Efficacy of Lactobacillus salivarius (L28) to control foodborne pathogens in a variety of matrices by Jorge Franco, B.S. A Thesis In Food Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCES Approved Kendra Nightingale, Ph.D. Chair of Committee Leslie Thompson, Ph.D. Alejandro Echeverry, Ph.D. Alexandra Calle, Ph.D. Mark Sheridan Dean of the Graduate School May, 2020 Copyright 2020, Jorge Franco Texas Tech University, Jorge Franco, May 2020 ACKNOWLEDGMENTS I would like to thank my parents Jorge F. and Patricia L. as well as my sisters Patricia F. and Pamela F. for supporting me throughout my life. All of the love and encouragement from y’all helped me continue with my education. I love and appreciate all of you, come visit more often! I would like to extend my gratitude towards the employees of the International Center for Food Industry Excellence (ICFIE), who have helped me get through hard times in and out of the lab. Our lab manager, Miss Tanya, deserves a special thanks for always being around to help with a positive attitude and a smile. I would also like to thank Dr. Mindy Brashears for being my first PI when I was an undergraduate student and giving me the opportunity to explore food science research. I would like to pay my special regards to Dr. Kendra Nightingale, my committee chair, for giving me the opportunity to advance my education and for sharing your knowledge with your students. I would like to recognize Dr. Leslie Thompson and Dr. Alejandro Echeverry for being a part of my committee and being supportive. I would like to thank Savannah F., perhaps the person I grew closest with throughout my masters. We have grown to lift and depend on each other while always remembering to have a fun time. A very warm thank you to both of my dogs Bastion and Sadie, who have helped me de-stress with cuddles and kisses. Lastly, I would like to extend my gratitude to my boyfriend Adam for all of the love and support, you have been my rock for the last four-and-a-half years, I love you. ii Texas Tech University, Jorge Franco, May 2020 TABLE OF CONTENTS ACKNOWLEDGMENTS _____________________________________________ ii ABSTRACT ________________________________________________________ v LIST OF TABLES __________________________________________________ viii LIST OF FIGURES __________________________________________________ ix CHAPTER I ________________________________________________________ 1 LITERATURE REVIEW _____________________________________________ 1 Food Safety _________________________________________________________ 1 Lactic Acid Bacteria (LAB) _____________________________________________ 3 Salmonella spp. ______________________________________________________ 7 Listeria Mmnocytogenes _______________________________________________ 9 Escherichia coli O157:H7 _____________________________________________ 12 Project Justification __________________________________________________ 14 REFERENCES _____________________________________________________ 15 CHAPTER II ______________________________________________________ 22 REDUCTION OF LISTERIA MONOCYTOGENES ATTACHMENT ON STAINLESS STEEL UTILIZING LACTOBACILLUS SALIVARIUS (L28) AT DIFFERENT TEMPERATURES AND CONCENTRATIONS AND ITS EFFICACY AS A PRE-TREATMENT TO PREVENT FUTURE ATTACHMENT ____________________________________________________ 22 ABSTRACT _______________________________________________________ 22 Introduction ________________________________________________________ 23 Materials and Methods ________________________________________________ 26 Stainless Steel Coupon Preparation __________________________________ 26 Inoculum Preparation _____________________________________________ 26 Inoculum Preparation for Treatment _________________________________ 27 Inoculum Application for Pre-treatment ______________________________ 28 Stainless Steel Coupon Processing __________________________________ 29 Statistics model _________________________________________________ 29 Results and Discussion ________________________________________________ 30 Stainless Steel LAB Treatment 25°C and 12°C _________________________ 30 Stainless Steel LAB Pre-treatment 25°C and 12°C ______________________ 32 Conclusion _________________________________________________________ 34 Tables and Figures ___________________________________________________ 35 REFERENCES _____________________________________________________ 41 iii Texas Tech University, Jorge Franco, May 2020 CHAPTER III _____________________________________________________ 43 FATE OF SALMONELLA SPP. PRESENT IN RAW CHICKEN FAT FOR PET KIBBLE COATING IN TANDEM WITH LACTOBACILLUS SALIVARIUS (L28) BIOCONTROL AGENT ___________________________ 43 ABSTRACT _______________________________________________________ 43 Introduction ________________________________________________________ 44 Materials and Methods ________________________________________________ 46 Inoculum Preparation _____________________________________________ 47 Inoculation and Sample Storage_____________________________________ 48 Inoculation and Sample Storage Enumeration of Salmonella and Lactobacillus salivarius in Product Sample Portions ____________________________________ 49 Statistics model _________________________________________________ 50 Results and Discussion ________________________________________________ 50 Pet Kibble ______________________________________________________ 50 Chicken Fat ____________________________________________________ 51 Conclusion _________________________________________________________ 52 Tables and Figures ___________________________________________________ 54 REFERENCES _____________________________________________________ 58 CHAPTER IV ______________________________________________________ 60 DISPLACEMENT OF SALMONELLA AND ESCHERICHIA COLI O157:H7 ON CACO-2 CELLS BY LACTOBACILLUS SALIVARIUS (L28) __________ 60 ABSTRACT _______________________________________________________ 60 Introduction ________________________________________________________ 61 Materials And Methods _______________________________________________ 63 Cell Culture Line ________________________________________________ 63 Subculture Procedure _____________________________________________ 64 Bacterial Strains _________________________________________________ 64 Caco-2 Experimentation___________________________________________ 65 Statistics Model _________________________________________________ 66 Results And Discussion _______________________________________________ 67 Conclusion _________________________________________________________ 68 Tables And Figures __________________________________________________ 70 REFERENCES _____________________________________________________ 72 iv Texas Tech University, Jorge Franco, May 2020 ABSTRACT Listeria monocytogenes (L. monocytogenes), Salmonella, and Escherichia coli O157:H7 (E.coli O157:H7) all pose a significant threat to food safety across the United States (U.S). Policies in place help diminish cross-contamination onto foodstuffs, but prevalence of these organisms has not diminished. Lactic acid bacteria (LAB) have been regarded as Generally Recognized as Safe by the U.S. Food and Drug Administration (FDA), allowing them as food additives. The antimicrobial properties of LAB are well known, but innovative ways to utilize LAB strains in the food industry need to be further explored. The objective of this study was to assess Lactobacillus salivarius (L28)’s ability to inhibit foodborne pathogens in a variety of matrices, which include stainless steel, dry pet food, and human colorectal epithelial adenocarcinoma cells (Caco-2). The experiment was divided into three parts; 1.) Reduction of L. monocytogenes attachment on stainless steel utilizing L28 at different temperatures and concentrations and its efficacy as a pre-treatment to prevent future attachment, 2.) Examination of the fate of Salmonella spp. present in raw chicken fat for pet kibble coating in tandem with L28 biocontrol agent, and 3.) Examination of the displacement of Salmonella and E. coli O157:H7 on caco-2 cells by L28. During the first phase of the project, stainless steel coupons were inoculated with L. monocytogenes for 24h at 12°C and 25°C, the coupons were treated with various concentrations of L28 for 24h. Consequently, L28 significantly (P<0.05) inhibited L. monocytogenes attachment at both temperatures and at most concentrations. After the reduction was noted, stainless steel coupons were pre-treated with L28 at the same concentrations and temperatures, then inoculated with L. v Texas Tech University, Jorge Franco, May 2020 monocytogenes for an hour. All pre-treatment concentrations significantly prevented L. monocytogenes attachment at 25°C, only the high concentration overnight cultures were able to provide significant inhibition at 12°C. For the second phase of the project, raw chicken fat was inoculated with a five- strain cocktail of Salmonella to mimic Salmonella contamination. The Salmonella inoculated chicken fat was assigned to one of two treatments in which half was inoculated with L28, and the other half served as the control. The chicken fat was applied onto the kibble and homogenized in a tumble drum, the target concentration of L28 was 106 CFU/lb of kibble. The kibble was allowed to dry for 4h, then the samples were stored at 25°C for 72h. Time points were plated for Salmonella enumeration at 0h, 4h, 24h, 48h, and 72h for the chicken fat and