UUnniivveerrssiittyy ooff MMaassssaacchhuusseettttss AAmmhheerrsstt SScchhoollaarrWWoorrkkss@@UUMMaassss AAmmhheerrsstt Doctoral Dissertations Dissertations and Theses August 2015 PPhhyyssiiccoocchheemmiiccaall AAnndd TTooxxiiccoollooggiiccaall AAsssseessssmmeenntt OOff AAnnttiimmiiccrroobbiiaall ε--PPoollyyllyyssiinnee--PPeeccttiinn CCoommpplleexxeess Cynthia L. Lopez Pena University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/dissertations_2 Part of the Food Chemistry Commons, Food Microbiology Commons, Nutrition Commons, Other Food Science Commons, Polymer Chemistry Commons, and the Toxicology Commons RReeccoommmmeennddeedd CCiittaattiioonn Lopez Pena, Cynthia L., "Physicochemical And Toxicological Assessment Of Antimicrobial ε-Polylysine- Pectin Complexes" (2015). Doctoral Dissertations. 380. https://doi.org/10.7275/6915705.0 https://scholarworks.umass.edu/dissertations_2/380 This Open Access Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks@UMass Amherst. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. PHYSICOCHEMICAL AND TOXICOLOGICAL ASSESSMENT OF ANTIMICROBIAL ε-POLYLYSINE-PECTIN COMPLEXES A Dissertation Presented By CYNTHIA LYLIAM LOPEZ PENA Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2015 DEPARTMENT OF FOOD SCIENCE © Copyright by Cynthia L. Lopez Pena 2015 All Rights Reserved PHYSICOCHEMICAL AND TOXICOLOGICAL ASSESSMENT OF ANTIMICROBIAL ε-POLYLYSINE-PECTIN COMPLEXES A Dissertation Presented by CYNTHIA LYLIAM LOPEZ PENA Approved as to style and content by: ________________________________ D. Julian McClements, Chair ________________________________ Hang Xiao, Member ________________________________ Lynne McLandsborough, Member ________________________________ Richard Wood, Member ________________________________ Eric A. Decker, Department Head Department of Food Science DEDICATION To my parents, who encouraged my scientific curiosity from a young age (and put up with many messes in the kitchen during my scientific quests) and inspired me to follow my dreams. ACKNOWLEDGMENTS I would like to begin by thanking my advisor, Dr. D. Julian McClements, for providing me with the opportunity to pursue a doctoral degree as a member of his lab, as well as his patience and guidance through this learning experience. It was truly an honor to learn from and work with one of the most innovative, talented, and influential Food Scientists in the world. The knowledge and skills I obtained during my doctorate are truly invaluable, and will undoubtedly continue to benefit my professional career and personal life for many years to come. I would also like to thank the exceptional faculty members I had the pleasure to work with during my stay at UMass: Dr. Hang Xiao, for his advice and guidance during the planning and execution of the toxicology studies; Dr. Lynne McLandsborough for her support, insight, and heartening words; Dr. Richard Wood for his openness to discuss ideas and provide extremely helpful advice; and Dr. David Sela not only for the valuable contribution he provided to the toxicology study, but also for his continuous mentorship and advice regarding research and the professional world. As a member of the McClements lab, I had the joy of meeting and working with and alongside many talented and great people from all over the world. I got to learn about many different cultures, learn a few words in other languages, and, most importantly, formed friendships with delightful people. I would like to thank past and present lab members, especially Izlia, Laura, Bicheng, Alessandro, Ben, Bingjing, Iris, Gam, Felix, Becca, Tanu, Ying, Alison, Mauricio, Bengü, Cheryl, Amir, and Jen for v making working in the lab such an enjoyable and unforgettable experience. I would particularly like to thank Jean not only for being a fantastic lab manager, but also for her unwavering friendship. Her advice, help, support, and cheery personality had a deep impact on my experience at UMass. Although I only collaborated with him for a short period of time, Mingyue’s hard work and dedication on the animal studies was crucial to the success of the project. I would also like to thank Xiaomeng for the extremely important role she played in the DNA portion of the animal study. Without them, the toxicology study would have been impossible to complete. There were a number of events that occurred during my doctorate that would not have been resolved without Fran and Deby’s help. I really appreciate them taking the time from their busy schedules to assist me with those administrative hurdles. I would like to thank my friends, particularly Melissa, Dania, Glenda, Nathalie, Sean, Nate, Shashank, Virginia, Anto, Chanelle, Imelda, Jeff, and Upasana not only for their continuous friendship, but also for their constant support, words of encouragement, and comic relief. I would especially like to thank Luis for being such a fantastic friend. My stay at UMass would not have been the same without his friendship. My greatest gratitude and appreciation is to my parents. Not only have they