Analysis of factors affecting volatile compound
formation in roasted pumpkin seeds with selected ion
flow tube mass spectrometry (SIFT-MS)
THESIS
Presented in Partial Fulfillment of the Requirements for the Degree Master
of Science in the Graduate School of The Ohio State University
By:
Tessa Bowman
Graduate Program in Food Science and Technology
The Ohio State University
2011
M.S. Committee:
Dr. Sheryl Barringer, Advisor
Dr. James Harper
Dr. John Litchfield
© Copyright by
Tessa Bowman
2011
Abstract
Pumpkin (Cucurbita pepo and maxima) seeds are uniquely flavored and
commonly consumed as a healthy roasted snack. The objective was to determine
dominant volatiles in raw and roasted pumpkin seeds, and the effect of seed coat,
moisture content, fatty acid ratio, total lipids, reducing sugars and harvest year on volatile
formation. Sensory was conducted to evaluate overall liking of seed variety and texture.
Seed processing included: extraction from the fruit, dehydration and roasting (150ᵒC).
Oil extraction was done using soxhlet method, fatty acid profile using GC-FID and
reducing sugars using 3,5 dinitrosalycylic acid reagent and uv spectroscopy. Headspace
analysis of seeds was performed by a Selected Ion Flow Tube Mass Spectrometer (SIFTMS). Volatiles dominating in raw pumpkin seeds were lipid aldehydes, ethyl acetate, 2,
3-butandione and dimethylsulfide. Compounds contributing to roasted aroma include
alkylpyrazines, and Strecker and lipid aldehydes. Overall, hull-less seeds had higher
volatile lipid aldehydes along with Strecker aldehydes. Seeds dehydrated to a moisture
content of 6.5% before roasting had higher initial and final volatile concentrations than
seeds starting at 50% moisture. Higher oil content resulted in higher lipid aldehyde
formation during roasting and a moderate correlation between FFA ratio and
corresponding lipid aldehyde was found. Harvest year (2009 vs. 2010) had a significant
ii
impact on volatile formation in hull-less seeds, but not as much as variety differences. No
significant correlation was found between reducing sugars and volatile formation.
Sensory showed that hull-less seeds were liked significantly more than hulled seeds.
Practical Applications
Elucidation of the aromatic flavor development during roasting with a SIFT-MS
provides information on flavor release and offer better control during processing.
Knowledge of volatiles in raw and roasted pumpkin seeds and effects of seed coat,
moisture content, seed composition and harvest date will allow for better control over the
production/ storage/transportation process and a more educated decision during selection
of a variety for production of pumpkin seeds in the snack food industry.
iii
Dedicated to my family
iv
Acknowledgements
First and foremost I would like to thank my advisor, Dr. Sheryl Barringer. She
taught me how to write academic papers, how to manage my projects and most
importantly, how to learn from my mistakes. She is by far the most patient professor I
have had the opportunity to learn from; and I will always be grateful for her constant
support and practical guidance.
Second, I would like to thank my committee members Dr. James Harper, for
introducing me to food science research as an undergraduate and Dr. John Litchfield, for
his continual interest and advice on my research.
Thank you to Jim Jasinski and Paul Courtright for their support and confidence in
me and for making the many hours spent in the pilot plant enjoyable.
I must thank my dad, Steve Bowman, and mom, Charel Bowman, for sacrificing
so much to make me whom I am today. They have taught me the most important lessons
in life through their unconditional love, encouragement and confidence in me at times
when I needed them the most.
Lastly, thank you to my friends and lab mates for their friendship and all of their
support and encouragement.
Mustache
v
Vita
Nov 25, 1986.......................................................................Born- Columbus, OH
2005 – 2009 ........................................................................B.S. Food Science &
Technology .........................................................................
2009-2011 ..........................................................................M.S. Food Science &
Technology
Ohio State University,
Columbus, OH
Publication
Research Publication:
Analysis of factors affecting volatile compound formation in roasted pumpkin seeds with
selected ion flow tube mass spectrometry (SIFT-MS)
Field of Study
Major Field: Food Science & Technology
vi
Table of Contents
Abstract .......................................................................................................................................... ii
Practical Applications .................................................................................................................... iii
Acknowledgements ....................................................................................................................... v
Vita ................................................................................................................................................ vi
List of Tables .................................................................................................................................. x
List of Figures ................................................................................................................................ xi
Chapter 1: Introduction ................................................................................................................... 1
Chapter 2: Literature Review .......................................................................................................... 3
2.1 Pumpkin Seed Background ................................................................................................... 3
2.1.2 Pumpkin Seed Components ............................................................................................ 4
2.2 Flavor .................................................................................................................................... 5
2.2.1 History of the Study of Flavor ........................................................................................ 5
2.2.2 Heat Induced Flavor Reactions....................................................................................... 6
2.2.3 Pumpkin Seed Flavor...................................................................................................... 8
2.3 Maillard Browning Volatiles................................................................................................. 9
2.3.1 Strecker Aldehydes ......................................................................................................... 9
vii
2.3.2 Lipid Oxidation Aldehydes .......................................................................................... 10
2.3.2 Pyrazines ....................................................................................................................... 12
2.3.3 Furans ........................................................................................................................... 15
2.3.4 Sulfur compounds ......................................................................................................... 16
2.3.5 Ketones ......................................................................................................................... 17
2.3.6 Alcohols ........................................................................................................................ 19
2.4 Analytical Methods for Headspace Analysis ...................................................................... 19
2.4.1 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) ............................................. 20
Chapter 3: Materials and Methods ................................................................................................ 27
3.1 Volatile Analysis ................................................................................................................. 27
3.1.1 Seed preparation ........................................................................................................... 27
3.1.2 Roasting............................................................................................................................ 28
3.1.3 Sample preparation ....................................................................................................... 29
3.1.4 Sift-MS ......................................................................................................................... 29
3.2 Total Lipid Quantification ................................................................................................... 32
3.3 Fatty Acid Ratio .................................................................................................................. 32
3.4 Total Reducing Sugars ........................................................................................................ 33
3.5 Sensory Analysis ................................................................................................................. 34
Chapter 4: Results and Discussion ................................................................................................ 36
viii
4.1 Volatile Compounds in Raw Pumpkin Seeds ..................................................................... 36
4.2 Effect of Roasting on Final Volatile Formation .................................................................. 41
4.3 Effect of Seed Coat on Volatile Formation ......................................................................... 43
4.4 Effect of Moisture Content on Volatile Formation ............................................................. 48
4.5 Effect of FFA Composition on Corresponding Lipid Aldehyde Formation ....................... 50
4.6 Effect of Reducing Sugars................................................................................................... 52
4.7 Effect of Harvest Year on Volatile Concentration .............................................................. 52
4.7 Sensory Analysis ................................................................................................................. 54
Chapter 5: Conclusions ................................................................................................................. 58
Chapter 6: References ................................................................................................................... 59
Apendix A: Additional Figures ..................................................................................................... 66
ix
List of Tables
Table
Page
Table 1 Pumpkin seed varieties labels. ............................................................................. 28
Table 2 Reaction kinetics of the volatile compound measured in the headspace of
pumpkin seeds ................................................................................................................... 31
Table 3 Raw and roasted volatiles (µg/L) of 5 seed varieties from 2009 harvest. Different
letters in the same row are significantly different ............................................................. 38
Table 4 Raw volatiles (µg/L) of 15 seed varieties grown only in 2009. Different letters in
the same row are significantly different............................................................................ 39
Table 5 Roasted volatiles (µg/L) of 15 seed varieties grown in 2009, roasted at 150 ᵒC for
40 min. Different letters in the same row are significantly different ................................ 40
Table 6 Raw and roasted volatiles (µg/L) of 5 seed varieties from 2010 harvest ............ 47
Table 7 Fatty acids (g) per 100 g seed oil and total oil (%) .............................................. 51
Table 8 Percent volatile concentration (µg/ L) increase in 2009 seed varieties from raw to
roasted (150°C for 40 min) ............................................................................................... 66
x
List of Figures
Figure
Page
Figure 1 Formation of methylbutanal during roasting from seed varieties 2H, 3H, 14H,
10NH and 15NH (shown on right axis), harvested in 2010. Methylbutanal representative
of all Strecker aldehydes, pyrazines and furans. ............................................................... 44
Figure 2 Formation of pentanal during roasting from seed varieties 2H, 3H, 14H, 10NH
and 15NH, harvested in 2010. Representative of lipid aldehydes = (E)-2-heptenal, (E)-2hexenal, pentanal, 2-pentylfuran; and N-heterocyclic compounds= dimethylpyrazine, 2acetylpyrrole, 2-methylpyrazine ....................................................................................... 48
Figure 3 Effect of starting moisture content (y axis on right) on volatile formation of
methylbutanal during roasting of 10NH seeds, harvested in 2010. .................................. 49
Figure 4 Effect of total lipid (%) on average lipid volatile formation .............................. 51
Figure 5 Roasted volatiles of varieties 2H, 3H, 10NH, 14H and 15NH from 2009 and
2010................................................................................................................................... 53
Figure 6 Overall liking scores of five varieties of pumpkin seeds. .................................. 55
Figure 7 overall liking of seed varieties 2H, 3H, 10NH, 14H and 15NH all roasted for
both 30 and 15 min. .......................................................................................................... 55
Figure 8 Overall liking of seasonings on roasted 10NH seed variety............................... 57
Figure 9 Mechanistic view of hydroperoxide breakdown (Halliwell and Chirico 2011) . 67
Figure 10 Principles of Selected Ion Flow Tube Mass Spectrometry (Smith and Spanel
2005) ................................................................................................................................. 68
Figure 11 Schematic diagram Maillard Browning Reaction (Ames 1990) ...................... 69
Figure 12 Schematic diagram of lipid oxidation and hydrolysis (Whitfield 1992) .......... 70
Figure 13 Effect of roasting on volatiles formation on seed varieties 2H, 3H, 14H, 15NH
and 10NH .......................................................................................................................... 71
Figure 14 Effect of fatty acid on corresponding volatile formation ................................. 72
Figure 15 Effect of percentage reducing sugars on volatile formation of methylbutanal 73
Figure 16 Roasted volatiles of 15 seed varieties from 2009 harvest ................................ 74
Figure 17 Percentage of participants selecting top five seasonings that sounded appealing
on a roasted pumpkin seed from survey sent out electronically using survey monkey. ... 75
xi
Chapter 1: Introduction
Pumpkin seeds are an underutilized resource for healthy snack items in the food
industry. They have historically been used to produce oil, fortify breads, consumed as a
snack or even for medicinal purposes. The unique flavor of pumpkin seeds and pumpkin
seed oil is well known and enjoyed all over the world and contribute to the development
of aromatic flavor during the roasting process. After harvesting, the seeds are often used
as animal feed, ground up for fertilizer or even discarded. With increased public
awareness in sustainable agriculture, clean and efficient energy and waste management
technologies, pumpkin seeds have the opportunity to capture a new and emerging market
share in the snack food industry. Currently, pumpkin seeds are gaining momentum in the
snack food industry as a healthy alternative to other fried snacks.
The Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) is a newer analytical
technique which has the ability to identify and quantify trace gases at relatively low
levels. This has been utilized to detect volatile organic compounds (VOCs) concerning
environmental gas emission (Wilson and others 2003), medical diagnosis (Amann and
others 2007), container air analysis and most recently food, flavor and fragrance (Frank
2007). SIFT-MS has been used to monitor real time release of food volatiles related to
spoilage, oxidation (Davis and McEwin 2007), breath analysis (Hansanugrum and
1
Barringer 2009), enzyme activity (Azcarate and Barringer 2010) and thermally induced
flavor reactions (Huang and Barringer 2011).
Many studies have been conducted on volatile flavor formation during roasting
reactions in model food and various food matrices including coffee (Hashim and
Chaveron 1996), cocoa (Huang and Barringer 2011), peanuts (Brown and others 1973),
almonds (Cantalejo 1997) and pumpkin seeds (Siegmond and Murkovic 2004). During
roasting, volatile concentrations increase significantly which has been attributed to the
Maillard reaction, lipid oxidation and pyrolysis.
Flavor is such an essential part of the marketability of a food product that making a
consistent quality is of the utmost importance. This can be especially difficult to achieve
with the many variables in minimally processed foods, such as pumpkin seeds. Studying
and monitoring headspace volatile compounds with a SIFT-MS can provide information
on flavor release and offer better control over the production/ storage/transportation
process and even detect aromatic compounds associated with off-flavors.
Pumpkin seed varieties often differ in oil percentage, fatty acid ratio, reducing sugar
percentage, moisture percentage, and the presence or absence of a seed husk depending
on pumpkin breed and climate growth conditions. These differences can have a marked
impact on consumer perception and volatile flavor release during roasting. The objective
of this study was to study the effects of roasting, seed variety, moisture content, harvest
year and seed composition on flavor development. This will provide valuable information
needed in the snack food industry aiding in better control during processing.
2
Chapter 2: Literature Review
2.1 Pumpkin Seed Background
Pumpkins are the fruit of the species Cucurbita pepo and belong to the New
World squash family. Recent studies have shown that all current Cucurbita pepo varieties
can be traced back to a common ancestor originating in Southern Mexico. The seeds of
pumpkins are planted so that harvesting occurs around October. Pumpkin seeds
germinate fastest around 94 °F but can germinate between lower and upper temperatures
of 56 and 115°F. Proper ripening occurs within four months and can be determined by a
hard rind or after the first light frost.
