PROCESSING INDUCED CHANGES IN PHENOLIC CONCENTRATION AND

ANTIOXIDANT CAPACITY OF CORN FOR PRODUCTION OF CORN FLAKES

BY

ZHOU ZHONG

THESIS

Submitted in partial fulfillment of the requirements

for the degree of Master of Science in Food Science and Human Nutrition

with a concentration in Food Science

in the Graduate College of the

University of Illinois at Urbana-Champaign, 2015

Urbana, Illinois

Master’s Committee:

Professor Nicki Engeseth, Chair

Associate Professor Martin Bohn

Associate Professor Kent Rausch

ii

ABSTRACT

Epidemiological studies have suggested that regular consumption of breakfast cereals would

help in reducing the risk of obesity and cardiovascular disease. However, average nutritional

quality of ready-to-eat breakfast cereals have been declining since late the 1980s. This study was

designed to examine how phenolics and antioxidant capacity change during processing of corn

into flakes, and to evaluate the effects of processing condition, corn genotype, and growing year

on phenolics, with the ultimate goal of improving the nutritional quality of corn flakes. Three

commercial corn hybrids grown in three different years with three replicates per year were dry-

milled, cooked, baked, and toasted into corn flakes to determine the impact on phenolic content

and antioxidant capacity. Genotype and processing effects were significant for phenolics, while

the yearly effect was not. Cooking and toasting were the two critical processing points to induce

phenolic changes. Total phenolic content significantly decreased during cooking, and

significantly increased again during toasting. Significant losses of antioxidant capacity were

observed during dry-milling and pressure cooking. Baking and toasting did not further alter the

antioxidant capacity of corn. Significant linear correlations were observed between antioxidant

capacity and phenolics in corn, while a higher correlation was observed for bound phenolics

rather than soluble phenolics. Total phenolic content of corn was enhanced with higher cooking

pressure, but decreased with extended pressure cooking time. Increased baking time and

temperature both induced significant increases in phenolics. Extended toasting time could also

liberate more bound phenolic compounds to increase total phenolic content, but higher toasting

temperature did not facilitate in faster liberation. Our findings will provide a guide to optimize

processing conditions and select proper corn hybrids for higher phenolic content and antioxidant

capacity in corn flakes.

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ACKNOWLEDGEMENTS

This thesis could not be down without all the help and guidance provided by my advisor, Dr.

Nicki Engeseth. She is so kind and always willing to provide generous help whenever I meet

problems in my research project or in my life. She lets me know what a good scientific

researcher is and helps me to develop skills to become a scientific researcher. I really appreciate

all her patient modifications and valuable suggestions for this thesis. It is my great fortune and

pleasure to work with her.

I also would like to thank my committee members, Dr. Martin Bohn and Dr. Kent Rausch for

their suggestions and guidance. Dr. Martin Bohn generously provided the corn samples needed

in this project, and I appreciate this. Dr. Kent Rausch is so nice that he always responds quickly

when I need him.

I want to thank Gurshagan, Nicole, and Carrie, who work with me in the Kellogg’s project.

Thanks Gurshagan for sharing the processing method to produce corn flakes, thanks Nicole for

providing me all the equipment and ingredients needed in flaking, and thanks Carrie for

packaging the corn samples and sending them to me. It is impossible for me to finish my

research without their effort and help.

Many thanks also to my labmates Jeff, Ziyi, and Eliana. Jeff was always willing to help when

I was in trouble. If I had some confusion in my research and looked for his help, he always tried

to answer it for me patiently. Ziyi assisted me in the ORAC assays for antioxidant capacity of

corn and I appreciate that.

I am so fortunate to meet a group of best friends in Champaign. I feel like we are a big family.

My two-year life in Champaign cannot be so colorful without them. Special thanks are due to

Haiqing, who always stands behind me and provides support and assistance when I am in need.

iv

Finally, I would like to thank my parents for supporting me to study abroad. They are always

my strong backing when I come across any difficulties, giving me warm words and encouraging

me to pursue what I want. Their love and faith in me are my motivation to keep walking forward.

v

TABLE OF CONTENTS

INTRODUCTION………………………………………………………………………………...1

CHAPTER 1: LITERATURE REVIEW………………………………………………………….3

1.1. Corn – As a Major Component of Human Diet………………………..……………….3

1.2. Lipid Oxidation and Natural Antioxidants.…................................................................10

CHAPTER 2: PHENOLIC CONTENT OF CORN AS AFFECTED BY GENOTYPE,

GROWTH YEAR, AND CORN FLAKE PROCESSING STAGES………………………....…28

2.1. Introduction…................................................................................................................28

2.2. Materials and Methods………………………………………………………..…….…29

2.3. Results…………………………………………………………....................................32

2.4. Discussion.......................................................................................................................35

2.5. Tables.............................................................................................................................42

2.6. Figures…........................................................................................................................48

CHAPTER 3: ANTIOXIDANT CAPACITY OF CORN AS AFFECTED BY GROWTH YEAR

AND CORN FLAKE PROCESSING STAGES.…………………………………………...........54

3.1. Introduction……………………………………..………………………......................54

3.2. Materials and Methods…………………………………………..…….........................55

3.3. Results…...………………..…..………………..……...................................................57

3.4. Discussion.……...……………………………….…......................................................59

3.5. Tables..…………………………………………...………..…………...........................64

3.6. Figures………………………………………………………..………..........................65

CHAPTER 4: EFFECT OF PROCESSING CONDITIONS ON THE PHENOLIC CONTENT

OF CORN FOR PRODUCTION OF CORN FLAKES.…………………………........................66

4.1. Introduction.…...………..…………………………………………………..................66

4.2. Materials and Methods.…….…………………………………………….....................67

4.3. Results…………..……………………………………………………..........................68

4.4. Discussion……..………………………………………………………........................74

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4.5. Tables…………….……………...………………..…………………...........................81

4.6. Figures………………………………..………………………………..........................83

CHAPTER 5: CONCLUSIONS AND FUTURE STUDIES.........................................................86

5.1. Conclusions………..……..……...……………………………………….....................86

5.2. Recommendations for Future Studies………………………..…………………..……88

BIBLIOGRAPHY.…………………………….…………………………………........................90

APPENDIX A. ANALYSIS OF VARIANCE FOR MOISTURE CONTENT, PHENOLICS,

AND ANTIOXIDANT CAPACITY OF CORN HYBRIDS GROWN IN THREE YEARS AT

FIVE PROCESSING STAGES……….……………………………………..............................118

1

INTRODUCTION

Corn (Zea Mays) is one of the most widely cultivated crops all over the world, whose global

production is higher than rice and wheat (Chaudhary et al., 2014). Epidemiological studies

suggest that regular consumption of breakfast, especially cereals, would help in reducing the

risks of obesity and cardiovascular disease (Geliebter et al., 2015). Corn flakes are a major kind

of ready-to-eat breakfast cereals with various nutrients, which are popular worldwide. However,

the average nutritional quality of ready-to-eat breakfast cereals has declined in the market since

the late 1980s. A database which monitored the nutritional profiles of over 150 top cereal brands

indicated a 16% decrease in nutritional quality of cereals produced in 2011, compared to 1988

(Wang et al., 2015). Hence, it is necessary to improve the nutritional value of ready-to-eat corn

flakes to meet the nutritional needs of consumers.

Apart from providing macro- and micro-nutrients in daily diets, corn also acts as an abundant

source of bioactive compounds, most of which possess antioxidant activity to enhance human

health. Major antioxidants in corn include carotenoids (Kurilich & Juvik, 1999; Kopsell et al.,

2009), phytosterols (Piironen et al., 2000; Yoshida & Niki, 2003), tocopherols (Kurilich & Juvik,

1999), ascorbic acid (Dewanto et.al., 2002), anthocyanins (Li et al., 2008; Pedreschi & Cisneros-

Zevallos, 2007), and other phenolic compounds (Pedreschi & Cisneros-Zevallos, 2007; Ramos-

Escudero et al., 2012). Phenolics were cited as the major contributors to antioxidant capacity in

corn (Velioglu et al., 1998; Adom & Liu, 2002; Hu & Xu, 2011). According to Adom & Liu

(2002), phenolic content of corn was higher than in rice, wheat, and oats. Phenolics provide a

wide variety of potential health-enhancing functions, such as antimutagenic activity (Cardador-

Martínez, 2002; Pedreschi & Cisneros-Zevallos, 2006), anticarcinogenic property (Owen et al.,

2000), anti-inflammatory effects (Jiang & Dusting, 2003), microbial inhibition (Nohynek et al.,

2

2006), and prevention of coronary heart disease (Morton et al., 2000). Therefore, enhancing

phenolic contents and antioxidant activities is important in improving the overall nutritional

quality of corn flakes.

Few studies have focused on the phenolic compounds and antioxidant activity of corn flakes,

while several studies have been conducted on the nutritional values of oat, rye, wheat, and

parsley flakes (Ewaidah & Alkahtani, 1992; Luthria, 2008; Sumczynski et al., 2015). Previous

studies examined the effect of processing on phenolics and antioxidant capacity in corn

(Dewanto et al., 2002; Parra et al., 2007; Lopez-Martinez, 2012; Harakotr et al., 2014). Dewanto

et al. (2002) indicated that cooking significantly elevated the free phenolic content and

antioxidant capacity in sweet corn. However, phenolics and antioxidant capacity in corn were

significantly reduced during processing for production of masa, tortillas, and tortilla chips (Parra

et al., 2007). More information is needed on the effect of the corn-flaking process on phenolics

and antioxidant capacity of corn.

Hence, the objectives of this study were to examine how phenolics and antioxidant capacity

change during the corn-flaking procedure and to evaluate the effects of processing conditions on

phenolics, with the goal of improving the nutritional quality of corn flakes. Three commercially

available corn hybrids grown in three years were used to investigate how genotype and growing

year influence phenolics and antioxidant capacity in corn during processing. The hypotheses of

this research are that processing will impact on phenolic concentration and antioxidant capacity

of corn, and corn flakes with higher phenolic content can be obtained with optimized processing

conditions. Since phenolic compounds may be disrupted under thermal treatment, we would

expect lowered antioxidant capacity of corn after flaking. Higher pressure and temperature with

extended treatment time may help to liberate more phenolics in corn.

3

CHAPTER 1: LITERATURE REVIEW

1.1. Corn – As a Major Component of the Human Diet

Corn (Zea Mays) is one of the most widely cultivated crops, providing foods for majority of

the population worldwide; corn provides multiple nutrients, as well as a variety of bioactive

phytochemicals that are beneficial to health. There are multiple varieties of corn. Among them,

white and yellow kernel corn are the most common species, while other pigmented corn such as

multicolored, black, red, purple, and blue corn are only produced in small amounts for some

specific foods or for decoration (Abdel-Aal et al., 2006; Žilić et al., 2012). Corn composition,

concentration of potentially-bioactive components, and other properties are greatly affected by

corn varieties and growing conditions (Lopez-Martinez, 2009; Luo & Wang, 2012).

1.1.1. Structure and Components of Corn Kernel

The corn kernel is comprised of the tip cap, pericarp, germ, and endosperm, with a

composition percentage of 0.8-1.0%, 5-6%, 10-11%, and 82-83%, respectively (Singh et al.,

2014). Endosperm is the largest fraction of corn kernel, which is mainly comprised of starch

(70%), protein (8-10%), and a small amount of fat (Lawton & Wilson, 1987; Prasanna et al.,

2001; Chaudhary et al., 2014). The carbohydrate content of germ (33-36 %) is relatively lower

than the other parts, but it contains high levels of protein (19%), oil (33-36 %), and ash (10%). A

high content of carbohydrate is present in pericarp (93-94%), while the contents of the other

components (3-4% protein, 0.7-0.9% oil, and 0.7-1.1% ash) are relatively low (Hopkins et al.,

1903).

4

1.1.2. Processing of Corn into Flake Products

Corn is processed into a variety of products in the food industry, among which, ready-to-eat

cereal products like corn flakes are popular. Whole kernels are first dry milled to seperate the

corn fractions of pericarp, germ and endosperm (Rausch et al., 2009). Flaking grits are then

obtained from the endosperm fraction, and are further cooked, delumped, dried and tempered,

flaked, and toasted into flakes (Fast & Caldwell, 2000).

U.S. No. 1 or No. 2 yellow dent corn is the common source for producing flaking grits

(Caldwell and Fast, 1990). To prevent the interference of fibers during processing and guarantee

consistent properties in final flake products, the pericarp fraction is removed during dry milling

(Culbertson, 2004). Germ is the part with highest oil content. Removal of germ fraction could

help prevent the final products from oxidative rancidity (Culbertson, 2004).

Formulation of corn flakes typically involves adding the ingredients of salt, sugar, and malt

syrup with water to flaking grits (Caldwell et al., 1990; Culbertson, 2004). The main objective of

adding salt in the formulation is flavor improvement. Sugar is added to increase sweetness. Malt

syrup, with its abundance of reducing sugars, proteins, and free amino acids, is of great

importance for color and flavor development induced by non-enzymatic browning (Kujawski,

1990, as cited in Culbertson, 2004). During cooking, typical reactions of starch gelatinization,

Maillard browning, Strecker degradation, and lipid oxidation will occur (Culbertson, 2004). Soft

and translucent grits with golden brown color will be obtained after complete cooking (Caldwell

& Fast, 1990; Culbertson, 2004). During tempering, retrogradation starts for the gelatinized

starch in cooked grits, resulting in increased firmness and resistance to digestion. Maillard

reaction, as well as caramelization of sugars such as sucrose will occur during toasting to further

5

generate the desired color and flavor of the final flake product. Compounds able to inhibit lipid

oxidation are also generated during toasting (Culbertson, 2004).

Limited studies exist on the phenolic compounds in corn flakes; however, significant

nutritional information is provided about oat, rye, wheat, and parsley flakes (Ewaidah &

Alkahtani, 1992; Luthria, 2008; Sumczynski et al., 2015). Hence, more research was needed that

focused on the changes of phenolics and antioxidant capacity during the corn-flaking procedure.

1.1.3. Bioactive Compounds in Corn and Their Health Benefits

Apart from providing macro- and micro-nutrients in daily diets, corn also acts as an abundant

source of bioactive compounds, including carotenoids (Kopsell et al., 2009), phytosterols

(Ostlund et al., 2002), resistant starch (Luo & Wang, 2012), and a range of phenolic compounds

(Pedreschi & Cisneros-Zevallos, 2007; Ramos-Escudero et al., 2012). These phytochemicals,

due to their specific properties, have diverse health-promoting effects in humans.

1.1.3.1. Carotenoids

Carotenoids are a family of organic pigments, including red, orange, and yellow colors (Luo

& Wang, 2012) that are located in chloroplasts and chromoplasts of plants (Žilić et al., 2012).

The yellow-orange color of corn endosperm is contributed by carotenoids, which can be split

into two classes, carotenes and xanthophylls (Panfili et al., 2004; Parra et al., 2007). Lutein,

zeaxanthin, and β-cryptoxanthin are classified as xanthophylls (Figure 1.1) and have been

identified with, α- and β-carotene (Figure 1.2) as dominant carotenoids in corn (Hu & Xu, 2011;

Panfili et al., 2004; Žilić et al., 2012). Carotenoid concentration and composition differ

significantly amongst different varieties of corn. According to a study conducted on four types of

6

waxy corn with different colors, the total carotenoid content in yellow normal corn was the

highest, followed by yellow waxy corn, black corn, and white corn, ranging from 1.24 to 39.61

μg/g dry weight (Hu & Xu, 2011). Another study of 44 sweet and dent corn lines also reported

significant variability among corn types from 0.15-33.11 μg/g dry weight (Kurilich & Juvik,

1999).

α-Carotene

β-Carotene

Figure 1.1: Structures of Major Carotenes in Corn

Carotenoids are antioxidants that play a crucial role in radical-initiated processes (Palozza &

Krinsky, 1992). Additionally, α- carotene, β-carotene, β-cryptoxanthin and other carotenoids

with at least one integrated nonoxygenated β-ionone ring also possess provitamin A activity

(Panfili et al., 2004), which means they are able to be metabolized in the gastrointestinal (GI)

tract to form vitamin A (Luo & Wang, 2012). Several epidemiological studies have linked rich

serum/adipose tissue carotenoid levels with reduced disease risk, including coronary heart

disease (Kritchevsky, 1999), lung cancer (Michaud et al., 2000), cataract development (Knekt et

al., 1992), and age-related macular degeneration (Carpentier et al., 2009), indicating that daily

consumption of carotenoids is beneficial for human health. Since human bodies are not able to

biosynthesize carotenoids (Luo & Wang, 2012), ingesting carotenoids from carotenoid-rich

7

foods such as fruits, vegetables, and whole grains is necessary. Among grains, corn has a higher

carotenoid concentration than wheat, oats and rice (Panfili et al., 2004).

Lutein

Zeaxanthin

β-Cryptoxanthin

Figure 1.2: Structures of Major Xanthophylls in Corn

1.1.3.2. Phytosterols

Phytosterols are mainly found in seeds of crops and cereals as secondary metabolites

(Bartłomiej et al., 2011). Corn oil contains more abundant phytosterols compared to the other

commonly used commercial oils (Weirauch et al., 1978). Phytosterols can be classified into three

major classes: 4-desmethylsterols, 4,4-dimethylsterols, and 4-monomethylsterols (Grunwald,

1975). Common phytosterols in corn oil such as sitosterol, stigmasterol, and campesterol (Figure

1.3) are all from the class of 4-desmethylsterols (Luo & Wang, 2012). Phytosterols in cereal

8

grains may also be conjugated with fatty acids, phenolic acids, carbohydrates, and acylated

carbohydrates (Lagarda et al., 2006; Bartłomiej et al., 2011).

β-Sitosterol Stigmasterol

Campesterol Cholesterol

Figure 1.3: Structures of Major Phytosterols in Corn Oil, Compared to Cholesterol

Ostlund et al. (2002) demonstrated the cholesterol lowering activity of dietary phytosterols in

corn oil upon consumption of physiological concentrations. Epidemiological studies also

indicated a 10% lowering effect on the low density lipoprotein (LDL) cholesterol level in human

blood serum by ingesting 1-3 g phytosterols every day (Katan et al., 2003; Lagarda et al., 2006).

The similarity of phytosterol and cholesterol structures contributes to the cholesterol-lowering

effect of phytosterols (Figure 1.3) (Luo & Wang, 2012). Phytosterols function in the intestine,

being able to inhibit cholesterol absorption and reduce LDL-cholesterol (Ling & Jones, 1995),

thus may help prevent atherosclerotic disease (Moghadasian & Frohlich, 1999; Ostlund et al.,

2002).

9

1.1.3.3. Phenolics

Phenolics are a class of compounds possessing one or more hydroxyl groups (-OH) bound

directly to one or more aromatic rings (Liu, 2004). Plant phenolic compounds are generally

classified as phenolic acids, flavonoids, tannins, and the less common categories, stilbenes and

lignans (Dai & Mumper, 2010). In corn kernels, phenolic acids and flavonoids are the most

predominant forms of phenolics (Žilić et al., 2012). According to Sen et al. (1994), free phenolic

acids and flavonoids were concentrated in pericarp, aleurone layer, and germ fractions of corn.

Žilić et al. (2012) also indicated that the total phenolic content in corn is significantly correlated

to its pericarp portion per kernel weight (R2=0.87).

Total phenolic contents of corn vary from genotype to genotype (Žilić et al., 2012). Corn

with dark colors such as red, blue and purple contain higher phenolics than light colored corn

types (Hu & Xu, 2011; Žilić et al., 2012). Growth stage is also an influencing factor on phenolic

content. During maturation, a significant decrease in total phenolic content is observed in corn

(Xu et al., 2010). Phenolic contents in corn, whether free, soluble-conjugated or insoluble bound

form, are higher than those in rice, wheat, and oats (Adom & Liu, 2002).

