LLoouuiissiiaannaa SSttaattee UUnniivveerrssiittyy LLSSUU DDiiggiittaall CCoommmmoonnss LSU Master's Theses Graduate School 2016 SSpprraagguuee DDaawwlleeyy RRaattss WWeerree AAbbllee TToo FFeerrmmeenntt PPuurriififieedd RReessiissttaanntt SSttaarrcchh AAnndd WWhhoollee GGrraaiinn SSttaarrcchh OOnn MMooddeerraattee AAnndd HHiigghh FFaatt DDiieettss Justin Lamont Guice Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Life Sciences Commons RReeccoommmmeennddeedd CCiittaattiioonn Guice, Justin Lamont, "Sprague Dawley Rats Were Able To Ferment Purified Resistant Starch And Whole Grain Starch On Moderate And High Fat Diets" (2016). LSU Master's Theses. 2734. https://digitalcommons.lsu.edu/gradschool_theses/2734 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. SPRAGUE DAWLEY RATS WERE ABLE TO FERMENT PURIFIED RESISTANT STARCH AND WHOLE GRAIN STARCH ON MODERATE AND HIGH FAT DIETS A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in The School of Nutrition and Food Sciences by Justin Lamont Guice B.S., Louisiana State University, 2013 August 2016 ~ To those who believe I am capable of much more than I give myself credit for ~ ii ACKNOWLEDGMENTS I would like to genuinely thank several persons who were instrumental in my development into a consummate professional. First, I would like to thank my mentor and advisor, Dr. Michael J. Keenan, whom without his vast knowledge, patience, and selflessness, I would not be forging this path today. Secondly, I would like to express my thanks and gratitude to my advisory committee members, Drs. Jack Losso, Georgianna Tuuri, and Jianping Ye, who all agreed to advise me to help make my research and this document a powerful example of what students are capable of when we are given the opportunity and tools to succeed. Next, I must thank Ms. Anne Raggio, who not only helped me gain competency in traditional and exotic laboratory techniques, but helped me solve the myriad of “problems” I tended to unearth throughout my graduate career. I must thank my former and current lab mates, Dr. Felicia Goldsmith and Dr. Diana Carvajal–Aldaz, who showed me how to be an adept researcher, and Mr. Ryan Page and Dr. Diana Obanda who helped me clarify thoughts and ideas. Similarly, I owe thanks to Dr. Diana Coulon for her expansive knowledge and logistics about animal research that has aided me tremendously in the studies I’ve conducted. To the many people who have entered my life and made me a better person today by having at least a modicum of faith in me, I thank you all. While I cannot mention you all, please know that I appreciate your contributions to my development. Finally, Dr. Courtney Passow, thank you for always having been there for me. Thank you for your support and encouragement. I don’t feel as if I’ll ever be able to show you the amount of gratitude, respect and love I have for you and that you deserve. Certainly though, I will try. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................. iii ABBREVIATIONS ........................................................................................................................ v ABSTRACT .................................................................................................................................. vii CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1. Significance of Research.............................................................................................. 1 1.2. Objectives .................................................................................................................... 2 CHAPTER 2: LITERATURE REVIEW ........................................................................................ 4 2.1. Resistant Starch ............................................................................................................ 4 2.2. Whole grains ................................................................................................................ 6 2.3. Fat ................................................................................................................................ 8 2.4. Factor Comparisons ..................................................................................................... 9 CHAPTER 3: A STUDY OF THREE INDEPENDENT DIETARY FACTORS IN SPRAGUE DAWLEY RATS: RESISTANT STARCH, WHOLE GRAIN AND FAT (MODERATE, 30%, OR HIGH, 42%) ........................................................................................................................... 11 3.1. Introduction ................................................................................................................ 11 3.2. Materials and Methods ............................................................................................... 14 3.3. Results ........................................................................................................................ 19 3.4. Discussion .................................................................................................................. 