The Effects of Prebiotic Fiber Inulin on Satiety and Gastrointestinal Tolerance and Prebiotic Properties of a Yeast Fermentate in Vitro

The Effects of Prebiotic Fiber Inulin on Satiety and Gastrointestinal Tolerance and Prebiotic Properties of a Yeast Fermentate In Vitro

A Dissertation SUBMITTED TO THE FACULTY OF UNIVERSITY OF MINNESOTA BY

Rachel Mottet

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

Joanne L. Slavin, PhD, RD, Advisor Marcia I. Endres, PhD, Co-advisor

August 2020

Acknowledgments

I first wish to express my thanks to my academic advisor, Dr. Joanne Slavin. I am extremely grateful for her support, guidance, and the opportunity that I had to be one of her graduate students. She kept the interests of myself and other students at heart and continuously presented our group with opportunities to grow professionally and academically. I would also like to thank my co-advisor, Dr. Marcia Endres, for going the extra mile to make sure I was getting the most from my program. Dr. Slavin and Dr.

Endres made a huge difference in my experience at the U of M and it was a true privilege to work with them both. Thanks to Dr. Susan Raatz, Dr. Rene Korzcak, and Dr. Kerry

Kuhle for playing a role in my academic success. Your time, feedback, and advice along the way were greatly appreciated.

I would like to thank my family members who have supported and encouraged me throughout my PhD program. To my amazing mother, Sue Zwick, and my incredible boyfriend, Yuan-tai Hung, words can hardly express how important you were to me throughout this journey. I am so grateful for your constant support. I will also never forget my first day back at school in my 30’s when my mom told me, “Be nice and you’ll make some new friends!” I suppose a mom never stops wanting her kids to be successful in making friends at school, no matter how old they are! Thank you for always being there for me.

Thank you to my fellow grad students who have been friends, supporters, and sounding boards through this shared challenge. Glenda Pereira, Rylee Ahnen, Michelle

DeBoer, Devan Catalano, Amanda Reiter, and Amanda Grev, I would not have wanted to go through this experience with anyone else!

Last, but not least, a huge thank you to those at Purina who were part of this journey. I would never have been able to do this without Kent Phalen, my former manager, who was instrumental in getting Purina on board with my academic aspirations.

Thank you, Kent, and Paul Homb, for giving me flexibility and support while I was on your team. To the equine nutritionist I have looked up to since day one, thank you Dr.

Karen Davison for being an amazing mentor and giving me great advice when I needed it most. To those who offered your friendship and support, thank you Angela Cramer,

Andrea Passe and Kaylyn Birchmeier. You have been amazing coworkers and friends!

Dedication

This dissertation is dedicated to all the academicians and equine nutritionists who have inspired me along the way. I have been fortunate to have had excellent professional mentors throughout my academic career and I thank all of those who guided and encouraged me.

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Abstract

The prevalence of chronic non-communicable diseases continues to rise in the

United States. Cardiovascular disease (CVD), type 2 diabetes, obesity, hypertension, and associated inflammatory conditions threaten the health and wellbeing of the population.

One dietary strategy to combat this trend is the promotion of dietary fiber intake. Dietary fiber offers numerous health benefits with consumption including the reduced risk of certain cancers and CVD, maintenance of healthy blood glucose levels, promotion of healthy bodyweight through enhanced satiety, and promotion of healthy blood lipid profiles. Prebiotics are a subcategory of fiber which offer all mentioned health benefits in addition to selective stimulation of beneficial gut bacteria, immuno-protective properties, attenuation of inflammatory pathways, and promotion of epithelial barrier integrity.

Inulin is an extensively studied prebiotic fiber and is most often seen as an isolated powder derived from chicory roots. Inulin is also found abundantly in Jerusalem artichokes, and to a lesser extent in onions, bananas, garlic, leeks, and wheat. Jerusalem artichokes offer a whole food fiber source which can be incorporated into meals or blended into beverages. However, little work has been done evaluating the gastrointestinal tolerance (GIT) and satiety properties of Jerusalem artichokes. Given a recent consumer interest of incorporating more healthful whole foods into the diet,

Jerusalem artichokes serve as an available, affordable, whole food vegetable offering a rich source of prebiotic fiber and health benefits upon consumption.

The objective of our first study was to compare two forms of inulin, an isolated powder and a whole food source (Jerusalem artichokes), and to identify their effects on

satiety and GIT when blended into a breakfast smoothie. We hypothesized that

Jerusalem artichokes would promote greater satiety and be better tolerated when compared to inulin powder. In a randomized, single-blind, crossover designed study, 26 participants (13 females, 13 males) fasted for 12 hours and then consumed a chocolate breakfast smoothie with inulin powder or Jerusalem artichoke puree mixed in. A plain smoothie without fiber was used as a control. The primary outcome was to observe satiety using a visual analog scale (VAS) to assess hunger, satiety, fullness, and then assess prospective food intake. The secondary outcome was to observe GIT through surveys probing the presence and severity of common adverse gastrointestinal symptoms.

Satiety was generally not different based on treatment group within four hours of smoothie consumption, with one exception at the 60-minute timepoint where those consuming Jerusalem artichokes indicated greater feelings of fullness than those receiving the inulin powder or control smoothie (P = 0.016). No significant differences in GIT were observed other than a slight increase in reported flatulence for both treatment groups when compared to a control at the 30-minute timepoint (P = 0.042). These data indicate that neither treatment promoted greater satiety than a control, and that both sources of inulin were well tolerated without producing adverse gastrointestinal symptoms in healthy adults.

As efforts to identify new prebiotics continue, the objective of our second study was to measure prebiotic properties of a yeast fermentate in an in vitro system. Yeast

(Saccharomyces cerevisiae) is used widely in human and animal nutrition for the contribution of micronutrients, antioxidants, amino acids, and other bioactive compounds. The fiber content of yeast fermentates has been shown to have prebiotic

effects and the ability to alter the gut microbiota in a positive direction for gut health in several animal species, although data with human subjects is lacking.

We hypothesized that a yeast fermentate would selectively stimulate the growth of Bifidobacteria and Lactobacillus, while also increasing short chain fatty acid (SCFA) production in an in vitro simulated human microbiome environment. In our trial, human fecal samples were used with practical doses of a yeast fermentate, 0.5 g/L and 1.5 g/L, to

+ compare SCFA, lactate, Bifidobacteria, Lactobacillus, ammonium (NH4 ) and branched

SCFA production. Maltodextrin was a placebo treatment. The yeast fermentate demonstrated prebiotic properties by beneficially altering microbiome activity and increasing Lactobacillus, butyrate, and propionate in a dose-dependent manner with the strongest effects being observed for the highest test dose (P < 0.05). This research supports that a yeast fermentate has prebiotic activity. Further, the changes we observed in gut microbiota and levels of SCFAs may be the mechanism by which yeast fermentate improves immune response and gut health seen in animal species.

The results from these two studies provide valuable data towards the continued evaluation of prebiotic compounds. Even though we did not see an effect on satiety from the two inulin sources tested, we found that Jerusalem artichokes were well tolerated and easy to blend into a beverage. With the current interest of incorporating healthy whole foods into the diet, this information will be valuable to consumers and provide a new option for achieving greater fiber intake. Our yeast fermentate data provide evidence of prebiotic properties in vitro using human fecal donors, which indicates a likelihood of prebiotic health benefits for humans upon consumption that are currently demonstrated in animal species. And while there are currently a limited number of supporting studies to

officially classify yeast fermentates as prebiotics for humans, these data strengthen that argument.

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Table of Contents Acknowledgments...... i

Dedication ...... iii

Abstract ...... iv

List of Tables ...... x

List of Figures ...... xi

List of Abbreviations ...... xiii

CHAPTER ONE: LITERATURE REVIEW ...... 1

Introduction ...... 1

Dietary Fiber ...... 3

Physiochemical Properties ...... 6

Prebiotic Fiber ...... 8

Satiety and Satiation ...... 12

Measurement ...... 13

Mechanisms ...... 15

Gastrointestinal Tolerance ...... 18

Measurement ...... 19

Inulin ...... 20

Prebiotic Properties ...... 21

Satiety ...... 22

Gastrointestinal Tolerance ...... 24

Yeast ...... 26

Conclusions ...... 29

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CHAPTER TWO: THE EFFECTS OF TWO INULIN SOURCES ON SATIETY AND GASTROINTESTINAL TOLERANCE ...... 33

Summary ...... 33

Introduction ...... 34

Methods...... 37

Results ...... 41

Discussion ...... 42

CHAPTER THREE: YEAST HYDROLYSATE HAS PREBIOTIC PROPERTIES IN AN IN VITRO SYSTEM ...... 50

Summary ...... 50

Introduction ...... 51

Methods...... 53

Results ...... 55

Discussion ...... 61

Conclusions ...... 78

References ...... 80

Appendices ...... 91

List of Tables

Figure 2-1. Nutrient composition of breakfast smoothies ...... 45

Figure 2-2. Nutrient composition of breakfast smoothie ingredients ...... 46

List of Figures Figure 1-1. Satiety Cascade ...... 31

Figure 1-2. Bifidogenic Effect ...... 32

Figure 2-1. Sensation ratings of hunger over time ...... 47

Figure 2-2. Sensation ratings of satisfaction over time ...... 47

Figure 2-3. Sensation ratings of fullness over time ...... 48

Figure 2-4. Sensation ratings of prospective consumption over time ...... 48

Figure 2-5. Symptom occurrence and severity with total score indicating the sum of severity scores for each symptom ...... 49