Phenolic compounds display a wide variety of potential health-enhancing functions, mainly

related to antimutagenic activity (Cardador-Martínez, 2002; Pedreschi & Cisneros-Zevallos,

2006), anticarcinogenicity (Owen et al., 2000), anti-inflammatory effects (Jiang & Dusting,

2003), microbial inhibition (Nohynek et al., 2006), and prevention coronary heart diseases

(Morton et al., 2000). However, it is also noted that under certain conditions, phenolic

compounds can act as pro-oxidants rather than antioxidants (Duthie et al., 2000; Morton et al.,

2000). This is achieved by chelating metals and altering their catalytic activity, thus free radical

formation is enhanced and oxidation damage is induced (Decker, 1997).

10

1.2. Lipid Oxidation and Natural Antioxidants

1.2.1. Mechanisms of Lipid Oxidation

Oxidation of polyunsaturated fatty acids in cell membranes occurs through free radical chain

reactions, by both enzymatic and auto-oxidative peroxidation (Torel et al., 1986). Chain

reactions and lipid peroxidation may become uncontrolled with an over-abundance of free

radicals, which may lead to health problems such as atherosclerosis and cancer (Cook &

Samman, 1996). The rate of lipid autoxidation depends on factors such as fatty acid composition,

degree of unsaturation, and the partial pressure of oxygen (Belitz et al., 2009). Three stages are

involved in the mechanisms of free-radical induced lipid peroxidation: initiation, propagation,

and termination (Torel et al., 1986; King et al., 1993).

Initiation: RH → R∙

Propagation: R∙ + O2 → ROO∙

ROO∙ + RH → ROOH + R∙

Termination: ROO∙ + ROO∙ → ROOR + O2

R∙ + R∙ → RR

ROO∙ + R∙ → ROOR

R∙ + H∙ → RH

In initiation, a hydrogen atom is abstracted from unsaturated fatty acids, and lipid free

radicals are formed (Cook & Samman, 1996; Frankel, 2012). The rate of initiation may be

affected by factors such as temperature, light, and metals (King et al., 1993). Lipid peroxidation

is initiated by free radicals and singlet oxygen that are generated in biological systems. Hence,

free radical scavengers or singlet oxygen quenchers may help to prevent the initiation of lipid

peroxidation (Torel et al., 1986). Initiation is followed by propagation, in which lipid radicals

11

reacts with oxygen to form peroxy radicals (ROO∙). The lipid peroxy radical is able to abstract a

hydrogen atom from a fatty acid to generate a monohydroperoxide (ROOH) and a new free lipid

radical (R∙) to propagate the chain of reaction (King et al., 1993; Cook & Samman, 1996; Belitz

et al., 2009). This hydrogen abstraction is the lowest step, which controls the rate of radical

generation (Belitz et al., 2009). Thus, it becomes selective to abstract the most weakly-bound

hydrogen in an unsaturated fatty acid (Frankel, 2012). However, with accumulation of

hydroperoxides, the reaction would accelerate and hydroperxide molecules break down into free

radicals via metals, heat, and ultraviolet light (Schaich et al., 2013). Peroxy radical scavengers

are able to break the chain reaction in propagation to inhibit lipid oxidation (Torel et al., 1986).

When the rate of radical quenching becomes faster than chain expansion, lipid free radicals tend

to generate stable non-radical products, which is the termination stage (Schaich et al., 2013).

Lipid oxidation occurring in foods will result in off-odors and flavor fade. It will also induce

membrane degradation and lipid structural changes, which may cause turgor loss and texture

degradation of foods (Schaich et al., 2013). Apart from its detrimental effects on foods, lipid

peroxidation is also related to human diseases such as cancer and atherogenesis (Frei et al., 1988).

Antioxidants can be used to limit the degree of lipid oxidation, which will be further discussed in

the following section.

1.2.2. Mechanisms of Antioxidant Activity

Free radicals are atoms, molecules or ions with one or more unpaired electrons, with the

potential to damage proteins, lipids, carbohydrates, and nucleotides (Slater, 1984; Halliwell,

1987). Such damage caused by oxidizing species are related to negative health problems such as

heart diseases (Halliwell, 1987; Tsutsui, 2011), fibrosis (Cheresh et al., 2013), reperfusion injury

12

(Zweier & Talukder, 2006), cancer and aging (Ames, 1989). Lipid peroxidation-induced damage

can be controlled by antioxidants, which interfere with free radicals. Antioxidants can be

classified into three major groups by different mechanisms of action (Schaich et al., 2013).

The most prominent group of antioxidants acts as radical scavengers. A hydrogen atom

transfer reaction can be induced by these antioxidants to scavenge free radicals as follows

(Leopoldini et al., 2004).

Hydrogen atom transfer: ROO∙ + AH → ROOH + A∙

ROO∙ + AH2 → ROOH + AH∙

AH∙ + R∙ → A + RH

The radical A∙, which is generated by this kind of chain-breaking antioxidant, is stable and

will not further propagate the radical chains (Halliwell, 1995; Schaich et al., 2013). Antioxidants

such as ascorbic acid, quinones, and some phenols can also change the reactive radicals into

unreactive ions through single electron transfer (Schaich et al., 2013), thus to quench the free

radicals.

Single electron transfer: ROO∙ + ArOH + e- → ROO

- + ArOH∙

+

ROO∙ + A - e- → ROO

+ + A

-

The second type of antioxidants acts to prevent production of free radicals rather than

scavenge them. These antioxidants function as metal chelators and complexers, being able to

block all the metal orbitals (Schaich et al., 2013). Production of free radicals is then inhibited,

with their metal catalysts chelating to the antioxidants (Duthie et al., 2000; Morton et al., 2000).

Antioxidants, particularly carotenoids are also capable of scavenging singlet oxygen to inhibit

radical production (Stahl & Sies, 2005).

13

The third type of antioxidant is related to environmental factors that can limit lipid oxidation.

If controlled properly, factors such as temperature, pH, moisture content, light, and

environmental gases can slow the rate of lipid oxidation, thus reducing the radical load (Schaich

et al., 2013).

1.2.3. Potential Antioxidants in Corn

Multiple beneficial functions are attributed to bioactive phytochemicals in corn, among

which antioxidant activity is one of the most important properties. Potential antioxidants in corn

include carotenoids (Kurilich & Juvik, 1999; Kopsell et al., 2009), phytosterols (Piironen et al.,

2000; Yoshida & Niki, 2003), tocopherols (Kurilich & Juvik, 1999), ascorbic acid (Dewanto et

al., 2002), anthocyanins (Li et al., 2008; Pedreschi & Cisneros-Zevallos, 2007), and other

phenolic compounds (Pedreschi & Cisneros-Zevallos, 2007; Ramos-Escudero et al., 2012). Corn

contains a large variety of phenolics, which are the major and most effective contributors of

antioxidant activity in corn (Velioglu et al., 1998; Hu & Xu, 2011). Hence, due to their

significant role in corn bioactivity, phenolic compounds in corn and their antioxidant capacity

will be mainly discussed in the following sections.

1.2.3.1. Phenolic Compounds in Corn

A variety of phenolic compounds have been identified in corn, among which, phenolic acids

and flavonoids are the two forms that are most common (Žilić et al., 2012).

Phenolic acids are aromatic acid compounds, which can be classified into two categories:

hydroxybenzoic acids and hydroxycinnamic acids (Figure 1.4) (Liu, 2004). Based on different

substitutions on the aromatic ring, different types of phenolic acids are obtained (Table 1.1 &

14

1.2). A majority of the phenolic acids in corn are in bound forms. According to Žilić et al. (2012),

76-91% of total ferulic and p-coumaric acids in corn are insoluble-bound. The free phenolic

acids are primarily found in the outer layer of pericarp, while the bound ones are mainly

esterified to cell wall components (Dykes & Rooney, 2007). In general, phenolic acids that have

been identified in different maize types and preparations include tannic, gallic, o-coumaric,

caffeic, vanillic, ferulic, cinnamic, protocatechuic, p-coumaric, salicylic, and chlorogenic acids

(Hu & Xu, 2011; Ramos-Escudero et al., 2012; Pandey, 2013). The composition of phenolic acid

varies among different corn varieties.

Benzoic acid Cinnamic acid

Figure 1.4: Basic Structures of Phenolic Acids (From Liu, 2004)

Phenolic acids can function as strong antioxidants, scavenging free radicals and other ROS

which are associated with some aging-related human diseases such as chronic cardiovascular

diseases and cancer (Yu et al., 2002; Yu et al., 2003; Kampa et al., 2004). Within the two

categories of phenolic acids, hydrocinnamic acids are found to possess higher antioxidant

activity than hydroxybenzoic acids; this is attributed to the CH=CH-COOH group in their

structures (White and Xing, 1997; Emmons et al., 1999; Andreasenm et al., 2001; Kim et al.,

2006).

15

Table 1.1: Benzoic Acid and Its Derivatives (Adapted from Liu, 2004)

Benzoic acid derivatives Substitutions

R1 R2 R3

p-Hydroxybenzoic H OH H

Protocatechuic H OH OH

Vannilic CH3O OH H

Syringic CH3O OH CH3O

Gallic OH OH OH

Table 1.2: Cinnamic Acid and Its Derivatives (Adapted from Liu, 2004)

Cinnamic acid derivatives Substitutions

R1 R2 R3

p-Coumaric H OH H

Caffeic OH OH H

Ferulic CH3O OH H

Sinapic CH3O OH CH3O

Flavonoids are a group of phenolic compounds located in the pericarp (Dykes & Rooney,

2007), which are responsible for the different colors of corn (Žilić et al., 2012), including orange,

red, yellow, blue, violet and ivory (Harborne, 1976). A major portion (91%) of the flavonoids in

corn is in bound form (Adom & Liu, 2002). The basic structure of flavonoids possesses two

aromatic rings (A & B), which are linked through three carbons in an oxygenated heterocycle

ring (Figure 1.5). Depending on the structural differences in heterocycle C ring, flavonoids are

divided into six groups: flavonols, flavones, flavanols, flavanones, anthocyanidins, and

isoflavonoids (Figure 1.6) (Liu, 2004). The antioxidant activity of flavonoids also mainly

depends on the position and number of hydroxyl groups in their structures (Noroozi et al., 1998).

Anthocyanins are the major flavonoids especially in dark-colored corn, whose dominant

compounds include cyaniding-3-glucoside, pelargonidin-3-glucoside, peonidin-3-glucoside and

their acylated counterparts (Pedreschi, 2007; Lopez-Martinez et al., 2009; Žilić et al., 2012).

Other major flavonoids present in corn are identified as kaempferol, hesperitin, quercetin,

leucopelargonidin, naringenin, and their derivatives (Dykes & Rooney, 2007; Pedreschi, 2007;

16

Ramos-Escudero, 2012). According to Adom & Liu (2002), the total flavonoid content in corn is

the highest among wheat, oats, and rice.

Figure 1.5: The Basic Structure of Flavonoids (from Liu, 2004)

Figure 1.6: The Structures of Major Classes of Flavonoids (from Liu, 2004)

Health benefits related to flavonoids include antioxidant activity (Noroozi et al., 1998),

preventing atherosclerosis (Naderi et al., 2003), decreasing capillary fragility and permeability

(Bentsáth et al., 1936), treating progressive neurodegenerative disorders (Weinreb et al., 2004),

anticancer (Fink et al., 2007), anti-inflammatory (Rathee et al., 2009), anti-allergic (kawai et al.,

2007), and gastroprotective properties (Mota et al., 2009). The health-enhancing effects of

17

flavonoids primarily result from their antioxidant activity. It is reported that flavonoids could

inhibit low-density lipoprotein (LDL) oxidation attributed to its antioxidant capacity, thus may

be helpful in preventing coronary artery disease (Naderi et al., 2003). In another study conducted

by Noroozi et al. (1998), the antioxidant activities of a series of flavonoid compounds were

examined, and they were found to exert protective effects towards oxygen radical generated

DNA damage in human lymphocytes.

Among the multiple phenolic compounds identified in corn, two most predominant ones,

ferulic acid and anthocyanin, which belong to the group of phenolic acids and flavonoids

respectively, will be discussed.

1.2.3.1.1. Ferulic Acid

Ferulic acids are the most abundant phenolic compounds in corn (Hu & Xu, 2011), which

mainly exists in bound form (> 93%) (Adom & Liu, 2002; Lopez-Martinez, 2009). A majority of

the ferulic acids in corn are bound to cell wall components as esters (Lozovaya, 2000). Adom &

Liu (2002) conducted a study comparing ferulic acid content in corn to those of wheat, rice, and

oats, and found corn contained the highest level at 906 μmol/100 g. Different ferulic acid

contents are present among different corn types. According to Lopez-Martinez (2009), orange

corn had the highest total ferulic acid content among 18 corn genotypes with different colors.

Figure 1.7: The Structure of Ferulic Acid

18

The methoxy and hydroxyl groups in the structure of ferulic acid (Figure 1.7) can donate

electrons to quench free radicals, and the unsaturated C-C double bond on the side chain can also

act as attack site for free radicals, which contribute to the antioxidant potency of ferulic acid

(Srinivasan et al., 2007; Luo & Wang, 2012). The carboxylic acid in the ferulic acid structure is

also able to conjugate with the bilayer of lipids to prevent lipid peroxidation (Luo & Wang,

2012). Ferulic acid is more bioavailable than the other phenolics, and remains in blood longer to

scavenge free radicals (Srinivasan et al., 2007). Due to its antioxidant potency, ferulic acid is

able to display a wide variety of health-beneficial effects, such as anti-inflammatory (Sakai et al.,

1997; Ou et al., 2003), antidiabetic (Ohnishi et al., 2004), anticancer (Asanoma et al., 1993;

Kawabata et al., 2000), hypotensive (Suzuki et al., 2002), neuroprotective (Kanski et al., 2002),

cholesterol-lowering (Kim et al., 2003), and anti-atherogenic effects (Perfilava et al., 2005, as

cited in Srinivasan et al., 2007).

1.2.3.1.2. Anthocyanin

Anthocyanins are a group of water-soluble vacuolar pigments responsible for orange, red

purple and blue colors of plants, which belong to a class of flavonoids (Giusti & Wrolstad, 2003;

Abdel-Aal et al., 2006). Anthocyanins are glycosides of anthocyanidins, and the structures of

common anthocyanins are shown in Figure 1.8 and Table 1.3. Mainly located in the aleurone

layer and pericarp, anthocyanins contribute to the color of corn kernels (Betran, 2000; Moreno et

al., 2005). The colors of anthocyanin compounds are also affected by the pH of the solution

(Moreno et al., 2005).

19

Figure 1.8: The Basic Structure of Common Anthocyanins (From Abdel-Aal et al., 2006)

Anthocyanins are the major free phenolic compounds in corn (Lopez-Martinez et al., 2009).

Anthocyanin composition in corn depends primarily on corn type and maturity stage (Hu & Xu,

2011). Pigmented corn species like black, purple, red, and blue phenotypes have higher

anthocyanin levels than orange, yellow, and white ones (Lopez-Martinez et al., 2009), since in

these light-colored corn species, carotenoids are the major pigments (Kurilich et al., 1999).

Among multiple corn types with different colors, purple corn is the one with the highest

anthocyanin content (Abdel-Aal et al., 2006). In the study of Pedreschi et al. (2007), anthocynins

in Andean purple corn extract accounted for 77.7% of the total phenolics. According to Žilić et al.

(2012), major anthocyanins that have been identified in corn include cyanidin-3-glucoside,

cyanidin-3,5-diglucoside, cyanidin-3-rutinoside, cyanidin-3,6-ethylmalonylglucoside,

pelargonidin-3-glucoside, pelargonidin-3,5-diglucoside, peonidin-3-glucoside, and delphinidin-

3-glucose. Scarlet red corn contains the highest number (27) of anthocyanin compounds (Abdel-

Aal et al., 2006). Cyanidin, pelargonidin, and peonidin are the major anthocyanins in corn,

among which, cyanidin is the most predominant form (Abdel-Aal et al., 2006; Žilić et al., 2012),

accounting for 73-87% of total anthocyanins (Moreno et al., 2005). Two forms of anthocyanins

20

exist in corn are acylated and nonacylated, and the proportion of acylated anthocyanins in

degermed corn kernels is between 55.2% and 63.3% (Moreno et al., 2005).

Table 1.3: Structures of Common Anthocyanins (Adapted from Abdel-Aal et al., 2006)

Anthocyanins Substitutions

R1 R2

Pelargonidin-3-glucoside H H

Cyanidin-3-glucoside OH H

Delphinidin-3-glucoside OH OH

Peonidin-3-glucoside OCH3 H

Petunidin-3-glucoside OCH3 OH

Malvidin-3-rutinoside OCH3 OCH3

Besides the color attributes, anthocyanins are also correlated to several health-enhancing

effects, such as antioxidant capacity (Satue-Gracia et al., 1997; Hu & Xu, 2011; Žilić et al.,

2012), preventing obesity (Tsuda et al., 2003; Jay aprakasam et al., 2006), anti-inflammatory

(Wang et al., 1999), ameliorating hyperglycemia (Tsuda et al., 2003), reducing colon cancer risk

(Zhao et al., 2004; Jing et al., 2008), suppressing tumor cells (Kamei et al., 1995), treating

circulatory disorders (Bettini et al., 1985), and maintaining normal vascular permeability (Wang

et al., 1997). Hence, anthocyanins can work as potential functional food ingredients and

functional colorants (Abdel-Aal et al., 2006).

1.2.3.2. Existence Forms of Phenolic Compounds in Corn

Phenolic compounds in plants generally exist in three forms: soluble free, soluble conjugated

and insoluble bound forms (Adom & Liu, 2002; Žilić et al., 2012). Phenolics in free form are

mainly flavanols, while the bound ones mostly consist of phenolic acids (Montilla, 2011). Unlike

phenolics in fruits and vegetables that are mainly present in free or soluble conjugated forms

(Vinson, 1998; Vinson, 2001), phenolics in corn tend to be in the bound form (Montilla, 2011).

According to the study conducted by Adom & Liu (2002), 85% of phenolics in corn are bound.

21

Thus, total antioxidant capacity is closely associated with phenolic content in bound extractions

of grains, while a low correlation is presented for free extracts (Adom & Liu, 2002). Additionally,

bound phenolics are able to survive digestion in the GI tract, reach the colon, and thus pose

beneficial health effects in preventing cancers (Adom & Liu, 2002).

Corn phenolics mainly occur as esters, less commonly as ethers, covalently bound to cell

wall components (Lozovaya, 2000; Sun et al., 2012). Ferulic acid, which is the most

predominant phenolic in corn (Hu & Xu, 2011), is generally esterified to form covalent cross-

links with cell wall components such as polysaccharides (Meyer et al., 1991; Wallace & Fry,

1994), including glucuronoarabinoxylans, xyloglucans and pectins (Wong, 2006), glycoproteins

(Fausch et al., 1963), polyamines (Legaz et al., 1998; Ou & Kwok, 2004; Underwood, 2012),

and lignin (Meyer et al., 1991). Different phenolic acids in plants tend to be bound with different

cell wall components. A majority of p-coumaric acids in plant are bound to lignin (Ralph, 2010),

while ferulic acids are prone to bind with polysaccharides (Ishii, 1997). This linkage is supported

by hydrogen bond formation between the phenolic hydroxyl group and the oxygen atoms from

glycosidic linkages of polysaccharides, hydrophobic interactions, as well as covalent bonds

between phenolic compounds and polysaccharides (Saura-Calixto, 2011).

Food processing methods like fermentation and malting, extrusion cooking, and alkaline

hydrolysis can facilitate release of bound phenolics (Acosta-Estrada, 2014). For analytical

purposes, enzymatic (Fazary & Ju, 2007), ultrasound (Peričin et al., 2009), microwave (Jokić et

al., 2012), thermal (Montilla, 2011), acid and alkaline hydrolyses (White et al., 2010) can be

used to break the ester bond and liberate bound phenolics. Alkaline hydrolysis is one of the most

common method to obtain the cell wall-linked bound phenolics in grains (Adom & Liu, 2002;

Dewanto et al., 2002; Kim et al., 2006; Parra et al., 2007; Žilić et al., 2012), reducing the loss of

22

corn ferulic acids from 78% to 4.8% compared with acid hydrolysis (Krygier et al., 1982). After

liberation, the released phenolics are capable of being extracted through specific solvent systems

(Acosta-Estrada, 2014).

