alpha amylase activity and its effects on the …
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The Pennsylvania State University
The Graduate School
ALPHA AMYLASE ACTIVITY AND ITS EFFECTS ON THE BREAKDOWN AND LIKING OF STARCH
THICKENED FOODS
A Thesis in
Food Science
by
Modesto Steiner Robles
© 2020 Modesto Steiner Robles
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2020
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The thesis of Modesto Steiner Robles was reviewed and approved by the following:
John E. Hayes Associate Professor of Food Science Thesis Adviser
Gregory R. Ziegler Professor of Food Science
Josephine Wee Assistant Professor of Food Science
Darrell W. Cockburn Assistant Professor of Food Science Robert F. Roberts Professor of Food Science Head of the Department of Food Science
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Abstract
Salivary alpha amylase is an enzyme that begins the process of digestion when starch-
based foods are introduced to the oral cavity. Prior work suggests that α-amylase activity
strongly effects the breakdown of starch in the mouth soon after coming in contact with it.
Much work has been in vitro systems and model systems. Here, my work looks at real foods
that individuals eat from a grocery store, and not model systems (e.g., starch solutions) that are
not a part of a normal diet. I tested 174 participants from the Penn State area in two separate
studies. In the first study, I typed participants into one of four Mouth Behavior groups, and
collected several food related personality measures. Participants were then given yogurt
samples that were either cornstarch or pectin thickened to eat; time to swallow and liking
ratings were collected. In the second study, I collected saliva from the participants and
measured both their copy number for the gene that encodes alpha amylase gene (AMY1) and
their α-amylase activity. Then I measured their salivary flow by having participants spit into a
vial for 5 minutes. Participants were then instructed to chew and expectorate yogurt and
pretzel samples upon which viscosity and visual cohesion tests were run. I found that Mouth
Behavior was not correlated with the specific personality measures tested here. I also saw that
when participants were allowed to eat without restriction, the only thing that correlated with
how quickly participants finished a starch thickened yogurt was how quickly they finished a
pectin thickened yogurt. That is, people who eat one yogurt quickly will likely eat another
yogurt quickly. And even when eating was controlled so that participants chewed at a steady
rate, a participant’s time to spit for one yogurt was highly correlated to their time to spit for
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another yogurt, regardless of their amylase activity. For a solid food (pretzels), I found that
when photographs of expectorated pretzel boluses were rated for a low to high cohesion by
research staff, salivary flow rate appeared to have a large effect on how cohesive a bolus
looked upon expectoration, while there was no evidence of an effect of amylase activity. In
summary, it appears that α-amylase activity can have an effect on starchy foods, but this effect
may be overstated in literature based on model systems. While large differences can be
observed when using model systems and starch solutions, but when it comes to eating real
foods that are more than just starch, there may not be that big of a difference.
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Table of Contents
List of Tables .......................................................................................................................... vi
List of Figures .........................................................................................................................vii
Acknowledgements .............................................................................................................. viii
Chapter 1: Literature Review .................................................................................................. 1 1.1: Starch origin and food applications ..........................................................................................1 1.2: Salivary Composition and Flow ................................................................................................3 1.3: Alpha Amylase Production and Uses ........................................................................................4 1.4: Copy Number ..........................................................................................................................6 1.5: Personality traits that may relate to food choices: Sensation and Variety Seeking ................... 10 1.6: Mouth Behavior .................................................................................................................... 12
Chapter 2: Salivary and personality differences as potential explanations for different mouth behavior styles ..................................................................................................................... 14
2.1: Introduction .......................................................................................................................... 14 2.2: Materials and Methods ......................................................................................................... 15 2.3: Results .................................................................................................................................. 17 2.4: Discussion ............................................................................................................................. 21 2.5: Conclusions ........................................................................................................................... 23
Chapter 3: Amylase activity and its effects on the breakdown and perception of starch thickened foods .................................................................................................................... 24
3.1: Introduction .......................................................................................................................... 24 3.2: Materials and Methods ......................................................................................................... 25 3.3: Results .................................................................................................................................. 28 3.4: Discussion ............................................................................................................................. 41 3.5: Conclusion ............................................................................................................................ 44
Chapter 4: Overall Conclusions and Future Work .................................................................. 46
References ............................................................................................................................ 48
Appendix A: VARSEEK scale (Variety Seeking) ....................................................................... 52
Appendix B: AISS (Arnett Inventory of Sensation Seeking) .................................................... 53
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List of Tables Table 2-1 Table of Jeltema/Beckley Mouth Behavior counts and Percentage ............................. 18
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List of Figures Figure 1-1 Diagram of (a) amylose and (b) amylopectin with branch point at the O6 position,
taken from Starch and Chemistry (BeMiller & Whistler, 2009) .............................................. 2
Figure 1-2 Diet and AMY1 copy number' (a) shows a comparison of the copy number distribution between high and low starch diets. (b) a cumulative distribution plot of copy number for the study populations. Figure taken from Perry (2007) ...................................... 7
Figure 2-1 Histogram of cornstarch yogurt time to swallow ........................................................ 19
Figure 2-2 Histogram of pectin yogurt time to swallow ............................................................... 19
Figure 2-3 Correlation between Pectin and Cornstarch yogurts time to swallow in seconds ..... 20
Figure 2-4 Correlation between AISS and VARSEEK ..................................................................... 21
Figure 3-1 AMY1 copy number variance....................................................................................... 29
Figure 3-2 Histogram of Amylase Activity ..................................................................................... 30
Figure 3-3 Correlation between AMY1 copy number and alpha amylase activity ....................... 31
Figure 3-4 Histogram of salivary flow (ml/min) ............................................................................ 32
Figure 3-5 Histogram of Total Amylase (U/min). Total amylase is a measure of the total amount of amylase that flows in a participants mouth as they produce saliva. ............................... 32
Figure 3-6 Correlation between Salivary flow and cornstarch yogurt expectoration viscosity. .. 34
Figure 3-7 Correlation between Salivary flow and Pectin yogurt expectoration viscosity. ......... 34
Figure 3-8 Correlation between Total amylase and the time to spit of cornstarch thickened yogurt .................................................................................................................................... 35
Figure 3-9 Correlation between Total amylase and Time to spit of pectin thickened yogurt. .... 36
Figure 3-10 Correlation between Time to spit and liking of cornstarch thickened yogurt .......... 37
Figure 3-11 Correlation between Time to spit and liking of pectin thickened yogurt ................. 37
Figure 3-12 Correlation between Liking and Amylase activity in cornstarch thickened yogurt... 38
Figure 3-13 Correlation between Liking and Amylase activity in pectin thickened yogurt .......... 38
Figure 3-14 Correlation between time to spit of both Cornstarch and Pectin thickened yogurts................................................................................................................................................ 39
Figure 3-15 Correlation between Salivary flow and pretzel bolus ratings ................................... 40
Figure 3-16 Correlation between Total Amylase and Pretzel Bolus ratings ................................. 40
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Acknowledgements
I’d like to firstly thank my advisor, John Hayes, without who’s guidance, support, and patience I would have had no chance of getting this done. Your mentorship was invaluable to me and I’ve learned so much from you that I can only offer you my sincerest thanks. To all my committee members, Josephine Wee, Darrell Cockburn, Gregory Ziegler. Your feedback and support was invaluable. You took the time from your busy schedules to meet with me and answer my questions and help me in my work, and for that I am truly thankful. My Mother and Father who have supported me all my life without ever asking anything in return. You who were my first teachers and who I’ve been learning from ever since. To my friends at Penn State who made Grad School so very much enjoyable, I could not have asked for better people to spend my time with. You truly made me look forward to coming into the office every day. All the administration and support staff at Penn state who were always available to help. Peter Hanchar, Svend Pederson, Kira Rigg, Tiffany Murray, Beth Tepsic. I wouldn’t have been able to do this without all of the amazing help you’ve given me.