Figure 2-6. Symptom occurrence and severity reported as number of individuals experiencing each symptom by treatment ...... 49

Figure 3-1. Absolute pH over 48-hours of fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 66

Figure 3-2. pH change over a 48-hour fermentation period of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 67

Figure 3-3. Gas production over a 48-hour fermentation period of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 68

Figure 3-4. Average total SCFA production (mM) during different time intervals (0-6h, 6-24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 69

Figure 3-5. Average acetate production (mM) during different time intervals (0-6h, 6- 24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 70

Figure 3-6. Average propionate production (mM) during different time intervals (0-6h, 6-24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 71

Figure 3-7. Average butyrate production (mM) during different time intervals (0-6h, 6- 24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 72

Figure 3-8. Average branched SCFA production (mM) during different time intervals (0- 6h, 6-24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 73

Figure 3-9. Average lactate production/consumption (mM) during different time intervals (0-6h, 6-24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 74

Figure 3-10. Average ammonium production (mg/L) during different time intervals (0- 6h, 6-24h, 24-48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 75

Figure 3-11. Average Bifodobacterium levels (16S rRNA gene copies/mL) on different time points (0h and 48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L)...... 75

Figure 3-12. Average Lactobacillus levels (16S rRNA gene copies/mL) on different time points (0h and 48h) upon fermentation of RenewX at two different concentrations (0.5 and 1.5 g/L) ...... 76

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List of Abbreviations AgRP agouti related peptide CART cocaine- and amphetamine-regulated transcript CCK cholecystokinin CNS central nervous system CVD cardiovascular disease DP degree of polymerization FOS fructo-oligosaccharide g grams GIT gastrointestinal tolerance GLP-1 glucagon-like peptide-1

H2 hydrogen ITF inulin-type fructan NDC non digestible carbohydrate

+ NH4 ammonium NPY neuropeptide Y OFS oligofructose PMN polymorphic nuclear POMC pro-opiomelanocortin PP pancreatic peptide PYY peptide tyrosine tyrosine qPCR quantitative polymerase chain reaction SCFA short chain fatty acid scFOS short chain fructo-oligosaccharide SHIME simulator of the human intestinal microbial ecosystem VAS visual analog scale

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CHAPTER ONE

LITERATURE REVIEW

Introduction

Nutrient consumption resulting in a physiological benefit to the host is currently at the forefront of public health research. More specifically, new technologies enabling insight into the biological interactions between dietary fiber and the gut microbiome continue to strengthen our ability to define and categorize the activities of different types of fibers (Sankar et al., 2015). Dietary fiber includes carbohydrate polymers naturally found in plants which are neither digested nor absorbed in the foregut and are subject to fermentation by microbiota of the large intestine and colon (Holscher, 2017; Slavin &

Green, 2007). Not all fibers undergo fermentation, however, those which are fermented play a role in microbiota metabolic activities and bacterial community composition.

Further, when fermentable fibers exert a beneficial physiological effect such as growth promotion of beneficial bacteria, they may be classified as prebiotic fibers (Slavin, 2013).

While most prebiotics are fibers, not all fibers are prebiotics (Slavin et al., 2013).

A prebiotic may also be defined as “a nondigestible compound that, through its metabolization by microorganisms in the gut, modulates composition and/or activity of the gut microbiota, thus conferring a beneficial physiological effect on the host” (Bindels et al., 2015). Lactobacilli and Bifidobacteria are beneficial microorganisms with increases in their incidence often being measured for prebiotic classification (Slavin,

2013).

Inulin is well accepted as a prebiotic fiber and is found abundantly in chicory and to a lesser extent in agave, garlic, onions, leeks, asparagus, Jerusalem artichoke and bananas. A serving of 5-10 grams (g) of inulin per day has repeatedly demonstrated bifidogenic effects which confer physiological benefits to the host (Gibson et al., 1995;

Rumessen & Gudmand-Hoyer, 1998). Purified inulin is added to many foods and beverages in the U.S. and is often produced industrially from chicory roots. Inulin is commonly used as a low-calorie sugar replacer or bulking agent with a net energy of 1 kcal/g (Roberfroid et al., 1993; Bruhwyler et al., 2009). This fiber provides texture in the mouth similar to fat making it ideal for use in candy, chocolate, baked goods, ice cream, and breakfast cereals (Davidson & Maki, 1999).

Yeast fermentates are widely fed to production livestock for prebiotic qualities and are currently being explored for their prebiotic capability in humans. Yeast fermentates are produced under anaerobic conditions where Saccharomyces cerevisiae microorganisms are deprived of oxygen and subsequently produce beneficial metabolites including proteins, peptides, organic acids, antixoxidants, and other micronutrients

(Jayachandran et al., 2018). Beta-glucan and mannans are fibers which provide structure in fermentates, with both compounds having known immunomodulatory effects seen in rats and livestock (Ducray et al., 2019; Price et al., 2010; Medina et al., 2002).

Preliminary studies involving human subjects indicate a beneficial microbiota

(Bifidobacteria) shift in vivo and in vitro in response to a yeast fermentate (Pinheiro et al., 2017; Possemiers et al., 2013, Jensen et al., 2008). However, further work is necessary for yeast fermentates to be accepted as prebiotics in human nutrition. The

following sections explore dietary fiber and the previously mentioned prebiotics, inulin and yeast fermentate, as they relate to the research covered in this dissertation.

Dietary Fiber

Dietary fiber offers a wide range of health benefits including reduced risk of cardiometabolic disease, blood glucose attenuation, weight management, and a reduced risk of certain cancers (Slavin, 2013). Current daily fiber intake recommendations are 25 g for women and 38 g for men, although, fiber has been a nutrient of concern since 2005 with over 95% of Americans falling short of meeting these recommendations (Slavin,

2008). Average consumption of fiber by adults in the U.S. is approximately 17 g/day, which falls behind European countries with average fiber consumption of 20 g/day

(USDA, 2015-2016). When compared to nations which favor more fiber-based meals, the U.S. and Europe lag far behind a traditional African diet which can reach over 50 g of fiber per day and is associated with a greatly reduced risk of colon cancer (O’Keefe et al.,

2015).

Low fiber diets seen in developed nations are often attributed to a typical Western diet consisting of high fat, sugar, starch, and animal protein, with low amounts of fiber.

This low fiber diet is thought to be a major contributor to the high incidence of chronic non-communicable diseases including CVD, obesity, and type 2 diabetes (Makki et al.,

2018). Low fiber intake may also be associated with gut microbiota dysbiosis, which is a more recent finding with emerging science and technology indicating the link between dietary fiber and a healthy gut microbiome. Thus, a major initiative of the public health professional community remains communicating the benefits of dietary fiber to promote

a healthy lifestyle. A few actions that can be taken to support this initiative include incorporating diet and fiber education into elementary schools, developing media and messaging to consumers on flavorful fibers that are easily incorporated in the diet, and by continuing to educate consumers on how to read and understand food labels. Before these actions can be most effective, however, public health agencies have work to do in presenting a consistent, clear definition of what dietary fiber is (Oliver et al., 2014).

The definition for dietary fiber has been greatly debated by regulatory agencies for decades, with no common definition currently in existence. In the past, dietary fiber was defined based on the presence of noncarbohydrate components in a food including cellulose, hemicellulose, lignin, and pectin. Currently, a variety of analytical methods are used for measuring fiber in whole foods and synthetic fibers, although acceptable assays for nutritional labels are inconsistent amongst regulatory agencies throughout the world.

And while advancements in analytical techniques have improved the precision of identifying and measuring fibers, some researchers are now pushing to define fibers based more on their physiological activity versus their physiochemical properties.

Several other factors have contributed to the challenge in agreement upon a common definition for fiber. For example, fiber is not defined by a single chemical group or similar group of compounds as other nutrients are. The term “dietary fiber” covers a wide range of compounds eliciting differing physiological or health benefits, with some effects being unique to specific physiochemical properties that can vary within a type of fiber (Jones, 2014). The way dietary fiber is consumed isolated versus part of a meal, solid or liquid, also impacts its function within the body; creating further difficulty in defining what exactly dietary fiber is, what it does, and what level is needed in the diet.

The Codex Alimentarius Commission defines dietary fiber as “carbohydrate polymers with ten or more monomeric units, which are neither digested or absorbed in the human small intestine and belong to the following categories: (i) edible carbohydrate polymers naturally occurring in foods as consumed, (ii) edible carbohydrate polymers which have been obtained from food raw materials by physical, enzymatic, or chemical means and which have a beneficial physiological effect demonstrated by generally accepted scientific evidence, and (iii) edible synthetic carbohydrate polymers which have a beneficial physiological effect demonstrated by generally accepted scientific evidence”

(CAC, 2009). This definition has been used, although slightly modified, by China,

Brazil, Canada, New Zealand, Europe, and Australia to include carbohydrates with 3-10 monomeric units (Jones 2014).

