely decrease in glyphosate injury to GR soybean. However, management based on growth stage and weed population density will need to be considered if this strategy is undertaken. On the other hand, a strategy to decrease the use of glyphosate may involve the use of preemergent herbicides or the mixture of residual herbicides with glyphosate in burndown

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management, thus increasing the long-term weed control allowing farmers to spray the lowest glyphosate rate as possible. Chapter 9 – Plants are naturally exposed to a variety of environmental and physiological stress situations, such as the oxidative stress associated to growth and development. Soybeans require Fe as a nutrient, in a concentration higher than 10-8 M in solution, to meet their needs. Fe plays critical roles in biological processes, including photosynthesis, respiration and nitrogen assimilation. However, Fe has a dark side because it is toxic to the cell due to its capacity of being an active catalyst for the generation of reactive oxygen radical species that causes lipid peroxidation, DNA strand breaks, oxidation of proteins, and degradation of other biomolecules. Fe homeostasis is maintained by the coordinated regulation of its transport, utilization and storage. Fe traffic has to be strictly controlled, and ferritin is one of the main proteins involved. Moreover, ferritin can prevent Fe toxicity because of its ability to sequester several thousand of Fe atoms in their central cavity in a soluble, non-toxic bio-available form. However, anytime Fe uptake exceeds the metabolic needs of the cell and the storage capacity is overwhelmed, a low molecular weight Fe pool, referred to as the labile iron pool is increased. The Fe in this pool represents the catalytic active Fe, and its size needs to be closely restricted, mostly in chloroplasts and mitochondria since besides being important sites for Fe utilization, they also are specific places for free radical production through the electron transfer chains. The general aspects linked to oxidative metabolism in soybean embryonic axes both, under physiological and stress conditions, will be analyzed mostly in the early stages of growth. Fe function, its cellular distribution, and its participation in free radical reactions leading to cellular damage will be described and also analyzed under Fe overload condition. An integrative overview of these topics will provide information that could be the key to elaborate strategies to improve soybean development, and also human nutrition through biofortification strategies. Chapter 10 – Soybean (Glycine max (L.) Merrill) is one of the most important sources of edible oil and protein, resulting in special interest in the genetic improvement of this crop. The velvetbean caterpillar (Anticarsia gemmatalis Hübner) is a major soybean pest. It causes extremely high levels of defoliation when infestation is heavy and can severely damage axillary meristems, with a single caterpillar being able to consume up to 110 cm2 of soybean foliage. Various authors have reported that A. gemmatalis can be controlled by the deltaendotoxin (δ-endotoxin) produced by some strains of Bacillus thuringiensis. Several DNA delivery methods and plant tissues have been used in developing transgenic soybean plants. Such techniques include particle bombardment of shoot meristems, embryogenic suspension cultures, and Agrobacterium tumefaciens-mediated T-DNA delivery into cotyledonary nodes derived from five- to seven-day old seedlings, mature seeds, immature zygotic cotyledons, somatic embryos derived from immature cotyledons or embryogenic suspension cultures. Earlier studies have indicated that soybean regeneration is genotype-specific with differences in their responses to in vitro culture and transformation. There is no efficient transformation system for a wide range of soybean cultivars, which explains why very few reports on genetic transformation of commercial soybean cultivars are available. There are two major routes to improve embryogenic culture-based soybean regeneration and transformation protocols with the goal of increasing the recovery of transgenic fertile lines. One option is to screen large numbers of new soybean cultivars and genotypes for embryogenic potential, while another option is to use existing protocols and cultivars coupled with traditional breeding programs