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Chapter 1: Literature Review
1.1: Starch origin and food applications
Starch is a carbohydrate resource that plants accumulate and store for future survival
and productivity needs (Da Silva, Qin, Debuse, & DeJong, 2017). It is capable of being digested
by humans and is found in many cultivated plants, most notably, rice, potatoes, and corn. The
earliest evidence of the cultivation and consumption of starch rich foods dates back 11-12
millennia, in clusters of sites along the Yangtze River in the Hubei and Hunan provinces of China
(Normile, 1997). While multiple plants are used for commercial production of starch, the main
sources are corn (maize), rice, wheat, potatoes, and cassava. The choice of crop depends on the
climate of the area, with corn being popular in temperate and subtropical zones, cassava in
tropical regions, rice in wet and inundated places, and potatoes in cold climates (Carvalho,
2013). Although starch has additional nonfood applications (Augustyn, 2019), they will not be
addressed in this thesis.
Starch is composed of two main macromolecules, amylose and amylopectin (Whistler &
Daniel, 1984). Amylose is a linear (1→4)-linked polysaccharide of D-glucose monomers.
Amylopectin is also a linked polysaccharide but has α-(1→6) branch points and is the major
component of the starch granule. Starch is a semicrystalline structure composed of stacks of
alternating crystalline and amorphous lamellae (Jenkins, Cameron, & Donald, 1993).
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Figure 1-1 Diagram of (a) amylose and (b) amylopectin with branch point at the O6 position, taken from Starch and Chemistry (BeMiller & Whistler, 2009)
Starting around the 1930s, chemists began to develop different types of starch-based
products. For instance, waxy corn starch, noted for its high amounts of amylopectin, was
discovered in China in the early 1900s and brought to the United States. It wasn’t until the
1940s that geneticists in Iowa developed this into a high-yield hybrid. As of 1996, an estimated
600,000 acres of waxy corn was grown in the United States (Schwartz & Whistler, 2009). Other
types of starches include high-amylose corn starch (which has an amylose content range
between 50% to 80% depending on the cultivar and differs from regular corn starch that
contains roughly 25% amylose) and is used primarily by candy manufacturers. Finally,
chemically modified starches are designed to give certain foods improved shelf life, texture, as
well as heat and acid stability (Schwartz & Whistler, 2009).
Prior to heating, starch is insoluble and can only absorb a small amount of water.
Processing causes starch granules to swell and solubilize to different degrees, depending on the
amount of heating and mechanical shearing (Mason, 2009). However once starch is heated,
gelatinization begins. Starch gelatinization can be defined as the transition of starch granules
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from an ordered state to a disordered one, where the semicrystalline nature of their structure
is reduced and a viscous solution is formed (Ratnayake & Jackson, 2008).
Starch can play multiple different roles depending on the food system. For example, in
battered or breaded foods, starch can act as an adhesive, while in fried and baked foods starch
can be used to enhance crisping. In beverages and creamers, starch provides emulsification and
stabilization. In cakes and meats, starch can be used to enhance moisture retention, and in
gravies, pie fillings, and soups, it can be used as a thickener (Mason, 2009). Starch thickening is
caused by starch granules growing due to thermal processing. Potato, tapioca, and modified
waxy maize are the most used thickeners due to their comparative stability to textural changes
(BeMiller & Whistler, 2009).
1.2: Salivary Composition
Saliva is a clear, mildly acidic mucoserous secretion that is a mixture of fluids from
various salivary glands (Humphrey & Williamson, 2001). Saliva is produced by three primary
glands, the parotid, submandibular, and sublingual glands – as well as numerous minor glands
(Rohleder & Nater, 2009). It is a dilute liquid that is composed of over 99% water, normally has
a pH between 6-7, and contains many components including, but not limited to bicarbonates,
phosphates, proteins, mucins, calcium, and immunoglobulins (Humphrey & Williamson, 2001).
Salivary amylase is found in parotid saliva as well as palatine secretions (Humphrey &
Williamson, 2001). A major role of salivary -amylase is to attack the two macromolecules,
amylose and amylopectin, and break the (-1,4) linkages found in them, converting the
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amylose into maltotriose, maltose, and glucose, and amylopectin into glucose and -limit
dextrins (Smith & Morton, 2010b).
1.3: Alpha Amylase Production and Purpose
α-amylase is the primary digestive enzyme found in human saliva (Smith & Morton,
2010a). Critically, it splits the glycosidic linkages found in amylose and amylopectin, thereby
beginning the process of starch digestion while food is still in the mouth. α-amylase continues
to act on the food even after it has entered into the stomach. This enzyme eventually stops
functioning when the pH becomes too low due to the gastric acid in the stomach. Pancreatic
amylase then takes over the process of starch digestion when the food enters into the small
intestine (Smith & Morton, 2010b).
In the mouth, higher amounts of α-amylase has been shown to have noticeable effects
on texture perception of starch thickened custards including decreased thickness, creaminess,
and fatty after feel (de Wijk, Prinz, Engelen, & Weenen, 2004). In addition to influences on
texture perception, high α-amylase activity has been correlated with reduced vanilla flavor
sensation, possibly due to an increased breakdown of the custard leading to reduced surface
area and decreased flavor release (Engelen et al., 2007). In addition to texture and flavor
differences, increased amylase has been shown to accelerate the release of volatiles from
starch solutions (Ferry, Hort, Mitchell, Lagarrigue, & Pàmies, 2004). Model systems have also
shown that α-amylase effects different starches at different rate, with waxy maize starch
reducing in viscosity and releasing volatiles quicker and to a greater extent than wheat starch
(Ferry et al., 2004). In human psychophysical studies, individual differences in salivary α-
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amylase have also been shown to play a role in starch hydrolysis as well as taste perception of
starch (Lapis, Penner, Balto, & Lim, 2017b).
Amylase is produced by the epithelial acinar cells of the exocrine salivary glands
(Rohleder & Nater, 2009). These same cells produce the majority of the liquid component of
saliva and provide this liquid with salivary proteins, like amylase. While there are several
different glands that produce saliva (i.e. the parotid, submandibular, and sublingual glands),
these glands differ in how much amylase they produce, with the parotid gland being
responsible for roughly 80% of the amylase production (Rohleder & Nater, 2009). In the mouth,
salivary α-amylase is regulated by parasympathetic nerves in several ways: a) through α-
amylase being released from parasympathetically innervated glands (namely the palate and
sublingual), b) salivary α-amylase secretion through the sympathetic system is amplified by
parasympathetic activity, and c) increased salivary flow rate heightens the concentration of α-
amylase as its secretion from acinar cells are stimulated (Bosch, Veerman, de Geus, & Proctor,
2011). Parasympathetic innervation is when cranial nerves stimulate salivary glands and leads
to increased secretion of salivary fluid. Stimulation of sympathetic nerves leads to a short-term
effect where salivary glands release saliva particularly high in α-amylase in addition to other
stored proteins (Smith & Morton, 2011). Augmented secretion is when parasympathetic and
sympathetic stimulations secrete more amylase then either produce separately (Proctor &
Carpenter, 2007).