Generally pumpkin seeds are a waste by product in the food industry. However, many
countries utilize the seeds as a nutritious food source. Pumpkin seeds can be ground,
fermented, roasted or eaten raw. Consumed as either part of a meal or as a healthy snack
high in fiber, protein, healthy fats and minerals, it is common to roast, salt and flavor
pumpkin seeds.
Pumpkin seeds can be categorized into two groups based on the presence or
absence of a hard outer shell. A hull-less pumpkin seed is the result of a single recessive
gene, producing a parchment like seed coat caused by a reduction of lignin and cellulose
in the hypodermin, sclerenchyma and parenchyma tissues of the seed coat (Fruhwirth and
3
Hermetter 2007). In 1934, Tschermak-Seysenegg reported a mutation, in which
the pumpkin seed coat was reduced to a thin skin (Loy 1990). The seed was further bred
and used to permit more efficient extraction of oil or eaten directly without the need to
remove the hull.
2.1.2 Pumpkin Seed Components
Depending on variety, pumpkin seeds are comprised of approximately 35-59%
oil, 59% protein and minor amounts of carbohydrates and fiber. The relative fatty acid
composition is not only affected by variety, but also by growth conditions, and is
comprised of about 98% linoleic, oleic, stearic and palmitic acids (Murkovic and others
1996). Due to a precursor-product relationship, oleic acid is always negatively correlated
with linoleic acid; therefore, pumpkins harvested late in a season (experiencing colder
temperatures) will have higher levels of linoleic acid and less oleic acid (Fruhwirth and
Hermetter 2007). According to a study conducted by Al-Khalifa (1996) the fatty acid
composition of pumpkin seeds can vary 11.8-13.1% palmitic, 6.0-6.3% stearic, 26.234.9% oleic, 43.1-53.2% linoleic and 0.9-0.12% linolenic acid.
Pumpkin seeds have been reported to be deficient in sulfur containing amino acids
(cysteine and methionine), but relatively higher in rare amino acids such ascitrullin, mcarboxyphenylalnine, β-pyrazolalanine, γ-aminobutyric acid and ethylasparagine
(Fruhwirth and Hermetter 2007). Trace amounts of vitamins A and E, and the minerals
potassium, magnesium, calcium, magnesium, zinc, iron, selenium, copper and
molybdenum have also been detected in pumpkin seeds. High enough levels of iodine
and selenium were measured in pumpkin seeds to cover the recommended daily value for
4
adults (Kreft and others 2002). This becomes especially beneficial to underdeveloped,
malnutritioned countries. Additionally, pumpkin seeds contain secoisolariciresinol
diglucoside, an anti-oxidant phytochemical that has been shown to protect against
hormone-dependent cancers, arthrosclerosis, diabetes, and to have modifying effects on
blood cholesterol levels (Murkovic 1996).
2.2 Flavor
2.2.1 History of the Study of Flavor
The study of flavor is important to the overall perception of a final food product,
however it is very difficult to quantify. An instrument may measure levels of volatile
compounds, but cannot distinguish which contribute most to an overall aroma. In
addition, it has been theorized that the human nose has a threshold detection limit of
approximately 10-19 moles, which surpasses any instrument used currently (Heath 1986).
Also, a low level of a compound may contribute to the identification of a final food more
than the next, which cannot be distinguished by an instrument. Pyrazines have been
known to contribute significantly to the overall aroma of roasted foods, such as coffee,
cocoa beans and seeds. Their threshold limit has been detected in a range from 1.8- 120
ppm, depending on the polarity of the solvent (Hashim and Chaveron 1996). Through
sensory testing the low levels of the pyrazines were shown to be very important to the
overall aroma and determination of optimal roasting temperatures (Hashim and Chaveron
1996).
Another complication with the study of flavor in foods is interactions with other
constituents in the food matrix. Hashim and Chaveron (1996) found that thresholds of
5
pyrazines in water were significantly lower than in oil, and contributed it to Pyrazinelipid interactions. Because of this, coffee beans with higher lipid content had lower levels
of pyrazine compounds formed during roasting. Another study on the interactions
between protein content and volatile flavor released indicated that with an increase in
protein content vanillin levels decreased (Kuhn and others 2006). Kuhn and others (2006)
contributed most volatile flavor-protein interaction to hydrophobic and hydrogen bonding
interactions.
2.2.2 Heat Induced Flavor Reactions
There are mainly two classes of browning, enzymatic browning and
nonenzymatic browning. Within nonenzymatic browning there are three major groups:
caramelization, ascorbic acid degradation and Maillard browning reaction.
Enzymatic browning occurs because of the existence of polyphenoloxidase
(PPO). PPO catalyzes the conversion of phenolic compounds to produce quinones, which
polymerization further to yield dark, insoluble polymers referred to as melanins.
Caramelization is a dehydration reaction that usually occurs during the heating of
carbohydrate rich foods at relatively high temperatures. The reaction is utilized for both
its fragmentation reactions, to produce flavor, and polymerization reaction, to produce
color.
Ascorbic acid degradation is a complex reaction in a food system that is
dependent on many factors, including pH, temperature, and concentration of oxygen,
sugar and salt, and the presence of amino acids, enzymes and metal catalysts. Ascorbic
6
acid easily oxidizes to dehydroascorbic acid and then hydrolyses to 2, 3-diketogulonic
acid where it no longer has vitamin C activity. Further oxidation, dehydration and finally
polymerization take place to form an array of products, including browning pigments.
Lastly, the Maillard browning reaction is an important reaction in thermally
processed foods that produces countless flavor compounds; and if allowed to continue
results in polymerization to brown pigments, also known as melanoidins.
2.2.2.1 Maillard Browning
Maillard browning reaction plays a vital role in the generation of flavors for many
roasted, baked, grilled and otherwise cooked foods. It involves the reaction between a
reducing sugar and an amino acid. This results in a loss of water and an imine that is able
to cyclize and produced an N glycoside. Instead of an isomerization, an Amadori
rearrangement may take place, forming a 1-amino-1-deoxyketose. These products (N
glycosides and Amadori products) are intermediates formed during the Maillard reaction.
Next fragmentation occurs and degrades Amadori products to deoxyosones. These
compounds generate many flavor compounds. 1-deoxyosone gives secondary products
such as furanoses, pentoses and hexoses, while 3-deoxyosone gives secondary products
which include pyrroles, pyradines and formylpyrroles. With increasing heat and/or time,
these products can react further to give different pigments to the final food product. Next
is Strecker degradation which occurs between a dicarbonyl compounds (deoxyosones)
and amines. This reaction involves a transamination and produces aminoketones,
aldehydes and carbon dioxide. Two amino ketones can then condense and react to form
7
pyrazines. Compounds are then polymerized to form melanoidins, brown pigment found
in the Maillard reaction.
2.2.3 Pumpkin Seed Flavor
Pumpkin seeds are often disliked because of a thick fibrous outer shell that can
make mastication difficult and un-enjoyable. Pumpkin seeds can be made more palatable
by de-hulling the seeds, roasting, seasoning or even using alkaline maceration to soften
the shell (Caramez and others 2008). Pyrolysis is the roasting of foods to create flavor
changes that make a food that is more desirable to consumers. Flavor precursors are
lipids, sugars, proteins and amino acids, most typically containing aspartic, glutamic
acids, asparagines-glutamine, phenylalanine and histidine (Buckholz and Daun 1981). As
pumpkin seeds are roasted important volatile organic chemical compounds are produced
via pathways such as Maillard browning, Strecker degradation, and lipid oxidation
(Siegmund and Murkovic 2004). Roasting has been classified into four categories for
coffee beans through sensory tests; slight (100-120°C), normal (120-140ᵒC), strong (140160ᵒC), and over-roasted (>160ᵒC) (Ramli and others 2006). Depending on the roasting
conditions chosen certain volatile chemicals may be higher or lower than others
(Siegmund and Murkovic 2004). These volatile compounds can and have been correlated
with sensory analysis to determine optimal roasting conditions and human perception of
measured flavor volatile chemical concentrations (Leunissen and others 1996).
Roasting is a critical step in the formation of these important compounds. The
main chemical compounds that contribute to the roasted flavor of pumpkin seeds are
pyrazines, aldehydes, alcohols, sulfur compounds and furan derivatives (Siegmond and
8
Murkovic 2004). Pyrazines and aldehydes are among the two most important categories
formed during roasting that contribute to a desirable roasted flavor (Ramli and others
2006), and have also been shown to contribute to flavor stability (Braddock and others
1995). Flavor stability can be caused by a wide variety of chemical and physical
processes, which involves the degradation or masking of characteristic flavors associated
with a given food.
2.3 Maillard Browning Volatiles
2.3.1 Strecker Aldehydes
Strecker degradation often occurs between dicarbonyls (deoxyosones) and αamino amines, resulting in the decarboxylation of the amino acid. This can yield a
Strecker aldehydes and α-amino ketones which contain one less carbon than the original
amino acid, or depending on the oxidizing agent, a free ammonia (Yaylayan 2003). αAmino ketones and ammonia, via different pathways, can further react to form pyrazines.
Because of their volatility, Strecker aldehydes have been thought to contribute
significantly to thermally processed food aroma. Aldehydes derived from Streker
degradation increased drastically during roasting, especially at temperatures above 100
ᵒC. These included 2- methylpropanal, 3- methylbutanal, 3- methylbutanal and
phenylacealdehyde (Siegmond and Murkovic 2004).
Yaylayan (2003) proposed four alternative pathways for the formation of a
Strecker aldehydes from amino acids; these include 1) from the addition of high
temperature alone; 2) through oxidative decarboxylation, heat and mild oxidizing agents;
9
3) from α-dicarbonyl assisted oxidation and lastly 4) from Amadori Rearrangement
Products (Hofmann and Scheiberle 2000).
Among the Strecker aldehydes 2-methylpropanal (2-MP), 2-methylbutanal (2MB) and 3-methylbutanal (3-MB), derived from the amino acids Valine, Leucine and
Isoleucine, have been correlated with a malty flavor and amino-like aroma that
contributes significantly to the aroma in roasted foods (Maeztu and others 2001). Slight
differences in their aromas have been found where 2-methylbutanal was described to be
more fermented and fruity whereas 3-methylbutanal is more pungent with a fruity flavor
(Kim and others 2000). Kim and others (2000) also found that in Perilla seeds roasted at
different temperatures 150-190ᵒC all contained relatively high amounts of 2-MP, 2-MB
and 3-MB. Their odor thresholds of 2-MP, 2-MB and 3-MB have been measured at 8.0,
4.9 and 3.2 mg/L in water and 48.3, 69.7 and 13.7 mg/L in oil respectively (Granvogl and
others 2006). Other aldehydes have been correlated with sensory attributes such as
phenylacetaldehyde which produces a flowery note and is generated from the amino acid
phenylalanine (Hofmann and Scheiberle 2000).
2.3.2 Lipid Oxidation Aldehydes
The main unsaturated fatty acids that make up the lipid content in food are oleic,
linoleic, linolenic and arachidonic acids. These fatty acids can easily oxidize in the
presence of oxygen and a catalyst causing the formation of free radicals. The three steps
associated with the reaction are initiation, propagation and termination. During initiation
a hydrogen atom is removed at the α-methylene group to form an alkyl radical. The
generation of the free radical is slow and is affected by the presence (or absence) of light,
10
heat, metals, enzymes and many others. During propagation radicals react and transfer
their unpaired electrons to other compounds and for termination to occur, two radicals
must combine to end the reaction. As a result, lipid hydroperoxides are formed and
because they are so unstable are quickly broken down. This occurs by the removal of a
hydroxyl group to form an alkyloxy radical which breaks down to form by-products such
as esters or aldehydes, among other compounds.
Heat induced lipid oxidation is very important since an increase in temperature
not only causes fatty acid hydrolysis to increase but also to become more random,
resulting in a less controllable reaction (Whitfield 1992). The two most susceptible fatty
acids to hydrolysis are linolenic and arachidonic acids followed by linoleic acid and then
oleic acid. A few of the typical aldehydes derived from each fatty acid are nonanal and
heptanal from oleic acid; hexanal and pentanal from linoleic acid; propanal and (E)-2hexenal from linolenic and pentanal, (E)2-heptenal and hexanal (at much high
concentrations) from arachidonic acid (Varlet and others 2007; Whitfield 1992). The odor
descriptors of select lipid aldehydes are as follows: pentanal - chemical, wine, sweet and
fruity; hexanal - grass cut, green, pungent; heptanal - herbaceous, fishy, fatty, pungent;
nonanal - green, tallow, citrus, fatty and soapy (Varlet and others 2007). Lipid aldehydes
have been reported to contribute to the raw and roasted aroma of peanuts (Brown and
others 1973), Perilla seeds (Kim and others 2000), pumpkin seeds (Siegmund and
Murkovic 2004), almonds (Vazquez-Araujo and others 2008); cocoa beans (Ramli and
others 2005); and mate (a type of tea) (Kawakami and Kobayashi 1991). Alternatively,
lipid aldehydes have also been used to measure degree of rancidity by several methods
11
including GC-HPLC-anitbody techniques, fluorescence and GC-MS (Halliwell and
Chirico 2011). Nonetheless, aldehydes from lipid oxidation that contribute to the aroma
of roasted pumpkin seeds included pentenal, hexanal and 2-heptenal, and increase during
roasting conditions (Siegmond and Murkovic 2004).
