ABSTRACT 
 
 
 
 
Title of Document: PROMOTING THE PRODUCTION AND 
CONSUMPTION OF WHEAT-BASED 
FUNCTIONAL FOODS RICH IN 
ANTIOXIDANTS 
  
 Marla West Luther, Doctor of Philosophy, 2008. 
  
Directed By: Associate Professor Liangli (Lucy) Yu 
Department of Nutrition and Food Science 
 
 
The present study evaluated the effects of solid state yeast treatment and 
thermal processing on the extractability and in-vitro bioavailability of antioxidants of 
wheat bran and whole-wheat pizza crusts, and developed possible sample outreach 
materials for promoting the consumption on whole-wheat functional foods.  The first 
section of this research analyzed the effect of solid-state yeast treatment on the 
extractable antioxidant properties of wheat bran.  Wheat bran was treated with ten 
commercially available food grade yeasts at two doses under solid state conditions. 
Antioxidant properties were evaluated by measuring total phenolic content, and 
radical scavenging capacities against the cation ABTS, peroxyl (ORAC), DPPH 
(RDCS), and hydroxyl (HOSC) radicals.  Results showed that under the selected 
conditions, yeast strain LBE.11 was able to increase ORAC and HOSC radical 
scavenging by 50% and 67%, respectively.   
 
The second part of this study evaluated the impact of bran particle size, 
fermentation time, and baking time and temperature on the 100% ethanol extractable 
antioxidant capacities of whole wheat pizza crust.  Results showed that pizza crusts 
produced with reduced particle size bran maintained antioxidant capacity throughout 
thermal processing.  At 18 hrs of fermentation RDSC and TPC values of the pizza 
crusts were increased by 17% and 23%, respectively.  Results showed that increasing 
baking temperature increased RDSC values by 14 and 17% for pizza crusts prepared 
with Lakin and Trego variety wheat, respectively.  The third part of this study 
analyzed the in-vitro bioavailability of wheat antioxidants in the baked pizza crusts.  
The antioxidant properties were evaluated by measuring total phenolic content, and 
radical scavenging capacities against the cation ABTS, peroxyl (ORAC), dpph 
(RDCS), and hydroxyl (HOSC) radicals.  Results showed that increasing baking 
temperature increased the in-vitro bioavailable TPC by up to 54%.  Pizza crusts with 
higher available antioxidants may have a greater level of bio-available antioxidants.   
In the final segment of this research a tri-fold was developed for promoting 
the consumption of whole wheat foods.  The tri-fold was designed to include food-
specific knowledge and consumption consequences knowledge in addition to recipes, 
directions, and nutritional facts.  Integrating research findings into outreach materials 
may be an effective way to increase functional food consumption.  Results from these 
studies indicate that there are numerous ways to increase antioxidant levels in the 
diet.     
 
 
 
 
 
 
 
 
 
 
 
PROMOTING THE PRODUCTION AND CONSUMPTION OF WHEAT-
BASED FUNCTIONAL FOODS RICH IN ANTIOXIDANTS 
 
 
 
By 
 
 
Marla West Luther 
 
 
 
 
 
Dissertation submitted to the Faculty of the Graduate School of the  
University of Maryland, College Park, in partial fulfillment 
of the requirements for the degree of 
Doctor of Philosophy 
2008 
 
 
 
 
 
 
 
 
 
 
Advisory Committee: 
Professor Liangli (Lucy) Yu, Chair 
Professor Thomas Castonguay 
Professor Mickey Parish 
Professor David Lei 
Dr. Jun-Jie Yin 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Marla W. Luther 
2008 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 ii 
Acknowledgements 
 This achievement is the result of kindness, encouragement, direction, and 
support from peers, instructors, friends, and family.  Thank you to my fellow 
labmates, in particular, Kequan (Kevin) Zhou, John Parry, Jeff Moore, Zhihong 
Cheng, and Margaret Slavin who have assisted me throughout my graduate 
experience at the bench as well as insight into new solutions and ideas.  You not only 
gave of your time and knowledge, but created a friendly environment that I have 
enjoyed working and learning in.  Dr. Liangli (Lucy) Yu, thank you for giving me this 
opportunity and for your guidance and assistance.  I would also like to thank my 
committee members for their assistance and support in completing my graduate work.  
Dr. Thomas Castonguay, Dr. David Lei, Dr. Mickey Parish, and Dr. Jun-Jie Yin.  My 
heart-felt gratitude also goes out to my family for helping me to keep things in 
perspective and laugh often.  I would be remiss not to thank my parents Lonnie and 
Mina Luther by name for their sound guidance, unselfish love, unconditional support, 
and continual prayers.   
 iii 
Table of Contents 
 
Acknowledgements....................................................................................................... ii 
List of Tables ................................................................................................................ v 
List of Figures.............................................................................................................. vi 
Wheat as a Commodity............................................................................................. 1 
Wheat Consumption.............................................................................................. 1 
Wheat Fractions .................................................................................................... 2 
Wheat Flours......................................................................................................... 2 
Health Promoting Properties of Whole Grains ......................................................... 5 
Epidemiological Studies ....................................................................................... 5 
Dietary Fiber ......................................................................................................... 6 
Antioxidant Activity & Energy Balance............................................................... 9 
Government Approved Health Claims................................................................ 11 
Whole Wheat Product Market ............................................................................ 12 
Functional Foods..................................................................................................... 13 
Functional Foods and Nutraceuticals.................................................................. 13 
Functional Food Outlook .................................................................................... 13 
Nutraceutical Components.................................................................................. 15 
Antioxidants............................................................................................................ 17 
Antioxidants and Human Health......................................................................... 17 
Methods of Antioxidant Analysis ....................................................................... 19 
Wheat Antioxidants ................................................................................................ 19 
Antioxidants in Wheat Fractions ........................................................................ 19 
Effect of Storage & Processing on Wheat Antioxidants..................................... 20 
Antioxidants in Wheat Based Food Products ..................................................... 21 
Effect of Solid State Reactions on Antioxidants..................................................... 24 
Solid State Reactions .......................................................................................... 24 
Nutritional Outreach ............................................................................................... 25 
Outreach Development Tools ............................................................................. 25 
U.S. Food Assistance Programs: Education & Nutrition.................................... 27 
Impact of Nutrition Knowledge on Health ......................................................... 33 
Food, Economics & Health................................................................................. 34 
Literature Cited ....................................................................................................... 35 
Chapter 2: Effect of Yeast Strain on the Antioxidant Properties of Wheat Bran 
Following Solid-State Treatments .............................................................................. 48 
Abstract................................................................................................................... 48 
Introduction............................................................................................................. 48 
Materials and Methods............................................................................................ 51 
Results and Discussion ........................................................................................... 57 
Literature Cited ....................................................................................................... 65 
Chapter 3: Effects of Thermal Treatment, Fermentation, and Bran Particle Size on the 
Ethanol Extractable Antioxidant Properties of Whole-Wheat Pizza Crust ................ 68 
Abstract................................................................................................................... 68 
Introduction............................................................................................................. 69 
 iv 
Materials and Methods............................................................................................ 70 
Results and Discussion ........................................................................................... 76 
Conclusion .............................................................................................................. 91 
Literature Cited ....................................................................................................... 92 
Chapter 4: In-vitro Bioavailability of Wheat Antioxidants in Baked Pizza Crust...... 95 
Abstract................................................................................................................... 95 
Introduction............................................................................................................. 96 
Materials and Methods............................................................................................ 98 
Results and Discussion ......................................................................................... 105 
Conclusion ............................................................................................................ 110 
Literature Cited ..................................................................................................... 111 
APPENDIX A. Yeast Log Growth Curve* ...................................................... 113 
Chapter 5:  Development of a Tri-fold to Increase Whole Wheat Pizza Crust 
Consumption in College Students............................................................................. 114 
Abstract................................................................................................................. 114 
Introduction........................................................................................................... 114 
Tri-fold Development ........................................................................................... 117 
Pizza Recipes .................................................................................................... 120 
Health Claims.................................................................................................... 120 
Conclusion ............................................................................................................ 121 
Literature Cited ..................................................................................................... 122 
 
 
 
 
 
 
 
 
 
 
 v 
List of Tables 
 
Table 1.1 New product introductions with a ?whole grain? claim. 
Table 3.1 Effect of Bran Mesh Size on Pizza Crust Antioxidant Properties for Two 
Hard Wheat Varieties 
Table 3.2 Effect of Fermentation Time on Pizza Crust Antioxidant Properties for 
Two Hard Wheat Varieties 
Table 4.1 Fed-state synthetic digestive fluid composition 
 
 vi 
List of Figures 
Figure 1.1 U.S. Per Capita Consumption by Disappearance (USDA 2008). 
Figure 1.2 U.S. Weighted Average Farm Price per Bushel (USDA 2008). 
Figure 1.3 Macronutrient Comparison of King Arthur Commercially Available 
Flours (King Arthur 2008). 
Figure 1.4 Effects of enzyme treatment on antioxidants in wheat bran. 
Figure 2.1 Effect of solid state yeast treatment on TPC  of soft red wheat bran.   
Figure 2.2 ABTS?+ scavenging by solid state yeast treated soft red wheat bran. 
Figure 2.3 Effect of solid state yeast treatment on ORAC of soft red wheat bran.   
Figure 2.4 Effect of solid state yeast treatment on HOSC of soft red wheat bran.  
Figure 2.5 Effect of solid state yeast treatment on the DPPH scavenging capacities of 
soft red wheat bran.   
Figure 3.1 Effect of thermal treatment on the TPC of whole wheat pizza crust.   
Figure 3.2 Effect of thermal treatment on ABTS?+ of whole wheat pizza crust.   
Figure 3.3 Effect of baking time and temperature ORAC capacities of whole wheat 
pizza crusts.   
Figure 3.4 Effect of baking time and temperature on the DPPH scavenging capacities 
of whole wheat pizza crusts.   
Figure 3.5 Effect of Baking Time and Temperature on Soluble Free Ferulic Acid of 
Whole Wheat Pizza Crusts.  
Figure 3.6 Effect of Baking Time and Temperature on Soluble Conjugated Ferulic 
Acid of Whole Wheat Pizza Crusts.  
Figure 3.7 Effect of Baking Time and Temperature on Insoluble Bound Ferulic Acid 
of Whole Wheat Pizza Crusts.  
Figure 4.1 In-vitro Digestion.  
Figure 4.2 In-vitro bioavailable total phenolic contents (TPC) of baked pizza crusts.   
Figure 4.3 In-vitro bioavailable ABTScircle3+ scavenging capacities of baked pizza crust.   
Figure 4.4 In-vitro bioavailable ORAC scavenging capacities of baked pizza crust.   
Figure 4.5 In-vitro bioavailable HOSC scavenging capacity of antioxidants from 
whole wheat pizza crust.   
 vii 
Figure 5.1 Hierarchy of Knowledge, Psychomotor Skill, and Food Related Sensory 
Attributes Leading to Increased Consumption of Functional Foods.   
Figure 5.2 Functional Whole Wheat Pizza Tri-fold ? Side A 
Figure 5.3 Functional Whole Wheat Pizza Tri-fold ? Side B 
 
 
 1 
Chapter 1: Literature Review 
Wheat as a Commodity 
Wheat Consumption 
Wheat (Triticum spp.)  was one of the first crops to be cultivated.  A global 
dietary staple, wheat is an important cereal grain in most temperate zones and has 
been adapted to many types of cuisine.  Wheat comprises one-third of the total grain 
crop world wide (Slavin 2001). U.S. consumption of wheat based products is 117.5 
grams per day, over 70% of the total grain intake (USDA 2007).  Consumption of 
corn, rice and oats followed at 27, 17.9 and 3.05 grams per day, respectively.  Barley 
and rye are each consumed at less than 0.5 grams per day (USDA 2007).  
Multiple factors including diet trends 
and price have impacted wheat 
consumption. One pound of wheat produces 
approximately 0.98 pound of whole-wheat 
flour but only 0.74 pound of refined flour 
(USDA 2006).  Per capita U.S. wheat 
consumption (Figure 1.1) is currently rising 
after experiencing fluctuations peaking in 
1997 at 146.8 lbs. per person and dropping 
to 134.2 lbs. per person in 2005.  
Meanwhile, contrast cost of wheat 
production has increased and the weighted-
130
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Figure 1.2: U.S. Weighted Average 
Farm Price per Bushel (USDA 2008) 
Figure 1.1: U.S. Per Capita Consumption 
by Disappearance (USDA 2008) 
 2 
average farm price (Figure 1.2) has increased steadily, most noticeably increasing 
170% between 2006 and 2008 (USDA 2008).   
 
Wheat Fractions 
Wheat kernels or berries are often milled into three fractions: the germ, 
embryo, and bran.  The wheat endosperm is comprised primarily of starch with some 
protein and accounts for about 82% of the kernel weight (Ardnt & Luther 2008).  The 
endosperm functions as a source of energy used in the germination and early growth 
of a new plant (Fulcher & Duke 2002).  The bran is comprised of several cell layers 
and accounts for about 15% of the kernel weight.  The bran is also primarily 
carbohydrate with a larger fraction (40%) of fiber as well as protein, ash and lipid.  
Germ is comprised of carbohydrate with fiber (13%), protein, the highest level of 
lipid in wheat, and ash.  In addition, several vitamins, minerals, and other 
phytonutrients, are found in the germ. (Ardnt & Luther 2008)   
 
Wheat Flours 
Flours 
Protein content of different flours is generally the most important 
consideration in baking and flour selection.  Flours of variable protein content are 
available for specific baking purposes (Figure 1.3).  Higher protein content is 
preferred when stronger dough is needed, that is, when dough needs to rise and hold a 
structure.  Rising for yeast products occurs when yeast metabolizes sugars and 
produces carbon dioxide.  When enough gluten is produced the dough will stretch and 
 3 
hold the carbon dioxide gases with-in the dough causing it to inflate.  Common pan 
bread needs approximately 11.5% protein in order to rise.  The wheat grain contains 
approximately 12% proteins; however flour producers can use cultivars characterized 
by high gluten yields, and milling and drying techniques to vary the end protein 
content and vital gluten levels in flour (Borght et al. 2005). 
Improved wheat cultivar characterization and milling technologies have 
brought about specialty pastry and pasta flours.  Whole wheat pastry flour is produced 
from the soft varieties with lower protein content than hard varieties and is a better 
selection for flaky pastries.  Whole durum wheat is produced under the same 
requirements as whole wheat flour but use durum and red durum wheat (21 CFR 
Section 137.225).  Durum wheat is generally used for pasta and noodles due to its 
high protein content.  In all flours the percent moisture is altered during production of 
whole wheat flour through a process known as tempering so that the final moisture 
content does not exceed 15% (21 CFR Section 137.200).   
All-Purpose 
1%
81%
3%
1%
14%
Fat
Carbohydrate
Dietary Fiber
Sugars
Protein
Whole Wheat
2%
66%
14%
2%
16%
Cake & Pastry Flour
1%
84%
2%
2%
11%
Figure 1.3: Macronutrient Comparison of King Arthur Commercially 
Available Flours (King Arthur 2008) 
 4 
Different classes of wheat are used to produce commercially available whole 
wheat, white whole wheat, and whole wheat pastry flours.  Five of the six wheat 
classes: Hard Red Wheat, Hard Red Spring, Soft Red Winter, Hard White, and Soft 
Wheat may all be used to produce whole wheat flour (21 CFR. Section 137.200).  
White whole wheat flour is produced from white varieties of wheat that do not have 
the genes to produce the typical brown/red bran color.  Whole wheat bread baked 
with white varieties also score higher among panelists in terms of flavor and mouth-
feel (Taylor et al. 2005).  White and red varieties are considered nutritionally 
equivalent.  The Whole Grains Council states that white varieties have fewer phenolic 
compounds that give the wheat a milder flavor (WGC 2008).  In contrast multiple 
studies indicate that while there are strain, environmental and storage impacts on 
antioxidant activity no studies to date have found a trend to indicate that white or red 
varieties are significantly different (Yu et al. 2002a, Yu & Zhou 2004a, Zhou & Yu 
2004b).   
 
Whole Wheat Flour 
Whole wheat flour, also known as graham flour, is flour produced in 
proportion to the original natural composition of the wheat kernel.  Wheat kernels are 
composed of three fractions: bran, germ, and endosperm.  Refined flour consists of 
only the endosperm with the bran and germ removed.   The requirements for whole 
wheat and refined flours are set by the Food & Drug Administration, Department of 
Health and Human Services in The Code of Federal Regulations, Sections 137.200 
and 136.105 respectively (21 CFR).   
 5 
 These requirements use particle size and ash to standardized flours.  Removal 
of the bran is possible as it is less easily reduced in size and therefore may be sifted 
out.  For whole wheat flour, cleaned wheat is ground so that 90% passes through a 
2.36 mm sieve and 50% will pass through an 850 ?m sieve (FDA 2007).  Ash 
content, the nonvolatile inorganic compounds, generally considered the mineral 
content in wheat, is determined by an AACC (Analytical Association of Cereal 
Chemists) method in which samples are incinerated overnight in a muffler furnace at 
600?C. (FDA, 2007)  Whole wheat flours have larger particles sizes and higher ash 
contents due to the presence of the bran fraction. 
 
Health Promoting Properties of Whole Grains 
Epidemiological Studies 
Epidemiological studies provide support that whole foods, such as those 
prepared with whole grains, have a greater effect against a broad range of diseases 
than isolated components (Slavin 2003).  A meta-analysis of 12 studies demonstrated 
that regular intake of whole grain foods was capable of a 26% reduction in risk for 
coronary heart disease (CHD), possibly because of their ability to decrease serum 
LDL- cholesterol and triglycerides (Anderson et al. 2000).   Women in the Iowa 
Women?s Health Study and Harvard Women?s Health Study with the highest 
consumption of whole grains (approximately three daily serving) experienced 22 & 
27 % reduced risk of type II diabetes, respectively (Jones et al. 2002).   Similarly, 
meta-analysis of 45 well designed studies found whole grains to be protective against 
cancer in 43 of the studies (Slavin 2000).  Interestingly, while at least one report by 
 6 
Arts and Hollman has challenged the role of antioxidants for their protective role 
against disease, no studies to date have reported an adverse effect to the consumption 
of whole grain with the exception of persons with disease complications such as 
celiac disease (Arts & Hollman 2005).   
 
