Gelatinization and Molecular Properties of Organic and Conventional Rice and Spelt Starches

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Pauline S. Ie, B.S.

Graduate Program in Food Science and Nutrition

The Ohio State University

2011

Master's Examination Committee:

Dr. Yael Vodovotz, Advisor

Dr. Kurt W. Koelling

Dr. Steven J. Schwartz

Pauline S. Ie

2011

Abstract

Gelatinization properties of organic and conventional rice and spelt starches were evaluated. Commercial organic and conventional rice starches were obtained from a supplier. Organic and conventional spelt was planted side by side in six replicated plots in Wooster, OH and the starch was extracted using protease digestion method.

Differential scanning calorimetry (DSC) was used to analyze starch gelatinization temperature range. Dynamic rheological measurement was applied to study the changes in complex viscosity and loss tangent during heating. High-performance size exclusion chromatography (HPSEC) was performed to elucidate the starch molecular composition.

DSC showed that the organic rice starch had a significantly lower gelatinization temperature range than the conventional rice starch (56-66°C for organic, 60-72°C for conventional rice starch). Rheological analysis displayed similar onset temperatures for both organic and conventional rice starches, but a higher peak temperature for the organic rice starch (65°C and 93°C for organic, 64°C and 91°C for conventional starch). The peak complex viscosity was lower for the conventional (2.8 Pa.s) than for the organic rice starch (5.1 Pa.s), suggesting varying structural properties, such as molecular composition,

ii as confirmed by the HPSEC. HPSEC showed that the organic rice starch contained a lower amylose level and a significantly higher molecular weight of amylopectin.

For the spelt starch, DSC showed that the gelatinization temperature range was

57°C - 69°C with a peak temperature of 62°C on average. There was no statistical difference in gelatinization properties between organic and conventional spelt starches.

Plot locations were found to be the driving factor of some of the observed differences.

Rheologically, no significant difference was observed in the onset and peak temperatures of increase in complex viscosity. Variation in the rheological behavior among different plot locations were more pronounced than that between the two growing conditions. The percent mass fraction of amylose in the spelt starch was in the range of 35.7-54.5%.

There was no significant difference in the molecular weight of amylose and amylopectin irrespective of the plot locations. Significant difference was found between the amylopectin Mw of organic and conventional spelt starches when analyzed collectively, which also correlated positively with the gelatinization enthalpy.

Therefore, the organic rice starches studied may be used to substitute for the conventional counterpart only along with suitable adjustment in formulation or processing parameters to achieve equivalent products. The organic spelt starches may replace the conventional one when gelatinization behavior is considered.

iii

Dedicated to my parents, Lioe Sui Fa and Leonard Idris Ie.

Acknowledgments

This thesis would have not been possible without the guidance of my graduate committee members. Therefore, I would like to sincerely thank Dr. Yael Vodovotz, Dr.

Kurt W. Koelling, and Dr. Steven J. Schwartz for providing countless advice and feedbacks for my coursework and research project. I would especially express my utmost gratitude to Dr. Yael Vodovotz, my graduate advisor for her constant support, lending her valuable knowledge and guidance throughout my Master‟s study.

Additionally, I would like to thank Dr. Hamaker, Dave Petros, Madhuvanti Kale, and Deepak Bhopatkar from the Whistler Center for Carbohydrate Research at Purdue

University for their technical expertise and great assistance in the starch molecular composition analysis using the Size Exclusion Chromatography and Multiple Angle

Laser Light Scattering. I would also like to thank Dr. Deborah Stinner and Dr. Larry

Phelan from the Organic Food and Farming Education and Research Program, Ohio

Agriculture Research and Development Center for their kind provision of the spelt grain samples and helpful advice.

Last but not least, I would like to express my sincerest appreciation for all my lab mates for the great insight and tremendous help throughout my study. Specifically, I would like to thank Jennifer Ahn-Jarvis for her technical expertise in sample preparation and analysis, as well as her wealth of knowledge in statistical analysis. I would like to also thank Amber Simmons and Luca Serventi for passing on their proficiency in thermal analysis. I would like to thank Sunny Modi for introducing me to rheological analysis.

Finally, I would like to thank Erica Fisher and Junnan Gu for their continuous support.

Vita

August 25th, 1986 ...... Born in Jakarta, Indonesia

2001-2004 ...... SMAK1 BPK Penabur Jakarta, Indonesia

2004-2008 ...... B.S. Science, Department of Food Science

and Human Nutrition, University of Illinois

at Urbana-Champaign

2009 to present ...... Graduate Research Associate, Department

of Food Science and Technology, the Ohio

State University

Publications

Van Camp, D., Ie, P., Muwanika, N., Vodovotz, Y., Hooker, N. 2010. The paradox of

organic ingredients. Food Technology 64(11):20-29.

Crockett, R., Ie, P., Vodovotz, Y. 2011. How do xanthan and hydroxypropyl

methylcellulose individually affect the physicochemical properties in a model gluten-

free dough. Journal of Food Science 76(3):E274–E282.

vii

Crockett, R., Ie, P., Vodovotz, Y. 2011. Effects of soy protein isolate and egg white

solids on the physicochemical properties of gluten-free bread. Food Chemistry

129(1):84-91.

Fields of Study

Major Field: Food Science and Nutrition

viii

Table of Contents

Abstract ...... ii

Acknowledgments...... v

Vita ...... vii

Publications ...... vii

Fields of Study ...... viii

List of Tables ...... xiii

List of Figures ...... xv

List of Symbols and Abbreviations...... xviii

Chapter 1: Introduction ...... 1

1.1 Organic Foods: Regulation, Growing Condition, and Research ...... 1

1.2 Starch: Composition and Functionality in Foods ...... 5

1.2.1 Rice Starch ...... 12

1.2.2 Spelt Starch ...... 14

1.3 Importance of Thermal Analysis ...... 16

1.3.1 Differential Scanning Calorimetry (DSC) Principle ...... 17

1.3.2 The Application of DSC Measurement in Starch Research ...... 20

1.4 Importance of Rheological Analysis ...... 21

1.4.1 Steady and Dynamic Shear Flow Measurements ...... 23

1.5 Importance of the Evaluation of Molecular Composition ...... 26

1.5.1 Comparison of Available Techniques ...... 27

1.5.2 The Principle of Size Exclusion Chromatography (SEC) Coupled with Multiple

Angle Laser Light Scattering (MALLS) ...... 29

Chapter 2: Statement of the Problem ...... 33

Chapter 3: Objectives ...... 35

Chapter 4: Gelatinization Behavior of Commercial Organic and Conventional Rice

Starches Assessed by Thermal and Rheological Analyses ...... 37

4.1 Introduction ...... 37

4.2 Materials and Methods ...... 41

4.2.1 Materials ...... 41

4.2.2 Gelatinization Study ...... 41

4.2.3 Thermogravimetric Analysis ...... 43

4.2.4 Chromatographic Analysis ...... 43

4.2.5 Statistical Analysis ...... 45

4.3 Results and Discussion ...... 45

4.3.1 Thermal Analysis ...... 45

4.3.2 Rheological Analysis ...... 48

4.3.3 Starch Molecular Composition ...... 53

4.4 Conclusion ...... 57

Chapter 5: Gelatinization Behavior of Organic and Conventional Spelt Starches Assessed by Thermal and Rheological Analyses ...... 58

5.1 Introduction ...... 58

5.2 Materials and Methods ...... 62

5.2.1 Spelt Growing Methods ...... 62

5.2.2 Starch Isolation ...... 63

5.2.3 Gelatinization Study ...... 65

5.2.4 Chromatographic Analysis ...... 66

5.2.5 Statistical Analysis ...... 68

5.3 Results and Discussion ...... 68

5.3.1 Thermal Analysis ...... 68

5.3.2 Rheological Analysis ...... 72

5.3.3 Molecular Composition Analysis ...... 76

5.4 Conclusions ...... 82

Chapter 6: Conclusions ...... 83

References ...... 85

Appendix A: Rheological Behavior of Conventional and Organic Corn Starches ...... 94

Materials ...... 95

Methods ...... 95

Gelatinization Study ...... 95

Gelled Starch Study ...... 96

Results ...... 97

xii

List of Tables

Table 1.1 Gelatinization temperature of several common native starches (Schoch 1956;

Tester and Morrison 1990a; Wang and others 2010; Wilson and others 2008)

...... 11

Table 1.2 Common shear rates used in food processing (Loh 1992) ...... 22

Table 4.1 Gelatinization properties of rice starches using DSC (mean ± standard

deviation)……………………………………………………………….…...46

Table 4.2 Onset temperature, peak temperature, and peak complex viscosity of rice

starches during gelatinization using the rheometer (mean of triplicate ±

standard deviation)…………………………………………………………...50

Table 4.3 Mass fractions and molecular weights (Mw) of rice starch components analyzed

by HPSEC (mean ± standard deviation)…..…………………………………53

Table 5.1 Gelatinization properties of spelt starches using DSC (mean ± standard

deviation)………………………………………………………………..…...69

xiii

Table 5.2 Onset temperature, peak temperature, and peak complex viscosity of spelt

starches during gelatinization using the rheometer (mean of triplicate ±

standard deviation)…………………………………………………………...74

Table5.3 Percent mass fractions and molecular weights (Mw) of spelt starch components

analyzed by HPSEC (mean ± standard deviation). Peak 1 represents

amylopectin, and Peak 2 represents amylose……………….……………….78

xiv

List of Figures

Figure 1.1 Structure of amylose and amylopectin (Tester and others 2004) ...... 6

Figure 1.2 Alternating amorphous and crystalline regions within a starch granule (Image

source: http://www.ceb.cam.ac.uk/pages/pfg-alumni-nitin-nowjee.html) ..... 9

Figure 1.3 Gelatinization and pasting properties of starch granules (Srichuwong and Jane

2007) ...... 10

Figure 1.4 Phyiscal transformation of starch structures (Jane 2004) ...... 11

Figure 1.5 A diagram showing a DSC furnace. (Image source:

http://pslc.ws/macrog/dsc.htm) ...... 18

Figure 1.6 An example of DSC themrogram for semi-crystalline materials (Kaletunç and

Breslauer 2003) ...... 19

Figure 1.7 Schematic of the principle of SEC. (Image source:

http://www.postech.ac.kr/chem/poly/research/chrom.htm) ...... 29

Figure 1.8 Schematic of chromatographic analysis attached in series to MALLS and RI.

(Image source: http://www.wyatt.com/theory/theory/understanding-light-

scattering-and-chromatography-mode.html) ...... 30

Figure 4.1 DSC thermogram showing gelatinization temperature range of conventional

and organic rice starches………………….……………………………..46

Figure 4.2 Complex viscosity (η*) of conventional and organic rice starches as a

function of temperature (25–95°C) and holding at 95°C for 5 min (1 Hz, 0.5

% strain)…………………………………………………...……………….49

Figure 4.3 Tan δ of conventional and organic rice starches as a function of temperature

(25–95°C) and holding at 95°C for 5 min. (1 Hz, 0.5 % strain)…………...52

Figure 4.4 HPSEC Chromatographs from (A) MALLS and (B) Refractive Index

detectors of conventional and organic rice starches. Peak 1 represents

amylopectin, and Peak 2 represents amylose………………………………54

Figure 5.1 Diagram showing six-replicate plots of conventional (CS) and organic spelt

(OS) on the field…………………………………………………………....63

Figure 5.2 Gelatinization profiles of (A) conventional and (B) organic spelt starches

using DSC………………………………………………………………….70

Figure 5.3 Complex viscosity (η*) of (A) conventional and (B) organic spelt starches as

a function of temperature (25–95°C) and holding at 95°C for 5 min (1 Hz,

0.5 % strain)…………………….………………………………………….73

Figure 5.4 Chromatographic profile of CS4 and OS4, representative of each conventional

and organic spelt starches. Peak 1 represents amylopectin, and Peak 2

represents amylose…………………..……………………………………..77

xvi

Figure A.1 Complex viscosity (η*) of conventional and organic corn starches as a

function of temperature (25–95 °C) and holding at 95 °C for 5 min (1 Hz, 0.5

% strain)…………………………………………………………………….97

Figure A.2 Storage modulus (G’) and loss modulus (G”) during frequency sweep of

gelatinized conventional and organic corn starches (25°C, 0.5% strain).…98

xvii

List of Symbols and Abbreviations

IFOAM International Federation of Organic Agricultural Movements

ISO International Organization for Standardization

NOP National Organic Program

NOSB National Organic Standards Board

CFR Code of Federal Regulations

DSC Differential Scanning Calorimetry

To Onset Temperature

Tp Peak Temperature

Tc Conclusion Temperature

°C Degree Celcius

ΔH Change in Enthalpy

J Joule g Gram

W Heat Flow

TGA Thermogravimetric Analysis

xviii

G’ Elastic (Storage) Modulus

G” Viscous (Loss) Modulus

η* Complex Viscosity

η*p Peak Complex Viscosity

Tan δ Loss Tangent (Ratio of Viscous to Elastic Modulus)

LVR Linear Viscoelastic Region

µm Micrometer

Hz Hertz

Pa Pascal

Pa.s Pascal Second

SEC Size Exclusion Chromatography

HPSEC High Pressure Size Exclusion Chromatography

MALLS Multipe Angle Laser Light Scattering

RI Refractive Index

Mw Molecular Weight (Weight Average)

DMSO Dimethyl Sulfoxide dn/dc Change in Refractive Index over Change in Concentration

xix

Chapter 1: Introduction

1.1 Organic Foods: Regulation, Growing Condition, and Research

Organic foods are defined as products that have been produced in compliance with the principles and practices of organic agriculture, which is aimed at a food production system that maintains the sustainability of the social, ecological, and economic systems (Bourn and Prescott 2002). Organic food products are typically certified according to the standards set forth by organic certifying agencies, which institute their own standards for production and certification processes. Many of these agencies are accredited by the International Federation of Organic Agricultural

Movements (IFOAM) or International Organization for Standardization (ISO), or audited by government agencies, which provide them with the capability of verifying their standards and operating systems (Bourn and Prescott 2002). The regulation of organic agricultural practices, food production and handling requirement is detailed in 7 CFR

205, entitled the National Organic Program (NOP) (e-CFR, 2011).

The organic food industry has grown rapidly occupying greater shelf space in the produce and dairy sections in major U.S. retail grocery stores, while also increasing prominence in the eggs, meats, breads, grains, and beverages section (Dimitri and

Oberholtzer, 2009). The sales of organic foods reached $22.9 billion in 2008 since the commencement of the NOP in 2002 (OTA 2009). Both the number and variety of organic food consumers have grown, though they cannot be classified into a particular race and income, or determined by the presence of children in the household (Dimitry and

Oberholtzer 2009). The propeller of the organic market growth varies with different age groups and from country to country with common reasons including safety (concerns for own health due to pesticide residues in food), freshness, health benefits, nutritional value, effect on environment, and flavor (Bourn and Prescott 2002).

NOP established a “National List”, a list of agricultural and non-agricultural ingredients that are not readily available in organic form and are therefore exempted for use in the non-organic form in foods labeled as “organic” and/or “made with organic” under specified regulation in 7CFR205.605 and 606. (e-CFR 2011). The “National List” was purposed to facilitate the development of multi-ingredient organic food products, while reducing the potential limitation of insufficient supply of organically produced minor ingredients. It was created under the assumption that producers of minor ingredients would benefit from the “incentive” and ingredient inclusion in the List would

“drive innovation” of organic alternatives (NOP 2000; NOSB 2009). However, this guideline has triggered controversy in the organic industry, since organic alternatives in many cases have been developed for particular ingredients, and yet organic food

2 manufacturers are reluctant in switching from minor conventional ingredients to the organic counterpart due to claims of commercial unavailability and unequal functionality

(Van Camp and others 2010).

Since the launch of the National List only one agricultural substance (rice starch) has been completely removed from the List as the use exemption for this product expired on June 21, 2009 (although the use of some others has been restricted), and 38 substances have been added. Several items on this list, such as corn starch, may be available; yet, manufacturers claim that organic versions do not provide equivalent functionality (NOSB

2005). In this case, a petition for corn starch removal from the National List in 2004 experienced a failure due to its lack of commercial availability (NOSB 2005), although a relatively broad range of organic corn starches have been developed since then.