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1.4: Copy Number
In humans, copy number variation (CNV) of the salivary amylase gene (AMY1) is
positively correlated with levels -amylase found in saliva (Mandel, des Gachons, Plank,
Alarcon, & Breslin, 2010; Perry et al., 2007). Copy number variation can be measured both with
fibre-FISH and qPCR (Carpenter et al., 2015). In a foundational study, Perry and colleagues
(2007) reported that human populations have historically had a large variation in dietary starch
intake, and when these populations are divided into high and low starch consuming
populations, the AMY1 copy number differs significantly between the two populations,
suggesting the possibility of positive natural selection for the CNV. Specifically, European,
Japanese, and Hadza hunter gatherers who consume high amounts of starch rich foods were
shown to have at least 6 AMY1 copies, at a rate nearly twice that seen in the low starch
population group, which consisted of Biaka and Mbuti hunter gatherers, Datog pastoralists, and
Yakut pastoralist/fishers. The AMY1 gene is also expressed in other nonhuman animals and
there is evidence that the gene copy number increases in mammals with diets high in starch
such as dogs, pigs, and rats (Pajic, 2019). In humans, subsequent work showed that high copy
number variation correlated with faster and significant decreases in perceived starch viscosity
both in vivo and in vitro (Mandel et al., 2010). It should be noted that while AMY1 copy number
is significantly correlated with salivary amylase production, more recent data suggest it does
not explain the majority of observed variation between people (Carpenter, Mitchell, & Armour,
2017).
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Figure 1-2 Diet and AMY1 copy number' (a) shows a comparison of the copy number distribution between high and low starch diets. (b) a cumulative distribution plot of copy number for the study populations. Figure taken from Perry (2007)
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1.5:Salivary Flow
Salivary flow is a measure of the rate that saliva is produced in an individual’s mouth.
There are two common ways to measure salivary flow: stimulated and unstimulated.
Unstimulated saliva is secreted continuously in the absence of any external stimulation. This is
contrasted to stimulated saliva which occurs when saliva is secreted as a response to
masticatory or gustatory stimulation (Mackie & Pangborn, 1990). Unstimulated salivary flow
rates in healthy individuals are on average .3-.4 ml/min while stimulated flow rates average
closer to 1.7 ml/min (Edgar, 2012).
Multiple factors that influence unstimulated salivary flow rate have been identified,
including hydration levels, body position, light exposure, and circadian rhythm. When it comes
to stimulated salivary flow, the main additional stimuli are mechanical (dealing with the process
of chewing and manipulating objects in one’s mouth) and gustatory (dealing with smells, tastes,
or other sensory stimuli) (Edgar, 2012). When measuring flow rates in human participants, it is
important to standardize as many of these factors as possible: e.g., collecting saliva at the same
time each day, avoiding collection soon after eating, making sure everyone is properly hydrated
before testing, and that lighting is consistent between sessions. Relative to water, mastication
of parafilm has been shown to increase salivary flow rate and amylase secretion, and
mastication of food increases salivary flow even more than parafilm. Additionally, as salivary
flow increases due to mastication, so does amylase secretion, so even as salivary flow rate is
increases, amylase concentration in the saliva stays roughly constant (Mackie & Pangborn,
1990).
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When collecting stimulated and unstimulated saliva there are several collection
methods that have different pros and cons depending on what the researcher is trying to
measure. In a review summarizing the benefits and drawbacks of various collection methods,
(Navazesh, 1982) describes the four most common approaches: draining, spitting, suction, and
absorbent material. Draining involves the participant tilting their head to allow saliva to flow
freely from between their lips into a test tube without additional stimulus. Spitting is similar to
draining, except that participants collect the saliva between their lips and then expectorate as
needed. Suction involves the experimenter placing a plastic tube connected to a vacuum pump
under the participants tongue and secretions being vacuumed into the waiting test tube.
Finally, the absorbent material approach involves participants placing dental cotton rolls into
their mouths under their tongue near their sublingual glands and two others in their upper
vestibules near their parotid glands. At the end of the trial the swabs are removed and weighed
(M. Navazesh & Christensen, 1982).
Gustatory stimulation can be brought about by citric acid solutions placed on the tongue
or continuously infused into the mouth (Edgar, 2012)(Navazesh, 1982) inducing a stimulated
salivary flow. Mechanical stimulation typically uses inert gum bases, sugar free candy, or
parafilm, which are chewed to stimulate salivary flow and then expectorated along with the
saliva at the end of testing (Mahvash Navazesh & Kumar, 2008). While each stimulus has
different pros and cons regarding ease of testing, change in salivary flow is consistent on a
relative basis between different stimuli. In other words, a participants flow rates remain
consistent in relation to other participants when multiple collections are run despite their
personal flow rates changing between tests (Gavião, Engelen, & Van Der Bilt, 2004).
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Recent work on stimulated salivary flow suggests that salivary flow is also affected by
cognitive factors and is not merely a reflex. Participants who were given the same stimulus (a
ready to drink tea) solution but told it was a different stimulus (rabbit hair extract, or tea)
expectorated different amounts of saliva, suggesting salivary flow may involve top down
processes (Running & Hayes, 2016).
1.6: Personality traits that may relate to food choices: Sensation and Variety Seeking
The concept of sensation seeking was first developed in 1979 by Marvin Zuckerman with
help from Hans and Sybil Eysenck (Zuckerman, Eysenck, & Eysenck, 1978). Zuckerman described
the trait as the need that people have to new, complex, and diverse sensations as well as the
willingness to take both social and physical risks to obtain such experiences (Zuckerman, 1979).
In subsequent years it was applied to many different behaviors, including drug use (Satinder &
Black, 1984), as well as alcohol use (Schwarz, Burkhart, & Green, 1978). It remains widely used
even today, but in 1992, Jeffrey Arnett published a new instrument to measure the same
underlying trait, to overcome flaws with the earlier questionnaire. First, Arnett did away with
the forced choice responses that made it difficult for responders who felt that both or neither
of the options applied to them. Second, some items dealt with strenuous activities like skiing or
mountain climbing, raising the question if differences in responses were due to sensation
seeking or simply physical strength. Third, some items were outdated (e.g., “I would like to
make friends in some of the ‘far-out’ groups like artists or ‘hippies’”). Finally, many items on
the scale explicitly asked about drug and alcohol use, as well as sexual behavior. As these
questions dealt directly with the kinds of behaviors that the scale was being used to study, it is
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possible that that the relationships between the sensation seeking and behaviors like drug and
alcohol use were merely artifacts due to inclusion of questions about these same
behaviors.(Arnett, 1994). Thus, Arnett described the guidelines that influenced his updated
scale: it should focus on novelty and intensity, be constructed in a graded agree/disagree
format, not contain age or strength related items, and should not contain illegal or other norm-
breaking behavior (Arnett, 1994). Sensation seeking, as measured with Arnette’s Inventory (the
AISS), has been correlated with the liking of both spicy meals and the burn from spicy foods as
well as yearly intake (Byrnes & Hayes, 2016).
As a trait, variety seeking is based on the concept of an Optimal Stimulation Level, which
states that "individuals need a certain idiosyncratic level of stimulation in their lives to function
effectively. Whenever the actual level of stimulation does not correspond with the OSL,
individuals are hypothesized to engage in stimulation-regulating behavior (exploratory
behavior) to restore the correspondence” (Van Trijp & Steenkamp, 1992). Van Trijp created the
Variety Seeking Scale (VARSEEK) in order to study OSL specifically in relation to a consumer’s
desire for variety in food consumption. As with sensation seeking (AISS), variety seeking
(VARSEEK) has been shown to correlate with spicy food preference (Nolden & Hayes, 2017).
Other personality traits besides sensation and variety seeking have also been shown to
correlate with food liking; specifically, high sensitivity to reward, low food neophobia, low
sensitivity to disgust all were associated with preferring burning food samples (Spinelli et al.,
2018).