The Institute of Medicine (IOM) provides definitions for dietary fiber, functional fiber and total fiber for fiber in the food supply (Slavin, 2013). According to the IOM, dietary fiber consists of nondigestible carbohydrates (NDC) and lignins which are intrinsic and intact in plants; functional fiber includes isolated, NDC which contribute to physiological benefit to humans; total fiber is the sum of dietary fiber and functional fiber

(IOM, 2001). The American Association of Cereal Chemists describes dietary fiber as,

“The edible part of plants or analogous carbohydrates, that are resistant to digestion and absorption in the human small intestine with complete or partial fermentation in the large intestine. Dietary fiber includes polysaccharides, oligosaccharides, lignin, and associated plant substances. Dietary fibers promote beneficial physiological effects including laxation, and/or blood cholesterol attenuation and/or blood glucose attenuation” (AACC,

2001). While other definitions exist from differing regulatory agencies, adoption of a

single definition and synchrony in messaging would be beneficial to improve consumer understanding and potentially improve fiber intake thereby potentially reducing adverse health outcomes associated with poor fiber intake (Jones 2014).

Physiochemical Properties

Fiber is further differentiated by physiochemical properties including solubility, viscosity and fermentability. Insoluble fibers comprise the structural elements of plants and are associated with laxation, water holding capacity, and fecal bulking (Titgemeyer et al., 1991). Examples of insoluble fibers include cellulose, hemicellulose, and lignin.

Soluble fibers describe those which form a mixture with water and are nearly completely broken down by bacteria in the large intestines. Pectins, gums, beta glucans, inulin, and psyllium are examples of soluble fibers. The property of viscosity measures the resistance of flow and the gel forming properties of a fiber. Higher viscosity in a fiber is linked with a reduction in the motivation to eat, forming an association between this characteristic with satiety and bodyweight (Slavin, 2007).

Fibers can be further classified as fermentable or non-fermentable. Fermentable fibers (ex. beta-glucans, pectins, guar gum, and inulin), as opposed to non-fermentable fibers (ex. cellulose), are complex carbohydrates that pass through the gastrointestinal tract intact making them available to gut microbe enzymes favoring saccharolytic fermentation. Fermentation is the incomplete oxidation of a substance in the absence of oxygen which occurs because glycosidic linkages are largely indigestible by human enzymes. Less than 20 glycosidases in humans have been identified (ex. salivary amylase, pancreatic amylase, and disaccharidases on the brush border of the intestines)

and these enzymes target digestible poly- and disaccharides in the foregut (Cantarel et al.,

2012).

Generally accepted health benefits of fermentable fibers include a decrease in risk factors associated with metabolic syndrome (obesity, diabetes, hypertension and hyperlipidemia), prolonged gastric emptying resulting in satiety and a decrease in overall energy intake, and these fibers can treat and prevent chronic diseases including certain cancers, irritable bowel syndrome, and colitis (Sheppach et al., 2001; Grabitske & Slavin,

2010). Soluble fibers, versus insoluble, are most commonly fermented due to their ability to swell and dissolve which makes them more accessible to microbial enzymes (Talaro,

2006). The capability of a microorganism to metabolize a type of fiber varies based on the number of enzymes they have available. Some microbiota have the capability to metabolize one or a few polysaccharides whereas others are able to metabolize dozens

(Martens et al., 2011; Scott et al., 2014).

Degree of polymerization (DP) and branching makes a difference in the location and rate of fermentation. Short chain fibers are fermented closer to the foregut (ileum and ascending colon) more rapidly, whereas less soluble fibers are fermented distally in the tract and at a slower rate (Holscher, 2017). For example, a short chain fructo- oligosaccharide is fermented within 4 hours of ingestion, whereas a highly branched fructan (ex. agave inulin) begins fermenting 4 hours post ingestion and peaks at 6 hours postprandially (Holscher et al., 2014). Chicory derived inulin with a linear structure and high DP has peak fermentation 8 hours post ingestion (Brighenti et al., 1999).

Fiber fermentation yields SCFAs acetate, propionate and butyrate, and gases CO2,

H2 and CH4. Approximately 90-99% of SCFAs are absorbed or utilized by gut

microbiota (Ruppin et al., 1980). SCFAs provide energy to cells throughout the body, with butyrate serving as a key energy source for enterocytes and colonocytes in addition to having anti-inflammatory actions (Donohoe et al., 2011; Holscher, 2017). Propionate is predominantly utilized for glucose production in the intestines (via gluconeogenesis) or travels to the liver to serve as a substrate for hepatic gluconeogenesis (Cummings et al.,

1987; De Vadder et al., 2014). Acetate is an energy pathway substrate that can be measured in peripheral circulation and has the ability to cross the blood-brain barrier

(Perry et al., 2016). SCFAs collectively play a role in epithelial barrier integrity, lipid metabolism, glucose homeostasis, and immuno-modulatory functions (Koh et al., 2016).

Prebiotic Fiber

Not all fibers are prebiotics, but most prebiotics are fibers (Slavin, 2013). A few examples of accepted prebiotics include inulin, wheat dextrin, polydextrose, beta-glucan, galactooligosaccharide, and fructans. The concept of prebiotic fiber was first introduced in 1995 and the definition for what constitutes a prebiotic has continued to evolve ever since. Originally, a prebiotic was defined as a “non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves health” (Gibson &

Roberfroid, 1995). As advances in research approaches and sequencing methods have led to a greater understanding of associated health benefits and microbiota shifts outside of the scope of original target populations, a less limited prebiotic definition continues to take shape. Currently, a fiber must demonstrate the following to be classified as a prebiotic: resistance to gastric acidity, evades hydrolysis by mammalian enzymes,

undergoes fermentation by intestinal microbiota, and selectively stimulates the growth of beneficial gut microbiota, thus, conferring physiological benefit(s) to the host (Gibson et al., 2004; Bindels et al., 2015). The interest in prebiotics has gained traction by consumers in recent years as a strategy to positively influence gut microbiota; as it is well accepted that prebiotics modulate beneficial gut microorganisms that are linked to health benefits (Vandeputte et al., 2017).

When testing a fiber for prebiotic properties, both in vivo and in vitro models may be utilized. In vivo study of fiber fermentation is difficult due to the invasive nature of colon studies in human models, and due to the complex nature of the colon including changes in microbial populations in different regions. Cannulated animal models can provide insight into the dynamic environment of the colon, however, data may be limited in its extrapolation to human subjects. Estimations of fiber fermentation can be made by measuring fiber consumed and fiber remaining in fecal samples, although this method is not without limitation as bacterial cell walls from feces are isolated in fiber analysis methods. SCFA analysis of feces provides an estimate of those produced in the colon, however, due to the great extent of SCFA absorption in the lumen (90-99%) their content in feces does not provide the full picture of fermentation. For these reasons, SCFA production from fiber fermentation is best measured in vitro, with a major benefit being the elimination of absorption losses.

Fiber fermentation is commonly measured in vitro by utilizing human fecal samples or by inoculating rats with human fecal flora (Makivuokko et al., 2006;

Henriksson et al., 2006). Carlson et al (2018) reviewed the fiber components that have been shown to have prebiotic properties in in vitro systems and found most support for

oligosaccharides, particularly fructo-ologosaccharides and galacatosaccharides.

Additionally, fiber supplements including partially hydrolyzed guar gum, inulin, acacia gum, and other soluble fibers have published studies supporting that they are fermented resulting in a decrease in pH, greater production of SCFAs, and changes in microbiota, especially bifidobacteria and lactobacillus.

A model which closely mimics the GI environment, the Simulator of the Human

Intestinal Microbial Ecosystem (SHIME), can be highly useful for in vitro study of prebiotics. This system has been validated and used for in vitro experiments for over 25 years with a key advantage being its GI chambers that are modeled to represent conditions found in a human GI tract (Marzorati et al., 2009). This model can evaluate changes in pH, gas production, metabolites and microbiota changes while simaultaneously allowing for natural reactions of the complex intestinal microbrial ecosystem to take place (Molly et al., 1993).

Experiments testing prebiotic potential of a compound most often include measurement of pH changes, SCFA production, gas production, and microbiota changes including inhibition of growth of undesirable bacteria while simultaneously promoting growth of beneficial bacteria. To be considered a beneficial bacteria, a microbe must inhibit the growth of harmful bacteria, decrease gas production, improve nutrient digestion, improve bowel function, enhance immunity, and not produce toxins

(Roberfroid et al., 2010; Hughes et al., 2003). Negative fermentation effects which can disqualify a compound from prebiotic status include carcinogen production, diarrhea, infection, and intestinal putrefaction (Gibson & Roberfroid, 1995; Hughes et al., 2003).

These negative effects are most often promoted by bacterial species which favor

proteolytic fermentation, over saccharolytic fermentation, and yield toxic compounds

+ including NH4 , phenols, cresols, sulfides, amines and branched SCFA (Hughes et al.,

2003). Thus, selective stimulation of saccharolytic fermentation favoring species is a key element to achieving beneficial status.

Known species with preference to saccharolytic fermentation include genera

Bifidobacterium, Ruminococcus, Lactobacillus, Bacteroides, Eubacterium and Clostrium

(Roberfroid et al., 2010). Bifidobacteria and lactobacilli are two genera most commonly recognized as beneficial bacteria, with shifts in bifidobacteria most often seen in prebiotics due to a larger concentration residing in the colon (Slavin, 2013).