In organic farming, the use of synthetic chemicals as fertilizer, pesticides, and herbicides are generally prohibited, but the use of animal manure (e.g. chicken litter), urban sewage, and composts from plant materials are allowed (Xu 2000); whereas in conventional growing, synthetic chemicals, i.e. urea and nitrogen, are normally employed as fertilizers. Studies have shown little supporting evidence for differences in the nutrient content (except for nitrate) and microbial safety between organic and conventional foods

(Bourn and Prescott 2002).

In a review of 19 studies comparing the content of ingredients in cereal and cereal products, Woese and others (1997) found that the protein content in cereals (wheat and rye) obtained from organically produced or fertilized crops was lower than that from the conventional products. This lower protein content in the organic wheat product led to a

3 reduced baking quality, specifically the loaf volume of bread. Significantly lower protein content was also reported in organically grown rice cultivars as compared to conventional counterparts, as expected due to the lower nitrogen concentration in the organic fertilizer

(Champagne and others 2007). This resulted in changes in texture, causing an increase in slickness and a slight decrease in roughness and hardness of cooked rice. Researchers further suggested that an increase in protein content with nitrogen application may be associated with a decrease in amylose content (Champagne and others 2009), which may then affect the functionality of the starch component. These studies support the hypothesis that the different growing conditions between organic and conventional cereal crops may alter the functional properties of the resultant ingredients used in food products.

While many studies in the realm of organic foods focus on the impact of organic food production on nutritional content, pesticide residue, and sensory quality, as well as consumer purchasing trends (Batte and others 2007, Bourn and Prescott 2002, Dimitri and Oberholtzer 2009, Woese and others 1997, Worthington 2001), research studying the effect of organic crop growing on functional properties of foods is limited. Specifically, to our knowledge there has been no study directly comparing the physicochemical properties of conventional and organic starches. Champagne and others (2007) compared the physicochemical properties of conventional and organic rice cultivars and reported that the differences in pasting and cooking quality of the rice were mostly attributed to the differing protein content, rather than the organic management alone. However, the functional properties of the starch component were not specifically analyzed. A research

4 by Guerreiro and Meneguelli (2009) investigated the viscoelastic properties of organic waxy corn starches treated with heat and acid, but did not compare it against a native conventional waxy corn starch.

The concern of ingredient removal from the National List due to functionality issue, accompanied with the different growing conditions between organic and conventional crops possibly resulting in varying molecular composition and/or structure, led us to hypothesize that a difference in functionality between organic and traditional starches may exist.

1.2 Starch: Composition and Functionality in Foods

Similar to glycogen, a storage form of energy in animals, starch is a storage form of energy in plants. It is also the major source of calories in humans‟ and domestic animals‟ diets (Zhong and others 2006). It is widely used in food and non-food applications due to its physical properties, such as its ability to thicken or stabilize products, to enhance texture, as well as to form a gel. These properties are normally achieved through the changes it experiences upon heating in the presence of excess water

(Collado and Corke 2003).

Starch is mainly composed of two types of polymeric chains of glucose within the granules, namely amylose and amylopectin. Amylose is mostly a linear chain of glucose with α-1,4 linkages, having approximately 200 to 2000 anhydroglucose units (Wurzburg

1972). Amylopectin chain contains α-1,4 linkages at the linear portion and α-1,6 linkages

5 at branched points, with 15 to 25 anydroglucose units in each branch (Wurzburg 1972).

The percentages of these two polysaccharides vary according to the plant source of the starch. The relative proportions of amylose and amylopectin based on weight in native cereal starches are 72-82% for amylopectin and 18-33% for amylose (Buléon and others

1998). The „waxy‟ starches are composed of mainly amylopectin (up to 99%) with less than 15% amylose, while the „high‟ amylose starches contain greater than 40% amylose

(Tester and others 2004). Molecular weight of amylose ranges from 3 x 105 to 9 x 105 Da, while that of amylopectin is between 10 x 106 and 333 x 106 Da (Buléon and others

1998), largely depending on the botanical origin. The molecular structures of amylose and amylopectin are displayed in Figure 1.1.

Figure 1.1 Structure of amylose and amylopectin (Tester and others 2004)

Due to its linearity and the presence of hydroxyl groups, amylose molecules are generally oriented parallel to one another promoting association of one chain with another through hydrogen bonding (commonly known as retrogradation). Hence, at low concentration, its water-attracting capability is decreased. However at high concentration, gelling occurs due to inhibition of partial orientation by steric factors (Wurzburg 1972).

On the other hand, due to the presence of branching, amylopectin molecules are less mobile and thus less likely to associate with one another to cause retrogradation.

Consequently, dispersion of starch rich in amylopectin (i.e. waxy starches) produces an aqueous solution with good clarity and stability, as well as resistance to gelling over time

(Wurzburg 1972).

The shape and size, as well as the size distribution and association as individual or clusters, of starch granules are characteristic of the botanical source of the starch (Tester and others 2004). Under a microscope, the molecular size and shape of starch granules can be effectively observed, allowing identification for the botanical origin of starches.

Diameter of the granules ranges from 3 to 100 microns. Being the smallest granules, rice starch has polygonal-shaped granules with diameter ranging from 3 to 8 microns

(Wurzburg 1972). Whereas, corn and sorghum starches are composed of a mix of polygonal and rounded granules with diameter ranging from 5 to 25 microns. Tapioca starch has rounded and kettle-drum-like granules with diameter between 5 to 35 microns.

Wheat starch has a flat and round clustered appearance, with two size ranges: 2-10

7 microns and 20-35 microns. Having the largest granules (15-100 microns in diameter), potato starch appears elliptical and egg-like shaped (Wurzburg 1972).

Starch granules often contain non-starch materials such as moisture, lipid, protein and mineral. The moisture content of starches typically ranges from 10 to 12% for cereal starches and from 14 to 18% for root and tuber starches (Tester and others 2004). The lipid content in cereal starches is generally composed of internal lipids

(lysophospholipids [LPL] and free fatty acids [FFA]) and surface lipids (triglycerides, glycolipids, phospholipids, and FFA) (Tester and others 2004). The internal lipids content is usually directly proportional to the amylose fraction, where fatty acid chains reside in the hydrophobic center within an amylose helix. Starches normally contain less than

0.6% protein located on the surface of granules. Proteins and lipids can potentially direct the functionality of starch. Many types of minerals (calcium, magnesium, potassium, sodium, and phosphorus) are present within starches in minute quantities (less than

0.4%). They all insignificantly contribute to starch functionality, except for phosphorus, which has three main forms in starch: phosphate monoesters, phospholipids, and inorganic phosphates (Tester and others 2004). Potato starch contains essentially no lipids with phosphate monoester content greater than 0.1%, which is typically bound to specific sites of amylopectin (Tester and others 2004).

Starch is semi-crystalline in nature with alternating crystalline and amorphous regions (Figure 1.2). The crystalline portion is due to the ordered structure of double- helical amylopectin; whereas the amorphous region consists of branched points of amylopectin. The nucleus around which the granule radially grows is called the hilum.

Starch granules from particular botanical origins, such as potato starch, also exhibit striation marks, indicated as concentric lines around the hilum (Wurzburg 1972). Under polarized light microscope, starch granules exhibit a phenomenon well-known as birefringence, evidenced by the appearance of “Maltese crosses” around the hilum.

Figure 1.2 Alternating amorphous and crystalline regions within a starch granule (Image source: http://www.ceb.cam.ac.uk/pages/pfg-alumni-nitin-nowjee.html)

Starch is water insoluble because starch granules are generally too large to be dissolved. When starch is mixed in excess water at room temperature, starch granules absorb some water, but limited amount of swelling occurs. When heat is applied to the starch dispersion, gelatinization occurs, characterized by a rapid granular swelling and the loss of molecular order manifested in irreversible changes in properties, such as crystallites melting, loss of birefringence, amylose leaching, and solubilization

(Scrichuwong and Jane 2007) (Figure 1.3). The progression of transformation of starch granules in the presence of heat and water is illustrated in Figure 1.4. During gelatinization, the semi-crystalline starch becomes amorphous and the viscosity of the system increases. Following gelatinization, pasting occurs, which involves granular swelling, leaching of starch components from granules (mainly amylose), and eventually a total disruption of the granules, causing a decrease in viscosity. Gelatinization and pasting are influenced by starch concentration, rate of heating, and shear force applied

(Srichuwong and Jane 2007).

Figure 1.3 Gelatinization and pasting properties of starch granules (Srichuwong and Jane

2007)

Swelling Heating Starch Granules Swollen Granules Gelatinized Starch

Shrinking Temp > Tg Heat and Shearing Storage Cooling Retrograded Starch Gel Starch Paste Heating

Figure 1.4 Phyiscal transformation of starch structures (Jane 2004)

Starch botanical source and molecular structures, such as amylose molecular size and amylopectin branch chain lengths, as well as amylose to amylopectin ratio, also affect physical properties of starch pastes and gels (Collado and Corke 2003), i.e. the temperature range and peak viscosity of gelatinization. Table 1.1 summarizes the gelatinization temperature ranges of several common commercial starches.

Table 1.1 Gelatinization temperature of several common native starches (Schoch 1956,

Tester and Morrison 1990a, Wang and others 2010, Wilson and others 2008)

Starch Plant Source Gelatinization temperature range (°C) Corn 62 - 70

Waxy Corn 63 - 72 Sorghum 68 - 75

Potato 56 - 68 Tapioca 59 - 70

Wheat 50 - 68 Rice 53 - 85

Spelt 54 - 76

1.2.1 Rice Starch

Rice is an important cereal grain in many parts of the world. In Southeast Asia and Africa combined, rice consumption encompasses more than one fifth of the calorie intake worldwide (Vlachos and Arvanitoyannis 2008). Rice is one of the most cultivated crops in many continents of the world (Asia, Pacific, Latin America, Carribean, and

North Africa) even in the recent centuries. Belonging to the genus Oryza, the two most cultivated rice species are Oryza sativa and Oryza glaberima (Vlachos and

Arvanitoyannis 2008). The consumption of rice in the U.S. has also been increasing, as it is becoming more commonly used not only as direct food, but also in processed foods and beer (Nathan 1993). The escalating demand for rice may be attributed to consumers‟ perception of rice as healthy food, the improving convenience of rice preparation, the tastiness of rice when eaten with many entrees, and the ease in adaptation of rice by- products and/or components (i.e. broken, rice bran, and starch) to consumer uses and as ingredients in food products (Nathan 1993).

Rice is composed of mainly carbohydrates and proteins, along with several vitamins and minerals. Protein content in rice ranges between 4.5-15.9% in O. sativa varieties, and 10.2-15.9% in O. glaberima varieties. The mineral component comprises of iron, zinc, calcium, phosphorus, copper, manganese, molybdenum, potassium, and magnesium. Rice is also a good source of thiamine, riboflavin, and niacin. The carbohydrate portion is composed of mainly two polysaccharide starch components,

12 amylose and amylopectin. The amount of amylose in the starch is largely dependent on the rice variety (Vlachos and Arvanitoyannis 2008).

Rice grain can be processed to yield valuable products for food and industrial applications such as starch, proteins, and oil. Starch is one of the most important commodities obtained from rice. Apart from its major use as an ingredient in foods (i.e. as stabilizer, thickener, and filler) or conversion to other functional ingredients (such as high fructose and high maltose syrups, glucose, and trehalose), studies have suggested its potential use as biofuel upon its conversion to bioethanol (Akoh and others 2008).

Rice starch possesses several distinctive characteristic, such as its smaller granule size (3-10 microns) compared to starches from other botanical sources and varying gelatinization temperatures (for different varieties) (Chatakanonda 2000). Many traditional Asian products, such as noodles, desserts, and snacks, utilize native rice starch and flour as the main ingredients.

Due to the rapid growth of organic food products, the demand for organic rice has also been increasing, reflected on the land acreage for organic rice production of approximately 32,000 acres, a 188% increase since 1995 (Champagne and others 2007).

Organic rice seems to convey to the consumers the perception of safer, fresher, healthier, and better-tasting alternative compared to the conventionally grown rice, although this perception has not been substantiated. In a study comparing the physicochemical and sensory qualities of several rice cultivars, Champagne and others (2007) reported that the protein content of organically grown rice was lower than those grown with the typical

100% N rate, as expected. The differences observed in pasting and cooked textural

13 properties were thus attributed to the variation in protein content, rather than the organic management alone.

Research comparing the physicochemical properties of conventional and organic rice starches is limited. However, as suggested by Champagne and others (2007), rice growing condition, such as fertilization and cultural practices may have an effect on the amylose content of the rice cultivars, which consequently may influence functional properties of the starch component. For example, an increase in protein content with nitrogen application was associated with a decrease in amylose content in rice (Prakash and others 2002).

1.2.2 Spelt Starch

Spelt (Triticum aestivum subsp. spelta) is a type of ancient wheat that was commonly cultivated and used in bread making during the fifth century in the European countries (Abed-Aal and Rabalski 2008, Bonafaccia and others 2000). Its production in the U.S. experienced a major decline in the early 1900‟s due to unpredictable yield, low test weight, limited availability of adapted cultivars, and the inefficient use of time for dehulling (Wilson and others 2008). Its production mainly is centered in the Midwest, especially Ohio, along with many other minor producers, for instance, Pennsylvania,

Kansas and North Dakota (Wilson and others 2008). In recent years, however, interest in this ancient wheat has increased due to the continuous effort of spelt millers to market spelt grain and use it in food products (Ranhorta and others 1996), such as bread,

14 breakfast cereal, and pasta (Abdel-Aal and others 1999, Abdel-Aal and others 1998,

Bonafaccia and others 2000, Macaroni and others 2002), as well as its rising use in organic foods (Abed-Aal and Rabalski 2008).

Spelt crop is attractive to farmers because it thrives in unfavorable climatic and soil conditions, such as the cold weather and low precipitation. This owes to many factors, such as the presence of tough hull that provides protection against soil-borne pathogens (Kema and Lange 1992, Riesen and others 1986) allowing for germination

(Ruegger and others 1990), and the ability to better utilize nutrients in harsh growing conditions (Moudrý and Dvořáček 1999). Spelt also contains consistently higher protein content than common wheat (Ranhorta and others 1996, Wilson and others 2008). Due to these factors, spelt is often considered for organic agriculture, which involves no use of synthetic fertilizers, genetically modified materials, or pesticides (Abdel-Aal and

Rabalski 2008). The current use of organic spelt in organic foods is also driven by consumers‟ perception of well-being upon consumption of spelt products (Abdel-Aal and

Rabalski 2008).

Starch, being the main component of grains including spelt, is the chief energy source in the diet. It is also widely used as key ingredient in dictating the physicochemical properties of finished food products. A study of spelt starch properties by Wilson and others (2008) found that amylose contents of all spelt starches tested were consistently higher than that of the wheat control by about 30%. The researchers also observed that the onset of gelatinization temperature for several of the cultivars studied were significantly elevated compared to that of the wheat control, though the peak and

15 conclusion temperatures as well as enthalpy change of gelatinization were not significantly different. The gelatinization temperature range for the spelt starch was approximately between 53.6 - 75.7°C. Additionally, the spelt starches also gave significantly higher pasting peaks and final viscosities than the wheat starches when studied using the rapid visco-analyzer (Wilson and others 2008).

Abed-Aal and Rabalski (2008) compared the nutritional properties of baked products made from organic spelt to the ones from common wheat and found that organic spelt flour and dough exhibited a higher amount of resistant starch and lower rate and extent of starch digestion, but no difference observed after baking compared to the common wheat products. Bodroža-Solarov and others (2009) studied the functional properties of organic spelt flours in comparison to wheat flour and found that the organic spelt contained significantly higher protein and gluten content than wheat. They also observed that some spelt cultivars were more suitable than others for bread-making purposes. However, these studies did not specifically compare the organic spelt to its conventional counterpart, nor did it focus on the functionality of the starch component.

Therefore, a need exists to study these factors due to the increasing interest of the grain and the organic market in general.

