1

Author: Filbrandt, Katelyn R.

Title: Effect of Pumpkin Seed Oil Cake on the Textural and Sensory Properties of White Wheat

Bread

The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School

in partial completion of the requirements for the

Graduate Degree/ Major: MS Food & Nutritional Sciences

Research Adviser: Hans Zoerb, Ph.D.

Submission Term/Year: Spring, 2012

Number of Pages: 70

Style Manual Used: American Psychological Association, 6th

edition

I understand that this research report must be officially approved by the Graduate

School and that an electronic copy of the approved version will be made available through

the University Library website

I attest that the research report is my original work (that any copyrightable materials

have been used with the permission of the original authors), and as such, it is automatically

protected by the laws, rules, and regulations of the U.S. Copyright Office.

My research adviser has approved the content and quality of this paper.

STUDENT:

NAME Katelyn R. Filbrandt DATE: 02/22/12

ADVISER: (Committee Chair if MS Plan A or EdS Thesis or Field Project/Problem):

NAME Hans Zoerb, PhD DATE: 2/22/12

---------------------------------------------------------------------------------------------------------------------

This section for MS Plan A Thesis or EdS Thesis/Field Project papers only

Committee members (other than your adviser who is listed in the section above)

1. CMTE MEMBER’S NAME: DATE:

2. CMTE MEMBER’S NAME: DATE:

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This section to be completed by the Graduate School

This final research report has been approved by the Graduate School.

Director, Office of Graduate Studies: DATE:

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Filbrandt, Katelyn R. Effect of Pumpkin Seed Oil Cake on the Textural and Sensory

Properties of White Wheat Bread

Abstract

Pumpkin seed cake (PSC) is an underutilized by-product from pumpkin seed oil

extraction. It is mainly used for an addition to animal feed. It could be a potential ingredient for

increasing the antioxidant, protein and unsaturated fatty acid content in food products, such as

bread. The pumpkin seed cake bread was made in an attempt to match a control wheat bread.

The PSC bread dough had a water/flour absorption ratio of 90/120 to reach 550 PUs

(promylograph units) and the control had a water/flour absorption ratio of 79/120 to reach 530

PUs. A significant difference was found between the heights of the PSC bread (54.65mm) and

control bread (77.07mm) indicating that the control bread had better gas holding properties than

did the PSC bread. Results for the texture analysis for the control were a modulus of

1.093millapascals (mPa) and a maximum compressive stress of 0.242mPa. The results for the

PSC bread indicated that it had a firmer crumb than the control, with a modulus of 2.733mPa and

a maximum compressive stress of 0.796mPa. Over 100 panelists participated in a sensory test

and evaluated both a piece of the control bread and a piece of PSC bread. There was no

significant difference in the sensory test for the perceived aftertaste rating (p<.5). However,

there was a significant difference for the nutty flavor, overall flavor, overall texture, and overall

acceptance ratings (p < 0.05). Therefore, bread can be made with PSC, but may need

reformulation to improve consumer preference acceptability.

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Acknowledgments

I would like to thank God, Dr. Hans Zoerb, Dr. Lamin Kassama, Dr. Carolyn Barnhart,

Dr. Renee Howarton, Dr. Cynthia Rohrer, Connie Galep, Ken Seguine and Jay Gilbertson from

Hay River Foods, Menomonie Market Food Co-op, parents, family, and friends.

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Table of Contents

.................................................................................................................................................... Page

Abstract ............................................................................................................................................2

List of Tables ...................................................................................................................................7

List of Figures ..................................................................................................................................8

Chapter I: Introduction .....................................................................................................................9

Statement of the Problem ...................................................................................................10

Research Objectives ...........................................................................................................10

Assumptions of the Study ..................................................................................................11

Definition of Terms............................................................................................................11

Limitations of the Study ....................................................................................................11

Methodology .......................................................................................................................11

Chapter II: Literature Review ........................................................................................................12

History of Pumpkin and Pumpkin Species ........................................................................12

Oil Pumpkin Cultivation Factors .......................................................................................13

Processing of Pumpkin Seed and Oil Extraction ...............................................................14

Physiochemical Characteristics of Pumpkin Seed Oil .......................................................16

Chemical Properties of Pumpkin Seed Oil ........................................................................17

Oxidative Stability .............................................................................................................18

Composition of Pumpkin Seeds and PSC ..........................................................................27

Health Claims of Pumpkin Seed Oil ..................................................................................28

Bread ..................................................................................................................................28

Dough Rheology ................................................................................................................29

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Pumpkin Seed Cake in Bread ............................................................................................32

Bread Rheology .................................................................................................................32

Sensory Analysis of Bread .................................................................................................33

Chapter III: Methodology ..............................................................................................................35

Flour Water Absorption .....................................................................................................35

Development of the Control Dough/Bread ........................................................................36

Bench Top Bread Manufacture ..........................................................................................37

Data Collection ..................................................................................................................41

Data Collection Procedures ................................................................................................46

Chapter IV: Results and Discussion ..............................................................................................47

PSC Proximate Analysis ....................................................................................................47

Flour Moisture Absorption ................................................................................................47

Final Dough Formulae .......................................................................................................49

Bread Volume and Dimensions .........................................................................................51

Bread Texture Data ............................................................................................................52

Sensory Analysis ................................................................................................................52

Appearance ........................................................................................................................55

Moisture Content ...............................................................................................................55

Fat Analysis .......................................................................................................................56

Chapter V: Conclusion ...................................................................................................................57

Volume and Texture Relationship .....................................................................................57

Sensory ...............................................................................................................................58

Sensory and Texture Relationship .....................................................................................59

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Economic Value .................................................................................................................59

Recommendations ..............................................................................................................59

References ......................................................................................................................................61

Appendix A: Bread Sensory Analysis Scorecard ..........................................................................69

Appendix B: Institutional Review Board Approval ......................................................................70

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List of Tables

Table 1: Fatty Acid Profile of Selected Oils…………..………………………………………....18

Table 2: Tocopherols in Pumpkin Seed Oil…………..…………………………………….…....25

Table 3: Carotenoids in Pumpkin Seed Oil………………………………………………………25

Table 4: Antioxidants in Pumpkin Seed Oil……………………………………………………..26

Table 5: Pumpkin Seed Composition……………………………………………………………27

Table 6: Final Formulae for Control Bread and PSC Bread …………………………………….39

Table 7: Estimates of Proximate Composition of Macro Ingredients in PSC…………………...47

Table 8: Farinograph Data from Flour/Water Absorption , Control Bread Dough and PSC

Bread Dough………………..………………………………………………………......49

Table 9: Final Benchtop Formulae for Control Bread and PSC Bread………………………….50

Table 10: Dimensions of Bread Loaves for Control and PSC…………………………………...51

Table 11: Rheology (Modulus and Maximum Compressive Stress Values) from the Instron®

exture Analyzer for the Control Bread Samples and with PSC Samples……………...52

Table 12: Sensory Evaluation Results of Control and PSC Bread………………………………54

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List of Figures

Figure 1: Lipid Oxidation for Linoleic Acid in the Initiation Step…………………….………..20

Figure 2: Lipid Oxidation for Linoleic Acid in the Propagation Step…………………………...21

Figure 3: Example of Lipid Oxidation in the Termination Step………………………..............22

Figure 4: Phenol Ring, Tyrosol, Vanillic Acid, Caffeic Acid, Vanillin, Luteolin and

Sinapic Acid…………………………………………………………………………..24

Figure 5: Antioxidant Capacities of Styrian Pumpkin Seed Oils and Other Edible Oils………..26

Figure 6: Farinogram of Hard Wheat and Parameters…………………………………………...31

Figure 7: Farinograms of Three Flours and Their Mixing Characteristics………………………31

Figure 8: Bread Dough Mixing in Promylograph Box…………………………………………..37

Figure 9: Control Dough Proofing Stages……………………………………………………….40

Figure 10: PSC Dough Proofing Stages…………………………………………………………40

Figure 11: A Single Column, Table Top Load Frame Texture Analyzer……………………….42

Figure 12: A 0.5” Steel Probe Forced into the Center Crumb of the Bread…………………….43

Figure 13: Tray Setup of bread Samples Given to Each Panelist for Evaluation………………44

Figure 14: Accelerated Solvent Extractor……………………………………………………….45

Figure 15: Farinograph of Flour/Water Absorption……………………………………………..48

Figure 16: Farinograph of Control Bread Dough………………………………………………..48

Figure 17: Farinograph of PSC Bread Dough…………………………………………………...49

Figure 18: Flavor and After Taste Sensory Results for Control and PSC Bread………………..53

Figure 19: Texture and Acceptance Sensory Results for Control and PSC Bread………………54

Figure 20: Middle Vertical Cross Section of Control Bread Crumb…………………………….55

Figure 21: Middle Vertical Cross Section of PSC Bread Crumb………………………………..55

Figure 22: Pumpkin Seed Oil Form PSC by the ASE…………………………………………...56

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Chapter I: Introduction

The components of pumpkin seeds (oil and cake) can be valuable as additives in bread,

because they provide a good source of protein, are rich in antioxidants, and are high in

monounsaturated and polyunsaturated fatty acids. When pumpkin seed oil is expelled from the

seed, a byproduct, pumpkin seed cake, is produced (Peričin, Krimer, Trivić, & Radulović, 2009;

El-Soukkary, 2001). Pumpkin seeds primarily consist of protein and fat providing a

concentrated energy source (Caili, Huan, & Quanhong, 2006).

Protein from food is one of the main components needed to be consumed each day for a

healthy diet. Pumpkin seed proteins contain a high amount of tryptophan (Zdunczyk,

Minakowski, Frejnagel & Flis, 1999). With the addition of pumpkin seed cake and oil to bread,

the nutritional content of bread could be increased and the disposal of the cake (PSC) minimized

at the same time (El-Soukkary, 2001).

Antioxidants are found in many foods, especially green plants and highly colored fruit. A

network of antioxidants, possessing different chemical properties, from food may provide proper

protection against stress oxidation. Taking antioxidant supplements like ascorbic acid does not

protect against oxidative stress-related diseases as well as antioxidants that come from food

(Halvorsen, Carlsen, Phillips, Bøhn, Holte, Jacobs Jr., & Blomhoff, 2006). Research has shown

that consuming foods rich in antioxidants can increase cognitive and behavioral abilities

especially as people age (Willis, Shukitt-Hale, & Joseph, 2009). Therefore, consuming foods

rich in antioxidants may provide an increased value to human nutrition than do supplements.

Since pumpkin seed oil and cake are rich in antioxidants like tocopherols and carotenoids, they

offer the opportunity to create a new kind of specialty bread that contains this ingredient.

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Unsaturated fats including monounsaturated and polyunsaturated fatty acids are

recommended for a healthy daily diet rather than the consumption of saturated fats. Pumpkin

seed oil contains high amounts of unsaturated fats including Omega 3 (linolenic) and Omega 6

(linoleic) essential fatty acids (Murkovic, & Pfannhauser, 2000).