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for introgressing transgenic traits into other genotypes. The Brazilian soybean cultivar IAS5 has commonly been used in genetic improvement programs and is recommended by the Brazilian Agricultural Ministry for commercial growing in the Brazilian states of Goiás, Minas Gerais, Paraná, São Paulo, and Rio Grande do Sul. This cultivar has shown good reliable response in embryogenic systems. In this Chapter will be described the development of transgenic soybean with resistance to A. gemmatalis larvae. Somatic embryos of the IAS5 cultivar growing on semi-solid medium were transformed with a synthetic Bacillus thuringiensis δ-endotoxin crystal protein cry1Ac gene by particle bombardment. The cry1Ac transgene insertion pattern in the transgenic plants was analyzed in the primary transformants. Transmission and expression of the transgene were also characterized in the T1, T2, and T3 generations. No significant yield reduction was observed in transgenic plants. The cytogenetical analysis showed that transgenic plants present normal karyotype (2n=40). The transgenic plants were also evaluated by in vitro and in vivo assays for resistance to A. gemmatalis. Two negative controls (non-transgenic IAS 5 and homozygous gusA isoline) were used. In a detached-leaf bioassay, cry1Ac plants exhibited complete efficacy against A. gemmatalis, whereas negative controls suffered significant damage. Whole plant-feeding assay data confirmed very high protection of cry1Ac plants against velvetbean caterpillar while non-transgenic IAS 5 and homozygous gusA isoline exhibited 56.5 and 71.5% defoliation, respectively. The bioassays indicated that the transgenic plants were highly toxic to A. gemmatalis, offering a potential for effective insect resistance in soybean. Chapter 11 – Soybean oil is the most consumed vegetable oil in the world, representing 30% of the consumption in the worldwide market. During the production of soybean oil, soybean deodorizer distillate (SODD) is produced as byproduct of a deodorization step. It represent about 3% of refined oil or 0.6% of soybean seed as feed in the refining process. Recent interest in the exploitation of SODD is due to its content of economically-valuable bioactive compounds. It has been suggested as an alternative to marine animals as natural source of squalene and as a good raw material for the production of fatty acid steryl esters (FASEs), free phytosterols, and tocopherols. The aim of this chapter is to discuss the isolation and separation techniques, and analysis methods of these bioactive compounds. SODD typically contains high level of free fatty acids (FFAs) and acylglycerols depending on the conditions of the oil refining process, and the efficient elimination of them is crucial for the enrichment of the bioactive compounds. There are several different methodologies for the elimination of FFAs and acylglycerols: hydrolysis, esterification, transesterification, distillation, crystallization, adsorption, and liquid-liquid extraction. The development of new isolation techniques has gained increasing importance in chemical, food, and pharmaceutical industries, due to the imposed environmental regulations and the necessity of minimizing energy requirement. The analysis methods of the bioactive compounds is also a challenging problem. Few analytical techniques are available to provide a detailed analysis. It will be of great importance if a method to identify individual compounds in SODD can be established. Chapter 12 – The authors previously showed that the soybean curd residue has efficient nutrients for higher production of a lipopeptide antibiotic, iturin A in solid-state fermentation (SSF) of Bacillus subtilis, and this cultured solid can be applied to soil to control plant diseases. The high production of iturin A in SSF may be due to biofilm formation of B. subtilis on the surface of the soybean curd residue. In order to prove this speculation, B. subtilis was grown both in an artificial air membrane surface (AMS) reactor where a biofilm was formed on the membrane and by liquid submerged cultivation (SmF) in suspended

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culture by using the same nutrients. The iturin A production in AMS reactor was significantly higher than that in SmF and the iturin A productivity of each cell in AMS reactor was more enhanced than that in SmF. The cells from both cultures were analyzed by 2-dimensional electrophoreses and proteome analysis. As the cells were under local nutrient depletion in biofilm, some proteins associated with nutritional depletion and biofilm formation such as alkyl hydroperoxide reductases (AhpC and AhpF), 2-oxoglutarate dehydrogenase, and translocation-dependent spore component (TasA) proteins were specifically or strongly expressed in the cells in the ASM reactor. Moreover, the genes responsible for iturin A production (ituB and degQ) were highly transcribed by the cells in the AMS reactor. These results suggest that typical characteristics of biofilm formed on the soybean curd residue triggered the high iturin A production in biofilm by B. subtilis via elevated expression of specific genes. Chapter 13 – Proteins play an important role in many biological processes. The authors compare commonly deployed methods used for extraction of soybean proteins. In addition, the authors also discuss separation and identification of different classes of soy proteins (storage, allergens, and anti-nutritional) using current proteomic technologies. Soybean proteins are used in human food in a variety of forms such as baby formula and protein isolate concentrate because of unique physiochemical properties. Soybean storage proteins are grouped into two types, beta-conglycinin and glycinin based on their sedimentation coefficients. Three allergen proteins, Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K represent the major seed allergens. In addition to the above two protein classes, several proteins in soybean seeds are considered to be anti-nutritional (Kunitz trypsin inhibitor and lectins). Protein extraction was carried out by different methods and separation of proteins was achieved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Individual proteins were characterized by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) coupled with liquid chromatography mass spectrometry (LC-MS/MS) analysis of tryptic fragments. The application of these proteomic tools can be extended to the analysis of proteins separated using different matrices. Chapter 14 – This review provides a scientific assessment of current knowledge of health effects of soybean oil (SO). SO is one of the few non-fish sources of omega-3 fatty acids and the primary commercial source of vitamin E in the U.S. diet. SO contains high levels of polyunsaturated fatty acids (PUFAs, 61 %), including the two essential fatty acids, linoleic and linolenic, that are not produced in the body. Linoleic and linolenic acids helps the body's absorption of vital nutrients and are required for human health. The PUFAs positively affect overall cardiovascular health, including reducing blood pressure and preventing heart disease. Epidemiological evidence has suggested an inverse relationship between the consumption of diets high in vegetable fat and cardiovascular risk factors such a blood pressure, although clinical findings have been inconclusive. In an experimental animal model of diabetes SO has been described to ameliorate lipid profile and improved glycemic control. Soybean oil contains natural antioxidants which remain in the oil even after extraction. Soybean oil also contains lecithin which lowers blood levels of cholesterol. As a result, the replacement of saturated fats with reasonable amounts of polyunsaturated fats, such as those found in soybean oil, may be useful in the dietary guidelines but the health claims remain controversial.