Dietary Fiber 
Dietary fiber is associated with decreased risk of cardiovascular disease, 
diabetes, and intestinal cancer in response to reducing plasma and LDL-cholesterol, 
glycemic and insulin response delay, and increasing stool bulk (Schneeman 1999).  
Epidemiological studies have demonstrated that dietary fibers play a pivotal role in 
modulating food intake, body weight, glucose homeostasis, plasma lipid profile, and 
cardiovascular disease (Koh-Banerjee & Rimm 2003).  Independent association of 
higher fiber intake with lower BMI or reduced food intake has been established 
(Kimm 1995, Pasman & Saria 1997, Burton & Freeman 2000, Cani et al. 2006, 
Melanson et al. 2006).  Fiber may play a unique role in weight management for 
women.  Women who consumed higher fiber diets were strongly correlated with 
lower BMI and that among these women; dietary fat intake had no relationship with 
BMI unless coupled with a high-fiber diet.  Furthermore, different types of fiber and 
fiber in different forms may differ in their effectiveness to modulate weight loss.  
(Howarth et al. 2005) 
Fiber supplementation studies on weight control indicate that fiber types may 
differ in their effectiveness.  Soluble guar gum fiber was found to decrease energy 
intake by free living obese women by 19% over a one week period (Pasman & Saris 
 7 
1997).  In contrast, in one study neither fermentable or non-fermentable fibers had an 
effect on energy intake nor body fatness with an increase of 28 grams fiber per-day 
by fiber supplementation with fiber isolates over a three week period.  In both fiber 
supplementation studies and bread formulas, authors suggest that the use of the fiber 
supplement vs the fiber of intact plants and the cellular structures associated with 
such dietary fiber in foods may have resulted in the unexpected results.  (Holt et al. 
2001; Howarth et al. 2003)  Similarly, a separate study on the effects of dairy 
products and fiber on weight loss in obese adults found that subjects who consumed 
more high-fiber foods was greater than the weight loss of subjects who consumed 
fiber supplements (Thompson et al. 2005).  When at least 34 grams of fiber per day 
were consumed, a 3-6% decrease in energy absorption was observed.  To be effective, 
the fiber may need to be an integral part of food rather than in supplemental form.  
(Howarth et al. 2005) 
Fiber rich foods may require more chewing, take longer to eat, and larger 
portion size of high fiber vs. high fat foods may induce satiety sooner.  Reduced 
palatability and increased chewing effort and time may alter cephalic and gastric 
responses leading to satiety hormones reaching the hypothalamus earlier and reducing 
food intake.  (Burton-Freeman 2000, Holt et al. 2001)  Soluble fibers may elicit their 
strongest effect in bulking properties.  MRI images demonstrate that alginates form 
lumps in the stomach which created a significantly greater sense of fullness when 
compared with a control (Hoad et al. 2004).  While bulking properties are often cited 
as the primary fiber mechanism for satiety, highly insoluble wheat bran based 
breakfast cereal induced greater satiety than a high solubility psyllium gum breakfast 
 8 
cereal (Delargy et al. 1997).  Satiety from rice-based, wheat-based and rice-pulse 
combinations were higher for fermented and whole fraction based preparations than a 
white bread standard (Pai et al. 2005).  Weight loss from fiber-rich whole-grain 
cereals was also shown to maintain or increase the intake of total and insoluble fiber, 
vitamin B-6, and magnesium, indicating that hypocaloric diets may be used as an 
effective means of weight loss without causing incomplete nutrition (Kimm 1995, 
Melanson et al. 2006).  
High fiber foods may also regulate glycemic index and alter satiety hormone 
levels.  Insulin sensitivity has been shown to decrease in whole grain diets compared 
with medium glycemic load compared with high glycemic load refined grain diets 
(Periera & Liu 2003).  In healthy men the insoluble fiber content in high-fiber cereal 
reduced appetite, food intake, and improved glycemic response to meal consumption 
consumed 75 minutes post treatment (Samra & Anderson 2007).  Low glycemic 
index diets have also been correlated with satiety with increased levels of 
cholecystokinin (CCK) independent of the glycemic response (Koh-Banerjee & 
Rimm 2003, Swinburn et al. 2004).   Elevated plasma CCK levels patterns have been 
recorded in both low fat, high fiber diets as well as high fat, low fiber diets.  Proximal 
fermentation of viscous fibers in the distal colon produce short chain fatty acids, an 
effect possibly caused by a delay in fat absorption (Burton-Freeman 2000; Cani et al. 
2006).  Likewise, ghrelin levels are decreased after carob fiber consumption possibly 
related to alterations in lipid metabolism (Gruendel et al 2007).  Similarly ghrelin and 
PYY levels were blunted after wheat fiber yet levels were not blunted for oat-fiber, 
however hunger scores were not significantly different (Weickert et al. 2006).    
 9 
 
Antioxidant Activity & Energy Balance 
Correlation between antioxidant activity and energy balance has recently, 
albeit in limited amount, been studied.  Antioxidants are thought to alter satiety 
effects by; lowering blood glucose response, reducing lipid oxidation, impeding 
glucose uptake by cells, and modulation of digestive enzymes.  Specifically, wheat 
bran, known to contain antioxidants, improves glucose tolerance over pectin 
administration, possibly by regulation of oxidative stress.  Vitamin E plays a role in 
insulin metabolism and the prevention or alleviation of hyperinsulinemia. (Koh-
Banerjee & Rimm, 2003)  Administration of green tea polyphenols in diabetic (db/db) 
mice results in increased levels of yet unidentified pancreatic polypeptides previously 
found to reduce food intake and increase energy expenditure (Tsuneki et al 2004).  In 
contrast, body weight was restored in alloxan-induced diabetic Sprague-Dawley rats 
when treated with feruloyl oligosaccharides.  The in vivo antioxidant restoration 
capacity is cited for mitigating the oxidative damage responsible for the degeneration 
of pancreatic ?-islet cells, exerting hypoglycemic activity by stimulating insulin 
release. (Ou et al 2007)   This is a key finding in the treatment of hyperglycemia and 
the prevention of atherosclerosis in addition to energy balance.   
Polyphenols may increase lipid oxidation and energy expenditure in humans 
and mice.  Lipid oxidation was significantly increased for a liquid meal challenge 
when a high fiber polyphenol rich Carob fiber supplement was given in human 
subjects.  Interestingly, acylated levels of ghrelin, a meal inducing hormone, were 
decreased indicating that carob fiber may influence the regulation of energy intake 
 10 
and body weight. (Koebnick et al. 2006, Gruendel et al. 2006) In contrast, a separate 
study on Wistar rats found that hypothalamic reactive oxygen specie production was 
required to elicit a satiating effect during hypertriglyceridemia.  Food intake during 
hypertriglyceridemia is reduced by GSH-EE, a potent ROS scavenger, and trolox, a 
water soluble vitamin E analog, indicating that ROS-sensitive mechanisms may be 
sufficient to promote satiety.  In metabolic diseases such as obesity and diabetes, 
ROS may be chronically elevated and therefore hypothalamic sensing of fat through 
ROS targets for hypertriglyceridemia-induced satiety may be a potential redox-
regulated system useful for regulating energy homeostasis. (Benani et al. 2007)  
Polyphenolics may reduce energy intake by impeding glucose intake into 
cells.  Green tea polyphenolics such as Epicatechin gallate inhibit the transportation 
of glucose by binding to the glucose transporter in an antagonist like manner 
(Kobayashi et al. 2000).  Similarly, isolated forms of (-)-epigallochatechingallate, (-)-
epichatechingallate, and epigallochatechin were found to be effective against both 
sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) 
in Caco-2 cells (Johnston et al. 2005).  A separate study utilizing New Zealand black 
mice found that epigallocatechin gallate (EGCG) attenuated diet-induced obesity.  
While the mechanism was not clear, it was hypothesized that higher fecal energy 
levels in EGCG treated mice indicates reduced diet digestibility, while there may 
have also been a metabolic effect on the liver and white fat.  (Klaus et al. 2005) 
Glucose transporters 1 and 4 may be inhibited by genistein, quercetin, myricetin, 
morin, rhamnetin, and isorhamnetin, and flavonoids, including catechin, respectively.  
Thus, at concentrations of 10-100?M quercetin, myricetin, and catechin-gallate dose 
 11 
dependently inhibited the uptake of methyl-glucose in rat adipocytes. (Strobel et al. 
2005)   
Intestinal activity of polyphenols against starch degradation may also reduce 
energy intake.  Plant polyphenolic extracts have demonstrated comparable 
effectiveness against ?-glucosidase and maltase activity as acarbose and voglibose, 
two synthetic inhibitors used to control non-insulin dependent diabetes mellitus.  
While in vitro activity is limited due to bioavailability, diacylated anthocyanins are 
particularly effective against ?-glucosidase, most likely due to competitive binding 
although the structure-activity relationship has not been fully explained.  The binding 
of digestive enzymes, particularly ?-amylase by polyphenolic-rich plant extracts may 
explain in part the insulin-like action of polyphenolics. (McDougall & Stewart 2005)  
Thus, polyphenolics through a variety of mechanism are potential natural alternative 
to assist in energy balance through diet-mediated means.      
 
Government Approved Health Claims 
The nutritional value of wheat and other whole grains has lead to multiple 
whole grain food intake recommendations.  The 2005 Dietary Guidelines for 
Americans report recommends increased consumption of whole grains as part of a 
health-promoting diet, stating a diet rich in whole grain foods may reduce the risk of 
coronary heart disease, type 2 diabetes and certain cancers and help with weight 
management (Marquart et al. 2003).  It recommends that half of daily grain intake 
should be from whole grains, which equates to three ounce-equivalent servings daily 
for a 2,000 calorie diet (USDA 2005).  Increased consumption of whole-grain foods 
 12 
is being campaigned for in the UK with ?Whole Grains for Health? and in Australia 
with ?Go Grains?.  Unfortunately, whole grain consumption remains low with less 
than 10% of American adults meeting the recommended intake (Cleveland et al. 
2000).  Whole grain food consumption may increase with consumer education and 
allowed health claims such as the FDA approved the health claim for whole-grain 
products containing at least 51% whole grain by product weight and meeting other 
criteria in the U.S (FDA 1999).   
 
Whole Wheat Product Market 
The whole-grain market in the United States is on the rise with inclusion of 
whole wheat in products such as crackers and breads (Carlson et al. 2005).  In 
addition to the FDA approved health claim, merchants creating whole-grain foods 
may also signify their product with the whole grain stamp.  The stamp was launched 
by the Whole Grains Council, a group of millers, manufacturers, scientists, and chefs 
have advocated for research on whole grains as well as their use and identification in 
the marketplace.  Additional sources report a 168% increase from 2004 to 2005 in 
frozen whole grain prepared foods, followed by whole grain pasta, whole grain 
cereal, and whole grain bread and baked foods. (The Whole Grain Council 2007)     A 
study of British adults found current whole grain consumption levels similar to those 
in the United States.  Of the small percentage of whole grains consumed 40% came 
from breads, and about one-third from whole grain breakfast cereals (Lang et al. 
2002).  The remaining portion came from whole grain pasta, rice, and other cereals.  
 13 
Development of more desirable whole wheat products and recipes may increase 
consumer attitudes toward these products.   
 
 
Functional Foods 
Functional Foods and Nutraceuticals 
Functional foods are defined by the International Food Information Council 
(IFIC 2007) as, ?foods or dietary components that may provide a health benefit 
beyond basic nutrition.?  Nutraceuticals is a term often used synonymously with 
functional foods or as a broader term that incorporates functional foods as well as the 
functional ingredient in a supplemental form.  In Japan functional foods have been 
regulated for over 15 years as Foods for Specialized Health Use (FOSHU) (Warfel et 
al. 2007).  Regardless of the title, functional foods are generally defined as foods 
which contain biologically active components (often termed bioactive compounds) 
which may exert beneficial physiological health effects and reduce the risk of chronic 
disease.  As research to identify beneficial health components of foods increases, 
many traditional foods may be labeled or modified to become functional foods.   
 
Functional Food Outlook 
Functional foods and beverages have a bright outlook as the largest single 
domestic market in the US with an estimated $29 billion in 2007 sales according to 
Global Industry Analysts (Douaud 2007).  The market in Japan, the only nation to 
legally define functional foods, has grown from $1 billion in 1997 to $6 billion in 
 14 
2006 (Warfel et al. 2007).  Canada has shown a similar trend with health promoting 
products comprising six out of seven food and beverage product categories with the 
greatest growth in sales rates (Cranfield et al. 2006).  Thus, the market demand has 
created new opportunities in the agricultural and food sectors and encouraged new 
innovations, while potentially promoting the well-being of its consumer base.  
According to Mintel Global New Products Database (Mintel 2007), launches 
of new food products with a "whole grain" claim have grown considerably since 
2000.  Launches of new whole grain products increased over 900% from 2000 to 
2006 reaching nearly 1,400 new products (Mintel 2007).  Bakery products, breakfast 
cereals, snacks, and side dishes accounted for most new products (Table 1.1).  
Despite increased availability of foods made with whole grains and strong 
recommendations to increase whole grain consumption, Americans currently 
consume an average of one daily serving of whole grains, which is one-third of the 
recommended intake.  
 15 
Table 1.1 New product introductions with a ?whole grain? claim. 
Category 2000 2001 2002 2003 2004 2005 2006 
Baby Food 3 3 6 1 8 0 20 
Bakery 80 109 149 205 309 291 570 
Beverages 0 0 0 1 2 2 3 
Breakfast 
Cereals 
31 80 68 98 138 192 347 
Confectionery 3 2 1 1 2 1 0 
Dairy 3 4 4 5 3 3 10 
Dessert & Ice 
Cream 
0 0 0 0 0 2 2 
Fruit & 
Vegetables 
0 0 0 0 2 0 1 
Meals & 
Meal Centers 
7 3 12 9 25 20 65 
Pet Food 1 1 0 0 6 7 14 
Processed 
Fish, Meat, 
Egg 
7 1 0 2 8 2 2 
Sauces & 
Seasonings 
0 1 1 1 1 6 9 
Side Dishes 16 18 45 33 44 93 117 
Snacks 3 22 15 30 58 83 227 
Soup 0 0 1 0 3 1 0 
Spreads 0 0 0 1 0 1 0 
Total 154 244 302 386 609 704 1387 
* Table taken from Arndt and Luther 2008 
Less than 10% of adult Americans meet the recommended whole grain consumption 
level (Cleveland et al. 2000).  The 2010 goal of the ?Healthy People Goals and 
Objectives for the Nation? by the U.S. Department of Health and Human Services is 
that at least 50% of Americans would consume three whole-grain servings daily.   
 
Nutraceutical Components 
Compounds found in food products, such as carotenoids, phenolics, fatty 
acids, dietary fiber, plant sterols, and pre-/pro-biotics, support health.  Bioactive 
 16 
compounds, such as carotenoids and phenolics, protect health through antioxidant 
activity against highly reactive free radicals and oxygen species.  Protection of body 
proteins, lipids, and DNA can play a vital role in preventing degenerative diseases 
such as heart disease, cancer, cataracts, brain dysfunction, and arthritis (Miller et al. 
2000).  Essential fatty acids (EFA) are fatty acids which are required from the diet 
and play an important role in growth, skin health and neurological function (IFIC 
2007).  There is a lot of potential for new reduced cost EFA functional foods 
containing fatty acids such as omega-3 long chain polyunsaturated fatty acids, as 
EFA?s are found in high-value foods such as fish and walnuts.  Such bioactive 
compounds play a key role in the production and protection of body cells and their 
components.   
Plant sterols and pre/pro-biotics play a major role in the gastrointestinal tract.  
Plant sterols are thought to lower serum cholesterol as their structure closely 
resembles cholesterol inhibiting its absorption while not being readily absorbed 
themselves (Jenkins et al. 1999).  A final example is the use of pre- and pro-biotics 
for healthy gut function.  Probiotics are viable microbial strains that beneficially 
affect the host, and prebiotics are non-digestible food components that selectively 
stimulate the growth and activity of selective bacteria.  Intake of pre- and pro-biotics 
may boost the immune system, reduce rotavirus-induced diarrhea, alleviate symptoms 
of lactose intolerance, and reduce the risk of colon cancer.  These examples are a few 
of what is currently known about the effects of different types of bioactive 
components and their effect on human health. 
 17 
Antioxidants 
Antioxidants and Human Health 
Correlation between oxidative stress and reactive oxygen species (ROS) or 
reactive nitrogen species (RNS) with chronic aging or disease states has increased 
interest in the role of dietary antioxidants to prevent these conditions and promote 
health.  Many definitions for antioxidants can be found.  The Halliwell and 
Gutteridge broad definition for antioxidants is ?any substance that when presented at 
low concentrations, compared to those of an oxidizable substrate, significantly delays, 
or inhibits, oxidation of that substrate? and has remained a prominent definition used 
in the sciences (Gutteridge & Halliwell 1999).  Of growing familiarity is the Institute 
of Medicine proposed definition, ?A dietary antioxidant is a substance that 
significantly decreases the adverse effects of reactive oxygen species, reactive 
nitrogen species, or both on normal physiological function in humans? (Food and 
Nutrition Board 1998).  Antioxidant research is important in the field of food science 
for both their potential to stabilize food systems and their potential health benefits. 
Perhaps the strongest evidence for the role of antioxidants in human health are 
the epidemiological studies indicating that a diet rich in foods containing antioxidants 
such as fruits, vegetables, and other plant derived foods decreases the risk of certain 
chronic diseases (Wattanapitayakul & Bauer 2001, Stanner et al. 2003, Wilcox et al. 
2008).  A comprehensive review of the intervention trials containing more than 1,000 
subjects studying the correlation between antioxidants and cardiovascular disease 
concluded that, ?people with a high occurrence of CVD often have low intakes or 
plasma levels of antioxidant nutrient? (Stanner et al. 2003).  The Nurses? Health 
 18 
Study and the Health Professionals? Follow-up Study found a 4% reduction in CHD 
for each 1-serving/day increase in the intake of fruits and vegetables when comparing 
the highest (?8.0 servings/day) and lowest (?3.0 servings/day) quintiles (Kris-
Etherton et al. 2002).  Supplementation with single or combinations of antioxidant 
have not been found to prevent CVD (Kris-Etherton et al. 2002).   
Vitamin E intervention studies are divided with the Nurse?s Health Study 
supplementing for ?2 years and at specific doses reducing the relative risk to 0.66, 
while the more recent Physicians Health Study found no significant impact of 
Vitamin E and CVD relative risk, and a MEDLINE meta-analysis does not support 
the routine use of vitamin E for the prevention of CVD (Rimm et al. 1996, 
Vivekananthan et al. 2003, Wilcox et al. 2008).  The difference between 
epidemiological study and intervention study observations may be due to the 
differences in the populations studied, or that the follow-up time required to identify 
clinical benefits of Vitamin E supplementation may be decades long, as antioxidants 
are thought to be most effective at combating ROS-induced atherosclerotic lesion 
formation (Wattanapitayakul et al. 2001, Steinhubl 2008).  Vitamin E serves as one 
example of the multiple bioactive components and intake conditions that may affect  
their effect on health that have been studied (Fang et al. 2002, Kris-Etherton et al. 
2002, Padayatty et al. 2003, Polidori 2003, Frei 2004).  Due to the expensive nature 
of intervention studies, the quest to understand the mechanism of action of 
antioxidants have lead to in vitro and in vivo model development to quantify foods 
with antioxidant activity (Nandakumar et al. 2008). 
 19 
Methods of Antioxidant Analysis 
Oxidative defense mechanisms, protection from free radicals, is a primary 
mechanisms investigated to combat aging and age-related diseases (Wilcox et al. 
2008).  Free radicals are molecules with an unpaired electron in the outer orbital 
shells, creating generally unstable and highly reactive conditions (Fang et al. 2002).  
Therefore, it is possible to measure antioxidant activity of foods, and food 
components by exposing them to reactive oxygen species.  Mechanisms by which 
antioxidants are postulated to scavenge these free radicals include hydrogen atom 
transfer (HAT), single electron transfer (ET or SET), and sequential proton loss 
electron transfer (SPLET) (Moore & Yu 2008).  Detailed descriptions of these radical 
scavenging capacity assays are available in Shahidi & Hos? book Antioxidant 
Measurement and Applications and more specifically Moore & Yu?s chapter  
Methods for Antioxidant Capacity Estimation of Wheat and Wheat-Based Food 
Products, in Wheat Antioxidants (Shadihi & Ho 2005, Moore & Yu 2008).  
 