Apart from insoluble phenolic forms that are bound to cell wall components, phenolics can

also be esterified with low molecular mass compounds like ethanol, or other phenolics through

carboxylic and hydroxyl groups in their structures (Sun, 2012). Phenolic compounds are also

able to be covalently conjugated with mono- and oligosaccharides (Ou & Kwok, 2004). These

soluble phenolics in corn, including free and conjugated fraction (ex. esterified and glycosylated

phenolics), can be solvent extracted directly. Commonly used solvents include ethanol (Adom &

Liu, 2002; Santiago, 2007; Montilla, 2011), methanol (Bacchetti et al., 2013), and acetone

(Krygier et al., 1982). Since conjugated phenolic compounds are still able to be oxidized, they

can be quantified together with free phenolics as a soluble fraction, through Folin-Ciocalteu

colorimeric reaction (Adom & Liu, 2002).

Free phenolics contribute little to the total phenolic content in corn. Only less than 0.6% of

ferulic acids are in free form (Adom & Liu, 2002). Free phenolic compounds identified in corn

include protocatechuic, vanillic, p-coumaric, o-coumaric, and ferulic acids (Hu & Xu, 2011;

Žilić et al., 2012). As corn matures, some free phenolics may be metabolized or synthesized into

bound phenolic compounds, thus causing a significant decrease in free phenolics of corn during

maturation (Hu & Xu, 2011).

1.2.3.3. Antioxidant Properties of Corn Phenolics and Potential Health Benefits

Phenolic compounds can act as antioxidants, scavenging reactive oxygen species (ROS) and

23

reactive nitrogen species (RNS) which mainly include superoxide, hydroxyl, peroxyl, alkoxyl,

peroxynitrite and oxides of nitrogen (Morton et al., 2000). ROS and RNS may derive from the

human body, in which are metabolically produced, or of exogenous origin such as ultraviolet

radiation, cigarette smoke, and air pollutant exposure (Morton et al., 2000). A state of oxidative

stress may be reached if the balance is perturbed between ROS and RNS generation and

antioxidant defense system in human body (Halliwell, 1994), thus damage will be produced to

cells and in vivo biological molecules (Morton et al., 2000).

Phenolics in corn possess considerable antioxidant capacity in vitro, and are able to delay the

cellular damage caused by oxidative stress (Rice-Evans et al., 1997; Adom & Liu, 2002; Alía et

al., 2006a; Hu & Xu, 2011; Žilić et al., 2012). Adom & Liu (2002) reported that the total

antioxidant activity of corn is 181.42 μmol Vitamin C equiv/g, which is higher than that of wheat,

rice, and oats. A large variation is found in antioxidant activity among different genotypes of

corn, due to their differences in phenolic profiles (Jacobo-VelÁzquez & Cisneros-Zevallos, 2009;

Lopez-Martinez et al., 2009). Corn with dark colors possesses higher antioxidant capacity than

light colored ones (Žilić et al., 2012). Several studies have indicated the positive relationship

between corn phenolic content and its antioxidant capacity. According to Adom & Liu (2002),

the total antioxidant activity of corn is closely related to the phenolic content (R2=0.983),

especially for bound phenolics (R2=0.991). Bacchetti et al. (2013) also demonstrated a

significant positive correlation between corn total phenolics and oxygen radical absorbance

capacity (ORAC) values. For a certain phenolic compound, its antioxidant activity is correlated

to available hydroxyl groups and their positions in its structure (Rice-Evans et al., 1996; Jacobo-

VelÁzquez & Cisneros-Zevallos, 2009). Common methods used to measure antioxidant capacity

in corn include total oxyradical scavenging capacity (TOSC) assay (Adom & Liu, 2002), ORAC

24

method (Li et al., 2007; Bacchetti, et al., 2013), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay

(Mohsen & Ammar, 2009; Sarepoua et al., 2013). Choosing different assays will also alter the

measured antioxidant capacity of corn (Žilić et al., 2012).

The major pathway for antioxidant action of phenolics is free radical scavenging, in which

the free radical chain reaction is broken (Morton et al., 2000). Thus, some negative oxidative

effects such as free radical mediated cytotoxicity and lipid peroxidation are inhibited (Sonee et

al., 2004; Alía et al., 2006a). Several studies have shown the capability of phenolic compounds

in scavenging ROS and RNS such as peroxyl radicals (Chimi et al., 1991), alkyl peroxyl radicals

(Nakao et al., 1998; Sawa et al., 1999), superoxide (Kashima, 1999), hydroxyl radicals (Chimi et

al., 1991; Kashima, 1999), nitric oxide radical (Vanacker et al., 1995), and peroxynitrite (Heijnen

et al., 2001).

The other mechanisms related to phenolic antioxidant activity include metal ion chelating,

enzyme regulation, oxidative damage repair, and peroxide metabolizing (Duthie et al., 2000;

Morton et al., 2000). Phenolics are capable of chelating divalent metal ions that work as catalysts

in ROS production, such as Fe and Cu, thus Fenton’s reaction is inhibited and the formation of

free radicals is prevented. It has also been reported that phenolics are able to modulate a variety

of enzymes such as lipoxygenase, cyclooxygenases, phosphatidyl-3-kinase, glutathione reductase,

glutathione peroxidase, superoxide dismutase and catalase (Kato et al., 1983; Lipkin et al., 1999;

Murakami et al., 2002; Alía et al., 2006a; Alía et al., 2006b). When a proper dose of a certain

phenolic compound is given, some changes may be occurred towards the activity and/or gene

expression of the antioxidant enzyme system in cells (Alía et al., 2006a). These antioxidant

enzymes play an important role in defense against oxidative stress. Catalase catalyzes the

conversion of H2O2 into H2O, and glutathione reductase enables the oxidized glutathione to be

25

reduced back to its original form (Goldberg et al., 1987; Ursini et al., 1995; Alía et al., 2006a).

Glutathione participates in the intracellular antioxidant defense mechanism, being capable of

scavenging free radicals and other ROS (Wu et al., 2004). According to Murakami et al. (2002),

by treating HepG2 cells with epigallocatechin 3-O-gallate (EGCG), the cytotoxicity induced by

H2O2 is significantly reduced, and oxidative enzymes such as Mn-superoxide dismutase and

glutathione peroxidase are enhanced. Similarly, another study conducted by Alía et al. (2006a)

indicated that higher dose of quercetin, a flavonoid compound, could induce a significant

increase in glutathione concentration within cells, and thus strengthen the antioxidant defense

system.

Attributed to their antioxidant capacity, phenolics are considered to have the potential to

prevent several chronic diseases. Phenolic compounds in corn possess anti-mutagenic capacity,

which is achieved by blocking or scavenging mutagen electrophiles and inhibiting enzymes

involved in mutagen activation (Pedreschi & Cisneros-Zevallos, 2006). Moreover, various

phenolic compounds are able to suppress proliferation of several types of cancer cells in vitro,

including ovary (Scambia et al., 1994; Peng et al., 2008), colon (Agullo et al., 1994; Yi et al.,

2005), breast (Mehta et al., 1997; Kampa et al., 2004), lung (Caltagirone et al., 1997), and liver

cancer cells (Tanaka et al., 1993; Chi et al., 1997). One of the major mechanisms for the anti-

carcinogenic effect of phenolic compounds is related to their antioxidant capacity, through which

oxidative DNA damage caused by ROS and RNS as potential carcinogens is inhibited (Duthie et

al., 2000), cellular oxidative stress is reduced, and cell-overgrowth is prevented (Duthie et al.,

1997; Noroozi et al., 1998; Alía et al., 2006a). Apart from direct ROS and RNS scavenging

capacity, phenolic compounds are also able to alter procarcinogenic metabolism by suppressing

phase I enzymes and enhancing phase II enzymes that have potential to inhibit tumor

26

development (Maliakal & Wanwimolruk, 2001; Schwarz & Roots, 2003; Ramos, 2008).

Additionally, phenolics are found to have inhibitory effect against cancer by inducing apoptosis

in a selective manner (Nair et al., 2004; Ramos, 2008). Although the anti-cancer effects of

phenolics are noted in a number of in vivo and animal studies, epidemiological evidence is not

well defined, and even some contradictory results are shown (Hertog et al., 1994; Duthie et al.,

2000; Spormann et al., 2008).

Another major health benefit of phenolics is related to their function in preventing heart

disease. LDL is susceptible to be oxidized by free radicals (Schuh et al., 1978; Duthie et al.,

2000), thus may contribute to atherogenesis (Steinberg, 1997). However, phenolic compounds

are able to suppress LDL oxidation and play a protective role in heart disease, through possible

mechanisms such as scavenging oxidizing species and binding LDL protein (Frankel et al., 1993;

Ishikawa et al., 1997; Geleijnse et al., 1999; Kerry & Abbey, 1999; Wang & Goodman, 1999;

Chang et al., 2000; Duthie et al., 2000).

1.2.3.4. Impact of Processing on Phenolic Compounds

During some food processes such as cooking, alkaline hydrolysis, and fermentation, bound

phenolics are able to be released into soluble ones (Acosta-Estrada et al., 2014). Since ferulic

acid is the most predominant phenolic acid in corn, it is typically used as a marker to show the

processing effects on phenolic components. Under heat treatment, insoluble-bound ferulic acids

in corn tended to be released into solubilized feruloylated oligosaccharides and free ferulic acids;

thus, bound phenolic content decreased during processing while free and conjugated phenolics

increased (Dewanto et al., 2002). The majority of the released bound phenolics were in soluble-

conjugated form before enzymatic treatment, chemical treatment or severe heat treatment

27

(temperature > 180 °C) was applied to liberate them into free form (Saulnier et al., 2001;

Dewanto et al., 2002). According to Parra et al. (2007), bound ferulic acid in corn was released

into free and soluble-conjugated ferulic acids during lime-cooking for production of corn masa.

However, with masa baked into tortillas and fried into tortilla chips, ferulic acids were

complexed again. In the study conducted by Đorđević et al. (2010), total phenolic contents of

buckwheat, wheat germ, barley, and rye all significantly increased after fermentation with L.

rhamnosus or S. cerevisiae.

Although phenolics are enhanced during fermentation, other processing operations may

result in degradation of phenolic compounds. The nixtamalization process used for production of

masa, tortillas, and tortilla chips induced a significant decrease in total phenolics and antioxidant

activities of corn (Parra et al., 2007). Cooking of corn decreased total phenolic content with

increased cooking time (Dewanto et al., 2002). Boiling destroyed more phenolic and antioxidant

compounds than steam cooking (Harakotr et al., 2014). The loss in phenolic compounds and

antioxidant activity of corn during cooking resulted from degradation or decomposition of

antioxidant compounds and leaching or diffusion of these compounds into cooking water

(Harakotr et al., 2014). During extrusion cooking of corn meals with blueberry and grape juice

concentrates, 74% of anthocyanins were degraded (Camire ei al., 2002). Some oxidative and

hydrolytic enzymes can be liberated from the cell wall when it is destroyed through some

processing methods, thus antioxidants in foods may be disrupted (Chism & Haard, 1996).

28

CHAPTER 2: PHENOLIC CONTENT OF CORN AS AFFECTED BY GENOTYPE,

GROWTH YEAR, AND CORN FLAKE PROCESSING STAGES

2.1. Introduction

Phenolics, a group of powerful bioactive compounds present in corn, are a class of

compounds possessing one or more hydroxyl groups (-OH) bonded directly to one or more

aromatic rings (Liu, 2004). Phenolic compounds in plants generally exist in three forms: soluble-

free, soluble-conjugated, and insoluble-bound (Adom & Liu, 2002; Žilić et al., 2012). Unlike

phenolics in fruits and vegetables that are mainly present in free or soluble conjugated forms

(Vinson, 1998; Vinson, 2001), phenolics in corn tend to be in the bound form (Montilla, 2011).

According to Adom & Liu (2002), 85% of phenolics in corn are bound. For whole corn kernels,

phenolic acids and flavonoids are the most predominant forms of phenolics (Žilić et al., 2012).

Free phenolic acids and flavonoids were concentrated in the pericarp, aleurone layer, and germ

fractions of corn (Sen et al., 1994), while bound phenolic acids are mainly esterified to cell wall

components (Dykes & Rooney, 2007). Total phenolic content of corn varied from genotype to

genotype (Žilić et al., 2012), and were higher than that in rice, wheat, and oats (Adom & Liu,

2002). Phenolic compounds display a wide variety of potential health-enhancing functions, such

as anti-mutagenicity (Cardador-Martínez, 2002; Pedreschi & Cisneros-Zevallos, 2006), anti-

carcinogenicity (Owen et al., 2000), anti-inflammation (Jiang & Dusting, 2003), microbial

inhibition (Nohynek et al., 2006), and prevention of coronary heart disease (Morton et al., 2000).

Hence, phenolic content is an important nutritional factor of corn products.

Corn flakes are a major component of corn products in the market of ready-to-eat breakfast

cereals. U.S. No. 1 or No. 2 yellow dent corn is the common source for producing flaking grits,

which could be further processed into corn flakes (Caldwell and Fast, 1990). Corn whole kernels

29

are first dry milled to have the corn fractions of pericarp, germ and endosperm separated (Rausch

et al., 2009). Flaking grits are then obtained from the endosperm fraction, and further cooked,

delumped, dried and tempered, flaked, and toasted into flakes (Fast & Caldwell, 2000). A large

number of studies have been conducted to analyze phenolic compounds in various corn types;

however, few studies have been focused on the phenolics in corn flakes.

The objectives of this study were to investigate the effects of genotype, growing year, and

processing stage on phenolic content in corn, and to have an overall understanding of how

phenolics change during the corn-flaking procedure. Significant changes in phenolics are

expected during production of corn flakes.

2.2. Materials and Methods

2.2.1. Corn Sample and Sample Preparation

Three commercially available yellow dent corn hybrids were used in this study. These

hybrids were grown at the University of Illinois at Urbana-Champaign and harvested in 2009,

2010, and 2011. Three replications of each hybrid were collected from the corn samples

harvested each year. The whole kernels were dry milled to obtain flaking grits using methods

described by Rausch et al. (2009). Before phenolic analyses, the whole kernel and flaking grit

samples were ground into powder using a coffee grinder and dried at 60°C in an drying oven

(Fisher Scientific, Fair Lawn, NJ) until they reached constant weight.

2.2.2. Chemicals and Reagents

Folin-Ciocalteu reagent, gallic acid, and sodium carbonate were purchased from Sigma

Chemical Company (St. Louis, MO). Sodium Chloride, sodium hydroxide, hydrochloric acid,

30

hexane, ethyl acetate, methanol, and acetone were obtained from Fisher Scientific (Fair Lawn,

NJ). Ethanol was purchased from Decon Labs (King of Prussia, PA). Salt, sugar, and molasses

were purchased from a local supermarket.

2.2.3. Production of Corn Flakes

Flakes were produced based on the methods described by Kandhola (2015). The formulation

for cooking was created by mixing 2 g salt, 6 g sugar, and 2 g molasses with 200 mL double

deionized water. This formulation (200 mL) was added to 100 g flaking grits and mixed in a jar.

The mixture was cooked in a pressure cooker (Wisconsin Aluminum Foundry Co., Inc., WI) at

15 psi for 1 hr, and the top layer of cooked grits was removed using a spatula once cooking

finished. The solid cooked grits were baked in a convection oven (Oster, China) at 107.2°C for

50 min, to reduce the moisture content of the cooked grits from 60 to 50%. Samples were stirred

in middle of baking time to ensure even drying. Baked grits were kept at room temperature for

30 min to equilibrate moisture. The dough of baked grits was pressed through the flaking

machine, and cut into 1 inch square pieces using a pizza cutter. Raw flakes were kept at room

temperature for 16 hrs and toasted in the convection oven at 204.4°C for 80 s to produce toasted

flakes. Samples were removed at each processing stage, dried at 60°C until they reached constant

weight, and ground into powder using a coffee grinder, prior to analysis.

2.2.4. Extraction of Soluble Phenolic Compounds

Methods to extract soluble phenolics in corn were modified based on the protocol described

by Adom & Liu (2002). Corn sample (2.5 g) was extracted by blending with 10 mL 80% chilled

ethanol using a Fisher vortex (Fisher Scientific, NY). Samples were homogenized using a

31

Polytron homogenizer (Kinematica AG, Switzerland) for 3 min and centrifuged at 1935 g for 17

min. Supernatant was removed and the residues were extracted one more time. Two supernatants

were combined and evaporated to 5 mL under nitrogen at 45°C using an analytical evaporator

(Organomation Associates Inc., MA), and reconstituted with double deionized water to a final

volume of 10 mL. This solution would be cloudy. It was added with 5 mL 4M sodium chloride,

vortexed 2 min, and centrifuged (1935 g, 10 min). Supernatant was removed and kept in the

refrigerator until use. Duplicate analysis was conducted for each sample.

2.2.5. Extraction of Bound Phenolic Compounds

Bound phenolics were extracted from corn using a method modified from the protocol

described by Adom & Liu (2002). Corn samples (0.5 g) were blended with 5 mL 80% chilled

ethanol using a Fisher vortex to extract soluble phenolics. Samples were further homogenized

using a Polytron homogenizer for 2 min and centrifuged at 1935 g for 10 min. Supernatant was

discarded, and extraction was repeated. Residues were digested with 10 mL of 2M sodium

hydroxide under nitrogen, and shaken for 1 hr. Hydrochloric acid (2 mL, 12N) was added to

neutralize the solution. Hexane (5 mL) was added, mixture was vortexed, and centrifuged (1935

g, 10 min) to remove lipids. The solution was further extracted 5 times with 5 mL ethyl acetate.

Ethyl acetate fractions were combined and evaporated to dryness under nitrogen at 45°C using an

analytical evaporator, and reconstituted in 5 mL 50% methanol. Bound phenolic extracts were

stored at 4 °C until use. Duplicate analysis was conducted for each sample.

2.2.6. Determination of Phenolic Content

Corn phenolic content was determined based on the Folin-Ciocalteu colorimetric method

32

described by Singleton et.al. (1999). Corn samples were diluted with 50% methanol. A 10%

dilution was made for bound extracts of whole kernels and flaking grits samples, while a 20%

dilution was made for all the soluble extracts, and bound extracts of cooked grits, baked grits,

and toasted flakes samples. Gallic acid was used as a standard. Diluted extracts (250 µL) and

gallic acid solutions were oxidized with 200 µL Folin-Ciocalteu reagent, and neutralized with

550 µL sodium carbonate. The solutions were vortexed and incubated at 42°C (9 min).

Absorbance was measured at 765 nm using a spectrometer (Spectronic Instruments, USA) after

the solutions were cooled in the dark. Results were expressed as micrograms gallic acid

equivalents (GAE) per gram sample.

2.2.7. Statistical Analysis

Analysis of variance (ANOVA) was performed to determine the effect of genotype, growing

year, biological replication and processing stage on phenolic content of corn samples using the

generalized linear mixed model (GLMM). Genotype and processing stage were treated as fixed

effects, while growing year and replicate nested within year were treated as random effects.

Mean values were compared using the Tukey’s multiple comparison test at 5% level. All

analyses were performed using Statistical Analysis System (SAS) software, version 9.4 (SAS

institute, Cary, NC).

2.3. Results

2.3.1. Moisture Content

The moisture content of whole corn kernels was 8.57% (Table 2.1). After dry-milling into

flaking grits, the moisture content increased to 8.89%. Grits absorbed moisture during cooking;

33

thus, after this process, cooked grits reached a high moisture content of 63.67%. Cooked grits

were then dried during baking, which resulted in a decrease in moisture content to 53.71%.