Bifidobacteria and lactobacilli produce lactate and acetate which cause a drop in pH inhibiting the growth of pathogenic species. For instance, the inhibition of harmful species including Escherichia coli and Clostridium has been demonstrated with bifidobacterial dominance (Okazaki et al., 1990). Emerging research recognizes beneficial contributions of other species including Faecalibacterium and Roseburia, and with progress being made in sequencing methods it is likely more beneficial species will be added in the future.

The mechanisms of action by which prebiotics promote health are often through immunomodulatory properties and the production of beneficial metabolites. Prebiotics, including beta-glucans and inulin-type fructans (ITFs), can activate receptors on immune cells triggering neutrophil phagocytosis (Goodridge et al., 2010). ITFs influence immune function by inducing anti-inflammatory cytokines while serving as a receptor for gut dendritic cells (Vinolo et al., 2011). Beneficial metabolites produced by prebiotic fermentation include folate, secondary bile acids, indoles, SCFAs, and trimethylamine-N-

oxide, with all having known health benefits (Enam & Mansell, 2019). Further, improvement of inflammatory bowel disease, anti-cancer properties, improvement of lipid profiles, and antidepressant effects have all been demonstrated by supplementation with prebiotics (Enam & Mansell, 2019).

Satiety and Satiation

A benefit of dietary fiber is appetite control and prevention of obesity through calorie displacement due to satiation (Slavin, 2005). Numerous studies have indicated that a diet high in fiber and whole grains is related to lower body weight and less overall energy intake when compared to a diet low in fiber and whole grains (Du H et al., 2010;

Tucker & Thomas, 2009; Davis et al., 2006). The effect of increasing dietary fiber by 14 g per can result in a 10% reduction in ad libitum energy intake, as demonstrated by satiety studies (Howarth et al., 2001). Fiber intake with resultant stomach distention, changes in gut hormones and fermentation can be responsible for this feeling of fullness after an eating episode (Mela, 2005). Further, fiber can promote satiety and weight loss by increased time and efforts of mastication, lower energy density in high fiber foods compared to high fat foods, and a feeling of fullness from bulking and viscous properties

(Slavin, 2007). These mechanisms also include modulation of gut hormones involved in appetite regulation (Cani et al., 2009).

The words satiety and satiation are often used interchangeably but have two distinct roles in appetite regulation. Satiation refers to appetite satisfaction that occurs during a meal and leads to the cessation of eating. Cessation of eating most commonly occurs for the reasons of a full stomach sensation or a decrease in the reward or

pleasantness of a food (Hetherington, 2006; Tuomisto et al., 1998). Satiety is the resultant low drive to eat, which is when hunger is suppressed due to finishing a meal

(Slavin & Green, 2007). Satiety influences timing of the next meal whereas satiation influences the size of the next meal.

Satiety and satiation are complex areas of study involving brain signals and psychological and physiochemical stimuli. Energy density, macronutrient composition, physical structure and sensory qualities influence satiation and satiety (Blundell et al.,

2010). Expression of appetite (the internal driving force behind ingestion of food) is also influenced externally by environmental factors including time of day, sensory stimuli, sensory hedonics, social pressure, environmental factors, and boredom (Rozin, 1996).

Measurement

Several formulas have been developed to quantify satiety of individual foods.

The satiety quotient, satiety efficiency and satiety index procedures calculate the energy- satiety ratio of foods to assess their potential to promote satiety and prevent overconsumption (Kissileff, 1984; Holt et al., 1995; Green et al., 1997). The satiety quotient relates fullness to the energy content of a food consumed and has found some use in describing satiety by food, exercise, or the biology of an individual (Green et al.,

1997). While these quotients are limited in their applicability to actual satiety, the satiety quotient has some strength when used with fixed meal size. Although, limitations exist due to a lack of a linear relationship between energy and returning hunger after eating

(Drapeau et al., 2005; Drapeau et al., 2007).

The most common and validated method for evaluating satiety by researchers is through a VAS. A VAS records subjective feelings of fullness and appetite sensations.

Subjects are asked to indicate their hunger, fullness, desire to eat and satisfaction on respective 100-mm lines with opposing feelings on anchored ends (Flint et al., 2000).

For instance, a subject will be asked to rate their hunger by placing an ‘X’ on the 100-mm line with the far left anchor “I am not hungry at all” and the far right anchor “I have never been more hungry.” A measurement can then be taken from where the subject indicates their hunger on the line.

Measurement of satiety can be difficult as it is influenced by type and size of meals consumed in the 24 hours prior to assessment, how much the participant likes the food being offered, caloric density of the meal, and activity levels of the subject prior to consuming the test meal. Taste and overall pleasantness perceptions of test foods can influence eating including emotional eating, social eating, and convenience or availability of foods (Blundell et al., 2010). Subjects also experience cognitive signals to terminate a meal, in particular, their knowledge of “how much” of a certain type of food they usually consume to be full. For instance, subjects may know they usually eat two pieces of pizza to feel comfortably satiated, thus their consumption choices will be influenced by their typical portion size. To control for these variables, researchers may cut experimental foods into irregular shapes, ask that subjects fast for a period prior to test diet consumption, ask that subjects keep a food diary before or after test meals to determine changes/differences in energy intake, and/or utilize a crossover design in a controlled and consistent environment with each subject serving as their own control (Brunstrom, 2007).

For measurement of satiation, energy intake can be measured from an ad libitum meal. Ad libitum meals are offered as a buffet or as a homogenous meal consisting of food(s) that are acceptable to the subject population. Energy intake is calculated from an ad libitum meal by measuring the food consumed and subtracting the discarded food

(Venti et al., 2010). This method provides greater accuracy than 24-h recall food diaries, as subjects are often not good at reporting their own intake (Venti et al., 2010).

While energy intake is a fair indicator of satiation, it cannot be overlooked that sensory factors majorly influence intake and palatability has a strong effect on food intake (de Graaf et al., 2005). Because it is understood that many sensory and cognitive variables influence satiation, it is crucial that subjects be in a uniform state of satiety, if possible, for research projects involving energy intake measurement. Researchers may also opt to implement a pre-load diet for better uniformity (Blundell et al., 2010).

Mechanisms

Processes including mastication, saliva production, gastric emptying, gastric distention, gut hormone secretion, transit time and fermentation contribute to satiety and satiation signals. The most important mechanism for overall appetite regulation is the gut-brain-adipose tissue axis which enables communication between the digestive tract and the central nervous system (CNS). The gastrointestinal tract is considered the largest endocrine organ in the body. Discussed in this section are various physiological markers which contribute to appetite regulation. A graphic connecting these biomarkers with satiety and satiation is referred to as the Satiety Cascade and is provided at the end of this chapter (Figure 1) (Blundell et al., 1987; Mela, 2006).

The hypothalamus serves at the regulatory center of appetite and neuronal activity is influenced by circulating hormones (Chaudhri et al., 2008). It was originally thought that the lateral hypothalamic area was the “hunger center” and the ventromedial hypothalamic nucleus was the satiety center, however it is now understood that integration within these two centers occurs and interaction with the brainstem, vagus nerve, and other cortical centers are involved (Suzuki et al., 2010).

Gut hormones and adipose tissue signals travel through the bloodstream and influence appetite pathways in the brain by acting on the neuropeptide Y (NPY) and agouti related peptide (AgRP) and pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) pathways (Smitka et al., 2013). Signals from gut hormones including glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK), peptide tyrosine tyrosine (PYY), ghrelin, insulin, pancreatic peptide (PP), and leptin are all received in the arcuate nucleus of the hypothalamus and influence NPY/AgRP and

POMC/CART activity. These signals effect two subpopulations of neurons oppositely affecting feeding behavior and maintenance of energy homeostasis through orexigenic and anorexigenic effects (Smitka et al., 2013). Orexigenic signals which co-express neuropeptide Y (NPY) and agouti related peptide (AgRP) result in food intake, whereas anorexigenic signals co-express pro-opiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) result in inhibition of eating (Smitka et al.,

2013). Interpretation of these signals relies on peripheral signals from the vagus nerve and gut indicating gastric distension and gut hormone levels (Bailey, 2008). These signals are interpreted by the brainstem through receptors for various gut hormones and act directly on the hypothalamus.

Gut hormones with a well-defined role in satiety and appetite regulation include

PYY, PP, GLP-1, glucagon, ghrelin, CCK, leptin and insulin (Stanley et al., 2005). PYY causes a reduction in food intake and has a low circulating level when fasting and is elevated for several hours after eating a meal (Batterham et al., 2003). Studies in rats with PYY infusion showed lower food and intake and body weight gain, which was also shown in humans (Berntson et al., 1993). Further studies in mice showed delayed gastric emptying and increased energy expenditure with infused PYY, although these studies were not repeatable in humans (Tucker et al., 1996, Chelikani et al., 2004). PP also reduces food intake and is secreted by pancreatic Islets of Langerhans. In the case of both PYY and PP, peripheral administration in humans results in anorectic effects, whereas CNS administration stimulates food intake (Eberlein et al., 1989). This difference is hypothesized to be attributed to differences in receptor presence at active sites.

GLP-1 is a proglucagon derived peptide released by enteroendocrine L cells in the intestines. GLP-1 is co-secreted with PYY and stimulates insulin secretion. Similar to previously mentioned hormones, GLP-1 levels rise following a meal for 1-2 hours and drops during a fasted state. GLP-1 delays gastric emptying and inversely reduces food intake (Cummings & Overduin, 2007).

