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Structural Elucidation, Mode of Action, Fermentation and Application in Commercial Apple Juice

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Structural Elucidation, Mode of Action, Fermentation and Application in Commercial Apple Juice Brevibacillin, an Antimicrobial Lipopeptide Discovered from Genus Brevibacillus: Structural Elucidation, Mode of Action, Fermentation and Application in Commercial Apple Juice Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xu Yang, B.S. Graduate Program in Food Science and Technology The Ohio State University 2017 Dissertation Committee: Dr. Ahmed E. Yousef, Advisor Dr. Farnaz Maleky Dr. Hua Wang Dr. Monica Giusti Copyrighted by Xu Yang 2017 Abstract Bacteria are capable of causing food spoilage and foodborne illness which lead to substantial economic losses and public health problems. All food processing steps cannot completely eliminate these microbial contaminations; therefore, antimicrobial agents are usually added into foods for post-processing control. However, resistance has been developed by some bacteria to the widely-used antimicrobial agents, such as potassium sorbate and sodium benzoate. In addition, consumers are now more concerned about the safety of chemical preservatives used in foods, and are more in favor of clean label products. Considering this dilemma, new safe and natural antimicrobial agents are needed in the food industry. On the other hand, the resistance of pathogenic bacteria has become a worldwide threat: in early 2016, a woman in Pennsylvania was reported to carry a “superbug” — a strain of Escherichia coli resistant to colistin. The emergence of this “superbug” induced a global health concern. In an earlier report provided by the US Centers of Disease Control and Prevention (CDC), more than two million people suffered from antibiotic-resistant bacteria with 23,000 deaths annually, and these estimates didn’t even include people who die from other diseases exacerbated by antibiotic-resistant bacteria. Considering all these risks, both in food industry and clinical field, the need for new antimicrobial agents is vital. ii The genus Brevibacillus was reclassified from Bacillus brevis cluster in 1996, based on 16S rRNA gene sequences and further phylogenetic analyses. A variety of antimicrobial compounds were produced from the former B. brevis cluster and the latter genus Brevibacillus, including gramicidin S and tyrocidine, which are applied for antibiotic treatment. However, few papers reviewed antimicrobial compounds produced from the genus Brevibacillus and its previous B. brevis cluster, despite the fact that the genus is a rich source for novel antimicrobial compounds. In the current study, we summarized antimicrobial compounds produced both from the genus Brevibacillus and its previous classification B. brevis cluster. These bioactive compounds belong to diverse structural groups: bacteriocin, lipopeptide, cyclic peptide and polyketides. The major focus was on the structure of these antimicrobial compounds, since some of these agents produced by Brevibacillus spp. share structural similarities that were overlooked by researchers. For example, BT peptide, BL-A60, and bogorol are made up of peptides with structural similarity that was revealed only recently. We also summarized the potential applications of antimicrobial compounds from Brevibacillus in various biological systems as antibiotics, feed additives or biological control agents. Brevibacillin is a new antimicrobial compound discovered in this study. It is produced by Brevibacillus laterosporus OSY-I1 and combats antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant strains of Enterococcus faecalis (VRE) and Lactobacillus plantarum. Brevibacillin was produced from isopropanol extracts of OSY-I1 cells, purified by high-performance liquid chromatography (HPLC), and structurally analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). MS and NMR results uncovered brevibacillin as a linear lipopeptide consisting of 13 amino acids, with an N terminus C6-fatty acid iii chain (FA), 2-hydroxy-3-methylpentanoic acid. The lipopeptide has a molecular mass of 1583.0794 Da and contains three modified amino acid residues: α,ß- didehydrobutyric acid, ornithine, and valinol. The minimum inhibitory concentrations of brevibacillin against a selected panel of Gram-positive bacteria were comparable to that of vancomycin, and these indicators included foodborne pathogenic and spoilage bacteria. Brevibacillin demonstrated high stability under heat or various pH condition: it showed no sign of degradation at 80°C for 60 min and retained at least 50% antimicrobial activity when held at room temperature for 22 hours in acidic or alikaline condition (pH 3.0 and pH 9.0 buffer, respectively). In addition to structural