2.3.2 Pyrazines
Pyrazines are characteristic of a roasted nutty aroma and in low concentrations
have been shown to be very desirable (Leunissen and others 1996). They are formed
through Maillard browning reactions and have been reported to increase exponentially at
temperatures higher than 120 ᵒC but lower than 160 ᵒC in roasted pumpkin seeds
(Siegmond and Murkovic 2004). The pyrazine compounds that contribute the most to
roasted flavor in pumpkin seeds are 2-methylpyrazine, dimethylpyrazine, 2-ethylpyrazine
and 2-ethyl-5, 6-dimethylpyrazine (Siegmond and Murkovic 2004).
Pyrazines have been identified in the headspace of many roasted seeds, beans and
nuts, and are considered to be an important flavor component in thermally processed
foods. At low levels, pyrazines have been associated with a sweet, nutty and typical
roasted aroma. Siegmund and Murkovic (2004) identified five pyrazines, 2methylpyrazine, dimethylpyrazine, 2-ethylpyrazine, 2-ethyl-5(6)-methylpyrazine and 2ethyl-3, 6-dimethylpyrazine, in the headspace of roasted pumpkin seeds, and theorized to
significantly contribute to the overall aroma of roasted pumpkin seeds.
Pyrazines are generated from the condensation reaction of two α-aminocarbonyl
compounds with the formation of a dihydropyrazine, which oxidizes spontaneously to the
12
corresponding pyrazine (Adams and others 2008). Koehler and Odell (1970) used
isotopic labeling of glucose to show that the alkyl substitution on methyl and
dimethylpyrazines are governed by the dicarbonyl fragment and the amino acid only
contributes the amine (Koehler and Odell 1970). Weenen and others (1994) conducted a
study using C-labeled sugars to elucidate pyrazine formation and found that the reaction
of asparagines with hexoses, trioses or glycolaldehydes generates methylpyrazines, with
2, 5-pyrazine predominating. Alternatively, Amrani-Hemaimi and others (1995) used the
labeled amino acids glycine and alanine in a model reaction with sugar to determine their
contribution to pyrazine formation. It was shown that glycine and alanine not only act as
the nitrogen source, but also can contribute alkyl side chains to some of the
alkylpyrazines. Shigematsu and others (1977) described another pathway for pyrazine
formation involving model reactions of glucose and other carbohydrates with ammonium
hydroxide as a source of ammonia, showing an alternative source of nitrogen.
Alternatively, Shu (1998) demonstrated pyrazine formation can occur during thermal
processing from the decarboxylation and dehydration of amino acids without a
carbohydrate source. In Yaylayan’s (2003) review of Strecker degradation and Amadoria
rearrangement it was concluded that pyrazine formation is the result of nitrogen
containing compounds derived from either α-dicarbonyls via Strecker degradation or
from α-hydroxy carbonyls via Amadori Rearrangement.
Although many studies have been conducted in simplified and complex food
systems, it only highlights the complexity of the Maillard reaction due to limitless
combinations of variables. The many factors that affect the Maillard reaction in turn
13
affect flavor formation. Each flavor formation pathway has an individual activation
energy, and at different temperatures certain compounds will be formed faster than others
(Heath 1986). Cantalejo (1997) concluded that the quantity of pyrazines depended more
on time and temperature of roasting than precursors by influencing the reaction kinetics.
Therefore, at higher temperatures pyrazines are seen in much higher concentrations.
Jayalekshmy and others (1991) showed that with increasing temperatures pyrazines
(methyl and dimethyl) increased in roasted coconuts, reaching a maximum between 130160 ᵒC.
Alternatively, Jousse and others (2002) showed that the type of precursors and pH
of the system affect the nature of products formed. The precursors in the food system, or
the amino acid and sugar composition, have been shown to strongly affect compounds
formed and total yield (Heath 1986). In a model system of aspartic acid and glucose at
elevated temperatures, it was found that with increasing concentrations of ammonia the
levels of pyrazines, pyradines and pyrroles also increased (Bohnenstengel and Baltes
1992). As far as the pH of the food system, it has been shown that pyrazines are more
easily formed in neutral or alkaline conditions than acidic (Pripis-Nicolau and others
2000). Adams and others (2008) used model reactions of 20 different amino acids with 1,
3-dihydroxyacetone, as a precursor to an important carbohydrate fragment in pyrazine
formation. The only pyrazine formed with all 20 amino acids was 2, 5-methylpyrazine,
showing the effect of amino acid type on pyrazine compounds formed. Kim and others
(2000), showed that pyrazines significantly increased with increasing temperatures from
150-190°C in roasted Perilla seeds with 2-methylpyrazine and 2, 5(6)-dimethylpyrazine
14
predominating at higher temperatures. Ramli and others (2006) and Hashim and
Chaveron (1996) both utilized pyrazine levels and ratios to determine degree of roasting
in roasted cocoa and coffee beans respectively.
2.3.3 Furans
Furans are cyclic ethers found in many heat treated foods, especially in coffee at
6.5% of total volatiles (Merritt and others 1963) and heat treated foods sold in jars and
cans (Crews and Castle 2007). Some furans have been reported to contribute burnt,
sweet, bitter, cooked meat and coconut-like flavor in foods (Vazquez-Araujo and others
2008). Many model studies have been done to better understand its formation
mechanism, linking it to sugar amine reactions (Maillard reaction), sugar degradation,
unsaturated fatty acids and ascorbic acid degradation (Hasnip and others 2006).
Locas and Yaylayan (2004) studied the formation of furan in simple sugar,
sugar/amine and acid systems heated to 250ᵒC. Ascorbic acid formed the highest levels
of furan followed by dehydroascorbic acid. Heated sugars and sugar/amine systems did
not produce very high levels with the exception of erythrose. Another study conducted by
Umano and others (2005) compared volatile formation between heated glucose, heated
cysteine and heated glucose/ cysteine. Furan compounds were formed in highest amounts
in glucose alone, but also in glucose/ cysteine in lower levels.
Furans have been listed as a potential human carcinogen by the FDA in 2005 and
issued an action plan for furans in food. It was determined that furans may act as an
indirect carcinogen based on animal tests. Although results are currently inconclusive, the
15
FDA has not recommended a change in diet (Food Safety Magazine 2007). A study
conducted in response to this statement found furan compounds to be formed from fatty
acids with the highest level from linolenic acid with ferric chloride followed by linolenic
acid alone (Becalski and Seaman 2005). From the same study, furan was found to form
from ascorbic acid and its derivatives; furan was found to form at the highest levels from
dehydroascorbic acid with ferric chloride followed by isoascorbic acid alone. 2pentylfuran was found to increase during roasting in the headspace of pumpkin seed and
was contributed to the oxidation of fatty acids (Siegmund and Murkovic 2004). Other
furan compounds such as 2-methylfuran, 2-acetylfuran and 5-methylfurfural, were found
to increase as well in the headspace of toasted almonds (Vazquez-Araujo and others
2008).
2.3.4 Sulfur compounds
Sulfur volatile compounds occur either naturally in foods or are a result of
processing or storage conditions. These volatile compounds contribute directly or
indirectly to food aroma especially since their thresholds are relatively low. For example,
the threshold in water of dimethylsulfide is 3x10-4 ppm and methional is 2x10-4 ppm
(Shankaranarayana and others 1974). Dimethylsulfide, methanethiol and methional are
considered to have the highest flavor impact of the sulfur compounds (Harper and others
2010) and have been identified in cocoa, roasted peanuts, popcorn, potato (Martin and
Ames 2001) and roasted pumpkin seeds (Siegmund and Murkovic 2004).
Dimethylsulfide and methional are derived from the amino acid methionine, one
of the essential amino acids. Methionine has been found to oxidize to methionine
16
sulfoxide easily, especially with heat treatment and/or oxidized lipids (Yu and Ho 1995).
When six different sugars were reacted with methionine and cysteine, methional was
found to be the main product formed (Shigematsu and others 1977). More recently, Yu
and Ho (1995) conducted a similar study on the thermal degradation of methionine and
methionine sulfoxide and found methional to predominate from methionine with and
without glucose, whereas dimethyl disulfide and dimethyl trisulfide predominated from
methionine sulfoxide with and without glucose. While higher concentrations of
methional, dimethyl disulfide and dimethyl trisulfide were formed in the presence of
glucose, the compounds could be formed from the amino acid methionine alone via the
Shigematsu reaction, an alternative to Maillard reaction. Strecker degradation of
methionine alone has also been proposed as a pathway for methional formation made
possible by the oxidative decarboxylation of methionine to form an imine and then
hydrolysis (Yu and Ho 1995). As methional breaks down further it forms methanethiol
which can oxidize to various dimethylsulfides (Yu and Ho 1995).
2.3.5 Ketones
Ketones have relatively high thresholds and therefore, at levels beneath their
threshold, don’t contribute directly to the aroma of roasted foods. Ketones impart a wide
variety of odor descriptors such as buttery from 2, 3-butanedione and 2, 3-pentandione,
banana/ slightly spicey from 2-heptanone and fungus or hay-like from 1-octenone
(Schenker and others 2002). Maillard browning, thermal degradation of sugars and lipid
oxidation are the three pathways that have been proposed for the formation of ketones in
thermally processed foods. In carbon labeled systems 2, 3-butanedione was found to form
17
through cleavage of the D-glucose moiety (Yaylayan and Keyhani 1999). Alternatively,
2, 3-pentanedione has been found to form both from glucose and from the combination of
glucose and alanine (Yaylayan and Keyhani 1999). In roasted coffee beans, high
temperature short time (HTST - 260°C for 160 sec), a maximum concentration (40 mg/kg
dm) of 2, 3-butanedione was attained for 45 s but quickly degraded. Similar behavior was
found for lower temperature longer time (LTLT - 228°C for 660 sec) but the maximum
concentration was not as high and the degradation was less rapid (Baggenstoss and others
2008). 2, 3-butanedione has also been found to form from 1-deoxyglycosone, derived
from sugar fragments, and also from glucose (Hollnagel and Kroh 1998). In another
labeling study, 3-hydroxy-2-butanone was found to be formed from glucose directly from
the reduction of 2, 3-butanedione contributing to the possibility of disproportionation
with R-hydroxycarbonyl compounds (Wnorowski and Yaylayan 2000). 2-butanone was
found in the highest concentration in the headspace of perilla seed oil and was
contributed to thermal degradation of sugars rather than by lipid oxidation (Kim and
others 2000).
Several ketones including acetone, 2-butanone, 2-pentanone and 2-heptanone,
were detected in the headspace of oil from raw and roasted Spanish and Runner peanuts
(Brown and others 1973). Most of the ketones increased in concentration from raw to
roasted peanuts. Similar ketones were detected in the highest levels in the headspace of
heated glucose alone and much lower levels in systems with glucose and cysteine, but
were not detected from cysteine alone (Umano and others 1995). This could indicate that
18
compounds formed from sugar degradation products, such as ketones, undergo secondary
reactions with amines to form heterocyclic flavor compounds (Umano and others 1995).
2.3.6 Alcohols
Alcohols are another flavor compound group that is very volatile. Like lipid
aldehydes, they are formed from the breakdown of hydroperoxides, but are also generated
from the oxidation of their corresponding aldehdyes. 2-methyl-1-butanol, 1-pentanol, 1hexanol and phenylethanol were found in the headspace of pumpkin seeds (Siegmund
and Murkovic 2004). 2-methyl-1-butanol imparts alcoholic, banana like, vinous odors,
has a threshold of 65mg/L and has been theorized to be produced during fermentation of
sugars by yeasts but also from the oxidation of 2-methylbutanal (Yamashita and others
1976). Pentanol is most likely an oxidation product of pentanal, imparts a fruity, and
raspberry-like odor and has a threshold of 64 µg/L (Yamashita and others 1976).
Hexanol, imparting a green, fatty note, has a much lower threshold of 4 µg/L and has
been theorized to form from both the oxidation of hexanal and the oxidation of linoleic
and linolenic acids (Yamashita and others 1976).
2.4 Analytical Methods for Headspace Analysis
Analyzing the volatile head space of roasted pumpkin seeds can help in the
understanding of flavor formation at varying temperatures to eventually optimize quality
and overall flavor acceptance, or to potentially artificially recreate aroma and flavor
profiles. Several methods have been established to analyze volatile compounds
including; gas chromatography mass spectrometry (GC-MS), atmospheric pressure
ionization mass spectrometry (API-MS) and proton transfer reaction mass spectrometry
19
(PTR-MS); however these methods can be limited by the inability to detect the real time
release of volatiles or inability to distinguish isomers (Xu and Barringer 2009).
2.4.1 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS)
Selected Ion Flow Tube- Mass Spectrometry is a mass spectrometry technique for
the analysis of headspace in real time by utilizing chemical ionization of sample trace
gases by selected ions during a time period along a flow tube. It has been utilized as a
highly sensitive, real time technique that can distinguish isomers and does not require
sample preparation to quantify volatile organic compounds found in the head space above
a sample (Xu and Barringer 2009).
2.4.1.1 Principles of SIFT-MS
Principles of SIFT-MS can be explained with the help of Figure 10. Precursor
ions are generated by a microwave discharge source. The ions pass into an upstream
chamber where a quadrupole mass filter selects only the reagent ions, H3O+, NO+ and
O2+, to proceed. These ions are used because they do not react with major components of
air, mainly N2, O2, H2O, Ar and CO2, but react with volatile compounds (Spanel and
Smith 1999). At a known velocity, the ions pass through a Venturi inlet into an inert
carrier gas (helium and argon). The trace gases from the sample enter the reaction
chamber at a controlled rate and react with the precursor ions to form product ions. The
product ions then enter into a downstream chamber to be filtered by a second quadrupole
mass filter. Finally, a particle multiplier detects the ions at the selected mass and the
count rate is passed to the instrument computer for processing.