1.3 Importance of Thermal Analysis

Gelatinization is one of the most important processes that starch must undergo before it can impart its functionality in foods, such as thickening, gelling and ingredient-

16 binding. Several methods have been used to evaluate starch gelatinization in foods, such as the estimation of maltose (Roberts and others 1954), determination of iodine blue complex (Roberts and others 1954), observation of polarizing patterns under microscope

(Tester and Morrison 1992, Yeh and Li 1996), and differential scanning calorimetry

(DSC). Among these methods, DSC has been widely utilized in the past 20 years to evaluate thermal properties of starch, particularly its gelatinization properties (Donovan

1979, Eliasson 1980, Wang and others 2010 Xie and others 2006, Yamin and others

1999, Yu and Christie 2001).

1.3.1 Differential Scanning Calorimetry (DSC) Principle

By definition, DSC is a form of thermal analysis methods that can be used to observe and analyze conformational and phase transitions due to a change in thermal energy, monitored as a function of temperature or time at a certain heating rate (Kaletunç and Breslauer 2003). In DSC analysis, two pans (or crucibles), one containing the sample and another serving as a reference, are heated together in the furnace (Figure 1.5). Upon heating, the temperatures of the sample and reference increase, based on the heat capacity of the materials. When endothermic transition occurs, the thermal energy supplied is absorbed by the sample and thus, its temperature increase lags behind that of the reference. On the other hand, when exothermic transition occurs, thermal energy is given off by the sample and thus, the temperature of the reference cell lags behind that of the sample. DSC measures the temperature difference observed between the two cells during

17 heating and report it as heat flow as a function of temperature (Kaletunç and Breslauer

2003).

Figure 1.5 A diagram showing a DSC furnace. (Image source: http://pslc.ws/macrog/dsc.htm)

Starch is a semi-crystalline material containing alternating crystalline region

(owing to the ordered structure of double-helical amylopectin) and amorphous region

(consisting of branched points of amylopectin) (Wurzburg 1972). When the crystalline region experiences ordering or disordering, peaks can be observed in the DSC thermogram (Figure 1.6). Endothermic peak represents the heat absorption causing the disordering of the crystalline region, i.e due to disentanglement of amylopectin molecules. This phenomenon in starch is often referred to crystallite melting transition, and is a well-known characteristic observed during gelatinization. Exothermic peak, conversely, represents heat release, i.e. due to re-ordering of starch molecular components during cooling, known as recrystallization (Kaletunç and Breslauer 2003). 18

The area under the peak corresponds to the enthalpic change (ΔH) that the material undergoes (Xie and others 2006).

A transition in the amorphous region can also be observed in starch preceding the endothermic or exothermic transitions; this transition is referred to as the glass transition.

Glass transition is detected as a sharp change in heat capacity (Figure 1.6) and represents a change in the molecular mobility of the starch components. It has been confirmed that in semi-crystalline materials, glass transition into the rubbery state must occur before endothermic or exothermic events can take place (Kaletunç and Breslauer 2003).

Figure 1.6 An example of DSC themrogram for semi-crystalline materials (Kaletunç and

Breslauer 2003)

1.3.2 The Application of DSC Measurement in Starch Research

The study of starch gelatinization is critical in both controlling processing parameters during the manufacture of starch-containing foods and understanding the mechanical properties of starch-based materials (Xie and others 2006). DSC measurements generally provide information regarding the transition temperatures and enthalpies of starch gelatinization (Xie and others 2006). The analysis of these properties can be performed in a relatively short time. One limitation to the use of DSC is that reported results may not be consistent due to the complexity of thermal properties of starch and variable measurement conditions. Therefore, several factors, such as pan selection, sample preparation and measurement conditions, must be taken into account and kept constant to obtain reproducible results (Xie and others 2006).

Nevertheless, DSC has been accepted as the most suitable method to study starch gelatinization. Several previous studies found that gelatinization properties of starch is dependent upon the botanical source of starch, specifically the amylose/ amylopectin content, molecular structures, granular morphology, degree of polymerization, chain length distribution, and the amount of minor components such as lipids and phospholipids, as well as the pre-treatment it receives (Srichuwong and Jane 2007, Wang and others 2010, Yamin and others 1999, Jayakody and others 2007, Tester and Morrison

1990a). For example, Liu and others (2006) found that the enthalpy change of gelatinization of amylopectin-rich starch (i.e. waxy starch) is greater than that of amylose-rich starch, most likely due to the presence of larger amount crystallinity in

20 amylopectin-rich starch. Furthermore, a low gelatinization temperature is usually observed for starch containing a large amount of short-chain amylopectin molecules

(Jane and others 1999).

External factor, such as moisture content, is also found to affect the shape of the thermal transition and the extent of gelatinization. In excess water, starch gelatinization peak occurs as a single symmetric endotherm (Xie and others 2006). However, a reduction in moisture content results in a biphasic endotherm with broad trailing shoulder. Further reduction in water causes the peak to shift to a higher temperature

(Sahai and Jackson 1999). Another endothermic peak may be observed at even higher temperature provided that starch is conditioned in approximately 50% water, which can be attributed to the annealing of amylopectin crystallites (Liu and others 2006).

Additionally, an endotherm at yet higher temperatures may be observed, owing to the melting of amylose-lipid complex (Andreev and others 1999). Due to its prevalence in the study of starch gelatinization and its relative ease and efficient use, DSC was employed in our study.

1.4 Importance of Rheological Analysis

The processing of starch-containing food usually involves complex heat transfer and fluid flow, in which the food is exposed to heating, cooling, and pumping at a wide range of shear rates (Loh 1992). Table 1.2 displays the typical shear rate ranges encountered in common operations in food processing. The way material behaves during

21 processing is strongly affected and dictated by its rheological properties. Therefore, knowing and understanding the rheological behavior of fluid is critical to predict how it would behave during processing at certain shear rates. Unique flow curves representing each fluid sample can be generated from a rheological study, which can provide useful information for industrial applications (Steffe 1996).

Table 1.2 Common shear rates used in food processing (Loh 1992)

Process or equipment Shear rate (s-1) Batch mixing 10-100 Pumping 30-500 Filling through nozzle 50-200 Plate heat exchanger 50-500 Scrape surface cooker 10-200 Jet cooker/ atomization 1000-100 000 Cooking extruder 500-50 000

The heating of starch dispersion leading to gelatinization induces substantial rheological changes, i.e. increases in apparent viscosity of the starch (Lagarrigue and

Alvarez 2001). By definition, rheology is the study of flow and deformation of matter, particularly, how matter responses to the applied stress or strain (Steffe 1996). One common method to characterize rheological behavior of starch dispersions is by the use of Barbender Visco/Amylograph. Although commonly used in the food industry as a quality control instrument to measure viscosity during gelatinization, the Brabender

Visco/Amylograph fails to provide reliable measurements due to its complex geometry, making it difficult to characterize the flow behavior of fluid. Additionally, a uniform

22 shear rate within the sample is difficult to achieve (Lagarrigue & Alvarez 2001). An alternative to the Visco/Amylograph is the Rapid Visco Analyzer. However, this instrument performs on the same principle as the Visco/Amylograph and cannot relibably be compared to other rheological measurements.

The rotational rheometer is a preferred method to study of the rheological properties of starch. The rotational rheometer has several advantages such as the use of fixed shear rates and temperatures, the ability to capture flow behavior, and the use of a relatively small amount of sample. Though it has some potential limitations, such as sedimentation and evaporation, they can normally be overcome by the use of turbulent shear conditions at initial stage of gelatinization and the use of solvent trap, respectively

(Lagarrigue & Alvarez 2001). Various geometries can be applied when using the rotational rheometer, including the concentric cylinder, cone and plate, and parallel plates.

1.4.1 Steady and Dynamic Shear Flow Measurements

There are two common rotational procedures to monitor gelatinization: the steady shear flow study and dynamic testing. Steady shear flow study evaluates the relationship between shear rates and shear stress (or shear viscosity), whereas dynamic measurements can be used to follow the viscoelastic changes during starch gelatinization. The steady shear flow study can be used to characterize the rheological properties of all fluids, whether or not they exhibit viscoelastic behavior. However, in order to evaluate complex

23 behaviors that cannot be described by steady shear functions alone, analyzing the elastic property using the dynamic (unsteady) shear flow may be necessary in some applications

(Steffe 1996).

Various studies have employed the steady shear flow measurement to understand the evolution of sample‟s apparent viscosity during gelatinization (Alvarez 1998, Loh

1992). Loh (1992) evaluated the effect of differing shear rates on the pasting property of native tapioca starch on a Haake rotational viscometer, equipped with a parallel plate set at varied gaps and rotational speeds. In general, he found that shear rates and gap settings used did not influence the gelatinization temperature, although shear rates did affect the apparent viscosity.

Dynamic rheological measurements have also been widely conducted to characterize the property changes of model starch systems during gelatinization (Eliasson

1986, Hsu and others 2000, Lii and others 1996, Tattiyakul and Rao 2000, Wang and others 2010, Yang and Rao 1998). Tattiyakul and Rao (2000) and Yang and Rao (1998) analyzed the change in complex viscosity (η*) versus temperature of cross-linked waxy maize and native corn starch dispersions, respectively, using a rheometer with a parallel plate geometry. These two studies produced complex-viscosity versus temperature master curves by plotting the reduced complex viscosity (a superposition of η* scaled by a frequency shift factor) over the course of temperature change at different oscillatory frequencies (ω) (Tattiyakul and Rao 2000, Yang and Rao 1998). The modified Cox-Merz rule (Eq. 1) was applied to describe the relationship between η* and apparent viscosity

(ηa) at different shear rates:

(Eq. 1)

In general, they observed that the plots of complex viscosity-frequency and apparent viscosity-shear rate resulted in two linear curves almost parallel to each other, and thus based on the Cox-Merz rule, complex viscosity may be used to estimate values of apparent viscosity. Additionally, Yang and Rao (1998) found that the two variables in the

Cox-Merz rule (C and α) were not affected by temperatures, and that the complex viscosity of corn starch dispersions during gelatinization was independent of the heating rates tested (1.6-6.0°C min-1).

A preliminary dynamic rheological study was conducted to evaluate the change in complex viscosity of conventional and organic corn starches during gelatinization (see materials, methods, and Figure A.1 in Appendix A). As expected, η*increased to a maximum point and then decreased, for both starches. The two starches underwent a different trend in rheological changes as temperature was increased, suggesting varying structural properties within the starch. Wang and others (2010) evaluated the changes in viscoelastic behavior of ten rice starch dispersions during gelatinization (heated from 20–

100°C). They observed the initial increase of the storage and loss moduli (G’ and G” respectively) as temperature increased until maximum values, after which both the dynamic moduli decreased during continuous heating. The initial rise in G’ was attributed to progressive swelling of starch granules until they were tightly packed, amylose leaching, and eventually formation of gel network (Eliasson 1986, Lii and others 1996,

Hsu and others 2000). The subsequent decrease in the dynamic moduli corresponded to the unraveling of amylopectin, resulting in the destruction of gel.

Additionally, the rheological behavior of gelled conventional and organic corn starches was also studied. Based on a strain sweep test (result not shown), a strain of

0.5% was selected from the Linear Viscoelastic Region (LVR) of both starches and set for the subsequent frequency, temperature, and time sweep experiments (see Figure A.2 in Appendix A). Frequency sweep study shows a dependence of the dynamic moduli on frequency, indicating a slight thickening effect at greater oscillatory shear. The elastic modulus, G’, of both starches was greater than the viscous modulus, G”, throughout the frequency range, which is indicative of a gel (Steffe 1996).As in previous studies

(Guerreiro and Meneguelli 2009, Rao and others 1997), the corn starches had a weak-gel behavior, in which the G’ is less than 10 times higher than G” (Ikeda and Nishinari

2001).

1.5 Importance of the Evaluation of Molecular Composition

The understanding of starch functionality in starch-containing foods is often supported by the study on the amylose to amylopectin ratio and the molecular weight

(Mw) of amylose (El-Khayat and others 2003, Sasaki and Matsuki 1998, Tester and

Morrison 1990a, Varavinit and others 2003, Zhong and others 2006). Oftentimes, differences observed in the thermal and rheological experiments may be explained by the knowledge of the starch molecular composition. For example, Varavinit and others

(2003) found that gelatinization temperatures of rice starches from different cultivars were so highly positively correlated with the percentage amylose that the gelatinization

26 temperatures may be used to predict the amylose levels, as they suggested. Additionally, the low-amylose rice starch was observed to affect the pasting properties, i.e. highest peak viscosity and lowest setback viscosity and pasting temperatures (Varavinit and others 2003). Another study by Sasaki and Matsuki (1998) found that the swelling power of wheat starch was negatively correlated with the amylose levels, and positively correlated with the amount of long chains of amylopectin in the granules.

1.5.1 Comparison of Available Techniques

Several techniques have been used to analyze the molecular composition of starch. The iodine-binding method is commonly used in amylose content determination

(Gerard and others 2001, Varavinit and others 2003, Zhu and others 2008). In particular, the iodine-binding procedure with dual wavelengths (510 and 620 nm) has many benefits since it provides relatively good reproducibility and applicability to rather complex food systems (Zhu and others 2008). However, this method is time-consuming and complicated as it requires a precise control and the generation of standard curves.

Although not as widely utilized, DSC can be used to specifically analyze the enthalpy changes of the complex between amylose and lipid (L-α-

Lysophosphatidylcholine from egg yolk) and thus quantify amylose content (Gerard and others 2001, Mestres and others 1996). This method provides a convenient way to test a small number of samples and is also specific and thus, not susceptible to variation due to the presence of non-starch components in food. However, it poses several limitations,

27 such as inability to do simultaneous measurement at a time and the necessity of standard curve construction (Zhu and others 2008).

Another conventional method to quantify amylose and amylopectin levels is by the complexation-with-Concanavalin-A method using the commercial Megazyme amylose/ amylopectin kit (Megazyme, Ireland) (Gerard and others 2001, Labuschagne and others 2007). In this method, Con A reagent is used to precipitate amylopectin.

Amylose content is determined by obtaining the ratio of glucose residue present after hydrolysis by amyloglucosidase to that in the initial sample before removal of amylopectin, analyzed through a colorimetric measurement (Gerard and others 2001).

Advantages of this method are that no standard curve generation is involved and amylose content can be directly determined. Nevertheless, it is tremendously time-consuming, requiring precise control, while also not applicable to complex food matrices due to increased variability (Zhu and others 2008).

Finally, the size exlusion chromatography (SEC) has also been used to determine amylose levels, and simultanouesly evaluate other molecular properties, such as molecular weight and molar mass (Han and others 2005, Kasemsuwan and others 1995,

Jane and Chen 1992, Patindol and others 2003, Yokoyama and others 1998, Zhang and others 2006, Zhong and others 2006, Zhu and others 2008). The use of SEC provides several benefits such as ease of operation and ability to produce straightforward responses from simple calculation based on the peak areas of the chromatographic profile. It is, nonetheless, not as highly applicable to samples other than pure starch and may be susceptible to the issue of sample dispersion, especially with non-starch

28 components (Zhu and others 2008). Gerard and others (2001) compared several amylose determination methods (iodine-binding, concanavalin A, DSC, and SEC methods) and concluded that only SEC gave the most accurate results among all methods tested, since the other methods tended to give elevated apparent amylose levels.

1.5.2 The Principle of Size Exclusion Chromatography (SEC) Coupled with Multiple

Angle Laser Light Scattering (MALLS)

Size exclusion chromatography (SEC) (also known as gel-filtration or gel- permeation chromatography) technique can be used to fractionate starch based on its size, measured as the hydrodynamic volume or radius (Gerard and others 2001, Syahariza and others 2010). This is possible due to the vast difference in the molecular weight (Mw) and structure of amylose and amylopectin (Kobayashi and others 1985). Thus, SEC is a well- known method in evaluating the Mw and Mw distribution of carbohydrate polymers.

Large molecules, such as amylopectin, tend to occupy the void volume in the interstitial space of gel columns (Kobayashi and others 1985) and elute from the column faster than smaller molecules. Smaller molecules travel through longer paths in the column because they tend to stay for longer periods in the pore matrix and thus, elute at later time (see

Figure 1.7).

Figure 1.7 Schematic of the principle of SEC. (Image source: http://www.postech.ac.kr/chem/poly/research/chrom.htm)

The analysis of Mw of starch using SEC poses several drawbacks, such as the necessity of calibration standards and the unavailability of Mw standard in the range of

6 Mw of starch molecules, with the highest available Mw standard being about 2 x 10

(Zhong and others 2006). In order to overcome this challenge, the High Pressure Size

Exclusion Chromatography (HPSEC) equipped with both MALLS and differential refractive index (RI) detectors have been frequently used to quantify the Mw of starch without the need for standard curve generation (Han and others 2005, Yokoyama and others 1998, Zhang and others 2006, Zhong and others 2006) (see Figure 1.8).