Baked grits were tempered and processed into raw flakes, and the subsequent toasting process

resulted in a decrease in moisture content to 6.04%. The moisture content of corn was

significantly influenced by corn-flaking processing stages, while effects of genotypes, years, and

replicates within years were not detected (Appendix A.1).

2.3.2. Soluble, Bound and Total Phenolics

There were significant main effects of genotypes and processing stages for soluble, bound,

and total phenolics, but the effects of years and replicates within years were not significant

(Table 2.2). Significant interactions of genotype × processing stage, and genotype × replicate

within year were observed. Genotype × processing stage × replicate within year interaction was

also significant. The complete ANOVA tables for soluble, bound, and total phenolics were listed

in the Appendix A.

The soluble phenolic contents obtained represent free as well as soluble-conjugated phenolics.

To assess the impact of processing on soluble phenolics, mean soluble phenolic contents during

the corn-flaking procedure were calculated by combining hybrids A-C (Table 2.3 & Figure 2.1).

There was no significant difference in soluble phenolic content between whole kernels and

flaking grits. After the grits were cooked, a decrease in soluble phenolics occurred. Subsequent

baking did not affect soluble phenolic content in grits. However, after baked grits were processed

into raw flakes and toasted, the soluble phenolic content of the toasted flakes reached 710 µg

GAE/g, which was significantly higher than those of all the other processing stages. Soluble

phenolic content of commercial hybrid C was significantly higher than hybrid B (Figure 2.2).

34

There was no difference presented between hybrid A and B or C. No difference was detected in

soluble phenolic content among corn grown in 2009, 2010, and 2011 for all five processing

stages (Table 2.4 & Figure 2.3).

There was no difference in bound phenolic content between corn whole kernels and flaking

grits (Table 2.5 & Figure 2.4). The cooking process induced a decrease in bound phenolic

content of corn grits. Subsequent baking and toasting processes did not further alter bound

phenolic content in corn grits. Bound phenolic content of corn hybrid C was higher than that of

hybrid B in all processing stages (Figure 2.5). The difference between hybrid A and B, or A and

C was not significant. There was no difference in bound phenolic content among corn grown in

2009, 2010, and 2011 (Table 2.6 & Figure 2.6).

Whole kernels contained the highest total phenolic content among processed corn samples

from five processing stages (Table 2.7 & Figure 2.7). The total phenolic content of flaking grits

did not differ from whole kernels. After grits were cooked, the total phenolic content decreased,

and remained unchanged during baking. The subsequent toasting process induced an increase in

total phenolic content, as a result of increased soluble phenolics. Corn hybrid C contained higher

total phenolic content than hybrid B (Figure 2.8). Total phenolics of hybrid A did not differ from

hybrid B or C. There was no difference in total phenolics among corn grown in 2009, 2010, and

2011 for all five corn-flaking processing stages (Table 2.8 & Figure 2.9).

2.3.3. Percentage of Bound Phenolics

For the percentage of bound phenolics, significant main effects of processing stages and

replicates within years were observed (Table 2.2). Bound phenolic percentage was not

significantly influenced by different corn hybrids and growing years. Interaction of genotype ×

35

processing stage × replicate within year was significant. The complete ANOVA tables for bound

phenolic percentage were listed in the Appendix A.

During the corn-flaking procedure, percent bound phenolics decreased with bound phenolics

released into soluble ones (Table 2.9 & Figure 2.10). The bound phenolic percentages in corn

whole kernels and flaking grits were not different. The cooking process helped release bound

phenolics, resulting in reduced bound phenolic percentages compared to flaking grits. No

difference in bound phenolic percentage was observed between cooked and baked grits. After

grits were toasted, another decrease in bound phenolic percentage occurred; the percentage

reduced to a final value of 64.49%. There was no significant genotype effect for percentage of

bound phenolics. The only difference occurred in flaking grits, with bound phenolic percentage

of hybrid C significantly higher than A and B (Table 2.9). No difference among growing years

was observed for all five processing stages (Table 2.10).

2.4. Discussion

2.4.1. Effects of Genotype, Growing year, and Processing Stage on Phenolics in Corn

Variations in soluble, bound, and total phenolics were all significant among different corn

commercial hybrids across three growing years and five processing stages (Table 2.2).

Commercial hybrid C contained the highest phenolic content, while hybrid B was the lowest.

The non-significant year effect for soluble, bound, and total phenolics indicated that the traits of

corn were consistent across different growing years. According to study conducted by Harakotr

et al. (2015), in which 49 genotypes of waxy corn were tested, genotype was a more significant

source of variation in total phenolic content and antioxidant activity than growing season.

Similar results were also reported by Chander et al. (2008) and Mahan et al. (2013). The

36

interaction of genotype × year was not significant by every individual processing stage, which

indicated that phenolic contents of different corn hybrids growing in one year were able to

predict their phenolic performances in other growing years (Harakotr et al., 2015). Hence,

breeding of corn for higher phenolic concentration was feasible, with similar relative

performance in phenolics across years (Knievel et al., 2009; Harakotr et al., 2015).

Variations in soluble, bound, and total phenolic contents were also significantly influenced

by corn-flaking processing stages. Strong interaction of genotype × processing stage was

observed for soluble, bound, and total phenolics, indicating that different corn hybrids may

respond differently with phenolic changes during corn-flaking. This can be explained by the

different phenolic content (Lopez-Martinez et al., 2009; Žilić et al., 2012), phenolic composition

(Hu & Xu, 2011; Ramos-Escudero et al., 2012; Pandey, 2013), and phenolic distribution (Dykes

& Rooney, 2007) of different corn hybrids, which may result in varied responses during thermal

processing. Processing stage also contributed the most to the total variations in bound phenolic

percentages. This was because bound phenolics tended to be released into soluble phenolics

under thermal treatment, which resulted in a decrease of bound phenolic percentage during

processing (Dewanto et al., 2002).

Significant variations for phenolics were observed among replicates within years of

individual hybrids (Appendix A.5, A.9 & A.13), while a significant processing stage × replicate

within year interaction was also observed within each hybrid. Additionally, the interaction of

genotype × replicate within year was significant among all the hybrids, which indicated that

hybrid ranking in phenolics was altered by different replicates. These suggested that corn

genotypes used in our study may not come from a widely diverse genetic background. More

37

replicates involved in our analyses may help in improving the estimates of genotype, year, and

processing stage effects (Connor et al., 2005).

2.4.2. Changes in Phenolics during Corn-Flaking Procedure

Total phenolic contents of whole kernels of yellow dent corn were 2390, 2161, and 2657 µg

GAE/g of sample in our study (Table 2.7), separately for commercial hybrid A, B, and C, which

were in good agreement with results obtained by Adom & Liu (2002) which was 2645 µg GAE/g.

Commercial hybrid C contained the highest total phenolic content among the three corn hybrids.

Percentage of bound phenolics varied across corn hybrids from 71.22 to 76.65% (Table 2.9),

which was lower than the 85% presented in Adom & Liu (2002), but higher than the 69.2%

published by Sosulski et al. (as cited in Dewanto et al., 2002).

Corn flaking grits were obtained from whole kernels through a dry-milling process, in which

fractions of pericarp and germ were removed (Rausch et al., 2009). Endosperm, germ, and

pericarp account for 82-83%, 10-11%, and 5-6% of the corn whole kernel, respectively (Singh et

al., 2014). Phenolic acids and flavonoids are the two major forms of phenolic compounds in corn

(Žilić et al., 2012). Free phenolic acids are primarily located in the outer layer of pericarp, while

the bound phenolic acids are mostly esterified to cell wall components in the endosperm (Dykes

& Rooney, 2007; Singh et al., 2014). Flavonoids are also mainly found in cereal pericarp (Dykes

& Rooney, 2007). These were demonstrated in the study conducted by Sen et al. (1994), in

which a microspectrofluorometer was used to detect phenolic acids and flavonoids in corn

kernels. The results showed that free phenolic acids as well as flavonoids were intensively

presented in the pericarp, aleurone layer, and germ fractions of corn, while only a few traces

were observed in the endosperm. This is credible since the endosperm consists of numerous cells,

38

with phenolic acids bound to the cell wall components (Singh et al., 2014), and may contain low

concentrations of free phenolic acids to be detected through fluorescence. According to Adom &

Liu (2002), free, soluble-conjugated, and bound ferulic acids account for 0.10%, 0.99%, and

98.91% of total ferulic acids in corn kernels, respectively. After removal of pericarp and germ

during dry-milling, even less free phenolics would remain in the corn flaking grits. Hence, we

have reason to believe that the soluble phenolic content of flaking grits determined in our study

mainly represents soluble-conjugated phenolics in corn. Although a large proportion of free

phenolics were removed during dry milling, there was still no significant difference in the overall

soluble and bound phenolic contents between whole kernels and flaking grits (Table 2.3 &

Table 2.5). Significant differences were only observed within hybrid A and B. This can be

explained by the low proportion of pericarp (5-6%) and germ (10-11%) in whole kernel (Singh et

al., 2014), and the low contribution of free phenolics to total phenolics in corn (Adom & Liu,

2002).

A significant decrease occurred in both soluble and bound phenolic content when the corn

flaking grits were pressure cooked (Table 2.3 & Table 2.5). These phenolic values were

expressed as µg GAE per gram sample. The addition of other ingredients such as water, sugar,

salt, and molasses to flaking grits for the cooking process would reduce the proportion of corn in

the subsequent processed corn samples. If calculating phenolic content of cooked grits on a corn

basis instead of a sample basis, no difference in soluble phenolics was observed between flaking

grits and cooked grits. However, there was a significant decrease in bound phenolics. Under mild

heat treatment in cooking, insoluble-bound phenolics tended to be released primarily into

soluble-conjugated phenolics, while a little amount was liberated into free phenolics (Dewanto et

al., 2002). Hence, the decrease in bound phenolic content of cooked grits in our study can be

39

explained by the released bound phenolics during heat treatment. The unchanged soluble

phenolic content of cooked grits indicated possible loss of phenolic compounds during pressure

cooking. The loss may be due to the breakdown of phenolics under thermal treatment (Harakotr

et al., 2014). In the study conducted by Dewanto et al. (2002), increased soluble phenolics were

observed with corn kernels cooked without any ingredients added. Therefore, we hypothesize

that removed pericarp and germ fractions of corn may have protective effects on phenolics

during heat treatment. Additionally, phenolics may also react with the ingredients added to the

cooking system to cause the loss. During cooking, typical reactions of starch gelatinization,

Maillard browning, Strecker degradation, and lipid oxidation will occur (Culbertson, 2004).

Phenolics in corn may act as free radical scavengers to delay lipid oxidation, and thus may cause

loss in phenolic content.

After cooked grits were baked to reduce moisture content, the percentage of bound phenolics

slightly decreased. However, the change did not reach a significant level (Table 2.9). According

to Parra et al. (2007), when corn masa was baked into tortillas, slight increases in soluble

phenolics and decreases in bound phenolics were observed, although not significant. It was also

reported by Li et al. (2007) that baking at 177°C for 20 minutes did not affect total phenolic

content in purple wheat bran. Our results in baking were consistent with their work. In the study

of Dewanto et al. (2002), for thermal processing at 100°C, a significant release of bound

phenolics was observed. With increased temperature, the level of bound phenolics releasing

significantly increased. Baking was conducted under a temperature of 107.2°C which was higher

than 100°C, so we propose that release of bound phenolics may occur together with degradation

of solubilized phenolic compounds, which finally resulted in the unchanged bound phenolic

percentage.

40

After baked grits were further flaked and toasted at 204.4°C, a significant decrease in bound

phenolic percentage from 70.03% to 64.49% was observed, with a 31.30% increase in soluble

phenolics (Table 2.3 & Table 2.9). According to Saulnier et al. (2001), at a heat treatment over

180°C, auto-hydrolysis reaction occurred and thus a significant amount of free phenolics were

released from maize bran. Our results were in good agreement with theirs. While soluble

phenolic content increased, bound phenolic content in flakes remained unchanged during

toasting, which resulted in increased total phenolics (Table 2.5 & Table 2.7). These indicated

that the alkaline hydrolysis in bound phenolic extraction method may not be able to release all

the insoluble-bound phenolics linked to cell wall materials. During thermal treatment at 204.4°C

for toasting, cell wall matrix was disintegrated, and more firmly bound phenolics were exposed

and able to be extracted through alkaline hydrolysis for analysis. Increased total phenolics in

wheat, buckwheat, corn, and oat sprouts were also observed by Randhir et al. (2008) during

thermal processing, and they suggested that released bound phenolic acids from the breakdown

of cellular constituents and cell walls might be the possible reason. Another study on the effects

of thermal processing on selected legume sprouts and seedlings also indicated enhanced total

phenolic content and antioxidant activity under thermal treatment (Randhir et al., 2009). Hence,

we suppose that toasting may also increase bioavailability of phenolic compounds to pose more

health benefits to the human body while consuming corn flakes.

The overall changes in soluble, bound, and total phenolics during corn-flaking were shown in

Table 2.11 and Figure 2.11. In conclusion, significant changes in phenolics for corn flakes

production occurred during cooking and toasting. Total phenolic content decreased during

cooking with both reduced soluble and bound phenolics, and increased again during toasting

41

with increased soluble phenolics and more exposed bound phenolics. The results accorded with

our hypothesis that processing would induce changes to phenolics in corn.

42

2.5. Tables

Table 2.1: Mean Moisture Content (MC%) of Commercial Corn Hybrids during Five Processing

Stages a,b

Processing

Stages Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Stage Mean 8.57±0.38D 8.89±0.54

C 63.67±0.55

A 53.71±0.68

B 6.04±0.21

E

a Values expressed as mean ± standard error of three corn hybrids growing in three growing years

with three replications per year, and with duplicate phenolic analysis per replication. b

Means in the same row followed by the same letter are not different (P<0.05).

Table 2.2: Analysis of Variance for Soluble, Bound, and Total Phenolic Content, and Bound

Phenolic Percenrage (BP%) of Different Corn Hybrids Growing in Different Years at Different

Processing Stages a,b

Source DF Pr > F

Soluble Phenolics Bound Phenolics Total phenolics BP%

Genotype 2 0.0318 0.0249 0.0241 0.1651

Processing Stage 4 <.0001 <0.0001 <0.0001 <0.0001

G×P 8 0.0100 0.0091 0.0097 0.1305

Year 2 0.5631 0.2675 0.2989 0.4761

Rep(Y) 6 0.2079 0.2552 0.2964 0.0293

G×Y 4 0.2054 0.0787 0.0921 0.3432

G×Rep(Y) 12 0.0002 <0.0001 <0.0001 0.3004

P×Y 8 0.5109 0.4843 0.4716 0.6098

P×Rep(Y) 24 0.0700 0.4981 0.4102 0.3103

G×Y× P 15 0.2120 0.4452 0.5247 0.1444

G×P×Rep(Y) 46 <.0001 <0.0001 <0.0001 <0.0001

Residual 132 - - - - a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

43

Table 2.3: Mean Soluble Phenolic Content of Three Commercial Corn Hybrids during Five Processing Stages (µg gallic acid

equivalents/g sample) a,b,c

Hybrid Processing Stages

Hybrid Mean Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

A 620±13B,a

594±36B,ab

532±36C,ab

521±50C,ab

751±41A,a

604±53 ab

B 618±12A,a

510±6B,b

477±10B,b

491±8B,b

589±26A,b

537±37b

C 613±32B,a

669±13B,a

606±27B,a

611±23B,a

790±10A,a

658±45a

Stage Mean 617±2B 591±46

B 538±37

C 541±36

C 710±62

A

a Values expressed as mean ± standard error of three growing years with three replications per year, and with duplicate phenolic

analysis per replication. b

Means in the same row followed by the same letter (capital) are not different (P<0.05). c Means in the same column followed by the same letter (small) are not different (P<0.05).

Table 2.4: Mean Soluble Phenolic Content of Corn Growing in Three Years at Five Processing Stages (µg gallic acid equivalents/g

sample) a,b,c

Year Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

2009 629±24B,a

568±52B,a

498±30C,a

497±40C,a

691±47A,a

2010 616±18B,a

598±52B,a

548±45B,a

549±47B,a

725±59A,a

2011 608±14B,a

608±44B,a

569±45B,a

577±40B,a

716±89A,a

Stage Mean 617±2B 591±46

B 538±37

C 541±36

C 710±62

A

a Values expressed as mean ± standard error of three corn hybrids with three replications which are duplicate analyzed.

b Means in the same row followed by the same letter (capital) are not different (P<0.05).

c Means in the same column followed by the same letter (small) are not different (P<0.05).

44

Table 2.5: Mean Bound Phenolic Content of Three Commercial Corn Hybrids during Five Processing Stages (µg gallic acid

equivalents/g sample) a,b,c

Hybrid Processing Stages

Hybrid Mean Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

A 1770±99A,ab

1649±132B,b

1282±120C,ab

1268±97C,ab

1315±151C,a

1457±136ab

B 1541±57A,b

1423±18A,b

1130±30B,b

1092±22B,b

1117±58B,a

1261±119b

C 2044±93A,a

2125±98A,a

1482±69B,a

1452±81B,a

1450±80B,a

1711±198a

Stage Mean 1785±145A 1732±207

A 1298±102

B 1271±104

B 1294±97

B

a Values expressed as mean ± standard error of three growing years with three replications per year, and with duplicate phenolic

analysis per replication. b

Means in the same row followed by the same letter (capital) are not different (P<0.05). c Means in the same column followed by the same letter (small) are not different (P<0.05).

Table 2.6: Mean Bound Phenolic Content of Corn Growing in Three Years at Five Processing Stages (µg gallic acid equivalents/g

sample) a,b,c

Year Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

2009 1707±79A,a

1589±187A,a

1195±113B,a

1185±69B,a

1212±69B,a

2010 1765±187A,a

1811±254A,a

1288±64B,a

1264±101B,a

1246±113B,a

2011 1883±190A,a

1797±203A,a

1412±150B,a

1364±279B,a

1424±174B,a

Stage Mean 1785±145A 1732±207

A 1298±102

B 1271±104

B 1294±97

B

a Values expressed as mean ± standard error of three corn hybrids with three replications which are duplicate analyzed.

b Means in the same row followed by the same letter (capital) are not different (P<0.05).

c Means in the same column followed by the same letter (small) are not different (P<0.05).

45

Table 2.7: Mean Total Phenolic Content of Three Commercial Corn Hybrids during Five Processing Stages (µg gallic acid

equivalents/g sample) a,b,c

Hybrid Processing Stages

Hybrid Mean Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

A 2390±109A,ab

2244±168B,b

1814±150D,ab

1789±146D,ab

2066±1856C,ab

2061±152ab

B 2162±53A,b

1934±22B,b

1608±25C,b

1583±25C,b

1706±75C,b

1798±142b

C 2657±63A,a

2794±106A,a

2088±92B,a

2063±101B,a

2241±90B,a

2369±194a

Stage Mean 2403±131A 2324±252

A 1837±139

C 1812±139

C 2004±157

B

a Values expressed as mean ± standard error of three growing years with three replications per year, and with duplicate phenolic

analysis per replication. b

Means in the same row followed by the same letter (capital) are not different (P<0.05). c Means in the same column followed by the same letter (small) are not different (P<0.05).

Table 2.8: Mean Total Phenolic Content of Corn Growing in Three Years at Five Processing Stages (µg gallic acid equivalents/g

sample) a,b,c

Year Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

2009 2336±103A,a

2157±238B,a

1693±143D,a

1682±106D,a

1903±103C,a

2010 2381±171A,a

2409±304A,a

1836±107B,a

1812±148B,a

1970±167B,a

2011 2492±184A,a

2405±244A,a

1981±192C,a

1941±201C,a

2140±263B,a

Stage Mean 2403±131A 2324±252

A 1837±139

C 1812±139

C 2004±157

B

a Values expressed as mean ± standard error of three corn hybrids with three replications which are duplicate analyzed.

b Means in the same row followed by the same letter (capital) are not different (P<0.05).

c Means in the same column followed by the same letter (small) are not different (P<0.05).