Ghrelin is the only gut hormone with known orexigenic effects. Levels of this hormone increase prior to a meal and fall after eating (Moran & Dailey, 2011). CCK is produced from the I cells of the small intestine and is associated with inhibition of food intake (Zelissen et al., 2005). The release of CCK is regulated by the presence of nutrients in the lumen, particularly fat and protein. Work with CCK antagonists have

been shown to reduce satiety and increase food intake (Beglinger et al., 2001). While the nature of how these hormones influence appetite, satiety and satiation is complex, an understanding how these hormones interplay with cognitive signals is valuable when designing an experiment measuring satiety.

Gastrointestinal Tolerance

Fermentable fibers can cause GI upset following consumption. Due to the inability of mammalian enzymes to digest dietary fiber, adverse GI symptoms can be seen with fermentable fiber intake including flatulence, distension, bloating, loose stool, diarrhea, acid reflux, colic, reduced appetite and increased stool frequency (Livesey,

2001; Grabitske & Slavin, 2009; Holscher et al., 2014). Gas production from the fermentation process can contribute to symptoms including flatulence, bloating, distension, and general abdominal discomfort. Diarrhea or increased fecal bulk can occur if non-digestible carbohydrate intake exceeds fermentation capacity (Marteau & Flourie,

2001). Certain symptoms can also be attributed to an osmotic effect from a greater number of undigested carbohydrate molecules in the large intestines causing an increase in water to balance osmotic pressure of intestinal contents (Grabitske & Slavin, 2009).

Due to discomfort of subjects as a result of these symptoms, research is not only aimed at identifying fermentable fibers with the greatest health benefits, but also at identifying tolerable upper levels of individual fermentable fibers to decrease the likelihood of consumer rejection.

Chemical characteristics of a fiber including molecular weight, DP, structure branching, and solid versus liquid presentation will affect GI tolerance. For example, due

to the rapid transit time of liquids, fermentable fiber offered in foods are as a meal are generally better tolerated than liquids (Livesey, 2001; Jenkins et al., 1980). There are unique host characteristics that also affect an individual’s GI response to a fermentable fiber. Host factors including age, gender, underlying health conditions (IBD, IBS, celiac disease, etc.), medications (antibiotics, caffeine, laxatives), physical activity, and average fiber intake will influence gastric emptying, intestinal enzyme activity, gut motility and composition of microbiota (Grabitske & Slavin, 2010). Research studies reduce confounding variables by creating exclusion criteria and by having a similar subject population (lifestyle, body mass index, eating habits, etc.).

Measurement

GI tolerance symptoms can be measured objectively by monitoring diarrhea, defecation frequency, flatulence, stool consistency and abdominal distention. Collection of feces is a tool commonly used with composition and weight being recorded measurements. Isolation of nutrients from feces can indicate nutrient malabsorption, notably carbohydrate presence, although this presence does not provide clues about fermentation in the large intestine. Abdominal distention can be measured by changes in an abdominal girth, and although rarely used, colonic gas production can be measured.

Common practice for measuring GI tolerance is through validated subjective symptom questionnaires probing the presence and degree of bloating, flatulence, stomach noises, and other symptoms associated with digestive upset (Bovenschen et al., 2006).

Ranking systems vary by study and a subject may be asked to rate their symptoms as none, mild, moderate, or severe, or on a numeric scale (0-6) with 0 being none to 6 being

unbearable. GI tolerance surveys are most often administered repeatedly over a span of hours posttest food or beverage consumption. While questionnaires can provide valuable feedback regarding GI tolerance, a limitation exists in controlling for outside variables including how a participant perceives their own symptoms (Yao et al., 2013). The information gathered by GI tolerance surveys indicates the quality of subject experience and is therefore very useful for future use of the product in a food or meal.

Breath hydrogen (H2) is another measurement for fermentation. This measurement is useful as expired H2 only comes from microbial fermentation and approximately 14% of H2 produced in the gut makes it to the lungs (Levitt, 1969). This measurement is commonly seen in fermentable fiber food studies to evaluate postprandial rate and peak of fermentation.

Inulin

Fructo-oligosaccharides (FOS) and inulin are prebiotic fibers belonging to the carbohydrate subgroup ITFs. The chemical structure consists of β-D-fructosyl groups linked by (2→1) glycosidic bonds, ending with a (1↔2) α-D-glucosyl group (Kelly,

2008; Ronkart et al., 2007). DP ranges from 2 to 60 monomers with an average of 10-12 units. Short chain FOS (scFOS) are often used as low-calorie sweeteners (30% sweetness of sucrose, 1-2 kcal/g) and long chain inulin works well as a fat replacement and texture modifier (Kelly, 2008; Carabin & Flamm, 2001). Physiochemical properties including solubility and viscosity are influenced by solvent temperature, molecular weight and DP.

In general, ITFs are categorized as soluble, highly fermentable fibers that are non- or lowly-viscous (Nair et al., 2010).

Fructans can be extracted from several plant families including Liliaceae,

Amaryllidaceae, Gramineae and Compositeae, or synthesized by enzymatic transfer of fructosyl groups from sucrose. The most common sources for industrial production are chicory and Jerusalem artichoke, which are part of the Compositeae family. Naturally occurring ITFs are found in smaller quantities in onions, bananas, garlic, asparagus, oats, wheat, and leeks (Slavin, 2005). Average ITF intake by Americans is 1-3 g per day and

3-11 g per day by Europeans (Coussement, 1999; Bonnema et al., 2010; Carabin &

Flamm, 1999).

Prebiotic Properties

Through fermentation, inulin selectively stimulates growth of health-promoting bifidobacteria and to a lesser extent, lactobacilli (Kleessen et al., 2002). A bifidogenic effect (Figure 2) has been shown at a minimum of 5 g per day for inulin and oligofructose, and at 9 g per day for long chain inulin (Bouhnik et al., 2007; Harmsen et al., 2002). Bouhnik and colleagues (2007) demonstrated this effect in a double-blind, randomized, placebo-controlled, parallel group study with 39 adults over an 8-week period. Researchers collected fresh stool samples and analyzed bacterial counts and fecal bacterial enzymatic activity. Results indicated increases in bifidobacteria (P < .0001) in the inulin group in addition to decreased β-glucuronidase activity (P = .001). The decrease in β-glucuronidase was significant as this enzyme is believed to play a role in activation of carcinogenic substances in the lumen of the colon.

In a double-blind, placebo controlled, cross-over study, 34 participants with low dietary fiber intake (<18 g/day for women, <22 g/day for men) or high dietary fiber

intake (>25 g for women, >30 g/day for men) were supplemented for 3 weeks with 16 g/day of an ITF prebiotic (Healey et al., 2018). Prebiotic intervention led to increases in

Bifidobacterium (P < .001) and decreases in Coprococcus, Dorea and Ruminococcus.

Researchers found the gut microbiota in the low fiber intake group was more resistant to change with the supplemented prebiotic, indicating that overall higher dietary fiber intake can lead to a greater microbiota response from a supplemented prebiotic. Further in vivo studies with doses of ITFs ranging from 13-20 g per day of have revealed increases in fecal SCFAs, lactate, acetate, and decreased microbial proteolytic fermentation resulting in lowered production of p-cresol, anomia and iso-SCFA (van Nuenen et al., 2003;

Grasten et al., 2003; Ten Bruggencate et al., 2006). These data are consistent with a number of other in vitro studies which report ITF supplementation leading to increased production of SCFAs (Scott et al., 2014; Jung et al., 2015).

Satiety

Feelings of fullness increase satiety and reduce energy intake, which are beneficial for controlling bodyweight. Studies evaluating the effect of ITFs on satiety, energy intake, or both, have been performed with inconsistent results. A randomized, double blind, placebo controlled, short term study tested the effect of 16 g/day of ITFs on food intake and appetite (Salmean, 2017). The ITF supplement was dissolved into artificially flavored water and consumed by 40 female participants daily between 7 and 8 a.m. with breakfast. Subjects reported GI symptoms and completed a VAS at 20, 50, 95,

155, 200, and 245 minutes post breakfast. The fiber group reported increased bloating and flatus compared to the treatment group (P = 0.029). The fiber treatment group also

showed a significant reduction in desire to eat, increased fullness, increased feelings of satisfaction, and decreased food consumption at the lunch meal. These findings are consistent with a study by Cani and associates, who performed a single-blind, crossover, placebo-controlled experiment supplementing 16 g of oligofructose (OFS) or a placebo of maltodextrin for 2 week periods, followed by a 2 week washout period (2006).

Supplemented treatment and control were divided into two portions of 8 g that were consumed in a beverage with breakfast and dinner, respectively. VAS scores were taken at 30, 60, 120, 180, and 240 minutes post breakfast, lunch, and dinner. OFS significantly increased satiety (P = 0.04) following the breakfast meal with treatment. No differences were observed following a lunch meal. OFS significantly increased satiety (P = 0.04), reduced hunger (P = 0.04) and reduced prospective food consumption (P = 0.05) following a dinner meal with treatment.