Figure 1.8 Schematic of chromatographic analysis attached in series to MALLS and RI.

(Image source: http://www.wyatt.com/theory/theory/understanding-light-scattering-and- chromatography-mode.html)

Light scattering is a non-invasive absolute technique that can be used to characterize polymers without requiring outside calibration standards. Light scattering technique can be applied to determine Mw by the Rayleigh ratio, which is the ratio of scattered light intensity at a certain angle to the intensity of the incident light, as a function of the scattering angle and starch concentration (Wyatt Technology Inc. 2010).

When light, containing certain electric field, passes through a material, the matter will partially scatter light due to oscillating charges at the same frequency of the incoming light. The degree of light scattering relies on the material‟s polarizability, which can be measured by the change in refractive index of the solution with the change in concentration of solutes, known as dn/dc (Wyatt Technology Inc. 2010). The intensity of the scattered light depends on the molar mass and also the angle of scattered light with 31 respect to incident light, which is reliant upon the molecular size. Therefore, by measuring the intensity of the scattered light, the molar mass and molecular size of the material can be calculated (Wyatt Technology Inc. 2010).

Another challenge in the use of SEC is the difficulty in completely dissolving starch molecular components in aqueous solutions. Polysaccharides in starch are slightly polar, but the presence of abundant hydrogen-bonding makes it difficult to be solubilized in neutral solution (Huber and Praznik 2004). Solubilization of these components can only be performed at high temperatures and high pH, which may result in molecular degradation yielding lower molecular size, or elevated molar mass due to incomplete disassociation (Yokoyama and others 1998). Dimethyl Sulfoxide (DMSO) is a polar aprotic solvent with the ability to interact with and dissolve both polar and non-polar compounds. DMSO can form stronger hydrogen bond with starch, thus breaking down hydrogen bonds that exists inter- and intra-molecularly, as well as between starch and water, thus dispersing starch molecules (Zhong and others 2006). Therefore, DMSO or

DMSO solutions have been used to dissolve starch components in the preparation for chromatographic analysis (Kobayashi and others 1985, Zhang and others 2006), and are sometimes used as the mobile phase in SEC experiment (Stone and Krasonski 1981,

Yokoyama and others 1998). The addition of small amount of water to DMSO has been proven to increase the solubility of starch. Particularly, Jackson (1991) found that at

DMSO to water ratio of 9:1, greatest dispersiblity was achieved for corn starch.

Chapter 2: Statement of the Problem

The organic food industry has grown rapidly since the commencement of

National Organic Program (NOP) by USDA in 2002, reaching a $22.9 billion sale in

2008 (OTA 2009). NOP established a “National List”, a list of agricultural and non- agricultural ingredients that are not readily available in organic form and are therefore allowed to be used in the non-organic form in foods labeled as “organic” and/or “made with organic” under specified regulation in 7CFR205.605 and 606. (e-CFR 2011). The creation of the “National List” was intended to facilitate the development of multi- ingredient organic food products, while reducing the potential limitation of insufficient supply of organically produced minor ingredients. It was created under the assumption that producers of minor ingredients would benefit from the “incentive” and ingredient inclusion in the List would “drive innovation” of organic alternatives (NOP 2000, NOSB

2009). However, this guideline has triggered controversy in the organic industry, since organic alternatives in many cases have been developed for particular ingredients, and yet organic food manufacturers are reluctant in switching from minor conventional ingredients to the organic counterpart due to claims of commercial unavailability and

33 unequal functionality (Van Camp and others 2010). Meanwhile, research studying the effect of organic crop growing on functional properties of foods is limited. Specifically, to our knowledge there has been no study directly comparing the physicochemical properties of conventional and organic starches, a prevalent functional ingredient in food products. Therefore, a research comparing the functionality of conventional and organic starches becomes crucial.

Chapter 3: Objectives

It was hypothesized that the different growing conditions used for organic and conventional rice and spelt may alter the molecular composition and thereby the gelatinization properties of the starch component. Therefore, the overall objective of our study was to evaluate the functional properties of organic and conventional rice and spelt starches in terms of thermal and rheological behaviors and to analyze their molecular composition. The following aims were identified.

Aim 1. To evaluate the functional properties related to gelatinization of organic and conventional rice starch in terms of thermal and rheological behaviors.

 To determine the gelatinization temperatures using the differential scanning

calorimetry.

 To investigate the change in rheological properties during gelatinization using the

controlled-stress rheometer.

 To determine the amylose and amylopectin contents and molecular weight using

Size Exclusion Chromatography coupled with Multiple Angle Laser Light

Scattering and Refractive Index detectors.

Aim 2. To evaluate the functional properties related to gelatinization of organic and conventional spelt starch using the same techniques as in aim 1.

Chapter 4: Gelatinization Behavior of Commercial Organic and Conventional Rice

Starches Assessed by Thermal and Rheological Analyses

4.1 Introduction

Organic food is defined as products that have been produced in compliance with the principles and practices of organic agriculture, which is aimed at a food production system that maintains the sustainability of the social, ecological, and economic systems

(Bourn and Prescott 2002). The organic food industry has grown rapidly ($22.9 billion sales in 2008) since the launch of the National Organic Program (NOP) in 2002 (OTA

2009). In particular, the demand for organic rice has amplified substantially, suggested by the increase in its production by 188% from 1997 to 2001 (Greene and Kremen 2001).

The study by Champagne and others (2007) showed no significant difference between organic and conventional rice cultivars in cooking and processing quality. However, to our knowledge, there is currently no research directly comparing the functional properties of organic and conventional rice starches, although suppliers of organic starches claim no difference between the two. Manufacturers utilizing conventional rice starch as an

37 ingredient may want to substitute it with the organic equivalent to attain organic certification and thus, such study would help support suppliers‟ claim and advance organic product development.

Starch is prominent in food products due to the functionality it imparts to foods, such as thickening, gelling, stabilizing and ingredient-binding. Starch, being the main component of rice flour, is a popular ingredient in many Asian traditional products, such as noodles, crackers, desserts, and fermented foods (Chatakanonda 2000). The processing of starch-containing food commonly involves complex heat transfer and fluid flow, which lead to property changes within the sample due to exposure to different temperatures and shearing rates. Oftentimes, it involves partial or complete gelatinization of the starch. In general, not until starch is gelatinized will it be able to perform as a thickener, stabilizer, and ingredient-binder.

Differential Scanning Calorimetry (DSC) has been utilized to evaluate thermal properties of starch, particularly its gelatinization properties (Wang and others 2010,

Donovan 1979, Eliasson 1980, Yamin and others 1999, Yu and Christie 2001). DSC measurement generally provides information regarding the transition temperatures and enthalpies of starch gelatinization (Xie and others 2006). Gelatinization properties of starch is dependent upon the botanical source of starch, specifically the amylose and amylopectin content, molecular structures, granular morphology, degree of polymerization, chain length distribution, and the amount of minor components such as lipids and phospholipids, as well as the pre-treatment it receives (Srichuwong and Jane

2007, Wang and others 2010, Yamin and others 1999, Jayakody and others 2007, Tester and Morrison 1990a).

Dynamic rheological measurements have also been conducted to characterize the property changes of model starch systems during gelatinization (Tattiyakul and Rao

2000, Yang and Rao 1998, Hsu and others 2000, Lii and others 1996, Wang and others

2010, Eliasson, 1986). Tattiyakul and Rao (2000) and Yang and Rao (1998) analyzed the change in complex viscosity (η*) versus temperature of cross-linked waxy maize and native corn starch dispersions, respectively, using a rheometer with a parallel plate geometry. In these studies, the modified Cox-Merz rule (Eq. 1) was applied to describe the relationship between η* and apparent viscosity (ηa) at different shear rates:

(Eq. 1)

In general, they observed that the plots of complex viscosity-frequency and apparent viscosity-shear rate data display as two linear curves almost parallel to each other, and thus based on this rule, complex viscosity may be used to estimate values of apparent viscosity (Tattiyakul and Rao 2000, Yang and Rao 1998).

Wang and others (2010) evaluated the changes in viscoelastic behavior of ten

20% (w/w) rice starch dispersions during gelatinization (heated from 20–100°C). They observed the initial increase of the storage and loss moduli (G’ and G” respectively) as temperature increased until maximum values, after which both the dynamic moduli decreased during continuous heating. The initial rise in G’ was attributed to progressive swelling of starch granules until they were tightly packed, amylose leaching, and eventually formation of gel network (Eliasson 1986, Lii and others 1996, Hsu and others

2000). The subsequent decrease in the dynamic moduli corresponds to the unraveling of amylopectin, resulting in the destruction of the gel.

The understanding of starch functionality in starch-containing foods is often supported by the study on the amylose to amylopectin ratio and the molecular weight

(Mw) of amylose (Zhong and others 2006). Oftentimes, differences observed in the thermal and rheological experiments may be explained by the knowledge of the starch molecular composition. In this study, size exclusion chromatography (SEC) technique was used to fractionate the starch based on its size. The multi-angle laser-light scattering

(MALLS) and the refractive index (RI) detectors served to obtain Mw information and to study the concentrations of different components in starch. This chromatography technique has been used elsewhere in previous research to study the molecular properties of starch (Patindol and others 2003, Kasemsuwan and others 1995, Jane and Chen 1992,

Zhang and others 2006, Zhong and others 2006, Zhu and others 2008). Therefore, the objective of this study was to compare gelatinization properties of commercially available organic and conventional rice starches using thermal and rheological analyses, followed by the analysis of their molecular composition using SEC.

4.2 Materials and Methods

4.2.1 Materials

Remy Dr - Conventional rice starch (A&B Ingredients, Fairfield, NJ) and Remy

O-Dr - Organic rice starch (A&B Ingredients, Fairfield, NJ) were compared for their gelatinization properties. The total moisture content of the conventional and organic rice starches were 10.92% and 10.21%, respectively.

4.2.2 Gelatinization Study

4.2.2.1 Thermal Analysis

Differential Scanning Calorimetry (DSC) Q100 (TA Instruments, New Castle,

DE), previously calibrated using indium, was used to determine the gelatinization temperature range of the starch. Starch samples of 2 -3 mg were weighed into a stainless steel pressure pans and water was added in 1:4 starch to water ratio. The sample pan was hermetically sealed (Perkin Elmer Instruments LLC, Shelton, CT) and placed against an empty reference pan inside the DSC cell previously flushed with nitrogen gas. Samples were equilibrated at 25°C, heated to 100°C at a 5 °C/ minute rate, and held at 100°C for 2 minutes. They were then cooled quickly to 25°C, held for 3 minutes, and rescanned to

100°C at the same rate. The first temperature scan was performed to determine the

41 gelatinization temperature range, while the second scan was to confirm that all starch was completely gelatinized in the first run. Analysis was performed in triplicate for all samples. To, Tp, Tc, and ΔH were obtained and indicate respectively the onset, peak, and conclusion temperatures, and change of enthalpy of gelatinization.

4.2.2.2 Rheological Analysis

Each type of starch was mixed in distilled water to prepare 5% starch dispersions

(w/w). The slurry was continuously stirred with a magnetic stirrer at 650 rpm for approximately 30 minutes at room temperature to hydrate the sample and to obtain a homogenous sample before subsequent analysis. Gelatinization using the rheometer was performed following the procedure described by Tattiyakul and Rao (2000) with a few modifications. A starch dispersion of approximately 0.64 ml was applied to the bottom plate of an AR 2000ex Rheometer (TA Instruments, New Castle, DE) with parallel plate geometry (40 mm dia.) and a 500 µm gap. A solvent trap was used to minimize water evaporation, and standard conditions of 1 Hz and 0.5% strain (previously determined to be in the Linear Viscoelastic Region) were applied. A temperature ramp from 50 – 95°C at a ramp rate of 2.1°C/min was conducted, followed by a time sweep for 5 minutes at

95°C. The effect of temperature change and holding time at 95°C on complex viscosity

(η*) and loss tangent (tan δ) were investigated in triplicated measurements.

4.2.3 Thermogravimetric Analysis

Starch samples were analyzed for total moisture content using the

Thermogravimetric Analysis (TGA). The moisture content of starch slurry (5%) before and after the rheological experiment was also evaluated to determine if evaporation occurred. Samples (6 - 7 mg) was placed on the center of previously-zeroed platinum pans for TGA Q5000 apparatus (TA Instruments, New Castle, DE) and scanned from

25°C to 150°C at 10°C/min ramp. The weight loss curve, attributed to moisture loss, was obtained, and the percentage change in weight was calculated for total moisture.

4.2.4 Chromatographic Analysis

4.2.4.1 Dispersion of Starch Components

Starch sample (125 mg) was added into a 50 mL tube. Five mL of 90% Dimethyl

Sulfoxide (DMSO) was added onto the starch and the sample was boiled for one hour with continuous stirring. It was then removed from heating and stirred overnight at room temperature. Ethanol (100%, 25mL) was added to precipitate the starch. The sample was centrifuged at 5000 RPM for 10 minutes and the supernatant was decanted. Ethanol precipitation and centrifugation were repeated three more times to remove residual

DMSO, after which the sample was vacuum-dried to remove traces of ethanol prior to

SEC analysis.

4.2.4.2 High Performance Size Exclusion Chromatography (HPSEC)

The HPSEC analysis was performed as described in previous studies (Han and others 2006, Zhang and others 2006). Dried starch was re-suspended in water at a 1 mg/ ml concentration. The suspension was vortexed and boiled in a household pressure cooker (Cuisinart Electric Pressure Cooker) for 30 minutes. After rigorous vortexing, the sample was run through a syringe fitted with a nylon filter of 5 μm pore size and injected into the HPSEC system previously washed twice with double-distilled water. The system consisted of an HR 16/50 column (Sephacryl S 500 HR by GE Healthcare, Piscataway,

NJ) and a pump (model LC-10AT vp, Shimadzu Corp., Columbia, MD), attached to the

MALLS (Dawn Heleos-II at 685 nm GaAs laser diode, Wyatt Technology Corp., Santa

Barbara, CA) and RI detectors (Optilab rEX, Wyatt Technology Corp., Santa Barbara,

CA). The eluent was 0.02% NaN3 (w/v) aqueous solution with a flow rate of 1.3 ml/min.

The collection time was set at 120 minutes. Light scattering intensities were collected at

18 angles. Angles from detector 7-14 were used in our analysis. Fraction peaks were processed by ASTRA software (v.3.4.14 Wyatt Technology Corporation, Santa Barbara,

CA) for Mw and percent mass fractions. The second-order Berry model was used for curve fitting. Mw and percent mass fraction calculations were based on the mobile phase refractive index of 1.331 and the dn/dc value of 0.146. The analysis was performed in duplicate.

4.2.5 Statistical Analysis

Statistical analysis was performed using Minitab 16 (Minitab Inc., State College,

Pennsylvania, USA). Data pertaining to the thermal, rheological, and compositional properties of organic and conventional rice starches were compared using a two-tailed t- test and significant differences were measured at a significant level of 0.05.

4.3 Results and Discussion

4.3.1 Thermal Analysis

The thermal properties of rice starches were analyzed using the DSC with two temperature scans as described previously (Figure 4.1). The result of the second scan are not included because all starch was completely gelatinized (no melting peak) during this scan. Significant difference was found in the gelatinization temperature range between organic and conventional rice starches (p < 0.05) (Table 4.1), with the conventional starch having higher onset, peak, and end temperatures of gelatinization.

-0.42

Conventional rice starch -0.44 Organic rice starch

-0.46

-0.48

-0.50

Heat (W/g)Flow - Exo up -0.52

-0.54 50 60 70 80

Temperature (°C)

Figure 4.1 DSC thermogram showing gelatinization temperature range of conventional and organic rice starches.

Table 4.1 Gelatinization properties of rice starches using DSC (mean ± standard deviation).