46

Table 2.9: Mean Percentage of Bound Phenolics (BP%) of Three Commercial Corn Hybrids during Five Processing Stages a,b,c

Hybrid Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

A 74.01±0.82A,a

73.36±0.58AB,b

70.42±1.12C,a

70.91±0.49BC,a

63.38±1.49D,a

B 71.22±1.33AB,a

73.61±0.19A,b

70.30±0.93B,a

68.94±0.50B,a

65.49±1.17C,a

C 76.65±1.89A,a

76.02±0.73A,a

70.83±0.55B,a

70.23±0.74B,a

64.58±1.10C,a

Stage Mean 73.96±1.57A 74.33±0.85

A 70.52±0.16

B 70.03±0.58

B 64.49±0.61

C

a Values expressed as mean ± standard error of three growing years with three replications per year, and with duplicate phenolic

analysis per replication. b

Means in the same row followed by the same letter (capital) are not different (P<0.05). c Means in the same column followed by the same letter (small) are not different (P<0.05).

Table 2.10: Mean Percentage of Bound Phenolics (BP%) of Corn Growing in Three Years at Five Processing Stages a,b,c

Year Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

2009 72.92±0.06A,a

73.52±0.69A,a

70.22±0.65B,a

70.34±0.79B,a

63.58±1.44C,a

2010 73.72±2.49A,a

74.87±1.01A,a

70.23±1.00B,a

69.70±0.34B,a

63.30±0.71C,a

2011 77.24±1.32A,a

74.59±1.02A,a

71.10±0.91B,a

70.04±1.09B,a

66.58±0.17C,a

Stage Mean 73.96±1.57A 74.33±0.85

A 70.52±0.16

B 70.03±0.58

B 64.49±0.61

C

a Values expressed as mean ± standard error of three corn hybrids with three replications which are duplicate analyzed.

b Means in the same row followed by the same letter (capital) are not different (P<0.05).

c Means in the same column followed by the same letter (small) are not different (P<0.05).

47

Table 2.11: Mean Soluble, Bound, and Total Phenolics, and Percentage of Bound Phenolics

(BP%) of Three Commercial Corn Hybrids during Five Processing Stages (µg gallic acid

equivalents/g sample) a,b

Phenolics Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Soluble 617±2B 591±46

B 538±37

C 541±36

C 710±62

A

Bound 1785±145A 1732±207

A 1298±102

B 1271±104

B 1294±97

B

Total 2403±131A 2324±252

A 1837±139

C 1812±139

C 2004±157

B

BP% 73.96±1.57A 74.33±0.85

A 70.52±0.16

B 70.03±0.58

B 64.49±0.61

C

a Values expressed as mean ± standard error of three hybrids grown in three years with three

replications per year, and with duplicate phenolic analysis per replication. b

Means in the same row followed by the same letter are not different (P<0.05).

48

2.6. Figures

Figure 2.1: Soluble Phenolic Contents of Corn during the flaking procedure.

Figure 2.2: Soluble Phenolic Contents of Three Corn Hybrids during the Corn-Flaking Procedure.

200

250

300

350

400

450

500

550

600

650

700

750

800

850

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Solu

ble

Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

0

100

200

300

400

500

600

700

800

900

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Solu

ble

Ph

en

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Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

Hybrid A

Hybrid B

Hybrid C

49

Figure 2.3: Soluble Phenolic Contents of Corn Grown in Three Years during the Corn-Flaking

Procedure.

Figure 2.4: Bound Phenolic Contents during the Corn-Flaking Procedure.

0

100

200

300

400

500

600

700

800

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Solu

ble

Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le 2009

2010

2011

400

550

700

850

1000

1150

1300

1450

1600

1750

1900

2050

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Bo

un

d P

he

no

lic C

on

ten

t (µ

g ga

llic

acid

e

qu

ival

en

ts/g

sam

ple

)

50

Figure 2.5: Bound Phenolic Contents of Three Corn Hybrids during the Corn-Flaking Procedure.

Figure 2.6: Bound Phenolic Content of Corn Grown in Three Years during the Corn-Flaking

Procedure.

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Bo

un

d P

he

no

lic C

on

ten

t (µ

g ga

llic

acid

e

qu

ival

en

ts/g

sam

ple

Hybrid A

Hybrid B

hybrid C

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Bo

un

d P

he

no

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on

ten

t (µ

g ga

llic

acid

e

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ts/g

sam

ple

2009

2010

2011

51

Figure 2.7: Total Phenolic Contents during the Corn-Flaking Procedure.

Figure 2.8: Total Phenolic Contents of Three Corn Hybrids during the Corn-Flaking Procedure.

500

700

900

1100

1300

1500

1700

1900

2100

2300

2500

2700

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Tota

l Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

0

300

600

900

1200

1500

1800

2100

2400

2700

3000

3300

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Tota

l Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

Hybrid A

Hybrid B

Hybrid C

52

Figure 2.9: Total Phenolic Content of Corn Grown in Three Years during the Corn-Flaking

Procedure.

Figure 2.10: Percentage of Bound Phenolics of Corn during the Flaking Procedure.

0

300

600

900

1200

1500

1800

2100

2400

2700

3000

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Tota

l Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le) 2009

2010

2011

58.00

60.00

62.00

64.00

66.00

68.00

70.00

72.00

74.00

76.00

78.00

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Pe

rce

nta

ge o

f B

ou

nd

Ph

en

olic

s (%

)

53

Figure 2.11: Soluble, Bound, and Total Phenolic Contents of Corn during the Flaking Procedure.

0

250

500

750

1000

1250

1500

1750

2000

2250

2500

2750

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

Soluble

Bound

Total

54

CHAPTER 3: ANTIOXIDANT CAPACITY OF CORN AS AFFECTED BY GROWTH

YEAR AND CORN FLAKE PROCESSING STAGES

3.1. Introduction

Potential antioxidants in corn include carotenoids (Kurilich & Juvik, 1999; Kopsell et al.,

2009), phytosterols (Piironen et al., 2000; Yoshida & Niki, 2003), tocopherols (Kurilich & Juvik,

1999), ascorbic acid (Dewanto et al., 2002), anthocyanins (Li et al., 2008; Pedreschi & Cisneros-

Zevallos, 2007), and other phenolic compounds (Pedreschi & Cisneros-Zevallos, 2007; Ramos-

Escudero et al., 2012). According to Adom & Liu (2002), total antioxidant activity of corn was

181.42 μmol Vitamin C equiv/g, which was higher than that of wheat, rice, and oats. Phenolics

in corn possess considerable antioxidant activity in vitro, and are able to delay the cellular

damage induced by oxidative stress (Rice-Evans et al., 1997; Adom & Liu, 2002; Alía et al.,

2006a; Hu & Xu, 2011; Žilić et al., 2012). Total antioxidant activity of corn is closely related to

phenolic content (R2=0.983), especially for bound phenolics (R

2=0.991) (Adom & Liu, 2002). A

significant positive correlation between corn total phenolic content and oxygen radical

absorbance capacity (ORAC) was also established (r=0.83) by Bacchetti et al. (2013).

Attributed to their antioxidant capacity, phenolics are considered to have the potential in

preventing several chronic diseases. It is reported that phenolic compounds in corn have anti-

mutagenic capacity, which is achieved by mechanisms of blocking or scavenging mutagen

electrophiles and inhibiting enzymes involved in mutagen activation (Pedreschi & Cisneros-

Zevallos, 2006). Moreover, various phenolic compounds are able to suppress the proliferation of

several types of cancer cells in vitro, including ovarian (Scambia et al., 1994; Peng et al., 2008),

colon (Agullo et al., 1994; Yi et al., 2005), breast (Mehta et al., 1997; Kampa et al., 2004), lung

(Caltagirone et al., 1997), and liver cancer cells (Tanaka et al., 1993; Chi et al., 1997). Also,

55

phenolic compounds are able to suppress LDL oxidation and have a protective impact on heart

disease, through possible mechanisms such as scavenging oxidizing species and binding LDL

protein (Frankel et al., 1993; Ishikawa et al., 1997; Geleijnse et al., 1999; Kerry & Abbey, 1999;

Wang & Goodman, 1999; Chang et al., 2000; Duthie et al., 2000).

As a major health-enhancing function, antioxidant activity of corn has been widely studied;

however, no study has been focused on the antioxidant activity in corn flakes. Hence, the

objectives of this chapter were to investigate how antioxidant capacity is altered during the corn-

flaking procedure, and examine the growing year effect on antioxidant capacity in corn. Since

there is removal of phenolic concentrated corn fractions during dry-milling and there are

potential losses in phenolics under thermal treatment, decreased antioxidant capacity was

expected during the corn-flaking procedure.

3.2. Materials and Methods

3.2.1. Sample Preparation

Commercial corn hybrid A grown in 2009, 2010, and 2011 with three replications per year

was used in this study. Samples of whole kernels, flaking grits, cooked grits, baked grits, and

toasted flakes were collected from each corn-flaking processing stage. Samples were dried and

ground into powder using a coffee grinder.

3.2.2. Chemicals and Reagents

AAPH (2,2’-azobis (2-methylpropionamidine) dihydrochloride), Trolox ((±)-6-hydroxy-

2,5,7,8-tetramethylchromane-2-carboxylic acid) and fluorescein were purchased from Sigma-

Aldrich (St. Louis, MO). Potassium phosphate monobasic was obtained from Fisher Scientific

56

(Fair Lawn, NJ), and potassium phosphate dibasic trihydrate was obtained from Acros Organics

(Fair Lawn, NJ).

3.2.3. Determination of Antioxidant Capacity

A modified oxygen radical absorbance capacity (ORAC) assay was used to determine the

antioxidant capacity of corn samples, based on the original method described by Cao & Prior

(1998) and modified by Dávalos et al. (2004) and Wu et al. (2004). ORAC assays were carried

out in a FL600 microplate fluorescence reader (BioTek Inc., VT) with AAPH (12 mM) as

peroxyl radical generator, fluorescein (70.3 nM) as fluorescent probe, and trolox (0-4 μM) as

antioxidant standard. Trolox was prepared in AWA (acetone: water: acetic acid = 140:59:1); all

other reagents were prepared in 75 mM phosphate buffer, pH 7. Extracts from 0.10 g corn

samples were evaporated to dryness and reconstituted in 10 mL AWA. Another 15% dilution

was made to the corn extracts (in 10 mL AWA) before ORAC assays.

Fluorescein (120 μL) and diluted corn extract or trolox standard solution (20 μL) were added

to a 96 well plate, and pre-incubated at 37°C (15 min). To avoid positional errors, a set layout

was used for filling wells (Table 3.1). Reaction was started by adding 60 μL AAPH solution

rapidly to each well using two multi-channel pipets, and the microplate was placed in the

fluorescence reader maintained at 37°C to monitor fluorescence changes. Peroxyl radicals,

generated from thermal degradation of AAPH, would induce fluorescein oxidation. Antioxidant

capacity was expressed as the degree of inhibition of fluorescence loss by scavenging peroxyl

radicals (Bacchetti et al., 2013). Fluorescence was measured every min for 80 min (emission

wavelength 515 nm and excitation wavelength 493 nm). The plate was automatically shaken for

3 s at intensity 3 before every reading. Area under the fluorescence decay curve (AUC) values

57

were calculated and expressed as micromoles Trolox Equivalents (TE) per gram sample.

Triplicate analyses were conducted for each sample.

3.2.4. Statistical Analysis

Analysis of variance (ANOVA) was performed to determine the effect of growing year,

biological replication and processing stage on antioxidant capacity of corn samples using the

generalized linear mixed model (GLMM). Processing stage was treated as fixed effects, while

growing year and replication nested within year were treated as random effects. A generalized

linear model (GLM) was used to conduct ANOVA for antioxidant capacity of corn samples at

each processing stage, separately. Mean values were compared using the Tukey’s multiple

comparison test at 5% level. Regression analysis between phenolic content and antioxidant

capacity was also conducted. Analyses were performed using the Statistical Analysis System

(SAS), version 9.4 (SAS institute, Cary, NC).

3.3. Results

3.3.1. Growing Year and Processing Effects on Antioxidant Capacity

Effects of processing stages and replicates within years were both significant for antioxidant

capacity of corn, but the year effect was not significant (Table 3.2). Interaction of processing

stages by replicates within years was significant, while the interaction between processing stages

and years was not. The complete ANOVA tables for antioxidant capacity were listed in the

Appendix A.

Differences in antioxidant capacity were observed between corn grown in year 2009 and

2010 or 2011 for every individual processing stage (Table 3.3 & Figure 3.1). Antioxidant

58

capacity of corn hybrid A grown in 2009 was lower than corn grown in 2010 at processing stages

of flaking grits, cooked grits, baked grits, and toasted flakes. There was no difference in

antioxidant capacity between whole kernels grown in 2009 and 2010. Antioxidant capacity of

corn grown in 2009 was also lower than corn grown in 2011 at all processing stages, with

exception of baked grits. No difference was observed between corn grown in 2010 and 2011,

except for the whole kernel stage, in which corn grown in 2011 had higher antioxidant capacity

than that of grown in 2010. Due to the different yearly ranking in antioxidant capacity of

different processing stages, the overall yearly effect was not significant while that for every

individual processing stage was significant.

Analysis of processing impact on antioxidant capacity was presented with years combined

(Table 3.3). When whole kernels were dry milled into flaking grits, a decrease in antioxidant

capacity occurred. Another reduction in antioxidant capacity was observed when the flaking grits

were cooked at 15 psi for 60 minutes to produce cooked grits. The subsequent corn-flaking

process did not further change the antioxidant capacity of corn samples; there was no difference

in antioxidant capacity among cooked grits, baked grits, and toasted flakes.

3.3.2. Correlations between antioxidant capacity and phenolic content

Phenolic compounds, especially bound phenolics, are significant contributors to the

antioxidant capacity in corn (Adom & Liu, 2002). Significant correlations were observed

between antioxidant capacity and soluble, bound, and total phenolics in corn (Table 3.4). There

was a weak overall correlation (R2=0.11) between antioxidant capacity and soluble phenolic

content of corn from five processing stages, while a high correlation (R2=0.66) was noted

59

between antioxidant capacity and bound phenolic content. Antioxidant capacity was also highly

correlated to the total phenolic content in corn (R2=0.60).

For individual processing stage analysis, no significant linear relationship was observed

between antioxidant capacities and soluble, bound, and total phenolic contents in corn whole

kernels (Table 3.4). High linear correlations were observed with soluble phenolics and

antioxidant capacity in corn flaking grits (R2=0.64) and cooked grits (R

2=0.70). Correlations

became weaker in baked grits (R2=0.45) and toasted grits (R

2=0.32). Linear correlations between

antioxidant capacity and bound phenolics in flaking grits (R2=0.75), cooked grits (R

2=0.70),

baked grits (R2=0.63), and toasted flakes (R

2=0.61) were all significant. Significant linear

relationships were also observed between total phenolics and antioxidant capacity in flaking grits

(R2=0.76), cooked grits (R

2=0.76), baked grits (R

2=0.59), and toasted flakes (R

2=0.62).

3.4. Discussion

3.4.1. Effects of Growing Year and Processing Stage on Antioxidant Capacity

Commercial hybrid A was used in this study to predict the effects of growing year and

processing stage on antioxidant capacity of corn. Similar to phenolics, variation in antioxidant

capacity was also significantly influenced by corn-flaking processing stage (Table 3.2), since

there is a large contribution of phenolics to antioxidant capacity of corn (Adom & Liu, 2002).

The influence of growing years on antioxidant capacity was not significant, which was consistent

with results obtained by Harakotr et al. (2015), who found that growing season showed small

effect on antioxidant activity of waxy corn. However, substantial year-to-year variation occurred

in every individual processing stage (Table 3.3). For the original whole kernels, antioxidant

capacity of corn grown in 2011 was higher than in 2009 and 2010. Different climate conditions

60

among different growing years, such as different ultraviolet radiation, temperature, water stress,

and mineral nutrient availability may influence the phenolic content and antioxidant activity of

corn (Connor et al., 2002; Jing et al., 2007). The non-significant processing stage × year

interaction indicated that the trend of changes in antioxidant capacity of corn during corn-flaking

was able to predict their performances in other years (Harakotr et al., 2015). Variation for

antioxidant capacity was significant among replicates within years, and a substantial processing

stage × replicate within year interaction was also observed, which indicated that the effects of

growing year and processing stage may be better predicted with more biological replications

(Connor et al., 2005). Additionally, since a significant interaction of genotype × processing stage

was observed in phenolics, more hybrids from diverse genetic background would be needed to

have a comprehensive understanding of the processing effects on antioxidant capacity in corn.

3.4.2. Changes in Antioxidant Capacity during Corn-Flaking Procedure

ORAC assay was used in our study to measure the potencies of antioxidants to protect

biological molecules from being attacked by free radicals, which could represent the antioxidant

activities of antioxidants (Zhou & Yu, 2004; Li et al., 2007). Our results showed that mean

ORAC values of corn whole kernels was 50.06±2.00 μmol TE/g sample (Table 3.3), which was

consistent with the values of 45.00 and 48.89 μmol TE/g sample reported by Li et al. (2007) for

two typical corn types, but much higher than that reported by Bacchetti et al. (2013). After whole

kernels were dry-milled into flaking grits, the antioxidant capacity decreased significantly. This

may be due to the loss of antioxidants located in the pericarp and germ such as anthocyanins and

free phenolic acids; these two fractions were removed during dry milling (Sen et al., 1994;

Betran, 2000; Moreno et al., 2005).

61

Continuous decrease in antioxidant capacity occurred when the flaking grits were cooked,

which was in agreement with results reported by Harakotr et al. (2014). According to Dewanto et

al. (2002), total antioxidant capacity of cooked yellow sweet corn decreased with extended

cooking time, as a result of increased antioxidant capacity of soluble phenolic extracts and

decreased antioxidant capacity of bound phenolic extracts. In the present study, the decrease in

antioxidant capacity during cooking was attributed to both decreased soluble and bound phenolic

contents. Harakotr et al. (2014) indicated that loss in corn antioxidant activity during cooking

was mainly attributed to the breakdown of antioxidant compounds. Antioxidants may also

participate in reactions that occur during cooking, such as scavenging free radicals generated in

lipid oxidation (Culbertson, 2004), and thus induce a loss in total antioxidant capacity.

There was no difference in antioxidant capacity between cooked grits and baked grits, due to

the unchanged phenolic content during baking. However, the antioxidant capacity of toasted

flakes still remained unchanged from baked grits, while a significant increase in total phenolics

was observed during toasting (Table 2.7). This may be explained by the results of research

conducted by Ohta et al. (1994) that soluble-conjugated ferulic acids possessed stronger

antioxidant activity than the released ferulic acids. The temperatures treated for cooking and

baking would induce the release of bound phenolics into soluble ones; however, most of the

released phenolics were in soluble-conjugated form (Dewanto et al., 2002). During toasting, a

temperature of 204.4°C was applied, under which auto-hydrolysis reaction occurred, and soluble-

conjugated phenolics were liberated into free form (Saulnier et al., 2001). Thus, although total

phenolics of corn flakes increased during toasting, the antioxidant capacity did not arise with

increased phenolics due to the lowered antioxidant capacity of released free phenolic acids (Ohta

et al., 1994).

62

In conclusion, cooking was the critical point for antioxidant capacity changes during corn-

flaking procedure. Antioxidant capacity decreased during dry-milling and cooking, and remained

unchanged during baking and toasting.