Statement of the Problem

Pumpkin seed oil cake (PSC), a byproduct of pumpkin seed oil processing, is currently

only used for an addition to animal feed. The nutritional properties of PSC provide a significant

rationale to use it as an alternate ingredient in a food product application. Rather than disposing

of a byproduct, an alternative is to capture its value by adding PSC to bread to fortify protein

content for a more nutritious white bread (Vaštag, Popović, Popović, Krimer, & Peričin, 2010).

This study was designed to formulate a new bread product with PSC. After characterizing

the material properties of the two breads (control and PSC), the PSC bread may present different

characteristics than control bread. Using PSC in bread may serve as a healthy substitute for

vegetable shortening and/or a protein enhancement. In addition, it may result in solutions for

alleviating the waste disposing cost of the byproduct.

Research Objectives

The primary research objective is to make quality bread using pumpkin seed cake as a

source of antioxidants, protein, and monounsaturated and polyunsaturated fats. The second

objective is to characterize bread for a material test on the texture analyzer and correlate those

with the sensory properties of the bread. Finally, it will be important to quantify standard white

wheat bread, the control, and bread with pumpkin seed cake in order to compare the two breads.

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Assumptions of the Study

The PSC came from the species Cucurbita pepo and the variety is a variation of the

Styrian oil pumpkin.

Definition of Terms

Farinograph. A type of recording mixer that measures and records the resistance over

time by a dough against mixing blades operating at a constant speed (rpm) and a constant

temperature; a promylograph is a smaller version of the farinograph (Delcour & Hoseney, 2001).

Rheology. The study of how materials deform, flow, or fail when force is applied

(Delcour & Hoseney, 2010).

Limitations of the Study

The oil pumpkin variety is unknown because this is proprietary information of the Hay

River Pumpkin Oil Foods. An untrained sensory panel was used for the sensory analysis

evaluation.

Methodology

An acceptable control dough/bread will be formulated and produced. The control dough

will be characterized using a promylograph. An attempt will be made to formulate and produce

a PSC dough with similar characteristics that mimic the control dough. A dough and bread will

be designed with PSC. Following baking of the breads, the control and the PSC bread will be

compared for their rheological and sensory properties. The rheological properties will be

quantified using compression testing. The sensory properties will be measured through a

randomized subjective sensory test.

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Chapter II: Literature Review

There are many underutilized agricultural byproducts with high nutritional value that can

be used in food products or other industrial applications. Currently, only a small fraction of

agricultural byproducts are used in food (Nyam, Long, & Che Man, 2009). Pumpkin seed cake

(PSC), the material remaining after pumpkin seed oil has been removed from the seed is

typically sold for incorporation into animal feed or spread on land as fertilizer (Vaštag et al.,

2010). As a good source of antioxidants, protein and unsaturated fatty acids, PSC can be used to

fortify food products, such as bread, cereals, and cereal bars that promote healthy nutrition.

(Xanthopoulou, Nomikos, Fragopoulou, & Antonopoulous, 2009).

History of Pumpkins and Pumpkin Species

The pumpkin is a pepo type fruit - berries with a hard, thick rind - in the Cucurbitaceae

family that includes cucumbers and squash. Pumpkins are traditionally defined as round in

shape and orange in color, and like other winter squash, have long vines and a flowering stage.

Other members of the Cucurbitaceae family include muskmelon, watermelon, cucumbers, and

gourds (Marr, Schaplowsky, Carey, & Ted, 2004). Cucurbita pepo (common field pumpkins),

C. maxima (many of the large-fruited winter squashes and pumpkins), C. moschata (winter

squash and some types of pumpkins), and C. mixta (green-striped cushaw) are all different

pumpkin species in the genus Cucurbita (Kemble, Sikora, Zehnder, & Bauske, 2000). The

pumpkin most commonly used for pies, puddings, casseroles and decoration is C. pepo.

Pumpkins are thought to have originated in southern Mexico and were introduced to

Europe along with maize and chocolate by Spanish explorers (Fruhwirth & Hermetter, 2008). It

has been cultivated in Europe and Asia for centuries. A particular variety of pumpkin, C. pepo

subspecies pepo var. Styriaca, was developed in Styria, Austria and is used exclusively for the

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production of green pumpkin seed oil (Nakić, Rade, Škevin, Štrucelj, Mokrovčak, & Bartolić,

2006). These pumpkins originated from a series of mutations that resulted in green colored seeds

with stunted outer hulls and are now known as C. pepo var. styriaca (Fruhwirth & Hermetter,

2008). The Styrian pumpkin is used almost exclusively for the production and export of

pumpkin seed oil (Nakić et al., 2006). Compared to the seeds with a lignified seed coat from

conventional pumpkins, the seeds from the Styrian pumpkin have a thin membranous seed coat,

are completely edible and are becoming a popular snack in many countries (Cucurbit Network

News, 2004). The lack of a traditional hull also makes extracting oil from the Styrian pumpkin

seed much simpler and more economical.

The Styrian oil pumpkin is one of the few oil seed crops grown in Austria and remains

the third most important field fruit in Styria (Fruhwirth & Hermetter, 2008; Ruckenbauer, 1999).

In 2006, there were 12,500 hectares used for growing oil seed pumpkins in Austria located in the

Styrian region with a total of 11,110 tons of pumpkin produced. One liter of Styrian pumpkin

seed oil requires 2.5kg pumpkin seeds, equivalent to 30-40 Styrian oil pumpkins (Fruhwirth &

Hermetter, 2008).

Oil Pumpkin Cultivation Factors

Styrian oil pumpkins are harvested in mid autumn, and it takes 5.5 lbs of dried pumpkin

seeds to make 1 liter of pumpkin seed oil (Fruhwirth & Hermetter, 2008). Light sandy loam soil

appears to be the best growth medium. Soil rich in humus and organic compounds are most

suitable. Rotating pumpkin crops with legumes or clover results in better production than

cucumbers since pumpkins share diseases with them. Various diseases can be controlled by

organic fertilizing. Careful sowing of nongerminated seeds or germinated seeds or transplants

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for optimal yield can be achieved either with a pneumatic corn seeder or manually (Bavec,

Mlakar, Rozman, & Bavec, 2007). Growing conditions can affect the phenolic content in plants.

Pumpkin Seeds

Styrian oil pumpkin seeds contain 35% protein, primarily albumin and globulin proteins.

They also contain between 35-55% oil (Deimel, 2007). The oil is also high in phenolic acids,

phytosterols, Vitamins E and A, and mixed carotenoids.

Processing of Pumpkin Seed Oil Extraction

The oil from C. pepo var. styriaca pumpkins is used in culinary preparations such as

salad dressings, as a replacement for other oils in meal preparations, and in neutraceutical and

supplement preparations (Applequist, Avula, Schaneberg,Wang, & Khan, 2006). Unlike the

Cucurbita pepo from South Eastern Europe, Africa, and Asia, which produce a light yellow or

clear seed oil, Styrian pumpkin seed oil is dark green (Fruhwirth & Hermetter, 2008). The oil is

green with a hint of red because the seeds contain tocopherols like protochlorophyll and

protopheophytin (Fruhwirth, & Hermetter, 2007). Pumpkin seed oil has a high amount of

unsaturated essential fatty acids that oxidize quickly with the addition of heat (Murkovic &

Pfannhauser, 2000). Unsaturated fatty acids reduce the oxidative stability, cause foaming, and

reduce the smoke point (Damodaran, Parkin, & Fennema, 2007).

Pumpkin seed oil is generally extracted by expelling the oil from the seeds mechanically.

Both hulled and hull-less pumpkin seeds can be used for oil production. The oil from the hull-

less seeds is much easier to extract and more commonly processed. The hull surrounding

common pumpkin seeds has been replaced by a membranous seed coat as in the Styrian pumpkin

seeds making the oil easier to remove mechanically. The oil from Styrian pumpkin seeds is dark

green compared to the lighter colored oils from C pepo.

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The traditional method of expelling oil from pumpkin seeds is accomplished by roasting

the seed pulp prior to mechanically expelling the oil (Nakić, et al., 2006). After seeds are

removed from the pumpkin, they are dried down to 5-7% moisture. The dried seeds are ground

and steeped in a salt brine (Fruhwirth & Hermetter, 2008). The increased moisture from the salt

brine protects the flavor of the oil during the roasting step (Fischerauer, 1996). The ground seed

pulp is roasted at 212-248°F for 60 minutes (Fruhwirth & Hermetter, 2008). During the roasting

process, proteins are denatured to facilitate liberation of the oil. Oil is pressed out of the seeds at

temperatures above 120°F and between 4,351 psi to 8,702 psi. At this point oil is cooled and

packaged for sale. Optimal packaging for pumpkin seed oil is a dark colored bottle, to retain the

beneficial components that can be lost to heat, light, and oxygen (Fruhwirth & Hermetter, 2007).

While natural, Styrian pumpkin seed oil processed by the traditional method is not

considered virgin oil because of the roasting step (Fruhwirth & Hermetter, 2008). This statement

reflects the German Society of Fat Science (DGF) definition of virgin oils (Matthäus & Spener,

2008). Pumpkin seed oil is classified somewhere between a virgin oil and a refined oil

(Fruhwirth & Hermetter, 2008). The Food and Agriculture Organization (FAO) (1999) defines

virgin oils and cold pressed oils as the following:

Virgin oils are obtained, without altering the nature of the oil, by mechanical procedures,

e.g. expelling or pressing, and the application of heat only. They may have been purified

by washing with water, settling, filtering and centrifuging only.

Cold pressed oils are obtained, without altering the oil, by mechanical procedures

only, e.g. expelling or pressing, without the application of heat. They may have been

purified by washing with water, settling, filtering and centrifuging only.

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Pumpkin seed oil is also produced using a simple cold press without roasting or chemical

solvents (Bavec et al., 2007). The pumpkin seeds are ground in a screw press at temperatures

less than 120°F. The oil is expelled under pressure. While this produces a more pristine oil,

extraction efficiency is reduced and much of the oil remains in the seed pulp, a byproduct of the

process (Gorjanović, Rabrenović, Novaković, Dimić, Basić, & Sužnjević, 2011). Botantical Oil

Innovations (Spooner, Wisconsin) manufactures pumpkin seed oil using a cold press expelling

technology called NatureFRESH-Cold Press™. Because the oil is extracted from the seed once,

the pumpkin seed oil produced by this method can be classified as extra virgin oil. The solid

material remaining after expelling the pumpkin seed oil is pumpkin seed cake (PSC). Pumpkin

seed cake has a dark musty green color with a texture typical of almond paste and still contains

significant amounts of oil. It has generally been used as fertilizer or hog feed. A second

pressing of the seed cake can extract additional oil, but generally results in lower quality salad

oils (Fruhwirth & Hermetter, 2008). Hay River Foods (Prairie Farm, Wisconsin) is a producer of

organic pumpkin seed oil and is seeking alternative food applications for the remaining PSC.

Physiochemical Characteristics of the Pumpkin Seed Oil

Physical Properties.