Wheat Antioxidants 
Antioxidants in Wheat Fractions 
Phytonutrients including phenolic acids, flavonoids, coumarins, polyphenols, 
phytates, terpenes, carotenoids, tocopherols, and tocotrienols are all antioxidant 
compounds found in wheat (Shahidi 2004).  These antioxidant compounds may act to 
improve health by scavenging free radicals, chelating transition metals, quenching 
singlet-oxygen molecules, acting as a reducing agent, and defense enzyme system 
activators for biological systems (Moore et al. 2005, Leenhart et al. 2006).   
 20 
Antioxidants are concentrated in the outer parts of the kernel, particularly the 
endosperm aleurone layer, pericarp, nucellar envelope and germ. Some bioactives 
such as zeaxanthin have been reported at higher levels in the germ fraction while 
some components such as carotenoids and lutein are distributed throughout the whole 
grain (Leenhardt et al. 2006).  When compared with the endosperm fraction, 
combined bran and germ fractions contain 15-18 times more total phenolics, 52-70 
times more ferulic acid, and on average 4 times more lutein, 12 times more 
zeaxanthin, and 2 times more cryptoxanthin (Zhou et al. 2004b, Adom et al. 2005, 
Moore et al. 2005).  The total antioxidant capacity (TAC) as measured by ?mole 
Trolox equivalents/100 g wheat portion, found that wheat germ had the highest TAC 
value with an average of 8,400 followed by wheat bran and refined wheat flour 
fractions at 3,500 and 1,450 ?mole Trolox equivalents/100 g, respectively (Arndt & 
Luther 2008).   
 
Effect of Storage & Processing on Wheat Antioxidants 
A multitude of factors may impact the final antioxidant activity in wheat 
samples and final wheat based foods.  Environmental factors including temperature, 
solar radiation and growing location have been shown to significantly impact 
antioxidant properties (Yu et al. 2003, Zhou et al. 2004a, Zhou et al. 2004c).  
Genotype has also been found to significantly impact antioxidant levels in some 
wheat varieties.  Chinese black-grained wheat has higher antioxidant activity than 
white and blue varieties (Li et al. 2007).  A study of Maryland-grown soft wheat also 
found significant differences between varieties suggesting that it may be possible to 
 21 
produce soft wheat varieties with higher antioxidant activity through cultivation 
(Moore et al. 2005).   
 Post harvest activities may also significantly impact final antioxidant activity 
in wheat foods.  Cheng et al. reported on the effects of mesh size on the availability of 
wheat antioxidants indicated that storing whole grains as opposed to smaller fractions 
is preferable for maintaining natural antioxidants (Cheng et al. 2006).  To date, 
multiple studies confirm that wheat antioxidants are asymmetrically distributed 
throughout the wheat kernel (Zhou et al. 2004a, Gallardo et al. 2006, Liyana-
Pathirana & Shahidi 2006, Liyana-Pathirana & Shahidi 2007).   A significant portion 
of wheat?s antioxidants are located in the bran fraction (Zhou et al. 2004b, Liyana-
Pathirana & Shahidi 2007) Zhou and others studied milling size and concluded 
microionization of the aleurone fraction improved the availability of the wheat 
antioxidants (Cheng et al. 2006).     
 
Antioxidants in Wheat Based Food Products 
Losses of vitamins and antioxidants have been reported in breadmaking and 
production of other wheat based foods.  A study on French and wheat breadmaking 
found that there were significant losses in tocopherols and tocotrienols during the 
dough formation and baking stages (Wennermark et al. 1992).  Oxygenation during 
dough production and heat destruction during baking are speculated causes for 30% 
decreases in Vitamin E content during bread preparation (Leenhart et al. 2006).  
Carotenoid losses in the kneading stage varied by species and were directly correlated 
with lipoxygenase activity (Leenhardt et al. 2006) . 
 22 
In bread wheat, the carotenoid content decreased 66% in whole and 40% in 
refined grain dough after kneading.  By comparison, there was less than 10% loss in 
carotenoids in whole grain einkorn dough after kneading.  Einkorn has a much higher 
carotenoid content and lower lipoxygenase activity compared to bread wheat.  
Carotenoid losses during fermentation and baking were about 10% and 36-45% 
respectively and did not differ appreciably between wheat species. (Leenhardt et al. 
2006)  These losses are higher carotenoid losses of approximately 20% for bagels and 
cakes, 30% for cookies, and total carotene losses of 4-15% in bread products and 18-
23% in crackers in other baking studies (Ranhotra et al. 1995, Park et al. 1997).   
Fermentation processes, such as those used in sourdough bread production, 
may impact antioxidant levels and bioavailability.  Katina and others found that 
extracts of flour that have undergone sourdough fermentation have increased folate 
and total phenolic content compared with unfermented flour (Katina et al. 2005).   
Alternatively, alkylresorcinols (ARs) were diminished in sourdough wheat bread as 
opposed to wheat flour ; however extraction of ARs may not have been complete due 
to AR-starch complexed within the food matrix (Ross et al. 2004).  In a whole wheat 
bread system, sourdough fermentation resulted in greater reduction in phytate (62%) 
compared to yeast fermentation (38%), while increasing the solubility of magnesium 
and phosphorus (Lopez et al. 2001).  Thus, phytonutrient composition of dough?s 
may vary at different stages of bread formation and in the final product.   
Baking conditions may also alter antioxidant content.  Increasing baking time 
and temperature increases antioxidant activity in the bread crust, and lowers 
antioxidant activity in the bread crumb portion (Lindenmeier et al. 2004).  Toasting 
 23 
bread has also been found to increase antioxidant content 134-367% in bagels, French 
bread, white bread and whole wheat bread compared to their un-toasted counterparts 
(Halvorsen et al. 2006).  Maillard reaction products have received significant 
attention as they may produce potential carcinogens; however, study of bakery 
product fractions also indicate that areas subject to the browning reaction have higher 
antioxidative potential in contrast to their corresponding inner counterparts 
(Lindenmeier et al. 2004). 
The most important factor in determining the antioxidant capacity of wheat 
products is the use of whole wheat rather than refined wheat flour.  Halvorsen and 
others found that the antioxidant content of whole wheat bread is more than 1.9 times 
higher compared to refined wheat bread (Halvorsen et al. 2006).  Unfortunately, 
straight bran supplementation was found to weaken the dough structure resulting in 
smaller bread loaves with poorer crumb quality (Salmenkallio-Marttila, et al. 2001).  
Miller et al. found the same trend with higher antioxidant content in whole grain 
cereals and breads compared to their refined grain equivalents (Miller et al. 2000).  
Similarly, the average antioxidant content of ready to eat (RTE) breakfast cereals was 
found to be higher than selected fruits and vegetables with the exception of berries, 
and whole grain cereal containing raisins was only slightly lower than berries.  
Antioxidant activity measured in ?moles Trolox equivalents/100 g dry cereal ranged 
from 1,300 to 3,900 among 17 different refined, whole grain and bran-based RTE 
cereals, with bran-based RTE cereals higher that of whole wheat cereals. (Miller et al. 
2000).  Baublis and others studied the antioxidant effects of wheat bran-, whole grain 
wheat- and refined wheat-based RTE cereals using aqueous extracts prepared from 
 24 
crushed cereal samples.  Both whole wheat and wheat bran-based RTE breakfast 
cereals were shown to inhibit lipid oxidation promoted by iron and peroxyl radicals 
while the refined wheat RTE cereal aqueous extract showed little antioxidant 
capacity.  (Baublis et al. 2000) 
 
Effect of Solid State Reactions on Antioxidants 
Solid State Reactions 
Solid state fermentation utilizes food grades bacteria or yeast at a low 
moisture content to release or produce beneficial components.  Phenolic acids, major 
contributors to the antioxidant properties in wheat, principally exist insolubly bound 
to cell wall material in the bran fraction.  Phenolic acids in the bound form are not 
readily bioavailable, with only 3% of ingested bran phenolics ingested over a 24 hour 
period in humans. (Moore et al. 2007)  Multiple studies evaluating wheat variety, 
growing conditions, post harvest treatment, particle size, temperature, enzyme 
treatment, and yeast treatment demonstrate a capacity to affect antioxidant activity 
and potential bioavailability of antioxidants (Zhou et al. 2004a, Yu & Zhou 2004,  
Moore et al. 2006).  As shown in Figure 1.4, enzyme treatment of wheat bran was 
able to convert vanillic acid, syringic acid, and up to 50% of the existing ferulic acid, 
the primary phenolic acid in wheat, from insoluble bound to the soluble bound form 
(Moore et al. 2006b).  Barley and wheat malt treated with brewers yeast also 
demonstrated a release of ferulic acid during fermentation through enzymatic activity 
(Coghe et al. 2004).   
 25 
Fig. 1.4 Effects of enzyme treatments (a) Vicozyme L and (b) Ultraflow L on the 
phenolic composition of Akron Wheat bran.  Results are expressed as micrograms of 
individual phenolic acids per gram of wheat bran on a dry weight basis.  The mean 
values of duplicate tests are reported.  Vertical bars represent the standard deviation 
and values marked by the same letter are not significantly different (P < 0.05).  
(Moore et al 2006) 
 
 
Nutritional Outreach 
Outreach Development Tools 
Effective development, distribution, and analysis of nutritional outreach programs is 
a complex project requiring the knowledge and skills from nutritional scientists, dieticians, 
public health specialists, informatics specialists, and educators among others.  Therefore, this 
segment will not attempt to cover all of the considerations for these fields, but rather to give a 
complete overview of the essential components used in the development of a specific food 
science, nutraceutical based outreach program.  These essential components are survey 
methodology, message framing, literacy and language, and technology.   
 
 26 
Outreach Materials 
The science of communication has recently been tapped to assist in using 
technology for nutrition outreach.  Tailored messages that speak personally to 
targeted audiences have a significantly greater chance of making an impact on health 
(Wilson 2007).  Unfortunately, funding for public health campaigns pales in 
comparison with food industry advertising dollars and therefore consumers have more 
exposure to nutritionally dubious food products than health information (Johnson-
Taylor et al. 2007).  New technologies such as telenutrition, the use of interactive 
compressed video technology to deliver medical nutrition therapy, may also prove 
fruitful.  Telenutrition allows urban dieticians to counsel patients? long distance.  
Thereby, specialists can reach patients in remote areas, eliminating travel, and extra 
financial and time expenditure.   (McCabe et al. 2001)  
 Language and culture are also key factors important for effective nutrition 
outreach.  Hispanics are the single largest ethnic group in the United States and 
Hispanic children are three times more likely to be overweight when compared with 
white companions (Mennella et al. 2006).  It is well accepted that many factors 
influence food selection.  Significant differences in early flavor and food experiences 
exist between Hispanic children when compared to non-Hispanic children.  Dietician 
awareness may assist in the development of evidence based strategies to improve 
Hispanic children?s eating habits and lower their risk of becoming overweight and 
developing other chronic diseases.  (Mennella et al. 2006)  In addition, review of 
Spanish-language versions of the U.S. household food security survey module found 
a ?lack of semantic equivalence across languages; lack of conceptual equivalence 
 27 
across cultures; and lack of normative equivalence across societies.? Thus, direct 
translation of materials may not be as effective as materials tailored for culture as 
well as language (Harrison et al. 2003).     
U.S. Food Assistance Programs: Education & Nutrition 
Domestic food assistance programs are essential for promoting health and 
fighting hunger in the United States.  In the 2008 Farm Bill, the U.S. Congress 
allocated an additional $10.361 billion for nutrition programs to combat rising food 
prices and growing food insecurity (House Committee on Agriculture 2008).  The 
bulk of this funding is distributed through three programs: the Supplemental Nutrition 
Assistance Program (SNAP) formerly the Food Stamp Program, The Emergency 
Food Assistance Program (TEFAP), and the Fresh Fruit and Vegetable Program 
under the Richard B. Russell National School Lunch Act (Rosenbaum 2008).  These 
programs and their subsidiaries are challenged with the twofold task of feeding 
America?s hungry while sustaining or improving nutrition, specifically preventing 
further increases in obesity.  For the purpose of this paper, a focused review of 
literatures on the SNAP program, and their evidence for cooking activity inclusion 
follows.   
 The Supplemental Nutrition Assistance Program (SNAP) is the largest of the 
federal food assistance and nutrition programs.  This program serves the nations 
poorest who have a gross and net income below 130% and 100% of the poverty line, 
respectively (Landers 2007, Bergeson 2008).  Advantages of SNAP are; eligibility is 
need based and all individuals meeting the financial guidelines can receive benefits, 
the thrifty meal plan outlines inexpensive, nutritionally adequate diets, and many 
 28 
participants are working while receiving food stamps as the stamps are intended to 
supplement food purchases.  Food stamps are intended to allow participants to obtain 
a low-cost nutritionally adequate diet.   
The idea of restricting food purchases with food stamps has been debated 
since 1950 when the House version of the program bill would have prohibited the 
purchase of soft drinks and so-called luxury foods (Landers 2007).  To date, with the 
exception of alcohol and imported foods, no restrictions exist.  Of great concern is 
also what participants are not purchasing.  SNAP participants were less likely than 
income-eligible nonparticipants to purchase fruits and vegetables, albeit most 
Americans fail to meet the recommendations for these foods (Guthrie et al. 2007).  
 The most compelling evidence for a change in the SNAP program is the 
health status of participants.  Most studies indicate that SNAP participants have a 
higher risk of being overweight and obese as compared with low-income 
counterparts, particularly for females (Gibson 2003, Gibson 2006, Webb et al. 2008).  
Recently, when compared to the national average, both male and female SNAP 
participants have achieved equivalent weight status to the general population, mostly 
due to increase in obesity and overweight of nonparticipants (Guthrie et al. 2007).  
With 26 million low-income Americans participating in SNAP, policy and education 
changes to the program could yield significant benefits in reduced medical costs, lost 
wages, and increased productivity.  
Shortcomings of SNAP to improve the nutritional status lead to the official 
food stamp nutrition education amendment of 1981.  This amendment includes, ?[to] 
encourage the purchase of nutritious foods, the Secretary is authorized to extend food 
 29 
and nutrition education to reach food stamp program participants, using methods and 
techniques developed in the expanded food and nutrition and other programs 
(Landers 2007).?  The Expanded Food and Nutrition Education Program (EFNEP) is 
an education program under the USDA.  The mission of EFNEP is to: ?assist limited-
resource audiences in acquiring the knowledge, skills, attitudes, and changed 
behavior necessary for nutritionally sound diets, and to contribute to their personal 
development and the improvement of the total family diet and nutritional well-being 
(USDA-CREES 2008).? In 2002, the Food Stamp Nutrition Education (FSNE) 
National Report was published through the Cooperative Extension/Land-Grant 
University System.  The development team identified four areas that would lead to 
increased access to nutrition education and nutritious foods (Little & Newman 2002).  
This report outlines the four focus areas for food stamp participants: quality/physical 
activity, food security, shopping behavior/food resource management, and food safety 
(Guthrie et al. 2006).   
 The USDA Thrifty Food Plan (TFP) not only provides a basis for Food Stamp 
allotments, but an economic food plan for good shopping behavior & food resource 
management.  The TFP has 15 ?market baskets?, essentially shopping lists which 
meet the Dietary Guidelines for Americans and cost no more than the Food Stamp 
allotments.  These market baskets are largely based upon raw food items which lower 
costs; however they increase food preparation time, kitchen equipment, and cooking 
knowledge.  A limited study of SNAP participants found some participants lacked 
basic cooking skills and were unfamiliar with cooking from a recipe (Rose 2007).  
Americans, particularly young adults generally have low levels of food preparation 
 30 
knowledge and are therefore unable to purchase and prepare foods for healthy meals 
(Michaud et al. 2007).  SNAP participants, and others with limited income may be 
less likely to try new recipes or make suggested dietary changes as these changes 
would risk spending valuable resources on foods that their families may dislike, not 
eat, or recipes that may not turn out (Michaud et al. 2007).  Hands on cooking 
activities have successfully increased cooking skills, confidence, and eating habits of 
preschool children and their families, college students, and WIC participants (Bensley 
et al. 2006, Condrasky et al. 2006, Dougherty & Silver 2007, Michaud et al. 2007).   
 Confounding the change from a provisional based program to an education 
program is the development of a process for validating outcome measures relevant to 
the target areas (Guthrie et al. 2006, Taylor-Powell 2006, Townsend 2006, Dollahite 
et al. 2008).  Program evaluation, necessary under the Government Performance and 
Results Act of 1993, and emphasized in a 2004 U.S. Government Accounting Office 
report, while complex, may help pull the nutrition community together and highlight 
promising and proven programs (Guthrie et al. 2006, Taylor-Powell 2006).  While 
education remains optional for both States and SNAP participants, similar nutrition 
outreach programs such as EFNEP with strong emphasis in education have recorded 
both health and expense benefits.   
  