20
Kinetic studies of ion reactions using selected ion flow tubes have been done for
many years (Adams and Smith 1976) which has provided a large kinetics database from
thousands of ion-neutral reactions (Smith and Španĕl 2005). This database holds
information on how rapidly an analyte reacts with a precursor ion (which is also referred
to as the rate coefficient for the reaction), the products of the reaction and their relative
abundances. Hence why, determination of individual reaction rate coefficients and the
product ion branching ratios are necessary for quantification of VOCs. For the reaction
A+ + B → D+ + E, a rate law is defined as equation 1 where k is the bimolecular rate
coefficient, t is the time and [A+] and [B] are respective concentrations of the precursor
ion and the VOC (Equation 1) (Frank 2007).
(1)
With these known rates, the volatile concentrations [M] can be instantly determined
from the ratios of the product to precursor ion count rates (Ip/I) and time (t), which is
defined by the known flow velocity of the helium carrier gas according to the following
equation (Spanel and Smith 1999):
(2)
SIFT-MS utilizes soft chemical ionization, instead of electron impact ionization
used in GC-MS, resulting in less compound fragmentation and a simpler mass spectrum.
This, in combination with three precursor ions, instead of only one precursor ion used in
PTR-MS, ultimately reduces compound interference issues because only one or two
product ions result from each VOC in the sample (Spanel and Smith 1999).
2.4.1.2 Mass Scan vs SIM Scan
The Syft Technologies Voice100™ SIFT-MS can be operated in two modes as
follows:
21
Full mass scans: Mass scans are used to identify unknown compounds in the
sample but also allows concentrations to be derived, by providing data on product masses
for each reagent ion (Harper and others 2010). As a sample is introduced into the carrier
gas at a steady flow rate, a complete mass spectrum is obtained by sweeping the detection
quadrupole ion over a selected mass-charge ratio (m/z) range 15 to 200 Daltons. The
mass and ion count rates are fed into the online computer to obtain a mass spectrum
(Smith and Spanel 2005).
Selected Ion Mode (SIM): SIM is used to target specific compounds for
quantitative analysis down to parts per billion by volume (ppbv) (Harper and others
2010). Only the count rate of the precursor ions and those of selected product ions are
monitored; achieved by switching the downstream mass spectrometer between the masses
of all the primary ions and the selected product ions and measuring each for a short
programmed time interval (Smith and Spanel 2005). This allows for a more precise
quantification and at lower concentrations than a mass scan.
2.4.1.3 Ion Chemistry
Depending both on the reagent ion and the chemical nature of the analyte, five
different types of reactions can occur. Consequently, a mass scan is needed to determine
suspected compounds within a given sample. Currently the SYFT library has data for
more than 500 VOCs, providing product masses for each reagent (Harper and others
2010).
Proton Transfer (H+):
22
H3O+ + Analyte  Analyte.H+ + H2O
A large majority of H3O+ reactions with organic compounds proceed via proton
transfer producing MH+, a stable ion. The advantage of the proton transfer reactions is
that it usually results in only one or two product ions (Smith and Spanel 2005). However,
MH+ ions sometimes partially or totally dissociate eliminating an H2O molecule resulting
in [M – OH]+ hydrocarbon ion (Spanel and Smith 1999).
Charge Transfer (reagent removes a charge from analyte):
O2+ + Analyte  Analyte+ + O2
NO+ + Analyte  Analyte+ + NO
The charge transfer can only occur if the ionization energy (IE) of M is less than
the IE of NO (9.26 eV) and O2 (12.06 eV) molecules (Smith and Spanel 2005). Since the
IE of O2 is relatively higher than most organic molecules it is a valuable precursor for a
variety of compounds. However, reactions with O2 can sometimes result in fragmented
ions making analysis difficult; consequently, O2+ ions are most valuably for the analysis
of small inorganic molecules where only a single product ion results (Spanel and Smith
1999). Alternatively, with a lower IE from NO, the reaction usually involves aromatic
hydrocarbons, low molecular weight alkanes and organosulfur molecules (Spanel and
Smith 1999).
Dissociative charge transfer:
O2+ + Analyte  Fragment+ + Neutral fragments + O2
23
This reaction involves a charge transfer and the formation of fragments. It almost
always takes place with O2+ but can occasionally occur with NO+ for compounds with
low ionization energy (Smith and others 2003).
Association:
NO+ + Analyte + M  Analyte.NO+ + M
H3O+ + Analyte + M  Analyte.H3O+ + M
This reaction involves a three body collision of a reagent ion, and analyte and a
carrier gas reaction. This process occurs when the proton affinity of the acceptor
molecule is less than the proton affinity of H2O or when proton transfer is endothermic
(Smith and Spanel 2005).
Hydride extraction:
NO+ + Analyte  [Analyte-H]+ + HNO
Hydride extraction involves the transfer of a hydride ion to the analyte, which
results in a single product ion and a HNO molecule (Smith and Spanel 2005). This
reaction most often occurs with saturated aldehydes and primary and secondary alcohols
(Smith and Spanel 2005).
A controlled amount of gas is introduced into the carrier gas through a mass flow
meter through an entry port in order to determine the rate coefficient and the products
ions for the reaction of the injected ions (Smith and Španĕl 2005). Once the loss of the
precursor ion current and the increase of the product ion count rates are observed the rate
24
coefficient for the reaction can be calculated. More than one product ion sometimes
results from an ion–molecule reaction, but can be overcome by determining the
branching ratios (Smith and Adams 1987).
2.4.1.4 Calibration and Validation of SIFT-MS
While a GC-MS calibrates by using a set of dilution of known concentration of a
reference substance used within an analytical sequence, SIFT-MS is capable of
automatic, online validation is completed with dilution of the gas mixtures benzene,
ethylbenzene, toluene, m-xylene, o-xylene and p-xylene within 15 min. Because SIFTMS quantifies based on the flow rate of sample gas, count rates of precursor and analyte
ion(s) and rate constant for precursor-analyte reaction, calibration involves obtaining rate
constants and product ratios and is only necessary once for a particular model of SIFTMS (Syft Technologies Ltd 2007).
2.4.1.5 Application of SIFT-MS in Food Science
The SIFT technique was developed over 30 years ago (Adams and Smith 1976)
but has been utilized in a variety of different fields of study including environmental
science, biological science, human physiology, clinical science and food science. The
application of trace gas analysis in food science has added value by utilizing the reactions
of H3O+, NO+ and O2+ in evaluating food quality control and quantifying and measuring
food flavors and emissions.
The advantages of apply the SIFT technique in analysis of food flavors are
partially due to its use of three ions instead of one, allowing isomers to be distinguished
(Smith and Spanel 2005). The applications of SIFT-MS have been reported on real time
25
release of volatile emissions from vegetables and fruits. Spanel and Smith (1999) showed
that that trans-2- and cis-3-hexenal could be distinguished based on their different
reactions with H3O+ and NO+ . In addition, benzaldehyde’s and vanillin’s reaction are the
same with H3O+ but with the use of NO+, they were able to be distinguished (Spanel and
Smith 1999). Davis and McEwin (2007) used the SIFT-MS to correlate concentrations of
propanal, hexanal, acetone, and acetic acid in the headspace above olive oil to oil
oxidation. The SIFT-MS has also been used for real time analysis of tomatoes to evaluate
flavor release during chewing (Xu and Barringer 2009), comparison between tomatillo
and tomato volatile compounds (Xu and Barringer 2010) and the effect of temperature on
lipid-related volatile production (Xu and Barringer 2010). The effect of milk on the
deodorization of malodorous breath after garlic ingestion was studied using SIFT-MS
(Hansanugrum and Barringer 2009), along with volatiles from cocoa (Huang and
Barringer 2010) crushed garlic and cut onion (Spanel and Smith 1999) and Swiss cheese
(Harper and others 2010). Additionally, SIFT-MS has been used to study the effect of
enzyme activity and frozen storage conditions on jalapeno pepper volatiles (Azcarate and
Barringer 2010), strawberry volatiles (Ozcan and Barringer 2011) and apple aroma
(Scotland and Barringer 2010). Recently, the SIFT-MS technique has been applied to dry
fermented sausage for real time detection and quantification of 31 volatiles (Olivares and
others 2010).
26
Chapter 3: Materials and Methods
3.1 Volatile Analysis
3.1.1 Seed preparation
Fifteen varieties of pumpkins were grown and harvested in October 2009 in
Springfield, Ohio (Table 1). In 2010, five of the fifteen varieties were grown and
harvested from the same research farms (Table 1). Pumpkin seeds were removed from the
pumpkins by slicing the pumpkin in half and scooping out the insides. Seeds were
cleaned and pulp was removed with a large colander by hand. All seeds, except for those
excluded to determine effect of starting moisture content on volatile formation during
roasting, were dried in a in a commercial grade dehydrator (160 liter, Cabelas, Sydney,
NE, USA). Temperature was set to 29 - 37°C for 24-30 h. Seeds were packaged in
ziplock freezer bags and stored at -10°C until roasted.
27
Table 1 Pumpkin seed varieties labels.
Variety name
Dickinson Field
Pik-a-piea
Small sugara
Baby Pam
Hybrid Pam
Touch of Autumn
Golden Hubbard
Baby Green Kitchenette
Uchiki Kuri
Snack Jacka
Sweet Meat
Blue Hubbard
Blue Magic
Mystic Plusa
Syrian (New Zealand) a
a
Seed Code
1H
2H
3H
4H
5H
6H
7H
8H
9H
10NH
11H
12H
13H
14H
15NH
Properties
seed coat
seed coat
seed coat
seed coat
seed coat
seed coat
seed coat
seed coat
seed coat
hull-less
seed coat
seed coat
seed coat
seed coat
hull-less
Usage
old processing type, flesh
Flesh
Flesh
Flesh
jack-o-lantern type
jack-o-lantern type
Flesh
Flesh
Flesh
seed type
Flesh
Flesh
Flesh
jack-o-lantern type
seed oil type
Seed varieties selected to be grown again in 2010 and used in sensory analysis.
3.1.2 Roasting
Seeds were removed from the freezer 30 min prior to roasting. A Blue M ®Stabiltherm oven (White Deer, Pennsylvania U.S.) was preheated to 150°C with an upper limit
set to 175°C. The oven was allowed to preheat to reach set temperature for at least 20
min. 50 g raw was spread evenly in a thin layer on a 38 x 25 cm nonstick baking sheet.
Roasting time was started once the sheet was placed in the oven. Seeds were roasted for
40 min then removed. Replicates were done by roasting multiple 50 g batches at the same
time on different shelves. For studies on effect of seed coat, moisture content and harvest
date, seed were roasted for 40-60 min and samples were removed at 5 or 10 min intervals,
depending on the study. Roasted seeds were immediately prepared for measurement.
28
3.1.3 Sample preparation
Seeds were ground with a Waring Pro™ Seven Speed Blender (Torrington, CT,
USA) for 30 sec on medium speed in a ratio of 50 g raw or roasted seed to 100 g water.
The slurry was poured into a 500ml PYREX® media/laboratory bottle with a wide mouth
funnel. Silicone septa and cap were immediately screwed in place. The ground seeds
were placed in a 50°C water bath and equilibrated for 90 min. Time elapsed between final
roasting time and scanning was no greater than 110 min.
3.1.4 Sift-MS
The bottles taken from the 50°C water bath were placed in a Styrofoam container
during scanning to retain heat. Scans were taken for 1 min using a method developed for
identification of pumpkin seed volatiles with the SYFT Voice 100 (Syft™ Technologies
Ltd, Middleton, New Zealand). Table 2 shows the reaction kinetics used in the method to
measure volatiles in the headspace above pumpkin seeds. A long needle was inserted
through the septa into the bottle so that the tip of the needle was approximately 2.5 cm
above the ground pumpkin seed slurry. This step is necessary to allow air to enter the
bottle, which prevents a vacuum from forming. The septa was pierced by a stainless steel
needle connected directly to the SIFT-MS and inserted approximately 2.5 cm into the
bottle. Inlet and outlet temperatures were set to 120°C.Validations were run at least once
before scanning samples. Validations are automatic and are completed within 15 min.
Automatic validations involve measurement of exact concentrations of the gas mixtures
benzene, ethylbenzene, toluene, M-xylene, O-xylene and P-xylene. Calibration of SIFT-
29
MS is necessary once for each Syft model built because it is based on the reaction
kinetics and product ratios of the precursor ions and VOCs (Syft Technologies Ltd 2007).