Color. Pumpkin seed oil is not suitable for cooking because of its dark greenish color and

foaming characteristics (Murkovic & Pfannhauser, 2000). This oil has a nutlike, toasty aroma

resulting from pyrazine formation during the roasting of the pumpkin seeds (Joebstl,

Bandoniene, Meisel, & Chatzistathis, 2010; Murkovic & Pfannhauser, 2000). However, these

flavors are absent from pumpkin seed oil produced by the cold press method. Styrian pumpkin

seed oil is dark green with tints of red, but the color deteriorates by exposure to sunlight and

oxygen. The pumpkin seed oil is best stored in dark colored bottles in a cool area. The green

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and red pigments in Styrian pumpkin seed oil are due to tetrapyrrol compounds like

protochlorophyll and protopheophytin located in the pumpkin seed’s inner seed-coat tissues

(Fruhwirth & Hermetter, 2007). The other three families of pigments are carotenoids,

polyphenolic compounds, and alkaloids (Fruhwirth & Hermetter, 2008). The dark green color is

indicative of antioxidants in the oil (Gorjanović et al., 2011). Animals and humans are unable to

synthesize these pigments; therefore, the pigments need to come from food including a variety of

different colored fruits and vegetables (Ruckenbauer, 1999). In addition to the pigments that

give the oil its color, there are compounds that provide aroma of the Styrian pumpkin seed oil

that include the oxidation products of polyunsaturated fatty acids. Pyrazene derivatives are

responsible for the flavor in the oil (Fruhwirth & Hermetter, 2007).

Chemical Properties of Pumpkin Seed Oil

Fatty Acids. Typical pumpkin seed oil is comprised of mostly unsaturated fatty acids,

primarily linoleic acid, with lesser amounts of oleic acid. Its fatty acid profile is similar to that of

soy bean oil with the exception of linolenic acid, which is very low in pumpkin seed oil (Table

1). (Fruhwirth & Hermetter, 2007; Murkovic, Piironen, Lampi, Kraushofer, Sontag, 2004;

Murkovic & Pfannhaus , 2000).

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Table 1

Fatty Acid Profile of Selected Oils

Fatty Acid Styrian Pumpkin Hay River Foods

b Olive Oil

c Canola Oil

d Soybean Oil

e

Seed Oila Pumpkin Seed Oil

Linoleic 35.6-60.8% 40-57% 9.4% 22.7% 52.0%

Oleic 21.0-46.9% 20-32% 72.5% 55.8% 26.7%

Linolenic <2.0% 13-19% 0.6% 12.5% 7.6%

Palmitic 9.5-14.5% 6-13% 12.1% 4.5% 8.9%

Stearic 3.1-7.4% 3-8% 2.6% 1.5% 3.9%

a(Murkovic, et al., 2004)

b(Botanical Oil Innovations, 2011a)

c (Dubois, Breton, Linder, Fanni, & Parmentier, 2007)

d (Vaisey-Genser, Malcolmson, Ryland, Przybylski, Eskin, & Armstrong, 1994)

e (Gryglewicz, Grabas, & Gryglewicz, 2000)

As a non-refined oil, Styrian PSO contains up to 2% non-triacylglycerides, for example

fatty alcohols, carotenoids, tocopherols and tocotrienols, phytosterols, and phenolic compounds

(Andjelkovic, Van Camp, Trawka, & Verhé, 2010). The levels of each depend upon growing

location, climate, and ripeness (Murkovic & Pfannhauser , 2000). Hay River Foods claim that

the pumpkin seed oil extracted from their particular variety of pumpkins contains significant

amounts of linolenic acid, an omega-3 fatty acid, making it much more like soybean oil in both

physical and nutritional properties. However, Styrian pumpkin seed oil does not appear to

oxidize as rapidly as soybean oil.

Oxidative Stability

Unsaturated, especially polyunsaturated oils, can oxidize and form unpleasant tasting by-

products. In food, lipid oxidation is generally regarded as the interaction of fatty acids with

oxygen that results in the decomposition of the fatty acids to small, volatile compounds that

19

produce unpleasant flavors. The result is known as oxidative rancidity. Lipid oxidation occurs

through the following steps.

Initiation. A hydrogen is abstracted from a methylene group that is located between

double bonds in a polyunsaturated fatty acid chain or one methylene group to either side of the

double bond in a monounsaturated fatty acid chain (Figure 1). What remains is the carbon from

the methylene group with only one hydrogen, forming an alkyl radical (L˙). This is the point at

which the double bond(s) shift because the radical delocalizes on the chain. Formation of

conjugated double bonds occurs in polyunsaturated fatty acids because there are two or more

double bonds in the chain. The formation of radicals during lipid oxidation in the initiation step

occurs at a higher rate for polyunsaturated than monounsaturated fatty acids because of the

higher number of double bonds in the chain. The carbon-hydrogen bond is weaker on the

methylene carbon than those on the adjacent carbons making the hydrogen easier to abstract

(Damodaran, Parkin, & Fennema, 2007).

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Figure 1. Lipid Oxidation for linoleic acid in the initiation step (from Damodaran, Parkin, &

Fennema, 2007).

Propagation. Oxygen is involved in the propagation step of lipid oxidation (Figure 2).

The alkyl radical on the fatty acid from the initiation step can react with triplet oxygen

(atmospheric oxygen) in the propagation step to form a peroxyl radical (LOO˙). The peroxyl

radical can abstract a hydrogen from another fatty acid to form a fatty acid hydroperoxide

(LOOH) plus another alkyl radical on the second fatty acid. The total number of hydroperoxides

that will form on fatty acid depends on the number of isomers that an unsaturated fatty acid can

form (Damodaran et al, 2007).

21

Figure 2. Lipid Oxidation for linoleic acid in the propagation step (after Damodaran, Parkin, &

Fennema, 2007).

Termination. The termination step in lipid oxidation occurs when there is a reaction

between a peroxyl radical and an alkyl radical from two separate unsaturated fatty acids. The

result of the radical interaction is a nonradical species. A generalized mechanism is shown in

Figure 3. Alkyl radical reactions can occur in low oxygen environments. The more unsaturated

the oil, the faster it will oxidize because there are more “methylene hydrogens available for

abstraction from the fatty acid” (Damodaran et al, 2007).

22

Figure 3. An example of lipid oxidation in the termination step under conditions of low oxygen

concentrations (after Damodaran, Parkin, & Fennema, 2007).

Oxidation of fatty acids is measured by the amount of peroxides or the amount of ketones

and aldehydes present in the oil. Peroxides are formed early in the oxidative process and do not

present any off-flavors. Ketones and aldehydes occur later in oxidation and are the volatile

chemicals responsible for the unpleasant flavors. There are two widely recognized methods used

to test the oxidative stability of oils, the Schaal Oven test and the Active Oxygen Method (AOM)

(Tan, Man, Selamat, &Yusoff, 2002). Both subject the fat or oil to conditions that favor rapid

oxidation followed by analysis to quantify the formation of either peroxides or ketones and

aldehydes. Other more rapid measurements include conductivity or spectrophotometric analysis.

The composition of the oil is one of the most important factors influencing stability of the

oil to oxidation. Since unsaturated lipids are the first fats to oxidize, and pumpkin seed oil is

similar to soybean oil with its oleic, linoleic, and linolenic fatty acid content, one could assume

that pumpkin seed oil would be similar in its oxidative stability. However, pumpkin seed oil has

23

a higher oxidative stability than its fatty acid profile would indicate and has a higher oxidative

stability than refined rapeseed oil, crude linseed oil, crude Camelina sativa oil, crude primrose

oil, crude borage oil, and crude amaranth oil (Szterk, Roszko, Sosińska, Derewiaka, & Lewicki,

2010). Given the fact that the fatty acid profile of the pumpkin seed oil predicted that the oil

would have a low oxidative stability, other factors or components in the oil inhibit oxidation.

Cold pressed oils have higher phenolic content than oils that are chemically extracted, or result

from processes using roasting (Robbins, 2003; Andjelkovic, Van Camp, Trawka, & Verhé,

2010). Higher phenolic acid content increases the oxidative stability of unsaturated fatty acids.

The high level of phenolic acids also contributes to the sensory properties (flavor and color) and

nutrition of pumpkin seed oil (Robbins, 2003).

Phenolic Acids. Phenolic acids are a subclass of phenolics and classified as phenols with

one carboxylic acid function. Polyphenols and simple phenols are two classes of phenolics.

Flavonoids have at least two phenol subunits and tannins contain three or more phenol subunits,

both of which are polyphenols. The distribution of phenolics in most plant food consists of two-

thirds flavonoids and most of the remaining portion as phenolic acids. The phenolic acids in

food contribute to color, sensory qualities, and nutritional and antioxidant properties in food

(Robbins, 2003).

Phenolics function as antioxidants by scavenging free radicals (Robbins, 2003). Phenols

may reduce free radical-mediated cellular damage (Parry, Hao, Luther, Su, Zhou, & Yu, 2006).

The majority of the phenolics that are present in pumpkin seed oil include tyrosol, vanillic acid,

caffeic acid, vanillin, luteolin, and sinapic acid (Figure 4) (Andjelkovic, et al., 2010). Peričin,

Krimer, Trivić, & Radulovi (2009) found that there is a higher concentration of phenolic acids in

the oil cake meal than in the seed.

24

Phenol Ring Tyrosol Vanillic acid

Caffeic acid Vanillin Luteolin

Sinapic acid

Figure 4. Phenol Ring, Tyrosol, Vanillic acid, Caffeic acid, Vanillin, Luteolin, and Sinapic acid

are the major phenolics in pumpkin seed oil (Andjelkovic, et al., 2010).

Antioxidants. Pumpkin seed oil also contains high amounts of vitamin E in the form of

α-tocopherols, γ-tocopherol, δ-tocopherol and tocotrienols (Nakić et al., 2006). The tocopherols

found in Styrian pumpkin oil include α-tocopherol (35.3μg/g oil), γ-tocopherols (360.5μg/g oil)

and δ-tocopherol (7.6μg/g oil) (Table 2) (Murkovic & Pfannhauser, 2000). Carotenoids and

phytosterols are also abundant in pumpkin seed oil (Table 3 and Table 4) (Szterk et al, 2010).

24

Phenol Ring Tyrosol Vanillic acid

Caffeic acid Vanillin Luteolin

Sinapic acid Figure 4. Phenol Ring, Tyrosol, Vanillic acid, Caffeic acid, Vanillin, Luteolin, and Sinapic acid are the major phenolics in pumpkin seed oil (Andjelkovic, et al., 2010).

Antioxidants. Pumpkin seed oil also contains high amounts of vitamin E in the form of

α-tocopherols, γ-tocopherol, δ-tocopherol and tocotrienols (Nakić et al., 2006). The tocopherols

found in Styrian pumpkin oil include α-tocopherol (35.3μg/g oil), γ-tocopherols (360.5μg/g oil)

and δ-tocopherol (7.6μg/g oil) (Table 2) (Murkovic & Pfannhauser, 2000). Carotenoids and

phytosterols are also abundant in pumpkin seed oil (Table 3 and Table 4) (Szterk et al, 2010).