 Cooking Ability & Health 
Increasing cooking knowledge, skills, and behavior may help improve healthy 
eating behaviors (Marquis et al. 2001, Levy & Auld 2004, Michaud et al. 2007).   
Estimates from the 1990?s predicted that by the year 2000 the majority of U.S. adults 
 31 
would have become dependent upon pre-prepared microwavable meals as to no 
longer be able to cook a meal for themselves (Beck 2007).  The debate is further 
complicated by the use of commercial foods at different stages of preparation.  For 
example, only 22% of weeknight meals prepared by middle-class parents in Los 
Angeles used little to no commercial foods; however, 53% demonstrated some 
independent cooking ability to prepare a meal not dominated by commercial foods 
(Beck 2007).  The debate over the decline of cooking skills, cooking interventions, 
and commodity kitchens all give insight into the relationship of kitchen habits and 
health today.   
Michaud et al. provide a thorough explanation of cooking skills and behaviors 
as a combination of mechanical cooking skills, perceptual skills, planning skills, and 
academic knowledge of food safety, nutrition, and culinary arts (Michaud et al.  
2007). No studies have been reported that encompass all of theses elements; however 
cooking skills and academic knowledge of nutrition and food safety have been 
researched.  Cooking confidence and ability have been identified as key factors in 
obtaining healthy foods (Condrasky et al. 2006, Michaud et al. 2007).  Young adults 
that purchased their own food and prepared meals at home had improved diet quality 
over there counterparts who did so less frequently (Larson et al 2006a, Larson et al 
2006b).  Among college students, 14% and 15% of male and female undergraduate 
students respectively self reported having little or no cooking experience and 23% 
and 18% of male and female 18-23 years old students, respectively, self reported 
having inadequate cooking skills (Hertzler & Bruce 2002, Larson et al. 2006a).   
 32 
Pilot studies of cooking classes to improve the use of commodity foods for 
low-income families found that participants increased use of commodities and had a 
more positive attitude towards eating correctly (Auld & Fulton 1995, McLaughlin et 
al. 2003, Stead et al. 2004, Larson et al. 2006a, Larson et al. 2006b, Wrieden et al. 
2007).  Collective kitchens are a second approach to nutrition and food safety 
education that may also empower participants (Marquis et al. 2001, DeHaven et al. 
2004, Engler-Stringer & Berenbaum 2005, Engler-Stringer & Berenbaum 2006, 
Wrieden et al. 2007).  In addition to providing food to food insecure populations, 
collective kitchens have provided valuable insights into cooking-related needs such as 
cookware, literacy levels, language, perceived and actual cost barrier, and 
transportation among others (Kempson et al. 2003).  Collective kitchens programs 
exist in different models; however, even non-funded health programs in faith based 
organizations have been shown to improve health and be more effective at reaching 
some at-risk populations (DeHaven et al. 2004). 
Exploratory trials of collective kitchens emphasizing community-based food 
skills indicate a small but positive effect on food choice and confidence in food 
preparation.  All such studies indicate the need for flexibility of such studies for 
reasons such as: poverty, food access, taste preference, literacy rates, and anxiety.  
Popular topics among participants in the UK were sauces, budget cooking, and soup.  
Younger respondents were more likely to be interested in less traditional dishes, yet 
overall vegetarian cooking along with vegetables, healthy cooking, and fish were the 
least popular topics. (Stead et al. 2004)  Cooking intervention with adults in socially 
deprived areas of Scotland lead to a 12% decrease in convenience foods, an 18% 
 33 
increase in ability to follow a recipe, an 8% increase in cooking from basic 
ingredients, a 27% increase in cooking lentil soup, and a 22% increase in cooking 
white sauce confidence in a 6-month follow-up compared with controls (Wrieden et 
al. 2007).  All studies indicate that cooking intervention, particularly after multiple 
interventions, lead to increased nutrition, however word choice, recipe choice, and 
societal awareness are all important factors in determining the success of the 
intervention.     
Impact of Nutrition Knowledge on Health 
The American diet has become increasingly energy-rich and nutrient poor, a 
diet that the World Health Organization has linked with the world-obesity epidemic.  
The relationship between nutritional knowledge and eating behavior remains 
controversial.  Multiple studies including one by the USDA found that improving 
nutrition knowledge has a small but measurable impact on improved diet quality 
among certain individuals.  Other studies have not found a correlation between 
nutrition knowledge and diet selection or weight control indicating that knowledge 
alone may be insufficient to elicit diet related behavior change. (Frazao & Allshouse 
2003, Klohe-Lehman et al. 2006)  Outreach materials to improve dietary selection 
may require a combination of strategies and interventions, tailored to the needs of 
specific people groups (Frazao & Allshouse 2003).   
 
 
 34 
Food, Economics & Health 
Cost is a major factor influencing food purchases.  However, short term 
economical purchasing of energy-dense diets high in refined grains, sugars, and fats 
may lead to diseases that are costly to treat, reduce longevity, diminish quality of life, 
and may be fatal.  The highest rates of obesity and diabetes in the United States are 
found among minorities and working poor.  (Drewnowski & Darmon 2005)  
Approximately 64% of American adults are considered obese.  Direct costs for obese 
individuals in 2003 for medical expenditures were $75 billion, 36% higher than their 
non-obese counterparts.  When indirect costs such as loss of work, are included the 
severe economic impact that the obesity epidemic may incur is quite clear.  (Seidell, 
2003)  Consumer education and the development of economical, health promoting 
foods is imperative to provide all economic groups with the opportunity to prevent 
the onset of diet related diseases. 
 Dollahite et al calculated that for every dollar spent under EFNEP there was a 
benefit-to-cost ratio ranging from $9.58 - $0.20 for direct and indirect benefits.  
Taking into consideration benefits such as future health care saving and avoiding or 
delaying loss of productivity the Virginia EFNEP program reported a $10.63 per 
$1.00 spent by avoiding or delaying diet-related conditions. (Dollahite et al. 2008)  
Positive cost-benefit analysis and willingness-to-pay ratios are important for a 
program with costs of $124 - $234 per year or $388 per participant with a mean cost 
per graduate (minimum of six lessons) of $849 (Burney & Haughton 2002, Dollahite 
et al. 2008).     Direct outcomes of EFNEP indicate that educating decision makers 
could reduce food expenditures while increasing nutrition and reducing food 
 35 
insecurity.  EFNEP participants demonstrated the ability to reduce food expenditures, 
improved food planning, increased nutrition label use, increases in specific nutrients 
such as iron, vitamins C and B-6, and fiber,  and decreases in salt use.   (Burney & 
Haughton 2002)  Research is required to analyze the effects of functional food 
education and its? potential health and economic impact. 
 
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 48 
Chapter 2: Effect of Yeast Strain on the Antioxidant Properties 
of Wheat Bran Following Solid-State Treatments 
Abstract 
Wheat bran contains a significant portion of the bioactive components in 
wheat.  This study assessed the ability of 10 food grade yeasts to increase the 
extractable antioxidant activity of wheat bran in order to increase its health beneficial 
properties.  Bran samples were treated with yeasts at two doses, 5.6 x 104 and 1.8 x 
105 yeast cells per g wheat bran, for low and high doses, respectively.  Treated bran 
samples were then analyzed for their ability to increase antioxidant capacity when 
compared with an inactivated control of yeast and a blank of sterile water over 72 h at 
ambient temperature.  Under the specified conditions yeast strain LBE.11 showed the 
greatest potential to increase antioxidant capacity, increasing ORAC and HOSC 
radical scavenging by 50% and 67%, respectively.  Results suggest the possibility to 
develop viable postharvest methods for enhancement of wheat bran health properties. 
 
Introduction 
Epidemiological studies show that a diet rich in foods containing antioxidants 
such as fruits, vegetables, and other plant derived foods decreases the risk of certain 
chronic diseases (Wattanapitayakul & Bauer 2001, Stanner et al. 2003, Wilcox et al. 
2008).  Antioxidants broadly include ?any substance that when presented at low 
concentrations, compared to those of an oxidizable substrate, significantly delays, or 
inhibits, oxidation of that substrate? (Gutteridge & Halliwell 1999).  The 
 49 
physiological relevance of antioxidants is their protection from free radicals, 
molecules with an unpaired electron in the outer orbital shell, termed the oxidative 
defense mechanism which prevents damage to biomolecules (Wilcox et al. 2004, 
Fang et al. 2002, Wilcox et al. 2008).    
 Wheat comprises one-third of the total grain crop world wide (Slavin 2001). 
At 117.5 grams per day, wheat based products account for over 70% of the total U.S. 
grain intake (USDA 2007).  A multitude of antioxidant compounds have been found 
in wheat including phenolic acids, flavonoids, coumarins, polyphenols, phytates, 
terpenes, carotenoids, tocopherols, and tocotrienols (Shahidi 2004).  When tested 
against free radical systems, wheat and wheat bran extracts have demonstrated 
scavenging of the DPPH, ABTS cation, peroxyl, superoxide anion, and hydroxyl 
radicals, inhibition of oxidation of fish and corn oil, human low-density lipoprotein 
cholesterol and DNA, and chelation of transition metal Fe2+ (Yu et al. 2002a, Yu et 
al. 2002b, Yu et al. 2004, Zhou & Yu 2004,  Zhou et al. 2004b, Moore et al. 2005, Yu 
et al. 2005, Liyana-Pathirana & Shahidi 2006,   Moore et al. 2006a, Anson et al. 
2008).  The majority of these compounds and radical scavenging capacity is located 
in the bran fraction (Zhou et al. 2004b, Liyana-Pathirana & Shahidi 2006).  Should 
whole grain consumption continue to increase, enhancing the bioavailability of these 
antioxidants through post harvest treatment may increase the overall healthfulness of 
whole grain foods. 
Fermentation of agricultural commodities with bacteria, mold, and yeasts has 
been investigated to develop functional ingredients by increasing bioavailability of 
pre-existing bioactive compounds or enrichment through metabolism products 
 50 
(Bartolome et al. 2003, Fernandez-Orozco et al. 2007, Moore et al. 2007, Lee et al. 
2008).  Increased bioavailability is possible through the use of enzymes, including 
those produced by microorganisms, such as xylanases, arabinase, ?-L-arabinosidase, 
?-gluconases, and cellulases that are predicted to break the ester link between 
phenolic acids and cell wall materials (Bartolome et al. 2003, Moore et al. 2006a).  
Solid state fermentation of cassava peels and wheat bran demonstrated the potential 
for protein enriched food ingredients (Oboh 2006, Moore et al. 2007).   
The capacity of a select strain to enhance the nutritive or mechanical 
properties of raw commodities is well known (Wang and Fields, 1978; Ejiofor et al 
1996, Mathew and Abraham 2005, Plessas et al 2007).  To date, no known study has 
been conducted to analyze the effect of multiple commercially available food grade 
strains of yeast under industry relevant solid state conditions.  Yeasts naturally 
contain cellular antioxidant defenses under stressful conditions.  Antioxidant 
activities  and activities of catalase and superoxide dismutase (SOD), the associated 
enzymes, were found to be yeast strain specific, either increasing or decreasing, when 
adapting to hydrogen peroxide exposure (Bayliak et al. 2006).  The aim of the present 
work was to study the effect yeast strain and dose on the extractable antioxidant 
activities of soft red wheat bran.   
  
 
 
 
 51 
Materials and Methods 
Chemicals & Reagents 
Wheat bran was generously donated by The Mennel Milling Company 
(Fostoria, OH).  2,2-diphenyl-1-picryhydrazyl radical (DPPH?), 2,2?-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS),  6-hydroxy-2,5,7,8-
tetramethyl-chroman-2-carboxylic acid (trolox), disodium ethylenediaminetetracetate, 
and 2,2?-bipyridyl gallic acid were purchased from Sigma-Aldrich (St. Louis, MO).  
2,2?-Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako 
Chemicals, USA, Inc. (Richmond, VA).  Petrifilm Yeast and Mold plates were 
purchased from 3M (St. Paul, MN).  YPD broth and agar were purchased from Fisher 
Scientific and prepared as directed (Pittsburg, PA).  All other chemicals and solvents 
were of the highest commercial grade and used without further purification. 
 
Yeast & Wheat Bran Samples 
 Our laboratory has recently established a library of the commercially available 
yeasts, including those traditionally used for baking, beer, and wine production.  
Yeasts were streaked onto YPD plates to remove additives and ensure single strain 
selection.  Log growth studies were conducted for 80 strains of yeast to determine the 
time frame that the yeast strains are actively growing under selected conditions (See 
Appendix a).  These log growth cycle tests demonstrated a consistent window of time 
in which yeast strains were in logarithmic growth.  Multiple strains were then 
combined and run over duplicate dates to ensure the period of exponential growth 
was not altered significantly by different ages of yeasts.  Ten yeasts samples were 
 52 
selected for solid state treatment.  These included: Hodgson Mill All Natural Active 
Dry Yeast ? Whole Grain, Saf-instant Instant Yeast, Sourdoughs International Italian 
(Camaldoli) Sourdough Starter, Wyeast Bavarian Wheat Blend, Brewferm Blanche, 
Lallemand Diamond Lager Yeast, Saflager S-04, BM DYW74, Lalvin D 47, and Red 
Star Cote des Blancs labeled as LBR.04, LBR.08, LBR.21, LBE.10, LBE.11, 
LBE.13, LW.01, LW.12, and LW.17, respectively.  Bran was ground to a 40 mesh 
size using a 3600 Laboratory Mill from Perten Instruments (Huddinge, Sweden).   
 
Yeast Preparation 
 Individual yeast strains were used to inoculate 10 mL YPD broth and grown at 
30 ? C for 48 hrs.  One loop of yeast cells was transferred to 10 mL fresh YPD broth 
and incubated for a second 48 hrs. at 30 ?C.  200 ?L were transferred to 50 mL of 
YPD broth and grown to the pre-determined log phase.  Yeasts were centrifuged 2 
minutes at 2000 rpm at 30 ?C, the supernatant decanted and yeast cells washed with 
10 mL sterile water at 30 ?C.  The optical density was then adjusted with sterile water 
to an absorbance of 0.5 at 600 nm.  Yeast dosages will be prepared with the high dose 
being 2 mL of 0.5 O.D. yeast preparation, a low dose of 1 mL 0.5 O.D. yeast 
preparation and 1 mL sterile water, and a control of 2 mL of the high dose inactivated 
by microwave. 
 
Yeast Counts 
 Yeast counts were conducted on the yeast treatments prior to the solid state 
treatment and on the treated wheat bran following the solid state treatment.  For the 
 53 
yeast treatments, serial dilutions of the high, low, and control were made using sterile 
water.  For post-treatment counts, 0.01 g of solid state yeast treated bran were 
weighed out and used for a serial dilution series. One mL of each serial dilution was 
plated according to manufacturers? directions on 3M yeast and mold count petrifilm.  
Yeast counts were read after 5 days of incubation at 20 ?C.   
Solid State Yeast Treatment 
 Soft red winter wheat bran was ground to pass through a #40 mesh and 5 g 
measured into sterile 50 mL beakers.  A total volume of 2 mL yeast treatment was 
added to the bran, while simultaneously being stirred gently for 15 seconds.  Yeast 
treatments were conducted in triplicate in addition to a negative control containing 5 
g bran and 2 mL of sterile water.  Beakers were covered with parafilm and incubated 
72 hours at ambient temperature.  Following incubation, 0.01 g treated wheat bran 
was removed for post-reaction yeast count.  The remaining solid state treated bran 
thermally inactivated by microwave, ground to pass through a #40 mesh and stored at 
-80 ?C until further analysis.   
 
Sample Extraction Procedures 
Samples re-ground following treatment to a uniform #40 mesh size were 
extracted using 50 % acetone at a ratio of 1 gram material (dry weight basis) per 10 
mL solvent, overnight. Samples were centrifuged and the supernatants collected.  
Samples were stored under nitrogen in the dark at 20 ?C until further analysis.   
 
 54 
Total Phenolic Content 
Total phenolic content was measured using the Folin-Ciocalteu reagent 
according to a laboratory procedure (Yu et al 2002a).  The final reaction mixture 
contained 100 ?L of sample extract, 500 ?L of the Folin-Ciocalteu reagent, 1.5 mL of 
20% sodium carbonate and 1.5 mL pure water.  Absorbance at 765 nm was recorded 
after two hours incubation at ambient temperature.  Gallic acid equivalence was 
calculated using a gallic acid standard curve.  TPC was determined in duplicate for 
each sample and then samples of the same yeast and treatment combined for analysis 
(n = 6). 
 
Radical Cation ABTS?+ Scavenging Capacity 
Sample extracts were tested against the ABTS?+ cation radical generated 
according to a laboratory protocol (Miller, Rice-Evans 1996, Zhou and Yu 2004).   
Briefly, an ABTS?+ solution was prepared by oxidizing a 5 mM aqueous solution of 
ABTS with manganese dioxide for 30 minutes at ambient temperature.  The solution 
was then filtered and adjusted with phosphate buffer to an absorbance of 0.7 at a 
wavelength of 734 nm.  A trolox standard is prepared with the same solvent with the 
blank consisting of 1 mL phosphate buffer and 80 ?L water blank.  Likewise the final 
reaction mixture contained 1 mL of the ABTS?+ solution , and 80 ?L of the sample of 
trolox standard.  Reaction mixtures were vortexed 30 seconds and a reading recorded 
at 1.5 minute.  Data is reported as ?moles of trolox equivalents per gram solid state 
yeast treated bran calculated using the trolox standard curve.   
 55 
 
Oxygen Radical Absorbance Capacity (ORAC) Assay 
ORAC was conducted on sample extracts using the fluorescent probe 
flourescein (FL) according to a laboratory protocol (Huang et al 2002, Zhou and Yu 
2004).  Trolox standards were prepared in the same solvent as the samples and AAPH 
and FL solutions were prepared with 75 mM phosphate buffer (pH 7.4).  The final 
reaction mixture per well contained 225 ?L of 81.63 nM FL solution, 30 ?L blank 
(50% acetone), standard, or sample solution, and 25 ?L 0.36 M AAPH solution.  
Therefore, each well contained a constant total volume of 280 ?L.  Standards and 
samples were run in duplicate simultaneously utilizing a Victor2 multilabel counter 
(PerkinElmer, Turku, Finland).  The reaction mixture was held at 37 ?C and 
fluorescence measurements taken every minute at excitation and emission 
wavelengths of 485 nm and 530 nm respectively for 1.5 hours.  Results are expressed 
as trolox equivalents (TE) calculated using the relative area under the curve for the 
samples as compared to the trolox standard curve run simultaneously.  Samples of the 
same yeast and dose treatment were combined and reported as the mean (n = 6) and 
standard deviation.   
 
Hydroxyl Radical Scavenging Capacity (HOSC) Assay. 
Hydroxyl radical scavenging capacity is measured using by the hydroxyl 
radical generated by ferric iron (Fe3+) and hydrogen peroxide as described previously 
(Moore et al. 2006b).  Briefly a 92.8 nM solution of flourescein (FL) was prepared 
 56 
using phosphate buffer.  The final well concentration was 300 ?L per well consisting 
of 170 ?L FL solution, 30 ?L blank, standard, or sample, 40 ?L H2O2 and 60 ?L 
FeCl3.  Degradation of the FL solution is measured every minute using a Victor2 
multilabel counter (PerkinElmer, Turku, Finland).  Trolox equivalents are calculated 
using the area under the curve as compared with the trolox standard run 
simultaneously with the samples.  Data is reported as the mean (n = 6) of each 
treatment and standard deviations.   
 