3.4.3. Correlations between Antioxidant Capacity and Phenolic Content

Significant linear correlations were observed between antioxidant capacities and soluble

(R2=0.11), bound (R

2=0.66), and total phenolic contents (R

2=0.60) (Table 3.4). Since the

majority of phenolics in corn were in bound form, antioxidant capacity was closely associated

with bound phenolic content, while a low correlation was presented for soluble phenolics, which

was consistent with the results reported by Adom & Liu (2002). The highest correlations

between antioxidant capacity and soluble, bound, and total phenolics were observed in flaking

grits and cooked grits. After the grits were further processed, correlations between bound

phenolics and antioxidant capacity slightly decreased due to the gradually released bound

phenolic compounds during thermal processing. The significant decrease in correlations between

soluble phenolics and antioxidant capacity in baked grits and toasted grits may result from the

lowered antioxidant activity of free phenolics released from soluble-conjugated phenolics (Ohta

et al., 1994). The presence of antioxidants other than phenolics in the pericarp and germ, and the

different proportions of these antioxidants in corn kernels may be the reason of the low

correlations presented between antioxidant capacity and whole kernel phenolic contents. Another

reason might be the limited variation between whole kernel samples used for the correlation

analysis, which were from the same corn hybrid with similar original phenolic contents. Samples

collected from different hybrids with various phenolic contents are better to be used for the

correlation analysis between antioxidant capacity and phenolic content in future studies.

63

Additionally, we also observed that for samples with the same total phenolic content, whole

kernel samples always possessed higher antioxidant capacity than the other processed samples,

while antioxidant capacity of toasted grits were always lower than the other corn samples. This

may be explained by the decreased contribution of other antioxidants such as ascorbic acids to

total antioxidant capacity during thermal processing, in which they were destroyed significantly

(Dewanto et al., 2002).

64

3.5. Tables

Table 3.1: Set Layout for Filling 96-Wells Microplate a,b

Blank Blank 1 2 3 4 4 3 2 1 Blank Blank

Blank 1 A B C D D C B A 1 Blank

Blank 1 B C D A A D C B 1 Blank

Blank 1 C D A B B A D C 1 Blank

Blank 1 D A B C C B A D 1 Blank

a 1-4 represent trolox of 1-4 µM.

b A-D represent corn extract samples 1-4.

Table 3.2: Analysis of Variance for Antioxidant Capacity of Corn Hybrid A Growing in

Different Years at Different Processing Stages a

Source DF Mean Square Error DF F value Pr > F

ProcessingStage 4 1389.11 8 37.58 <0.0001

Year 2 637.49 5.12 3.56 0.1073

Rep(year) 6 189.56 24 3.98 0.0067

ProcessingStage×Year 8 36.97 24 0.78 0.6277

ProcessingStage×Rep(year) 24 47.66 90 7.42 <0.0001

Residual 90 6.42 - - - a Analysis of variance is performed using the generalized linear mixed model (GLMM).

Table 3.3: Antioxidant Capacity of Corn Hybrid A at Five Processing Stages in Different

Growing Years (μmol Trolox equiv/g sample) a,b,c

Growing

years

Processing Stages

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

2009 47.26±2.43A,b

36.51±1.50AB,b

28.75±1.01B,b

32.04±2.30B,b

30.25±2.11B,b

2010 48.97±1.13A,b

46.01±1.03B,a

36.27±1.38C,a

36.36±0.95C,a

38.01±0.96C,a

2011 53.95±1.16A,a

47.16±2.08B,a

34.89±1.95C,a

34.90±1.67C,ab

38.05±1.55C,a

Stage Mean 50.06±2.00A 43.23±3.38

B 33.30±2.31

C 34.43±1.27

C 35.43±2.59

C

a Values expressed as mean ± standard error of three replications per year with triplicate analysis.

b Means in the same row followed by the same letter (capital) are not different (P<0.05).

c Means in the same column followed by the same letter (small) are not different (P<0.05).

65

Table 3.4: Correlation Analysis of Antioxidant Capacity (AC) and Soluble Phenolics, Bound

Phenolics, and Total Phenolics of Corn Hybrid A from Different Processing Stages a,b,c

Phenolic Content Soluble Phenolics Bound Phenolics Total Phenolics

Total AC

Whole Kernel 0.0001 0.0007 0.0004

Flaking Grits 0.6392**

0.7545**

0.7578***

Cooked Grits 0.7039**

0.7018**

0.7633***

Baked Grits 0.4477* 0.6280

** 0.5853

**

Toasted Flakes 0.3208 0.6112**

0.6167**

Overall 0.1087* 0.6573

*** 0.6019

***

a Values listed as correlation coefficient R-square.

b *,**, and *** indicate significance at p<0.05, p<0.01, and p<0.001, respectively.

c For each processing stage, correlations are based on n = 10; For overall item, correlations are

based on n = 50.

3.6. Figures

Figure 3.1: Antioxidant Capacity of Corn during the Flaking Procedure.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

Whole Kernel Flaking Grits Cooked Grits Baked Grits Toasted Flakes

An

tio

xid

ant

Cap

acit

y (μ

mo

l Tro

lox

eq

uiv

/g s

amp

le)

2009

2010

2011

66

CHAPTER 4: EFFECT OF PROCESSING CONDITIONS ON THE PHENOLIC

CONTENT OF CORN FOR PRODUCTION OF CORN FLAKES

4.1. Introduction

Epidemiological studies have suggested that regular consumption of breakfast cereals would

help in reducing the risks of obesity and cardiovascular diseases (Geliebter et al., 2015). Corn is

a common source of grains to produce ready-to-eat breakfast cereals, whose phenolic content

was higher than rice, wheat, and oats (Adom & Liu, 2002). Corn contains a large variety of

phenolics, which are the major contributors of antioxidant activity in corn (Velioglu et al., 1998;

Hu & Xu, 2011).

Previous studies have been conducted to evaluate how processing impacted phenolics in corn.

Under thermal treatment, in-soluble bound phenolics tended to be released mainly into soluble-

conjugated phenolics. An enzymatic treatment, chemical treatment or a severe heat treatment

with a temperature higher than 180°C was needed to further liberate the soluble-conjugated

phenolics into free form (Saulnier et al., 2001; Dewanto et al., 2002). According to Parra et al.

(2007), bound ferulic acids in corn were released into free and soluble-conjugated ferulic acids

during lime-cooking for production of corn masa. However, with masa baked into tortilla and

fried into tortilla chips, ferulic acids were complexed again. Losses in phenolics may also occur

during thermal treatment due to degradation or decomposition of phenolic compounds (Harakotr

et al., 2014). More information was needed about the overall effect of processing on phenolics in

production of corn flakes.

The average nutritional quality of ready-to-eat breakfast cereals in the market has been

declining since the late 1980s (Wang et al., 2015). Improving the nutritional values of corn flakes

is needed to meet the needs of consumers. Hence, the objective of this chapter was to better

67

understand how processing conditions of corn-flaking impact phenolics, in order to provide

information for elevating the phenolic content in final flake product. Our hypothesis is that

optimization of processing conditions is feasible to obtain corn flakes with a higher phenolic

level. Higher temperature and pressure applied with extended time may help in liberating more

phenolics from the cell wall materials of corn.

4.2. Materials and Methods

4.2.1. Sample Preparation

Corn samples of commercial hybrid A grown in 2009 were used for this study. Flaking grits

samples were cooked under a pressure of 15 psi for 45, 60, 75, and 90 min, and cooked under a

pressure of 5 and 10 psi for 60 min. Samples cooked 60 min under a pressure of 15 psi were

baked at 225°F (107.2°C) for 30, 40, 50, and 60 min; and at 200°F (93.3°C), 250°F (121.1°C),

and 275°F (135.0°C) for 50 min. For toasting, samples cooked 60 min at 15 psi and baked at

225°F (107.2°C) for 50 min were subjected to different treatments. These baked samples were

toasted at 400°F (204.4°C) for 60, 70, 80, and 90s, and at 300°F (148.9°C), 350°F (176.7°C), and

450°F (232.2°C) for 80s. Three replications were conducted for each treatment. All samples were

dried and ground into powder using a coffee grinder.

4.2.2. Chemicals and Reagents

Folin-Ciocalteu reagent, gallic acid, and sodium carbonate were purchased from Sigma

Chemical Company (St. Louis, MO). Sodium chloride, sodium hydroxide, hydrochloric acid,

hexane, ethyl acetate, methanol, and acetone were obtained from Fisher Scientific (Fair Lawn,

68

NJ). Ethanol was purchased from Decon Labs (King of Prussia, PA). Salt, sugar, and molasses

were purchased from a local supermarket.

4.2.3. Extraction of Phenolic Compounds

Methods used to extract soluble and bound phenolic compounds in corn samples were

described in section 2.2.4 and section 2.2.5., respectively.

4.2.4. Determination of Phenolic Content

Methods for determination of phenolic content in corn were described in section 2.2.6.

4.2.5. Statistical Analysis

Analysis of variance (ANOVA) was performed to determine the effect of processing

conditions on phenolic content of corn samples using the generalized linear model (GLM). Mean

values were compared using the Fisher’s Least Significant Difference (LSD) test at 5% level.

Analyses were performed using the Statistical Analysis System (SAS), version 9.4 (SAS institute,

Cary, NC).

4.3. Results

4.3.1. Effect of Pressure Cooking Conditions on Phenolic Content in Corn

4.3.1.1. Effect of Pressure Cooking Time on Phenolic Content in Corn

The soluble phenolic content of raw flaking grits was 605 µg GAE/g sample (Table 4.1 &

Figure 4.1). After cooking at 15 psi for 45 minutes, the soluble phenolic content of these grits

was 636 µg GAE/g, indicating no significant change with cooking. With increased cooking time

69

at 15 psi for 60, 75, and 90 minutes, soluble phenolic content dropped to 629, 579, and 533 µg

GAE /g, respectively. The difference in soluble phenolics between cooking times of 45 and 60

minutes was not significant. However, significant reductions were noted when cooking time

increased to 75 and 90 minutes.

The bound phenolic content of raw flaking grits was 1726 µg GAE/g. After grits were

cooked at 15 psi for 45 minutes, the bound phenolic content dropped significantly to 1026 µg

GAE /g. There was an increase in bound phenolics when cooking time was extended from 45 to

60 minutes. However, with increased cooking time at 15 psi for 75 and 90 minutes, bound

phenolic content decreased significantly from that of the 60 minute cooked grits.

The total phenolic content of raw flaking grits was 2331 µg GAE/g. Subsequent cooking

process at 15 psi for 45 minutes induced a decrease in total phenolics of grits to 1662 µg GAE/g.

When pressure cooking time was increased from 45 to 60 minutes, a significant increase in total

phenolics was observed. However, after 60 minutes, extended cooking times of 75 and 90

minutes resulted in reduced total phenolic content.

There were no significant differences in bound phenolic percentages among flaking grits

cooked for 60, 75, and 90 minutes. The percentage of bound phenolics of grits cooked for 45

minutes was lower than the other cooked grit samples with longer cooking time.

4.3.1.2. Effect of Pressure Cooking Pressure on Phenolic Content in Corn

The soluble phenolic content of raw flaking grits was 605 µg GAE/g of sample (Table 4.2 &

Figure 4.2). After cooking for 60 minutes at 5 psi, soluble phenolic content of these grits

decreased to 462 µg GAE/g. However, with cooking pressure increased from 5 to 10 and then 15

psi, soluble phenolic content significantly increased again.

70

The bound phenolic content of raw flaking grits was 1726 µg GAE/g of sample. After

cooking for 60 minutes at 5 psi, bound phenolic content of these grits decreased to 883 µg

GAE/g. There was no difference in bound phenolic content between corn grits cooked for 60

minutes at 5 and 10 psi. However, a significant increase was observed when cooking pressure

was increased to 15 psi.

The total phenolic content of raw flaking grits was 2331 µg GAE/g. Subsequent cooking at 5

psi for 60 minutes resulted in a decrease in total phenolics to 1345 µg GAE/g of sample.

However, with cooking pressure increased from 5 to 10 psi, and from 10 to 15 psi, significant

increases in total phenolic content were observed.

There was no significant difference in bound phenolic percentage between grits cooked at 10

psi and 15 psi. The bound phenolic percentage of flaking grits cooked at 5 psi was significantly

higher than that of grits cooked at 10 and 15 psi.

4.3.2. Effect of Baking Conditions on Phenolic Content in Corn

4.3.2.1. Effect of Baking Time on Phenolic Content in Corn

After pressure cooking at 15 psi for 60 minutes, the soluble phenolic content of cooked grits

was 629 µg GAE/g (Table 4.3 & Figure 4.3). Subsequent baking at 225°F for 30 minutes

resulted in decreased soluble phenolic content in these cooked grits, but with extended baking

time, soluble phenolic content in grits increased again. There was no difference in soluble

phenolic content between grits baked for 30 and 50 minutes. Cooked grits baked for 40 minutes

had higher soluble phenolic content than 30 and 50 minute treatments. The soluble phenolic

content of cooked grits baked at 225°F for 60 minutes was higher than the other baking time

treatments.

71

The bound phenolic content of flaking grits cooked at 15 psi for 60 minutes was 1119 µg

GAE/g. These cooked grits were baked at 225°F for 30 minutes subsequently, and the bound

phenolic content decreased to 891.91 µg GAE/g. However, with increased baking time from 30

to 40 minutes and to 50 minutes, bound phenolic content of these baked grits increased again.

There was no difference in bound phenolic content between grits baked for 50 and 60 minutes at

225°F (107.2°C).

The total phenolic content of flaking grits cooked at 15 psi for 60 minutes was 1749 µg

GAE/g. After baking at 225°F for 30 minutes, the total phenolic content of these grits decreased

to 1303 µg GAE/g of sample. However, with baking time increased from 30 to 40 minutes, a

significant increase in total phenolic content of grits was observed. No difference in total

phenolic content was noted between grits baked for 40 and 50 minutes. When baking time was

extended to 60 minutes, another increase in total phenolic content was observed.

With baking time extended from 30 to 40 minutes, bound phenolic percentage significantly

decreased. There was no difference in bound phenolic percentage between grits baked for 40 and

60 minutes.

4.3.2.2. Effect of Baking Temperature on Phenolic Content in Corn

After pressure cooking at 15 psi for 60 minutes, the soluble phenolic content of cooked grits

was 629.21 µg GAE/g (Table 4.4 & Figure 4.4). Subsequent baking at 200°F for 50 minutes

resulted in decreased soluble phenolic content in these cooked grits to 399 µg GAE/g. There was

no difference in soluble phenolic content between grits baked at 200 and 225°F. However, with

increased baking temperature from 225 to 250°F, and to 275°F, significant increases in soluble

phenolic content were observed.

72

The bound phenolic content of flaking grits cooked at 15 psi for 60 minutes was 1119 µg

GAE/g. Subsequent baking at 200°F for 50 minutes induced a significant reduction in bound

phenolic content to 933 µg GAE/g. With baking temperature increased from 200 to 225°F, bound

phenolic content in grits significantly increased. After that, no difference in bound phenolic

content was observed among grits baked at 225, 250, and 275°F for 50 minutes.

The total phenolic content of flaking grits cooked at 15 psi for 60 minutes was 1749 µg

GAE/g. After baking at 200°F for 50 minutes, the total phenolic content of these corn grits

dropped to 1332 µg GAE/g of sample. With baking temperature increased from 200 to 225, 250,

and 275°F, significant increases in total phenolic content were observed between every two

treatments.

There was no difference in bound phenolic percentage between grits baked at 200 and 225°F.

Significant reductions in bound phenolic percentage were observed when baking temperature

was increased from 225 to 250 and 275°F.

4.3.3. Effect of Toasting Conditions on Phenolic Content in Corn

4.3.3.1. Effect of Toasting Time on Phenolic Content in Corn

The soluble phenolic content of grits cooked at 15 psi for 60 minutes and then baked at

225 °F for 50 minutes was 418 µg GAE/g, which was not different from the soluble phenolic

content obtained after they were toasted for 60 seconds at 400°F (Table 4.5 & Figure 4.5).

When toasting time was increased from 60 to 70 seconds, an increase in soluble phenolic content

was observed. No difference was noted between corn flakes toasted for 70 and 80 seconds. With

toasting time increase to 90 seconds, another increase in soluble phenolic content was observed.

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The bound phenolic content of grits cooked at 15 psi for 60 minutes and then baked at 225°F

for 50 minutes was 980 GAE/g of sample. After the baking process for 60 seconds at 400°F, the

bound phenolic content dropped to 906 GAE/g. When toasting time was increased from 60 to 70

seconds, an increase in bound phenolic content was observed. There was no difference in bound

phenolic content between corn flakes toasted for 70 and 80 seconds. When toasting time was

increased to 90 seconds, another increase in bound phenolic content was observed.

The total phenolic content of grits cooked at 15 psi for 60 minutes and then baked at 225°F

for 50 minutes was 1398 µg GAE/g, which was not different from the total phenolic content

obtained after they were toasted for 60 seconds at 400°F. With toasting time extended from 60 to

70 seconds, an increase in total phenolic content was presented. There was no difference in total

phenolic content between corn flakes toasted for 70 and 80 seconds. When toasting time was

increased to 90 seconds, another increase in total phenolic content was observed.

There was a decrease in bound phenolic percentage when toasting time was extended from

60 to 70 seconds. No difference in bound phenolic percentage was observed between flakes

toasted for 70 and 80 seconds. With toasting time extended from 80 to 90 seconds, another

reduction was noted in bound phenolic percentage.

4.3.3.2. Effect of Toasting Temperature on Phenolic Content in Corn

The soluble phenolic content of corn samples cooked at 15 psi for 60 minutes and then baked

at 225 °F for 50 minutes was 418 µg GAE/g (Table 4.6 & Figure 4.6). After toasting at 300°F

for 80 seconds, the soluble phenolic content of corn flakes increased to 492 µg GAE/g. With

increased toasting temperature from 300 to 350°F, the soluble phenolic content of corn flakes

significantly increased. No difference was noted between flakes toasted at 350 and 400°F. When

74

toasting temperature was increased from 400 to 450°F, another increase in soluble phenolic

content of corn flakes was observed.

The bound phenolic content of grits cooked at 15 psi for 60 minutes and baked at 225 °F for

50 minutes was 980 GAE/g. The subsequent toasting process at 300°F for 80 seconds did not

induce a difference to the bound phenolic content of corn samples. When the toasting

temperature increased from 300 to 350°F, bound phenolic content in corn flakes decreased

significantly. No difference was observed with continuously increased toasting temperature from

350 to 400 and 450°F.

The total phenolic content of grits cooked at 15 psi for 60 minutes and baked at 225°F for 50

minutes was 1398 µg GAE/g. After toasting at 300°F for 80 seconds, the total phenolic content

of these corn samples increased to 1525 µg GAE/g. However, with increased toasting

temperature from 300 to 350, 400, and 450°F, no difference in total phenolic content of corn

flakes was observed.

When toasting temperature was increased from 300 to 350°F, a significant reduction in

bound phenolic percentage was observed. There was no difference between corn flakes toasted at

350 and 400°F. Another decrease in bound phenolic percentage was observed when toasting

temperature increased from 400 to 450°F.

4.4. Discussion

4.4.1. Effect of Pressure Cooking Conditions on Phenolic Content in Corn

After flaking grits were cooked 45 minutes at 15 psi, the bound phenolic content decreased,

while soluble phenolic content remained the same (Table 4.1). This decrease in bound phenolic

percentage indicated the release of bound phenolics into free and soluble-conjugated phenolics

75

during thermal processing, which was consistent with what observed by Dewanto et al. (2002).

With extended pressure cooking time (except for 60 minutes), both soluble and bound phenolics

in corn declined, which was in good agreement with the results reported by Harakotr et al. (2014),

who found significant losses in phenolics during both boiling and steam cooking in purple waxy

corn. The increase in bound phenolics when grits were cooked from 45 to 60 minutes may be

explained by the disruption of cell wall matrix after long-time cooking that more firmly bound

phenolics were exposed and able to be measured. Dewanto et al. (2002) also described the

release of bound phenolic acids from the breakdown of cellular constituents during thermal

processing. After cooking 60 minutes, increased cooking time did not further alter the bound

phenolic percentage, which meant that release of bound phenolics no longer occurred. However,

losses in phenolics still occurred, which indicated the continuous breakdown of phenolic

compounds during thermal treatment (Harakotr et al., 2014). It may also indicate that the

treatment condition of 15 psi was not able to expose more firmly bound phenolics from cell wall

components after 60 minutes of cooking.