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Chapter 15 – The use of microbial and cell culture nutrient media based on hydrolyzed soybean meals has been increasing continuously, mainly for the production of human and animal pharmaceutical products, due to the risks related to the transmission of the prion diseases. Two kinds of bacteria have been studied: Haemophilus influenzae b (Hib), a bacterium that causes meningitides in children less than two years old, and Erysipelothrix rhusiopathiae (Erhu), the causative agent of swine erysipelas, a disease responsible for great worldwide losses in swine culture. The focuses of these projects are to develop vaccines based on polysaccharide against Hib and inactivated cells suspensions containing the protein SpaA, the main antigenic agent against Erhu infections, respectively. Several kinds of peptones, from animal and vegetal origin, were tested and the results concerning cells, polysaccharide or protein production were compared. For H. influenzae b cultivations, the following nutrients were used: i) from vegetal origin: Bacto Soytone (BD), HiSoy (Kerry), Soy peptone (Sigma); ii) from animal origin: Bacto casamino acids (BD) and casein acid hydrolysate (Sigma). Usually, E. rhusiopathiae is cultivated in complex media from animal origin. Two types of soybean peptones were tested: Bacto Soytone (BD) and soy peptone (Acumedia) as well as corn steep liquor (Milhocina from Corn Products, Brazil). The results for H. influenzae b showed that the peptone from vegetal origin promoted a 2~2.5 times better growth than that from animal origin. The best one was Bacto Soytone from BD, leading to OD540nm= 6.0 in shake flask and 16 in biorreactor. The production of polysaccharide was 296 mg/L in shake flask and 600 mg/L in bioreactor. Concerning E. rhusiopathiae, when cultivated in shaker flasks, an increase of 50% in biomass yield on glucose consumed and of 10% in the maximum specific growth rates (max) were observed with vegetal peptones. For E. rhusiopathiae bioreactor cultivation with animal peptone, a maximum OD420nm of 7.8 and max of 0.5 h-1 were achieved. However, these values were significantly higher, reaching 35 for OD420nm and 1 h-1 for max, with soybean peptone. The expression of SpaA was favored with soybeans peptone as nitrogen source as well. Production of vaccines usually involves cultivation of fastidious microorganisms in complex animal media. The results confirm that the soybean peptones can successfully replace the peptones from animal origin with improvements in biomass formation, growth rate, antigenic protein expression and yield of polysaccharide. Chapter 16 – Okara, a major by-product of the soymilk industry, is rich in proteins. The authors previous studies have shown that okara under physiological conditions may release potential bioactive peptides. Specifically, okara protein isolate has been digested sequentially with pepsin (60 min) and pancreatin (195 min) and the okara protein hydrolysates (OPHs) obtained at the end (255 min) of the in vitro digestion have been fractionated by ultrafiltration, characterized in terms of amino acids and Molecular Weight (MW) distribution, and tested for angiotensin-converting enzyme inhibition (ACE-I) and multifunctional antioxidant activities. As a result, a bioactive peptide from soybean lipoxygenase-1 with a calculated mass of 751.48 Da has been identified in the < 1 kDa ultrafiltrated (UF) fraction. On this frame, the ACE-I activity of the OPHs released during the first phase (pepsin) of the sequential in vitro digestion is the subject of this chapter. Further work focusing on the 60-