 Radical DPPH Scavenging Capacity. 
Free radical scavenging capacity of each sample was estimated following a 
previously reported procedure utilizing the stable 2,2-diphenyl-1-picryhydrazyl 
radical (DPPH?) (Yu et al. 2002b), by the high-throughput radical scavenging 
capacity assay (RDSC) (Moore et al. 2006a) .  The 200 ?M DPPH? working solution 
was freshly prepared from a 1 mM DPPH? stock solution in sample solvent.  Briefly, 
100 ?L of freshly made 200 ?M DPPH? working solution was combined with 100 ?L 
of solid state yeast treatment extract to start the radical-antioxidant reaction.  On the 
same plate was also a blank of pure sample solvent, and control of solvent and 200 
?M DPPH? working solution, and standards ranging from 7.8 to 250 mM Trolox. The 
absorbance at 517 nm was determined by Victor3 multilabel plate reader over 40 
minutes (Perkin-Elmer, Turku, Finland).  The total area under the curve was 
calculated and used to determine the % DPPH radical quenched and RDSC value (TE 
mmol/g).  Samples were run in duplicate and treatments of the same yeast and dose 
combined for reporting the mean (n = 6) and standard deviation.   
 57 
 
Statistical Analysis 
Data are reported as mean ? SD of the combined treatments with individual 
extracts run in duplicate.  Thus, data for the solid state treatment, pizza crust, and in-
vitro digestion model will be the mean ? SD for six values. One-way Analysis of 
Variance (ANOVA) and Tukey?s Honestly Significant Difference (HSD) tests were 
performed (Statistical Package for the Social Sciences (SPSS) for Windows, Version 
Rel. 10.0.5., 1999, SPSS Inc., Chicago, IL) to identify differences among means, with 
statistical significance declared at P ? 0.05.  
 
Results and Discussion 
Effects of Different Yeast Strains on the Antioxidant Properties of Soft Red 
Wheat Bran Under Solid State Treatment Conditions.  Solid state yeast treatments 
were conducted with 10 food grade yeasts (3 traditionally used for bread, 4 for beer, 
and 3 for wine) at two dose levels and compared with their controls, thermally 
inactivated high doses of the same yeast, and a negative control of sterile water which 
were all run under the same conditions.  The ten yeasts used are represented as 
LBR.04, LBR.08, LBR.21, LBE.03 LBE.10, LBE.11, LBE.13, LW.01, LW.12, and 
LW.17 in correspondence with their library identification.  Strains beginning with 
LBR, LBE, and LW are traditionally used in bread production, beer, and wine, 
respectively.  Solid-state treatment conditions were selected to test the selected strains 
under industry possible conditions.  Under these conditions, there were few 
 58 
differences between yeast strains and their abilities increase the extractable 
antioxidant components of soft red winter wheat bran.     
 
Yeast Counts  ? All controls were negative for yeast and mold.  Low doses of 
yeast contained approximately 2.8 x 10^5 cells per mL or 5.6 x 10^4 cells per g.  High 
doses of yeast contained approximately 9.0 x 10^5 cells per mL or 1.8 x 10^5 cells per 
g.  Following solid state treatment there was no significant difference in yeast count 
between yeast strain for low and high doses.  Low and high solid state yeast treatment 
wheat bran samples contained approximately 4.5 x 10^4, and 1.0 x 10^5 cells per g 
treated wheat bran, respectively.  
 
Total Phenolic Content (TPC) ? Figure 2.1 shows the total phenolic content 
of the controls and their corresponding yeast treatments.  Low and high dose yeast 
treatments under solid state conditions were not significantly different from their 
corresponding control.  It was interesting to note that the controls when analyzed 
independently differed significantly indicating that yeast themselves may differ in 
their extractable TPC.  The same observation was made previously in inactivated 
yeast controls in Lakin wheat bran (Moore et al. 2007).    
 59 
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ababc
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ab
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dbcd
ab
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ab
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ab
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bc
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ac
db
ab
ab
cda
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cd
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
Blank LBR.04 LBR.08 LBR.21 LBE.03 LBE.10 LBE.11 LBE.13 LW.01 LW.12 LW.17
Yeast Strain
TP
C 
(m
g 
GA
E/
g 
br
an
)
Control Low High
 Figure 2.1 Effect of solid state yeast treatment on the total phenolic content 
(TPC) of soft red wheat bran.  Results are expressed as milligrams of gallic acid 
equivalents (GAE) per gram of yeast treated wheat bran on a dry weight basis.  Blank 
represents wheat bran run concurrently with sterile water.  LBR.04, LBR.08, LBR.21, 
LBE.03, LBE.10, LBE.11, LBE.13, LW.01, LW.12, and LW.17 represent food grade 
yeasts from our laboratory yeast library.  Control is inactivated yeasts; low and high 
were treated with 5.6 x 104 and 1.8 x 105 cells per g wheat bran, respectively at 
ambient temperature for 72 h.  All measurements were conducted in duplicate with 
the mean value of the triplicate yeast dose treatment combined reported (n = 6).  
Vertical bars represent the standard deviation of each data point.  Values marked by 
the same letter are not significantly different (P < 0.05).   
 
 ABTS?+ Scavenging Capacity ? Figure 2.2 compares the ABTS?+ scavenging 
capacities of solid state yeast treated soft red bran with controls.  In contrast with the 
minimal differences between controls and select treatments in TPC there were no 
significant differences between dose or yeast treatments.    
 
 ORAC ? Figure 2.3 demonstrates the ability of some yeast strains to 
significantly increase ORAC values in wheat bran when compared to their control.  
ORAC measures the radical scavenging capacity of the physiologically relevant 
 60 
peroxyl radical, and may discontinue lipid peroxidation during the propagation step.  
All results are expressed as ?moles of trolox equivalents per gram of wheat bran on a 
dry weight basis.  Three yeasts significantly increased ORAC values including 
LBR.08, LBE.11, and LW.17.  These data indicate the potential for using non-
traditional bread yeasts in the treatment of bran and bran-based foods.  It needs to be 
pointed out that as in TPC analysis, the control ORAC values for the controls are 
significantly different.  More research is required to determine the contribution of 
yeasts on the ORAC assay. 
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Blank LBR.04 LBR.08 LBR.21 LBE.03 LBE.10 LBE.11 LBE.13 LW.01 LW.12 LW.17
Yeast Strain
AB
TS
+ (
????m
ol
 T
E/
g 
br
an
)
Control Low High
 
 
Figure 2.2. ABTS?+ radical  scavenging capacity of solid state yeast treated soft 
red wheat bran.  Results are expressed as Trolox equivalents (TE) per gram of solid 
state treated bran on a dry weight basis.  Blank represents wheat bran run 
concurrently with sterile water.  LBR.04, LBR.08, LBR.21, LBE.03, LBE.10, 
LBE.11, LBE.13, LW.01, LW.12, and LW.17 represent food grade yeasts from our 
laboratory yeast library.  Control is inactivated yeasts; low and high were treated with 
5.6 x 104 and 1.8 x 105 cells per g wheat bran, respectively at ambient temperature for 
72 h.  All measurements were conducted in duplicate with the mean value of the 
triplicate yeast dose treatment combined reported (n = 6).  Vertical bars represent the 
standard deviation of each data point.  Values were not significantly different (P < 
0.05).   
 61 
 
  
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efg
hi
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de
hi
efg
hi
ab
a
de
fgh
i
ab
cd
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ab
cd
efg
hi
ab
cd
efg
hi
de
fgh
i
de
fgh
i
gh
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de
fgh
i
i
ab
cd
efg
hi
efg
hi
ab
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ab
cd
gh
iefg
hi
bc
de
fgh
i
ab
cd
efg
h
ab
cd
efg
hi
de
fgh
i
fgh
i
de
fgh
iefgh
i
ab
cd
efg
0
20
40
60
80
100
120
Blank LBR.04 LBR.08 LBR.21 LBE.03 LBE.10 LBE.11 LBE.13 LW.01 LW.12 LW.17
Yeast Strain
OR
AC
 (????
m
ol
 T
E/
g)
Control Low High
 
Figure 2.3 Effect of solid state treatment on the oxygen radical absorbing 
capacities (ORAC) of soft red wheat bran.  Results are expressed as Trolox 
equivalents (TE) per gram of solid state treated bran on a dry weight basis.  Blank 
represents wheat bran run concurrently with sterile water.  LBR.04, LBR.08, LBR.21, 
LBE.03, LBE.10, LBE.11, LBE.13, LW.01, LW.12, and LW.17 represent food grade 
yeasts from our laboratory yeast library.  Control is inactivated yeasts; low and high 
were treated with 5.6 x 104 and 1.8 x 105 cells per g wheat bran, respectively at 
ambient temperature for 72 h.  All measurements were conducted in duplicate with 
the mean value of the triplicate yeast dose treatment combined reported (n = 6).  
Vertical bars represent the standard deviation of each data point.  Values marked by 
the same letter are not significantly different (P < 0.05).   
 
HOSC ? Figure 2.4 compares the ability of yeasts and their dose to enhance 
the hydroxyl radical scavenging capacity of wheat bran.  When all yeasts and doses 
are compared only LBR.21 and LBE.11 demonstrate a dose dependent increase in 
HO? scavenging capacity.  When each yeast strain is analyzed independently LBE.03 
and LW.12 also show a statistically significant increase in hydroxyl radical 
scavenging as compared to their inactivated control.  Remaining yeasts showed no 
 62 
significant increases in hydroxyl radical scavenging activity versus their 
corresponding controls.  It is important to mention the significant differences between 
the control values.   
 
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cd
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bc
de
fgh
i
ab
cd
ef
ab
cd
ab
cd
efg
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efg
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ab
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de
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i
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ij
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de
fgh
ij
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hi
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efg
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de
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l
ab
cd
efg
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de
fgh
ij
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ij
gh
ijk
l
a
bc
de
fgh
0
20
40
60
80
100
120
140
Blank LBR.04 LBR.08 LBR.21 LBE.03 LBE.10 LBE.11 LBE.13 LW.01 LW.12 LW.17
Yeast Strain 
HO
SC
 (????
m
ol
 T
E/
g)
Control Low High
 
Figure 2.4 Effect of solid state treatment on the hydroxyl radical scavenging 
capacities (HOSC) of soft red wheat bran.  Results are expressed as Trolox 
equivalents (TE) per gram of solid state treated bran on a dry weight basis.  Blank 
represents wheat bran run concurrently with sterile water.  LBR.04, LBR.08, LBR.21, 
LBE.03, LBE.10, LBE.11, LBE.13, LW.01, LW.12, and LW.17 represent food grade 
yeasts from our laboratory yeast library.  Control is inactivated yeasts; low and high 
were treated with 5.6 x 104 and 1.8 x 105 cells per g wheat bran, respectively at 
ambient temperature for 72 h.  All measurements were conducted in duplicate with 
the mean value of the triplicate yeast dose treatment combined reported (n = 6).  
Vertical bars represent the standard deviation of each data point.  Values marked by 
the same letter are not significantly different (P < 0.05).   
 
DPPH ? Figure 2.5 compares the DPPH radical scavenging capacity of all 
yeasts at different doses and to that of the control and the blank.  Results indicate that 
no yeasts were able to significantly increase DPPH scavenging capacity of soft red 
wheat bran.  Similar to the other test results, the controls were significantly different, 
 63 
indicating that the yeasts themselves might have different DPPH? scavenging 
components.   
 
b
de
fghdef
ghc
de
f
cd
efg
h
hig
hi
de
fgh
i
de
fgh
de
fgh ghic
d
cd
e
ighi
de
fgh
i
de
fgh bc
de
fgh fghi
b
a
de
fgh
i
gh
i
gh
i
i
cd
efg
cd
efg
cd
efg
h
efg
hi
b
0
0.1
0.2
0.3
0.4
0.5
0.6
Blank LBR.04 LBR.08 LBR.21 LBE.03 LBE.10 LBE.11 LBE.13 LW.01 LW.12 LW.17
Yeast Strain
RD
SC
 (T
E 
?m
ol/
g)
Blank Low High
 
Figure 2.5 Effect of solid state yeast treatment on the DPPH scavenging 
capacities of soft red wheat bran.  Results are expressed as Trolox equivalents (TE) 
per gram of solid state treated bran on a dry weight basis.  Blank represents wheat 
bran run concurrently with sterile water.  LBR.04, LBR.08, LBR.21, LBE.03, 
LBE.10, LBE.11, LBE.13, LW.01, LW.12, and LW.17 represent food grade yeasts 
from our laboratory yeast library.  Control is inactivated yeasts; low and high were 
treated with 5.6 x 104 and 1.8 x 105 cells per g wheat bran, respectively at ambient 
temperature for 72 h.  All measurements were conducted in duplicate with the mean 
value of the triplicate yeast dose treatment combined reported (n = 6).  Vertical bars 
represent the standard deviation of each data point.  Values marked by the same letter 
are not significantly different (P < 0.05).   
 
 
 Conclusion 
These results show the challenge of using solid state yeast treatment to 
increase the potential antioxidant properties of soft red wheat bran.  An important 
observation of this study is that solid state treatment may change the sensory 
properties of wheat bran, specifically strong scents, often fruity, were observed for 
 64 
both dose levels.  Slight genetic differences between Saccharomyces cerevisiae 
strains may be responsible for slight variances in fermentation rate, production of 
flavor chemicals, and nutrient preferences (Maloney & Foy 2003).  This suggests that 
higher levels of yeasts may not be appropriate for all food-based applications, and 
that solid state yeast treatment warrants further research to develop bran-based foods 
with improved acceptability.    
 
 65 
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 68 
Chapter 3: Effects of Thermal Treatment, Fermentation, and 
Bran Particle Size on the Ethanol Extractable Antioxidant 
Properties of Whole-Wheat Pizza Crust 
 
Abstract 
This study investigated the impact of bran particle size, fermentation time, and 
baking time and temperature on the 100% ethanol extractable antioxidant capacities 
of whole wheat pizza crust.  Trego and Lakin wheat varieties were used to prepare 
pizza crusts which were then analyzed for total phenolic content, ferulic acid content, 
and radical scavenging capacities against the peroxyl (ORAC), hydroxyl (HOSC), 
DPPH (RDSC) and cation ABTS radicals.  Bran particle size did not significantly 
alter extractability of antioxidant properties.  18 hrs. of fermentation resulted in a 16.5 
and 22.6% increase in RDSC and TPC values of 100% ethanol extracts of whole 
wheat pizza crusts.  Three cooking combinations of 7 minutes at 400 ?F, 14 minutes at 
400 ?F, and 7 minutes at 550 ?F showed possible effects on available antioxidant 
properties in baked pizza crusts.  Interestingly, the ratio of soluble free, soluble 
conjugated and bound ferulic acid in the pizza crusts was not significantly altered 
during thermal processing.  Results suggest possible increases in ethanol extractable 
antioxidants in whole wheat pizza crusts by improving food preparation conditions. 
   
 69 
Introduction 
Epidemiological studies show that a diet rich in foods containing antioxidants 
such as fruits, vegetables, and other plant derived foods decreases the risks of certain 
chronic diseases (Wattanapitayakul & Bauer 2001, Stanner et al. 2003, Wilcox et al. 
2008).  Oxidative defense mechanisms, protection from free radicals, is a primary 
mechanisms investigated to combat aging and age-related diseases (Wilcox et al. 
2008).  Recent in-vitro studies have demonstrated that antioxidants found in wheat 
are capable of scavenging free radicals such as the hydroxyl, peroxyl, superoxide 
anion, DPPH, and cation ABTS radicals, chelate transition metals such as ionizing 
Fe2+, and prevent lipid peroxidation (Yu et al. 2002, Zhou et al. 2004a,  Zhou et al. 
2004b, Zhou & Yu 2004, Adom et al. 2005, Moore et al. 2005).   
 Wheat (Triticum spp.) is an important commodity for U.S. consumers.  It 
comprises 71 % of grain consumption and is one of the most commonly consumed 
grains globally (USHHS & USDA 2005).  The most important factor in determining 
the antioxidant capacity of wheat products is the use of whole wheat rather than 
refined wheat flour (Halvorsen et al. 2006).  The presence of bran in breads however 
has created sensory challenges for producers including loaf volume, darker crumb, 
and different flavor profiles (Lai et al. 1989, Hung et al 2007).  One of the most 
widespread convenience foods worldwide, pizzas may make whole wheat pizza crust 
a good candidate to increase whole wheat consumption (Coppola et al 1998).   
Food processing to develop higher quality whole wheat products may impact 
their antioxidant properties.  Dough prepared with finely ground bran was found to be 
superior to dough prepared with conventionally ground bran.  However, reduced 
 70 
particle size reduces antioxidant stability during storage (Lai et al 1989, Cheng et al 
2006a).  Previous studies conducted in our laboratory showed that particle size had no 
effect on the 50% acetone antioxidant properties extractable in pizza crusts (Moore et 
al. 2008).  A few studies have evaluated the affects of baking conditions on 
antioxidant levels in loaf breads, however limited data is available on the affects of 
processing on pizza dough properties and baked pizza crusts (Coppola et al. 1998, 
Lindenmeier et al. 2004, Halvorsen et al. 2006).  Therefore, the objective of this study 
was to evaluate the effects of bran particle size, fermentation time, and thermal 
baking conditions on the ethanol extractable antioxidant properties and phenolic acid 
content of pizza dough and baked pizza crusts.  
 
Materials and Methods 
Chemicals & Reagents 
2,2-diphenyl-1-picryhydrazyl radical (DPPH?), 2,2?-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid diammonium salt (ABTS),  6-hydroxy-2,5,7,8-
tetramethyl-chroman-2-carboxylic acid (trolox), flourescein (FL), FeCl3, and 2,2?-
bipyridyl gallic acid were purchased from Sigma-Aldrich (St. Louis, MO).  2,2?-
Azobis(2-amidinopropane) dihydrochloride (AAPH) was purchased from Wako 
Chemicals, USA, Inc. (Richmond, VA).  All other chemicals and solvents were of the 
highest commercial grade and used without further purification. 
 
 
 71 
Wheat Flour Samples & Preparation  
 Bran from hard winter wheat varieties, Trego and Lakin, were provided by Dr. 
Scott Haley of the Department of Soil and Crop Science, Colorado State University, 
Fort Collins, CO 80523.  Samples had been grown in 2004 under typical agronomic 
practices for wheat production in eastern Colorado.   Harvested grains were cleared of 
all non-grain debris present by seed cleaners and stored under ambient conditions.  A 
Brabender Quadromat Junior experimental mill was used to grind grain samples and 
separate the bran fraction.  A coffee grinder was then used to further grind the bran 
fraction to 20- and 80- mesh.  Bran and non-bran fractions were then recombined in 
ratio to produce two varieties of whole wheat flour, each at two bran mesh sizes.     
 