On the other hand, soluble and bound phenolic contents both significantly increased with

enhanced cooking pressure (Table 4.2), indicating that a proper cooking pressure would help in

liberating more bioavailable phenolics from the cell wall complex. According to Butz et al.

(2003), 30 minute pressurization with thermal treatment induced a 25% degradation of

antioxidant capacity in apple juice, while only 10% degradation occurred with thermal treatment

alone (Sanchez-Moreno et al., 2009). Although greater degradation of phenolics would occur

under more severe cooking conditions (Hiemori et al., 2009), the rate of bound phenolic

liberation was still higher than phenolic degradation, thus, there was an increase in phenolic

76

contents in our study. Thus, cooking at 15 psi for 60 minutes was found to be an optimal cooking

condition for producing higher phenolic contents in processed corn products.

4.4.2. Effect of Baking Conditions on Phenolic Content in Corn

Grits cooked 60 minutes at 15 psi were used to examine the effect of different baking

conditions on phenolic content in corn. In chapter 2, the results indicated no significant

difference in phenolics between cooked grits and baked grits (Table 2.15), and we hypothesized

that release of bound phenolics may occur together with the degradation of phenolic compounds,

which finally resulted in the unchanged phenolic contents during baking. These were confirmed

by the results showed in Table 4.3, in which a significant decrease was observed for both soluble

and bound phenolics in grits baked for 30 minutes at 225°F, compared to unbaked grits. Phenolic

contents significantly increased again with extended baking time. These indicated that a large

amount of phenolic compounds were degraded or decomposed under thermal treatment during

baking (Patras et al., 2010); however, after a long baking time, the cell wall network was

gradually disrupted (Zivanovic & Buescher, 2004), from which more bound phenolics were

liberated. After baking 30 minutes, the liberation rate became higher than degradation; this

resulted in increased phenolic contents with extended baking time.

With increased baking temperature, bound phenolic percentage decreased significantly

(Table 4.4), indicating that more bound phenolics were released into soluble phenolics under

higher heating temperature. During baking, phenolic antioxidants tended to be oxidized to

quinones and their polymers, while the esters and glucosides of antioxidants were prone to be

hydrolyzed (Nayak et al., 2015). Other reactions such as Maillard reaction, caramelization, and

Strecker degradation would also occur, which may result in the loss of phenolic compounds

77

during baking. As shown in Chen & Lin (2007), losses of total phenolic contents in yams

increased with enhanced heating temperature. However, with more firmly bound phenolics

liberating from cell wall networks with increasing temperature, phenolics still increased

significantly. Randhir et al. (2008) indicated that the increase in total phenolic content during

thermal processing may be the result of disassociation of conjugated phenolics followed by

polymerization or oxidation of phenolic constituents.

Although more phenolic compounds were obtained in processed corn samples with increased

baking temperature and baking time, the moisture content of baked grits may become too low to

be further processed into flakes if exposed to a high temperature for too long. Hence, moisture

contents should be also taken into account while choosing optimal baking conditions.

4.4.3. Effect of Toasting Conditions on Phenolic Content in Corn

During toasting, bound phenolics were further released into soluble phenolics with extended

toasting time (Table 4.5), while soluble-conjugated phenolics were also auto-hydrolyzed into

free phenolics (Saulnier et al., 2001; Dewanto et al., 2002). Increases in both soluble and bound

phenolic contents indicated the presence of alkali-stable bound phenolics in the cell wall matrix,

which were able to be liberated by thermal treatment. Hassan & Youssef (2012) also reported the

increases in total phenolics and antioxidant activity of white beans during toasting with extended

time, which was consistent with our results. The unchanged total phenolic contents with

increasing toasting temperature (Table 4.6) indicated that the amount of bound phenolics

liberated from the cell wall network highly depended on toasting time, while higher temperature

did not result in faster liberation. However, increased temperature did assist in releasing bound

phenolics that could be alkaline hydrolyzed during extraction into soluble phenolics, which

78

resulted in a lower bound phenolic percentage. Jannat et al. (2013) examined the effects of

roasting conditions on Iranian sesame seeds, and also found significantly increased total phenolic

and γ-tocopherol contents with enhanced roasting time and temperature, until the temperature

reached 200°C. Temperatures higher than 200°C would induce higher losses in phenolics.

During toasting, Maillard reaction may also occur. Additionally, the temperature was also

reached for caramelization of sugars (mainly sucrose) which were added as ingredients before

cooking, to further generate desired color and flavor for the final flakes (Culbertson, 2004).

Hence, color and flavor attributes of flakes were also important parameters while choosing the

optimum toasting time and temperature. Although extended toasting time is able to produce more

bioavailable phenolics, over-toasting may result in undesired brown color in flakes.

Solubilized phenolic compounds in foods are able to be absorbed in the human stomach and

small intestine (Acosta-Estrada et al., 2014), and enter the circulatory system to induce health-

enhancing functions such as preventing coronary heart diseases (Morton et al., 2000) and anti-

inflammation (Jiang & Dusting, 2003). High bioavailability in vivo was demonstrated for soluble

phenolics, while a fast absorption was observed in 15 minutes for hydrolyzed Trichilia emetica

phenolic extracts (Germanò et al., 2006). Bound phenolics are also bioavailable, and capable of

posing beneficial effects on human health (Pérez-Jiménez et al., 2009). It was demonstrated by

Andreasen et al. (2001) that esterases in the human gastrointestinal (GI) tract were able to release

the esterified diferulates in orally ingested cereal brans into free diferulic acid, which could be

absorbed and participate in peripheral circulation. Adom & Liu (2002) indicated that insoluble-

bound phenolics in grains may survive digestion of the upper GI tract and reach the colon, thus

pose beneficial health effects on preventing colon cancer. In humans, more than 95% of the total

release of feruloyl groups from wheat bran occurred in the colon, in which they were fermented.

79

Esterified feruloyl groups were solubilized through the activities of ferulic acid esterase and

xylanase from the microflora in the human GI tract (Kroon et al., 1997). Other phenolic acid

esters such as chlorogenic acids that were esters of caffeic and quinic acids could also be

released through colonic microflora (Plumb et al., 1999). Hence, soluble and bound phenolics

function towards different targets in human body to promote different health effects. Whether

higher temperature is needed to release more bound phenolics into soluble ones during toasting

depends on different demands for health-promoting effects.

Alkaline hydrolysis is the most common method to release the cell wall-linked bound

phenolics in corn (Adom & Liu, 2002; Dewanto et al., 2002; Parra et al., 2007; Lopez-Martinez

et al., 2009; Žilić et al., 2012), reducing the loss of corn ferulic acids from 78% to 4.8%

compared with acid hydrolysis (Krygier et al., 1982). However, increases in total phenolics

during thermal processing in our study indicated that alkaline hydrolysis may still not be able to

release all the bound phenolics from the cell wall components, thus the bound phenolics reported

may be underestimated. Future studies are needed to examine a more effective way to

disintegrate the cell wall matrix, and have the insoluble-bound phenolics in corn liberated and

prone to be extracted. On the other hand, since those firmly-bound alkali-stable phenolics, as

well as other heat-resistant phenolic cross-links may not be able to be released and absorbed in

human body (Scalbert & Williamson, 2000), the bound phenolic contents obtained through

alkaline hydrolysis in our study may represent the bioavailable bound phenolics present in corn.

In this study, we only examined the effects of processing conditions on phenolics in corn.

The effects of processing conditions on antioxidant capacity in corn need to be further

investigated. Based on the results obtained in chapters 2 and 3, we predict that higher cooking

pressure would facilitate an increase in antioxidant capacity of corn, while longer cooking time

80

may cause loss in antioxidants. Increased baking temperature and time may enhance the

antioxidant capacity of corn; however, toasting with higher temperature or longer time may not

make changes to the antioxidant capacity of corn flakes.

81

4.5. Tables

Table 4.1: Effect of Pressure-Cooking Time on Phenolic Content in Corn (µg gallic acid

equivalents/g sample) a,b

Pressure Time Soluble Phenolics Bound Phenolics Total Phenolics BP%c

15 psi

0 min 605±52a 1726±6

a 2331±58

a 74.10±1.58

a

45 min 636±2a 1026±14

c 1662±16

c 61.72±0.25

c

60 min 629±10a 1119±16

b 1749±26

b 64.01±0.12

b

75 min 579±10b 1027±6

c 1606±14

c 63.93±0.34

b

90 min 533±11c 972±4

d 1505±15

d 64.59±0.37

b

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

Table 4.2: Effect of Pressure-Cooking Pressure on Phenolic Content in Corn (µg gallic acid

equivalents/g sample) a,b

Time Pressure Soluble Phenolics Bound Phenolics Total Phenolics BP% c

60 min

5 psi 462±5c 883±18

b 1345±22

c 65.64±0.26

a

10 psi 548±6b 937±17

b 1486±18

b 63.09±0.49

b

15 psi 629±10a 1119±16

a 1749±26

a 64.01±0.12

b

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

Table 4.3: Effect of Baking Time on Phenolic Content in Corn (µg gallic acid equivalents/g

sample) a,b

Temp Time Soluble Phenolics Bound Phenolics Total Phenolics BP% c

225 °F

0 min 629±10a 1119±16

a 1749±26

a 64.01±0.12

d

30 min 411±6d 892±6

d 1303±12

d 68.43±0.23

b

40 min 465±18c 933±11

c 1398±21

c 66.73±0.91

c

50 min 418±12d 980±12

b 1398±24

c 70.10±0.33

a

60 min 511±10b 1001±12

b 1512±21

b 66.20±0.21

c

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

82

Table 4.4: Effect of Baking Temperature on Phenolic Content in Corn (µg gallic acid

equivalents/g sample) a,b

Time Temp Soluble Phenolics Bound Phenolics Total Phenolics BP% c

50 min

200 °F 399±5c 933±8

b 1332±12

d 70.06±0.19

a

225 °F 418±12c 980±12

a 1398±24

c 70.10±0.33

a

250 °F 521±11b 965±11

ab 1487±22

b 64.93±0.23

b

275 °F 595±13a 985±7

a 1579±21

a 62.36±0.35

c

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

Table 4.5: Effect of Toasting Time on Phenolic Content in Corn (µg gallic acid equivalents/g

sample) a,b

Temp Time Soluble Phenolics Bound Phenolics Total Phenolics BP% c

400 °F

0 s 418±12d 980±12

a 1398±24

c 70.10±0.33

a

60 s 451±7c 906±7

c 1356±14

c 66.77±0.18

b

70 s 547±12b 950±14

b 1496±25

b 63.45±0.26

c

80 s 547±14b 947±11

b 1494±25

b 63.39±0.36

c

90 s 669±16a 985±10

a 1655±26

a 59.55±0.36

d

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

Table 4.6: Effect of Toasting Temperature on Phenolic Content in Corn (µg gallic acid

equivalents/g sample) a,b

Time Temp Soluble Phenolics Bound Phenolics Total Phenolics BP% c

80 s

300 °F 492±11c 1033±18

a 1525±28

a 67.74±0.20

a

350 °F 547±14b 951±10

b 1498±24

a 63.51±0.38

b

400 °F 547±14b 947±11

b 1494±25

a 63.39±0.36

b

450 °F 623±14a 932±6

b 1555±20

a 59.92±0.40

c

Whole kernels 561±7 1674±96 2236±103 74.91±0.86 a Values expressed as mean ± standard error of three replications.

b Means in the same column followed by the same letter are not different (p<0.05).

c BP% = Bound phenolics / Total phenolics × 100%

83

4.6. Figures

Figure 4.1: Effect of Pressure-Cooking Time on Phenolic Content in Corn (at pressure of 15 psi)

Figure 4.2: Effect of Pressure-Cooking Pressure on Phenolic Content in Corn (cooking for 60

min)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

0 min 45 min 60 min 75 min 90 min

Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id e

qu

ival

en

ts/g

sa

mp

le)

Soluble

bound

total

0

200

400

600

800

1000

1200

1400

1600

1800

2000

5 psi 10 psi 15 psi

Ph

en

olic

Co

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(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

Soluble

Bound

Total

84

Figure 4.3: Effect of Baking Time on Phenolic Content in Corn (at a temperature of 225 °F)

Figure 4.4: Effect of Baking Temperature on Phenolic Content in Corn (baking for 50 min)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 min 30 min 40 min 50 min 60 min

Ph

en

olic

Co

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(µg

galli

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sa

mp

le)

Soluble

Bound

Total

0

200

400

600

800

1000

1200

1400

1600

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200 ⁰F 225 ⁰F 250 ⁰F 275 ⁰F

Ph

en

olic

Co

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(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le)

Soluble

Bound

Total

85

Figure 4.5: Effect of Toasting Time on Phenolic Content in Corn (at a temperature of 400 °F)

Figure 4.6: Effect of Toasting Temperature on Phenolic Content in Corn (Toasting for 80 s)

0

200

400

600

800

1000

1200

1400

1600

1800

0 s 60 s 70 s 80 s 90 s

Ph

en

olic

Co

nte

nt

(µg

galli

c ac

id e

qu

ival

en

ts/g

sa

mp

le)

Soluble

Bound

Total

0

200

400

600

800

1000

1200

1400

1600

1800

300 ⁰F 350 ⁰F 400 ⁰F 450 ⁰F

Ph

en

olic

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nt

(µg

galli

c ac

id

eq

uiv

ale

nts

/g s

amp

le) Soluble

Bound

Total

86

CHAPTER 5: CONCLUSIONS AND FUTURE STUDIES

5.1. Conclusions

In this study, commercial corn hybrids A, B, and C that were grown in three different years

with three replicates per year were processed into corn flakes through five major processing

stages. The effects of genotype, growing year, and processing on phenolics and antioxidant

capacity in corn were examined. The hypotheses of this research are that processing will impact

on phenolic content and antioxidant capacity of corn, and corn flakes with higher phenolic

content can be obtained with optimized processing conditions.

This study of changes in soluble, bound, and total phenolics in three commercial corn hybrids

demonstrated significant variations among genotypes and processing stages, together with

significant genotype × processing stage and genotype × replicate within year interactions. The

non-significant year effect and genotype × year interaction indicated the feasibility of breeding

corn for higher phenolic concentration. More replicates may help in improving the estimates of

genotype, year, and processing stage effects.

The trend of phenolic changing during production of corn flakes was discussed in this study.

During thermal processing, bound phenolics tended to be released into soluble phenolics, and

more firmly bound phenolics in the cell wall matrix were able to be liberated. Cooking and

toasting were the two critical processing points to induce phenolic changes during production of

corn flakes. Total phenolic content significantly decreased during cooking with breakdown of

phenolic compounds, and significantly increased again during toasting with increased soluble

phenolics and more exposed bound phenolics from the disrupted cell wall network.

Corn commercial hybrid A was used to predict the effects of growing year and processing

stage on antioxidant capacity of corn. Significant variation in antioxidant capacity was presented

87

among five corn-flaking processing stages and replicates within years, but not among growing

years. More hybrids from diverse genetic background would be needed to have a comprehensive

understanding of the year and processing effects on antioxidant capacity in corn.

The ORAC assay was used to measure the antioxidant capacity of corn samples. Significant

losses in antioxidant capacity were observed during dry-milling and pressure cooking, due to the

removal of pericarp and germ fractions which contained concentrated free phenolic acids and

flavonoids, and the breakdown of antioxidants under thermal treatment. Baking and toasting did

not significantly alter the antioxidant capacity of corn. Hence, we conclude that processed corn

flakes had lower antioxidant capacity than raw corn kernels. Significant linear correlations were

observed between antioxidant capacities and soluble, bound, and total phenolic contents in corn.

However, antioxidant capacity was more closely related to the bound phenolics rather than

soluble phenolics. Correlations also varied for different corn-flaking processing stages.

The effects of processing conditions on phenolic content in corn were also examined in this

study. Total phenolic content in corn decreased with extended pressure cooking time, but were

enhanced with higher cooking pressure. Increased baking time and temperature would induce

significant increase in phenolics. Extended toasting time could also liberate more bound phenolic

compounds to increase total phenolic content, but higher temperature in toasting did not help in

faster liberation. Our findings may help in optimizing the processing conditions for higher

phenolic content and antioxidant capacity in corn flakes. Soluble and bound phenolics are both

bioavailable, but are absorbed in different parts of human GI tract. Hence, the ideal phenolic

form for ingestion depends on different demands of health-promoting effects.

88

5.2. Recommendations for Future Studies

1. Future studies are needed to examine a more effective way to measure the overall bound

phenolics in corn. We found in our study that alkaline hydrolysis that we used in bound phenolic

extraction may not be able to release all the firmly bound phenolics from the cell wall matrix.

Thus, bound phenolic contents reported may be underestimated. A new method is needed to

disintegrate the cell wall matrix, and have more insoluble-bound phenolics in corn liberated and

prone to be extracted.

2. More corn hybrids were better to be used in predicting the effect of corn-flaking

processing on antioxidant capacity. A significant interaction of genotype × processing stage was

observed in phenolics, which indicated that different corn hybrids may performed differently in

phenolic changes during corn-flaking procedure. Since the high correlation of phenolics and

antioxidant capacity, same interaction may also occur between genotype and antioxidant capacity.

Hence, more hybrids from diverse genetic background would be needed to have a comprehensive

understanding of the processing effects on antioxidant capacity in corn.

3. The effects of processing conditions on antioxidant capacity of processed corn could be

further examined in a future study. This research only explored how phenolics changed with

different treatment conditions during cooking, baking, and toasting. However, according to our

results, changes in antioxidant capacity during corn-flaking may perform differently from

phenolics. Hence, it will be better to see how antioxidant capacity was altered by different

processing conditions, and thus with more information to choose the optimal combinations.

4. An overall optimization of corn-flaking processing conditions could be done in future

studies to obtain higher phenolics and antioxidant capacity in final flake products. This study

only involved single factor experiments in Chapter 4, but response surface methodology could be

89

used to further optimize the corn-flaking processing conditions. Other nutrient changes during

processing such as resistant starch, fibers, and vitamins, and final product attributes such as color,

flavor, and crispness are also needed to be measured and taken into account in the overall

optimization.

5. Data obtained in this study needs to be compared with that of commercial corn flakes.

Since in this study, a lab scale method was used to produce corn flakes, differences in the final

attributes and nutritional levels may present between lab-produced corn flakes and commercial

corn flakes. Data comparison is needed to see if the results obtained in this study are applicable

in the industry.