25

Table 2

Tocopherols in Pumpkin Seed Oil

Tocopherol μg/g of oila

mg/kg oilb μmol/kg

α-tocopherols 35.3 26.8 ± 0.9

γ-tocopherol 360.5 216.3 ± 2.4

δ-tocopherol 7.6 19.2 ± 0.0

Total Tocopherols 625.6

a(Murkovic & Pfannhauser, 2000).

b(Botanical Oil Innovations, 2011b)

Table 3

Carotenoids in Pumpkin Seed Oil

Carotenoids μg/kg of oila

Beta Carotene 981

Lutein 272

Zeaxanthin 28.6

Cryptoxanthin 4917

a (

Botanical Oil Innovations, 2011)

26

Table 4

Antioxidants in Pumpkin Seed Oil

Antioxidant mg/100g of pumpkin seed oil

Phytosterols & Stanols 349.0 mgb

Δ7-sterols 70.4 mgb (as part of total phytosterols and stanols)

b(Szterk et al, 2010)

The antioxidant capacity in Styrian pumpkin seed oil exceeds that of olive oil, extra

virgin olive oil, sunflower oil, sunflower oil (high oleic), hemp seed oil, poppy seed oil (gray),

poppy seed oil (white), thistle oil, and walnut oil (Figure 5) (Fruhwirth & Hermetter, 2007). The

ORAC (Oxygen Radical Absorbance Activity) value is used as a measurement of antioxidants in

foods (United States Department of Agriculture, 2010). The ORAC value for pumpkin seed oil

is 110 μmoles Trolox equivalents/100g (Botanical Oil Innovations, 2011b).

Figure 5.Antioxidant levels of Styrian pumpkin seed oils compared to olive oil, extra virgin olive

oil, sunflower oil, sunflower oil, high oleic, hemp seed oil, poppy seed oil, poppy seed oil

(white), thistleoil, and walnut oil (Fruhwirth, & Hermetter, 2007).

27

Since pumpkin seed oil is extracted in a handcrafted, small scale process it is difficult to

produce a standardized oil or seed cake. This may be the reason why proximate analysis of fats

and proteins differ between publications (Fruhwirth & Hermetter, 2008).

Composition of Pumpkin Seeds and PSC

The primary components in pumpkin seeds include 35-55% oil and 30-40% protein

(Table 5). Additionally, the following vitamins are found in pumpkin seeds: Vitamin E (30

mg/100 g of pumpkin seeds), Vitamin A, Vitamins B1, B2, and B6, Vitamin C, and Vitamin D.

In 100 g of pumpkin seeds there are 550 to 610 calories (Deimel, 2007). Pumpkin seed cake is

the byproduct remaining after oil is physically expelled from pumpkin seeds (Popović, Peričin,

Vaštag, Popović, & Lazić, 2010). It still contains significant amounts of oil as well as high

levels of protein. It is even more appropriate for foods because the endemic antioxidants make

its use more universal.

Table 5

Pumpkin Seed Composition

Component %a

Oil 35-55%

Protein 30-40%

Carbohydrates 4-8%

Crude Fiber 2-4%

Minerals & Trace Elements 4-5%

a (Deimel, 2007).

28

Health Claims of Pumpkin Seed Oil

Since pumpkin seed oil is high in antioxidants, (Botanical Oil Innovations, 2011b) many

health claims accompany the consumption of this oil (Peričin, et al., 2009). Reports claim

consumption of pumpkin seed oil can help in the prevention and therapy of cardiovascular

disease, prostate cancer (benign prostatic hyperplasia-BPH), dysuria, urinary tract infections, and

digestive problems. Consumption of pumpkin oil contributes to smoothed skin and increased

energy (Botanical Oil Innovations, 2011b).

Tocopherols, hydrocarbons including squalene, sterols, alcohols, and phospholipids are

found in pumpkin seed oil and when the oil is consumed it can contribute to good health and

prevention of other diseases. It is the Δ7-sterols in pumpkin seed oil that are reported to support

health of the prostrate gland and bladder. Similar components in olive oil are also found in

pumpkin seed oil and may help protect against breast cancer (Nakić et al., 2006). Pumpkin seeds

and pumpkin oil have also shown to be antidiabetic, antifungal, antibacterial, anthelmintic,

antihypercholesterolemic, and anti-inflammatory (Caili, Huan, & Quanhong, 2006).

Bread

Bread by definition and standard of identity is made from wheat flour, water, yeast, and

sodium chloride. It may contain other ingredients including shortening, dough

oxidizing/reducing agents, emulsifiers, sugar, and other types of flour. Flour is the key to a

quality bread dough, and it is characterized by the amount and quality of protein in the flour. A

typical bread flour contains 71% carbohydrates, 13% protein, 1% lipids, and 14% water (Scanlon

& Zghal, 2001). The primary proteins in wheat flour are gliadin and glutenin, which make up

the gluten complex, the unique proteins that form the gas holding structure in bread dough.

During mixing the two proteins interact with each other through hydrophobic interactions and

29

disulfide links to form large polymeric sheets. The unique physical properties of the gluten

polymers result from a combination of the elastic properties of glutenin and the extensible

character of gliadin when they are hydrated during dough mixing. The result is a viscoelastic

quality in dough that allows dough to expand during leavening (proofing) and baking while

retaining the necessary pressure to hold the CO2 gas bubbles and build volume. Gliadin is also

responsible for the cohesiveness in dough (Delcour & Hoseney, Proteins of Cereals, 2010). The

carbohydrates in flour are starch and hemicellulose. Starch is compartmentalized in starch

granules that minimally hydrate during mixing but gelatinize during baking to support the final

structure. Wheat starch is about 25% amylose and 75% amylopectin (Miyazaki, Maeda, &

Morita, 2004).

The bread making process begins with mixing where the ingredients are blended and

hydrated and the gluten developed. Mixing also introduces air into the dough mass. The trapped

air becomes initiation sites for gas cell formation. When completely developed during mixing,

dough is a continuous, hydrated protein matrix surrounding starch granules and dispersed air

cells (Gallagher, Gormley, Arendt, 2003). After mixing, dough is proofed, and CO2 produced by

yeast migrates to the air cells causing them to expand, increasing the dough volume and

producing a foam. Gluten development is critical during the mixing of bread dough, because it

determines the volume of the baked bread, and is an important determinant of the crumb

structure in bread (Scanlon & Zghal, 2001).

Dough Rheology

As a viscoelastic material, the rheology of dough exhibits both solid or elastic properties

and liquid or flow characteristics (Tanner, Qi, & Dai, 2008; Weipert, 2008). Dough rheology

determines final bread quality. Because the rheological properties of dough are developed

30

during mixing when glutenin and gliadin interact to form the gluten matrix, measuring the

development as mixing progresses is important in optimizing dough texture. During mixing,

ingredients in dough hydrate and react with one another and the dough begins to increase in

viscosity. As viscosity increases, the resistance to mixing increases (Delcour & Hoseney, 2010).

While viscoelastic properties of a material are classically described by stress and strain

measurements, the baking industry has adopted empirical methods to define the rate at which

flour absorbs water and the amount of water it absorbs to reach a designated dough viscosity.

The most commonly used instrument is the farinograph (C.W. Brabender Instruments),

which simulates a mixer and measures the resistance or consistency (a function of viscosity) of

the dough as mixing proceeds. A resistance curve versus time is generated (Figure 6). The

curve describes a number of properties critical to processing dough and baking bread: the time to

reach maximum viscosity (hydration and development time), peak height (maximum viscosity),

stability of the dough (how the dough resists mixing), mixing tolerance and softening (how the

dough tolerated processing). The longer the stability time of the dough, the stronger the dough

(Figure 7), (Atwell, 2001). Dough rheology measurements are important because they are a

function of protein quality and amount, starch content, ash content and moisture. They also

quantify changes in dough as other ingredients like protein supplements, shortening and dough

conditioners are added to dough. They also allow quantification of flour differences and the

optimization of dough during baking (Tanner, Qi, & Dai, 2008).

Bread dough reacts to different strains and forces during processing. Mixing is a

relatively large force while dough proofing and rising in the oven is a small force, but both are

important in producing quality bread. To produce quality bread, dough must be developed to

31

respond to both the large and small deformation rates during mixing and baking. These

characteristics need to be recognized during testing and monitoring (Weipert, 1990).

Figure 6. Farinogram of hard wheat and parameters: dough development time, stability, mixing

tolerance, time to peak, and the degree of softening (Atwell, 2001).

Figure 7. Farinograms of three flours and their mixing characteristics: a) weak, b) medium, and

c) strong (Atwell, 2001).

32

Pumpkin Seed Cake in Bread

El-Soukkary evaluated a variety of pumpkin seed products as ingredients in pan bread.

He used raw pumpkin seed meal, roasted pumpkin seed meal, autoclaved pumpkin seed meal,

germinated pumpkin meal, fermented pumpkin seed meal, pumpkin seed protein concentrate,

and pumpkin seed protein isolate; all from Curcurbita moschata, variety Dickinson. All were

added to increase protein content. Pumpkin seed meal and pumpkin proteins all increased the

moisture absorption and development time of the bread dough. The dough with protein isolate

had the highest water absorption. In addition, pumpkin seed meal reduced the developed

dough’s stability during mixing. The nonfunctional albumin and globulin proteins in pumpkin

seeds bind water without contributing to dough development or stability of the dough, both of

which are detractors from bread dough quality. The dough stability time decreased, as did the

strength of the dough by addition of the pumpkin seed products (El-Soukkary, 2001).

Bread Rheology

During baking, dough is transformed into bread. From a material perspective, the

proteins dehydrate and denature, the starch gelatinizes and the dough shifts from a viscous

material to a more solid material. At the same time it is converted from a foam (discontinuous

air cells) to a sponge (continuity of air channels) as air cells rupture.

The textural characteristics of bread are measured by methods designed to describe

solids. These tests can be extension tests (tensile) where a sample is stretched and the force

required to make the material yield is recorded, or compression tests where the bread is squeezed

and the force is required to make the material yield is recorded. Other tests attempt to simulate

texture as perceived during chewing and swallowing bread. This is called texture profile

analysis (TPA) and employs both compression and extension methods to mimic forces in the

33

mouth. A simpler technique is a probe compression test where a probe with known diameter is

pushed into the bread at a constant rate to a defined distance. The maximum force necessary to

push the probe into the bread is a measure of bread firmness. One can also determine the

modulus relating to the stiffness of the bread. It is defined as the applied force per unit of area

(stress) to the relative deformation (strain) (Delcour & Hoseney, 2010).

Describing the mechanical behavior using the stress strain curve for bread is difficult as

the bread has a porous structure and lacks of homogeneity in the distribution of cells within the

bread. Bread has been analyzed for texture by compressive loading test (indentation), tensile

tests (extension), and small angle deformation tests (shear). The compressive loading test is

preferred over tensile tests in bread because of its simplicity, small sample size and validity in

evaluation of mechanical properties. Disadvantages of the tensile test in bread texture include

difficulty gripping the sample and adherence to sample size, shape and stiffness. An advantage

to tensile testing over the compression testing is the ease of interpreting results and determining

mechanical properties (Scanlon & Zghal, 2001).

Relative density is the “density of the cellular material, divided by the density of the solid

material” (Zghal, Scanlon, & Sapirstein, 2002). The relationship between relative density and

mechanical properties can be applied to bread. The density has a large impact on the mechanical

properties of the bread crumb.

