IDENTIFICATION, PROPERTIES, AND APPLICATION OF ENTEROCINS PRODUCED BY ENTEROCOCCAL ISOLATES FROM FOODS THESIS Presented in Partial Fulfillment of the Requirement for the Degree Master of Science in the Graduate School of The Ohio State University By Xueying Zhang, B.S. ***** The Ohio State University 2008 Master Committee: Approved by Professor Ahmed E. Yousef, Advisor Professor Hua Wang __________________________ Professor Luis Rodriguez-Saona Advisor Food Science and Nutrition ABSTRACT Bacteriocins produced by lactic acid bacteria have gained great attention because they have potentials for use as natural preservatives to improve food safety and stability. The objectives of the present study were to (1) screen foods and food products for lactic acid bacteria with antimicrobial activity against Gram-positive bacteria, (2) investigate virulence factors and antibiotic resistance among bacteriocin-producing enterooccal isolates, (3) characterize the antimicrobial agents and their structural gene, and (4) explore the feasibility of using these bacteriocins as food preservatives. In search for food-grade bacteriocin-producing bacteria that are active against spoilage and pathogenic microorganisms, various commercial food products were screened and fifty-one promising Gram-positive isolates were studied. Among them, fourteen food isolates with antimicrobial activity against food-borne pathogenic bacteria, Listeria monocytogenes and Bacillus cereus, were chosen for further study. Based on 16S ribosomal RNA gene sequence analysis, fourteen food isolates were identified as Enterococcus faecalis, and these enterococcal isolates were investigated for the presence of virulence factors and antibiotic resistance through genotypic and phenotypic screening. Results indicated that isolates encoded some combination of virulence factors. The esp gene, encoding extracellular surface protein, was not detected in any of the isolates. Phenotype of antibiotic resistance indicated that all isolates were resistant to kanamycin ii (25 µg/ml). Some isolates were also resistant to tetracycline (16 µg/ml). All isolates were sensitive to ampicillin, erythromycin, and chloramphenicol. Bacteriocins produced by E. faecalis OSY-RM1 and E. faecalis OSY-3E1, referred to as enterocins OSY-RM1 and OSY-3E1, respectively, were chosen for further characterization due to their strong antimicrobial activity against selected indicators. Results indicated that both enterocins were relatively heat stable, sensitive to protease, and stable over a wide pH range. Application of enterocin OSY-RM1 to inactivate selected spoilage and food-borne pathogenic bacteria was investigated. Enterocin OSY-RM1 (44.7 nisin-equivalents IU/ml), extracted by ammonium sulfate precipitation, had a bactericidal effect on L. monocytogenes Scott A and bacteriostatic effect on B. cereus ATCC 11778 in sterile milk. Although enterocin OSY-RM1 was not active against Escherichia coli K12, combination treatment of high pressure processing and enterocin OSY-RM1 caused significant inactivation of E. coli K12 in phosphate buffer saline. Through polymerase chain reaction (PCR) amplification and direct sequencing, the structural gene of enterocin OSY-RM1 was identified with similarity to published enterocin EJ-97. Unlike enterocin EJ-97 which is encoded on a plasmid, enterocin OSY- RM1 is likely encoded on bacterial chromosome since plasmid-cured E. faecalis OSY- RM1 retained its antimicrobial activity. iii Gene encoding enterocin OSY-3E1 was also identified by PCR amplification and direct sequencing and result indicted that its structural gene was similar to that of enterocin AS-48. Unlike enterocin AS-48 that is active against Gram-negative bacteria (i.e. E. coli and Shigella sonnei), enterocin OSY-3E1 has no inhibitory activity against Gram-negative bacteria tested. Enterocin OSY-3E1 was further applied to inactivate spoilage bacterium in food system. Result showed that enterocin OSY-3E1 (25,600 AU/ml), obtained from modified MRS broth containing 5% glucose, can effectively inactivate the spoilage bacterium, Leuconostoc mesenteroides ATCC 14935, in sterile milk. In conclusion, bacteriocins produced by food isolates, E. faecalis OSY-RM1 and OSY-3E1 have potential uses to inactivate spoilage and pathogenic bacteria. iv To my family whom I honor and love v ACKNOWLEDGMENTS I first wish to thank my advisor, Professor Ahmed E. Yousef, for his guidance, encouragements, and supports throughout this project. I have learned from him about hard work and critical thinking. I would like to extend my sincere thanks to Professor Hua Wang and Professor Luis Rodriguez- Saona, for serving as my committee members. I am grateful to Dr. Joy Waite for the proofreading of my thesis. I also would like to thank Dr. Zengguo He, Dr. Yoon Chung, and Yuan Yan, for their exceptional and invaluable cooperation and help during my research. I am also grateful for the help and support from the other members of our laboratory, including Dr. Luis Rodriguez-Romo, Mustafa Varma, Jennifer Perry, Joe Jones, and Amrish-Suresh Chawla. Finally, I wish to thank my parents, my sisters and my brother for their constant support on my study. I also want to express my great appreciations to my husband for his encouragement and support. vi VITA December 11, 1973……………………………….