Sample To (°C) Tp (°C) Tc (°C) ΔH (J/g) Conventional rice starch 60.1 ± 0.6 66.3 ± 0.0 72.2 ± 0.1 1.94 ± 0.04 Organic rice starch 56.6 ± 0.5 62.1 ±0.1 66.2 ± 1.4 1.99 ± 0.06 p-value1 0.0021 0.00014 0.018 0.092 1 p-value was obtained from independent two-tailed t-test. Significant difference was found in gelatinization temperatures between organic and conventional starches (p < 0.05).

Gelatinization temperature is influenced by many factors. Although the starches studied originate from the same type of botanical source and are both in the native form, gelatinization properties may still be dependent upon many aspects such as the amylose and amylopectin content, granular morphology, degree of polymerization, and chain length distribution, as well as the presence of minor components such as lipids and phospholipids (Srichuwong and Jane 2007, Wang and others 2010, Yamin and others

1999, Jayakody and others 2007, Tester and Morrison 1990a). Furthermore, a few of these properties, such as amylose and amylopectin content, may be affected by the growing method (conventional versus organic) including the fertilization practices

(Champagne and others 2007) and cultivar variety (Vlachos and Arvanitoyannis 2008).

As an example, in a previous study, a decrease in amylose content in rice was associated with an increase in protein content due to increased nitrogen application (Prakash and others 2002).

Wang and others (2010) studying the thermal properties of rice starches from ten cultivars observed that the onset, peak and conclusion temperatures, as well as enthalpy of gelatinization varied significantly. They suggested that this variation may have been caused by varying amounts of longer chains amylopectin and crystallinity within the starch granules. According to Yamin and others (1999), longer chains require a higher temperature, thus greater energy, to dissociate and disentangle the chain to initiate gelatinization than double helices with shorter chains. Agreeing with Wang and others

(2010), Tester and Morrison (1990b) studying three sets of waxy rice starches (low gelatinization temperature (GT) – 64-67°C, intermediate GT – 68-71°C, and high GT –

75-79°C), found that the high-GT starches had longer amylopectin chains and higher crystallinity than the low-GT starches. The present study may therefore suggest that the conventional rice starch contained more crystalline material and longer chain amylopectin than the organic rice starch, which needs to be confirmed by a further research. The specific relationship between gelatinization properties and amylose/ amylopectin contents as well as weight average molar mass (Mw) was addressed below.

4.3.2 Rheological Analysis

The rheological properties of the rice starches were analyzed using a dynamic rheological method. The complex viscosity (η*) changes of conventional and organic rice starches during gelatinization are shown in Figure 4.2. In general, η* rose to a maximum for both starches and then declined. The onset temperature of gelatinization for both starches was statistically similar (Table 4.2). However, the peak temperature was found to be significantly different, with the organic rice starch having a higher peak temperature, although the difference was only within 2°C. Organic rice starch also exhibited a significantly higher peak complex viscosity than the conventional starch

(Table 4.2), which was proposed to be due to the difference in molecular composition and is discussed in the subsequent section. Rheological behavior of starch dispersion during gelatinization are mostly ascribed to the granular structure and intergranular interaction, i.e. entanglement among starch molecules in neighboring granules as they become more

48 tightly packed (Lii and others 1996). Amylose is also thought to contribute to the rigidity of the gelatinized starch dispersion.

100 Conventional rice starch Organic rice starch 10

0.1 5 min, 95°C 0.01

0.001

Complex Viscosity (Pa.s) Complex Viscosity

0.0001 50 60 70 80 90 95 100 110

Temperature (°C)

Figure 4.2 Complex viscosity (η*) of conventional and organic rice starches as a function of temperature (25–95°C) and holding at 95°C for 5 min (1 Hz, 0.5 % strain).

Table 4.2 Onset temperature, peak temperature, and peak complex viscosity of rice starches during gelatinization using the rheometer (mean of triplicate ± standard deviation).

Sample To (°C) Tp (°C) η *p (Pa.s) Conventional rice starch 64.2 ± 0.8 90.5 ± 0.2 2.75 ± 0.66 Organic rice starch 65.0 ± 3.2 92.7 ± 0.8 5.07 ± 1.01 p-value1 0.70 0.041 0.043 1 p-value was obtained from independent two-tailed t-test. Significant difference was found in Tp, and η*p between conventional and organic starches (p < 0.05).

Furthermore, the organic and conventional rice starches underwent a different trend in the increase in η*. The conventional starch exhibited a two-stage increase in η*

(initial rapid increase, followed by a slower increase indicated by the shoulder in Figure

4.2), while the organic starch experienced a gradual increase in η* at a higher temperature. This observation may be attributed to differing reaction rate of starch components with water as well as the physical-transformation rate, such as the melting of crystalline regions (Kubota and others 1979), which may then suggest varying structural properties, such as molecular composition and chain length distribution within the starch granules.

According to Eliasson (1986), starch gel can be regarded as a suspension with starch granules as rigid fillers (dispersed phase) in a polysaccharide matrix of amylose leaching to the surrounding water (continuous phase). The increase in elastic and viscous moduli (G’ and G”) can be divided into two stages. The initial increase (around 60-75°C) is due to the progressive swelling of starch granules until they are fully and tightly 50 packed. The later increase leading to the final peak (around 75-95°C) can be attributed to an increased gel volume after amylose and some of the amylopectin leach out. Complex viscosity is a direct function of G’ and G” (Steffe 1996) and therefore, this explanation may justify the phenomenon observed for the conventional starch in the present study.

An increase in η* was observed for organic rice starch during holding at 95°C for

5 min, but not for the conventional rice starch. Total moisture content before and after rheological study was analyzed by the Thermogravimetric Analysis (TGA). TGA measurement gave total moisture content before and after gelatinization of 95.3% and

91.6%, respectively (results not shown). This indicates that the increase in η* during holding at 95°C was not due to evaporation, rather, may owe to molecular reordering of starch components. Hsu and others (2000) explained that this phenomenon may be attributed to leached low-molecular-weight amylopectin that interacts with amylose to reinforce the network.

Tan δ is a measure of viscous (G”) and elastic (G’) moduli and calculated as the ratio of G” to G‟. Tan δ is commonly evaluated in dynamic rheological studies of starch during heating to analyze its pasting and gelling behavior (Lii and others 1996, Hsu and others 2000). As seen in Figure 4.3, the tan δ for both organic and conventional rice starches were above 1 at low temperatures, indicating a liquid-like system, but dropped to below 1 at temperatures above 64°C for conventional or 63°C for organic starch, representing a gel-like material, as expected. These transition temperatures closely matched with the onset gelatinization temperature in thermal analysis signifying crystalline melting beyond this point. This agreed with previous finding by Lii and others

(1996) and Hsu and others (2000), which found that tan δ of rice starch was <0.15 when heated to above 80°C and a transition from sol to gel occurs. During this transition, a 3- dimensional network is formed by the leached amylose and reinforced by the swollen starch particles (Hsu and others 2000). At higher temperatures, an increase in tan δ was observed for both organic and conventional starches, indicating the destruction of gel structure due to extended heating (Hsu and others 2000, Tsai and others 2007), which was postulated to be due to the melting of remaining crystalline material in the granule

(Eliasson 1986) or disentanglement of amylopectin.

100

10 Conventional rice starch Organic rice starch

1 5 min, 95°C

Tan Delta Tan

0.1

0.01 50 60 70 80 90 95 100 110

Temperature (°C)

Figure 4.3 Tan δ of conventional and organic rice starches as a function of temperature

(25–95°C) and holding at 95°C for 5 min. (1 Hz, 0.5 % strain).

4.3.3 Starch Molecular Composition

High performance size exclusion chromatography was used to analyze the molecular composition of the starches. The refractive index detector elucidates the percent mass fractions of amylose and amylopectin in the starches, while the MALLS detector was used to determine their Mw (see Table 4.3 and Figure 4.4). It was previously suggested that peak 1 corresponds to amylopectin portion of the starch, while peak 2 represents amylose (Kobayashi and others 1985, Ratnayake and Jackson 2007).

Table 4.3 Mass fractions and molecular weights (Mw) of rice starch components analyzed by HPSEC (mean ± standard deviation).

Mass Fraction (%) Molecular Weight (g/mol) Sample Peak 12 Peak 22 Peak 1 Peak 2 1.23x108 ± 6.95x106 ± Conventional rice starch 54.4 ± 1.9 45.7 ± 1.9 4.03x106 6.70x105 3.71x108 ± 3.51x107 ± Organic rice starch 61.3 ± 4.1 38.7 ± 4.1 2.55x106 7.10x106 p-value1 0.27 0.27 0.009 0.11 1 p-value was obtained from independent two-tailed t-test. Significant difference was observed in the Mw of amylopectin (peak 1) between conventional and organic starches (p < 0.05). 2 Peak 1 corresponds to amylopectin and Peak 2 corresponds to amylose.

2.0 A Conventional Rice Starch 1.5 Organic Rice Starch

1.0

0.5

Detector Voltage (V) 0.0

10 20 30 40 50 60 70 Time (min)

Peak 1 Conventional Rice Starch B Organic Rice Starch

Peak 2

Differential Refractive Index

10 20 30 40 50 60 70

Time (min)

Figure 4.4 HPSEC Chromatographs from (A) MALLS and (B) Refractive Index detectors of conventional and organic rice starches. Peak 1 represents amylopectin, and

Peak 2 represents amylose.

There was no statistical difference found in the percent mass fraction of amylose and amylopectin in the conventional and organic rice starches, although the values suggested that organic rice starch contained a lower percentage of amylose than the conventional one. However, the organic rice starch displayed significantly higher Mw of amylopectin (p < 0.05) than the conventional counterpart. The result agrees with previous finding that the higher the amylose content of rice starch, the lower the Mw (Zhong and others 2006).

The variation in amylose/ amylopectin contents and molecular weights between the two starches may explain the significant differences observed in thermal and rheological properties of the starches. Previous studies reported that pasting peak viscosity of wheat and rice starches negatively correlated with their amylose content (El-

Khayat and others 2003, Varavinit and others 2003). In our study, the organic rice starch displayed significantly higher peak complex viscosity than the conventional starch, which may then be associated with the lower amylose content. Additionally, the significantly higher Mw of amylopectin in the organic starch, i.e. due to increased chain length or greater degree of polymerization, may contribute to further swelling of the starch and thus higher peak viscosity, as seen in a previous study (Jane and Chen 1992).

A study comparing the gelatinization temperatures of Thai rice cultivars found that there was a positive correlation between amylose content and onset, peak and conclusion temperatures of gelatinization (Varavinit and others 2003). This trend agrees with the results in our study, as the organic rice starch displayed significantly lower

55 gelatinization temperatures and had a lower amylose level compared to the conventional starch. Obanni and BeMiller (1997) suggested that starch granule which contains crystallites that requires less melting energy (i.e. smaller crystals or those containing defects) would melt first upon heating. Accordingly, the organic rice starch may contain crystal structures that required less energy to melt compared to the conventional starch.

The percentage mass fractions of amylose (39-46%) were higher than previously reported values of 16-26% for rice starch (Patindol and others 2003, Varavinit and others

2003). This anomaly may be explained by the fact that the presence of intermediate materials (i.e. fragmented amylopectin) could not be identified as a distinctive peak, as in other previous studies (Kobayashi and others 1985, Patindol and others 2003), and thus was not taken into account. Instead, this intermediate component may have been lumped together with the amylose fraction, increasing the percentage mass fraction of amylose.

Another possible explanation is that overestimation of amylose level may have occurred due to amylose being more easily dispersed in DMSO solution than amylopectin (Zhu and others 2008).

6 7 The Mw of amylose and amylopectin (6.95x10 to 3.51x10 Da for amylose and

1.23 x108 to 3.71 x108 Da for amylopectin) were generally higher than literature values

(6.17 x 104 Da for amylose and 1.49 x 107 Da for amylopectin) (Ratnayake and Jackson

2007). Large variability in the results may be ascribed to the existence of interaction among starch components, i.e. amylopectin-amylopectin or amylose-amylopectin (Zhong and others 2006) that may produce aggregates and thus yield a higher Mw reading.

Variation in Mw may also be attributed to the method for starch dissolution. It was found

56 that starch dissolution in DMSO/50 mM LiBr resulted in a lower starch Mw suggesting less aggregation than starch dissolved in 90%DMSO in water, which is the case in this study (Zhong and others 2006).

4.4 Conclusion

In conclusion, the significant differences observed in the thermal and rheological properties between the organic and conventional starches could be partly explained by the variation in amylose content and Mw. Therefore, the difference in functional properties may not be a direct result of the growing condition per se, but rather due to the inherent variation in molecular composition of the starch. This indicates that the organic rice starch studied may be used to substitute for the conventional counterpart along with adjustment in formulation or processing parameters. A future study is needed to evaluate the effect of organic versus conventional growing method, in a controlled manner, on the complete profile of the starch molecular composition including the degree of polymerization of amylose and amylopectin. It will also be worthwhile to explore the relationship between amylopectin branch chain lengths and the functional properties of the organically and conventionally grown starches.

Chapter 5: Gelatinization Behavior of Organic and Conventional Spelt Starches

Assessed by Thermal and Rheological Analyses

5.1 Introduction

(Bourn and Prescott 2002). The organic food industry has grown rapidly ($22.9 billion sales in 2008) since the launch of the National Organic Program (NOP) in 2002 (OTA

2009). Despite this dramatic growth, research comparing the functionality of organic and conventional ingredients is scarce.

Spelt (Triticum aestivum subsp. spelta) is a type of ancient wheat widely cultivated and used in bread making during the fifth century in Europe (Abed-Aal and

Rabalski 2008). It has been increasing in popularity in recent years due to the continuous effort of spelt millers to market spelt grain and use it in food products (Ranhorta and others 1996), such as bread, breakfast cereal, and pasta (Abdel-Aal and others 1998,

Abdel-Aal and others 1999, Macaroni and others 2002), as well as its rising use in organic foods (Abed-Aal and Rabalski 2008). Consumers often perceive a sense of well- being from the consumption of organic spelt products, which they claim to provide some health benefits (Abdel-Aal and Rabalski 2008).

The use of spelt in organic agriculture, which involves no use of synthetic fertilizers, genetically modified materials, or pesticides, is propelled by several factors, such as its ability to thrive in harsh weather, low precipitation, and unfavorable soil conditions (Wilson and others 2008). These valuable characteristics arise from the presence of a tough hull which provides protection against soil-borne pathogens (Riesen and others 1986, Kema and Lange 1992) and thus allows for germination under stress conditions (Ruegger and others 1990), and the ability to better utilize nutrients in harsh growing conditions (Moudrý and Dvořáček 1999).

Starch is the main component in many cereal grains including spelt and widely used as a key ingredient in foods dictating the physicochemical properties of finished products. A study of spelt starch properties by Wilson and others (2008) found that amylose contents of all spelt starches examined were consistently higher than that of the wheat control by about 30%. The researchers also observed that the onset of gelatinization temperature for several of the cultivars studied were significantly elevated compared to that of the wheat control, though the peak and conclusion temperatures as well as enthalpy change of gelatinization were not significantly different. The gelatinization temperature range for the spelt starch studied using the rapid visco-

59 analyzer was approximately between 53.6 - 75.7°C and resulted in significantly higher pasting peaks and final viscosities compared to wheat starches (Wilson and others 2008).

Abed-Aal and Rabalski (2008) compared the nutritional properties of baked products made from organic spelt to the ones from common wheat and found that organic spelt flour and dough contained a higher amount of resistant starch and lower rate and extent of starch digestion, but no difference was observed after baking compared to the common wheat products. Bodroža-Solarov and others (2009) studied the functional properties of organic spelt flours in comparison to wheat flour and found that the organic spelt contained significantly higher protein and gluten content than wheat. However, these studies did not specifically compare the organic spelt to its conventional counterpart, nor did it focus on the functionality of the starch component.

Gelatinization is one of the most important processes that starch must undergo before it can impart its functionality, such as thickening, gelling and ingredient-binding.

Differential Scanning Calorimetry (DSC) has been utilized to evaluate thermal properties of starch, particularly its gelatinization properties (Donovan 1979, Eliasson 1980, Wang and others 2010, Yamin and others 1999, Yu and Christie 2001). DSC measurements generally provide information regarding the transition temperatures and enthalpies of starch gelatinization (Xie and others 2006).