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1.7:Mouth Behavior
Mouth behavior is a hypothesized framework for understanding a possible major driver
of food choice, satisfaction and desire. First described in 2015, it states that individuals have
preferred ways to manipulate food in their mouths, and that it is this preference, rather than
the texture of food per se, that is a primary driver of food preference (Jeltema, Beckley, &
Vahalik, 2015). Jeltema and colleagues hypothesize that people can be divided into four groups
depending on their preferred way of manipulating food in their mouths. These are: Crunchers,
Chewers, Suckers, and Smooshers. Crunchers are forceful in their bite and prefer foods that
fracture upon biting. Chewers prefer foods that can be chewed for longer periods of time and
do not fracture upon biting. Suckers prefer harder foods that can be sucked on for a longer
time. Finally, Smooshers prefer soft foods such as puddings that do not require much mouth
activity but can be held in the mouth for a long time (Jeltema et al., 2015).
Subsequent research (Jeltema, Beckley, & Vahalik, 2016) indicated Mouth Behavior
groups had clear differences between each other when told to rate their liking of a variety of
food products. In addition, those authors argue that participants try to manipulate products
into a texture that could be eaten in a way they most desired that aligns with their Mouth
Behavior group. However, other, recent research suggests that neither texture liking, nor
certain physiological measurements (e.g., high bite force or high salivary flow rate) predict
membership any of the mouth behavior groups (Kim & Vickers, 2019).
1.8:Conclusions
The goals of this thesis can be broken down into two main parts. In the first study I
looked to see if Mouth Behavior type is influenced by salivary amylase, sensation, or variety
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seeking. In the second study, my goals can be broken down into several parts. First, does copy
number variation correlate with salivary alpha amylase activity? Secondly, how do salivary flow
and amylase activity affect the breakdown of pectin and cornstarch thickened yogurts in human
trials? Other work has been done on how amylase breaks down starch solutions and other
model systems, but more work needs to be done on foods that people eat and how they eat it.
Third, do differences in yogurt breakdown, amylase, or time to swallow affect liking? Finally,
what effects, if any, do salivary flow and amylase activity have on breakdown, cohesion, and
liking of a solid food, like a pretzel?
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Chapter 2: Salivary and personality differences as potential explanations for different mouth
behavior styles
2.1: Introduction
Jeltema and colleagues report individuals can be grouped into one of four preferred
‘mouth-behavior’ styles (Jeltema et al., 2016). The Jeltema Beckley Mouth Behavior (JBMB)
tool, an online questionnaire, sorts people into different mouth behavior groups, either
Crunchers, Chewers, Suckers, or Smooshers. In their classification scheme, Crunchers consist of
people who like to use their teeth to break foods and are more forceful in their bite, while
Chewers like foods that can be chewed longer and don’t fracture upon biting. Suckers prefer
foods that are manipulated in the mouth between the tongue and the roof of the mouth and
can be sucked on for an extended period of time, and Smooshers prefer soft foods that don’t
require much oral processing and would fill the mouth (Jeltema et al., 2016). Reportedly these
Mouth Behavior groups vary dramatically in regards to how they interact with food. Other
research by the same authors suggests that individuals in different Mouth behavior groups will
eat their food differently in order to create a texture that suits their Mouth Behavior (Jeltema,
Beckley, Vahalik, & Garza, 2020). Additionally, it is possible to discriminate a participants mouth
behavior group based on how their chewing changed between different foods at different
times with 68% accuracy (Wilson et al., 2018). Despite the evidence supporting Mouth Behavior
as a useful sorting tool, other research has suggested that a participants Mouth Behavior group
influences participant liking and perception less than one might expect. One study showed that
a participants mouth-behavior group is not predicted by texture liking or oral physiological
measurements (Kim & Vickers, 2019), which one would not expect if mouth behavior is largely
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measured using foods with very different textures. Additionally, preferred Mouth Behavior
does not necessarily relate to preferred oral texture perception (Cattaneo, Liu, Bech, Pagliarini,
& Bredie, 2020).
Previous work suggests differences in amylase activity may influence texture perception
(de Wijk et al., 2004; Mandel et al., 2010). Additionally, texture plays an important role when it
comes to product liking and rejection (Bridges, Smythe, & Reddrick, 2017). As the mouth
behavior task relates to preference of certain types foods, amylase activity may be a possible
explanation for mouth behavior classification.
Finally, behavioral measures of risk taking and sensation seeking were shown to possibly
reflect motivations for the liking of spicy foods (Byrnes & Hayes, 2016). Previous work has
shown that willingness to try different foods and sensation seeking (using VARSEEK and AISS to
measure) as well as low food neophobia (the avoidance or fear of new foods) in diet is
associated with capsaicin liking and intake (Byrnes & Hayes, 2016) (Nolden, 2017). Here, I was
interested in looking into whether or not these behavioral measures like sensation seeking or
variety seeking might help explain differences in JBMB groups.
The first hypothesis of this study is that there is a correlation between Mouth Behavior
styles and personality traits, such as variety seeking and sensation seeking. Additionally, I also
hypothesized individuals in different mouth behavior styles rate will rate the liking of starch or
pectin thickened foods differently.
2.2:Materials and Methods
Participants (n=103) were recruited from the Penn State campus and the surrounding
community. They had previously responded to an online test screener using Compusense Cloud
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(Guelph, ONT), sent to participants on an email listserv; the exclusion criteria included
participants with problems with taste or smell, smoking, difficulties swallowing, piercings,
dental work within the past month. Once participants came to the laboratory, they provided
informed consent via a click through question on the computer. All procedures were exempted
from Institutional Review Board review by professional staff in the Penn State University Office
of Research Protections under the wholesome foods/approved food additives exemption in 45
CFR 46.101(b). Participants were paid a small token incentive for their time.
Participants sat in booths under northern daylight illuminant (5k LED) with a computer
running the test in Compusense Cloud (Guelph, ONT). Compusense Cloud is a software that
facilitates the creation and running of computer-based testing. After providing consent,
participants were instructed via writing on the screen to put two mini pretzels (Hanover Butter
Snaps; Hannover, PA) in their mouth and start a timer on their computer. They then started
chewing as normal until they were ready to swallow, at which point they swallowed and
marked their completion time by stopping the timer on their computer. Participants then rated
the pretzels on a 9 point hedonic scale (Peryam & Pilgrim, 1957), where 1 was labelled Dislike
Extremely and 9 was labelled Like Extremely. Following this, participants completed the Arnett
Inventory of Sensation Seeking (AISS) (Arnett, 1994).
Participants then performed a different test with cornstarch or pectin thickened yogurts
that followed the same general procedure. For this test, they put a spoonful of yogurt in their
mouth, eating it as they would normally, and marked how long it took for them to swallow
using a timer on the computer. After swallowing, they rated liking on a 9-point hedonic scale.
Presentation orders for the cornstarch and pectin thickened yogurts were counterbalanced so
17
half of the participants sampled cornstarch first, followed by pectin yogurts, while the other
half the opposite. In between the two yogurt samples, participants completed out the variety
seeking tendency questionnaire (VARSEEK) (Van Trijp & Steenkamp, 1992). After the VARSEEK
questionnaire was completed participants went on to eat, time, and rate the yogurt then had
not sampled previously. After the second yogurt, they finished by completing the Jeltma
Beckley Mouth Behavior (JBMB) typing tool online and reporting their resulting group
classification into a box in Compusense.