30
Table 2 Reaction kinetics of the volatile compound measured in the headspace of
pumpkin seeds
Compound
(E)-2-heptenal
(E)-2-hexenal
1-hexanol
1-pentanol
1-penten-3-one
1-phenylethanol
2, 3-butanedione
2-acetylpyrrole
2-heptanone
2-methyl-1-butanol
2-methylpropanal
2-methylpyrazine
2-pentanone
2-pentylfuran
3, 5(6)-dimethyl-2ethyl pyrazine
acetoin
ammonia
benzaldehyde
butanone
dimethyl sulfide
dimethylpyrazine
Precursor
Ion
NO+
NO+
NO+
O2+
NO+
NO+
NO+
H3O+
NO+
H3O+
O2+
NO+
NO+
NO+
k (10-9
cm3/s)
3.9
3.8
2.4
2.8
2.5
2.2
1.3
3.3
3.4
2.8
3
2.8
3.1
2
m/z
Product Ion
Reference
111
97
101
42
114
106
86
110
144
71
72
94
116
138
C7H11O+
C5H9O+
C6H13O+
C3H6+
C5H8O.NO+
C6H10+
C4H6O2+
C6H7NO.H+
C7H14O.NO+
C5H11+
C4H80+
C5H6N2+
NO+.C5H10O
C9H14O+
Spanel and others 2002
Spanel and others 1997
Spanel and Smith 1997
Spanel and Smith 1997
Syft Technologies 2009
Wang and others 2004b
Spanel and others 1997
Syft Technologies 2009
Smith and others 2003
Wang and others 2004b
Spanel and others 2002
Syft Technologies 2009
Spanel and Smith 1997
Syft Technologies 2009
NO+
2.5
136
C8H12N2+
Syft Technologies 2009
+
NO
O2+
NO+
NO+
O2+
NO+
2.5
2.6
2.8
2.8
2.2
2.8
118
36
105
102
62
108
ethanol
NO+
1.2
45+63
+81
ethyl acetate
furan
furfural
hexanal
O2+
NO+
NO+
NO+
2.4
1.7
3.2
2.5
61
68
96
99
methanol
H3O+
2.7
33+51
+69
methional
methylbutanal
nonanal
pentanal
phenylacetaldehyde
tetramethylpyrazine
toluene
NO+
H3O+
O2+
O2+
NO+
NO+
NO+
2.5
3.7
3.2
3
2.5
2.5
1.8
104
87
138
44
120
136
92
31
+
C4H8O2.NO
NH3+
C7H5O+
NO+.C4H8O
(CH3)2S+
C6N2H8+
C2H6O+,
C2H5O+.H2O,
C2H5O+.2H2O
C2H5O2+
C4H4O+
C5H4O2+
C6H11O+
CH5O+,
(CH3OH)2.H+.(H20)2
, CH3OH.H+.(H2O)2
C4H8OS+
C5H10O.H+
C10H18+
C2H4O+
C8H8O+
C8H12N2+
C7H8+
Syft Technologies 2009
Spanel and Smith 1998b
Spanel and Smith1997
Spanel and others 1997
Spanel and Smith 1998a
Syft Technologies 2009
Spanel and Smith 1997
Spanel and Smith 1998c
Wang and others 2004a
Wang and others 2004a
Spanel and others 1997
Spanel and Smith 1997
Syft Technologies 2009
Michel and others 2005
Syft Technologies 2009
Spanel and others 1997
Syft Technologies 2009
Syft Technologies 2009
Spanel and Smith 1998c
3.2 Total Lipid Quantification
Seeds were chopped to a fine particle size for 20 min with a Hobart chopper
(Troy, OH U.S.). Approximately 2-4 g of the seed meal was weighed into a Florham
Park, NJ 22mm x 80mm Whatman cellulose extraction thimble. The lipid portion of the
seed meal was extracted with a Soxhlet apparatus using petroleum ether (boiling point 39
– 54 ᵒC) for 8 hr. Total lipid quantification was done by evaporating solvent with a rotary
evaporator set to 40ᵒC and weighing flat bottom flasks before and after extraction. All
chemicals were obtained through Fischer Scientific (Pittsburgh,PA, USA). The defatted
seed meal was used for total reducing sugar quantification.
3.3 Fatty Acid Ratio
Methylation:
The lipid portion, obtained via Soxhlet extraction, was used for fatty acid ratio
determination. Fatty acid determination consisted of the following steps: 0.5 g lipid was
weighed into a test tube, 10 ml of 4% methanolic sulfuric acid and 1 ml of benzene were
added, test tubes were vortexed, then boiled for 90 min. Test tubes were cooled to room
temperature and 1 ml HPLC water and 2 ml hexane were added and vortexed. One ml of
the supernatant was removed, pipetted into an HPLC vial then dried under nitrogen. Vials
were rehydrated with 0.5 ml iso-octane and capped.
GC-FID:
An Agilent Technologies (Wilmington, Delaware, USA) gas chromatograph
equipped with a flame ionizer detector was used to determine fatty acid ratios. The GC32
FID column was ordered from Agilent Technologies (Santa Clara, CA) product
specifications: HP-FFAP, 25 m, 0.32 mm, 0.50 µm. Minimum and maximum
temperatures were set to 110 and 240 ᵒC. Run time was set to a total of 30 min, with the
first 20 min step including the sample analysis and the last 10 min the column cleaning
step with a maximum temperature of 300 ᵒC.
3.4 Total Reducing Sugars
Extraction:
Approximately 2.0 g defatted seed meal was weighed into 50 ml centrifuge tubes
and mixed with hot 70% ethanol for 60 min. Tubes were centrifuged on high for 15 min
and the supernatant was removed and deposited in a round bottom flask for latter
analysis. This extraction step was repeated three times. The 70% ethanol was removed
with a rotary evaporator; the reducing sugars were re-hydrated with 20 ml warm HPLC
water.
Quantification:
Reducing sugar quantification was done with DNSA and UV spectroscopy.
DNSA reagent was made with 1 g 3, 5-dinitrosalicylic acid (DNSA), 20 ml 0.2 N NaOH,
30 g sodium potassium tartrate tetrahydrate (Rochelle Salt Fairlawn,NJ, USA) and 50 ml
distilled water. 2 ml of reducing sugar substrate and 2 ml DNSA were added to a test tube
and boiled for 10 min. Tubes were removed and allowed to cool. Concentrations were
measured using a Thermo Scientific Biomate6 UV-visible spectrometer (Redwood City,
California, USA) at 540 nm. Quantification was done by comparing to a standard curve.
33
3.5 Sensory Analysis
In 2009, a focus group was conducted with ten people in which 15 varieties of
pumpkin seeds were tasted and discussed. Seeds were presented and prepared as roasted
(250ᵒF for 30 min), oiled (1% by weight corn oil) and salted (15% by weight) snack
items. Ten of 15 seeds were eliminated based on appearance, texture, flavor and seed
yield. The five seed varieties selected were planted and harvested the following year
(2010).
Sensory analysis, consisting of three panels, was conducted on seeds from
pumpkin varieties harvested in 2010. The three sensory panels were conducted using The
Ohio State University Food Industry Center’s sensory lab. Evaluation of seeds was done
using Compusense® Five 5.2 sensory software. Results were also analyzed using
Compusense® (Guelph, Ontario, Canada).
Panel 1: Roasting conditions and seed variety.
Five varieties of seeds were used; each variety was roasted at 150ᵒC for both 15
and 30 min, totaling 10 samples per panelist. Eighty-eight panelists (64% female, 36%
male) were presented one sample at a time in 2 oz capped plastic cups in randomized
order. Panelists were asked to evaluate seeds for texture on a JAR (Just About Right)
scale from much too soft to much too crispy. Secondly, samples were scored based on
overall liking using a 9 point hedonic scale.
Panel 2: Seed Variety
All five seeds were roasted at 150ᵒC for 30 min and were roasted, oiled, salted
and presented all at once in randomized order. Seventy-six panelists were instructed to
34
taste all five then complete the questions. Seeds were evaluated using a 9 point hedonic
scale on overall liking, liking of the flavor and lastly, forced ranking.
Panel 3: Seed seasonings
Prior to panel 3, an online survey was distributed using survey monkey to
determine seasonings most preferred. Out of 100 responses (62% female and 38% male),
51% said they had previously purchased pumpkin seeds to eat as a snack item. The
seasonings that ranked the highest were then applied to roasted seeds to evaluate in panel
3.
All five seeds were roasted at 150ᵒC for 30 min and were roasted, oiled and
seasoned. Samples were evaluated by sevety-eight panelists (74% female and 26% male)
and presented all at once in randomized order. Seasonings, added to roasted seed at 15%
by weight, were garlic, BBQ, salted, vanilla cinnamon sugar and pumpkin pie spiced
Cargill Flavor Solutions (Cincinnati, Ohio USA) and were evaluated using a 9 point
hedonic scale on overall liking, liking of the flavor, amount of flavor intensity and lastly
forced ranking.
35
Chapter 4: Results and Discussion
4.1 Volatile Compounds in Raw Pumpkin Seeds
Fifteen pumpkin seed varieties were harvested in 2009 (Table 1). Seed varieties
consisted of two distinct traits; a majority of the seed varieties have thick, fibrous hulls
and are designated by their variety number and “H” for “HULL”. Two of the seed
varieties contain a single recessive gene that causes the development of a thin,
parchment-like seed coat (Loy 1990). Even though these seed varieties technically
contain a seed coat, they are often referred to as hull-less seeds and are designated with
their variety number and “NH” for “NO HULL” for simplicity.
Thirty-four volatile compounds were measured in the 15 raw seed varieties
(Tables 3, 4). Raw pumpkin seeds have a chewy texture and a subtle, sweet and nutty
flavor (Murray and others 2005). Raw seeds were generally higher in lipid related
volatiles, including (E)-2-heptenal, (E)-2-hexenal, hexanal and pentanal, when compared
to other groups of volatiles such as pyrazines or furan derivatives. Other compounds that
were relatively higher in raw seeds were ethyl acetate (sweet odor), 2, 3-butandione
(buttery odor) and dimethylsulfide (cabbage-like odor) (Tables 3, 4). Alternatively,
alcohols, including hexanol, 3-methylbutanol and 1-pentan-3-ol were the highest volatiles
compounds found in a variety of pumpkin seeds mostly used to make pumpkin seed oil,
by Siegmund and Murkovic (2004).
36
Raw volatiles that varied the most across seed variety were 2-pentanone, ethanol,
hexanal, methanol, methional and tetramethylpyrazine (Table 3, 4). While some volatiles
varied across seed variety, compounds in highest concentration remained high in all
varieties, compared to other volatiles within the seed variety. Raw seed variety 15NH had
the highest concentrations of all volatiles except 2-pentanone, 2-heptanone, butanone,
ammonia, methanol, acetoin and 2, 3-butanedione, followed by 10NH, compared to other
varieties. The volatiles in highest concentration in 15NH were (E)-2-heptenal, 2methylpyrazine, benzaldehyde, dimethyl sulfide, ethanol, ethyl acetate, methanol,
nonanal and pentanal, which were all above 10,000 µg/L (Tables 3, 4). An uncontrolled
variable worth mentioning was that 15NH was grown, harvested, processed and shipped
from New Zealand in 2009, therefore, higher volatile concentrations, especially lipid
aldehydes, are most likely due to unknown storage conditions, which could have
promoted lipid oxidation.