Hiel et al. (2019) found that subjects consuming a diet based on inulin-rich vegetables (15.67 ± 0.31 g fructans/day) showed greater satiety and a reduction in desire to consume salty, fatty or sweet foods throughout the day. Another study evaluating fat replacement with inulin incorporated 24 g of inulin into a sausage patty and fed it to subjects as a breakfast meal (Archer et al., 2004). Researchers found inulin patties resulted in lower daily energy intake when compared with lupin-kernel fiber and control patties. Based on the findings of these studies and supporting evidence from others, these researchers suggest that ITFs may be useful for weight management (Hume et al., 2017;

Whelan et al., 2006).

On the contrary, in a double-blind crossover study, Hess et al. (2011) evaluated the effect of scFOS on satiety and hunger. Subjects received 0, 5, or 8 g of scFOS mixed

in hot cocoa and served with a breakfast meal consisting of a bagel and cream cheese.

VAS was assessed at timepoints 15, 30, 45, 60, 90, 120, 180 and 240 post meal consumption and ad libitum food intake was measured at lunch. An additional treatment of scFOS was consumed 2 hours prior to the subject’s dinner meal, in the form of 3 chocolate chews. No significant differences were found in satiety ratings. However, one interesting finding with the 8 g dose of scFOS was reduced food intake in women and increased intake in men. A number of other studies with similar and higher doses of inulin or oligosaccharides have reported no differences in satiety or energy intake

(Karalus et al., 2012; Dewulf et al., 2013; Heap et al., 2016; Peters et al., 2009). Due to conflicting data on satiety and energy intake post ITF supplementation, further evaluation is necessary.

Gastrointestinal Tolerance

Increasing the content of fermentable fiber in the diet is associated with undesirable GI symptoms (Grabitske & Slavin, 2009). Thus, GI tolerance in response to supplementation with ITFs has been explored. Bonnema et. al (2010) examined GI tolerance to both short chain and long chain inulin at doses of 5 and 10 g per treatment dissolved into orange juice. In a randomized, double-blind, controlled crossover study, subjects participated in 5 visits where a breakfast fiber challenge consisting of a bagel, cream cheese and orange juice were consumed. Researchers reported that doses up to 10 g per day of native inulin and up to 5 g of short chain inulin were well tolerated in healthy young adults. Reports of mild to moderate flatulence were observed at the 10 g dose, although symptoms were mild and varied widely on an individual basis.

Two doses of chicory-derived inulin extract (5 g and 7.8 g) were evaluated at a in a double-blind crossover consisting of 18 subjects. The inulin supplement was dissolved into a morning coffee drink and compared against a sucrose control for three consecutive

6-day periods (Ripoll et al., 2010). A slight significant (P = 0.05) increase in abdominal discomfort was noted in the 7.8 g inulin group, although no other significant adverse symptoms were recorded. The same group of researchers completed a follow-up study with 35 participants who consumed a 5 g inulin supplement in an instant coffee drink twice a day for 4 weeks (Ripoll et al., 2010). Compared to a sucrose (8.1 g) placebo, no significant differences in GI symptoms were reported. Researchers concluded that short- and long-term consumption of 5 g of inulin dissolved in an instant-coffee drink are well tolerated by healthy subjects.

A double-blind, placebo controlled, crossover, dose-ranging randomized study evaluated GI tolerance of 3 ITFs at doses of 5-20 g over a 10-week period (Bruhwyler et al., 2009). Each ITF treatment dose had two test products with differing DP, with researchers hypothesizing that the higher DP compound would result in greater GI tolerance. Eighty-four healthy adult volunteers added packets of 5 g of ITFs to their morning beverage of water, tea, coffee, juice, or milk for 2 weeks, followed by a 2-week placebo washout period. GI tolerance was measured and flatulence at the 20 g dose was the only significant adverse symptom reported in this study, leading researchers to conclude that doses up to 20 g per day are well tolerated. Researchers found no difference in GI symptoms based on DP and noted that the shorter chain inulin was better tolerated than the longer chain, leading them to reject their hypothesis that longer chain oligosaccharides would be better tolerated. A number of other studies have demonstrated

favorable GI tolerance outcomes in response to ITF supplementation, although results should be interpreted carefully as different populations and unique individual microbiota communities can influence outcomes.

Yeast

Yeast is the term used to describe a unicellular fungus. There are hundreds of species of yeast with one of the most well-known in health and nutrition being

Saccharomyces cerevisae, also known as brewer’s yeast and baker’s yeast.

Saccharomyces, or "sugar mushroom" from Greek, is a member of the fungus kingdom that has been present on earth for at least 400 million years (Garcia-Mazcorro et al.,

2020). Saccharomyces has been widely studied and commonly used in food production because of its ability to ferment a wide range of carbohydrates. A major recent achievement of yeast research has been the determination of the complete metabolic pathways for amino acid utilization as carbon and nitrogen sources, amino acid biosynthesis, and the conversion of amino acids to other metabolites including nucleotides.

Probiotic properties of yeast have been reviewed and it is generally accepted that yeasts are probiotic (Czerucka et al., 2007). Studies have reported the use of yeasts

(Saccharomyces boulardii or Saccharomyces cerevisiae) as a potential bio-therapeutic agent (probiotic) for the treatment of microbes associated with diarrhea and colitis

(Kelesidis et al., 2012). Saccharomyces cerevisiae and Saccharomyces boulardii are clinically proven as a human probiotic to positively influence host’s health by antimicrobial and nutritional effects, inactivation of bacterial toxins, quorum sensing,

trophic effects, immuno-modulatory effects, anti-inflammatory effects, cell restitution and maintenance of epithelial barrier integrity (Moslehi et al., 2010). Members of

Saccharomyces genus can possess anti-bacterial properties, with anti-bacterial capability of Saccharomyces cerevisiae potentially being due to production of extracellular protease, secretion of inhibitory proteins, stimulation of immunoglobulin A, acquisition and elimination of secreted toxins, killer toxins, sulfur di-oxide, etc. (Gibson et al., 2017).

Saccharomyces cerevisiae fermentates have found wide use in animal nutrition as a prebiotic (Price et al., 2010; Medina et al., 2002). Yeast fermentates are produced under anaerobic conditions where Saccharomyces cerevisiae microorganisms are deprived of oxygen. In typical industrial yeast fermentate production, large vessels (or fermenters/bioreactors) facilitate the fermentation with carbon and nitrogen sources.

Followed by fermentation, the resulting fermentate is hydrolyzed, extracted and dried before packaging. Control points are set to ensure proper growth and prevention of contamination.

Following the standard yeast fermentation process, the yeast cells are hydrolyzed using food grade enzymes to produce commercial yeast fermentate. This product contains beneficial metabolites including proteins, peptides, organic acids, antixoxidants, and other micronutrients (Jayachandran et al., 2018). Fermentates also contain beta- glucans and mannans from the yeast cell wall, with both fibers having known physiological benefits contributing to immuno-modulatory properties and prebiotic effects (Ducray et al., 2019; Price et al., 2010; Medina et al., 2002).

Recent studies show promise for yeast fermentates in having anti-inflammatory properties, improving gastrointestinal discomfort, improving intestinal epithelial

integrity, and promoting beneficial gut microbiota (Jensen et al., 2015; Possemiers et al.,

2013; Pinheiro et al., 2017; Ducray et al., 2019). Possemiers et al. compared prebiotic properties of a proprietary Saccharomyces cerevisiae fermentate (EpiCor®) against established prebiotics inulin and FOS (2013). Cellulose was used as a negative control.

Utilizing the SHIME system, researchers identified stimulation of bifidobacteria and lactobacilli, a reduction in potential pathogens, changes in microbiota community, and a reduction in pro-inflammatory cytokines ins response to treatment with EpiCor. SCFA butyrate production was greater than FOS and control, which is significant as butyrate as can modulate expression of cytokines in epithelial and immune cells (Vinolo et al., 2011).

Because EpiCor performed similarly to other known prebiotics, a case could be made to push yeast fermentates as prebiotics for humans.

Anti-inflammatory effects of Saccharomyces cerevisiae fermentate (EpiCor®) were tested on human polymorphonuclear (PMN) cells in vitro (Jensen et al., 2015).

PMN cells comprise 50-70% of circulating white blood cells and are involved in the rapid inflammatory response and resolution of inflammation. PMN cells engage in pro- inflammatory responses including production of reactive oxygen species when under oxidative stress. Jensen et al. found a reduction in reactive oxygen species formation with EpiCor treatment compared to untreated PMN cells (P < 0.05).

In a single-blind, placebo-controlled crossover, the same group of researchers evaluated a histamine-induced inflammatory response on the arms of 12 human subjects

(Jensen et al., 2015). Subjects underwent a topical skin inflammation procedure and treatment of 0.01 mL of dried yeast fermentate and saline were applied to opposing forearms. Clinical results indicated significantly lower inflammatory responses and

lower subjective irritation scores with the dried yeast fermentate treatment compared to the control saline (P < .05). These data provide strength to preliminary work demonstrating anti-inflammatory properties of a yeast fermentate. It could also be hypothesized that a similar anti-inflammatory response occurs when the gut mucosa is exposed to a yeast fermentate, however the mechanisms for immuno-protective effects remains largely unknown.