elucidation and minimum inhibitory concentration (MIC) analysis of brevibacillin, mode of action of the novel antimicrobial compound was also investigated. Results of the study proved that brevibacillin disrupts S. aureus ATCC 6538 cytoplasmic membrane through depolarization, increasing membrane permeability and leakage of intracellular potassium. Therefore, cytoplasmic membrane is determined as one of the major targets. Additionally, results have also shown that brevibacillin can bind to lipoteichoic acid (LTA), a cell wall component, before disrupting cell membrane. In summary, we propose that the electrostatic attraction between anionic LTA and cationic brevibacillin induced the accumulation of the antimicrobial agent at cell surface. The accumulation of brevibacillin was then translocated into the cytoplasmic membrane to disrupt its integrity. In addition to LTA binding and membrane disruption, the possibility of one or multiple intracellular targets for brevibacillin may still exist. Production of brevibacillin in liquid medium with subsequent scale-up production in laboratory-scale fermenter was tested. The optimization of liquid medium was first experimented in test tubes to compare the yield of brevibacillin in liquid M9 medium iv supplemented with various nitrogen sources. A semi-synthetic medium was chosen and prepared in a laboratory-scale bioreactor to produce brevibacillin. Antimicrobial activity was detected after 8 hours of incubation in the fermenter and the presence of brevibacillin was proved by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis. The application of brevibacillin was tested in commercial apple juice to inhibit the growth of Alicyclobacillus acidoterrestris, both vegetative cells and spores. A. acidoterrestris is famous for causing spoilage and producing guaiacol, a smoky and medicinal compound to deteriorate product quality in a variety of fruit juices. Brevibacillin containing fermentate generated from the laboratory-scale bioreactor was applied into commercial apple juice for spoilage inhibition: the fermentate which contained brevibacillin at concentration of 0.089 μg/ml exhibited significant (p < 0.05) inhibition against A. acidoterrestris vegetative cells. In comparison, 0.81 μg/ml brevibacillin containing fermentate was required to inhibit the spoilage caused by spores of A. acidoterrestris. The capability of producing brevibacillin in semi-synthetic liquid medium and potent antimicrobial activity of the compound both indicated brevibacillin can potentially be applied as a juice additive to combat spoilage caused by A. acidoterrestris. v Acknowledgement My sincerest thanks go to my advisor, Dr. Ahmed Yousef. He provided the opportunity for me to work with the best group of researchers, and trained me from an individual who knows nothing about food microbiology, to a scientist capable of designing, conducting and publishing food microbiology related researches. I am proud to say that I have spent four and a half wonderful years in Dr. Yousef’s lab and have improved all aspects of my life. I would also like to offer my thanks to my committee members, Dr. Hua Wang, Dr. Farnaz Maleky and Dr. Monica Giusti, for their patience, guidance and encouragement. I am also grateful for my lab associates who have helped me through the years: Dr. Jennifer Perry, Dr. En Huang, Dr. Jin-Gab Kim, Dr. Baosheng Liu, Ebrahim Elkhtab, Dr. Ismet öztürk, Dr. Rui Li, David Kasler, Greg Culbertson, Mustafa Yesil, Nathan Morrison, Michelle Gerst, Yang Song, Emily Holman, Walaa Hussein, Joshua Kim, Alexandra Chirakos and Clifford Park. Special thanks to Dr. En Huang for being a great mentor to guide my research, provide valuable suggestion and feedback. Together we have conquered many difficult projects. Special thanks go to Greg Culbertson for training me all aspectic technique and looking over my shoulder during the lab course. Last but not the least, I would like to thank Dr. Liwen Zhang and Dr. Chunhua Yuan, the two of the best chemists on campus, who make my research a lot easier. vi I would also like to thank my family members. My parents, Jun Yang and Yanxia He who raised me and provided all they can to help me finish my degree. And my beloved grandmother, Jinying Dong, I will miss you for a lifetime. I would also like to say thank you to my wife Jingxin Guo, for putting up with me and encourage me when I was down. Thank you for your unconditional support! vii Vita July 17, 1989…………………………………………Born, Zhengzhou, Henan, China 2012…………………………………………...………B.S., Food Safety and Nutrition China Agricultural University, China 2012-present……………………………………………..Graduate Research Associate The Ohio State University Publications Yang, X., Huang, E., & Yousef, A. E. (2017). Brevibacillin, a cationic lipopeptide that binds to lipoteichoic
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