Dynamic rheological measurements have also been conducted to characterize the property changes of model starch systems during gelatinization (Eliasson 1986, Hsu and others 2000, Lii and others 1996, Tattiyakul and Rao 2000, Wang and others 2010, Yang and Rao 1998). Tattiyakul and Rao (2000) and Yang and Rao (1998) analyzed the change

60 in complex viscosity (η*) versus temperature of cross-linked waxy maize and native corn starch dispersions, respectively, using a rheometer with a parallel plate geometry. The modified Cox-Merz rule (Eq. 1) was applied to describe the relationship between η* and apparent viscosity (ηa) at different shear rates:

(Eq. 1)

In general, they found that based on this equation, η* may be used to estimate values of

ηa.

The understanding of starch functionality in starch-containing foods is often supplemented by the study on the amylose to amylopectin ratio and the molecular weight

(Mw) of amylose (Sasaki and Matsuki 1998, Tester and Morrison 1990, Varavinit and others 2003, Zhong and others 2006). In this study, size exclusion chromatography (SEC) technique was used to fractionate the starch based on its size. The multi-angle laser-light scattering (MALLS) and the refractive index (RI) detectors served to determine Mw and concentrations of different components in starch, amylose and amylopectin. This chromatography technique has been used elsewhere in previous research to study the molecular properties of starch (Jane and Chen 1992, Kasemsuwan and others 1995,

Patindol and others 2003, Zhong and others 2006, Zhu and others 2008).

Therefore, the objective of this study was to compare gelatinization properties and molecular composition of starches extracted from locally grown organic and conventional spelt using thermal, rheological and SEC analyses.

5.2 Materials and Methods

5.2.1 Spelt Growing Methods

Spelt (Oberkulmer, var.) was planted in fall 2009 in organic and conventional plots of the Ohio State University‟s Ohio Agriculture Research and Development

Center‟s long-term Organic Farming Systems Experiment (OFSE) located in Wooster,

OH. This experiment specifically compared conventional and certified organic management of agronomic crops in a randomized block design with six replicates. Soil type was Wooster Silt Loam. The organic plots have been certified organic under

USDA‟s National Organic Program regulations since 2002. All plots were plowed, disked and prepared for planting in a similar fashion. In the conventional plots, fungicide- treated seed was planted on October 22, 2009 into six-replicate plots (Figure 5.1).

Chemical fertilizer was applied during planting at the rate of 20 kg/ha. Spelt in conventional plots was top-dressed with 90 kg/ha of Urea on April 6, 2010. Fungicide

Express (tribenuron methyl) was sprayed at 24 mL/ha on April 4, 2010. In organic plots untreated seed was planted on October 22, 2009. Spelt in organic plots was fertilized during planting with an organically-approved partially composted poultry product at 2000 kg/ha and top-dressed with an additional 2000 kg/ha on April 18, 2010. Whole grain samples were collected on August 20, 2010 and stored in drying oven set at room temperature prior to starch analyses. The six field replicates were labeled as such: CS1,

CS2, CS3, CS4, CS5, CS6, OS1, OS2, OS3, OS4, OS5, and OS6 (CS signifies

62 conventional spelt; OS signifies organic spelt; 1-6 is the plot numbers representing replicates at six field locations), totaling 12 samples.

Figure 5.1 Diagram showing six-replicate plots of conventional (CS) and organic spelt

(OS) on the field.

5.2.2 Starch Isolation

Whole spelt grain was cracked using the Allis-Chalmers wheat mill (Allis-

Chalmers Co., Milwaukee, Wisconsin, USA) with a roller of 0.5 mm gap size. The starch was extracted from the grain following the protease method described by Reddy and Seib

(1999) and Wilson and others (2008) with slight modifications as follows. The cracked spelt grain (20 g) was added to 0.02M HCl (200 mL) at 4°C and held for 8-10 min.

Sodium metabisulphite (100 mg) and thiomersal (2 mg) were added to the mixture and its

63 pH was adjusted to 7.6 by the addition of tris(dyroxymethyl)aminomethane (2.5 g) and

1M HCl. Protease (Type XIV, Sigma Chemical Co., St. Louis, Missouri, USA, 100 mg) was dissolved in 0.02M HCl (12 mL) and held for 5 min at room temperature to denature

α-amylase. The protease solution was added to the slurry and the mixture was digested for 30 hr at 4°C with continuous stirring. The digest was then filtered through 425-µm,

150-µm and 74-µm wire mesh sieves connected in series and the softened mass was rubbed against the sieve and washed with water (2 x 20 mL). The filtrate was collected, while the overs were blended in a household blender with water for 30 sec. and re-filtered through the 150-µm and 74-µm sieves. The overs were then placed in a test tube and ground with a homogenizer for 30 sec. The grinding procedures were repeated once more and the mixture was filtered through the 74-µm sieve. All filtrates were combined and centrifuged at 2,500 x g for 10 min. The supernatant was decanted and the suspended starch was washed with water (3 x 20 mL), centrifuged, and the dark tailings were removed using a spatula. The starch was washed with 1% NaCl solution (20 mL) and water (3 x 10 mL). It was subsequently air-dried for about 48 hr. Starch recovery from the whole grain was 45% on average, which is relatively close to that from wheat grain using similar method with 54% on average (Reddy and Seib 1999).

5.2.3 Gelatinization Study

5.2.3.1 Thermal Analysis

Differential Scanning Calorimetry (DSC) Q100 (TA Instruments, New Castle,

DE), previously calibrated using indium, was used to determine the gelatinization temperature range of the starch. Starch samples of 2 -3 mg were weighed into a stainless steel pressure pans and water was added to the starch to in a 1:4 starch-to-water ratio. The sample pans was hermetically sealed (Perkin Elmer Instruments LLC, Shelton, CT) and let stand for 1 hr at room temperature to hydrate the starch. It was subsequently placed against an empty reference pan inside the DSC cell previously flushed with nitrogen gas.

Samples were equilibrated at 25°C and heated to 100°C at a 5°C/ min rate. Analysis was performed in triplicate for all samples. To, Tp, Tc, and ΔH were obtained and indicate respectively the onset, peak, and conclusion temperatures, and change of enthalpy of gelatinization.

5.2.3.2 Rheological Analysis

Each sample was mixed in distilled water to prepare 5% starch dispersions (w/w).

The slurry was continuously stirred for approximately 30 min at room temperature to hydrate and homogenize the sample. Gelatinization using the rheometer was performed following the procedure described by Tattiyakul and Rao (2000) with a few

65 modifications. A starch dispersion of 0.64 ml was applied to the bottom plate of an AR

2000ex Rheometer (TA Instruments, New Castle, DE) with parallel plate geometry (40 mm dia.) and a 500 µm gap. A solvent trap was used to minimize water evaporation, and standard conditions of 1 Hz and 0.5% strain (previously determined to be in the Linear

Viscoelastic Region) were applied. A temperature ramp from 50 – 95°C at a ramp rate of

2.1°C/min was conducted, followed by a time sweep for 5 min. at 95°C. The effect of temperature change on complex viscosity (η*) were investigated. Analysis was performed in triplicate.

5.2.4 Chromatographic Analysis

5.2.4.1 Dispersion of Starch Component

Starch sample (125 mg) was added into a 50 mL tube. Five mL of 90% Dimethyl

Sulfoxide (DMSO) was added onto the starch and the sample was boiled for one hr with continuous stirring. It was then removed from heating and stirred overnight at room temperature. Ethanol (100%, 25mL) was added to precipitate the starch. The sample was centrifuged at 5000 RPM for 10 min and the supernatant was decanted. Ethanol precipitation and centrifugation were repeated three more times to remove residual

DMSO, and subsequently was vacuum-dried to remove traces of ethanol prior to SEC analysis.

5.2.4.2 High Performance Size Exclusion Chromatography (HPSEC)

The HPSEC analysis was performed as described in previous studies (Han and others 2006, Zhang and others 2006). Dried starch was re-suspended in water at 2 mg/ ml concentration. The suspension was vortexed and boiled in a household pressure cooker (Cuisinart Electric Pressure Cooker) for 30 min. After rigorous vortexing, the sample was run through a syringe fitted with a nylon filter of 5 μm pore size and injected into the HPSEC system previously washed twice with double-distilled water. The system consisted of an SEC column (Sephacryl 500 HR by GE Healthcare, Piscataway,

NJ) attached to the MALLS (Dawn Heleos-II at 685 nm GaAs laser diode, Wyatt

Technology Corporation, Santa Barbara, CA) and RI detectors (Optilab rEX, Wyatt

Technology Corporation, Santa Barbara, CA). The eluent was 0.02% NaN3 in water with a flow rate of 1.3 ml/min. The collection time was set at 120 min. Fraction peaks were analyzed by ASTRA software (v.3.4.14 Wyatt Technology Corporation, Santa Barbara,

5.2.5 Statistical Analysis

Means and standard deviations were calculated and differences among the plot locations and growing methods were analyzed using the Multivariate Analysis and t-test on SPSS Statistics 19 (IBM Corp., Armonk, NY). Scheffe method was applied as a post- hoc test. Significant differences were determined when p ≤ 0.05. Correlation of starch molecular composition and physicochemical properties were calculated based on the

Pearson product-moment correlation coefficient (r) using Sigma Plot 11.0 (Systat, San

Jose, CA).

5.3 Results and Discussion

5.3.1 Thermal Analysis

DSC thermograms of organic and conventional spelt starches (Figure 5.2) indicate that there was slight variation in the gelatinization temperature of the starches (Table

5.1). In general, the range of gelatinization was between 56.7°C to 68.8°C with a peak temperature of 62.4°C in average. This temperature range is slightly higher than that of wheat starch (approximately 50-66°C) (Sasaki and Matsuki 1998) and agrees with previous research, which established similar observation (Wilson and others 2008).

Table 5.1 Gelatinization properties of spelt starches using DSC (mean ± standard deviation).

1 2 Sample To (°C) Tp (°C) Tc (°C) ΔH (J/g) CS1 57.2(±0.3)abc 62.7(±0.2)bcd 67.2(±0.3)abcd 2.05(±0.23)a CS2 58.0(±0.4)c 63.4(±0.6)d 68.4(±0.8)d 2.15(±0.11)a CS3 57.8(±0.3)bc 62.1(±0.6)abcd 67.5(±0.6)abcd 2.60(±0.26)a CS4 56.8(±0.2)a 61.5(±0.3)ab 66.6(±0.2)abc 2.49(±0.51)a CS5 57.2(±0.1)abc 62.8(±0.3)cd 68.2(±0.6)bcd 2.25(±0.08)a CS6 57.0(±0.3)ab 62.7(±0.4)bcd 68.4(±0.2)d 2.25(±0.09)a OS1 57.7(±0.2)bc 63.1(±0.4)cd 68.8(±0.8)d 2.23(±0.11)a OS2 56.7(±0.1)a 62.8(±0.0)bcd 68.3(±0.3)cd 2.46(±0.08)a OS3 57.5(±0.2)abc 62.1(±0.2)abc 66.1(±0.1)a 2.44(±0.28)a OS4 57.0(±0.3)ab 61.3(±0.1)a 66.5(±0.0)ab 2.51(±0.17)a OS5 57.3(±0.1)abc 62.1(±0.1)abcd 67.1(±0.1)abcd 2.26(±0.07)a OS6 57.6(±0.1)abc 61.7(±0.2)abc 66.4(±0.3)ab 2.53(±0.21)a 1 CS is conventional spelt; OS is organic spelt; 1-6 is the plot numbers. 2 Means followed by different letters significantly differ at p ≤ 0.05.

-0.15 A CS1 CS4 -0.20 CS2 CS5 CS3 CS6 -0.25

-0.30

-0.35

-0.40

-0.45

Heat Flow (W/g) - Exo up (W/g) - Exo Flow Heat

-0.50 40 50 60 70 80 90 Temperature (°C)

-0.15 B OS1 -0.20 OS2 OS3 -0.25 OS4 OS5 -0.30 OS6

-0.35

-0.40

-0.45

Heat Flow (W/g) up Heat Flow - Exo

-0.50 40 50 60 70 80 90 Temperature (°C) Figure 5.2 Gelatinization profiles of (A) conventional and (B) organic spelt starches using DSC. 70

Cooke and Gidley (1992) suggested that the change in enthalpy during gelatinization represents the degree of crystallinity and amylopectin double-helical order.

Tester and Morrison (1990) further suggested that the level of crystallite perfection is reflected in the gelatinization temperature. A higher temperature of gelatinization can be attributed to the higher amount of longer chain amylopectin, which requires greater thermal energy to dissociate (Wang and others 2010, Yamin and others 1999). In a previous study, it was found that gelatinization temperature of various wheat starches positively correlated with proportion of longer chain amylopectin (DP > 35), suggesting that longer chain amylopectin is able to form more stable crystallites in larger regions

(Sasaki and Matsuki 1998).

There was no clear trend suggesting significant difference in gelatinization properties between organic and conventional starches analyzed by DSC. Plot locations

(indicated by numbers 1 through 6) seemed to be the driving force in some of the observed differences. Gelatinization temperature is influenced by many factors. Although the starches studied originate from the same cultivar and are both in the native form, other factors that may have been affected by growing method, such as amylose and amylopectin content, granular morphology, degree of polymerization, and chain length distribution, as well as the presence of minor components such as lipids and phospholipids, may affect gelatinization behavior (Jayakody and others 2007,

Srichuwong and Jane 2007, Tester and Morrison 1990, Wang and others 2010, Yamin and others 1999). However, no such trend was observed in this study.

5.3.2 Rheological Analysis

The change in complex viscosity (η*) of spelt starches during heating (Figure 5.3) indicates that there were variations in the rheological properties of the starch from spelt crops planted within the same growing conditions at different plot locations. In general, the variation among plot locations were more pronounced than that between the two growing conditions. This difference may be attributed to the reaction rate of starch components with water as well as the physical-transformation rate, such as the melting of crystalline regions (Kubota and others 1979), which may then suggest varying structural properties, such as molecular composition and chain length distribution within the starch granules.

10 A

1 5 min, 95°C

0.1 CS1 CS2

(Pa.s)

* CS3  0.01 CS4 CS5 0.001 CS6

0.0001 40 60 80 95100 Temperature (°C)

10 B

1 5 min, 95°C

0.1 OS1 OS2

* (Pa.s) *

 0.01 OS3 OS4 OS5 0.001 OS6

0.0001 40 60 80 95100 Temperature (°C)

Figure 5.3 Complex viscosity (η*) of (A) conventional and (B) organic spelt starches as a function of temperature (25–95°C) and holding at 95°C for 5 min. (1 Hz, 0.5 % strain).

There was no significant difference in the onset temperature (To) of increase in η*

(average value of 64.4°C), except for OS1 (with significantly lower To, 62.1°C) and OS2

(with significantly higher To, 68.0 °C) (Table 5.2). Similarly, no significant difference was found in the peak temperature (average of 89.0 ± 2.8°C) among the six plot locations or between the two growing methods. Significant difference was observed in the peak complex viscosity (η*p) between CS5, OS2 (having lower η*p), and CS3 (having higher

η*p) potentially due to the variation in molecular composition (see below).

Table 5.2 Onset temperature, peak temperature, and peak complex viscosity of spelt starches during gelatinization using the rheometer (mean of triplicate ± standard deviation).

1 2 Sample To (°C) Tp (°C) η*p (Pa.s) CS1 63.4(±0.5)abc 89.9(±1.6)a 22.5(±4.9)ab CS2 67.5(±1.4)bc 91.9(±0.1)a 16.9(±1.8)ab CS3 64.2(±0.8)abc 88.8(±1.3)a 25.5(±4.7)b CS4 65.2(±3.2)abc 86.1(±5.4)a 14.9(±5.7)ab CS5 63.6(±0.2)abc 90.3(±0.3)a 10.8(±1.4)a CS6 64.1(±0.6)abc 90.3(±0.9)a 19.1(±0.5)ab OS1 62.1(±0.6)a 84.8(±3.2)a 18.6(±1.9)ab OS2 68.0(±2.3)c 91.8(±0.6)a 12.06(±1.2)a OS3 62.6(±0.4)ab 89.0(±1.8)a 17.8(±1.4)ab OS4 62.7(±0.3)ab 87.9(±1.2)a 19.2(±0.9)ab OS5 64.3(±0.1)abc 86.4(±1.2)a 14.2(±2.0)ab OS6 64.7(±1.4)abc 90.8(±0.5)a 14.9(±5.6)ab 1 CS is conventional spelt; OS is organic spelt; 1-6 is the plot numbers. 2 Means followed by different letters significantly differ at p ≤ 0.05.