Conclusions

Dietary fiber and prebiotics are influential in maintaining a healthy gut microbiome and promoting a healthy lifestyle. The focus of continued research in this area is identification of optimal sources of prebiotic fibers that result in the greatest feelings of satiety, minimal GI intolerance, and contribute the greatest health benefit to the host. Current gaps in prebiotic research include a lack of understanding regarding the most optimal form fed (whole food versus isolated, liquid or solid), inconsistent data on the most ideal physiochemical properties for each fiber source, inconsistent methods used across fiber studies making comparison difficult, and inconsistency in what qualifies as a prebiotic. The studies in this dissertation explore several of these key topics.

Given a recent consumer interest in consuming whole healthy foods as opposed to isolated nutrients, we first explore how a whole food source of inulin performs against a purified inulin form. This area of fiber research is very limited, particularly with the whole food we evaluated, Jerusalem artichokes. This research also touches on differing physiochemical properties of the same source of fiber, as it is known that the purified powder would be a mixture of short and long chain inulin, whereas the whole food source

would be predominantly long chain structures. Due to the impact of chain length on fermentation characteristics, this property can influence fiber consumption and consumer experience with eating. Thus, it was important for us to evaluate different inulin forms on GI tolerance and evaluate another key parameter for health promotion, satiety.

This dissertation also explores a compound that has gained popularity as a prebiotic in animal species but has yet to be widely used by humans. Yeast fermentates offer promise as a prebiotic with repeated demonstration of immunomodulatory mechanisms, anti-inflammatory properties, improvement of intestinal epithelial integrity, and a variety of other health benefits. Research utilizing animal models is robust but work with humans or human samples is limited. Our second study explores a yeast fermentate in a simulated human gastrointestinal model to measure its outcomes associated with prebiotic activity. Both studies have the common goal of furthering prebiotic research by investigating differing forms and variables at which these compounds may promote health.

Figure 1-1. Satiety Cascade.

Figure 1-2. Bifidogenic Effect.

CHAPTER TWO

THE EFFECTS OF TWO INULIN SOURCES ON SATIETY AND

GASTROINTESTINAL TOLERANCE

Summary

Inulin is an extensively studied prebiotic fiber and is most often seen as an isolated powder derived from chicory roots. The objective of this study was to compare two forms of inulin, an isolated powder and a whole food source (Jerusalem artichokes), and to identify their effects on satiety and GIT. We hypothesized that the Jerusalem artichokes would promote greater satiety and be better tolerated when compared to isolated inulin. In a randomized, single-blind, crossover study, 26 subjects (13 females,

13 males) fasted for 12 hours and then consumed a chocolate breakfast smoothie with inulin powder or Jerusalem artichoke puree mixed in. A plain smoothie without fiber was used as a control. The primary outcome was to assess subjective satiety using a VAS to assess hunger, satiety, fullness, and prospective food intake. The secondary outcome was to assess subjective gastrointestinal tolerance through surveys indicating the presence and severity of common adverse gastrointestinal symptoms. We found that satiety was not different based on treatment group within 4 hours of smoothie consumption, with one exception at the 60-minute timepoint where those consuming Jerusalem artichokes indicated greater feelings of fullness than those receiving the inulin powder or control smoothie (P = 0.016). No differences in GIT were observed other than a slight increase in reported flatulence for both treatment groups when compared to a control at the 30- minute timepoint (P = 0.042). These data indicate that neither treatment promoted

greater satiety than a control, and both sources of inulin were well tolerated without producing adverse gastrointestinal symptoms in healthy adults.

Introduction

Dietary fiber offers a wide range of health benefits including reduced risk of cardiometabolic disease, blood glucose attenuation, and a reduced risk of certain cancers

(Slavin, 2013). Another benefit of fiber consumption is appetite control and prevention of obesity through calorie displacement due to satiation (Slavin, 2005). Numerous studies have indicated that a diet high in fiber is related to lower body weight and less overall energy intake when compared to a diet low in fiber (Du H et al., 2010; Tucker &

Thomas, 2009; Davis et al., 2006). Thus, appetite regulation is an important area for evaluation in fiber studies as some fibers provide stronger satiating properties than others.

Current daily fiber intake recommendations are 25 g for women and 38 g for men, however, fiber is a nutrient of concern with over 95% of Americans falling short of these recommendations (Slavin, 2008). To account for this deficiency, some food manufacturers add functional fibers to their products. Inulin is a thoroughly researched and commonly isolated fiber added to many foods and beverages in the U.S. Naturally occurring inulin is found abundantly in chicory roots and Jerusalem artichokes, and in smaller quantities in onions, bananas, garlic, asparagus, oats, wheat, and leeks (Slavin,

2005). The most common source for industrial production of inulin is that isolated from chicory roots. In addition to its use as a fiber additive, inulin is often used as a low- calorie sugar replacer or bulking agent with a net energy of 1 kcal/g (Roberfroid et al.,

1993; Bruhwyler et al., 2009).

Inulin is well accepted as a prebiotic meaning it resists gastric acidity, evades hydrolysis by mammalian enzymes, undergoes fermentation by intestinal microbiota, and selectively stimulates the growth of beneficial gut microbiota, thus, conferring physiological benefit(s) to the host (Gibson et al., 2004; Bindels et al., 2015). A serving of 5-10 g of inulin per day has repeatedly demonstrated increases in beneficial microbiota bifidobacteria (Gibson et al., 1995; Rumessen & Gudmand-Hoyer, 1998). However, due to its fermentation property, adverse GI symptoms may be associated with inulin intake.

These symptoms include flatulence, distension, bloating, loose stool, diarrhea, and increased stool frequency (Holscher et al., 2014). Diarrhea or increased fecal bulk can occur if non-digestible carbohydrate intake exceeds fermentation capacity (Marteau &

Flourie, 2001). Certain symptoms can also be attributed to an osmotic effect from a greater number of undigested carbohydrate molecules in the large intestines causing an increase in water to balance osmotic pressure of intestinal contents (Grabitske & Slavin,

2009). Inulin doses of 15-30 g are most often associated with GI intolerance (Bruhwyler et al., 2009).

Measuring GI symptoms is an important element of fiber studies although it is difficult to compare results across studies due to a lack of consistency in assessment scales utilized. The most common practice for measuring GI tolerance is through validated subjective symptom questionnaires which probe the presence and degree of bloating, flatulence, stomach noises, and other symptoms associated with digestive upset

(Bovenschen et al., 2006). A validated, common GI symptom questionnaire was used in this study.

The purpose of this study was three-fold including subjective measurements of satiety and GI tolerance as well as evaluating a source of inulin that could easily be incorporated in the diet as a whole food. The basis for this is consumer interest in consuming healthful whole foods rich in fiber versus isolated healthy additives built into other foods or beverages. Therefore, we compared the commonly used isolated inulin against the lesser known whole food Jerusalem artichokes. Jerusalem artichokes are gaining attention as a functional food by demonstrating beneficial bacteria growth, which may be attributed to their inulin content of up to 50% in dried artichoke tubers (Nokkaew et al., 2018). Further, a recent study evaluating satiety and gastric emptying from consumption of a snack bar incorporating Jerusalem artichokes indicated prolonged subject satiety from this ingredient (Nokkaew et al., 2018).

We chose our treatment dose based on current food label claims indicating a

“good” source of fiber is 2.5 g, and an “excellent” source of fiber is 5 g. A dose of 7 g was selected as this dose is consistent with levels seen in snack bars and beverages incorporating inulin. We hypothesized that the Jerusalem artichokes would promote greater satiety and result in greater GI tolerance when compared to a purified inulin powder.

Methods

Subjects

Healthy men and women aged 18 to 65 years old with a BMI between 18.5 and 27 kg/m2 were recruited via flyers placed around the University of Minnesota St. Paul campus. Interested participants completed an online screening questionnaire to identify

inclusion and exclusion criteria. Exclusion criteria included smoking, non-regular breakfast or lunch consumption (≤4 days/week), veganism, lactose intolerance, women experiencing pregnancy or lactation, gastrointestinal conditions affecting digestion or absorption, use of antibiotics within a 3-month period from the start of the study, use of medication which could influence results including conditions for high blood pressure, high cholesterol, and diabetes. Exclusion factors also included individuals with a weight fluctuation of 10 or more pounds within the month prior to the study and individuals with restrictive eating habits identified as a score of 11 or more on the Three Factor Eating

Questionnaire (Appendix 1). All subjects demonstrated proficiency in spoken and written English and provided written consent after a review of study protocol and procedures prior to the start date.

Thirty subjects (15 male, 15 female) were originally enrolled in the study, 26 individuals (13 males, 13 females) completed the study. The number of participants for statistical significance was based on power calculations to achieve 80% power (α = 0.05) from similar satiety studies which calculated differences utilizing VAS scores.

Study design

The study design was reviewed and approved by the University of Minnesota

Institutional Review Board Human Subjects Committee. The trial was registered at clinicaltrials.gov as NCT04067362. In a randomized, single-blind crossover design, two inulin sources were compared for their effects on satiety and GI tolerance. Treatments included a breakfast smoothie with 0 g fiber, 7 g inulin from Jerusalem artichokes, or 7 g of purified inulin from chicory root (Table 2-1 & 2-2). The treatment dose was based on

what is characterized by the FDA as an excellent source of fiber (5 g or ≥ 20% of the daily value) (FDA, 2019). Treatment sequences were randomized and evenly divided by gender.