Sensory Analysis of Bread

The following order of sensory attributes is how a consumer perceives a food product:

appearance, aroma, texture, and flavor. The relationship between the ingredients and the

characteristics of the baked bread are important to understand for formulators in order to meet

consumer preferences (Kihlberg, Öström, Johansson, & Risvik, 2006). In addition, the objective

34

instrumental measurements of bread firmness can be correlated to the subjective sensory

properties (Scanlon & Zghal, 2001). The gluten protein matrix in wheat flour is important to the

quality of the bread. Recipe modifications are necessary when the wheat flour is not ideal

(Kihlberg et al, 2006) or when potentially non functional ingredients are added like pumpkin

seed cake.

The objective of this study is to baseline the effects of pumpkin seed cake (PSC) as a

partial replacement for shortening and as a protein supplement in bread. The effect of PSC on

dough development and consistency, and on the final quality and texture of bread will be

evaluated. Pumpkin seed cake will be added to white wheat bread to magnify the effects that this

ingredient may have in bread compared to the control bread. A baseline bread formula for

control bread and bread with PSC will be developed. A comparison between the control and

PSC breads will be measured using sensory analysis, texture analysis, bread measurements, and

crumb structure. The rheology of control bread dough and the PSC bread dough will be

analyzed for development time, stability, and mixing time.

35

Chapter III: Methodology

The objectives of this research were to determine if a novel bread could be developed

using pumpkin seed cake (PSC), a by-product from the extraction of pumpkin seed oil, as a

source of shortening and other bioactive ingredients. To develop an acceptable bread containing

PSC, it was necessary to understand how the addition of PSC affected the moisture absorption

and development of the dough, what formula adjustments to the PSC-containing dough were

necessary to match a control dough, and how PSC impacted the final flavor and texture of the

bread. The experimental design used the following approach:

1. Determine the moisture absorption of the bread flour to be used in the study as a baseline

for comparison with PSC-containing dough.

2. Establish a “control dough” using typical ingredients: flour, water, yeast, salt and

shortening.

3. Determine the moisture absorption of flour containing PSC so that formula adjustments

could be made to mimic the control dough.

4. Use the Promylograph to establish dough rheology and to determine optimum mix times

and flour/water ratios for the control dough and dough containing PSC.

5. Scale up the Promylograph mix parameters to a bench top mixer.

6. Make bread with and without PSC on a bench top scale for laboratory texture

analysis and sensory evaluation.

Flour Water Absorption

Water absorption in flour is defined as the amount of water (expressed as a percent, w/w

of the flour) necessary to balance the farinograph curve on the 500 BU line (Atwell, 2001). In

this study, water absorption was determined using a Promylograph (measured in PU’s) according

to a method modified from the one described by AACC Method 54-21(2000). The

36

Promylograph (measured in PU’s) has similar units to the Brabender farinograph. Flour (120g)

was placed in the Promylograph mixing bowl and water (~60ml) was added to the bowl using a

burette. Water was added until the resistance curve reached the 500PU line. This process

required two to three iterations to accurately determine the amount of water necessary. If the

rheology curve exceeded 500 BU, the amount of water was increased in the next run. If the

rheology curve was less than 500 BU, the amount of water was decreased in the following run.

The volume of water needed to reach 500 BU was recorded and calculated as a percent of the

flour. This result became the starting point for the flour/water ratio used to formulate the control

dough in the following experiments.

Development of the Control Dough/Bread

The control dough formula and mixing parameters were developed using the

Promyograph to insure uniform, consistent mixing and to measure dough rheology (Figure 8).

The starting formula for the control dough was obtained from the basic straight-dough, long

fermentation method (AACC Method 10-09.1, 2000). Dough (200g) was mixed for 16 minutes

in the Promylograph and the curves were analyzed for development time, peak, and stability

(AACC 54-21, 2000). Mixed dough was placed in small (200g), greased loaf pans and allowed

to proof for 1 hour 55 minutes at 88°F. They were baked at 425°F for 25 minutes, removed from

the pans, allowed to cool, and evaluated for volume and texture. Adjustments to the formula

were made to determine optimal dough rheology for acceptable bread quality. The rheological

parameters of dough producing acceptable bread were used as a starting point in designing dough

with pumpkin seed cake.

37

Figure 8. Bread dough mixing in Promylograph box.

Bench Top Bread Manufacture

A modified long fermentation method (AACC 10-09.1, 2000) was used to make 200 g

bread loaves for sensory and analytical testing. Mixing times and rheology data obtained using

the Promylograph were used to design mixing times for “bench top” production of control and

PSC-containing bread. Peak dough development was targeted at 550BU for both control and

PSC-containing dough.

Flour and nonfat dry milk were mixed together in a Kitchen Aid mixing bowl and a

depression was made for the remaining ingredients. The yeast was dissolved in half of the water.

Salt and sugar were dissolved in the remaining water in a separate bowl. The yeast solution was

38

added to the flour/nonfat milk mixture followed by the salt and sugar solution. The shortening

(or the PSC) was added last. The ingredients were mixed slowly (speed 2) using a dough hook

for 45 seconds to blend and incorporate the liquids into the dry ingredients to form a dough mass.

The bowl was scraped to insure all ingredients were incorporated and the dough was mixed at

high speed the remaining mixing time. The mixer was stopped at 1 min. 30 seconds, 3 minutes,

and 4 minutes to scrape the bowl. The total mixing time was 20 minutes for each batch. After

mixing, 200g of dough were placed into the promylograph mixing bowl to determine mix

rheology. If the reading was above 550 PU, another batch was made with additional water. If

the reading was below 550 PUs, water in the subsequent batch was decreased. Following this

series of experiments, the flour/water ratio and mix times for the bench top bread were

established.

The same procedure was carried out for the PSC bread. Pumpkin seed cake and pumpkin

seed oil were added to the formula to replace the vegetable shortening. The two pumpkin seed

ingredients were added last, before the mixing began. The formulae for the two breads listed in

Table 6.

39

Table 6

Final Formulae for Control Bread and PSC Bread

AACC Bread Control Bread PSC Bread Ingredient Supplier Baker’s %/% % %

Bread Flour Heartland Mills 100%/54.8% 50.8% 45.9% Water 60/37.5% 42.0% 37.7% Dry Active Yeast Red Star 3/1.6% 1.5% 1.4% Table Salt Morton 2/1.1% 1.0% 0.9% Sugar, granulated Sugar Crystal 4/2.2% 2.0% 1.8% Vegetable Shortening Crisco 3/1.6% 1.5% -- NFDM Dairy Americas 2/1.1% 1.0% 0.9% PSC Hay River Foods -- -- 10.9% Pumpkin Seed Oil Hay River Foods -- -- 0.4% Total 100.0% 100.0% 100.0%

Preparation of bread for sensory and analytical evaluation. Control and experimental

doughs were prepared. After mixing, dough temperature was recorded. The dough was rounded

and placed in a round, metal bowl coated with vegetable shortening (Figures 9 and 10). A damp

cloth towel was placed on top of the bowl, and it was placed in a proof box. The temperature in

the proof box was 88°F, and the relative humidity was 90%. The total proofing time was 115

minutes. After initial fermentation for 60 minutes, the dough was removed from the

fermentation bowl and placed on a table between two wooden blocks (0.5 inches in thickness).

A rolling pin was used to “punch” the dough by rolling over the dough two times in opposite

40

directions, each time beginning in the middle. The dough was divided into 200g pieces, rounded

and placed in small, greased baking pans. The pan size was 14.6cm x 7.6cm x 5.4cm. The pans

were placed in a large metal container. A damp cloth was placed on top to prevent moisture loss.

Resting dough was always covered with a damp cotton cloth to minimize changes in moisture.

Containers containing the panned, bread dough were placed in the proofing box and allowed to

proof for the remaining 55 minutes.

After Mixing After First Proof After Second Proof

Figure 9. Control dough proofing stages.

After Mixing After First Proof After Second Proof

Figure 10. PSC dough proofing stages.

Bread Baking and Storage. Proofed loaves were baked in a preheated 450°F oven for

25 minutes. Duplicate loaves were prepared for both the control and PSC-containing batches

and were baked simultaneously in the same oven. However, the control and PSC doughs were

baked at alternate times. After baking, loaves were removed and placed on a cooling rack at

41

room temperature for two hours. After two hours of cooling, each loaf was placed in a plastic

bag and sealed.

Data Collection

Bread Measurements. The height, width, and length of each loaf of bread were

measured using an electronic 6” digital caliper (Cen-Tech) 30 minutes after baking. Height and

width were measured at three different points on each loaf; the middle, 30mm from the right

edge, and 30mm from the left edge of the loaf. Only one measurement was taken for the length

of each loaf of bread.

Crumb Structure. The crumb structure was evaluated using a photo copier to generate a

black and white image of a vertical slice from the loaf. The loaf was cut vertically in the middle

with a serrated bread knife and a “photograph” was made of the exposed side by placing it face

down on a copy machine. Digital photos were also taken of the cross section and a side of each

loaf.

Texture Analysis. A single column, table top Load Frame Texture Analyzer (Series

3340, Instron® Corporation, Canton, Massachusetts) was used to analyze the firmness of the

bread (Figure 11). Texture of individual loaves was analyzed by forcing a 0.5” steel probe into

the center crumb of the bread (Figure 12). The instrument measured the force necessary to drive

the probe a specified distance into the crumb at a constant speed. Data was converted and

reported as shear stress as a function of shear rate. Maximum shear stress for each variable was

used to evaluate firmness. Texture analysis was conducted 24 hours after the bread was baked.

The top crust of the loaf was sliced off to create a level surface parallel to the loaf bottom and to

expose the center of the crumb for penetration tests. The same depth of top was removed from

each loaf. Two separate penetration tests were conducted on each loaf, each along the central

42

axis of the loaf, approximately 1/3 of the distance from each edge, taking care to avoid any

obvious holes or gaps in the crumb. The probe was set to penetrate 20.00mm into the bread at a

speed of 20.00mm/min. Each piece of bread was placed cut side up and centered under the

probe on the platform. The probe was lowered to the top of the bread’s surface, but not

touching. The load and distance were set to zero, and the test begun. The instrument recorded

force as a function of distance penetrated at a constant rate and converted the data to shear stress

as a function of shear rate.

Figure 11. A single column, table top Load Frame Texture Analyzer

43

Figure 12. A 0.5” steel probe forced into the center crumb of the bread

Sensory Evaluation. Three replicate batches of the control and PSC bread were made

for sensory analysis. Bread was prepared 24 hours prior to testing. The sensory panel was

comprised of 104 students and faculty members randomly recruited through class

announcements, e-mails, and fliers posted on campus. Each participant signed a consent form

informing them of the dietary restrictions, time commitment and payment, risks and benefits of

the study, confidentiality and his/her voluntary participation rights. Panelists evaluated the

control and PSC bread for overall texture, nutty flavor, overall flavor, overall acceptance and

aftertaste.

Samples for sensory analysis were produced identically to those used for texture analysis.

The ends of the loaves of bread were cut off and the bread was cut into slices. Each slice was cut

in half so that each piece had a crust on three of the four edges. Random sample codes were

assigned to each sample.