Dough & Pizza Crust Preparation 
Pizza dough was prepared by combining whole-wheat flour with water, 
honey, soybean oil, dry active yeast (Fleischmann?s, Fenton, Misssouri) and salt at 
w/w percentage ratios of 52.04, 33.24, 10.30, 2.34, 1.13, and 0.95 respectively.  
Dough was mixed using a KitchenAid stand mixer equipped with a dough hook on 
low speed for 4 minutes at ambient temperature.  Dough was divided manually into 
250 g pieces, rounded into balls, placed into stainless steel containers sprayed with 
Pam, and covered with plastic wrap.  Fermentation was conducted at 4 ?C for 0 
(Control), 18, and 48 hours.  Dough was tempered for 2 h prior to hand rolling into 12 
inch pizza crusts.  Crusts were hand transferred to aluminum pizza screens and baked 
at different time-temperature combinations in a conventional oven.  Combinations 
included 7 minutes at 400 ?F, 14 minutes at 400 ?F, and 7 minutes at 550 ?F.  
 72 
Following 30 minutes cooling time, and crusts and dough samples were frozen, freeze 
dried, and ground to 40-mesh for extraction.  Samples were stored in desiccators at 
ambient temperature in the bark until further analysis.     
 
Sample Extraction Procedures 
Freeze dried pizza crusts and dough (Control) were extracted using 100% 
ethanol at a ratio of 1 gram material (dry weight basis) per 10 mL solvent, overnight. 
Samples were centrifuged and the supernatants collected and extracts used determine 
total phenolic content, and ABTS cation, ORAC, and DPPH radical scavenging 
capacity.  Extracts were stored under nitrogen in the dark at 20 ?C until further 
analysis.   
 
Total Phenolic Content 
Total phenolic content was measured using the Folin-Ciocalteu reagent 
according to a laboratory procedure (Yu et al 2002).  The final reaction mixture 
contained 100 ?L of sample extract, 500 ?L of the Folin-Ciocalteu reagent, 1.5 mL of 
20% sodium carbonate and 1.5 mL pure water.  Absorbance at 765 nm was recorded 
after two hours incubation at ambient temperature.  Gallic acid equivalence was 
calculated using a gallic acid standard curve.  TPC analysis was conducted in 
duplicate with mean and standard deviation reported of triplicate thermal, mesh, or 
fermentation treatments combined (n = 6) .   
 
 73 
Radical Cation ABTS?+ Scavenging Capacity 
Sample extracts were tested against the ABTS?+ cation radical generated 
according to laboratory protocol (Miller & Rice-Evans 1996, Zhou and Yu 2004).   
Briefly, an ABTS?+ solution was prepared by oxidizing a 5 mM aqueous solution of 
ABTS with manganese dioxide 30 minutes at ambient temperature.  The solution was 
then filtered and adjusted with phosphate buffer to an absorbance of 0.7 at a 
wavelength of 734 nm.  A trolox standard is prepared with sample solvent with the 
blank consisting of 1mL phosphate buffer and 80 ?L water blank.  Likewise the final 
reaction mixture contained 1 mL of the ABTS?+ solution, and 80 ?L of the sample of 
trolox standard.  Reaction mixtures were vortexed 30 seconds and a reading recorded 
at 1.5 minute.  Data is reported as trolox equivalents (TE) per gram whole wheat 
pizza crust on a dry weight basis calculated using the trolox standard curve.  Samples 
of the same thermal treatment, fermentation time, or mesh size were combined and 
reported as the mean (n = 6) and standard deviation.   
 
Oxygen Radical Absorbance Capacity (ORAC) Assay 
ORAC was conducted on sample extracts using the fluorescent probe 
flourescein (FL) according to laboratory protocol (Huang et al 2002, Zhou and Yu 
2004).  Trolox standards and samples were prepared in the same solvent as the 
samples and AAPH and FL solutions were prepared with 75 mM phosphate buffer 
(pH 7.4).  The final reaction mixture per well contained 225 ?L of 81.63 ?M FL 
solution, 30 ?L blank (50% acetone), standard, or sample solution, and 25 ?L 0.36 M 
AAPH solution.  Therefore, each well contained a constant total volume of 280 ?L.  
 74 
Standards and samples were run in duplicate simultaneously utilizing a Victor2 
multilabel counter (PerkinElmer, Turku, Finland).  Prior to AAPH addition, FL and 
antioxidant extracts were held at 20 minutes at 37 ?C.  The reaction mixture was held 
at 37 ?C and fluorescence measurements taken every minute at excitation and 
emission wavelengths of 485 nm and 530 nm respectively for 1.5 hours.  Results are 
expressed as trolox equivalents (TE) calculated using the relative area under the curve 
for the samples as compared to the trolox standard curve run simultaneously.  
Samples of the same thermal treatment, fermentation time, or mesh size were 
combined and reported as the mean (n = 6) and standard deviation.   
 
Radical DPPH Scavenging Capacity (RDSC). 
Free radical scavenging capacity of each sample was estimated following a 
previously reported procedure utilizing the stable 2,2-diphenyl-1-picryhydrazyl 
radical (DPPH?) (Yu et al. 2002), by the high-throughput radical scavenging capacity 
assay (RDSC) (Cheng et al. 2006a).  The 200 ?M DPPH? working solution was 
freshly prepared from a 1 mM DPPH? stock solution.  Briefly, 100 ?L of freshly 
made 200 ?M DPPH? working solution was combined with 100 ?L of solid state 
yeast treatment extract to start the radical-antioxidant reaction.  On the same plate 
was also a blank of pure sample solvent, and control of solvent and 200 ?M DPPH? 
working solution, and standards ranging from 7.8 to 250 mM Trolox. The absorbance 
at 517 nm was determined by Victor3 multilabel plate reader over 40 minutes (Perkin-
Elmer, Turku, Finland).  The total area under the curve was calculated and used to 
determine the % DPPH radical quenched and RDSC value (TE mmol/g).  Samples 
 75 
were run in duplicate.  Samples of the same thermal treatment, fermentation time, or 
mesh size were combined and reported as the mean (n = 6) and standard deviation.   
 
Phenolic Acid Composition 
 Baked pizza crusts were analyzed for their soluble free, soluble conjugated, 
and insoluble bound ferulic acid contents using a previously reported laboratory 
procedure (Moore et al. 2005).  Acetone/methanol/water (7/7/6, v/v/v) was used to 
separate soluble and insoluble fractions.  The supernatant containing soluble and 
conjugated free ferulic acid were separated further by acidifying the solution (pH 2) 
and extracting soluble free ferulic acid into ethyl ether/ethyl acetate (1/1, v/v).  
Soluble conjugated ferulic acid was then hydrolyzed with NaOH, re-acidifying the 
solution to a pH of 2, and the free ferulic acid was collected by ethyl ether/ethyl 
acetate (1/1, v/v) extraction.  Bound ferulic acid in the solid residue was hydrolyzed 
with NaOH, supernatant acidified to a pH of 2 and the released ferulic acid was 
extracted with ethyl ether/ethyl acetate (1/1, v/v).  The ethyl ether/ethyl acetate was 
evaporated using a nitrogen evaporator and extract re-dissolved in HPLC grade 
methanol.  Samples were filtered using a 0.45 ?m membrane syringe filter and 
analyzed by HPLC using a Phenomenex C18 column (250 mm x 4.6 mm).  A 
gradient elution program was used to separate the phenolic acids.  Solvent A (acetic 
acid/H20, 2:98, v/v) and solvent B (acetic acid/acetonitrile/ H20, 2:30:68, v/v/v) were 
programmed from 10 ? 100% B in 42 minutes with a flow rate of 1.5 mL/min (Zhou 
and Yu 2004).  Identification and quantification of individual phenolic acids was 
accomplished by comparing the retention time and phenolic profile to that of known 
 76 
concentrations of the pure phenolic compounds.  HPLC analysis was conducted in 
duplicate and the value for the thermal treatment reported as the mean and the 
standard deviation.  
 
Statistical Analysis 
All treatments (bran size, fermentation time, baking time and temperature) 
were conducted in triplicate.  Data is reported as mean ? SD of the combined 
treatments with individual extracts run in duplicate.  One-way ANOVA and Tukey?s 
HSD tests will be performed (Statistical Package for the Social Sciences (SPSS) for 
Windows, Version Rel. 10.0.5., 1999, SPSS Inc., Chicago, IL) to identify differences 
among means, with statistical significance declared at P ? 0.05.  
 
Results and Discussion 
Effects of Bran Mesh Size on the Antioxidant Properties of Baked Whole 
Wheat Pizza Crust.    
To evaluate the effects of bran particle size on 100% ethanol extractable pizza 
crust antioxidant properties, pizza crusts were prepared with two varieties (Lakin and 
Trego) of wheat, each having their bran ground to mesh sizes of 20 and 80.  Table 3.1 
presents the results of antioxidant activity analysis of pizza crusts.  These results 
indicate that reducing particle size slightly increased the ABTS?+ scavenging 
capacity for the Lakin variety, while slight decreases were observed in the Trego 
variety although neither change was significantly different.  The significant increase 
 77 
in antioxidant activity was observed in the ORAC test where reduced particles size 
resulted in an 11.8% increase for the Trego variety.  Slight increases were also 
observed for DPPH? scavenging capacity although no significant differences were 
observed.  Total phenolic contents followed the same trend as the ABTS?+ 
scavenging capacity assay.   
 
 
 78 
Table 3.1. Effect of Bran Mesh Size on Pizza Crust Antioxidant Properties for Two Hard Wheat Varieties* 
Wheat Variety Mesh Size ABTS  
 (?mol TE/ g pizza  
crust dw) 
ORAC 
(?mol TE/g pizza  
crust dw) 
RDSC 
(?mol TE/g pizza  
crust dw) 
TPC 
(mg GAE/g pizza  
crust dw) 
Lakin 20 1.96 ? 0.08 a 1.83 ? 0.19 a 0.24 ? 0.03 a 0.34 ? 0.02 a 
Lakin 80 2.46 ? 0.32 ab 2.21 ? 0.38 a 0.25 ? 0.03 a 0.31 ? 0.00 a 
Trego 20 3.58 ? 0.46 b 2.63 ? 0.42 a 0.21 ? 0.01 a 0.32 ? 0.05 a 
Trego 80 2.74 ? 0.62 ab 3.58 ? 0.16 b 0.23 ? 0.02 a 0.36 ? 0.01 a 
*Pizza crusts baked at 204 ?C for 7 minutes.  TE and GAE stand for trolox equivalents and gallic acid equivalents, respectively.  All 
results are expressed on a per dry pizza dough or crust weight basis (dw).  ABTS stands for ABTS?+ scavenging capacity; ORAC 
stands for oxygen radical absorbing capacity; RDSC stands for relative DPPH? scavenging capacity; TPC stands for total phenolic 
content.  All sample extracts were analyzed in duplicate and reported mean values are the mean of the triplicate treatments ? the 
standard deviation (n = 6).  Values in the same test marked by the same letter are not significantly different (P < 0.05).   
 79 
 Whole grain consumption is widely accepted as having components that 
maintain health and help resist age related diseases (Kantor et al. 2001, USHHS & 
USDA 2005, Hung et al. 2007, Mellen et al. 2008).  Presence of bran and germ in 
wheat products to date is associated with decreased quality and sensory parameters 
such as lower loaf volume, darker crumb, and different flavor profiles (Lai et al. 
1989, Hung et al 2007).  Debranning flours completely may not be suitable for all 
applications either as debranning lowers the gluten index (Lijuan et al. 2007).  
Decreased particle size has been found to reduce antioxidant stability during storage, 
and antioxidant analysis for micro-ionized bran independently demonstrated that 
reduction of particle size may have the ability to increase antioxidant availability 
(Zhou et al. 2004b, Cheng et al. 2006b).  A combination of grinding and presoaking 
bran using water and sodium stearoyl lactylate was found to negate the effects of bran 
presence and maintain loaf volume (Lai et al 1989).  Slight increases in antioxidant 
activity were observed in whole wheat pizza crust extracts prepared with 80-mesh 
bran flour.  These results suggest that there were no significant decreases in 
antioxidant activity due to reduced particle size during pizza crust processing.  
Further analysis is required to determine the effects of bran particle size on the 
sensory attributes of non-loaf whole wheat products.    
 
Effects of Fermentation Time on the Antioxidant Properties of Whole Wheat 
Pizza Dough.    
Dough fermentation for the production of specialty bread products is an 
ancient technology.  Most yeasts utilized in the baking industry are members of the 
 80 
ascomycetous genus Saccharomyces with Saccharomyces exiguous and 
Saccharomyces cerevisiae used for sourdough, and modern baking yeasts, 
respectively (Maloney & Foy 2003).  Saccharomyces cerevisiae strains are effective 
at producing carbon dioxide and ethanol from sugars and are used for modern baking, 
and beer and wine production (Maloney & Foy 2003).  Carbon dioxide dissolves in 
the free water of dough and then expands in the gaseous form trapped by the gluten 
protein network, commonly known as dough rise (Poitrenaud 2004).  The leavening 
process including leavening time is critical to maintain overall quality including 2-5 
fold volume increase as well as acid production and rheological properties of baked 
products (Coppola et al. 1998).  It is therefore of interest to understand how pizza 
dough fermentation may influence other minor components such as extractable 
antioxidant properties.  
To evaluate the effects of fermentation time on 100% ethanol extractable 
pizza crust antioxidant properties, pizza crusts were prepared at a bran mesh size of 
20, formed into 250 g balls and fermented for 0, 18, and 48 hrs.  Table 3.2 shows the 
antioxidant property of pizza dough samples.  Results presented indicate that 
extended fermentation time of Lakin produced pizza doughs were either not 
significantly different from their control, or that antioxidant properties of the extracts 
were reduced.  Similarly, Vitamin E and carotenoids losses were previously reported 
due to oxygenation during dough production, kneading, lipoxygenase activity, and 
heat destruction (Leenhardt et al. 2006).  RDSC and TPC values from 100% ethanol 
extracts of whole wheat pizza crusts prepared with Trego wheat increased by 16.5% 
and 22.6% at 18 hrs. of fermentation over their controls, respectively.  Previous study 
 81 
of Trego wheat indicated that it has a unique capacity to inhibit lipid peroxidation in 
fish oil (Yu et al. 2002).  More research is required to analyze how different 
distribution of phenolics in wheat bran may contribute to the different levels of 
antioxidant activity and their degradation or release during food preparation. 
 
 82 
Table 3.2 Effect of Fermentation Time on Pizza Crust Antioxidant Properties for Two Hard Wheat Varieties* 
Wheat Variety Fermentation 
Time ( hrs.)  
ABTS (?mol TE/g 
pizza crust dw) 
ORAC (?mol TE/g 
pizza crust dw) 
RDSC(?mol TE/g 
pizza crust dw) 
TPC (mg GAE/g 
pizza crust dw) 
Lakin 0 3.28 ? 0.18 b 4.00 ? 0.16 ab 0.30 ? 0.01 c 0.36 ? 0.02 ab 
Lakin 18 3.08 ? 0.06 b 4.02 ? 0.18 ab 0.29 ? 0.00 bc 0.34 ? 0.03 ab 
Lakin 48 3.17 ? 0.04 b 3.30 ? 0.46 a 0.20 ? 0.01 a 0.35 ? 0.03 ab 
Trego 0 3.07 ? 0.12 b 4.60 ? 0.28 bc 0.26 ? 0.02 bc 0.31 ? 0.02 a 
Trego 18 2.39 ? 0.21 a  5.36 ? 0.51 c 0.27 ? 0.01 bc 0.38 ? 0.02 b 
Trego 48 2.46 ? 0.00 a 4.73 ? 0.71 bc 0.25 ? 0.03 ab 0.37 ? 0.00 ab 
*Pizza dough fermentation was conducted at 2 ?C, covered in greased stainless steel containers.  TE and GAE stand for trolox 
equivalents and gallic acid equivalents, respectively.  All results are expressed on a per dry pizza dough weight basis (dw).  ABTS 
stands for ABTS?+ scavenging capacity; ORAC stands for oxygen radical absorbing capacity; RDSC stands for relative DPPH? 
scavenging capacity; TPC stands for total phenolic content.  All sample extracts were analyzed in duplicate and reported as the mean 
of the triplicate treatments ? the standard deviation (n = 6).  Values in the same column marked by the same letter are not significantly 
different (P < 0.05).
 83 
   Effects of Baking Time and Temperature on the Antioxidant Properties of 
Baked Whole Wheat Pizza Crust.    
During food processing wheat antioxidant activity may be altered 
(Lindenmeier & Hofmann 2004, Leenhardt et al. 2006, Michalska et al. 2008).   
Increasing baking time and temperature increased antioxidant activity in the bread 
crust, but lowered antioxidant activity in the bread crumb portion (Lindenmeier & 
Hofman 2004).  Toasting bread has also been found to increase antioxidant content 
134-367% in bagels, French bread, white bread and whole wheat bread compared to 
their un-toasted counterparts (Halvorsen et al. 2006).  Maillard reaction products have 
received significant attention as they may be potential carcinogens, however study of 
bakery product fractions also indicate that areas subject to the browning reaction have 
higher antioxidative potential in contrast to their corresponding inner counterparts 
(Lindenmeier & Hofman 2004).  It is therefore of interest to analyze the impact of 
thermal treatment on the existing antioxidant compounds in wheat, as well as the 
impact of pizza crust as a baked item with a large surface area. 
 
Total Phenolic Content (TPC) ? Figure 3.1 shows that of the baking 
conditions evaluated Lakin wheat pizza crust total phenolic content (TPC) was more 
stable at shorter heat exposure while TPC of Trego wheat pizza crusts had slightly 
higher although neither increased time nor temperature combinations significantly 
altered TPC content.  TPC values in this study of whole wheat pizza crusts are 
comparable to those of 100% ethanol extracts of Ankor wheat grain (Cheng et al. 
2006b).  Therefore, including whole wheat and wheat bran into foods supports the 
 84 
notion that wheat may serve as a good source for phenolic compounds in functional 
foods as a major constituent of food formulas. 
 
bc
ab
bcc abcabc
a
abc
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal Treatment
TP
C 
(m
g 
GA
E/
g)
Lakin Trego
 Figure 3.1 Effect of thermal treatment on the total phenolic content of 100% 
ethanol extracts of whole wheat pizza crust.  Control represents dough samples not 
baked.  Baking time and temperature combinations consisted of 204 ?C for 7 minutes, 
204 ?C for 14 minutes, and 288 ?C for 7 minutes.  Results are expressed as gallic acid 
equivalents (GAE) per g of sample on a dry weight basis.  All measurements were 
conducted in duplicate with the mean value of the triplicate yeast dose treatment 
combined reported.  Vertical bars represent the standard deviation of each data point.  
Values marked by the same letter are not significantly different (P < 0.05).   
 85 
b
b
a
ab
b
bab
ab
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Control 400 ?C (7 min) 400 ?C (14 min) 550 ?C (7 min)
Thermal Treatment
AB
TS
?+
 S
ca
ve
ng
in
g 
Ca
pa
cit
y 
(????
m
ol
 T
E/
g)
Lakin Trego
 Figure 3.2 Effect of thermal treatment on scavenge capacity of the cation 
ABTS?+ radical content of 100% ethanol extracts of whole wheat pizza crust.  
Results are expressed as Trolox equivalents (TE) per gram of solid state treated bran 
on a dry weight basis.  Control represents dough samples not baked.  Baking time and 
temperature combinations consisted of 204 ?C for 7 minutes, 204 ?C for 14 minutes, 
and 288 ?C for 7 minutes.  All measurements were conducted in duplicate with 
triplicate samples of the yeast/dose combined.  Mean (n = 6) values are reported and 
standard deviations indicated by the vertical bars over each data point.  No significant 
differences were detected (P<0.05).  
 