….………Born, Inner Mongolia, China 1995………………….….………Associate degree, Shijiazhuang Medical College, China 2002………………B.S. The Medical Analysis, Third Military Medical University, China 2006 – present..………..…….…Graduate Research Associate, The Ohio State University FIELDS OF STUDY Major Field: Food Science and Nutrition Graduated Program vii TABLE OF CONTENTS Page Abstract …………………………………………………………………………… ii Dedication ………………………………………………………………………… v Acknowledgement ………………………………………………………………... vi Vita ………………………………………………………………………………… ii List of Tables ……………………………………………………………………… x List of Figures ……………………………………………………………………... xi Introduction ……………………………………………………………………….. 1 Chapters: 1. Literature Review ………………...................................................................... 8 Lactic acid bacteria (LAB) ............................................................................ 8 Enterococci .................................................................................................... 8 Bacteriocins ……………………………………………………………...... 9 LAB bacteriocins ………………………………………………………….. 10 Class I bacteriocins ……………………………………………………….. 10 Nisin ……………………………………………………………………...... 12 Class II bacteriocins ………………………………………………………. 13 Class III bacteriocins ………………………………………………….….. 14 Class IV bacteriocins ……………………………………………………... 15 Regulation of bacteriocin production …………………………………….. 15 Regulation of nisin biosynthesis ………………………………………….. 16 Isolation and purification of LAB bacteriocins …………………………… 17 Food biopreservation with LAB bacteriocins …......................................... 17 References …………………………………………………………………. 19 viii 2. Investigation of virulence factors and antibiotic resistance among bacteriocin-producing enterococci isolated from food products …………......... 36 Abstract …………………………………………………………………….. 36 Introduction ………………………………………………………………… 37 Materials and Methods ……………………………………………………... 38 Results and Discussion ……………………………………………………… 44 References…………………………………………………………………… 48 3. Enterocin OSY-RM1, a bacteriocin from Enterococcus faecalis: isolation and characterization of the peptide and structural gene ……............................... 57 Abstract ……………………………………………………………………... 57 Introduction …………………………………………………….…………… 58 Materials and Methods ………………………………………….................... 59 Results and Discussion ……………………………………………………… 68 References …………………………………………………………………… 73 4. Characterization of a bactreriocin produced by Enterococcus faecalis OSY-3E1 isolated from comtė cheese .................................................................. 89 Abstract ……………………………………………………………………... 89 Introduction …………………………………………………………………. 90 Materials and Methods ……………………………………………………… 91 Results and Discussion ……………………………………………………… 95 References……………………………………………………………………. 98 List of References …………………………………………………………………. 109 ix LIST OF TABLES Table Page 1.1 Overview of biochemically and genetically characterized Class II bacteriocins……………………………………………………….……… 31 2.1 PCR primers and product size for detection of virulence factors in enterococcal isolates …………………………………………………. 52 2.2 Antimicrobial activities of supernatants from overnight culture of Enterococcus faecalis isolates ………………………………………….. 53 2.3 Identified enterococcal isolates and their sources ……………………... 54 2.4 Occurrence of virulence factors from genotypic and phenotypic screening among Enterococcus faecalis isolates ………………………... 55 2.5 Resistance of Enterococcus faecalis strains isolated from foods to selected antibiotics ……………………………………………………... 56 3.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-RM1 bacteriocin …………………………… 79 3.2 Susceptibility of enterocin OSY-RM1 to heat, enzyme, and wide pH value range treatments ……..………… ………………………. 80 3.3 Different properties between enterocin OSY-RM1 and enterocin EJ-97 ………………….………………………………….… 81 4.1 Primers used in the PCR reactions for attempted amplification of Enterococcus faecalis OSY-3E1 bacteriocins …………………………….. 103 4.2 Susceptibility of enterocin OSY-3E1 to heat, enzyme, and wide pH value range treatments …………………………………….. 104 x 4.3 Antimicrobial activity and pH value of supernatants from MRS broth supplemented with various concentration