44

Each panelist received one control and one PSC piece of bread simultaneously on a single

tray (Figure 13). Samples were ordered randomly for each panelist. Panelists evaluated each

sample for intensity of nutty flavor, overall flavor, and aftertaste using a 1-5 hedonic scale. They

then evaluated each sample for liking of texture and overall acceptance using a 1-9 hedonic

scale. Each panelist was instructed to rate aftertaste by waiting 30 seconds after tasting each

sample. Compusense Sensory Software (Guelph, Ontario) was used to collect and analyze the

data. A bread sensory analysis scorecard is in Appendix A.

Figure 13. Tray setup of bread samples given to each panelist for evaluation.

45

Moisture Content. Moisture content of the pumpkin seed cake was determined by oven

drying and weight difference according to AACC Method 44-15A (2000). Triplicate samples of

PSC were weighed and placed in a convection oven (Model MO1450A/SA, Lindberg/Blue,

Asheville, North Carolina) for 24 hours at 103°C. Dried samples were weighed and weight loss

was calculated as percent moisture.

Fat Analysis. Eight, 2g samples of PSC were weighed and dried at 103°C for 24 hours to

less than 10% moisture in preparation for fat content in an Accelerated Solvent Extractor (ACE)

(Dionex, Salt Lake City, Utah) using hexane as a solvent (Figure 14). Dried samples were stored

in a desiccator prior to fat extraction. Percent fat is calculated as the grams of fat (recovered

from the hexane) divided by the original sample weight times 100. Weighed samples of PSC

were placed in the extraction vials with diatomaceous earth on the top and bottom of the sample

to fill the remaining space in the vial. Vials were sealed and wiped with Kim® wipes to remove

any oils and dust that may have collected on the outside and placed in the ACE carousel. Fat

extraction was conducted at 125°C and 1500 psi for 8 minutes.

Figure 14. Accelerated Solvent Extractor

46

Data Collection Procedures

The Statistical Program for Social Sciences (SPSS) version 17.0 (SPSS, 2010) was used

for the data analysis. Data for bread measurements, fat analysis, and moisture content was

analyzed by an independent sample t-test in SPSS. Compusense Sensory Software version 4.6

(Guelph, Ontario) was used to collect data for the sensory evaluation tests and computed

statistics on the data using the Tukeys HSD test. All tests were evaluated at a statistical

significance of p<0.05. A stress at maximum force was produced from the data collected by the

Instron®. The modulus (initial slope of the stress/strain curve), and maximum stress data were

analyzed in SPSS through an independent sample t-test.

47

Chapter IV: Results and Discussion

This research was designed to evaluate the effects of PSC on the texture of bread and to

determine if acceptable quality bread can be made using PSC. Bread was made with and without

PSC. It was analyzed for firmness using compression tests and specific sensory attributes using

an untrained sensory panel.

PSC Proximate Analysis

Data analysis gave a moisture content of 6% and a fat content of 37% in the PSC. The

protein (45%), carbohydrates (5%), and crude fiber (2.5%) were calculated by difference using

literature values (Table 7).

Table 7

Estimates of Proximate Composition of Macro Ingredients in PSC

Compound %

Moisture 6%

Protein 45%*

Fat 37%

Carbohydrates 5%*

Crude Fiber 2.5%*

*Calculated by difference using literature values (Deimel, 2007)

Flour Moisture Absorption

The control dough required 79.0ml of distilled water to reach the target level of 530 PU

in 7 minutes while the PSC-containing dough needed 90.0mL distilled water to reach the 550 PU

level in 8½ minutes (Figures 15-17), (Table 8). The Promylograph curves and increased

moisture levels suggest delayed development of gluten in the PSC dough resulting from slower

48

hydration of the proteins and an increased competition for water between the hydrophilic

elements in the dough. Pumpkin seed cake increases the total protein content of the dough by the

addition of primarily globulin type proteins, which are highly water soluble and nonfunctional

with respect to gluten. The soluble globulin proteins preferentially hydrate and limit the water

available to glutinen and gliadin proteins requiring additional water to reach the target BU.

Figure 15. Farinograph of flour/water absorption

Figure 16. Farinograph of control bread dough

49

Figure 17. Farinograph of PSC bread dough

Table 8

Farinograph Data from Flour/Water Absorption, Control Bread Dough, and PSC Bread Dough

Data Point Flour/Water Control PSC

Absorption Bread Dough Bread Dough

Highest PU’s 550 PU’s 530 PU’s 550 PU’s

Development Time (min.) 2 min. 7 min. 8 ½ min.

Stability (min.) 13+ min. 4 min. 1 ½ min.

Final Dough Formulae

Differences between the control and the PSC bread included the flour/water ratio, percent

of individual base ingredients, and the type of fat used (Table 9). Hypothetically, there was

4.433% total fat from pumpkin seed derived ingredients in the PSC bread. The total fat in PSC

50

was unknown before bread formulation. Vegetable shortening was added to the control and PSC

and pumpkin seed oil were added to the PSC bread. The dough development time or time to

peak and the stability time differed between the control and PSC bread. The control bread dough

had a 7 minute development time and a 4 minute stability time. The PSC bread dough had an 8.5

minute development time and a 1.5 minute stability time. The height thickness of the ink line on

the PSC bread dough farinograph was very short compared to the control bread dough

farinograph. These differences can account for the addition of an oil and the type of proteins in

PSC in the PSC bread dough.

Table 9

Final Benchtop Formulae for Control Bread and PSC Bread

AACC Bread Control Bread PSC Bread

Ingredient Supplier Baker’s %/% Baker’s %/% Baker’s %/%

Bread Flour Heartland Mills 100%/54.8% 50.8% 45.9%

Water 60/37.5% 42.0% 37.7%

Dry Active Yeast Red Star 3/1.6% 1.5% 1.4%

Table Salt Morton 2/1.1% 1.0% 0.9%

Sugar, granulated Sugar Crystal 4/2.2% 2.0% 1.8%

Vegetable Shortening Crisco 3/1.6% 1.5% --

NFDM Dairy Americas 2/1.1% 1.0% 0.9%

PSC Hay River Foods -- -- 10.9%

Pumpkin Seed Oil Hay River Foods -- -- 0.4%

Total 100.00% 100.0% 100%

51

Bread Volume and Dimensions

The average height, width, and length of loaves of control bread were

77.07mm x 76.10mm x 138.38mm compared to 54.65 x 74.33mm x 136.59mm for loaves with

PSC (Table 10). The only marked difference is the average height, which was considerably less

for bread with PSC. This results from the lower specific volume (ml/g) in bread made with PSC.

Length and width did not change because the loaves were constrained by the baking pans.

Volume was figured by multiplying the average length by the average width by the average

height of the breads. It was observed that PSC at 10.9% lowers the proof volume of dough

compared with the control. This could result from the interference of PSC in the development

and gas holding capacity of gluten or PSC could have inhibited the fermentation of yeast in the

dough resulting in less CO2 production.

Table 10

Dimensions of Bread Loaves for Control and PSC

Bread Sample Mean Height Mean Volume

Control 77.07mm 81149.22cc3

PSC 54.65mm 55409.53cc3

*p < .05

Improved gluten quality will increase the form ratio (height/width) of bread. Lower

quality gluten results in a reduced form ratio (Tronsmo, Faergestad, Schofield, & Magnus, 2003).

The amount of gluten in bread will also impact loaf volume of bread (Kihlberg et al., 2006).

Pumpkin seed cake has a low quality protein with respect to bread so the form ratio is adversely

affected. Pumpkin seed cake may also inhibit the development of gluten during mixing resulting

in a reduced quality gluten matrix.

52

Changes to consider for improvement of volume and dense crumb in the PSC bread may

be to adjust the mix time or the addition of ingredients to increase gas-holding properties such as

an oxidant, dough strengthener, or vital wheat gluten (Atwell, 2001).

Bread Texture Data

Relative maximum stress is indicative of firmness in bread, the greater the stress the

firmer the crumb. Maximum compressive stress in PSC containing bread was 0.796 mPa

compared with 0.242 mPa for the control bread (Table 11). The increase in firmness correlates

with the increase in the modulus (2.733) of PSC-containing bread over the modulus for the

control (1.093).

Table 11

Rheology (Modulus and Maximum Compressive Stress Values) from the Instron®

Texture

Analyzer for the Control Bread Samples and with PSC Samples

Descriptive Statistics

Calculated Data M SD

Modulus, Control 1.093mPa 0.340mPa

Modulus, PSC 2.733 mPa 0.798mPa

Maximum Compressive 0.242 mPa 0.040mPa

Stress, Control

Maximum Compressive 0.796 mPa 0.113mPa

Stress, PSC

*p < .05

Sensory Analysis

Nutty flavor was perceived as higher in the PSC bread, but overall flavor in the control

was preferred to the PSC bread (Figure 18). The nutty flavor associated with pumpkin seed cake

is not perceived as a positive in bread, even though many whole grain bread products contain

53

nuts and ingredients with a nut-like flavor. There was no significant difference in the perceived

aftertaste rating (p<.5). Subjects preferred the control bread (hedonic of 6.38) to the bread with

PSC (hedonic of 4.82) (Figure 19), (Table 12). These preference numbers were nearly identical

to the hedonics for overall texture suggesting that texture differences drove liking of these

breads.

Figure 18. Within each set of bars, different lower case letters are significantly different (p<.05).

2.20a

3.15a

2.74a

2.80b 2.71b 2.88a

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

Nutty Flavor Overall Flavor Aftertaste

Flavor and Aftertaste Sensory Results for Control and PSC Bread

Control #582

PuOC #849

54

Figure 19. Within each set of bars, different lower case letters are significantly different (p<.05).

Table 12

Sensory Evaluation Results of Control and PSC Bread

Descriptive Statistics

Attribute M SD

Nutty Flavor, Control 2.20 1.18

Nutty Flavor, PSC 2.80 1.12

Aftertaste, Control 2.74 1.21

Aftertaste, PSC 2.88 1.23

Overall Texture, Control 6.30 1.59

Overall Texture, PSC 4.66 1.91

Overall Flavor, Control 3.15 1.13

Overall Flavor, PSC 2.71 1.10

Overall Acceptance, Control 6.38 1.57

Overall Acceptance, PSC 4.82 2.03

*p < .05

6.30a 6.38a

4.66b 4.82b

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Overall Texture Overall Acceptance

Texture and Acceptance Sensory Results for Control and PSC Bread

Control #582

PuOC #849

55

Appearance

Figure 20 shows the crumb structure of the control bread. Figure 21 shows the crumb

structure of the PSC bread. The control appears to have smaller cell sizes and a finer crumb

structure than bread made with PSC. The top crust of the breads also differed. The control had a

rounded, raised top while the PSC bread had an even, flattened surface.

Figure 20. Middle vertical cross section of control bread crumb

Figure 21. Middle vertical cross section of PSC bread crumb

Moisture Content

The moisture analysis indicated that the PSC had a moisture content (MC) of 6.06%. This

moisture content is in range of the mean in research conducted on six hull-less pumpkin seed

varieties of which it was found to have a mean of 6.24 + 0.84% moisture content considering that

only oil is extracted from the seed (Nakić et al., 2006). Data analysis from another study reported

a moisture content in hull-less pumpkin seeds (C. pepo var. olinka) as 6.65% moisture

(Vujasinovic, Djilas, Dimic, Romanic, & Takaci, 2010).