provided me with unconditional love, support, encouragement, and words of wisdom throughout my life, but they are also the inspiration and driving force behind everything I do. vi ABSTRACT PHYSICOCHEMICAL AND TOXICOLOGICAL ASSESSMENT OF ANTIMICROBIAL ε-POLYLYSINE-PECTIN COMPLEXES MAY 2015 CYNTHIA LYLIAM LOPEZ PENA, B.S., CORNELL UNIVERSITY PhD., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor D. Julian McClements ε-Polylysine is an appealing FDA-approved, all natural antimicrobial biopolymer effective against a wide range of microorganisms. Its implementation is greatly limited by its strong cationic charge, which has been linked to instability in food systems, perceived astringency and bitterness, and the ability to inhibit lipid digestion. Previous studies have shown that controlled complexation of ε-polylysine with anionic pectin is able to prevent instability and astringency in simplified model food systems, while maintaining the antimicrobial character of polylysine. Isothermal titration calorimetry, micro-electrophoresis, microscopy, and turbidity analyses of the stability of electrostatic pectin-polylysine complexes in the presence of strongly anionic κ-carrageenan, and carrageenan-polylysine complexes in the presence of pectin at different mass ratios (pH 3.5) suggested that although polylysine-carrageenan interactions were much stronger, polylysine-pectin complexes maintained their stability in the presence of carrageenan. vii In vitro digestion models showed that electrostatic interactions between bile salts and polylysine, which have been suggested as the mechanism for lipase inhibition by polylysine (2ppm), were affected by components in the sample’s matrix. The implementation of an anionic (quillaja saponin) versus a non-ionic surfactant (Tween 20) in corn oil emulsions (2.5%w/w) showed a marked decrease of lipase inhibition, suggesting that electrostatic complexes formed by polylysine with other components prior to its exposure to bile salts in the small intestine may prevent the lipase-inhibiting polylysine-bile salts complex from occurring. Corn oil emulsions (2%w/w) stabilized by Tween 20 subjected to oral, gastric, and intestinal digestion in the presence and absence of mucin and polylysine (200ppm) demonstrated that polylysine forms electrostatic complexes with bile salt-stabilized mixed micelles, potentially decreasing lipid absorption and altering its metabolism. Complexes formed between polylysine and mucin prior to addition of bile salts showed a decrease in insolubilized oil after digestion, suggesting that interactions between polylysine and bile salts were somewhat inhibited. The influence of polylysine and pectin on the in vitro digestibility of animal feed either as individual components or as an electrostatic complex was assessed as part of a subchronic toxicity study. While pectin appeared to increase the rate and extent of lipid digestion, there did not seem to be any inhibition generated by polylysine. viii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ........................................................................................................................ v ABSTRACT ............................................................................................................................................. vii LIST OF TABLES ................................................................................................................................ xiii LIST OF FIGURES ................................................................................................................................ xiv CHAPTER 1. INTRODUCTION ................................................................................................................................ 1 1.1 Background ………………………………………………………………………….. 1 1.2 Objectives ........................................................................................................... 2 2. LITERATURE REVIEW .................................................................................................................... 5 2.1 Antimicrobial Agents in Food Systems ................................................... 5 2.1.1 Natural Antimicrobials ........................................................... 7 2.2 Regulation of Preservatives by the U.S. Food and Drug Administration ........................................................................................... 21 2.2.1 Toxicology Analysis .............................................................. 23 2.3 Gastrointestinal Tract and Experimental Models ............................ 24 2.3.1 Human Digestive Tract........................................................ 24 2.3.2 Digestion, Absorption, and Transport of Lipids ........ 29 2.3.3 In Vitro Simulations of the Digestive Tract ................. 35 2.4 Gut Microbiota ............................................................................................... 40 2.4.1 Gut Health ................................................................................ 41 ix