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min OPH released at the end of pepsin digestion is also described. Results indicated that pepsin digestion released OPHs showing relatively high inhibitory activity against ACE. The highest inhibition was exerted by the 5 min-OPH, showing an IC50 value of 1.523 ± 0.005 μg protein/μL. After ultra-filtration partitioning of the 60-min OPH, the effect of amino acid content and MW of UF fractions on ACE-I activity was discussed. The UF fraction containing the lower MW peptides showed the higher hydrophobic amino acid content and ACE-I activity. Concluding, the consumption of okara protein might exert health benefits on the basis of the bioavailability of the peptides released by pepsin. Further research on animal model is necessary to verify the tested potential ACE-I activity of these peptides released by in vitro pepsin digestion. Chapter 17 – S. pneumoniae is a pathogen that affects the human respiratory tract, causing otitis media, sinusitis, pneumonia, meningitis and sepsis. Pneumococcus is a fastidious anaerobic fermentative microorganism that produces mainly lactate. S. pneumoniae isolation and cultivation is commonly done using animal-sourced media, such as Brain and Heart Infusion, Todd-Hewitt, hydrolyzed casein derivates, etc., frequently supplemented with whole blood or serum. Although these media are appropriate for clinical diagnosis, they are unsuitable for vaccine production process, since they may cause prion related diseases. Hence, regulatory authorities such as FDA and WHO recommend replace all animal source medium whenever possible. In order to replace Todd-Hewitt (THY) and acid hydrolyzed casein (AHC) media, enzymatically hydrolyzed soybean meal (EHS) had been tested for pneumococcal vaccine production. The microorganism was cultivated in static flasks using three different media THY, AHC and EHS and in 10L-bioreactors using AHC or EHS media. In flasks, the biomass was 1.7 times higher in AHC and 2.3 times higher in EHS than in THY. In pH-controlled bioreactors, the biomass production using EHS was 2.5 times higher than that using AHC medium. Further improvement was obtained performing fed-batch culture using concentrated EHS in the feeding medium. The fed-batch strategy increased the biomass production twice when compared to the simple batch cultivation. This soy based medium was shown to be satisfactory for this fastidious bacterium and met the requirements for human vaccine production. Chapter 18 – The ―edamame‖ is a kind of soy food popular in Japan, and it is classified as a vegetable and not a grain crop as in the case of mature soybean seeds. Traditionally, the edamames were sold as a bunch of stems with pods in Japan. ―Eda‖ means stems, and ―Mame‖ means beans. The immature pods are boiled in salt water, and served after cooling. Edamames are usually taken as a snack or a relish with beer in Japan. It contains various nutrients, such as proteins, lipids, carbohydrates and minerals at similar levels to mature soybean seeds based on dry weight. In addition, edamames contain higher concentrations of vitamin A, C, K and folic acid than mature soybean seeds. There are many local varieties of Edamame soybean in Japan, and the cultivars for grain soybean are usually not delicious to eat as edamames. The flavor is excellent in edamame varieties but not in grain soybeans. Edamame varieties have a sweet taste and distinctive good flavor and aroma. The higher concentrations of sugars (e.g., sucrose) and free amino acids (e.g., alanine) are related to the good taste. The variety, cultivation methods and the stages of plants at harvest are very important factors for good quality. The bunch of stems with pods, or separated fresh pods in a plastic bag are marketed from early summer to late autumn in Japan. The market prices of the

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edamames mostly depend on the freshness, taste, brand name and seasons. The edamame plants are cultivated in the greenhouse, or in a tunnel of double plastic sheets to keep them warm and harvest in the late spring and early summer (May to June) with a high price. Then soybean plants are cultivated in open fields and harvested from July to November. The freshness is very important for the taste of edamames; therefore, many farmers harvest the edamame plants at midnight or early morning and market them on the same day. The wrapping technology and the transportation system were improved and the market distribution of the fresh edamames has expanded in recent years. In addition to fresh edamames, the import of frozen cooked edamames from foreign countries is increasing, and people can eat edamames all year around. Chapter 19 – Soybean, Glycine max (L.) Merr., is one of the major world crops, occupying large acreages of land. Soybeans are grown in most countries and have become essential due to a multitude of food products they provide, which include oil and high-protein defatted meal. In recent years, soybeans have also been under intensive study because of their multiple health-enhancing properties. Although soybean breeding programs have developed varieties with improved yields, quality and disease resistance, every year substantial losses in soybean production still occur due to pathogen attack. Therefore, the understanding of defensive responses of soybean to pathogens is important not only to increase crop yield, but also to predict the consequences of climate changes on agricultural ecosystems. Much research on soybean defensive responses has been done using modifications of the classical cut-cotyledon assay. As soybean cotyledons have simple architecture and cellular uniformity, and show very sharp and localized responses to incompatible isolates of pathogens or to endogenous/exogenous signaling molecules, they represent a nearly ideal model to study these responses under variable environmental conditions. The authors group has been successfully using this assay to screen highly defensive soybean cultivars, to characterize phytoalexin-inducing molecules (elicitors) derived from microbes and plants, to evaluate the role of nitric oxide in defensive isoflavonoid metabolism, and to analyze the effects of elevated CO2 atmospheres on soybean responses to elicitors. Chapter 20 – Soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that also includes barley peroxidase, peanut peroxidase, and widely studied horseradish peroxidase (HRP). There is increasing interest in SBP as an alternative to HRP for analytical applications because of its conformational stability and good stability at high temperature and in acidic media. In this review, the applications of SBP in analytical science are demonstrated, such as the applications in chemiluminescence immunoassays, colorimetric immunoassays, rapid chemiluminescent detection, identification, and enumeration of microorganisms using SBP-labelled peptide nucleic acid probe, DNA probe assays, direct electrochemistry of SBP, as well as electrochemical biosensors for the determination of hydrogen peroxide, organic peroxide, phenol, glucose, and lactate. Chapter 21 – Soybeans and soy products provide an excellent source of multiple macro and micronutrients. The objective of this communication was to provide a brief overview of soybean storage proteins composition and its uses in different food preparation. In addition, the authors briefly summarize the authors knowledge of isoflavones compounds in soybean and their health benefits as it relates to cancer and bone health as described in recent peerreviewed publications. Chapter 22 – Soybean meal (SBM) and soybean hull (SBH) are generated after recovery of soybean oil from soybean processing. The protein content of SBM can be as high as 53.5