 Radical Cation ABTS?+ Scavenging Capacity ? Figure 3.2 compares the 
ABTS?+ scavenging capacities of baked whole wheat pizza crusts.  Slight increases 
were observed for all baked pizza crusts compared to their dough controls with the 
exception of minimally processed Lakin pizza crust.  Lakin and Trego based pizza 
crusts showed increased ABTS?+ radical scavenging by 45.3 and 58.8% for extended 
baking time and 45.9 and 45.9% for increased temperature, respectively.  ABTS?+ 
radical scavenging increases were between 42 and 47% for previously reported 50% 
acetone extracts of baked whole wheat pizza crusts (Moore et al. 2008).  ABTS?+ 
 86 
radical scavenging capacity of wheat bran was previously shown to be temperature 
sensitive during wheat storage (Cheng et al. 2006b).  These data suggest that whole 
wheat foods as opposed to wheat bran independently may resist the loss of natural 
antioxidants under elevated temperatures.   
a
aa
bc
a
ab
ab
c
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal Treatment
OR
AC
 (????
m
ol
 T
E/
g)
Lakin Trego
 Figure 3.3 Effect of baking time and temperature on the oxygen radical 
absorbing capacities of whole wheat pizza crusts.  Results are expressed as Trolox 
equivalents (TE) per gram of pizza crust on a dry weight basis.  Control represents 
dough samples not baked.  Baking time and temperature combinations consisted of 
204 ?C for 7 minutes, 204 ?C for 14 minutes, and 288 ?C for 7 minutes.  
Measurements were conducted in duplicate with triplicate samples of the yeast/dose 
combined.  Mean (n = 6) values are reported and standard deviations indicated by the 
vertical bars over each data point.  No significant differences were detected (P<0.05).  
 
 
 Oxygen Radical Absorbance Capacity (ORAC) ? Figure 3.3 shows that time 
and temperature combinations had no significant impact on ORAC values however 
all baked crusts were significantly lower than their dough controls.  ORAC values for 
baked pizza crusts reported here are significantly higher than those reported for whole 
 87 
wheat bread on a per serving basis (Wu et al. 2004).  Taking loss of ORAC activity 
during processing into account, whole wheat pizza crust still has ORAC values which 
may have a positive effect on overall health when consumed as part of a healthy diet. 
 
Radical DPPH Scavenging Capacity (DPPH) ? Figure 3.4 shows the DPPH 
radical scavenging capacity of pizza crusts baked under specified thermal treatments 
and their control.  Results indicate that increasing temperature increased RDSC value 
by 31.8 and 29.8% for Lakin and Trego based pizza crusts, respectively.  In contrast, 
increasing baking time decreased RDSC values by 14.4 and 16.6% for Lakin and 
Trego based pizza crusts, respectively.  While increased temperature did not 
significantly increase RDAC values to their respective controls, values did not 
decrease indicating that shorter exposure to high temperatures may be suitable means 
of food processing to maintain DPPH scavenging capacity.   
 88 
 
e
ab
bcd
de
de
a
abc
cd
0.0
0.1
0.1
0.2
0.2
0.3
0.3
0.4
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal Treatment
RD
SC
 (????
m
ol
 T
E/
g)
Lakin Trego
 
Figure 3.4: Effect of baking time and temperature on the DPPH scavenging 
capacities of whole wheat pizza crusts.  Results are expressed as Trolox equivalents 
(TE) per gram of pizza crust on a dry weight basis.  Control represents dough samples 
not baked.  Treatments were baked in a conventional oven for their respective times 
and temperatures.  Measurements were conducted in duplicate with triplicate samples 
of the yeast/dose combined.  Mean (n = 6) values are reported and standard deviations 
indicated by the vertical bars over each data point.  No significant differences were 
detected (P<0.05).  
 
  
Ferulic Acid Content of Baked Pizza Crusts ? Figures 3.5- 3.7 contain the 
quantity of soluble free, soluble conjugated and bound ferulic acid from whole wheat 
pizza crusts prepared under selected baking conditions.  Significant increases in 
soluble free ferulic acid of 26.6% were demonstrated when pizza crusts were baked at 
204 ? C for 7 minutes as compared with their dough control.  It needs to be pointed 
out that pizza crusts prepared with the Trego wheat variety had a significant decrease 
in soluble free ferulic acid; however neither extending baking time nor increasing 
 89 
baking temperature demonstrated further reduction over basic baking conditions.  
Soluble conjugated ferulic acid was significantly decreased for all baking time and 
temperature combinations of pizza crusts prepared with the Trego variety but not for 
Lakin (Fig 3.6).  Insoluble bound ferulic acid remained unaffected by thermal 
processing (Fig. 3.7).  It is interesting to note that baked pizza crusts prepared with 
the Lakin variety had significantly more soluble conjugated ferulic acid, while baked 
pizza crusts prepared with the Trego variety had significantly more bound ferulic 
acid.  Previous studies of the Trego variety have indicated it?s unique potential to 
inhibit lipid peroxidation, which was not explained by its radical scavenging and 
chelating activities (Yu et al 2002).  These data indicate that wheat variety differences 
in antioxidant activity may be carried over into whole wheat functional foods.  
Figure 3.5: Effect of baking time and temperature on soluble free ferulic acid of 
whole wheat pizza crusts* 
bbc
c
a aaa
b
0.0
1.4
2.8
4.2
5.6
7.0
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal treatments
So
lub
le 
fre
e f
er
uli
c a
cid
 (?
g/g
)
Lakin
Trego
 
 
 
 90 
Figure 3.6: Effect of baking time and temperature on soluble conjugated ferulic 
acid of whole wheat pizza crusts* 
 
 
Figure 3.7: Effect of baking time and temperature on insoluble bound ferulic 
acid of whole wheat pizza crusts* 
 
Figures 3.5 ? 3.7* Results are expressed as Trolox equivalents (TE) per gram of 
pizza crust on a dry weight basis.  Control represents dough samples not baked.  
Baking time and temperature combinations consisted of 204 ?C for 7 minutes, 204 ?C 
for 14 minutes, and 288 ?C for 7 minutes.  Measurements were conducted in duplicate 
with triplicate samples of the yeast/dose combined.  Mean (n = 6) values are reported 
and standard deviations indicated by the vertical bars over each data point.  No 
significant differences were detected (P<0.05).  
aa
aa
bbbb
0.0
80.0
160.0
240.0
320.0
400.0
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal treatments
In
so
lub
le 
bo
un
d f
er
uli
c a
cid
 (?
g/g
)
Lakin Trego
bcbc
bcc
a
a
a
b
0.0
5.0
10.0
15.0
20.0
25.0
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal treatments
So
lub
le 
co
nju
ga
ted
 fe
ru
lic
 ac
id 
(?
g/g
)
Lakin Trego
 91 
 
Conclusion 
 Results from this study indicate that whole wheat pizza crusts maintain 
significant antioxidant activity following food processing.  The pizza crusts prepared 
from two varieties of wheat, Lakin and Trego, differed significantly in soluble free 
and bound ferulic acid indicating that significant differences in antioxidant activities 
of wheat variety may be carried over in finished products.  Reducing of bran particle 
size may not result in the deterioration of antioxidant activity in the pizza crust model 
suggesting that reduced particle size may be one means of improving whole wheat 
product sensory products, while controlling for the loss of natural antioxidants.  
Antioxidant activities were also generally stable through extended fermentation times 
indicating the possibility to develop functional wheat based foods with sufficient rise 
and unique flavor character profiles.  Further studies are required to understand the 
thermal processing effects on whole wheat food systems and to optimize conditions 
for desired sensory and health beneficial properties.    
 
 92 
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Cheng, Z.; Moore, J.; Yu, L. A high-throughput relative DPPH radical scavenging 
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Mellen, P.B.; Walsh, T.F.; Herrington, D.M. Whole grain intake and cardiovascular 
disease: A meta-analysis.  Nutrition, Metabolism & Cardiovascular Disease. 
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Michalska, A.; Amigo-Benavent, M.; Zielinski, H.; Dolores del Castillo, M. Effect of 
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Moore, J.; Luther, M.; Cheng, Z.; Yu, L. Effects of baking conditions, dough 
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Stanner, S.A.; Hughers, J.; Kelly, C.N.M.; Buttriss, J. A review of the 
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Zhou, K.; Laux, J. J.; Yu, L. Comparison of Swiss red wheat grain, and fractions for 
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 95 
Chapter 4: In-vitro Bioavailability of Wheat Antioxidants in 
Baked Pizza Crust. 
Abstract 
 This study evaluates the effect of baking time and temperature on the in-vitro 
bioavailability of wheat antioxidants in baked pizza crusts.  Pizza crusts were 
prepared under three baking conditions of 7 minutes at 400 ?F, 14 minutes at 400 ?F, 
and 7 minutes at 550 ?F.  After in-vitro digestion, the supernatant was separated from 
the solid residue.  Acetone was added to the supernatant for clarification and re-
centrifuged resulting in 37.5% acetone supernatant solutions.  The supernatant 
solutions were then analyzed for total phenolic content (TPC), and radical scavenging 
activity against peroxyl and hydroxyl anion and the ABTS?+ cation radicals.  The 
available total phenolic content ranged between 2.8 and 5.2 gallic acid equivalents 
(GAE) for dough and thermal treatment of 288 ?C for 7 minutes, respectively.  
Likewise the ABTS?+  scavenging capacity increased with thermal treatment with a 
range of 3.6 ? 4.5 ?moles trolox equivalents (TE)  per gram  pizza crust, oxygen 
radical absorbance capacity had a range of 112.2  ? 190.0 ?moles TE/g, and hydroxyl 
radical scavenging had a range of 77.4 ? 174.0 ?moles TE/g.  Pizza crust samples 
prepared under higher temperatures or longer baking times had higher in-vitro 
bioavailable TPC and ABTS?+ scavenging capacity.  In-vitro bioavailability of ORAC 
and HOSC antioxidant capacity assays also suggest that thermally treated samples 
might have greater available antioxidant activities than that of their controls.  
 96 
Understanding the bioavailability of antioxidants from the food matrix may assist in 
the development of physiologically active functional foods. 
 
Introduction 
It is now recommended that Americans consume at least 3 or more once-
equivalence of whole grains per day in order to reduce the risk of several chronic 
diseases and assist with weight management (USDA 2005).  Wheat is a prominent 
ingredient for the Western diet with intake of approximately 133 pounds per person 
annually in the United States (USDA-ERS 2006).  Epidemiological studies provide 
support that whole foods, such as those prepared with whole grains, have a greater 
effect against a broad range of diseases than isolated components (Slavin, 2003).   
Antioxidant research on Swiss red wheat has determined that the aleurone 
portion of the wheat kernel has the highest level of antioxidants followed by bran and 
whole grain (Zhou et al. 2004).  Multiple wheat varieties contain antioxidants with 
the ability to chelate Fe2+ and Cu2+, and quench anion, neutral, and cation free 
radicals (Zhou & Yu 2004, Zhou et al. 2004, Yu & Zhou 2004, and Zhou et al. 2005).  
This antioxidant activity may be due to the presence of phenolic compounds such as 
flavanoids, phenolic acids, and catechins as found in the bran of whole grains (Adom 
et al. 2005, Ross et al. 2004).  Phenolic acids are generally found in the bound form 
and it is unclear what portion of these phenolics is available for absorption in the 
digestive tract (Kern et al. 2003, Zhou et al. 2004, Price et al. 2008).   
 97 
Post harvest treatment and food processing may have an effect on the 
available antioxidants from wheat.  Storing wheat as a whole grain for long-term 
storage may help prevent the loss of natural antioxidants (Cheng et al. 2006).  For 
immediate use, reduction of particle size increases bioavailability of phenolics 
(Cheng et al. 2006).  Post harvest treatment of wheat bran with enzymes and yeasts 
have demonstrated the potential for solid state treatment to increase the proportion of 
soluble free antioxidants from their native bound form (Moore et al. 2006, Moore et 
al. 2007).  Not all compounds and types of processing increase antioxidant activity as 
demonstrated by Leenhart et al. who found that carotenoids levels decreased during 
kneading, fermentation, and baking (Leenhart et al. 2006).  Research conducted 
previously in our laboratory has shown that increasing baking time or baking 
temperature increases the total phenolic content and antioxidant activity of whole-
wheat pizza crusts (Moore 2008).   
Limited studies have been conducted to measure the in vivo impact of 
consuming whole wheat products or wheat bran.  In vitro research has shown that the 
total phenolic content (TPC) and total antioxidant activity (TAA) of wheat extracts 
was increased after simulated pH digested conditions (Liyanapathirana & Shadidi 
2004).   Following high bran cereal consumption limited ferulic and sinapic acids 
were taken up by humans and authors concluded that in this food model, bound 
phenolic acids were not absorbed (Kern et al. 2003).  Higher plasma uptake of 
phenolics was detected in untreated wheat bran indicates that wheat bran phenolics 
are relatively well absorbed with plasma total phenolic increases comparable to red 
wine and spinach (Price et al. 2008).  These two reports utilizing 6 and 17 human 
 98 
subjects are the only studies we know of analyzing the absorption of phenolics from 
wheat by humans.   
A prominent item in the western-diet, this study analyzes the effects of 
thermal treatment on in-vitro bioavailability of antioxidants from whole wheat pizza 
crusts.  In-vitro bioavailability of antioxidants was then measured by analyzing 
supernatants for their total phenolic content, and radical scavenging activity against 
the peroxyl, hydroxyl, and ABTS?+ radicals.   
 
Materials and Methods 
Digestive fluid chemicals; potassium chloride, magnesium chloride, D?
gluceronic acid, D(+)-glucosamine hydrochloride, bile from bovie and ovine, and 
pepsin as well as reagents mixed tocopherols, 2,2-diphenyl-1-picryhydrazyl radical 
(DPPH?), 2,2?-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt 
(ABTS),  6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (trolox), 
disodium ethylenediaminetetracetate, pancreatin, and 2,2?-bipyridyl gallic acid were 
purchased from Sigma-Aldrich (St. Louis, MO).  Urea, ammonium chloride, sodium 
phosphate, sodium chloride, calcium chloride, and potassium thiocyanate were 
purchased from Fisher Scientific and were used without further purification.  Sodium 
bicarbonate and sodium sulfate were purchased through J.T. Baker (Phillipsburg, NJ), 
D(+)-glucose and uric acid were purchased through Acros (Morris Plains, NJ).  Folin-
Ciocalteu reagent was freshly prepared in the laboratory.  All other chemicals and 
solvents were of the highest commercial grade and used without further purification. 
 99 
Preparation of pizza crust samples 
Pizza crusts were prepared from whole-grain flours of Trego and Lakin hard 
winter wheat varieties provided by Dr. Scott Haley in the Department of Soil and 
Crop Science, Colorado State University, Fort Collins, CO 80523.  Briefly, the bran 
fraction of the grain was separated and ground to a 20-mesh using a coffee grinder 
and recombined to create whole-grain flour.  Whole-grain flour, water, honey, 
soybean oil, dry active yeast (Fleischmann?s Fenton, Missouri), and salt were then 
combined on a w/w ratio of 52.04, 33.24, 10.30, 2.34, 1.12 and 0.95 grams 
respectively.  Dough was then mixed with a Kitchen-Aid stand mixer for 1 minute on 
low speed, followed by 3 minutes of hand kneading.  250 g pieces were then rounded 
and placed onto greased stainless steel containers and allowed to ferment 18 hrs. at 4 
?C.  Dough was incubated at room temperature for 2 hrs. prior to hand rolling into 12 
inch crusts.  Crusts were baked on greased aluminum pizza screens in a conventional 
oven at baking combinations of; 7 minutes at 208 ?C, 14 minutes at 208 ?C, and 7 
minutes at 288 ?C.  After cooling for 30 minutes baked samples as well as the control 
(unbaked fermented dough), were frozen, freeze-dried, and ground to a 40-mesh. 
 
In-vitro Digestion  
The purpose of the in-vitro digestion model is to simulate the digestion 
process in the gastrointestinal tract by replicating physiological conditions.  This 
model is based upon the models introduced by Oomen et al. (2003), Versantvoort et 
al. (2005), and the United States Pharmacopeia specifications for digestive fluids.  
 100 
Digestive fluids were prepared according to fed-state conditions (Table 4.1).  Nine 
grams of pizza crust equivalent was used per sample by re-hydration of the sample to 
25% moisture, the average moisture content of baked pizza crust.  Duplicate samples 
of 40-mesh, freeze dried pizza crust were weighed into 50 mL disposable test tubes.  
Figure 4.1 depicts the manner in which digestive fluids were added, mechanical 
treatment of samples, and the pH at which all stages occurred.  Water was added to 
simulate the salivary portion of digestion in lieu of alpha-amylase as preliminary 
studies found no significant difference in the antioxidant level available from samples 
treated with water compared to those treated with alpha-amylase (data unpublished, 
Moore 2006).  Samples were kept in the dark under nitrogen at ambient temperature 
for analysis.   
 
 
 
 
 
 
 
 
 101 
 
Table 4.1: Fed-state synthetic digestive fluid composition* 
 Gastric fluid Duodenal fluid Bile fluid 
Inorganic solutions 15.7 mL 2.99 M NaCl 40 mL 2.99 M NaCl 15 mL 2.99 M NaCl 
 3 mL 740.12 mM NaH2PO4 40 mL 1.01M NaHCO3 34.15 mL 1.01M NaHCO3 
  9.2 mL 1.20 M KCl 10 mL 58.78 mM KH2PO4 2.1 mL 1.20 M KCl 
 18 mL 151.04 mM CaCl2-2H20 6.3 mL 1.20 M KCl 0.075 mL 1.02 M HCl 
 10 mL 572.07 mM NH4Cl 10 mL 52.53 mM MgCl2-6H2O  
 6.5 mL 1.02 M HCl 0.18 mL 1.02 M HCl  
    
Organic solutions 10 mL glucose 4 mL urea 5 mL urea 
 10 mL glucuronic acid   
 3.4 mL urea   
 10 mL glucosamine HCl   
    
Enzymes & final 
solution add-ins 3200 mg pepsin 2500 mg pancreatin 15,200 mg 50% bile 
  9 mL CaCl2-H2O 5 mL CaCl2-H2O 
    
Fluid pH 1.3 ? 0.02 8.1 ? 0.02 8.1 ? 0.02 
 102 
 
Figure 4.1 In-vitro digestion.   
Total phenolic content 
Total phenolic content of the digested pizza crust samples was measured using 
the Folin-Ciocalteu reagent according to a laboratory procedure (Yu et al. 2002).  The 
final reaction mixture contained 100 ?L of digested pizza crust, 500 ?L of the Folin-
Ciocalteu reagent, 1.5 mL of 20% sodium carbonate and 1.5 mL pure water.  The 
absorbance at 765 nm was recorded after two hours incubation at ambient 
Digestion Component pH Mechanical Treatment
9 g pizza crust equivalent
6 mL water
12 mL gastric fluid
12 mL duodenal fluid
6 mL bile fluid
2 mL 1M NaHCO3
15 mL acetone
6.8
2-3
6.5-7
stir 30 s
vortex 30 s, incubate, shaking at 60 rpm 2 h
shake violently 4 times, invert tube 75 times, 
incubate 2 h, shaking at 60 rpm
centrifuge 5 min at 2750 rcf
collent supernatent
vortex, centrifuge 5 min at 2750 rcf
collent supernatent
antioxidant analysis of 
digested baked pizza crust
 103 
temperature.  A gallic acid standard was utilized to calculate the phenolic content.  
Duplicate measurements per digested pizza crust sample extract were taken. 
 