The VARSEEK questionnaire ask participants 6 questions that they rate on a 5-point scale
from “Completely Disagree” to “Completely Agree”. The AISS questionnaire asks 20 questions
on a 4-point scale from “Describes me very well” to “Does not describe me at all”. The JBMB
tool is a proprietary tool meant to asses a participants Mouth Behavior Group. Questions
include asking about a participants favorite and least favorite types of food, texture
preferences, and demographics.
2.3:Results
The mean chew time of each sample was calculated in seconds. Pretzel chew time had a
(M =18.70 sec, SD=7.20). Pectin thickened yogurt was (M = 3.63 sec, SD= 2.23) and cornstarch
thickened yogurt had a (M =3.64 sec, SD = 2.68). A paired t-test comparing the means of the
two yogurts failed to find any evidence of significant difference between the two conditions
(t(197)=.04, p=0.97). AISS scores (M = 54.771, SD = 7.396) were roughly consistent with scores
for another study published in 2016 drawn from the same participant pool (M=52.7, SE ±0.8)
18
(Byrnes & Hayes, 2016). VARSEEK scores (M = 20.91, SD = 5.15) were higher however when
compared another test run at Penn State in 2017 (M=16.4, SE±0.5) (Nolden & Hayes, 2017).
Table 2-1 Table of Jeltema/Beckley Mouth Behavior counts and Percentage
Mouth Behavior Count Percentage
Cruncher 34 32%
Chewer 54 51%
Sucker 2 2%
Smoosher 15 14%
To measure differences between Mouth Behavior classification and speed at which
participants chewed the yogurt or their behavioral traits, I used separate one-way ANOVA
models. I found that Mouth Behavior types were not significantly different in regards to
participant’s VARSEEK scores [(F(3,101) = 1.84, p=0.145], total AISS scores [(3,101) = 0.5,
p=0.683], pectin time to swallow, [F(3,98)=0.58, p=0.632], or cornstarch time to swallow
[F(3,99) = 0.02, p=0.995].
Looking at the histograms of the time to swallow for the different samples, we can see
that both the pectin and cornstarch thickened yogurts are highly right skewed, suggesting the
mean is higher than the median.
19
Figure 2-1 Histogram of cornstarch yogurt time to swallow
Figure 2-2 Histogram of pectin yogurt time to swallow
20
Next, I ran a correlation to see how much speed in chewing one yogurt correlated with
the other. As one might expect, they were highly correlated with (r=0.60, P<.001), suggesting
eating time might be a stable trait within a person across samples. Pretzel time to swallow was
not correlated with cornstarch yogurt time to swallow (r=0.141, P=0.173) but it was slightly
correlated with pectin yogurt time to swallow (r=0.203, p=0.05) I also tested for a correlation
between the two personality measures – AISS and VARSEEK –and they were moderately
correlated (r=0.42 p<.001).
Figure 2-3 Correlation between Pectin and Cornstarch yogurts time to swallow in seconds
21
2.4: Discussion
Here, I failed to find any relationship between Mouth Behavior groups and breakdown
speed of food inside of participants mouths. I think that this is due to some faults in our
methods that made it difficult to accurately measure these differences between people. First,
by using something like yogurt which naturally breaks down very quickly within the mouth, I
made it so that the time was just too brief to get an accurate rating. Previous work has been
done with much thicker starch solutions (Mandel, 2010) that took much longer to break down.
Figure 2-4 Correlation between AISS and VARSEEK
22
By instructing participants to chew and swallow as they would normally, this introduced too
large an amount of variance. The goal was to measure if a participant’s food breakdown rate
related to their mouth behavior. However, I failed to control for participants different eating
habits, such as not chewing the yogurt and simply swallowing it immediately. Studies have
shown that the rate individuals eat is highly consistent between meals of similar foods
(McCrickerd & Ford, 2017). For example, participants who eat thin porridge quickly are likely to
eat thick porridge quickly and vice versa. By having participants eat yogurts thickened with
either cornstarch or pectin, I hoped that differences in how quickly they finished would be due
to the amylase in their saliva. Additionally, as the pectin thickened yogurt started at a thicker
initial viscosity, we cannot say that the difference in mode and the angle of the slope in figure
2.3 is due to amylase activity. Second, by stirring the yogurt before distributing it to our
panelists, I may have unintentionally disturbed the structure that we were expecting the saliva
to break down, further decreasing the time each participant needed to chew the yogurt until it
was ready to swallow. In future studies, it may be a good idea to have participants mash their
yogurts in a consistent manner for there to be less of a variation due to differences in chewing
and swallowing behavior. A person’s Mouth Behavior group does not seem to influence the rate
at which they consume yogurt.
No effect was found regarding personality effects on Mouth Behavior type. This is likely
due to neither variety nor sensation seeking being dominant in any particular Mouth Behavior
group. Studies have shown how these personality measures influence the liking of chili
containing foods (Nolden & Hayes, 2017) (Byrnes & Hayes, 2016), however none of the 4 types
23
of Mouth Behavior (Smoosher, Sucker, Cruncher, Chewer) seem to be influenced by the
personality traits we studied in our participants.
2.5:Conclusions
The time it takes for participants to finish a spoonful of yogurt may be dependent on
eating styles different from eating preferences studied in the mouth behavior task. Participants
who reported to prefer foods that can be smooshed did not eat their spoonful of yogurt any
quicker than participants who rated themselves as preferring crunchy, chewy, or suckable
foods. The effect of amylase and salivary flow on swallow rate was not able to be measured due
to errors in data collection. However, this does however bring into question how much of an
effect α-amylase has on foods in the mouth when many participants are likely to swallow
yogurts after less than a second. In all prior literature, participants are instructed to hold the
sample in their mouth for a set period of time, which is important for looking at the effect of
amylase when starch is held in the mouth for 5 seconds (de Wijk, 2004), but not as important if
the majority of participants swallow in half that time. Mouth Behavior type does not seem to be
influenced by sensation or variety seeking behavioral measures. Further work needs to be
done to test participants in a more controlled manner.
24
Chapter 3: Amylase activity and its effects on the breakdown and perception of starch
thickened foods
3.1: Introduction
Starch is a polymer made of glucose. Salivary amylase is an enzyme secreted in the
mouth that can break long starch chains down into smaller fragments. This process begins
when people chew their food. Amylase activity varies between people due to multiple factors,
including, salivary flow, age, and most importantly copy number variation (Nater & Rohleder,
2009). Salivary α-amylase is encoded by the AMY1 gene. Copy number variation of the AMY1
gene reportedly ranges from 2 to 15 copies of the AMY1 gene (Mandel et al., 2010). These
differences may potentially be important for dietary behavior and food preferences; previously,
it has been shown that salivary amylase concentration is correlated with having more copies of
the AMY1 gene, potentially due to selection pressure (Perry et al., 2007). Anthropological data
suggests AMY1 copy numbers are higher in groups whose ancestors traditionally consumed a
high starch diet, as compared to low starch consuming hunter gatherers or fishing peoples
(Perry et al., 2007). This would suggest that the increased abundance of starchy foods would
lead to a selective pressure to digest it more efficiently. While the genetic variation of alpha
amylase has appeared to evolve independently in various populations across the globe, and
may relate to diet, whether such variation influences food sensations and preferences in a
modern food environment remains underexplored. Earlier studies have shown that individuals
are able to taste glucose polymers (like starch) (Pullicin, Penner, & Lim, 2017) but however
responsiveness did not differ with α-amylase activity (Lapis, Penner, & Lim, 2014). Later studies
have shown however that high α-amylase activity has been shown to correlate with starch
25
detection in cooked starch solutions (Lapis, Penner, Balto, & Lim, 2017a) but not raw solutions.