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to 56.2% moisture-free basis (mfb). Soybean meal also has a favorable amino acid composition compared with other plant proteins. Because of its high protein content, SBM is primarily used as a commercial animal feed product. Soybean hull contains about 85.7% carbohydrates, 9% protein, 4.3% ash, and 1% lipids and is used as a fiber supplement in animal feed. Despite the recognized value of SBM and SBH, these components have some inherent disadvantages. The dietary energy value of uncooked SBM is relatively low because of antinutrional factors such as proteinase inhibitors and phytate. Oligosaccharides in the carbohydrate fraction, particularly raffinose and stachyose, could lead to flatulence and abdominal discomfort. Further, the widespread availability of distiller grains due to increaseed ethanol production from corn and other cereal grains has saturated the high-protein animal feed market. Microbial bioprocessing of SBM and SBH, along with protein enhancement and removal of antinutritional factors, will result in an enhanced sulfur amino acid profile and additional nutrients, such as vitamin B12. This chapter focuses on SBM and SBH conversion to premium animal feed products and reviews the latest developments in nutritional enhancement via enzymatic and microbial bioconversion.

In: Soybeans: Cultivation, Uses and Nutrition Editor: Jason E. Maxwell

ISBN: 978-1-61761-762-1 © 2011 Nova Science Publishers, Inc.

Chapter 1

SOYBEAN SEED COMPOSITION AND QUALITY: INTERACTIONS OF ENVIRONMENT, GENOTYPE, AND MANAGEMENT PRACTICES Nacer Bellaloui1, Krishna N. Reddy2, H. Arnold Bruns2, Anne M. Gillen1, Alemu Mengistu3, Luiz H. S. Zobiole4, Daniel K. Fisher2, Hamed K. Abbas1, Robert M. Zablotowicz2 and Robert J. Kremer5 1

Crop Genetics Research Unit, USDA-ARS, Stoneville, MS, USA Crop Production Systems Research Unit, USDA-ARS, Stoneville, MS, USA 3 Crop Genetics Research Unit, USDA-ARS, Jackson, TN, USA 4 Center for Advanced Studies in Weed Science (NAPD), State University of Maringá (UEM), Paraná, Brazil 5 Cropping Systems and Water Quality Research Unit, USDA-ARS, Columbia, MO, USA

2

ABSTRACT Soybean [Glycine max (L) Merr.] seed is a major source of protein, oil, carbohydrates, isoflavones, and minerals for human and animal nutrition. About one-third of the world's edible oils and two-thirds of protein meal are derived from soybean seed. Thus, improving soybean seed composition and quality is key to improving human and animal nutrition. Seed composition refers to major constituents found within the seed including protein, oil, fatty acids, carbohydrates, isoflavones, and mineral content which determine seed nutritional value. Soybean seed quality refers to viability, germination, and seedling vigor which directly impact yield. Seed composition and quality are known to be genetically controlled, and significant variability in seed quality and composition exist due to differences in the gene pool. The physiological and biochemical mechanisms by which this variability is expressed are still not completely understood, but are known to be significantly influenced by genotype (G), environment (E), management practices (MP), and their interactions. Understanding the interaction of these factors and how they impact seed composition and quality is crucial for maintaining high yield and quality.

2

Nacer Bellaloui, Krishna N. Reddy, H. Arnold Bruns et al. This review highlights the current research on seed composition and quality as influenced by G, E, MP, plant diseases, and their interactions from physiological, biochemical, and genetic perspective. This review also highlights major challenges in soybean seed composition and quality improvement, and examines how current research techniques have been used for improving seed nutritional value for conventional uses such as food and feed or for specialty uses such as tofu, soymilk, and other soy products. This information is beneficial as a source in soybean breeding, physiology, and biochemistry research.

1. INTRODUCTION Soybean is one of the major world crops. In 2008, the world consumption of soybean was over 221 million metric tons of which approximately 50% came from the U.S. In the U.S, soybeans were planted on approximately 77 million ha in 2008 (Clemente and Cahoon, 2009). Soybean seed is a major source of protein, oil, carbohydrates, isoflavones, and minerals for humans and animals (Hou et al., 2009). Protein in soybean seed ranges from 34 to 57% of total seed weight, with a mean of 42%. The oil content ranges from 8.3 to 28%, with a mean of 19.5% (Wilson, 2004). The concentration of saturated fatty acids in soybean oil ranges from 10 to 12% palmitic acid (C16:0), and 2.2 to 7.2% stearic acid (C18:0) (Cherry et al., 1985). The mean concentration of unsaturated fatty acids in oil is 24% oleic acid (C18:1), 54% linoleic acid (C18:2), and 8.0% linolenic acid (C18:3) (Schnebly and Fehr, 1993). Higher oleic acid and lower linolenic acid are desirable traits for oil stability and longterm shelf storage. Another component of soybean seed composition is carbohydrates. Most of the carbohydrates are insoluble polysaccharides, including pectin, cellulose, hemicellulose, and starch (Liu, 1997). Soluble carbohydrates include monosaccharides (glucose and fructose), disaccharides (sucrose), and oligosaccharides (raffinose and stachyose) (Liu, 1997). Soybean seed contains 9 to 12% total soluble carbohydrates, which include 4 to 5% sucrose (C12H22O11), 1 to 2 % raffinose (C18H32O16), and 3.5 to 4.5% stachyose (C24H42O21) (Wilson, 1995). Many international and domestic soybean processors prefer soybean with at least 34% protein and 19% oil (Hurburgh et al., 1990). Although seed composition traits are genetically controlled, environmental conditions during seed development, especially seed-fill (R5-R6) stage, can lead to protein and/or oil deficits for processing and substandard seed protein concentrations (Rotundo and Westgate, 2009). Soybean seed quality is another essential component because it determines the germination rate, vigor, and ability of soybean seedlings to emerge and establish, and to avoid disease and other biotic and abiotic stresses. The soybean seed industry uses a standard germination test to determine seed quality. However, such a germination test may not provide information about seed vigor, beyond the ability of seed to germinate (Cartwright et al., 2009; Miller, 2009). Vigor assays were described in detail by Cartwright et al. (2009). Briefly, accelerated aging is achieved by stressing the seed before conducting the standard germination test. Initially the seeds are put into an incubation chamber at 41 ºC for 72 hours. Then, seeds are wrapped in special paper and placed under optimum conditions for two weeks. Seeds subjected to accelerated aging are also planted in the field to test their performance under field conditions (Cartwright et al., 2009). In Mississippi, U.S.A., the minimum germination for certified seed is 80%, and seedlots with less than 60% germination are declared illegal for sale (Keith and Delouche, 1999).