37
Table 3 Raw and roasted volatiles (µg/L) of 5 seed varieties from 2009 harvest. Different
letters in the same row are significantly different
RAW
2H
3H
10NH
263
b
18
b
3087
a
14 b
30 b
7b
162 a
55 c
19 c
157 b
17 c
171 a
1-pentanol
82 b
95 b
235 b
67 b
2890 a
481 a
213 d
563 a
274 c
421ab
1-penten-3-one
11 c
3c
33 b
15 c
73 a
157bc
22 d
324bc
51 d
73 d
1-phenylethanol
73 b
116 b
248 b
106 b
5557 a
585 c
196 d
1405 a
196 d
2160 a
2,3-butanedione
305 e
273 e
288 e
117ef
196ef
32621 a
8808 d
35638 b
7648 d
6130 d
b
b
b
b
a
b
c
a
c
485 a
3503
307
ce
272
72
78
f
67
581
b
453
b
15NH
18 b
392
b
14H
1-hexanol
1
45100
b
10NH
95
53
5
a
3H
(E)-2-hexenal
2
310
b
2H
b
11
33
b
b
15NH
18
2-acetylpyrrole
10
14H
b
(E)-2-heptenal
b
ROASTED
406
107
b
6097 a
103
f
1001 a
69
1c
2c
2c
26 c
0c
349 b
41 c
417 a
96 c
57 c
53 b
110 b
214 b
44 b
1161 a
4850 b
1044 e
6545 a
1557 e
4833 b
1b
1b
5b
1b
114 a
270bc
11 f
226 a
67 f
206ce
2-methylpropanal
39 b
90 b
133 b
46 b
2703 a
8964 a
2421 d
10584 a
2943 d
3287 d
2-methylpyrazine
12 b
8b
118 b
140 b
10297 a
1292 d
187 f
2627 a
417 e
1770 c
2-pentanone
53cd
61cd
89bc
44 d
81 b
5254 a
1399 d
6378 a
990 d
911 c
b
b
b
b
a
ab
d
a
cd
150bc
2-heptanone
2-methyl-1-butanol
2-methyl-3-ethylpyrazine
2-pentylfuran
acetoin
ammonia
benzaldehyde
butanone
dimethyl sulfide
dimethylpyrazine
2
2
6
2
909
189
14
256
40
172 c
539 c
266 c
51 a
587 b
301 d
226 d
16304 a
56 d
12300 b
11 c
43 c
23 c
806 a
0c
24 b
36 b
27 b
81ab
5b
247 c
418 b
949 b
105 b
22600 a
1779 d
581 e
4655 b
443 f
8153 a
30 b
58 c
85 b
23 c
120ab
7192 a
1692 c
7999 a
1692 c
1517 c
218 c
137 b
539 b
120 b
19233 a
19176 b
11202 d
24626 a
7023 d
14033 c
7b
5b
22 b
5b
1320 a
540ab
108 d
610 a
110 d
439 b
d
d
b
d
a
b
b
b
b
71700 a
ethanol
4985
ethyl acetate
1083 b
296 b
1596 b
173 b
16496 7 a
3614 b
1164 c
7416 a
1211 c
11867 a
8b
8b
8b
11 b
25 a
506 b
82 e
345 c
246 d
207 a
furfural
13 b
4b
47 b
24 b
199 a
382 b
53 d
599 a
125de
198 d
hexanal
36 c
17 d
161 b
27 d
294 a
467 a
119 d
523 a
167cd
343 b
a
9188 d
26567 b
27937 b
14892 c
47495 a
10545 c
52400 a
furan
methanol
methional
methylbutanal
24700
b
1833
29416
b
30054
47005
1756
94600
2868
602
20899
1157
10 e
10 e
88 c
7e
273 a
2013 d
345 e
3393 b
497 e
3690 b
b
b
b
c
a
b
e
c
e
6780ce
205
262
439
84
3383
10239
4013
13049
3977
nonanal
14 b
12 b
120 b
19 b
128833a
1153 c
193 c
6092 b
166 c
8937 a
pentanal
380 b
84 b
306 b
34 b
33867 a
5746 a
3556 b
6263 a
2624 c
2510 c
phenylacetaldehyde
25 b
18 b
41 b
14 b
300 a
202 d
81 d
832 b
71 d
1137 a
tetramethylpyrazine
4d
2d
8d
3d
339 a
113 c
22 c
326 b
33 c
569 a
41 b
21 b
193 b
13 b
4467 a
419 c
74 e
993 b
104 d
1947 a
toluene
38
Table 4 Raw volatiles (µg/L) of 15 seed varieties grown only in 2009. Different letters in
the same row are significantly different
1H
(E)-2-heptenal
(E)-2-hexenal
4H
31
b
164
b
5H
13
b
41
b
6H
35
b
188
b
7H
21
b
57
b
8H
12
b
151
b
9H
4
b
29
b
11H
19
b
50
b
12H
11
b
113
b
13H
13
b
6b
97
b
48 b
1-hexanol
21 b
8b
19 b
7b
5b
4b
11 b
6b
9b
3b
1-pentanol
190 b
86 b
91 b
90 b
88 b
32 b
78 b
101 b
77 b
34 b
6c
12 c
12 c
12 c
7c
4c
10 c
6c
4c
3c
1-phenylethanol
183 b
239 b
72 b
172 b
58 b
40 b
102 b
137 b
125 b
30 b
2,3-butanedione
1124
b
216 e
170ef
282 e
327 e
270 e
143 f
1385 a
614 c
380de
25 b
5b
26 b
9b
9b
2b
9b
6b
11 b
2b
c
a
c
b
c
c
c
c
c
1c
1-penten-3-one
2-acetylpyrrole
2-heptanone
5
54
1
30
3
5
3
11
1
73 b
54 b
63 b
53 b
59 b
39 b
70 b
49 b
48 b
31 b
2b
3b
1b
1b
0b
1b
3b
1b
1b
1b
2-methylpropanal
109 b
68 b
41 b
81 b
68 b
57 b
115 b
74 b
80 b
36 b
2-methylpyrazine
24 b
88 b
18 b
37 b
8b
14 b
32 b
10 b
13 b
9b
176 a
63cd
44 d
71cd
69cd
70cd
72cd
177 a
104bc
57cd
b
b
b
b
b
b
b
b
b
1b
2-methyl-1-butanol
2-methyl-3-ethylpyrazine
2-pentanone
2-pentylfuran
4
3
2
3
0
0
1
3
6
508 b
144 c
132 c
219 c
187 c
84 c
257 c
706 c
810 c
149 c
57 c
306 c
26 c
151 c
27 c
1277 a
12 c
117 c
51 c
145 c
694 b
244 b
239 b
326 b
202 b
110 b
388 b
431 b
446 b
94 b
butanone
56 c
68bc
26 c
75bc
50 c
44 c
114 a
39 c
59 c
20 c
dimethylpyrazine
10 b
10 b
11 b
8b
3b
4b
7b
8b
32 b
4b
dimethyl sulfide
616 b
234 b
239 b
290 b
531 b
157 b
189 b
316 b
183 b
117 b
d
c
c
c
d
d
d
d
d
2676 d
acetoin
ammonia
benzaldehyde
ethanol
4459
ethyl acetate
2454 b
378 b
1776 b
591 b
2164 b
118 b
322 b
684 b
807 b
387 b
6b
9b
8b
6b
7b
5b
10 b
4b
8b
6b
furfural
18 b
21 b
12 b
11 b
5b
3b
10 b
8b
8b
4b
hexanal
35 c
54 c
66 c
52cd
26 d
34cd
61 c
27 d
23 d
32cd
d
2816 8bc
d
1629 9cd
3256fe
d
7448 d
d
9430 d
furan
b
7798
7135
11371
2029
1099
13204
2379
5141
methanol
2614
methional
176 b
38de
12 e
43de
16de
21de
30de
52cd
32de
25de
b
b
c
c
b
c
c
b
b
103 c
methylbutanal
392
16465
165
242
1238 9
143
187
68
14668
152
220
18866
195
nonanal
79 b
29 b
20 b
32 b
14 b
12 b
12 b
46 b
46 b
11 b
pentanal
838 b
111 b
529 b
212 b
721 b
37 b
102 b
262 b
264 b
144 b
phenylacetaldehyde
42 b
55 b
22 b
35 b
10 b
12 b
14 b
18 b
19 b
9b
tetramethylpyrazine
23 d
8d
4d
8d
5d
9d
4d
120 b
69 c
6d
toluene
46 b
26 b
63 b
39 b
48 b
12 b
56 b
20 b
28 b
14 b
39
Table 5 Roasted volatiles (µg/L) of 15 seed varieties grown in 2009, roasted at 150 ᵒC for
40 min. Different letters in the same row are significantly different
1H
4H
b
5H
151
b
6H
7H
142b
384b
252e
188ef
164ef
241f
161ef
196ef
127f
c
bc
c
c
c
c
c
c
11c
1-hexanol
103
1-pentanol
526a
327c
492a
288bc
307bc
180c
265bc
302 a
281 c
218c
1-penten-3-one
162b
75d
194b
69d
78d
38e
114cd
53de
59abc
51de
1-phenylethanol
746c
413cd
579c
348d
293d
154d
350 c
221d
263de
231d
2,3-butanedione
3725a
9796d
2466b
8581d
1689c
484d
2026c
7000d
1270d
1080 0d
2-acetylpyrrole
289b
88c
286b
82c
134c
69c
149c
82c
124c
106 c
a
c
b
c
c
c
c
c
c
85 c
2-heptanone
438
161
271
132
144
51
161
26
169
13H
b
252e
45
14
12H
b
b
13
210
11H
b
429bc
26
88
9H
b
(E)-2-hexenal
22
199
8H
b
501
78
148
b
(E)-2-heptenal
25
418
b
67
17
82
5973ab
1392de
6172ab
1202e
2889c
1587de
2152de
1145de
1708de
2058cd
437a
41f
362ab
43f
173ef
104f
53 f
44f
62f
81 f
2-methylpropanal
9663a
2816d
7578b
2746b
4973c
1536e
459 c
2720d
4034d
3101 d
2-methylpyrazine
2294
b
ef
de
de
e
f
e
e
e
482ef
2-pentanone
5339a
1539d
4943a
1231a
2414c
909d
349 b
1042d
1773d
1717 d
2-pentylfuran
281a
34cd
231ab
39ab
104cd
41cd
61cd
50cd
66cd
65cd
1944c
1451cd
334d
1589d
249d
101d
32 d
279d
231d
228 d
49b
70b
44b
56b
23b
166a
3b
53b
3b
29 b
benzaldehyde
2485c
1089d
1660d
1003e
969e
384f
109 e
613f
869f
666 f
butanone
7942a
2152c
7628a
1862c
3880b
1379c
404 b
2424c
3023c
2559 c
b
d
ab
d
c
d
c
d
d
239 c
2-methyl-1-butanol
2-methyl-3ethylpyrazine
acetoin
ammonia
387
9372d
13529c
6173d
17172b
7581e
10574d
10895 e
ethanol
4431b
7246b
4047b
6932b
2166b
1679b
2070b
1375b
1942b
1426 b
ethyl acetate
4033b
1776c
3561b
1795c
2166c
1068c
2269c
1277c
1609c
1544 c
furan
449b
98d
668d
90e
300c
158d
202d
116d
176d
220 d
furfural
584a
122c
40b
123b
229c
90d
184d
99d
147d
134 d
hexanal
442a
219cd
424cd
223cd
242cd
160d
285cd
132d
196cd
174cd
32633b
15140c
20962c
12911c
15708c
6221d
18919c
9866c
14661c
11776 c
a
d
c
c
d
d
d
d
d
1446 c
4444
methylbutanal
11871b
4509e
8040bc
4349e
6399e
2469e
7272e
3339e
5161e
4377 e
nonanal
1668c
488c
1701c
500c
610c
184c
497c
388c
337c
292 c
pentanal
6789a
3254c
4169b
2949b
4103b
1719d
4510b
2696c
3603b
3089 c
phenylacetaldehyde
391c
204d
282c
162c
144d
115d
139d
118d
142d
131 d
tetramethylpyrazine
314
b
c
c
c
c
c
c
b
c
53 c
toluene
845c
136d
168 d
296d
117
505d
54
276d
40
54
225d
33
78c
65
190c
774
252
methional
60
1079
143
483
16786b
1090
22
274
11258d
1551
101
70
24847a
984
275
187
dimethyl sulfide
2745
91
736
466
934
542
404
dimethylpyrazine
methanol
114
1001
306
111d
807
159
4.2 Effect of Roasting on Final Volatile Formation
After roasting at 150ᵒC for 40 min, seed volatiles in highest concentration were
Strecker aldehydes, including methylbutanal (malty odor), 2-methylpropanal and
methional (potato-like odor), 2, 3-butandione, dimethylsulfide and pentanal (pungent,
almond-like odor) (Tables 3,5). Strecker aldehydes, formed by Strecker degradation of
amino acids during the Maillard reaction, are frequently important to roasted aroma.
Volatile compounds from these reactions have been studied in numerous thermally
processed foods and increase significantly from raw to roasted pumpkin seeds (Siegmond
and Murkovic 2004).
Roasting caused a significant increase in volatile concentrations in all varieties of
seed, except 15NH, where most raw volatiles were significantly higher (Table 3). All
volatiles for varieties 1-14 increased on average between 80-100% except the highly
volatile compounds: ammonia, ethanol and methanol, which generally decreased or
remained the same. Out of 34 total volatile compounds measured, 15NH showed a
decrease in 16 compounds with the top three compounds being pentanal, ethyl acetate
and nonanal, all decreasing over 1200% during roasting. Since pentanal has been used to
measure oxidation in heated milk based products (Romeu-Nadal and others 2004) and
nonanal has been used to measured oxidation in olive oil (Morales and others 1997), it is
a reasonable to assume that 15NH raw seeds were considerably oxidized and the roasting
process caused rapid volatilization of these abundant volatiles.
The lipid oxidation products that increased during the course of roasting pumpkin
seeds were pentanal, hexanal, 2-heptenal and nonanal. The same increase in oxidation
41
products was found in roasted pumpkin seeds by Siegmond and Murkovic (2004). While
concentrations well above their thresholds result in rancid flavors, lower concentrations
of lipid aldehydes, which are formed from the oxidation of fatty acids, contribute green,
grassy and slightly fruity flavor notes which contribute to the roasted aroma of pumpkin
seeds (Siegmond and Murkovic 2004).
In this study 2-methylpyrazine increased the most during roasting, followed by
dimethylpyrazine. In a previous study of pumpkin seeds, dimethylpyrazine increased to a
much higher extent than 2-methylpyrazine (Seigmond and Murkovic 2004). Pyrazines,
also formed during the Maillard reaction but at relatively lower levels than Strecker
aldehydes and lipid oxidation products, have low thresholds and contribute roasty, nutty
flavor notes at appropriate levels; they were also previously found to increase in roasted
pumpkin seeds (Siegmond and Murkovic 2004).
Siegmond and Murkovic (2004) also showed a significant decrease in 1-penten-3ol, 2-methylbutanol and hexanol during roasting, which they attributed to oxidation to
their corresponding aldehydes, which was not found in this study. We found that these
three compounds increased significantly during roasting, while their corresponding
aldehydes increased to an even greater extent for all seed varieties except 15NH,
indicating the same mechanism may be taking place.
Roasted seed volatiles varied by seed variety (Tables 3, 5). It is likely that seed
composition, especially volatile precursors, fluctuate from one seed variety to another.
Roasted volatiles were also found to be different between two varieties of peanuts,
Spanish and Runner (Brown and others 1973). If a correlation existed between raw and
42
roasted volatile concentrations, it would suggest that volatiles were formed primarily
before roasting from reactions such as sprouting, respiration or fermentation. But, as
expected, roasted seed volatile concentration did not depend on raw volatile
concentration, indicating that volatile formation was linked to volatile precursors, such as
those necessary for thermally induced reactions. Roasted volatile formation is mainly
attributed to Maillard browning in foods such as pumpkin seeds (Seigmond and
Murkovic 2004), peanuts (Brown and others 1973), perilla seeds (Kim and others 2000),
cocoa beans (Ramli and others 2006) and coffee beans (Schenker and others 2002). Since
these foods are relatively high in lipids, volatile formation has also been partially
attributed to lipid oxidation.
After the 2009 harvest of the 15 varieties, a focus group was conducted with 10
individuals to eliminate 10 varieties based on roasted texture, flavor and off-notes. Five
varieties were selected based on these attributes in order to produce an optimal snack
seed. These five varieties were planted, grown and harvested the following 2010 season.