Procedure

Subjects were asked to refrain from consuming alcohol, performing vigorous exercise or consuming fiber supplements for 24 hours prior to three scheduled study collections, which were spaced at least one week apart. Subjects were advised on how to record a food diary and asked to complete a 24-hour food diary prior to visits. Food diaries were analyzed with the Nutrition Data System for Research (NDSR, Nutrition

Coordinating Center, Minneapolis, MN, U.S.A.).

Subjects were fasted for 12 hours (8:00 p.m. – 8:00 a.m.) in preparation for each visit. For the first two study collection dates, subjects arrived at the testing site at 7:45 a.m. and remained in a quiet testing room for the full visit lasting approximately 4.5 hours. Subjects were able to use a personal laptop to work quietly or read during the testing period. Location and physical conditions of the test room were consistent for the first two visits.

Following arrival on visit 1, subjects were shown how to complete the VAS and

GI tolerance questionnaire. A breakfast smoothie containing an assigned treatment was provided at 8:00 a.m., with subjects being given 15 minutes to consume the beverage.

VAS were repeated at 15, 30, 45, 60, 90, 120, 180, and 240 minutes post-baseline.

Questions assessing palatability of the treatment were taken at minute 15 in the form of 4 additional VAS questions.

For the third study collection date, smoothies were picked up near campus or delivered to participants prior to 7:30 a.m. on the morning of the study date. Individuals were asked log into a webinar between 7:50 - 9:00 a.m. for prompts regarding baseline

VAS and GI tolerance questionnaires (7:55 a.m.), smoothie consumption (8:00 - 8:15 a.m.) and followup VAS and GI questionnaires in alignment with previous collection days.

Treatment

Breakfast smoothies looked the same and were similar in macronutrient content

(Table 2-1). The breakfast smoothie was made up of 1% chocolate milk (245 ml, Kemps

LLC, St. Paul, MN, U.S.A.) and reduced fat vanilla ice cream (85 g, Kemps LLC, St.

Paul, MN, U.S.A.). The inulin fiber treatment was added either as fresh, raw Jerusalem artichoke (38.8 total g, 7 g inulin) or purified chicory root fiber powder (7.7 g, Frutafit®

IQ, Sensus, The Netherlands, Europe). A hand blender was used to mix ingredients.

Subjects were given 15 minutes to consume the full smoothie.

Visual analog scales

Subjective appetite sensations were recorded by a VAS using a previously validated computerized 100-mm scale (Flint et al., 2000; Appendix 2). The questions were the same at each time point, with the exception of 4 additional palatability questions at minute 15. Questions were displayed in the same order, one at a time, with two opposing statements separated by a 0-100 mm scale. Assessed ratings included hunger (0

= I am not hungry at all; 100 = I have never been more hungry), satisfaction (0 = I am completely empty; 100 = I cannot eat another bite), fullness (0 = not at all full; 100 =

totally full), and prospective food consumption (0 = nothing at all; 100 = a lot). Subjects were asked to click a point on the scale which corresponded with their perceived appetite sensations. Subjects completed the assessment within 1 minute and had the option to review or change answers before they selected to save responses. Additional questions to assess palatability probed smell, taste and overall pleasantness with 0 mm indicating

“good”, and 100 mm indicating “bad”. Aftertaste was scored with 0 mm indicating

“much”, and 100 mm indicating “none”.

GI tolerance questionnaires

GI tolerance was assessed through subject indication of occurrence and severity of common adverse GI symptoms (Appendix 3). Symptoms probed included gas/bloating, nausea, flatulence, diarrhea, loose stools, constipation, GI rumbling, GI cramping (Grabitske & Slavin, 2009). Severity of individual symptoms was based on a

7-point scale (0 = none, 1=mild, 2=moderate, 3=quite a lot, 4=severe, 5=very severe,

6=unbearable). GI tolerance surveys were collected at baseline and were repeated at 30,

60, 120, 180, 240 minutes, 12 hours, and 24 hours post-baseline.

Statistical analysis

Treatments were compared using mixed-effects models (SAS Proc Mixed).

Models included fixed effects of sequence, period, and treatment and a random effect of subject (nested within sequence) to account for the within-subject correlation among repeated measurements. The interaction between treatment and period was tested and was dropped from the model as it was not significant. If the overall F test for the effect

of treatment was significant, pairwise comparisons were conducted to examine which treatment is different from which. No adjustment was made for multiple testing.

Analyses were performed in SAS 9.4 (SAS Institute, Cary NC). P values of less than

0.05 were considered statistically significant.

Results

Mean appetite VAS responses to different forms of inulin were the same based on treatment, with an exception at the 60-minute timepoint where those consuming

Jerusalem artichokes indicated greater feelings of fullness than those receiving the inulin powder or control smoothie (P = 0.016). There were differences in baseline VAS scores between treatments with the control group having a lower mean rating for hunger compared to the two inulin treatments groups (P < 0.005). Additional VAS questions to assess smell, taste and aftertaste were not different based on treatment (P ≥ 0.08). A question probing the overall pleasantness of the smoothie was different between the control group and inulin powder group, with the control group having a mean closer to

“good” versus “bad” anchor on the VAS (P = 0.024).

Very few differences in GI tolerance were observed. A slight increase in flatulence was reported by both treatment groups when compared to a control at the 30- minute timepoint (P = 0.042). An increase in flatulence was also observed at 12 hours post-smoothie consumption from the inulin powder treatment group compared to the control group (P = 0.01).

Discussion

These data do not support the hypothesis that Jerusalem artichokes increase satiety or improve GI tolerance greater than an isolated inulin powder mixed into smoothies. These results are consistent with previous research indicating challenges in finding differences in satiety in a healthy population of normal-weight adults in response to inulin or fructo-oligosaccharide supplementation (Hess et al., 2011; Karalus et al.,

2012; Dewulf et al., 2013; Heap et al., 2016; Peters et al., 2009). Challenges in satiety reporting may be due to the subjective nature of an individual’s perception of their own feelings of fullness and an inability to recognize internal satiety signals. Additionally, while the use of a VAS is the most commonly practiced method for obtaining satiety ratings and was used in this study, external factors including distractions, boredom, social factors in a test setting, and other confounding variables may contribute to error. Thus, these data may not be the strongest indicator of actual satiety and should be interpreted carefully. The body of data supporting satiety in a healthy population with inulin supplementation at similar doses cannot be overlooked and contributes to the equivocal nature of satiety data, particularly with inulin (Nokkaew et al., 2019; Salmean, 2017; Hiel et al., 2019; Hume et al., 2017; Whelan et al., 2006). Energy intake after a period of time following treatment consumption may have provided greater insight into true satiety and would be recommended for similar follow-up studies.

The form of our treatment as a smoothie may have contributed to a lack of difference in satiety ratings. Whole foods have a stronger effect on satiety than beverages (Mourao et al., 2007). Because it is more likely that an individual would consume Jerusalem artichokes as an ingredient mixed in a meal versus pureed and added

to a smoothie, our results could have differed had we offered the Jerusalem artichokes in their more likely consumed form.

We observed little difference in GI tolerance post treatment consumption other than a slight increase in flatulence at 30 minutes for both treatments and at 12 hours from the inulin powder smoothie. Increasing the content of fermentable fiber in the diet is associated with undesirable GI symptoms, so this side effect was not unexpected

(Grabitske & Slavin, 2009). However, the severity of this symptom was reported as mild and varied greatly on an individual basis, so it is reasonable to state that both treatments were just as well tolerated as a control. These data are consistent with previous GI tolerance research with inulin supplementation at similar doses indicating none or minimal GI intolerance (Bonnema et al., 2010; Ripoll et al., 2010).

While certain limitations existed with our study, our data are strengthened by our crossover design which allowed each subject to serve as his or her own control. The smoothie was also provided at a time that most subjects would typically consume breakfast and was made to match calories and macronutrient content typically found in a breakfast meal. Palatability ratings assessing smell, taste and aftertaste were not different based on treatment, which could be a useful way to promote the addition of fiber into a smoothie as it can easily be disguised into the taste of potentially more exciting ingredients. We can conclude that our results show promise for the promotion of

Jerusalem artichokes as a well-tolerated, whole food prebiotic fiber source that could be marketed towards consumers to improve fiber intake.

Table 2-1. Nutrient composition of breakfast smoothies.

Jerusalem Inulin Powder Artichoke Control Smoothie Smoothie

Total Carbohydrate (g) 58.8 59.5 52

Sugars (g) 47.7 44.5 44

Fiber (g) 7 7 0

Inulin (g) 7 7 0

Fat (g) 6.6 6.6 6.6

Protein (g) 12.7 12.3 12.3

Calories 344 332.2 316

Table 2-2. Nutrient composition of breakfast smoothie ingredients.

Jerusalem Milk Ice Cream Fruitafit Artichoke (245 ml) (85 g) (7.7 g) (38.8 g) Total 29 23 6.8 7.5 Carbohydrate (g)

Sugars (g) 27 17 3.7 0.5

Fiber (g) 0 0 7 7

Inulin (g) 0 0 7 7

Fat (g) 2.6 4 0 0

Protein (g) 8.3 4 0.8 0

Calories 176 140 28 16.2

Figure 2-1. Sensation ratings of hunger over time.

Hunger 10.00 9.00 8.00 * 7.00 6.00 5.00 4.00

VAS VAS (mm) Score 3.00 2.00 1.00 0.00 0 15 30 45 60 90 120 180 240 Time (minutes)