Rheological behavior of starch dispersion during gelatinization are mostly ascribed to the granular structure and intergranular interaction, i.e. entanglement among starch molecules in neighboring granules as they become more tightly packed (Lii and others 1996). Amylose is also thought to contribute to the rigidity of the gelatinized starch dispersion. According to Eliasson (1986), starch gel can be regarded as a suspension with starch granules as rigid fillers (dispersed phase) in a polysaccharide matrix of amylose leaching to the surrounding water (continuous phase). The increase in elastic and viscous moduli (G’ and G”) can be divided into two stages. The initial increase (around 60-75°C) is due to the progressive swelling of starch granules until they are fully and tightly packed. The later increase leading to the final peak (around 75-95°C) can be attributed to an increased gel volume after amylose and some of the amylopectin leach out. Complex viscosity is recognized as a direct function of G’ and G” (Steffe

1996). There were noticeably two distinctive stages that can be observed in most of the complex viscosity curves (Figure 5.3), and thus it can be accepted that these processes occurred during gelatinization.

Yang and Rao (1998) analyzed the change in η* of corn starch dispersion during heating and found a two-stage increase in η* over increasing temperature. Agreeing with

Eliasson (1986) they explained that the initial raise in η* can be attributed to swelling of starch granules resulting in increased interaction among the granules. There is also sometimes a noticeable slight decrease in η* following the first rise, which coincides with the conclusion temperature of DSC crystalline melting peak (Eliasson 1986). Increase in

η* occurs further upon heating due to continued swelling, causing the formation of gel

75 network, amylose leaching, and granule disruption; after which further heating results in granule rapture and decrease in η*, indicating the weakening of starch network (Yang and Rao 1998), as also observed in Figure 5.3.

5.3.3 Molecular Composition Analysis

HPSEC was applied to analyze the molecular composition of the starches. The refractive index and MALLS detectors elucidate the percent mass fractions and the Mw of amylose and amylopectin in the starches. Figure 5.4 displays representative chromatographs of the conventional and organic spelt starches (CS4 and OS4). It was previously suggested that peak 1 corresponds to amylopectin fraction of the starch, while peak 2 represents amylose (Kobayashi and others 1985, Ratnayake and Jackson 2007).

Calculated percent mass fraction and molecular weights of amylose and amylopectin are summarized in Table 5.3. Statistical analysis was not performed on percent mass fraction data since it only represents proportions of starch components, instead of absolute values.

Peak 1 CS4 OS4

Peak 2

Differential Refractive Index Refractive Differential

10 20 30 40 50 60 70

Time (min)

Figure 5.4 Chromatographic profile of representative conventional and organic spelt starches (CS4 and OS4). Peak 1 represents amylopectin, and Peak 2 represents amylose.

Table5.3 Percent mass fractions and molecular weights (Mw) of spelt starch components analyzed by HPSEC (mean ± standard deviation). Peak 1 represents amylopectin, and

Peak 2 represents amylose.

x Mass Fraction (%) Mw (g/mol) Sample Peak 1 Peak 2 Peak 1 Peak 2 CS1 58.9 41.1 1.36 x108 2.08 x107 CS2 45.5 54.5 2.84 x108 1.58 x107 CS3 53.1 46.9 2.72 x108 1.87 x107 CS4 57.9 42.1 4.15 x108 1.56 x107 CS5 63.8 36.2 2.45 x108 1.90 x107 CS6 53.3 46.8 1.89 x108 2.24 x107 OS1 60.9 39.1 3.22 x108 2.36 x107 OS2 62.3 37.7 3.88 x108 1.61 x107 OS3 62.5 37.6 3.63 x108 2.10 x107 OS4 64.3 35.7 3.86 x108 2.39 x107 OS5 52.0 48.0 2.44 x108 1.68 x107 OS6 56.3 43.7 3.74 x108 1.18 x107 x No significant difference was found in Mw of Peak 1 and Peak 2 among the 12 samples (p > 0.05). However, significant difference was found in Mw of Peak 1 when t-Test was performed to compare the overall organic versus conventional spelt starches (p≤ 0.05).

The percent mass fraction of amylose and amylopectin varied greatly in the spelt starches studied with a range of 35.7-54.5% of amylose. These values are higher than expected, compared to a previous study with amylose content of 30-33% among starches from different spelt cultivars, and 30-31% amylose in the same cultivar (Oberkulmer var.) (Wilson and others 2008). This discrepancy may be attributed to the differing methods in determining percent amylose, as Wilson and others (2008) measured it using the Megazyme amylose assay based on precipitation of amylopectin - Concanavalin A complex. Gerard and others (2001) compared several amylose determination techniques

(iodine-binding, concanavalin A, DSC, and SEC methods) in measuring percent amylose of starch from several botanical origins and found highly variable results among these methods. Another possible explanation for the elevated percent amylose in our study is that overestimation of amylose level may have occurred due to amylose being more easily dispersed in DMSO solution than amylopectin (Zhu and others 2008).

Additionally, this discrepancy may also be explained by the fact that the presence of intermediate materials (i.e. fragmented amylopectin) could not be identified as a distinctive peak, as in other previous studies (Kobayashi and others 1985, Patindol and others 2003), and thus was not taken into account. Instead, this intermediate component may have been included with the amylose fraction, increasing the percentage mass fraction of amylose.

Multivariate analysis did not determine any significant difference among the Mw of amylose and amylopectin in the 12 spelt starches, possibly due to the relatively high variability among the results. This large variability may be ascribed to the existence of interaction among starch components, i.e. amylopectin-amylopectin or amylose- amylopectin (Zhong and others 2006) that may produce aggregates and thus yield higher

Mw values. However, when spelt starches were grouped together based on their growing methods (conventional versus organic) regardless of their plot locations and analyzed using independent t-Test, significant difference was found in the Mw of amylopectin (p =

8 8 0.035), with average Mw of amylopectin of 2.57 x 10 and 3.46 x 10 g/mol for the conventional and organic starches, respectively.

The variation in amylose/ amylopectin contents and molecular weights between the two starches may explain the significant differences observed in thermal and rheological properties of the starches. It was expected that the significantly higher amylopectin Mw in the organic spelt starch would increase their gelatinization temperature and enthalpy over those of the conventional starch. Sasaki and Matsuki

(1998) studying the effect of wheat starch structure on its gelatinization property found that gelatinization temperature and enthalpy had positive correlations with the proportion of long-chain amylopectin (DP > 35). Nevertheless, there was no clear trend observed in the gelatinization temperatures of the 12 samples in this study (Table 5.1) as the significant differences were mostly due to plot locations instead of growing methods.

However, it is worth noting that the Mw of amylopectin correlated positively with the enthalpy of endothermic peak (r = 0.732, p = 0.00675), indicating a possibly higher level of crystallinity in the organic starch, although the enthalpy was not significantly different

(Table 5.1).

The significantly higher Mw of amylopectin in the organic starch, which may possibly be due to increased chain length or greater degree of polymerization, was also expected to contribute to further swelling of the starch and thus higher peak viscosity, as well as more extensive retrogradation, as seen in previous studies (Jane and Chen 1992,

Satsaki and Matsuki 1998). However, this effect was not observed in the peak complex viscosity between conventional and organic starches (Table 5.2), which may suggest that an even greater difference in the Mw of amylopectin may be necessary to yield a significant difference in complex viscosity. Previous findings showed that high Mw

80 fractions of amylopectin promotes further swelling (thus, increase in viscosity) at earlier time than the lower Mw counterparts (Mua and Jackson 1997, Satsaki and Matsuki 1998).

It was further suggested that the long chain of amylopectin is likely to interact to a great extent with amylose to synergistically affect pasting properties (Jane and Chen 1992).

However the significantly different Mw of amylopectin was not found to contribute to significant changes in gelatinization and pasting properties of the conventional and organic spelt starches.

Previous studies reported that pasting peak viscosity of wheat and rice starches negatively correlated with their amylose content, especially for low-amylose starch (El-

Khayat and others 2003, Varavinit and others 2003). However, this relationship was not observed in our study, as the correlation between peak complex viscosity and percent fraction amylose was weak (r = 0.223, p = 0.485). The pasting viscosity of starch can be influenced by many other factors pertaining to the molecular structure, such as the chain average degree of polymerization and branching, Mw, and percent crystallinity (Mua and

Jackson 1997). Specifically, Mua and Jackson (1997) found that amylopectin with low

7 7 Mw (approx. 7 x 10 – 8 x 10 ), high branching ratios (>1.5) and short branch chains (DP of 15-18) exhibited low peak viscosity and high peak temperatures during pasting of corn starch.

5.4 Conclusions

Based on the thermal and rheological analyses of the spelt starches, significant differences in the physiochemical properties were more pronouncedly driven by the plot locations, rather than the growing methods (conventional versus organic). There was no significant difference in the molecular composition of amylose and amylopectin when plot locations and two growing conditions were both considered as influencing factors.

Nonetheless, when each of the six conventional and organic starches was analyzed collectively regardless of the plot locations, the amylopectin Mw of the organic spelt starch was observed to be significantly higher than that of the conventional counterpart.

This significant difference, however, did not contribute to the differing rheological behavior between the conventional and organic starches. The amylopectin Mw was also found to correlate positively with the enthalpy of endothermic peak.

Therefore, the organic spelt starch studied may be used to substitute for the conventional starch when gelatinization behavior is considered. A future study is needed to evaluate the effect of organic versus conventional growing method, in a controlled manner, on the complete profile of the starch molecular composition including the degree of polymerization of amylose and amylopectin. It will also be worthwhile to explore the relationship between amylopectin branch chain lengths and the functional properties of the organically and conventionally grown starches.

Chapter 6: Conclusions

In conclusion, the significant differences observed in the thermal and rheological properties between the organic and conventional rice starches could be partly explained by the variation in amylose content and Mw. Therefore, the difference in functional properties may not be a direct result of the growing condition per se, but rather due to the inherent variation in molecular composition of the starch.

However, based on the thermal and rheological analyses of the spelt starches, significant differences in the physicochemical properties were more pronouncedly driven by the plot locations, rather than the growing methods (conventional versus organic).

There was no significant difference in the molecular composition of amylose and amylopectin when plot locations and two growing conditions were both considered as influencing factors. Nonetheless, when each of the six conventional and organic starches was analyzed collectively regardless of the plot locations, the amylopectin Mw of the organic spelt starch was observed to be significantly higher than that of the conventional counterpart. This significant difference, however, did not contribute to the differing

83 rheological behavior between the conventional and organic starches. The amylopectin Mw was also found to correlate positively with the enthalpy of endothermic peak.

Therefore, the organic rice and spelt starch studied may be used to substitute for the conventional starch when gelatinization behavior is considered. A future study is needed to evaluate the effect of organic versus conventional growing method, in a controlled manner, on the complete profile of the starch molecular composition including the degree of polymerization of amylose and amylopectin. It will also be worthwhile to explore the relationship between amylopectin branch chain lengths and the functional properties of the organically and conventionally grown starches.

References

Abed-Aal ESM, Hucl P, Sosulski FW. 1998. Food uses for ancient wheats. Cereal Foods World 43:763-767.

Abed-Aal ESM, Hucl P, Sosulski FW. 1999. Optimizing the bread formulation for soft spelt wheat. Cereal Foods World 44:480-483.

Abed-Aal ESM, Rabalski I. 2008. Effect of baking on nutritional properties of starch in organic spelt whole grain products. Food Chemistry 111:150-156.

Akoh C, Chang SW, Lee GC, Shaw JF. 2008. Biocatalysis for the production of industrial products and functional foods from rice and other agricultural produce. Journal of Agricultural and Food Chemistry 56:10445-10451.

Alvarez G. 1998. Evolution rhéologique d‟un texturant alimentaire au cours du procédé de fabrication. Fellowship report, Cornell University, Geneva, NY.

Andreev NR, Kalistratova EN, Wasserman LA, Yuryev VP. 1999. The influence of heating rate and annealing on the melting thermodynamic parameters of some cereal starches in excess water. Starch 51(11-12):422-429.

Batte MT, Hooker NH, Haab TC, Beaverson J. 2007. Putting their money where their mouths are: Consumer willingness to pay for multi-ingredient, processed organic food products. Food Policy 32:145-159.

Bonafaccia G, Galli V, Francisci R, Mair V, Skrabanja V, Kreft I. 2000. Characteristics of spelt wheat products and nutritional value of spelt wheat-based bread. Food Chemistry 68:437-441.

Bodroža-Solarov M, Mastilović J, Filipčev B, Šimurina O. 2009. Triticum aestivum spp. Spelta – the potential for the organic wheat production. Časopis za Procesnu Tehniku i Energetiku u Poljoprivredi 13(2):128-131.

Bourn D, Prescott J. 2002. A comparison of the nutritional value, sensory qualities, and food safety of organically and conventionally produced foods. Critical Reviews in Food Science and Nutrition 42(1):1-34.

Buléon A, Colonna, P, Planchot V, Ball S. 1998. Mini review – Starch granules: Structure and biosynthesis. International Journal of Biological Macromolecules 23:85- 112.

Champagne ET, Bett-Garber KL, Grimm CC, McClung AM. 2007. Effects of organic fertility management on physicochemical properties and sensory quality of diverse rice cultivars. Cereal Chemistry 84(4):320-327.

Champagne, ET, Bett-Garber KL, Thomson JL, Fitzgerald MA. 2009. Unraveling the impact of nitrogen nutrition on cooked rice flavor and texture. Cereal Chemistry 86(3):274-280.

Chatakanonda P, Varavinit S, Chinachoti P. 2000. Effect of crosslinking on thermal and microscopic transitions of rice starch. Lebensmittel-Wissenschaft und-Technologie 33:276-284.

Collado LS, Corke H. 2003. Starch properties and functionalities. In G Kaletunc & KJ Breslauer (Eds.), Characterization of cereals and flours (pp. 473-506). New York, NY: Marcel Dekker, Inc.

Cooke D, Gidley MJ. 1992. Loss of crystalline and molecular order during starch gelatinization: Origin of the enthalpic transition. Carbohydrate Research 227:103-112.

Donovan JW. 1979. Phase transition of the starch-water system. Biopolymers 18:263.

Dimitri C, Oberholtzer L. 2009. Marketing U.S. Organic Foods: Recent Trends from Farms to Consumers, Economic Information Bulletin No. 58, U.S. Department of Agriculture, Economic Research Service. http://www.ers.usda.gov/Publications/EIB58. Accessed march 30, 2011.

e-CFR National Organic Program. http://ecfr.gpoaccess.gov/cgi/t/text/text- idx?c=ecfr&sid=3f34f4c22f9aa8e6d9864cc2683cea02&tpl=/ecfrbrowse/Title07/7cfr205_ main_02.tpl. Accessed March 30, 2011.

El-Khayat G, Samaan J, Brennan CS. 2003. Evaluation of vitreous and starchy Syrian durum (triticum durum) wheat grains: the effect of amylose content on starch characteristics and flour pasting properties. Stärke 55(8):358-365.

Eliasson AC. 1980. Effect of water content on the gelatinization of wheat starch. Stärke 32: 270-272.

Eliasson AC. 1986. Viscoelastic behavior during the gelatinization of starch I. Comparison of wheat, maize, potato and waxy-barley starches. Journal of Texture Studies 17:253-265.

Gérard C, Barron C, Colonna P, Planchot V. 2001. Amylose determination in genetically modified starches. Carbohydrate Polymers 22:19-27.

Greene C, Kremen A. 2001. U.S. organic farming in 2000-2001: Adoption of certified systems. Economic Research Service, USDA: Washington,DC. http://www.ers.usda.gov/Publications/AIB780/. Accessed March 30, 2011.

Guerreiro LMR, Meneguelli FC. 2009. Influência do tratamento térmico e da acidez no comportamento reológico de amidos natives funcionais de milho cerosos orgânicos comerciais. Ciência e Tecnologia de Alimentos 29(2):412-419.

Han JA, Lim H, Lim ST. 2005. Comparison between size exlusion chromatography and micro-batch analyses of corn starches in DMSO using light scattering detector. Stärke 57:262-267.