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3

Smith et al. (2008) reported that ideal soybean genotypes for seed quality would have standard and accelerated aging germination percentages of ≥ 90%, and would have low incidences of Phomopsis longicolla, low incidences of wrinkled or shriveled seed, and hardseededness. In spite of the yield benefits under irrigated and non-irrigated conditions (Heatherly, 1999), the Early Soybean Production System (ESPS) is challenged by lower seed quality and substandard germination in the midsouthern United States (Mayhew and Caviness, 1994; Mengistu and Heatherly, 2006; Keith and Delouche, 1999; Smith et al., 2008). Therefore, further research is needed to select high seed quality traits that are adapted to ESPS. In the current review, we will summarize the research on soybean seed composition and quality, and highlight challenges and possible solutions to improve seed composition and quality.

2. EFFECT OF ENVIRONMENT AND GENOTYPE ON SEED COMPOSITION The inverse relationship between protein and oil, and protein and yield remains a major challenge to soybean breeders to select for higher yield and higher protein (Burton, 1985; Rotundo et al., 2009). Attempts to breed genotypes for increased protein without reducing grain yield were previously reported by Wilcox and Cavins (1995). They showed in successive backcross populations that oil concentration increased from 14.8% in BC1 to 17.4% in BC3, suggesting a pattern of oil concentration recovery (20.4 %) of Cutler 71 cultivar. They demonstrated that high seed protein can be backcrossed into a soybean cultivar to fully recover the yield, suggesting the absence of physiological barriers to combine high seed protein with high seed yield. Another challenge for the soybean seed industry is to develop a model to predict the effects of environmental variability across regions and locations to obtain consistent high seed quality (Rotundo and Westgate, 2009). Generally, variability of soybean seed oil between different geographic regions in the U.S.A. (Brenne et al., 1988), Europe and China (Vollmann et al., 2000) was not significant. However, protein was found to be higher in the southern soybean production region than the northern region of the U.S.A. (Brenee et al., 1988; Yaklich et al., 2002). The effect of environment on seed composition has been extensively studied (Maestri et al., 1998; Piper and Boote, 1999; Zhang et al., 2005; Dardanelli et al., 2006). The effect of maturity group (MG) and its interaction with the environment (E) on protein and oil was investigated in 3-yr multilocation soybean trials across Argentina (Dardanelli et al., 2006). Several MGs from II to IX were evaluated in 14 to 24 environments in each year, and the environment was found to be the major contributor to variation for protein and oil content except for 1 yr, and the main effect of MG was greater than the effect of MG × E interaction for oil content, and oil + protein content. They found that all environments produced high oil for MGs II, III, and IV, while the MG × E interaction was seen in two MG × E combinations that maximized protein (i.e., in some environments MG VI had the highest protein and in others, MG II and III yielded more protein). Dardanelli et al. (2006) suggested that high temperature during seed fill could explain the consistent pattern of higher oil content across seasons and environments in early MGs. These results are consistent with those found by Piper and Boote (1999), who studied the effect of mean daily temperature

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Nacer Bellaloui, Krishna N. Reddy, H. Arnold Bruns et al.

and cultivar on oil and protein concentration on 20 cultivars representing 10 MGs across 60 locations under temperatures from 14.6 to 28.7°C. They found a quadratic relationship between proteins and mean daily temperature during seed-fill, with higher concentrations of protein at temperatures below 20°C. In other studies, Maestri et al. (1998) found a negative correlation between oil content and mean temperature during seed maturation, but no effect was found on protein or fatty acids.