Conceivably, faster state change in the mouth may lead to increased liking for these foods (i.e.,
the theory of high dynamic contrast; see (Hyde & Witherly, 1993). Hyde and Witherly describe
dynamic contrast as how foods that are considered highly palatable are more likely to have
higher moment-to-moment sensory contrast or texture change. However, it also needs to be
noted that the majority of amylase variation comes from other sources beyond AMY1 CNVs
(Carpenter et al., 2017), which calls the ecological relevance of earlier reports (Mandel, 2010)
into question.
The present study was designed explore differences across people, in terms of the time
it takes for them to break down starch thickened food in the mouth, their liking of starchy
foods, and whether these relate to the amount and activity of alpha-amylase in their saliva. I
hypothesized that people with higher CNVs of AMY1 would produce more amylase, and
increased amylase activity would break down starchy foods more quickly, resulting in higher
liking of starchy foods in these individuals.
3.2:Materials and Methods
Participants (n=71; 15 men, 56 women) were recruited from the Penn State campus and
the surrounding community. They had previously responded to a screener and had been
screened for a lack of problems with taste or smell, smoking, difficulties swallowing, piercings,
dental work within the past month as well as a willingness to provide saliva samples, chew and
expectorate food, and fast from food and alcohol the morning prior to testing. Procedures were
approved by the local Institutional Review Board ID00010138; written informed consent was
obtained, and participants were given a small cash incentive for their time.
26
Participants were seated in an open room within the test facility with blinders between
them so that they would not be able to observe other participants. The researcher overseeing
data collection sat in front of the participants to ensure that test procedures were properly
followed.
Participants were asked to chew on a 1x1 inch square of parafilm for 2 minutes, using a
metronome (set to 40 beats per min) to ensure a fixed chewing rate. Prior work has shown that,
on average, people naturally chew at a much faster rate than this: 86.4 bpm ± 14.4bpm (Farooq
& Sazonov, 2016). However, this test was run at slightly less than half that speed to ensure that
participants would not have any trouble chewing at that rate consistently for an extended
period of time. Participants chewed for 5 min, spitting their saliva into the collection tube as it
filled up. Their saliva was collected in a centrifuge tube (and frozen at minus 20°C for later
analysis). Next, they were given two commercially available foods: yogurt thickened with pectin
(Mountain High, Carson, California) and yogurt thickened with cornstarch (Yoplait, Minneapolis,
Minnesota). They first chewed each ad libitum, swallowed, and rated each for liking on a 9-
point hedonic scale (Peryam & Pilgrim, 1957), rinsing with water in between. Presentation
order was counterbalanced across participants. Participants then took a 5-min break.
After the break, participants placed a spoonful of yogurt (thickened with either pectin or
cornstarch) and pressed it to the roof of their mouth (in time with a metronome, at 40 beats
per min). They were instructed to do this until the yogurt in their mouth reached a water like
consistency. They then expectorated this yogurt into a cup in front of them. This was repeated
5 more times until all samples were sampled (2 types of yogurt x 3 replicates for each), with a 2-
min break between each. For each sample, the time required to reach water like consistency
27
was recorded by the panelist on a tablet computer (Apple iPad Air, 9.7-inch display, Apple Inc.,
Cupertino, CA) in front of them; the 3 yogurt samples in each rep were pooled for viscosity
measurement.
Immediately after finishing their yogurt, yogurt expectoration samples were collected by
research staff, and an amylase inhibitor (0.1M acetate buffer composed of 9.82mL of 0.1 M
acetic acid and 0.177mL of 0.1 M sodium acetate) was added within one minute in order to
inhibit the further breakdown of any starches. Participants then took another 5-min break.
Finally, participants were given 3 mini pretzels (Snyder’s, Hanover, Pennsylvania), and
asked to chew at fixed rate (again, with a metronome set to 40 bpm), spitting the pretzels out
into a weight boat after 1 min. Photographs of the expectorated bolus were taken within 1
hour.
Following sample collection, the pooled expectorated yogurt was run through a Zahn
Cup (size 2) to measure the viscosity of the samples (Abbas, Abdulkarim, Saleh, & Ebrahimian,
2010). All samples were measured on the same day, and in the order as collected.
Saliva samples were first lightly centrifuged to remove air bubbles and measured to
obtain salivary flow rate before being frozen for storage. Later, DNA was extracted from
thawed saliva samples using a QIAamp DNA Mini Kit (ID:51304). Then the extracted DNA was
quantitated using a NanoDrop machine and standardized to 5ng/ul. Reactions were then run
through a real time PCR in triplicate in order to obtain copy number variation. The PCR ran for
one cycle at 95ºC for 2 minutes to start. Then it ran for35 cycles with three steps to 95ºC for 30
seconds to denature, 55ºC for 30 seconds to anneal, and finally 72ºC for 30 seconds to extend
the DNA polymerase. Finally, it ended the process at with one cycle at 72 ºC and dropped to 4
28
ºC upon completion. Additionally, saliva samples had their Alpha amylase levels measured
through a Salimetrics Salivary Alpha-Amylase Assay Kit (Salimetrics, State College, PA) following
the manufacturer’s instructions. The only modification was that I increased the dilution factor
from 1:200 to 1:400. This was done as the original dilution factor led to results that were too
high for the light spectrophotometer to properly measure.
The photographs of the expectorated pretzels were loaded into Compusense and
evaluated by research staff and other lab members (n=8) on a line scale to determine degree of
cohesiveness; the scale was anchored from least (1) to greatest (9). Cohesiveness was defined
for the raters (8 research staff and other lab members) with verbal definitions: low
cohesiveness was defined as appearing to be less digested with clearly recognizable pieces of
pretzels, while high cohesiveness was defined by having a more uniform consistency,
appearance, and texture. The group mean rating for each photograph was used as a variable in
subsequent analysis.
3.3:Results
Data from four participants were removed from all data analysis: two were removed for
not following testing procedure properly and two were outliers who chewed the yogurts
substantially longer than other participants, leaving a total of 67 participants in the dataset.
Copy number variation in the AMY1 gene was assessed for 60 participants, as 7 additional
participants had DNA samples that could not be extracted at a high enough purity for further
analysis. DNA purity was defined as a 260nm/280nm ratio of 1.8 on a NanoDrop
Spectrophotometer, consistent with standard guidelines (NanoDrop One User Guide, 2016). The
29
AMY1 copy number distribution is shown in Fig. 3-1 and has a range of 1-13 with a modal copy
number of 7 and 8. This matches expectations from previous research which had similar ranges
1-11 (Mandel et al., 2010), 2-18 (Carpenter et al., 2015), 2-15 with a mode of 6 (Carpenter et
al., 2017).
Figure 3-1 AMY1 copy number variance
I then looked at salivary amylase enzyme activity. As multiple freeze thaw cycles can
degrade amylase activity, samples that took more than three cycles for processing were
excluded from data analysis. Amylase activity was measured and was consistent with previous
studies in exhibiting considerable variation in amylase expression (Mandel et al., 2010). The
amylase activity ranged between 4.5 and 164 U/ml, this distribution is shown in Fig. 3-2.
Contrary to my hypothesis, no correlation was observed between AMY1 copy number and
amylase activity; this is shown in Fig. 3-3.
30
Figure 3-2 Histogram of Amylase Activity
31
Figure 3-3 Correlation between AMY1 copy number and alpha amylase activity
Salivary flow had a clear right tailed skew, as shown in Fig. 3-4, with a range from 0.44 to
3.54 ml/min. Salivary flow (ml/min) and amylase activity (U/ml) were multiplied together in
order to get an estimate of the total amount of amylase (U/min) present in the mouth during
chewing. This calculated value (U/min) is called Total Amylase for the remainder of the
document. The distribution of Total Amylase values is shown in Fig. 3-5.