90

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118

APPENDIX A: ANALYSIS OF VARIANCE FOR MOISTURE CONTENT, PHENOLICS,

AND ANTIOXIDANT CAPACITY OF CORN HYBRIDS GROWN IN THREE

YEARS AT FIVE PROCESSING STAGES

Appendix A.1: Analysis of Variance for Moisture Content (MC%) of Different Corn Hybrids

Growing in Different Years at Different Processing Stages a,b

Source DF Sum of Squares Mean Square Error DF F value Pr > F

Genotype 2 10.51 5.26 3.98 2.38 0.2088

Processing Stage 4 63260 15815 7.96 3037.94 <0.0001

G×P 8 41.74 5.22 14.65 2.07 0.1078

Year 2 22.98 11.49 4.61 2.30 0.2026

Rep(Y) 6 2.58 0.43 0.31 1.39 0.7336

G×Y 4 8.85 2.21 5.17 1.22 0.4058

G×Rep(Y) 12 15.31 1.28 38 0.64 0.7912

P×Y 8 41.55 5.19 4.28 3.26 0.1245

P×Rep(Y) 24 24.99 1.04 38 0.53 0.9505

G×Y× P 15 37.60 2.51 38 1.27 0.2705

G×P×Rep(Y) 38 75.30 1.98 0 1.78E12 -

Residual 0 2.68E-11 1.11E-12 - - - a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

Appendix A.2: Analysis of Variance for Soluble Phenolic Content of Different Corn Hybrids

Growing in Different Years at Different Processing Stages a,b

Source DF Sum of Squares Mean Square Error DF F value Pr > F

Genotype 2 672560 336280 3.99 9.24 0.0318

Processing Stage 4 1056245 264061 7.91 30.45 <.0001

G×P 8 198984 24873 15 4.01 0.0100

Year 2 68904 34452 4.67 0.65 0.5631

Rep(Y) 6 222369 37061 14.63 1.63 0.2079

G×Y 4 144494 36124 12.94 1.72 0.2054

G×Rep(Y) 12 235492 19624 46 4.27 0.0002

P×Y 8 69223 8652.88 15.73 0.94 0.5109

P×Rep(Y) 24 182699 7612.45 46 1.66 0.0700

G×Y× P 15 93156 6210.41 46 1.35 0.2120

G×P×Rep(Y) 46 211310 4593.69 132 3.09 <.0001

Residual 132 195948 1484.45 a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

119

Appendix A (continue)

Appendix A.3: Analysis of Variance for Soluble Phenolic Content of Different Corn Hybrids Growing in Different Years at Each

Processing Stage a,b

Source DF Whole Kernel Flaking Grits Cooked Grits

MSc F P MS F P MS F P

Genotype 2 271.47 0.02 0.9798 113658 12.15 0.0200 75018 9.81 0.0287

Year 2 1601.84 0.10 0.9059 8049.26 0.44 0.6659 23899 1.71 0.2525

Rep(Y) 6 6479.01 1.76 0.2057 15809 2.27 0.1069 9335.80 3.07 0.0465

G×Y 4 13181 3.57 0.0548 9352.69 1.34 0.3102 7649.84 2.51 0.0969

G×Rep(Y) 12 3689.58 1.00 0.4735 6963.62 3.17 0.0062 3042.43 6.61 <.0001

Residual 27 3704.28 - - 2193.70 - - 460.17 - -

Source DF Baked Grits Toasted Flakes

MS F P MS F P

Genotype 2 70483 5.34 0.0743 204648 10.59 0.0252

Year 2 29604 2.28 0.2409 5576.75 0.17 0.8493

Rep(Y) 6 5747.97 0.97 0.4859 31543 1.73 0.1969

G×Y 4 13201 2.22 0.1275 19324 1.06 0.4174

G×Rep(Y) 12 5937.04 11.94 <.0001 18216 22.39 <.0001

Residual 27 497.09 - - 813.66 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “Y”= “Year”.

c “MS” indicates “Mean Square”.

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Appendix A (continue)

Appendix A.4: Analysis of Variance for Soluble Phenolic Content of Processed Corn Hybrids Growing in Three Years a,b

Source DF 2009 2010 2011

MSc F P MS F P MS F P

Genotype 2 120604 6.10 0.0610 118174 6.75 0.0522 198055 9.17 0.0320

Processing Stage 4 127237 19.73 0.0003 93746 8.05 0.0066 71382 15.04 0.0009

G×P 8 5705.49 1.43 0.2591 18488 3.49 0.0160 14084 3.15 0.0321

Rep 2 2628.42 0.12 0.8910 52846 2.22 0.1906 55710 2.52 0.1976

G×Rep 4 19781 4.95 0.0087 17503 3.30 0.0374 21589 4.83 0.0117

P×Rep 8 6448.09 1.61 0.1980 11642 2.20 0.0857 4747.47 1.06 0.4395

G×P×Rep 16 4000.09 2.13 0.0237 5296.72 2.96 0.0021 4468.63 6.06 <.0001

Residual 45 1876.30 - - 1790.40 - - 736.82 - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”.

c “MS” indicates “Mean Square”.

Appendix A.5: Analysis of Variance for Soluble Phenolic Content of three Processed Corn Hybrids Growing in Different Years a

Source DF Hybrid A Hybrid B Hybrid C

MSb F P MS F P MS F P

Processing Stage 4 153399 28.98 <.0001 60590 13.31 0.0022 110817 10.05 0.0033

Year 2 104076 3.70 0.1111 467.21 0.9861 0.9861 1822.85 0.09 0.9109

Rep(year) 6 31658 3.60 0.0110 33010 <.0001 <.0001 13046 2.75 0.0353

Processing Stage×Year 8 5293.29 0.60 0.7675 4551.77 0.2344 0.2344 11021 2.32 0.0527

Processing Stage×Rep(year) 24 8804.78 6.99 <.0001 3128.22 0.0264 0.0264 4744.71 2.90 0.0010

Residual 45 1259.38 - - 1563.81 - - 1635.46 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “MS” indicates “Mean Square”.

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Appendix A.6: Analysis of Variance for Bound Phenolic Content of Different Corn Hybrids

Growing in Different Years at Different Processing Stages a,b

Source DF Sum of Squares Mean Square Error DF F value Pr > F

Genotype 2 8511879 4255939 3.99 10.69 0.0249

Processing Stage 4 13590125 3397531 7.88 105.66 <0.0001

G×P 8 1033273 129159 15 4.09 0.0091

Year 2 1690189 845095 4.52 1.79 0.2675

Rep(Y) 6 1315120 219187 11.53 1.52 0.2552

G×Y 4 1580119 395030 11.64 2.76 0.0787

G×Rep(Y) 12 1727707 143976 46 4.69 <0.0001

P×Y 8 257207 32151 7.88 1.03 0.4843

P×Rep(Y) 24 727649 30319 46 0.99 0.4981

G×Y× P 15 473239 31549 46 1.03 0.4452

G×P×Rep(Y) 46 1411184 30678 132 8.40 <0.0001

Residual 132 482064 3652 a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

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Appendix A.7: Analysis of Variance for Bound Phenolic Content of Different Corn Hybrids Growing in Different Years at Each

Processing Stage a,b

Source DF Whole Kernel Flaking Grits Cooked Grits

MSc F P MS F P MS F P

Genotype 2 651103 6.13 0.0872 2307770 21.20 0.0074 560556 7.61 0.0433

Year 2 232790 2.28 0.2865 276733 1.60 0.2825 213576 2.35 0.2315

Rep(Y) 6 57752 0.93 0.5152 114612 2.28 0.1062 72683 1.31 0.3247

G×Y 4 106235 1.71 0.2286 108860 2.16 0.1354 73645 1.33 0.3157

G×Rep(Y) 12 62291 6.86 <.0001 50350 15.82 <.0001 55532 26.60 <.0001

Residual 27 9079.31 - 3182.20 - - 2087.75 - -

Source DF Baked Grits Toasted Flakes

MS F P MS F P

Genotype 2 584262 7.77 0.0419 506877 2.88 0.1679

Year 2 144995 2.46 0.2976 233039 1.22 0.3818

Rep(Y) 6 34997 0.68 0.6669 66902 1.27 0.3391

G×Y 4 75209 1.47 0.2721 175949 3.35 0.0464

G×Rep(Y) 12 51212 21.11 <.0001 52572 25.18 <.0001

Residual 27 2426.10 - - 2087.69 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “Y”= “Year”.

c “MS” indicates “Mean Square”.

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Appendix A.8: Analysis of Variance for Bound Phenolic Content of Processed Corn Hybrids Growing in Three Years a,b

Source DF 2009 2010 2011

MSc F P MS F P MS F P

Genotype 2 1827487 4.02 0.1104 1823826 33.06 0.0033 2146421 14.37 0.0149

Processing Stage 4 4525616 45.87 <.0001 1481367 31.57 <.0001 921366 47.55 <.0001

G×P 8 454498 1.47 0.2430 112034 2.97 0.0302 22248 1.63 0.2054

Rep 2 762057 1.79 0.2927 110831 1.72 0.2966 165701 1.05 0.4248

G×Rep 4 909441 5.89 0.0041 55164 1.46 0.2591 149403 10.98 0.0003

P×Rep 8 197307 0.64 0.7351 46917 1.25 0.3358 19375 1.42 0.2691

G×P×Rep 16 618033 11.73 <.0001 37662 7.78 <.0001 13611 4.93 <.0001

Residual 45 148190 - - 4842.08 - - 2761.44 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”.

c “MS” indicates “Mean Square”.

Appendix A.9: Analysis of Variance for Bound Phenolic Content of three Processed Corn Hybrids Growing in Different Years a

Source DF Hybrid A Hybrid B Hybrid C

MSb F P MS F P MS F P

Processing Stage 4 997519 80.66 <.0001 618114 18.31 0.0008 2112520 42.76 <.0001

Year 2 1279249 5.14 0.0541 31289 0.56 0.6025 452052 2.13 0.1996

Rep(year) 6 257348 12.29 <.0001 49975 1.87 0.1316 206300 4.72 0.0026

Processing Stage×Year 8 12366 0.59 0.7758 33757 1.26 0.3130 49402 1.13 0.3788

Processing Stage×Rep(year) 24 20938 5.14 <.0001 26725 9.29 <.0001 43683 11.04 <.0001

Residual 45 4071.00 - - 2876.80 - - 3956.53 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “MS” indicates “Mean Square”.

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Appendix A.10: Analysis of Variance for Total Phenolic Content of Different Corn Hybrids

Growing in Different Years at Different Processing Stages a,b

Source DF Sum of Squares Mean Square Error DF F value Pr > F

Genotype 2 13905979 6952989 4.00 10.90 0.0241

Processing Stage 4 14967749 3741937 7.89 88.43 <0.0001

G×P 8 1207664 150958 15 4.03 0.0097

Year 2 2392720 1196360 4.29 1.62 0.2989

Rep(Y) 6 2106851 351142 12.00 1.39 0.2964

G×Y 4 2530188 632547 11.56 2.59 0.0921

G×Rep(Y) 12 2996491 249708 46 6.30 <0.0001

P×Y 8 338232 42279 8.09 1.05 0.4716

P×Rep(Y) 24 1018031 42418 46 1.07 0.4102

G×Y× P 15 561682 37445 46 0.94 0.5247

G×P×Rep(Y) 46 1823393 39639 132 6.48 <0.0001

Residual 132 807522 6118 a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

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Appendix A.11: Analysis of Variance for Total Phenolic Content of Different Corn Hybrids Growing in Different Years at Each

Processing Stage a,b

Source DF Whole Kernel Flaking Grits Cooked Grits

MSc F P MS F P MS F P

Genotype 2 626755 6.95 0.0748 3417112 19.99 0.0083 1045685 10.73 0.0247

Year 2 199939 3.14 0.3381 375422 1.30 0.3438 373175 2.98 0.1786

Rep(Y) 6 44064 0.61 0.7191 203489 2.40 0.0932 102737 1.37 0.3030

G×Y 4 90209 1.25 0.3441 170925 2.01 0.1567 97439 1.30 0.3257

G×Rep(Y) 12 72371 5.90 0.0002 84939 12.69 <.0001 75154 19.02 <.0001

Residual 27 12274 - - 6693.92 - - 3951.15 - -

Source DF Baked Grits Toasted Flakes

MS F P MS F P

Genotype 2 1046227 7.58 0.0436 1338822 4.48 0.0953

Year 2 302181 2.84 0.2628 268027 0.80 0.5062

Rep(Y) 6 50038 0.61 0.7177 134726 1.36 0.3072

G×Y 4 138039 1.69 0.2174 298907 3.01 0.0621

G×Rep(Y) 12 81855 19.95 <.0001 99400 23.39 <.0001

Residual 27 4103.82 - - 4249.18 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “Y”= “Year”.

c “MS” indicates “Mean Square”.

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Appendix A.12: Analysis of Variance for Total Phenolic Content of Processed Corn Hybrids Growing in Three Years a,b

Source DF 2009 2010 2011

MSc F P MS F P MS F P

Genotype 2 3392632 4.62 0.0914 2813793 22.77 0.0065 7251508 14.05 0.0155

Processing

Stage 4 5973454 43.26 <.0001 1540750 26.11 0.0001 3468982 25.73 0.0001

G×P 8 558843 1.52 0.2273 120540 2.27 0.0775 246180 2.08 0.1155

Rep 2 734006 1.03 0.4403 309720 2.39 0.2130 753405 1.35 0.3471

G×Rep 4 1469964 7.98 0.0010 123575 2.33 0.1006 1032228 15.26 <.0001

P×Rep 8 276180 0.75 0.6495 59020 1.11 0.4057 269692 1.99 0.1238

G×P×Rep 16 736930 8.21 <.0001 53103 6.13 <.0001 236819 4.29 0.0001

Residual 45 252457 - - 8655.71 - - 165558 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”.

c “MS” indicates “Mean Square”.

Appendix A.13: Analysis of Variance for Total Phenolic Content of three Processed Corn Hybrids Growing in Different Years a

Source DF Hybrid A Hybrid B Hybrid C

MSb F P MS F P MS F P

Processing Stage 4 1246594 75.02 <.0001 837310 20.89 0.0005 2034812 33.47 <.0001

Year 2 2064374 4.97 0.0591 39077 0.25 0.7847 511270 1.79 0.2455

Rep(year) 6 435393 11.95 <.0001 151045 4.75 0.0030 278358 5.27 0.0014

Processing Stage×Year 8 16616 0.46 0.8744 40082 1.26 0.3143 60802 1.15 0.3670

Processing Stage×Rep(year) 24 36431 5.34 <.0001 31804 6.30 <.0001 52808 8.24 <.0001

Residual 45 6821.82 - - 5049.61 - - 6410.14 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “MS” indicates “Mean Square”.

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Appendix A.14: Analysis of Variance for Percentage of Bound Phenolics (BP%) of Different

Corn Hybrids Growing in Different Years at Different Processing Stages a,b

Source DF Sum of Squares Mean Square Error DF F value Pr > F

Genotype 2 120.60 60.30 3.95 2.94 0.1651

Processing Stage 4 3356.53 839.13 7.87 66.28 <0.0001

G×P 8 216.98 27.12 15 1.93 0.1305

Year 2 97.52 48.76 5.54 0.85 0.4761

Rep(Y) 6 311.27 51.88 9.53 3.96 0.0293

G×Y 4 81.82 20.45 10.13 1.27 0.3432

G×Rep(Y) 12 137.05 11.42 46 1.22 0.3004

P×Y 8 101.36 12.67 12.35 0.81 0.6098

P×Rep(Y) 24 264.94 11.04 46 1.18 0.3103

G×Y× P 15 211.30 14.09 46 1.50 0.1444

G×P×Rep(Y) 46 431.48 9.38 132 5.78 <0.0001

Residual 132 214.15 1.62 a Analysis of variance is performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”; “Y”= “Year”.

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Appendix A.15: Analysis of Variance for Percentage of Bound Phenolics of Different Corn Hybrids Grown in Different Years at Each

Processing Stage a,b

Source DF Whole Kernel Flaking Grits Cooked Grits

MSc F P MS F P MS F P

Genotype 2 69.73 1.84 0.3006 39.09 11.26 0.0227 1.44 0.07 0.9305

Year 2 36.02 0.72 0.5356 8.69 2.18 0.3432 4.53 0.15 0.8678

Rep(Y) 6 21.84 2.50 0.0965 5.28 1.11 0.4130 20.44 2.29 0.1045

G×Y 4 37.84 4.33 0.0337 3.47 0.73 0.5901 19.60 2.20 0.1308

G×Rep(Y) 12 8.75 1.74 0.1300 4.77 2.28 0.0365 8.92 28.62 <.0001

Residual 27 5.04 - - 2.09 - - 0.31 - -

Source DF Baked Grits Toasted Flakes

MS F P MS F P

Genotype 2 17.93 2.11 0.2363 20.03 1.50 0.3264

Year 2 1.84 0.13 0.8795 59.33 1.97 0.2752

Rep(Y) 6 13.35 1.66 0.2129 35.19 1.91 0.1593

G×Y 4 8.48 1.06 0.4190 13.35 0.73 0.5906

G×Rep(Y) 12 8.02 15.30 <.0001 0.53 34.91 <.0001

Residual 27 0.52 - - - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “Y”= “Year”.

c “MS” indicates “Mean Square”.

129

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Appendix A.16: Analysis of Variance for Percentage of Bound Phenolics of Processed Corn Hybrids Grown in Three Years a,b

Source DF 2009 2010 2011

MSc F P MS F P MS F P

Genotype 2 3.21 0.26 0.7838 41.29 8.11 0.0392 36.14 2.15 0.2317

Processing Stage 4 281.23 26.38 0.0001 368.88 21.65 0.0002 235.25 43.42 <.0001

G×P 8 15.35 1.37 0.2808 29.39 3.33 0.0194 10.06 1.27 0.3335

Rep 2 101.58 8.56 0.0845 23.68 1.78 0.2869 30.38 2.11 0.2872

G×Rep 4 12.39 1.11 0.3872 5.09 0.58 0.6833 16.77 2.11 0.1333

P×Rep 8 10.66 0.95 0.5036 17.04 1.93 0.1250 5.42 0.68 0.7006

G×P×Rep 16 11.20 4.96 <.0001 8.82 5.16 <.0001 7.94 9.37 <.0001

Residual 45 2.26 - - 1.71 - - 0.85 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “G”=”Genotype”; “P”= “Processing Stage”.

c “MS” indicates “Mean Square”.

Appendix A.17: Analysis of Variance for Percentage of Bound Phenolics of three Processed Corn Hybrids Grown in Different Years a

Source DF Hybrid A Hybrid B Hybrid C

MSb F P MS F P MS F P

Processing Stage 4 320.89 31.04 <.0001 158.35 9.66 0.0056 435.02 30.21 <.0001

Year 2 42.98 1.64 0.2796 3.93 0.14 0.8754 53.72 1.77 0.2405

Rep(year) 6 25.98 2.59 0.0444 23.56 2.24 0.0777 25.25 2.71 0.0376

Processing Stage×Year 8 10.34 1.03 0.4409 16.40 1.56 0.2007 14.40 1.54 0.1948

Processing Stage×Rep(year) 24 10.03 7.30 <.0001 10.53 5.40 <.0001 9.33 5.96 <.0001

Residual 45 1.37 - - 1.95 - - 1.56 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “MS” indicates “Mean Square”.

130

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Appendix A.18: Analysis of Variance for Antioxidant Capacity of Corn Hybrid A Growing in

Different Years at Different Processing Stages a

Source DF Sum of

Squares

Mean

Square Error DF F value Pr > F

ProcessingStage 4 5556.45 1389.11 8 37.58 <0.0001

Year 2 1274.98 637.49 5.12 3.56 0.1073

Rep(year) 6 1137.38 189.56 24 3.98 0.0067

ProcessingStage×Year 8 295.74 36.97 24 0.78 0.6277

ProcessingStage×Rep(year) 24 1143.76 47.66 90 7.42 <0.0001

Residual 90 578.22 6.42 a Analysis of variance is performed using the generalized linear mixed model (GLMM).

131

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Appendix A.19: Analysis of Variance for Antioxidant Capacity of Corn Hybrid A Growing in Different Years at Each Processing

Stage a

Source DF Whole Kernel Flaking Grits Cooked Grits

MSb F P MS F P MS F P

Year 2 216.99 15.62 0.0001 307.91 44.40 <.0001 144.13 39.74 <.0001

Rep(year) 6 490.35 11.77 <.0001 71.09 10.25 <.0001 69.84 19.26 <.0001

Error 18 6.95 - - 6.93 - - 3.63 - -

Source DF Baked Grits Toasted Flakes

MS F P MS F P

Year 2 43.31 5.69 0.0121 181.51 25.90 <.0001

Rep(year) 6 84.95 11.17 <.0001 72.59 10.36 <.0001

Error 18 7.61 - - 7.01 - - a Analysis of variance was performed using the generalized linear model (GLM).

b “MS” indicates “Mean Square”.

Appendix A.20: Analysis of Variance for Antioxidant Capacity of Processed Corn Hybrid A Growing in Three Years a

Source DF 2009 2010 2011

MSb F P MS F P MS F P

Processing Stage 4 501.74 4.62 0.0317 318.41 44.43 <.0001 642.90 23.75 0.0002

Rep 2 79.92 0.74 0.5092 112.43 15.66 0.0017 376.35 13.90 0.0025

Processing Stage× Rep 8 108.72 9.72 <.0001 7.18 1.39 0.2400 27.07 9.23 <.0001

Residual 30 11.18 - - 5.16 - - 2.93 - - a Analysis of variance was performed using the generalized linear mixed model (GLMM).

b “MS” indicates “Mean Square”.