4.3 Effect of Seed Coat on Volatile Formation
The hull-less seeds 10NH and 15NH, from both years 2009 and 2010, had
significantly higher concentrations of most roasted volatiles compared to any of the seed
varieties containing a seed coat (Tables 3, 6). Methylbutanal formation during roasting is
typical of all volatile formation from seed varieties 2H, 3H, 10NH and 14H; and volatile
groups including Strecker aldehydes, pyrazines and furans from 15NH (Figure 1).
Methylbutanal, formed from Strecker degradation of the amino acids leucine and
isoleucine, is an important volatile compound contributing to the unique roasted aroma of
43
pumpkin seeds, and is representative of volatile formation behavior of most other
compounds, except certain volatiles from 15NH that had a drastic initial decrease in
concentration.
15NH
14H
7000
Concentration (µg/L)
16000
14000
3H
2H
6000
Concentration (µg/ L)
8000
12000
10NH
5000
10000
4000
8000
3000
6000
2000
4000
1000
2000
0
0
0
5
10
15
20
Time (min)
25
30
35
40
Figure 1 Formation of methylbutanal during roasting from seed varieties 2H, 3H, 14H,
10NH and 15NH (shown on right axis), harvested in 2010. Methylbutanal representative
of all Strecker aldehydes, pyrazines and furans.
Most volatiles from varieties 2H, 3H, 10NH and 14H increased linearly, peaked
and either leveled off or decreased (Figure 1). This excludes 15NH which had certain
volatile groups at high initial concentrations causing them to decrease during roasting.
During roasting, hulled seed varieties 2H, 3H and 14H showed a linear formation of
Strecker aldehydes, pyrazines and furans, while NH varieties had a more drastic increase,
with 10NH increasing much faster than 15NH (Figure 1). Seed variety 10NH had
significantly higher volatile formation, especially Strecker aldehydes, than any other seed
44
variety, including 15NH, from both years 2009 and 2010 (Figure 1). A possible
explanation for higher volatile concentration in hull-less seeds may be that because 10NH
and 15NH do not have a seed coat, heat is able to penetrate faster during roasting,
meaning the internal temperature of the seed is reaching 150 ᵒC faster and remaining
higher for a longer period of time. It is also possible that because of the presence of a
seed coat on 2H, 3H and 14H, they retain more moisture, despite the dehydration process.
In a typical hulled seed at a 6% moisture content, the seed hull has a moisture content of
10%, while the kernel is only about 4.5% (Teotia and others 1989). A higher moisture
content would drastically reduce kinetics of Maillard browning. 15NH had the highest
raw volatile concentrations, especially lipid aldehydes, of any seed variety (Table 6). The
seed varieties with a seed coat, 2H, 3H and 14H have much lower initial and final volatile
concentrations (Table 6).
Volatiles that decreased during roasting in 15NH were pentanal, ethyl acetate,
nonanal, (E)-2-heptenal, (E)-2-hexenal, dimethylpyrazine, 2-acetylpyrrole, 2methylpyrazine and 2-pentylfuran and are represented by pentanal (Figure 2). Seed
variety 15NH had significantly higher initial volatiles than any other seed variety,
especially lipid aldehydes including nonanal, (E)-2-heptenal and pentanal (Table 6).
Compared to other seed varieties, 15NH had the highest raw volatile concentrations of
(E)-2-heptenal, (E)-2-hexenal, dimethylpyrazine 2-acetylpyrrole, 2-methylpyrazine, 2pentylfuran, ethyl acetate, nonanal and pentanal (Table 6). During roasting these
compounds decreased significantly within the first 10 min in 15NH, before being
generated at higher concentrations than they were volatilizing at 20 min. When seed
45
variety 15NH was being extracted a noticeable amount of seeds had sprouted, leading to
the conclusion that harvest was done too late in the season. Sprouting has been shown in
pumpkin seeds to cause a decrease in total reducing sugars, which is suggestive of
multiple (non-thermal) reactions taking place, resulting in volatile formation of all
volatile compounds (Odoemena 1991).
46
Table 6 Raw and roasted volatiles (µg/L) of 5 seed varieties from 2010 harvest
RAW
2H
3H
ROASTED
10NH 14H 15NH 2H
10NH 14H 15NH
8b
18 b
12 b
13 b 819 a
38 c
22 c 708 b
27 c 3367 a
(E)-2-heptenal
13 c
19bc
89 b
17 c 3710 a
96 c
59 c 439 b
68 c 787 a
(E)-2-hexenal
b
b
b
b
a
c
c
b
23
44
30
30
466
7
5
106
5 c 148 a
1-hexanol
1b
1b
2b
3 b 396 a 104 c 104 c 632 a 129 c 397 b
1-pentanol
c
c
b
8
20
107
9 c 4857 a
29 b
22 b 268 a
18 b 112 b
1-penten-3-one
4b
11 b
22 b
4 b 242 a
54 c
34 c 952 b
40 c 1810 a
1-phenylethanol
31 c
41 c
92 b
30 c 476 a 1287 c 983 c 40433 a 1102 c 6603 b
2,3-butanedione
b
b
b
4
7
32
3 b 112 a
29 b
19 b 388 a
27 b 475 a
2-acetylpyrrole
17 a
20 a
23 a
19 a 934 a
7c
17 c 541 a
13 c
93 b
2-heptanone
b
b
b
b
a
b
b
a
b
6
7
8
7
62
528
301 7553
397 7020 a
2-methyl-1-butanol
6b
5b
5b
5b
13 a
11 c
8 c 424 a
8 c 273 b
2-methyl-3-ethylpyrazine
12 b
15 b 283 b
10 b 22633 a 484 c 436 c 12233 a 517 c 3783 b
2-methylpropanal
b
b
b
6403 13100 33233 11117 b 86667 a
57 c
55 c 3113 a
70 c 1079 b
2-methylpyrazine
111 b 113 b 236 a
56 b 2060 a 247 c 164 c 5310 a 180 c 1024 b
2-pentanone
b
b
b
6
4
2
6b
0a
19 c
13 c 370 a
20 c 195 b
2-pentylfuran
1b
4b
9b
3 b 127 a
29ab
24 b 4657ab
26 b 8213 a
acetoin
20467 a 21067 a 44733 a 14167 a 41400 a
3a
9a
3a
6a
6a
ammonia
b
b
b
b
a
c
c
b
c
5
14
107
5 3507
131
100 3323
98 6920 a
benzaldehyde
0a
0a
1a
0a
76 a 445 c 354 c 8163 a 412 c 1970 b
butanone
b
b
b
b
278
700 1783
379 64733 a
80 c
70 c 678 a
84 c 428 b
dimethylpyrazine
4b
6b
4b
7 b 123 a 2340 c 1590 c 21533 a 1587 c 12167 b
dimethyl sulfide
5b
0b
89 b
0 b 35933 a 732 c 637 c 10753 b 473 c 68133 a
ethanol
b
b
b
82
179
460
112 b 13033 a 1243 c 884 c 5650 b 872 c 9957 a
ethyl acetate
23 b
43 b
75ab
23 b 221 a
92 c
52 c 663 a
81 c 400 b
furan
b
b
b
b
a
b
b
a
28
89
346
45 9653
91
64
687
78 b 236 b
furfural
2b
3b
2b
2b
30 a
92 c
82 c 650 a
65 c 387 b
hexanal
6b
13 b
12 a
5 b 183 a 20567 c 12600 d 35500 b 13027 d 45167 a
methanol
b
b
b
57
92
147
65 b 6267 a 451 b 321 b 4443 a 306 b 4240 a
methional
1b
4b
10 b
1 b 115 a 916 c 663 c 14400 a 788 c 6310 b
methylbutanal
b
b
b
3
6
11
2 b 536 a
94 c
52 c 3033 b
49 c 6343 a
nonanal
13 b
27 b
49 b
17 b 2060 a 674 c 529 c 5747 a 577 c 2480 b
pentanal
0b
1b
2b
1b
7 a 110 c
77 c 411 b
74 c 1104 a
phenylacetaldehyde
b
b
b
b
a
c
c
b
38
224
23 2977
12
20
18
214
17 c 358 a
tetramethylpyrazine
0b
1b
0b
0 b 155 a
94 c
46 c 1015 b
51 c 1610 a
toluene
47
3H
14000
10NH
15NH
14H
2H
3H
Concentration (µg/L)
12000
10000
8000
6000
4000
2000
0
0
10
20
30
40
Time (min)
Figure 2 Formation of pentanal during roasting from seed varieties 2H, 3H, 14H, 10NH
and 15NH, harvested in 2010. Representative of lipid aldehydes = (E)-2-heptenal, (E)-2hexenal, pentanal, 2-pentylfuran; and N-heterocyclic compounds= dimethylpyrazine, 2acetylpyrrole, 2-methylpyrazine
4.4 Effect of Moisture Content on Volatile Formation
Typically pumpkin seeds are dried to about 6-7% moisture before roasting,
especially for the production of pumpkin seed oil. However, some sources recommend
roasting seeds wet, directly out of the pumpkin. While dehydrated seeds allow for easier
storage, this study was conducted to understand dehydration’s effects on volatile flavor
formation.
The 2010 seed variety 10NH was roasted both wet, with a starting moisture
content of 46-48%, and dehydrated, with a starting moisture content of 6-7%.
Commercially seeds are dried at 40 to 60°C until they reach a final moisture content of
8% to 10% (Bavec and others 2007). Methylbutanal is representative of all other volatiles
48
(Figure 3). Seeds starting with 6-7% moisture formed significantly higher concentrations
of all volatiles, except ammonia and ethanol, than seeds starting with 48% moisture
(Figure 3). All final pyrazine and Strecker aldehyde levels were between 10-40 times
higher in the dehydrated roasted seeds. The Maillard reaction occurs less readily in higher
moisture systems, mainly because reactants are diluted. Once the wet seed reached 6%
moisture, which is the same as the dehydrated seeds, methylbutanal began to be
generated faster than it was volatilizing (Figure 3). Methylbutanal is also formed in
highest concentration in extruded maize flour with 14% moisture (53 ng/10 g) compared
to 18% and 22%, with only 21 and 11 ng/10 g, respectively (Bredie and others 1998).
50%
48%
14000
45%
43%
Concentration (µg/ L)
12000
40%
Methylbutanal-WET
36%
Methylbutanal- DEHYD
10000
30%
Moisture-WET
27%
8000
35%
25%
Moisture-DEHYD
20%
6000
15%
15%
4000
6%
2000
7%
6%
10%
5%
4%
4%
4%
4%
4%
6%
5%
0
0%
0
10
20
30
Time (min)
40
50
Figure 3 Effect of starting moisture content (y axis on right) on volatile formation of
methylbutanal during roasting of 10NH seeds, harvested in 2010.
49
60
Moisture (%)
16000
4.5 Effect of FFA Composition on Corresponding Lipid Aldehyde Formation
Fatty acid composition and total lipid (%) varied significantly by seed variety
(Table 7), which is consistent with results from another study conducted on fatty acid
ratios in pumpkin seed varieties (Al-Khalifa 1996). The higher the number of double
bonds in a given fatty acid, the greater the degree of oxidation. Relative oxidation rates
of triplet oxygen with oleic, linoleate and linolenate are 1:27:77 (Lee and others 2003).
Moderate correlations were found (R2 = 0.87 and R2 = 0.49) between linoleic and oleic
acid concentrations and formation of hexanal and nonanal, respectively across different
seed varieties. Hexanal, described as fatty, green and oily is a main volatile originating
from Linoleic acid (13-LOOH) (Morales and others 1997). Nonanal, described as
pungent and fatty, is an abundant product derived from oleic acid. There was no
correlation between concentrations of linolenic acid and its corresponding volatile, (E)-2heptenal, R2 = 0.23. (E)-2-Hexenal, described as sweet, almond-like and green, is
typically an abundant product from linolenic acid (12/13-LnOOH) (Morales and others
1997). Total oil percentage did have a significant effect on the amount of volatile
compounds known to originate from lipids, which were hexanal, pentanal, nonanal, (E)2-hexenal, (E)-2-heptenal and 2-pentylfuran (Figure 4). This correlation could indicate
that there was minimal volatile formation until 35% total lipid where volatile content
increased exponentially (R2=0.71). Alternatively, this correlation could have been caused
because NH seed varieties have higher lipid content (42 and 45%) and therefore causing
total content of easily oxidized fatty acids to be higher. In a study on crushed hazelnuts
50
with varying amounts of lipids, a higher oil percentage resulted in higher concentration of
lipid aldehydes (Fallico and others 2003).
Table 7 Fatty acids (g) per 100 g seed oil and total oil (%)
14H
2H
3H
Palmitic
10.19 c
10.52 b
9.90 d
Stearic
7.39
a
7.21
a
6.22
b
6.82
Oleic
38.44
c
43.81
a
41.28
b
Linoleic
43.44
b
38.11
e
42.60
Linolenic
0.54
a
0.34
c
0.47
Total Oil
23.00 d
a
34.60 c
10NH
15NH
11.84 a
11.52 a
ab
6.73
34.79
d
40.74
b
c
46.56
a
41.01
d
ab
0.38
bc
0.36
c
34.70 c
45.40
ab
42.30 b
Different letters in the same row are significantly different.