56

Fat Analysis

The fat content on the pumpkin seed cake was analyzed using the Accelerated Solvent

Extractor and then dried in the moisture oven to remove the remaining hexane liquid. The lipid

content was 37% fat in the PSC. Figure 22 shows the remaining fat that was found in triplicate

samples of PSC, following evaporation of hexane.

Figure 22. Pumpkin seed oil extracted from PSC by the ASE

Albumin and globulin are the main proteins in Styrian oil pumpkin seeds (Fruhwirth &

Hermetter, 2007). In the bread study with wheat-flour and pumpkin seed product blends, all the

test blends containing pumpkin seed or pumpkin seed proteins had higher percent water

absorption and development times with lower stability times than the control dough without

pumpkin seed. The stability time is a function of the dough strength. Fermenting the PSC would

denature the proteins limiting their ability to absorb water. This allows gluten proteins an

opportunity to absorb water and develop the dough, thus resulting in a shorter development time

and longer stability (El-Soukkary, 2001). The PSC in this research was not treated or processed

after the pumpkin seed oil extraction before being added to the bread dough. The results

discussed in this paper reflect the data reported in the bread study using pumpkin products from

the Cucurbita moschata, variety Dickinson pumpkins.

57

Chapter V: Conclusion

An attempt was made to produce quality bread using pumpkin seed cake as a source of

antioxidants, protein, and monounsaturated and polyunsaturated fatty acids. The data from the

material properties and sensory analysis (except in overall aftertaste) was significantly different

between the control bread and the PSC bread. This research was conducted as a baseline to

determine if PSC could be used to make good quality bread. It was determined that further bread

formulation with the PSC to improve consumer acceptability is recommended. Utilizing PSC, a

by-product of expelling pumpkin seed oil, in food products needs further investigation to

determine if its nutritional properties offer an economical alternative to disposing PSC as

fertilizer on crop fields or mixing into animal feed.

The PSC inhibits gluten development by interfering with the interaction of glutenin and

gliadin during mixing. This interference was shown in the low proof height of the PSC dough

and the low bread loaf height in the PSC bread. Pumpkin seed cake becomes a non-functional

nutritional additive disrupting gas-holding capacity of gluten in bread. Pumpkin seed cake

affects the height, volume, crumb structure, textural properties, and sensory properties when

added to a yeast leavened white wheat bread.

Pumpkin seed cake still contains about 37% oil after expelling oil the first time. Oil

breaks down foams. Bread dough is a foam and this additional oil in the PSC may be

contributing to the final bread structure.

Volume and Texture Relationship

Even though the control and the PSC bread contained the same amount of wheat flour,

there was a higher percent of gluten in the control compared to the PSC bread. This resulted in

size differences between PSC bread and control bread. The PSC bread could not tolerate the

58

large and small deformation rates during mixing and baking. Therefore, the addition of wheat

gluten or dough strengtheners to the PSC bread may improve PSC bread volume and texture.

The PSC bread dough took a longer time to reach peak than the control bread dough. The

average height and average volume of the control bread compared to the PSC bread is greater

and significantly different. The difference in volume of the two breads was affected by ability of

the PSC to hold gas. The PSC bread did not rise as high as the control bread during fermentation

through baking. The crumb in the PSC bread is tighter and smaller than the crumb in the control

bread because the PSC bread has smaller air capacity holding properties.

Sensory

There was no significant difference in the perceived aftertaste rating between the control

bread and PSC bread. A more detailed and appropriate question could have been utilized

addressing bitter aftertaste rather than aftertaste. Both breads may have had an aftertaste either

pleasant or non-pleasant, but the question was referring to the intensity of taste. Also, the

participants may not have waited 30 seconds after tasting each sample.

The overall acceptance of PSC bread was significantly lower than the control bread. The

sensory attributes (appearance, aroma, texture, and flavor) that consumers subjectively use to

score a food product, all contribute to the consumer’s overall acceptance of the food product.

Texture and overall acceptance, the most important attribute in this study, was consistent with

material properties.

The bitter aftertaste would probably decrease and the consumer acceptance would

improve for the PSC bread if the PSC was used in a whole wheat bread. One of the reasons is

that consumers with an interest in nutrition eat whole wheat bread. Adding PSC to bread could

improve nutritional quality of the bread. Adding PSC to whole wheat or breads that contain

59

whole grains and seeds, could mask the taste and texture issues outlined in this study. Pumpkin

seed cake was added to a white wheat bread to magnify the differences between the PSC bread

and control bread. Future research could include adding pumpkin seed cake to a whole wheat

bread that contains other adjuncts like pumpkin seeds, nuts, raisins, etc. to disguise the bitterness

of PSC.

Sensory and Texture Relationship

The control bread and PSC bread were characterized using compression analysis. The

texture data was consistent with the sensory properties of the bread. The sensory and texture

relationship was negatively correlated; the higher the maximum stress, the lower the texture

preference in the sensory test. In the sensory test, texture drove the overall acceptance. Based

upon an average consumer acceptance, the PSC bread scored lower than the control bread in the

sensory test.

Economic Value

If an acceptable consumer bread product with PSC can be made, PSC can become a value

generating ingredient rather than a liability. Even if bread is not the best target food product for

PSC, this study suggests that PSC could be used in other more flavorful food applications such

as nutrition bars, pesto, or a products with whole grains.

Recommendations

Pumpkin seed meal (pumpkin seed cake with remaining oil removed) may work better in

the PSC bread and could replace some of the flour. Experimentation with the addition of wheat

gluten may enhance the texture, sensory, and volume properties. A new formula or a change in

the process may improve the gas holding capacity to increase the volume and improve the

consumer acceptability of the PSC bread texture. This would have a positive effect on the PSC

60

bread, because the height of the bread would increase. The pumpkin seed products could be

fermented before being added into the dough. This would result in a higher protein bread than

standard wheat bread. Ideally, the fermentation step would break down the proteins and decrease

the competition for water between the functional and non-functional proteins in dough

development.

61

References

American Association of Cereal Chemists (AACC). Approved Methods Committee. (2000).

(10th

Ed.), St. Paul, MN: AACC

Andjelkovic, M., Van Camp, J., Trawka, A., & Verhé,R., (2010). Phenolic compounds

and some quality parameters of pumpkin seed oil. European Journal of Lipid

Science and Technology. 112(2), 208-217. doi: 10.1002/ejlt.200900021

Applequist, W., Avula, B., Schaneberg, B., Wang, Y., & Khan, I. (2006). Comparative

fatty acid content of seeds of four Curcurbita species grown in a common

(shared) garden. Journal of Food Composition and Analysis, 19(6-7), 606-611.

doi: 10.1016/j.jfca.2006.01.001

Atwell, B. (2001). Wheat and flour testing: Wheat flour. (pp. 53-55). American Association of

Cereal Chemists. St. Paul, MN: AACC International, Inc.

Bavec, F., Mlakar, S., Rozman, C., & Bavec, M. (2007). Oil pumpkins: Niche for organic

producers. Reprinted from: Issues in new crops and new uses. 2007. J. Janick &

A. Whipkey (eds.). ASHS Press, Alexandria, VA. Retrieved from

http://www.hort.purdue.edu/newcrop/ncnu07/pdfs/bavec185-189.pdf

Botanical Oil Innovations. (2011a). NatureFRESH cold-press.™ Retrieved from

http://www.botanicoil.com/research/cold_press_process.htm

Botanical Oil Innovations. (2011b). Pumpkin seed oil from certified organic pumpkin seed.

Retrieved from http://www.botanicoil.com/datasheets/pumpkin%20seed%20oil.pdf

Caili, F., Huan S., & Quanhong, L. (2006). A review on pharmacological activities and

utilization technologies of pumpkin. Plant Foods for Human Nutrition, 61(2), 73-80. doi:

10.1007/s11130-006-0016-6

62

Cucurbit network news. (2004). Retrieved from http://www.cucurbit.org/oilpages/backgr.html

Damodaran, S., Parkin, K., & Fennema, O. (2007) Lipids. (4th

Ed.), Fennema’s Food Chemistry

(186-191). Boca Raton, FL: CRC Press.

Deimel, T. (2007). Important facts about award-winning styrian pumpkin seed oil and

your health. Retrieved from: www.deimel.biz/_biz/info/pumpkinoil.htm

Delcour, A., & Hoseney, R. (2010a). Proteins of cereals. (3rd

Ed.), Principles of Cereal Science

and Technology (pp. 58-59). American Association of Cereal Chemists. St. Paul, MN:

AACC International, Inc.

Delcour, A., & Hoseney, R. (2010b). Rheology of doughs and batters. (3rd Ed.), Principles of

Cereal Science and Technology (pp. 87-88). American Association of Cereal Chemists.

St. Paul, MN: AACC International, Inc.

Dubois, V., Breton. S., Linder, M., Fanni, J., & Parmentier, M. (2007). Fatty acid profile

of 80 vegetable oils with regard to their nutritional potential. European Journal of

Lipid Science and Technology, 109(7), 710-732. doi:10.1002/ejlt.200700040

El-Soukkary, F. (2001). Evaluation of pumpkin seed products for bread fortification.

Plant Foods for Human Nutrition, 56(4), 365-384.

FAO Corporate Document Repository. (1999) SECTION 1. Codex general standard for

fats and oils. Codex standard for edible fats and oils not covered by individual

standards. Retrieved from

http://www.fao.org/DOCREP/004/Y2774E/y2774e03.htm

Fischerauer, A. (1996). All about pumpkin seed oil. Retrieved from

www.pumpkinseedoil.com/procedure

63

Fruhwirth, G., & Hermetter, A. (2007). Seeds and oil of the Styrian oil pumpkin:

Components and biological activities. European Journal of Lipid Science and

Technology, 109(11), 1128-1140. doi:10.1002/ejlt.200700105

Fruhwirth, G., & Hermetter, A. (2008). Production technology and characteristics of

Styrian pumpkin seed oil. European Journal of Lipid Science and Technology,

110(7), 637-644. doi: 10.1002/ejlt.200700257

Gallagher, E., Gormley, T., & Arendt, E. (2003). Crust and crumb characteristics of gluten free

breads. Journal of Food Engineering. 56(2-3), 153-161. doi:10.1016/S0260-

8774(02)00244-3

Gorjanović, S., Rabrenović, B., Novaković, M., Dimić, E., Basić, Z., & Sužnjević.

(2011). Cold-pressed pumpkin seed oil antioxidant activity as determined by a DC

polarographic assay based on hydrogen peroxide scavenge. Journal of the

American Oil Chemists’ Society, 88(12), 1875-1882. doi:10.1007/s11746-011-

1863-3

Gryglewicz, S., Grabas, K., & Gryglewicz, G. (2000). Use of vegetable oils and fatty acid

methyl esters in the production of spherical activated carbons. Bioresource

Technology, 75(3), 213-218. doi:10.1016/S0960-8524(00)00054-7

Halvorsen, B., Carlsen, M., Phillips, K., Bøhn, S., Holte, K., Jacobs Jr., D., & Blomhoff, R.