32
Figure 3-4 Histogram of salivary flow (ml/min)
Figure 3-5 Histogram of Total Amylase (U/min). Total amylase is a measure of the total amount of amylase that flows in a participants mouth as they produce saliva.
Next, I looked at what effects salivary flow had on the viscosity of expectorated yogurts.
I tested for correlations between salivary flow and the two yogurts: salivary flow was not
correlated with cornstarch yogurt expectoration viscosity (r=0.026, p=0.839), however it was
33
highly correlated with pectin yogurt expectoration viscosity (r=-0.959, p<0.001). These
correlations are shown in Figures 3-6 and 3-7.
34
Figure 3-6 Correlation between Salivary flow and cornstarch yogurt expectoration viscosity.
Figure 3-7 Correlation between Salivary flow and Pectin yogurt expectoration viscosity.
35
Next, I tested whether Total Amylase correlated with the time it took for participants to
spit out their yogurts. As Total Amylase increased, the time to spit (TTS) for cornstarch
thickened yogurts decreased (r=-0.472, p<0.001); however, this effect was not significant for
the pectin thickened yogurts (r=-0.189, p=0.140). This can be seen in Figures 3-8 and 3-9 below.
Figure 3-8 Correlation between Total amylase and the time to spit of cornstarch thickened yogurt
36
Figure 3-9 Correlation between Total amylase and Time to spit of pectin thickened yogurt.
Next, liking of the yogurts was analyzed as a function of time to spit (TTS). The apparent
negative correlation between TTS and liking of cornstarch yogurts Fig 3-10 was not significant
(r=-0.227, p=0.064). Conversely, there was a positive and significant correlation between TTS
and liking of pectin yogurts Fig 3-11 (r=0.339, p=0.005). A paired t-test was then run on the
liking values of both yogurts: the liking of the cornstarch thickened yogurts (M=7.16, SD 1.4)
was significantly higher than that that of pectin thickened yogurt (M=4.2, SD 2.3, t(69) = 8.57, p
<0.001).
37
Curiously, liking in Cornstarch is correlated with amylase activity in starch thickened
yogurts Fig 3-12 (r=0.304, p=0.021), but not pectin thickened yogurts Fig 3-13 (r=-0.127,
p=0.345).
Figure 3-10 Correlation between Time to spit and liking of cornstarch thickened yogurt
Figure 3-11 Correlation between Time to spit and liking of pectin thickened yogurt
38
Also, the TTS of both yogurts were compared and they are highly correlated (r=0.537,
p<0.001), as can be seen below in Fig 3-14.
Figure 3-12 Correlation between Liking and Amylase activity in cornstarch thickened yogurt
Figure 3-13 Correlation between Liking and Amylase activity in pectin thickened yogurt
39
Figure 3-14 Correlation between time to spit of both Cornstarch and Pectin thickened yogurts.
Finally, I looked at Pretzel ratings. Salivary flow and Pretzel Bolus ratings were highly
correlated (r=0.467, p<0.001), while Total Amylase and pretzel Bolus ratings are not correlated
(r=0.079, p=0.561). Pretzel liking was also not correlated (r=0.007, p=0.959) with Amylase
Activity.
40
Figure 3-15 Correlation between Salivary flow and pretzel bolus ratings
Figure 3-16 Correlation between Total Amylase and Pretzel Bolus ratings
41
3.4: Discussion
Looking at the effect of α-amylase activity on foods, it was correlated with liking effects
on starch thickened yogurt but not on the pectin thickened yogurt. This aligns with my
hypothesis that amylase would affect the breakdown of starch thickened foods while not
having an effect on semisoft foods thickened with a non-starch thickener. This finding also
supports research by Ferry and colleagues, which shows that starch thickened products have
better flavor and taste perception when compared to non-starch thickened products due to
amylase activity (Ferry et al., 2004). However, it should be noted that the pectin thickened
yogurt was not as well liked overall and may have led to null results as the liking range was too
small. Here, I also found that salivary flow influenced the breakdown rate of pectin thickened
yogurt, but not the starch thickened yogurt. This supports my hypothesis that salivary flow and
mixing contributing to the thinning of non-starch thickened foods while the starch thickened
yogurts were affected by the amylase, and salivary flow was not as important to starch
breakdown as total amylase was. Individuals with high amylase activity did not have an
increased breakdown rate of cornstarch thickened yogurts. This finding conflicts with previous
research showing the impact that amylase activity had on chewed custards viscosity (de Wijk et
al., 2004). They found that custards with added amylase had increased starch breakdown. My
result may be due to yogurts being much thinner than custards to begin with; additionally,
participants either stopping before their yogurts reached a water like viscosity or chewing for
too long after they had already reached that point. By not having a clear stopping point for
participants there was increased variability in the study due to participants expectorating at
different viscosities when they should have been the same.
42
It is also important to note that the commercially available yogurts used here were not
matched for viscosity. The two yogurts were selected from commercial products on the market,
based on the thickener used. Unfortunately, I did not anticipate that the pectin thickened
yogurts would be significantly thicker than the cornstarch thickened yogurts. Presumably, this
explains, in part, why the viscosity and time to spit was on average much lower for the
cornstarch yogurts (M 117.02 ± 97.9) when compared to the pectin yogurts (M 164.6 ± 97.9).
Here, I find that liking is positively correlated with time to spit for the pectin thickened
yogurts, however this effect is not seen in the cornstarch thickened yogurts. One possible
explanation for why this might be is that participants who disliked the pectin thickened yogurts
spat it out quicker, while the ones who liked it more were more willing to continue to chew it
for longer periods of time. However, I decided against this explanation, as Fig 3-10 also shows
that the time to spit for both yogurts was highly correlated, suggesting that time to spit is not
meaningfully influenced by a participants liking of the yogurt. Looking into liking further, I saw
that amylase activity highly correlated with liking, but only for the starch thickened yogurts and
not the pectin thickened yogurts. This provides more evidence for the role of dynamic contrast
(Hyde & Witherly, 1993) as a driving role in the liking of yogurt, in other words moment to
moment sensory contrast sensory contrast can lead to increased liking. For the pectin yogurts,
the longer the participants took to chew the yogurt the more they liked it. Conversely for the
cornstarch thickened yogurts, the higher the amylase, and therefore the quicker it broke down,
the more the participants liked it. This may not have been seen in the cornstarch thickened
yogurts, as they started off thinner and all participants were able to feel the dynamic contrast,
and participants with higher levels of amylase activity felt it the quickest leading to increased
43
liking. Additionally, the liking data may be explained as participants with higher amylase broke
down the starch quicker lead to more in mouth viscosity in the cornstarch thickened yogurts,
and would explain why amylase only had a liking effect in the starch thickened yogurts and not
the pectin thickened ones. This is supported by the custard studies showing similar results in
products with more comparable initial viscosities (Ferry et al., 2004).
When it comes to the pretzels however, despite being starch thickened, we do not see
any evidence for α-amylase having an effect on pretzel breakdown. From Figure 3.15, salivary
flow appears to be the main driver of pretzel bolus cohesion. The reason for this may be that
amylase has a harder time absorbing into the solid pretzel and salivary flow and breaking it
down.
Both the pretzel and cornstarch thickened yogurt were made with modified cornstarch.
We know that amylase doesn’t affect raw and cooked starch in the exact same way (Lapis,
2017). As the yogurt and pretzel are cooked in completely different ways with different
ingredients and different starch amounts it is highly likely that amylase didn’t affect them the
same way either.