2500
Concentration (µg/L)
2000
R² = 0.71
1500
1000
500
0
20
25
30
35
Lipid (%)
40
45
Figure 4 Effect of total lipid (%) on average lipid volatile formation
51
50
4.6 Effect of Reducing Sugars
While reducing sugars were hypothesized to be the rate limiting factor for volatile
formation, our results showed that reducing sugars did not have a significant effect on
final concentrations of all Maillard volatiles (R2=0.44). This demonstrates the complexity
of thermally induced flavor reactions, and that correlation to a single factor is often very
difficult. Many other nonenzymatic reactions that do not depend on reducing sugars are
possible explanations for volatile formation including lipid oxidation, pyrolysis of sugars
and even caramelization. Reducing sugars in the five varieties from 2010 were in a very
small range, 1.4 to 2.4%. It is very likely that a correlation was not found because a large
enough difference was not found in reducing sugar concentration across seed varieties.
Since the ideal harvest date can vary based on genetic differences between
pumpkin varieties and 15NH was so much higher in raw volatiles, especially nonanal and
pentanal, it is reasonable to conclude that 15NH was harvested too late in the season and
seeds were oxidized (Loy 1990). The 15NH variety may have been harvested too late
causing the seeds to sprout. While this may explain higher volatile levels in 15NH raw
seeds, there was no significant correlation to reducing sugar percentage without 15NH.
4.7 Effect of Harvest Year on Volatile Concentration
There were significant differences in volatile concentrations between varieties 2H,
3H, 10NH, 14H and 15NH which were grown and harvested in both 2009 and 2010
(Table 4, 5, Figure 5). Hulled varieties (2H, 3H and 14H) varied from 2009 to 2010 more
significantly than the hull-less varieties (10NH and 15NH). In general, 2009 seed
varieties had higher concentrations of roasted volatiles than 2010. However, 10NH from
52
2009 has a lower concentration of methylbutanal than 10NH from 2010, most likely
caused by slight differences in seed composition. Seeds from 2009 also had higher
concentrations of raw volatiles except (E)-2-heptenal and 2-methylbutanol. Even though
the effect of harvest year is clear, the effect of seed variety, especially between NH and H
varieties, showed a much more significant effect. It is interested to point out that hullless varieties were consistent from 2009 to 2010 in forming significantly higher
concentrations of volatiles during roasting than hulled varieties, especially nonanal and
other lipid aldehydes. Seed variety 15NH’s raw volatiles were significantly higher in 26
out of 34 volatiles than any other seed variety. Compounds that 15NH did not show the
highest concentrations in were all ketones and methanol.
Figure 5 Roasted volatiles of varieties 2H, 3H, 10NH, 14H and 15NH from 2009 and
2010
53
4.7 Sensory Analysis
Five varieties of seeds were evaluated for texture using a Just-About-Right (JAR)
scale. While no significant difference was found between the roasting times (15 and 30
min), a noticeable difference was found between varieties. For both hull-less varieties,
10NH and 15NH, 72% of panelist for both varieties, reported the texture to be just about
right. However, in the hulled varieties, 2H, 3H and 14H, only 55, 50 and 38% of panelist
reported texture to be just about right.
Roasted seeds were evaluated in two separate panels for overall liking. From the
second panel, panelists preferred hull-less seeds 10NH and 15NH significantly more than
seed varieties 2H, 3H and 14H which contain a hull (Figure 6). This is especially
interesting because in the first panel seeds were also evaluated for overall liking and
15NH roasted for 30 min were described as rancid and oxidized, and were rated
significantly lower than all seeds except 14H roasted for 30 min (Figure 7). This is most
likely because the 15NH variety was extracted and processed on two different days, from
two different farms, leading us to believe that the harvest used in the first sensory study
54
was different than the batch used in the second sensory study.
a
7.5
a
Mean Score
7
6.5
6
b
b
3H
2H
b
5.5
5
4.5
4
15NH
14H
10NH
Seed Variety
Figure 6 Overall liking scores of five varieties of pumpkin seeds.
7.5
30 min
7
15 min
Mean Score
6.5
6
5.5
abc ab
5
4.5
c
ab
ab
a
a
ab ab
bc
4
15NH
14H
10NH
Seed Variety
3H
2H
Figure 7 overall liking of seed varieties 2H, 3H, 10NH, 14H and 15NH all roasted for
both 30 and 15 min.
55
It is difficult to compare ratings from the two panels, for several reasons. Firstly,
in panel one each sample (out of 10 total) was given one at a time compared to panel two,
the five samples were presented all at once. During a sensory panel, when people can
compare all seed samples at the same time they may realize that NH varieties are much
better than H varieties. Alternatively, lower rating in panel one could also have been
because people were over loaded with 10 samples. Lastly, in panel one, the seeds weren't
oiled and salted, but they were in the second panel. Since salt is used on many snack
items to bring out flavor, it is understandably that panelists ranked unsalted seed much
lower than salted.
Even though in panel one 15NH was ranked significantly lower than most other
seed varieties, in panel two there was no significant difference in overall liking between
hulled seed varieties 2H, 3H and 14H; and no difference between overall liking of hullless seed varieties 10NH and 15NH (Figure 7). From the sensory study, we cannot know
if seed texture, the presence or absence of a seed hull or the flavor was most important.
Especially considering the results from panel 2, it certainly appears that the absence of a
seed hull is the dominant factor in ranking for overall liking. There are some very
significant differences between 10NH and 15NH in terms of volatile concentrations, oil
percentage/ fatty acid ratio and color, yet that did not cause a difference in sensory rating.
It is worth adding, from having tasted them, the flavor is mild and there is not a strong
difference between them.
From the survey sent out, using survey monkey, on roasted pumpkin seed
seasonings, participants were given a list of 10 seasonings and told to pick the top 5
56
seasonings that sounded appealing on roasted pumpkin seeds, the top seasonings were
salted (84%), honey roasted (61%), pumpkin pie spiced (51%), garlic (42%), barbeque
(36%) and vanilla cinnamon sugar (36%). During preliminary testing honey roasted was
eliminated and vanilla cinnamon sugar was selected to take its place. Sun dried tomato
was eliminated based on external marketing data and seasoning availability.
Garlic, barbeque (BBQ), salted, cinnamon vanilla and pumpkin pie spiced
seasonings were evaluated on seed variety 10NH only. A 9 point hedonic scale was used
and showed that savory (BBQ and salted) seasonings were liked significantly more than
sweet seasonings (Figure 8). However, there was no significant difference between garlic
seasoning and sweet seasonings. There was no significant difference between savory
seasonings or between sweet seasonings (pumpkin pie spice and vanilla cinnamon sugar)
(Figure 8).
a
6.9
a
Mean Score
6.7
6.5
ab
6.3
6.1
5.9
5.7
b
b
5.5
Pum pie
spice
Van cinn
Garlic
BBQ
sugar
Seasoning Type
Salt
Figure 8 Overall liking of seasonings on roasted 10NH seed variety
57
Chapter 5: Conclusions
Raw seeds had higher concentrations of lipid aldehydes whereas roasted seeds
had higher concentrations of Strecker aldehydes. Roasted volatiles varied by seed variety
and did not depend on concentration of raw volatiles. Hull-less seeds had significantly
higher concentrations of some raw volatiles, and most roasted volatiles. Higher starting
moisture content in seeds resulted in less volatile formation during roasting. A moderate
correlation was found between fatty acid ratio and formation of corresponding volatiles
and total lipids and average lipid aldehyde formation, however it is difficult to know for
sure if this is because of the difference between hulled and hull-less seeds or the total oil
content. No correlation was found between reducing sugars and typical Maillard reaction
volatiles. Lastly, sensory analyses showed that hull-less seeds were liked significantly
more than seeds that contain a hull. This data provided a better understanding of factors
affecting volatile formation in roasted pumpkin seeds intended to be consumed as a snack
item.
58
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Apendix A: Additional Figures
Table 8 Percent volatile concentration (µg/ L) increase in 2009 seed varieties from raw to
roasted (150°C for 40 min)
(E)-2-heptenal
(E)-2-hexenal
1-hexanol
1-pentanol
1-penten-3-one
1-phenylethanol
2,3-butanedione
2-acetylpyrrole
2-heptanone
2-methyl-1-butanol
2-methyl-3-ethylpyrazine
2-methylpropanal
2-methylpyrazine
2-pentanone
2-pentylfuran
acetoin
ammonia
benzaldehyde
butanone
dimethylpyrazine
dimethyl sulfide
ethanol
ethyl acetate
furan
furfural
hexanal
methanol
methional
methylbutanal
nonanal
pentanal
phenylacetaldehyde
tetramethylpyrazine
toluene
1H
94
62
79
64
96
76
97
91
99
99
100
99
99
97
98
74
-17
72
99
98
98
-1
39
99
97
92
20
96
97
95
88
89
93
95
2H
95
69
67
83
93
88
99
96
100
99
99
100
99
99
99
43
53
86
100
99
99
-74
70
98
97
92
12
100
98
99
93
87
96
90
3H 4H
86 91
57 84
26 66
55 74
85 84
41 42
97 98
96 94
96 66
89 96
94 93
96 98
96 77
96 96
88 92
-139 90
-22 -337
28 78
97 97
95 91
99 98
-205 -8
75 79
91 91
93 83
86 75
-98 -9
97 96
93 96
94 94
98 97
78 73
89 87
71 91
5H
92
51
76
81
94
87
99
91
100
99
100
99
98
99
99
61
42
86
100
98
99
-76
50
99
97
85
-34
100
97
99
87
92
96
88
6H
86
78
69
69
82
50
97
89
78
96
97
97
91
94
93
86
-169
67
96
91
97
-64
67
94
91
77
4
96
97
94
93
79
85
86
66
7H
94
20
81
71
91
80
98
94
98
98
100
99
99
97
100
25
-16
79
99
99
96
6
0
98
98
89
-4
99
97
98
82
93
90
78
8H 9H 10NH 11H 12H 13H 14H 15NH
96 91 47 92 92 96 95 -640
82 79 42 30 51 62 83 -208
69 76 81 78 49 70 59
5
82 71 58 66 73 84 75 -587
89 91 90 88 94 93 72
1
74 71 82 38 53 87 46 -157
94 99 99 80 95 96 98 97
97 94 87 93 92 98 99 -622
90 98 99 84 99 99 73 100
98 97 97 96 97 98 97 76
99 94 98 98 99 99 99 45
96 97 99 97 98 99 98 18
92 95 96 96 97 98 66 -482
92 98 99 83 94 97 96 91
99 98 98 94 91 99 94 -506
16 21 98 -153 -251 35 9
95
-667 61 15 -121 -1480 -395 -901 100
71 65 80 30 49 86 76 -177
97 97 99 98 98 99 99 92
96 97 96 94 87 99 95 -200
97 99 98 96 98 99 98 -37
35 -538 -44 -73 -165 -88 -52 -32
89 86 78 46 50 75 86 -1290
97 95 98 96 96 97 96 88
96 94 92 92 94 97 81 -1
79 79 69 79 88 82 84 14
48 22
1
25 -29 20 13 49
98 97 97 93 96 98 99 93
97 98 97 93 96 98 98 50
93 98 98 88 86 96 89 -1342
98 98 95 90 93 95 99 -1249
90 90 95 85 87 93 81 74
74 94 98 61 56 88 90 40
84 70 81 82 79 91 88 -129
Figure 9 Mechanistic view of hydroperoxide breakdown (Halliwell and Chirico 2011)
67
Figure 10 Principles of Selected Ion Flow Tube Mass Spectrometry (Smith and Spanel
2005)
68
Figure 11 Schematic diagram Maillard Browning Reaction (Ames 1990)
69
Figure 12 Schematic diagram of lipid oxidation and hydrolysis (Whitfield 1992)
70
Figure 13 Effect of roasting on volatiles formation on seed varieties 2H, 3H, 14H, 15NH
and 10NH
71
Hexanala
700
roasted
Conc (µg/ L)
600
R² = 0.87
500
400
300
200
100
0
9.0
11.0
13.0
15.0
17.0
19.0
Linoleic acid (g)/ 100 g seed
7000
23.0
Nonanalb
6000
Conc (µg/ L)
21.0
roasted
5000
R² = 0.49
4000
3000
2000
1000
0
8.0
10.0
4000
16.0
18.0
(E)-2-heptenalc
3500
roasted
3000
Conc. (µg/ L)
12.0
14.0
Oleic acid (g)/ 100 g seed
R² = 0.23
2500
2000
1500
1000
500
0
0.1
0.1
0.2
0.2
Linolenic (g)/ 100 g seed
Figure 14 Effect of fatty acid on corresponding volatile formation
72
0.2
Conc. (µg/L)
2500
2000
R² = 0.71
1500
1000
500
0
20
25
30
35
40
45
50
Lipid (%)
Figure 15 Effect of percentage reducing sugars on volatile formation of methylbutanal
73
25000
Conc. (µg/ L)
20000
15000
10000
5000
0
2H
3H
14H 10NH
15NH
2009
2H
3H
14H
dimethyl sulfide
methylbutanal
pentanal
2-methylpyrazine
10NH
2010
Seed Variety
Figure 16 Roasted volatiles of 15 seed varieties from 2009 harvest
74
15NH
Seasoning Type
Salted
Honey Roasted
Pumpkin Pie Spice
Garlic
Sundried Tomato & Basil
Chipotle Lime
Vanilla Cinnamon Sugar
BBQ
Spicy Jalapeno
Ranch
84%
61%
51%
42%
39%
38%
36%
36%
30%
26%
0%
50%
Participants (%) selecting seasoning
100%
Figure 17 Percentage of participants selecting top five seasonings that sounded appealing
on a roasted pumpkin seed from survey sent out electronically using survey monkey.
75