2.1 Quantification of Partial Contribution of Environmental Factors and Genotypic Backgrounds to Total Seed Protein and Oil The source of variability of seed composition in the previous studies could be attributed to MG, genotype within MG, and their interactions. In those studies the genotype and MG were confounded because the genotypes of the same MG did not have a common genotypic background. Recently, Bellaloui et al. (2009d), worked on two sets of isolines (Clark and Harosoy isolines), where each set has the same genotypic background, but differ in maturity genes. They found that year, maturity, and year × maturity were significant (p 85% and reduced palmitic acid < 5%) across multiple environments in 2004 and 2005. They found that fatty acids were not influenced by environment, and the yield was not compromised under both irrigated and nonirrigated. They also found that the novel soybean event 335-13 seed characteristics, including total oil and protein, and amino acid profile were not altered. These findings are not in agreement with those reported by Bachlava et al. (2008); Bachlava and Cardinal (2009); Oliva et al. (2006) in that environment × genotype interactions were significant for oleic acid. Also, the findings of Graef et al. (2009) are not in agreement with those reported by Scherder

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Nacer Bellaloui, Krishna N. Reddy, H. Arnold Bruns et al.

and Fehr (2008) in that yield in soybean event 335-13 did not decrease across environment. Graef et al. (2009) reported stable expression of 86%–87% oleic acid content in the oil, with no negative effects on yield, maturity, plant height, lodging, seed weight or oil content. The development of the transgenic soybean events 294-5, 325-61, 333-7 and 335-13 has been previously reported (Buhr et al., 2002), and the parental genotype was either A3237 (Asgrow® Seed Company, Dekalb, IL, U.S.A.) or Thorne (McBlain et al., 1993).

2.3. Effects of Environmental Factors and Genotypes and their Interactions on Seed Carbohydrates Soybean seed soluble carbohydrates include disaccharides (sucrose), and the oligosaccharides (raffinose and stachyose). Raffinose and stachyose are undesirable seed quality traits because they have detrimental effects on the nutritive value of food and feed, causing flatulence or diarrhea in nonruminants (Liu, 1997). Therefore, soybean seed with low raffinose and stachyose is desirable because of increased feed energy efficiency, mineral uptake, and reduce flatulence for nonruminant animals (Obendorf et al., 1998). Soybean seed with high sucrose is desirable because it improves taste and flavor in tofu, soymilk, and nato (Hou et al., 2009). Hymowitz et al. (1972) evaluated 60 plant introductions and found a positive relationship between stachyose and seed protein, suggesting possible difficulties for breeding reduced stachyose concentration with high seed protein. However, Hartwig et al. (1997) suggested that there is a possibility for developing soybean germplasm with high seed protein and lower stachyose + raffinose in the meal. In their experiment, they evaluated sucrose, raffinose, and stachyose in 20 soybean cultivars and breeding lines with high oil and 20 breeding lines with high protein and found nonsignificant negative correlation between protein and stachyose + raffinose, suggesting sugars in soybean seed vary among soybean genotypes and between maturity groups (Hymowitz et al., 1972; Hymowitz and Collins, 1974; Hartwig et al., 1997; Hou et al., 2009). Hymowitz et al. (1972) evaluated protein, oil, and total sugars (sucrose, raffinose, and stachyose) in 60 selected soybean lines from maturity group (MG) 00 through MG IV. They found significant variability (in units of g constituent per 100 g seed) among genotype and between MGs (5.6 to 10.9 total sugars; 2.5 to 8.2 sucrose; 0.1 to 0.9 raffinose; and 1.4 to 4.1 stachyose. They showed a positive correlation between total sugar and sucrose and raffinose, but a negative correlation was found between sucrose and stachyose. Other researchers evaluated 195 soybean cultivars from maturity groups 00 through IV for total sugars, sucrose, fructose, raffinose, and stachyose, and found significant variability in these sugars (Hymowitz and Collins, 1974), and concluded that sufficient variability exists for sugar selection. Carbohydrates in soybean seed has been found to be significantly altered by temperature (Wolf et al., 1982), genotype (Pattee et al., 2000), maturity (Bellaloui et al., 2010), and their interactions (Bellaloui et al., 2010). For example, when soybeans were grown at 18/13 ºC (day/night) and high temperature (33/28 ºC) (day/night), a decrease of sucrose (from 8.1% to 3.6%, a decrease of 56%) and stachyose were observed at higher temperature, but glucose, fructose, and raffinose levels were unaffected at the highest temperature. Recently, Ren et al. (2009) found that the concentrations of sucrose, raffinose, and stachyose did not change in mature seed grown at 37/30 oC compared with the control seed grown in 27/28 oC. However,