(2006). Content of redox-active compounds (ie, antioxidants) in foods consumed in the

United States. The American Journal of Clincial Nutrition, 84(1), 95-135. Retrieved from

www.ajcn.org

64

Joebstl, D., Bandoniene, D., Meisel, T., & Chatzistathis, S. (2010). Identification of the

geographical origin of pumpkin seed oil by the use of rare earth elements and

discriminant analysis. Food Chemistry, 123(4), 1303-1309.

doi:10.1016/j.foodchem.2010.06.009

Kemble, J., Sikora, E., Zehnder, G., & Bauske, E. (2000). Guide to commercial pumpkin and

winter squash production. Alabama Cooperative Extension System. ANR-1041

http://www.aces.edu/pubs/docs/A/ANR-1041/ANR-1041.pdfF

Kihlberg, I., Öström, Ǻ., Johansson, L., & Risvik, E. (2006). Sensory qualities of plain white pan

bread: Influence of farming system, year of harvest and baking technique. Journal of

Cereal Science, 43(1), 15-30. doi:10.1016/j.jcs.2005.04.008

Marr, C., Schaplowsky, T., & Carey, T. (2004). Commercial vegetable production: Pumpkins.

Kansas State University Agricultural Experiment Station and Cooperative Extension

Service. Retrieved from www.oznet.ksu.edu

Matthäus, B., & Spener, F. (2008). What we know and what we should know about

virgin oils – a general introduction. European Journal of Lipid Science and

Technology, 110(7), 597-601.

Miyazaki, M., Maeda, T., & Morita, N. (2004). Effect of various dextrin substitutes for wheat

flour on dough properties and bread qualities. Food Research International, 37(1), 59-64.

doi:10.1016/j.foodres.2003.08.007

Murkovic, M., & Pfannhauser W. (2000). Stability of pumpkin seed oil. European

Journal of Lipid Science and Technology, 102(10), 607-611.

doi: 10.1002/1438-9312(200010)

65

Murkovic, M., Piironen, V., Lampi, A., Kraushofer, T., & Sontag, G. (2004). Changes in

chemical composition of pumpkin seeds during the roasting process for

production of pumpkin seed oil (Part 1: non-volatile compounds), Food

Chemistry, 84(3) 359-365. doi:10.1016/S0308-8146(03)00240-1

Nakić, S., Rade, D., Škevin, D., Štrucelj, D., Mokrovčak, Ž., & Bartolić, M. (2006).

Chemical characteristics of oils from naked and husk seeds of Cucurbita pepo L.

European Journal of Lipid Science and Technology, 108(11), 963-943. doi:

10.1002/ejlt.200600161

Nyam, K., Tan, C. Lai, O., Long, K., & Che Man, Y. (2009). Physicochemical properties and

bioactive compounds of selected seed oils. LWT - Food Science and Technology, 42(8),

1396-1403. doi:10.1016/j.lwt.2009.03.006

Parry, J., Hao, Z., Luther, M., Su, L., Zhou, K., & Yu, L. (2006). Characterization of cold-

pressed onion, parsley, cardamom, mullein, roasted pumpkin, and milk thistle seed oils.

Journal of the American Oil Chemists' Society, 83(10), 847-854. doi:10.1007/s11746-

006-5036-8

Peričin, D., Krimer, V., Trivić, S., & Radulović, L. (2009). The distribution of phenolic

acids in pumpkin’s hull-less seed, skin, oil cake meal, dehulled kernel and hull.

Food Chemistry, 113(2), 450-456. doi:10.1016/j.foodchem.2008.08.079

Popović, S., Peričin, D., Vaštag, Ž., Popović, L., & Lazić,V. (2010). Evaluation of edible

film-forming ability of pumpkin oil cake; effect of pH and temperature. Food

Hydrocolloids, 25(3), 470-476. doi:10.1016/j.foodhyd.2010.07.022

66

Robbins, R. (2003). Phenolic acids in foods: An overview of analytical methodology. Journal of

Agricultural and Food Chemistry, 51(10), 2866–2887.

doi: 10.1021/jf026182t

Ruckenbauer, P. (1999). Forming part of the IENICA project. Retrieved from University

of Agricultural Sciences Vienna, Institute of Agronomy and Plant Breeding

website www.ienica.net/reports/austria.pdf

Scanlon, M., & Zghal, M. (2001). Bread properties and crumb structure. Food

Research International, 34(10), 841–864. doi:10.1016/S0963-9969(01)00109-0

Szterk, A., Roszko, M., Sosińska, E., Derewiaka, D., & Lewicki, P., (2010). Chemical

composition and oxidative stability of selected plant oils. Journal of the American Oil

Chemists' Society, 87(6), 637–645. doi:10.1007/s11746-009-1539-4

Tan, C., Man, C., Selamat, J., & Yusoff, M., (2002) Comparative studies of oxidative

stability of edible oils by differential scanning calorimetry and oxidative stability

index methods. Food Chemistry, 76(3) 385-389.

doi:10.1016/S0308-8146(01)00272-2

Tanner, R., Qi, F., & Dai, S. (2008). Bread dough rheology and recoil I. Rheology. Journal of

Non-Newtonian Fluid Mechanics, 148(1-3), 33-40. doi:10.1016/j.jnnfm.2007.04.006

Tronsmo, K., Faergestad, E., Schofield, J., & Magnus, E. (2003). Wheat protein quality

in relation to baking performance evaluated by the Chorleywood bread process

and a hearth bread baking test. Journal of Cereal Science, 38(2), 205-215. doi:

10.1016/S0733-5210(03)00027-4

USDA (United States Department of Agriculture) Agricultural Research Service. (2010).

USDA database for the oxygen radical absorbance capacity (ORAC) of selected

67

foods, Release 2. Retrieved from

http://www.ars.usda.gov/SP2UserFiles/Place/12354500/Data/ORAC/ORAC_R2.p

df

Vaisey-Genser, M., Malcolmson, L., Ryland, D., Przybylski, R., Eskin, A., & Armstrong, L.

(1994). Consumer acceptance of canola oils during temperature-accelerated storage.

Food Quality and Preference, 5(4), 237-243.

Vaštag, Z, Popović, L., Popović, S., Krimer, V., & Peričin, D. (2010). Hydrolysis of

pumpkin oil cake protein isolate and free radical scavenging activity of

hydrolysates: Influence of temperature, enzyme/substrate ratio and time. Food

and Bioproducts Processing, 88(2-3), 277-282. doi:10.1016/j.fbp.2009.12.003

Vujasinovic, V., Djilas, S., Dimic, E. Romanic, R., & Takaci, A. (2010). Shelf life of

cold-pressed pumpkin (Cucurbita pepo L.) seed oil obtained with a screw press.

Journal of the American Oil Chemists’ Society, 87(12), 1497-1505. doi:

10.1007/s11746-011-1863-3

Weipert, D. (1990). The benefits of basic rheometry in studying dough rheology. Cereal

Chemsitry, 67(4), 311-317.

Willis, L. M., Shukitt-Hale, B., & Joseph, J. A. (2009). Modulation of cognition and

behavior in aged animals: role for antioxidant- and essential fatty acid-rich plant

foods. The American Journal of Clincial Nutrition, 89(5), 1602S-1606S.

Retrieved from www.ajcn.org

Xanthopoulou, M., Nomikos, T., Fragopoulou, E., & Antonopoulou,S. (2009). Antioxidant and

lipoxygenase inhibitory activities of pumpkin seed extracts. Food Research International,

42(5-6), 641-646. doi:10.1016/j.foodres.2009.02.003

68

Zdunczyk, Z., Minakowski, D., Frejnagel S., & Flis M. (1999). Comparative study of the

chemical composition and nutritional value of pumpkin seed cake, soybean meal and

casein. Food/Nahrung, 43(6), 392-395. doi:10.1002/(SICI)1521-

3803(19991201)43:6<392::AID-FOOD392>3.0.CO;2-2

Zghal, M., Scanlon, M., & Sapirstein, H. (2002). Cellular structure of bread crumbs and

its influence on mechanical properties. Journal of Cereal Science, 36(2), 167-176.

doi:10.1006/jcrs.2001.0445

69

Appendix A: Sensory Evaluation Form

Sensory Evaluation

Protein Fortified Bread

Please taste the sample on the left first and drink water. Wait for 30 seconds before proceeding to

tasting the next sample. Rank each sample according to the Hedonic Scale ratings.

Rate the sample based on its nutty/grainy flavor.

☐ ☐ ☐ ☐ ☐

1 2 3 4 5

Low Intensity High Intensity

What is your impression of the overall flavor quality of this sample?

☐ ☐ ☐ ☐ ☐

1 2 3 4 5

Disliked Liked

Aftertaste—Please wait at least 30 seconds after tasting the sample before selecting the

rating below.

☐ ☐ ☐ ☐ ☐

1 2 3 4 5

Low Intensity High Intensity

With respect to bread, what is your impression of the texture?

☐ ☐ ☐ ☐ ☐ ☐ ☐ ☐ ☐

1 2 3 4 5 6 7 8 9

Disliked Liked

Rate the sample according to your overall acceptance.

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1 2 3 4 5 6 7 8 9

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Appendix B: Institutional Review Board Approval

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Murkovic, M., Piironen, V., Lampi, A., Kraushofer, T., & Sontag, G. (2004). Changes in

chemical composition of pumpkin seeds during the roasting process for

production of pumpkin seed oil (Part 1: non-volatile compounds), Food

Chemistry, 84(3) 359-365. doi:10.1016/S0308-8146(03)00240-1

Nakić, S., Rade, D., Škevin, D., Štrucelj, D., Mokrovčak, Ž., & Bartolić, M. (2006).

Chemical characteristics of oils from naked and husk seeds of Cucurbita pepo L.

European Journal of Lipid Science and Technology, 108(11), 963-943. doi:

10.1002/ejlt.200600161

Nyam, K., Tan, C. Lai, O., Long, K., & Che Man, Y. (2009). Physicochemical properties and

bioactive compounds of selected seed oils. LWT - Food Science and Technology, 42(8),

1396-1403. doi:10.1016/j.lwt.2009.03.006

Parry, J., Hao, Z., Luther, M., Su, L., Zhou, K., & Yu, L. (2006). Characterization of cold-

pressed onion, parsley, cardamom, mullein, roasted pumpkin, and milk thistle seed oils.

Journal of the American Oil Chemists' Society, 83(10), 847-854. doi:10.1007/s11746-

006-5036-8

Peričin, D., Krimer, V., Trivić, S., & Radulović, L. (2009). The distribution of phenolic

acids in pumpkin’s hull-less seed, skin, oil cake meal, dehulled kernel and hull.

Food Chemistry, 113(2), 450-456. doi:10.1016/j.foodchem.2008.08.079

Popović, S., Peričin, D., Vaštag, Ž., Popović, L., & Lazić,V. (2010). Evaluation of edible

film-forming ability of pumpkin oil cake; effect of pH and temperature. Food

Hydrocolloids, 25(3), 470-476. doi:10.1016/j.foodhyd.2010.07.022