There are possible explanations for why the alpha amylase didn’t correlate with the
participants copy number. First, population diversity is a potential explanation, as this study
used participants at Penn State University and demographics were not collected. We know that
CNV’s can vary with regional genetics (Mandel et al., 2010), and all our participants were
gathered from the Penn State area. Second, circadian rhythm (Rohleder, Wolf, Maldonado, &
Kirschbaum, 2006) and dietary induction has a known effect on amylase production with
amylase levels being decreased in the morning and increasing until the evening. While I tried to
44
control for these factors it is possible that I did not go far enough and more effort should have
been put into having participants on the same diet and rhythm prior to saliva collection. Finally,
a possible explanation is that other outside factors that were not screened for, such as stress or
depression, that are known to impact the production of salivary alpha amylase affected the
results (van Stegeren, Rohleder, Everaerd, & Wolf, 2006). After the study was designed, it was
reported that a participants copy number is only a minor contributor to variation in salivary
amylase (Carpenter et al., 2017). While further research in the effects of α-amylase on starch
perception and liking is warranted, trying to focus on the genetic component seems less than
worthwhile.
3.5:Conclusion
Here, salivary flow was shown to have no effect on the viscosity of starch thickened
yogurt expectoration viscosity. However salivary flow is shown to have a strong effect on the
viscosity of pectin thickened yogurt viscosity. Total Amylase was negatively correlated with the
time to spit of cornstarch thickened yogurt, something that is not seen in pectin thickened
yogurt. As the yogurts were not matched for thickness further research should be conducted of
foods that are commonly eaten by people while controlling for consistent initial viscosity.
The liking of pectin thickened yogurts is correlated with their time to swallow, this is not
seen in cornstarch thickened yogurts. Cornstarch yogurt liking is correlated with amylase
activity, this effect is not seen in the liking of pectin thickened yogurts. Finally, salivary flow is
highly correlated with pretzel bolus cohesion ratings, while Total alpha amylase is not.
While failure to control stress and circadian rhythm may have led to me not finding a
correlation between AMY1 copy number variation and α-amylase activity further evidence
45
(Carpenter et al., 2017) has shown that it may be less important to human production of
amylase than previously believed and further research should focus more on the effects of
amylase activity on foods eaten in a normal manner. In addition, future work will should be
done to look into the breakdown of yogurts when they have been matched for thickness.
46
Chapter 4: Overall Conclusions and Future Work
There were two main problems with how Experiment 2 – the amylase genetics study –
was run. Firstly, by having the yogurts be of different initial thickness it became difficult to
distinguish what differences were due to the participants, and what were brought about due to
different initial viscosities. Secondly participants in the future should not be expected to be able
to consistently stop when the yogurts have reached a set consistency without significant prior
training. Instead, I would suggest having all participants chew for a concrete set period of time
before stopping and expectorating. This would lead to a more accurate measuring of viscosity
and take out the need to measure time to spit. Care must be taken to ensure that the given
time does not lead to significant floor or ceiling effects however.
I did find evidence that amylase had an effect on the breakdown of starch thickened
foods, but not as much as one might expect. Additionally, it seemed to only have a significant
effect on foods that were already soft and where it could easy mix into. It did not have a
perceptible effect on the starchy pretzels that either it may not have been able to mix into or
was cooked in a way that made amylase breakdown more difficult.
I also found that salivary flow had a larger effect on foods than initially expected.
Salivary flow was highly correlated with both pectin yogurt viscosity and had very noticeable
effects on pretzel bolus cohesion.
Finally, no correlation was found between copy number and amylase activity, this was
possibly due to outside factors effecting salivary amylase production that were not screened
for. Another possible explanation is that amylase starts to degrade after repeated freeze thaw
47
cycles, and the three freeze thaw cycles that my samples went through may have been enough
to render the results unreliable.
Future work will need to be done on testing yogurts of the same thickness as well as
redoing the Amylase activity and copy number tests.
48
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Augustyn, A. (2019). Starch Encylopaedia Britannica. March 14, 2019: Encyclopaedia Britannica, inc.
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Appendix A: VARSEEK scale (Variety Seeking)
1. When I eat out, I like to try the most unusual items, even if I am not sure I would like
them.
2. While Preparing food or snacks, I like to try out new recipes.
3. I think it is fun to try out food items one is not familiar with.
4. I am eager to know what kind of foods people from other countries eat.
5. I like to eat exotic foods.
6. Items on the menu that I am unfamiliar with make me curious.
7. I prefer to eat food products I am used to.
8. I am curious about food products I am not familiar with.
The items are rated on a five-point Likert scale with all categories labelled, ranging from
completely disagree (=1) to completely agree (=5). Item 7 was recoded before analysis.
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Appendix B: AISS (Arnett Inventory of Sensation Seeking) For each item, indicate which response best applies to you: A) describes me very well B) describes me somewhat C) does not describe me very well D) does not describe me at all 1. I can see how it would be interesting to marry someone from a foreign country. 2. When the water is very cold, I prefer not to swim even if it is a hot day. (-) 3. If I have to wait in a long line, I'm usually patient about it. (-) 4. When I listen to music, I like it to be loud. 5. When taking a trip, I think it is best to make as few plans as possible and just take it as it comes. 6. I stay away from movies that are said to be frightening or highly suspenseful. (-) 7. I think it's fun and exciting to perform or speak before a group. 8. If I were to go to an amusement park, I would prefer to ride the rollercoaster or other fast rides. 9. I would like to travel to places that are strange and far away. 10. I would never like to gamble with money, even if I could afford it.(-) 11. I would have enjoyed being one of the first explorers of an unknown land. 12. I like a movie where there are a lot of explosions and car chases. 13. I don't like extremely hot and spicy foods. (-) 14. In general, I work better when I'm under pressure. 15. I often like to have the radio or TV on while I'm doing something else, such as reading or cleaning up. 16. It would be interesting to see a car accident happen. 17. I think it's best to order something familiar when eating in a restaurant. (-)
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18. I like the feeling of standing next to the edge on a high place and looking down. 19. If it were possible to visit another planet or the moon for free, I would be among the first in line to sign up. 20. I can see how it must be exciting to be in a battle during a war. Novelty subscale 1. I can see how it would be interesting to marry someone from a foreign country. 3. If I have to wait in a long line, I'm usually patient about it.(-) 5. When taking a trip, I think it is best to make as few plans as possible and just take it as it comes. 7. I think it's fun and exciting to perform or speak before a group. 9. I would like to travel to places that are strange and far away. 11. I would have enjoyed being one of the first explorers of an unknown land. 13. I don't like extremely hot and spicy foods. (-) 15. I often like to have the radio or TV on while I'm doing something else, such as reading or cleaning up. 17. I think it's best to order something familiar when eating in a restaurant. (-) 19. If it were possible to visit another planet or the moon for free, I would be among the first in line to sign up. Intensity subscale 2. When the water is very cold, I prefer not to swim even if it is a hot day. (-) 4. When I listen to music, I like it to be loud. 6. I stay away from movies that are said to be frightening or highly suspenseful. (-) 8. If I were to go to an amusement park, I would prefer to ride the rollercoaster or other fast rides.
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10. I would never like to gamble with money, even if I could afford it.(-) 12. I like a movie where there are a lot of explosions and car chases. 14. In general, I work better when I'm under pressure. 16. It would be interesting to see a car accident happen. 18. I like the feeling of standing next to the edge on a high place and looking down. 20. I can see how it must be exciting to be in a battle during a war. Scoring: Combine responses to items, with A = 4, B = 3, C = 2, D = 1, so that higher score = higher sensation seeking. For items followed by (-), scoring should be reversed.