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combined sugars (sucrose+raffinose+stachyose) in seed grown under high temperature (37/30 o C) showed lower concentrations compared to the control seed (27/18 oC). Bellaloui et al. (2010) explained that the lack of change in sucrose, stachyose, and raffinose, found by Ren et al. (2009) under higher temperature (37/30oC), could be due to the sampling strategy since abnormally shaped or colored seed were discarded before analysis that may have reduced source of variability in the seed caused by temperature. Bellaloui et al. (2010) investigated the effect of temperature and maturity trait in two sets of near-isogenic lines of Clark and Harosoy for maturity genes. They found a negative linear relationship between days to maturity and the concentration of sucrose (r2=0.83 in 2004; r2=0.94 in 2005), stachyose (r2=0.51 in 2004; r2=0.51 in 2005), and combined sugars (sucrose+raffinose+stachyose) (r2=0.83 in 2004; r2=0.91 in 2005) in Clark isolines. There was a significant negative relationship between maturity and sugars in one year only for sucrose, stachyose, and combined sugars (Table 3). A significant positive relationship with sugars was found in one year, and the negative relationship in another year (Table 3). The Clark isolines showed a significant positive linear correlation between sucrose and raffinose, and sucrose and stachyose (Figure 1a,b), and stachyose and combined sugars (Figure 1c,d). No correlation was found between stachyose and raffinose (Figure 1c, d). A similar observation was recorded for the Harosoy isolines (Bellaloui et al., 2010). This observation was consistent in a two-year field experiment in both sets of near-isogenic lines. They also quantified the contribution of each component to the total sugars and concluded that the contribution of temperature or maturity to the total variability of sugars depended on type of sugar, and the contribution of maturity to total variability in sugars was greater than of temperature. Maturity × genotypic background × temperature interactions were also a major contributor (Bellaloui et al., 2010). Table 3. Regression analysis (level of significance of r2, coefficient of determination) of seed sucrose, raffinose, stachyose, and combined sugar on maturity (R8), and maximum temperature (MaxT) in all Clark and Harosoy near-isogenic lines in 2004 and 2005. The experiment was conducted at the Stoneville Delta States Research Center, MS, U.S.A. Table was extracted from Bellaloui et al. (2010) Isoline Set Clark Maturity MaxT Harosoy Maturity MaxT

Year

Sucrose

Raffinose

Stachyose

Combined sugars

2004 2005 2004 2005

***†(-) ***(-) *** (+) **(-)

ns ns ns ns

***(-) ***(-) *(+) *(-)

***(-) ***(-) **(+) **(-)

2004 2005 2004 2005

Ns **(-) Ns ***(-)

ns ns ns ns

ns **(-) ns **(-)

ns ***(-) ns ***(-)

* indicates significance at p ≤ 0.05, ** indicates significance at p ≤ 0.01, and *** indicates significance at p ≤ 0.001. (+) indicates positive relationships; (-) indicates negative relationships; ns indicates not significant.

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Nacer Bellaloui, Krishna N. Reddy, H. Arnold Bruns et al. 140

140

a

2004

c

2004

Clark near-isogenic lines

Clark near-isogenic lines

120

120

sucrose vs raffinose sucrose vs stachyose

stachyose vs raffinose stachyose vs combined sugars 100

Concentration (mg g-1)

Concentration (mg g-1)

100

r=0.967, p65%. The ranges of trypsin inhibitor activity are given in Table 1, 5-7 mg trypsin inhibitor/g of concentrate or 811 mg/g of concentrate protein. Soy protein isolates are processed to contain >90% protein. Depending upon the amount of washing of the precipitated curd as well as the trypsin inhibitor content of the starting soybeans, isolates contain trypsin inhibitor levels in the range of 1-30 mg/g or, because nearly all of this product is protein, the same range per gram of protein (Table 1). Oriental and other soybean foods are generally low in trypsin inhibitor (Table 2). Soy sauce, produced by enzymic or acidic hydrolysis of a mixture of soybeans and wheat, is low in trypsin inhibitor content at 0.3 mg/g of sample or 3.3 mg/g of protein equivalent. It is unlikely that these values represent activity from the protein inhibitors but the origin of the measured activity is uncertain. Miso is a product made by fermentation of soaked and steamed soybeans. Table 2 shows a value of 4 mg/g of sample or 23 mg/g of protein for miso. However, when lipids are removed from miso by extraction, the trypsin inhibitor level falls to 4 mg/g of protein and it will decrease further if the miso is heated. Tofu, which is made from heated soybean milk by coagulation of the proteins with a salt such as calcium sulfate followed by cooling, has a trypsin inhibitor content (9 mg/g of protein, Table 2) near to that as associated with a fully toasted soy flour (16 mg/g of protein, Table 1). Soy infant formula is made from soy protein isolate with the addition of other nutrients. The

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M.K.Tripathi and S.Mangaraj

trypsin inhibitor level in these products falls in the range of 0.3-3 mg/g of sample or 2-16 mg/g of protein. Di Pietro and Liener (1989) differentiated between the Kunitz and BowmanBirk inhibitor content of soybean flours, concentrates, isolates and a variety of foods. The Kunitz soybean trypsin inhibitor present was quantitated by rocket immunoelectrophoresis and the Bowman-Birk inhibitor was measured by chymotrypsin inhibition. Soy flours were found to contain from 1.1 to 19.6 mg/g and from