Han X, Ao Z, Janaswamy S, Jane J, Chandrasekaran R, Hamaker BR. 2006. Development of low glycemic maize starch: Preparation and Characterization. Biomacromolecules 7:1162-1168.

Hsu S, Lu S, Huang C. 2000. Viscoelastic changes of rice starch suspensions during gelatinization. Journal of Food Science 65(2):215-220.

Huber A, Praznik W. 2004. Analysis of molecular characteristics of starch polysaccharides. In P Tomasik (Ed.), Chemical and functional properties of food saccharides (pp. 349-369). Boca Raton, FL: CRC Press.

Ikeda S, Nishinari K. 2001. “Weak gel”-type Rheological properties of aqueous dispersions of nonaggregated κ-carrageenan helices. Journal of Agricultural and Food Chemistry 49(9):4436-4441.

Jackson DS. 1991. Solubility behavior of granular corn starches in methyl sulfoxide (DMSO) as measured by high performance size exclusion chromatography. Stärke 43:422-427. 87

Jane J. 2004. Starch: Structure and properties. In P Tomasik (Ed.), Chemical and functional properties of food saccharides (pp. 81-101). Boca Raton, FL: CRC Press.

Jane J, Chen J. 1992. Effect of amylose molecular size and amylopectin branch chain length on paste properties of starch. Cereal Chemistry 69(1):60-65.

Jane J, Chen YY, Lee LF, McPherson AE, Wong KS, Radosavljevic M, Kasemsuwan T. 1999. Effects of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chemistry 76:629-637.

Jayakody L, Hoover R, Liu Q, Donner E. 2007. Studies on tuber starches. II. Molecular structure, composition and physicochemical properties of yam (Dioscorea sp.) starches grown in Sri Lanka. Carbohydrate polymers 69(1):148-163.

Kaletunç G, Breslauer KJ. 2003. Calorimetry of pre- and postextruded cereal flours. In G Kaletunç, KJ Breslauer (Eds). Characterization of cereals and flours – Properties, analysis, and applications (pp. 1-35). New York, NY: Marcel Dekker, Inc.

Kasemsuwan T, Jane J, Schnable P, Stinard P, Robertson D. 1995. Characterization of the dominant mutant amylose-extender (Ae1-5180) maize starch. Cereal Chemistry 72(5):457-464.

Kema GHJ, Lange W. 1992. Resistance in spelt wheat to yellow rust II. Monosomic analysis of the Iranian accession 415. Euphytica 63: 219-224.

Kobayashi S, Schwartz SJ, Lineback DR. 1985. Rapid analysis of starch, amylose and amylopectin by high-performance size-exclusion chromatography. Journal of Chromatography 319:205-214.

Kubota K, Hosokawa Y, Suzuki K, Sosaka H. 1979. Studies on the gelatinization rate of rice and potato starches. Journal of Food Science 44(5):1394-1397.

Labuschagne MT, Geleta N, Osthoff G. 2007. The influence of environment on starch convent and amylose to amylopectin ratio in wheat. Stärke 59:234-238.

Lagarringue S, Alvarez G. 2001. The rheology of starch dispersions at high temperatures and high shear rates: a review. Journal of Food Engineering 50:189-202.

Lii CY, Tsai ML, Tseng KH. 1996. Effect of amylose content on the rheological property or rice starch. Cereal Chemistry 73(4):415-420.

Liu H, Yu L, Xie F, Chen L. 2006. Gelatinization of cornstarch with different amylose/amylopectin content. Carbohydrate Polymers 65(3):357-363. 88

Loh J. 1992. The effect of shear rate and strain on the pasting behavior of food starches. Journal of Food Engineering 16:75-89.

Marconi E, Carcea M, Schiavone M, Cubadda R. 2002. Spelt (Triticum spelta L.) pasta quality: combined effect of flour properties and drying conditions. Cereal Chemistry 79:634-639.

Mestres C, Matencio F, Pons B, Yajid M, Fliedel G, Montpellier. 1996. A rapid method for the determination of amylose content by using differential scanning calorimetry. Stärke 48(1):2-6.

Moudrý J, Dvořáček V. 1999. Chemical composition of grain of different spelt (Triticum spelta L.) varieties. Rostlinná Výroba 45: 533-538.

Mua JP, Jackson DS. 1997. Relationships between functional attributes and molecular structures of amylose and amylopectin fractions from corn starch. Journal of Agricultural and Food Chemistry 45:3848-3854.

Nathan C. 1993. Americans are eating more rice. Food Review 16(2): 19-25.

NOP. 2000. Final Rule. National Organic Program, Federal Register 65: 80548-80684. http://federalregister.gov/a/00-32257.

NOSB. 2005. Meeting transcripts, November 16. Washington, DC: National Organic Program. Updated July 22, 2006. http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template=TemplateN&n avID=NationalOrganicProgram&leftNav=NationalOrganicProgram&page=Nov2005Tran scripts&description=NOSB%20Meeting%20Transcripts:%20November%202005. Accessed March 30, 2011.

NOSB. 2008. Meeting transcripts, May 20. Washington, DC: National Organic Program. Updated Jun 23, 2008. http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template=TemplateN&n avID=NationalOrganicProgram&leftNav=NationalOrganicProgram&page=May2008Tra nscripts&description=NOSB%20Meeting%20Transcripts:%20May%202008&acct=nosb. Accessed March 30, 2011.

NOSB. 2009. Meeting transcripts, November 3-5. Washington, DC: National Organic Program. Updated Dec 3, 2009. http://www.ams.usda.gov/AMSv1.0/ams.fetchTemplateData.do?template=TemplateJ&na vID=FindMeetingInformationNOSBHome&rightNav1=FindMeetingInformationNOSBH ome&topNav=&leftNav=NationalOrganicProgram&page=NOSBMeetings&resultType= &acct=nosb. Accessed March 30, 2011. 89

Obanni M, BeMiller JN. 1997. Properties of some starch blends. Cereal Chemistry 74:431-436.

OTA. 2009. Organic Trade Association releases its 2009 organic industry survey. Greenfield, MA: Organic Trade Association. http://www.organicnewsroom.com/2009/05/organic_trade_association_rele_1.html. Accessed March 30, 2011.

Patindol J, Wang Y, Siebenmorgen T, Jane J. 2003. Properties of flours and starches as affected by rough rice drying regime. Cereal Chemistry 80(1): 30-34.

Prakash YS, Bhadoria PBS, Amitava R, Rakshit A. 2002. Relative efficacy of organic manure in improving milling and cooking quality of rice. International Rice Research Notes 27(1):43-44.

Ranhorta GS, Gelroth JA, Glaser BK, Stallknecht GF. 1996. Nutritional profile of three spelt wheat cultivars grown at five different locations. Cereal Chemistry 73: 533-535.

Rao MA, Okechukwu PE, Da Silva PMS, Oliveira JC. 1997. Rheological behavior of heated starch dispersion in excess water: role of starch granule. Carbohydrate Polymers 33:273-283.

Ratnayake WS, Jackson DS. 2007. A new insight into the gelatinization process of native starches. Carbohydrate Polymers 67:511-529.

Reddy, I., Seib, P.A. 1999. Paste properties of modified starches from partial waxy wheats. Cereal Chemistry 76(3), 341-349.

Riesen TH, Winzler H, Ruegger A, Fried PM. 1986. The effect of glumes on fungal infection of germinating seed of spelt (Triticum spelta L.) in comparison to wheat (Triticum aestivum L.). Journal of Phytopathology 115:318-324.

Roberts RL, Potter AL, Kester EB, Keneaster KK. 1954. Effect of processing conditions on the expanded volume, color, and soluble starch of parboiled rice. Cereal Chemistry 31:121-129.

Ruegger A, Winzeler H, Nosberger J. 1990. Studies on the germination behavior of spelt (Triticum spelta L.) and wheat (Triticum aestivum L.) under stress conditions. Seed Science and Technology 18:311-320.

Sahai D, Jackson DS. 1999. Enthalpic transitions in native starch granules. Cereal Chemistry 76(3):444-448.

Sasaki T, Matsuki J. 1998. Effect of wheat starch structure on swelling power. Cereal Chemistry 75(4):525-529.

Schoch TJ, Maywald EC. 1956. Microscopic examination of modified starches. Analytical Chemistry 28(3):382-387.

Srichuwong S, Jane, J. 2007. Physicochemical properties of starch affected by molecular composition and structures: A review. Food Science and Biotechnology 16(5):663-674.

Steffe, J.F. 1996. Rheological methods in food process engineering. East Lansing, MI: Freeman Press. Chapter 5: pp. 294-349.

Stone RG, Krasonski JA. 1981. Determination of molecular size distributions of modified corn starch by size-exlusion chromatography. Analytical Chemistry 53(4):736-737.

Syahariza ZA, Li E, Hasjim J. 2010. Extraction and dissolution of starch from rice and sorghum grains for accurate structural analysis. Carbohydrate Polymers 82:14-20.

Tattiyakul J, Rao MA. 2000. Rheological behavior of cross-linked waxy maize starch dispersions during and after heating. Carbohydrate Polymers 43:215-222.

Tester RF, Karkalas J, Qi X. 2004. Starch – composition, fine structure and architecture: Review. Journal of Cereal Science 39:151-165.

Tester RF, Morrison WR. 1990a. Swelling ang gelatinization of cereal starches. I. Effects of amylopectin, amylose, and lipids. Cereal Chemistry 67(6):551-557.

Tester RF, Morrison WR. 1990b. Swelling and gelatinization of cereal starches. II. Waxy rice starches. Cereal Chemistry 67(6): 558-563.

Tester RF, Morrison WR. 1992. Swelling and gelatinization of cereal starches. III. Some properties of waxy and normal nonwaxy barley starches. Cereal Chemistry 69:654-658.

Tsai ML, Li CF, Lii CY. 1997. Effects of granular structures on the pasting behaviors of starches. Cereal Chemistry 74:750-757.

Van Camp D, Ie P, Muwanika N, Vodovotz Y, Hooker N. 2010. The paradox of organic ingredients. Food Technology 64(11): 20-29.

Varavinit S, Shobsngob S, Varanyanond W, Chinachoti P, Naivikul O. 2003. Effect of amylose content on gelatinization, retrogradation and pasting properties of flours from different cultivars of Thai rice. Stärke 55(9):410-415.

Vlachos A, Arvanitoyannis IS. 2008. A review of rice authenticity/ adulteration methods and results. Critical Reviews in Food Science and Nutrition 48:553-598.

Wang L, Xie B, Shi J, Xue S, Deng Q, Wei Y, Tian B. 2010. Physicochemical properties and structure of starches from Chinese rice cultivars. Food Hydrocolloids 24:208-216.

Wilson JD, Bechtel DB, Wilson GWT, Seib PA. 2008. Bread quality of spelt wheat and its starch. Cereal Chemistry 85(5): 629-638.

Woese K, Lange D, Boess C, Bögl KW. 1997. A comparison of organically and conventionally grown foods – results of a review of the relevant literature. Journal of the Science of Food and Agriculture 74:281-293.

Worthington V. 2001. Nutritional quality of organic versus conventional fruits, vegetables, and grains. The Journal of Alternative and Complementary Medicine 7(2):161-173.

Wurzburg OB. 1972. Starch in the food industry. In TE Furia (Ed.), CRC handbook of food additives, 2nd ed. (pp. 361-395). Cleveland: The Chemical Rubber Co.

Wyatt Technology Inc. 2010. Understanding classical / static light scattering. http://www.wyatt.com/theory/rayleighscattering/understanding-classical-static-light- scattering.html. Accessed June 30, 2011.

Xie F, Liu H, Chen P, Xue T, Chen L, Yu L, Corrigan P. 2006. Starch gelatinization under shearless and shear conditions. International Journal of Food Engineering 2(5): 1- 29 (art. 6).

Xu HL. 2000. Nature Farming: History, principles and perspectives. Journal of Crop Production 3(1):1-10.

Yamin FF, Lee M, Pollak LM, White PJ. 1999. Thermal properties of starch in corn variants isolated after chemical mutagenesis of inbred line B73. Cereal Chemistry 76(2):175-181.

Yang WH, Rao MA. 1998. Complex viscosity-temperature master curve of cornstarch dispersion during gelatinization. Journal of Food Process Engineering 21:191-207.

Yeh AI, Li JY. 1996. A continuous measurement of swelling of rice starch during heating. Journal of Cereal Science 23:277-283.

Yokoyama W, Renner-Nantz JJ, Shoemaker CF. 1998. Starch molecular mass and size by size-exclusion chromatography in DMSO-LiBr coupled with multiple angle laser light scattering. Cereal Chemistry 75(4):530-535. 92

Yu L, Christie G. 2001. Measurement of starch thermal transitions using differential scanning calorimetry. Carbohydrate Polymers 46(2):179-184.

Zhang G, Ao Z, Hamaker BR. 2006. Slow digestion property of native cereal starches. Biomacromolecules 7:3252-3258.

Zhong F, Wallace Y, Wang Q, Shoemaker CF. 2006. Rice starch, amylopectin, and amylose: Molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of Agricultural and Food Chemistry 54:2320-2326.

Zhu T, Jackson DS, Wehling RL, Geera B. 2008. Comparison of amylose determination methods and development of a dual wavelength iodine binding technique. Cereal Chemistry 85(1):51-58.

Appendix A: Rheological Behavior of Conventional and Organic Corn Starches

Materials

AmiocaTM - a conventional waxy corn starch (National Starch, Bridgewater, NJ) and

Rapunzel corn starch (Edison, NJ – produced and packaged in Austria) were compared for their rheological properties.

Methods

Gelatinization Study

Five percent solution (w/w) was prepared by mixing the starch into distilled water. The slurry was continuously stirred with a magnetic stir bar on a stir plate for approx. 30 minutes at room temperature to hydrate the sample and to obtain a homogenous sample before subsequent rheological analysis.

Gelatinization using the rheometer was performed following the procedure described by Tattiyakul and Rao (2000) with a few modifications. A starch dispersion of

0.64 ml was transferred to the bottom plate of an AR2000 Rheometer (TA Instruments,

New Castle, DE) with parallel plate geometry (40 mm dia.) with a 500 µm gap. A solvent trap was used to minimize water evaporation, and standard conditions of 1 Hz and 0.5% strain (Linear Viscoelastic Region) were applied. The sample was exposed to a temperature ramp from 25 – 95 °C at a ramp rate of 2.1 °C/min, followed by a time

95 sweep for 10 minutes at 95 °C. The effect of temperature change and holding time at 95

°C on complex viscosity (ɳ*) was investigated. Analysis was performed in triplicate.

Gelled Starch Study

Five percent solution (w/w) was prepared by mixing the starch into distilled water. The dispersion was stirred with a magnetic stir bar and heated on a stir-hot plate to a temperature of 95 °C until a semi-solid gel was formed. The gel was removed from the heat, covered, and cooled down to room temperature prior to the rheological analysis.

An AR2000 Rheometer with a parallel plate geometry (40 mm dia.) and 1000 µm gap was used to conduct oscillatory testing comprising of strain and frequency sweeps. In each experiment, a fresh sample was used and before each testing the sample was equilibrated at 25 °C for 3 minutes. Percent strain for frequency sweep was chosen from the percent strain well within the linear viscoelastic range (LVR) during the strain sweep test at both 1 Hz and 10 Hz. The testing range in frequency sweep was 0.1 – 10 Hz. A solvent trap was used to eliminate water evaporation. Data points were recorded approximately every 10 seconds. Analysis was performed in triplicate.

Results

18 16 95°C, 5 min 14 Conventional corn starch Organic corn starch 12 10 8 6 4 2

Complex Viscosity (Pa.s) Viscosity Complex 0

40 60 80 95100

Temperature (°C)

Figure A.1 Complex viscosity (η*) of conventional and organic corn starches as a function of temperature (25–95 °C) and holding at 95 °C for 5 min (1 Hz, 0.5 % strain).

1000 1000

100 Conventional G' 100 Organic G' Conventional G" Organic G"

10 10

Loss Modulus, G" (Pa) Loss Modulus,

Storage Modulus, G' (Pa) G' Modulus, Storage

1 1 0.1 1 10

Frequency (Hz)

Figure A.2 Storage modulus (G’) and loss modulus (G”) during frequency sweep of gelatinized conventional and organic corn starches (25°C, 0.5% strain).