31 CHAPTER 4: CONCLUSION ..................................................................................................... 36 REFERENCES ............................................................................................................................. 38 VITA ............................................................................................................................................. 44 iv ABBREVIATIONS ABF%: Abdominal fat percent AMDR: Acceptable Macronutrient Distribution Range CON: Control CHD: Coronary Heart Disease EBW: Emboweled body weight ECW: Empty cecum weight ELISA: Enzyme–linked immunosorbent assay FAT: Fat factor GI: Gastrointestinal GLP1: Glucagon–like peptide 1 HAMRS: High–amylose maize resistant starch HF: High Fat HMWG: High amylose maize whole grain resistant starch IACUC: Institutional Animal Care and Use Committee MF: Moderate Fat v RS: Resistant Starch factor SEM: Standard Error of the Mean SCFA: Short chain fatty acid SD: Sprague Dawley rat WG: Whole Grain factor WWG: Waxy Whole Grain ZDF: Zucker Diabetic Fatty rat vi ABSTRACT Introduction: Whole grain (WG) and fat content of the diet have been previously shown to affect intestinal fermentation and phenotype conferred by high–amylose maize starch (HAM), a form of fermentable dietary fiber. The current study was designed to compare rodent gut health following consumption of whole grain and non–whole grain prebiotics on moderate fat (MF) and high fat (HF) diets using a 2x2x2 factorial design. Methods: MF and HF diets were prepared to contain the following diet sources: (1) control starch with no WG or RS [CON], (2) whole grain waxy corn flour with low RS [WWG], (3) purified HAM resistant starch (RS) [HAMRS], and (4) WG HAM flour rich in resistant starch (WG+RS) [HMWG]. The eight diet conditions were fed to Sprague Dawley rats for six weeks (n = 12 per group). After euthanasia, blood, cecal contents and cecal epithelial cells were collected and gastro–intestinal (GI) tract portions and fat pad (retroperitoneal, perirenal, and epididymal) weights recorded. Results: The presence of purified RS2 resulted in greater fermentation as part of the RS*WG interaction whereas no WG with high RS had the lowest pH of cecal contents. There was a main effect of RS with the high RS groups having the lowest abdominal fat percent of body weight. The presence of WG resulted in consistency of fermentation as groups with WG had similar levels of short chain fatty acids with MF and HF diets as reflected by WG*FAT. No RS*FAT effect was observed because of the WG consistency. Also, a greater butyrate production with WG was demonstrated by RS*WG. Results were primarily driven by two major effects, reflected by the presence of and lack of some significant differences. Purified RS fermented better on MF than HF diets. Diets with RS+WG show similar fermentation on both types of vii diets. These effects may be driven by RS1 vs. RS2 as high RS2 ferments better with MF diets, but presence of RS1 may ferment better with HF diets. viii CHAPTER 1: INTRODUCTION 1.1. Significance of Research Nutritional policies and recommendations regarding an adequate level of fiber (38g/day and 25 g/day for men and women respectively) [1] have not been successfully met as most Americans fail to meet even half the Adequate Intake [2]. New approaches must be employed to maximize nutritional benefits within this reduced fiber consumption profile. Fiber itself is not a singular substance and is understood to have complex chemical arrangements with a variety of functions. One function is the degree to which the fiber is fermentable. Fermentable fiber has a greater bioactive or biological effect than a non–fermentable fiber. It can act as a prebiotic to promote gut health by elevating the growth of beneficial bacteria, which increases the production of short–chain fatty acids [3–5]. The recommendations for dietary fat intake are within an acceptable macronutrient distribution range (AMDR) between 20% and 35% of energy [1], and the average dietary fat intake for Americans is approximately 33% of energy [6]. Studies examining the effects of fat intake on changes in the microbiota have focused on low fat (18–20% of energy) or very high fat (60–70% of energy) diets [7, 8]. The effects of high fat diets on the gut microbiota and the host have been characterized, with a reduction in fermentation as a primary result [9]. Simultaneously, there exists a void in the literature when examining the effects of a moderate fat diet on gut health. However, our lab group has begun to address this issue. In one study, we examined the effects of a moderate fat (26% of energy) diet, and found that low and moderate fat diets had similar effects on the fermentation of a non–whole grain resistant starch prebiotic fiber for reducing body fat [10] and improving bacterial population (unpublished data). However, a robust characterization of fermentation parameters produced from intake similar to what Americans consume (moderate fat versus high fat) does not currently exist. 1