Radical Cation ABTS?+ scavenging activity 
 The 37.5% acetone extracts of pizza crust were tested against ABTS?+ which 
was generated according to a laboratory protocol (Miller, Rice-Evans 1997, Zhou and 
Yu 2004).   Briefly, an ABTS?+ solution was prepared by oxidizing a 5 mM aqueous 
solution of ABTS with manganese dioxide 30 minutes at ambient temperature.  The 
solution was then filtered and adjusted with phosphate buffer to an absorbance of 0.7 
at a wavelength of 734 nm.  A trolox standard was prepared with 37.5% acetone and 
the blank consisted of 1mL phosphate buffer and 80 ?L water blank from the in-vitro 
digestion model.  Likewise the final reaction mixture contained 1mL of the ABTS?+ 
solution , and 80 ?L of the sample of trolox standard.  The reaction mixture was 
vortexed 30 seconds and a reading recorded at 1.5 minute.  Data is reported as trolox 
equivalents per gram of digested pizza crust as calculated using the trolox standard 
curve.   
 
Oxygen radical absorbing capacity (ORAC) 
ORAC was conducted for 37.5% acetone extracts of the digested pizza crust 
samples using the fluorescent probe flourescein (FL) according to laboratory protocol 
(Huang et al. 2002, Zhou and Yu 2004).  Trolox standards and samples were prepared 
 104 
in 37.5% acetone.  AAPH and FL solutions were prepared with 75 mM phosphate 
buffer (pH 7.4).  The final reaction mixture per well contained 225 ?L of 81.63 nM 
FL solution, 30 ?L blank (37.5% acetone), standard, or sample solution, and 25 ?L 
0.36 M AAPH solution.  A total volume of 280 ?L was constant for all wells.  
Standards and samples were run in duplicate simultaneously utilizing a Victor2 
multilabel counter (PerkinElmer, Turku, Finland).  The reaction mixture was held at 
37 ?C and fluorescence measurements taken every minute at excitation and emission 
wavelengths of 485 nm and 530 nm respectively for 1.5 hours.  Results were 
expressed as trolox equivalents (TE) calculated using the relative area under the curve 
for the samples as compared to the trolox standard curve run simultaneously.  
Samples of the same thermal treatment were combined and reported as the mean and 
the standard deviation.   
 
Hydroxyl Radical Scavenging Capacity 
Hydroxyl radical scavenging capacity was measured by oxidation by the 
hydroxyl radical generated by ferric iron (Fe3+) and hydrogen peroxide and described 
previously (Moore 2006).  Briefly, trolox standards were prepared in 37.5% acetone 
and pure acetone was used as the blank.  Distilled water was used to prepare an 3.43 
mM iron chloride (FeCl3) solution and a 0.1990 M H2O2 solution.  A 92.8 ?M 
solution of flourescein (FL) was prepared using phosphate buffer.  The final well 
concentration was 300 ?L per well consisting of 170 ?L FL solution, 30 ?L blank, 
standard, or sample, 40 ?L H2O2 and 60 ?L FeCl3.  Degradation of the FL solution 
was measured every minute using a Victor2 multilabel counter (PerkinElmer, Turku, 
 105 
Finland).  Trolox equivalents were calculated using the area under the curve as 
compared with the trolox standard run simultaneously with the samples.  Data is 
reported as the mean of the thermal treatments and their standard deviations.   
 
Statistical analysis   
Data were reported as mean and standard deviation for triplicate 
measurements.  Analysis of variance and Tukeys honestly significant difference tests 
were conducted (SPSS for Windows, Version Rel. 10.0.5., 1999, SPSS Inc., Chicago, 
IL) to determine differences among means.  Statistical significance was declared at P 
< 0.5.   
 
Results and Discussion 
Total Phenolic Content 
In-vitro bioavailable total phenolic content (TPC) of pizza crusts are 
expressed as mg gallic acid equivalents (GAE) per gram of pizza crust.  Baked pizza 
crust had significantly lower bioavailable TPC than the corresponding dough control, 
however extending baking time or increasing baking temperature increased in in-vitro 
bioavailable TPC (Figure 4.2).  Previous TPC analysis of 50% acetone extracts of 
baked whole wheat pizza crusts showed increased TPC values as thermal treatment 
increased (Moore et al. 2007).   
 106 
b
b
a
c
bb
a
c
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
TP
C 
(m
g G
AE
/g)
Lakin
Trego
 
Figure 4.2. In-vitro bioavailable total phenolic contents (TPC) of baked pizza 
crusts.  Results are expressed as milligrams gallic acid equivalents (GAE) per gram 
of sample on a dry weight basis.  Controls were unbaked pizza dough samples.  
Baking time and temperature combinations consisted of 204 ?C for 7 minutes, 204 ?C 
for 14 minutes, and 288 ?C for 7 minutes.  All measurements were conducted in 
duplicate and triplicate values of thermal treatment combined to report the mean (n = 
6) and standard deviation.  Vertical bars represent the standard deviation of each data 
point.  Values marked by the same letter are not significantly different (P < 0.05).   
 
Radical Cation ABTS?+ scavenging activity 
ABTS?+ scavenging activity ranged from 3.55 to 4.55 ?moles trolox 
equivalents (TE) per gram of pizza crust (Figure 4.3).  The greatest ABTS?+ 
scavenging activity was observed in the in-vitro digested pizza crust prepared with 
Lakin wheat at the increased thermal treatment of 288 ?C for 7 minutes and 204 ?C for 
14 minutes.  The ABTS?+ scavenging activity was statistically different between the 
two varieties, however the trend for the Trego variety was the same in that ABTS?+ 
 107 
scavenging activity increased with thermal treatment.  This finding is supported by 
previous data that found different varieties of Maryland grown soft wheat to have 
significantly different ABTS?+ scavenging activity (Moore et al. 2005).   
 
dd
ab
cd
bcab
a
bc
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
dough 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal Treatment
AB
TS
+ S
ca
ve
ng
ing
 C
ap
ac
ity
 
(????
m
ol 
TE
/g 
)
Lakin Trego
 
Figure 4.3 In-vitro bioavailable ABTScircle3circle3circle3circle3+ scavenging capacities of baked pizza 
crust.  Results are expressed as ?moles trolox equivalents (TE) per gram of sample 
on a dry weight basis.  Controls were unbaked pizza dough samples.  Baking time and 
temperature combinations consisted of 204 ?C for 7 minutes, 204 ?C for 14 minutes, 
and 288 ?C for 7 minutes.  All measurements were conducted in duplicate and 
triplicate values of thermal treatment combined to report the mean (n = 6) and 
standard deviation.  Vertical bars represent the standard deviation of each data point.  
Values marked by the same letter are not significantly different (P < 0.05).   
 108 
a
a
a
cd
d
ab
bc bc
0
50
100
150
200
250
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal treatment
OR
AC
 (T
E 
?m
ol
es
/g
)
Lakin
Trego
 
Figure 4.4 In-vitro bioavailable ORAC scavenging capacities of baked pizza 
crust.  Results are expressed as ?moles trolox equivalents (TE) per gram of sample 
on a dry weight basis.  Controls were unbaked pizza dough samples.  Baking time and 
temperature combinations consisted of 204 ?C for 7 minutes, 204 ?C for 14 minutes, 
and 288 ?C for 7 minutes.  All measurements were conducted in duplicate and 
triplicate values of thermal treatment combined to report the mean (n = 6) and 
standard deviation.  Vertical bars represent the standard deviation of each data point.  
Values marked by the same letter are not significantly different (P < 0.05).   
 
Oxygen radical absorption capacity (ORAC)  
The ORAC assay measured the capacity of in-vitro digested pizza crust 
samples against the peroxyl radical.  Samples with higher ORAC values may exert a 
protective effect for biomolecules against free radicals by donating a hydrogen atom.  
ORAC values are expressed as ?moles trolox equivalents (TE) per gram of pizza 
crust (Figure 4.4).  ORAC values were significantly higher at 190.0 and 170.8 
 109 
?moles TE/g for digested pizza crusts that had been baked at 288 ?C for 7 minutes for 
Lakin and Trego varieties, respectively than for other treatments.  It is significant to 
note that the ORAC for all digested, baked samples is greater than their 
corresponding dough samples with ORAC values of 112.2 and 132.6 ?moles TE/g  
for Lakin and Trego, respectively.  
 
Hydroxyl radical scavenging capacity (HOSC) values 
a ab
d
b
ab
bc cd
d
0
50
100
150
200
250
Control 204 ?C (7 min) 204 ?C (14 min) 288 ?C (7 min)
Thermal treatment
HO
SC
 (T
E 
?m
ol
es
/g
) Lakin
Trego
 
Figure 4.5 In-vitro bioavailable HOSC scavenging capacity of antioxidants from 
whole wheat pizza crust.  Results are expressed as ?moles trolox equivalents (TE) 
per gram of sample on a dry weight basis.  Controls were unbaked pizza dough 
samples.  Baking time and temperature combinations consisted of 204 ?C for 7 
minutes, 204 ?C for 14 minutes, and 288 ?C for 7 minutes.  All measurements were 
conducted in duplicate and triplicate values of thermal treatment combined to report 
the mean (n = 6) and standard deviation.  Vertical bars represent the standard 
deviation of each data point.  Values marked by the same letter are not significantly 
different (P < 0.05).   
 
 110 
Hydroxyl radical scavenging capacity values for the digested pizza crust 
samples are reported as ?moles trolox equivalents/g digested pizza crust on a dry 
weight basis (Figure 4.5).  Results corresponded with ORAC data as there were 
significant increases in HOSC as thermal treatment increased.  The highest HOSC 
values were 160.6 and 174.0 ?moles TE/g for digested pizza crusts that had been 
baked at 288 ?C for 7 minutes for Lakin and Trego varieties, respectively than for 
other treatments.  The lowest, yet significant HOSC values were the dough samples at 
77.4 and 97.3 ?moles TE/g for the Lakin and Trego varieties, respectively.   
 
Conclusion 
Results from this study demonstrate for the first time that in-vitro 
bioavailability of wheat antioxidants is increased when baking time or temperature 
was increased in baked whole wheat pizza crust.  Results indicate that a food with 
higher available antioxidants may have a greater level of bio-available antioxidants.  
More research is required to determine if in-vitro methods for antioxidant analysis are 
correlated in-vivo measures such as a decrease in blood oxidative status.  In addition, 
correlating the extractable components with bioavailability may lead to economic 
means of analysis to predict the health benefits from antioxidants in food systems.       
 
 111 
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Cheng, Z.; Su, L.; Moore, J.; Zhou, K.; Luther, M.; Yin, J.; Yu, L. Effects of 
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Moore, J.; Cheng, Z.; Hao, J.; Guo, G.; Liu, J.-G.; Lin, C.; Yu, L. Effects of solid-
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Moore, J.; Cheng, Z.; Su, L.; Yu, L. Effects of solid-state enzymatic treatments on the 
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Moore, J.; Luther, M.; Cheng, Z.; Yu, L. Effects of baking conditions, dough 
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Price, R.K., Welch, R.W., Lee-Manion, A.M., Bradbury, I., Strain, J.J. Total phenoli 
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Yu, L.; Zhou, K. Antioxidant properties of bran extracts from 'Platte' wheat grown at 
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Zhou, K.; Yin, J.; Su, L.; Haley, S.; Yu, L. Phenolic acid, tocopherols, and 
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 113 
APPENDIX A. Yeast Log Growth Curve* 
0
0.5
1
1.5
2
0 5 10 15
Time hrs.
Ab
so
rb
an
ce
 (6
00
nm
) Group12-Ave
Group13-Ave
Group14 Ave
Group15-Ave.
Group16-Ave.
Group17-Ave
Group18-Ave
Group19-Ave
Group20-Ave.
 
* Log growth curves were conducted for all yeasts.  Yeasts were later presumed to be 
in logarithmic growth between 6 and 10 hrs. following the same pre-treatment 
conditions.    
 114 
Chapter 5:  Development of a Tri-fold to Increase Whole Wheat 
Pizza Crust Consumption in College Students 
 
Abstract 
 Print materials are an effective means of distributing information.  A tri-fold 
was developed to promote the preparation and consumption of whole wheat pizza 
among college students.  Tri-fold information sheets including a whole wheat pizza 
dough recipe, three healthy recipe topping sets with nutritional information, and 
relevant health claims and nutritive definitions were developed.  Coupling health 
claims with popular foods items may be an effective way to increase nutritional 
quality of collegiate diets.      
 
Introduction 
The United States and other developed nations are recording alarming trends 
in obesity with cardiovascular disease and cancer remaining the top two causes of 
mortality.  The American Dietetic Association states that the ?total diet or overall 
pattern of food eaten is the most important focus of a healthful eating style (Nitzke & 
Freeland-Graves 2007).? Healthful recipes and directions can help overcome barriers 
to preparing meals at home such as poor cooking skills, time constraints, cost, and 
available ingredients (Cunningham 2007).  Hands on cooking activities have 
successfully increased cooking skills, confidence, and eating habits of preschool 
 115 
children and their families, college students, and WIC participants (Bensley et al. 
2006, Condrasky et al. 2006, Dougherty & Silver 2007, Michaud et al. 2007).   
Adolescents and young adults transitioning from parental supervision to 
independent living are developing their eating behaviors and dietary practices which 
will affect their health (Larson et al. 2007, Richards et al. 2006).  A study of young 
adults found that young adults that purchased their own food and prepared meals at 
home had improved diet quality over there counterparts who did so less frequently 
(Larson et al 2006a, Larson et al 2006b).  Targeting nutrition education at young 
adults and college students is particularly important as 19-34 yr. olds are less 
knowledgeable about diet-cancer connections, National Cancer Institute dietary 
recommendations, and composition of foods (Georgiou et al. 1997).   
 116 
 
Figure 5.1 Hierarchy of knowledge, psychomotor skill, and food related sensory 
attributes leading to increased consumption of functional foods.  Knowledge 
related attributes based upon the hierarchy of nutritional knowledge by Brian 
Wansink (Wansink et al. 2005). 
 
This addresses the critical issues, ?People do not know foods to eat for a 
balanced diet (Kim et al 2006).? Effective public health practice is dependent upon 
scientists and other health professional?s ability to appropriately communicate to 
diverse audiences, the majority of which are individuals outside the scientific 
community (Nelson 2002).  Figure 5.1 illustrates the belief that when multiple 
barriers are overcome such as nutritional knowledge, food preparation ability, and 
sensory preferences, functional foods are more likely to be consumed.  Materials that 
Consumption  
Consequences  
Knowledge 
Food-Specific  
Attribute 
Knowledge 
No Knowledge 
Sen
sor
y
Fo
od
 Pr
ep
ara
tio
n S
kill
Consumption 
 117 
allow participants to be interactive, prepare pizzas? at home, may encourage more 
skill development and behavior change than cognitive knowledge alone. 
 
Tri-fold Development 
Tri-folds were developed for easy print material distribution to encourage the 
production and consumption of whole wheat pizza (Figures 5.2 & 5.3).  Tri-folds 
were tested in house for general appearance acceptability and readability.  
Information included on the tri-fold was: whole wheat pizza crust recipe and 
directions, three recipe sets for final pizza products, nutrition facts, whole wheat 
health claims and suggested intake, and findings from our laboratory to promote food 
preparation practices that maintain or increase antioxidant availability. 
 118 
 
Figure 5.2 Functional Whole Wheat Pizza Tri-fold ? Side A 
 119 
 
 
 
Figure 5.3 Functional Whole Wheat Pizza Tri-fold ? Side B
 120 
 
Pizza Recipes 
Pizza recipes were developed in the Laboratory of Nutraceuticals & 
Functional Foods at the University of Maryland, College Park.  Suggested pizza 
dough fermentation and baking times were selected based on our tests to analyze the 
effects of processing on the antioxidant levels of whole wheat pizza crust.  The 
effects of baking time and temperature on sensory products were also tested in-house 
to ensure that quality of the final product.  Three pizza recipes: Sweet & Savory 
Slices (SS), Herb Garden Fresh Pizza (HG), and Creamy Pesto Pizza (CP) were 
developed and tested in-house for quality.  Nutritional facts were generated using 
Diet Analysis Plus 6.0 (Salem, OR) and reported on a per serving basis.  The goal of 
the variety of these recipes was to: provide recipes for which there was a degree of 
familiarity among college students (SS), provide a vegetable based healthier option 
(HG), and suggest alternative sauces to those who may have diet restrictions (CP).   
 
Health Claims 
Health claims are intended to, ?benefit consumers by providing information 
on healthful eating patterns that may help reduce the risk of heart disease, cancer, 
osteoporosis, high blood pressure, dental cavities, or certain birth defects 
(Arvanitoyannis & Houwelingen-Koukaliaroglou 2005).?  The nutritional impact of 
inclusions of health claims on products is not well established.  Consumers generally 
perceive health claims as helpful, are more likely to purchase items with health 
 121 
claims, and may make healthier dietary choices, however the accuracy and 
truthfulness of health and nutrition claims is widely questioned amongst all 
populations (Marietta 1999, Williams 2005, Misra 2007).  Nonetheless, health claims 
were provided in the tri-fold to provide readers with food-specific attribute 
knowledge and consumption consequences knowledge.   
 
Conclusion 
Outreach materials containing both consumption consequence knowledge 
(health claims) and food-specific attribute knowledge (antioxidants) may increase 
consumption over outreach materials with only one form of knowledge.  Simple 
recipes and directions for commonly consumed foods may increase at-home food 
preparation which has been correlated with increased nutritive value of foods.  This 
research indicates that creating outreach materials for functional forms of commonly 
consumed items may increase their adaptation.  More research is required to 
document the effect of recipe outreach methods for college students.     
   
 
 
 122 
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