Structures and Antifungal Activities in Foods by Nuanyi Liang a Thesis
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Hydroxy fatty acids: Structures and antifungal activities in foods by Nuanyi Liang A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Science and Technology Department of Agricultural, Food and Nutritional Science University of Alberta © Nuanyi Liang, 2020 Abstract Spoilage and mycotoxin production by mycelial fungi threatens global food security. Climate change and increasingly complex food value chains may exacerbate these concerns. Alternative or complementary antifungal agents for food and agricultural applications may alleviate problems associated with fungi. Hydroxy fatty acids (HFAs) are novel antifungal agents, however, due to the diversity in the molecular structures of hydroxy fatty acids from various sources, their structure-function relationships, modes of action, and potential applications, have not been fully investigated. In this work, several HFAs were extracted from two types of lactobacilli fermented cultures and four types of plant seed oils. HFAs were purified using solid-phase extraction (SPE) or high-speed counter-current chromatography (HSCCC). The purified HFAs were then characterized by LC-APPI-MS/MS and LC-ESI-MS/MS. Eight types of HFAs were tested to challenge the growth of common food-related fungi, including filamentous fungi and yeasts (Chapter 3-5). Structure-antifungal activity relationships demonstrated that the location of the hydroxy groups within a HFA determines its antifungal activity. Mono-HFAs with hydroxy group located at the middle of the fatty acid chain (C9-13) were found to have high anti-mold activities. Remarkably, filamentous fungi, but not yeasts, were sensitive to HFA with a hydroxy group in C9-13 location. To investigate the difference between HFA-resistant yeasts and -sensitive molds, the content of a cellular membrane fluidity modifier, ergosterol, was quantified. With one exception, the HFA-resistant yeasts tend to have high sterol content (Chapter 5). This structure-function relationship elucidated the application of HFA in preventing mold growth while allowing yeast viability and activities. This relationship also indicates that HFA mainly targets fungal strains with a relatively low ergosterol content, but other resistant mechanisms may also be involved. ii A representative HFA was applied in sourdough bread to determine their efficacy in situ (Chapter 6). The anti-mold effect of ricinoleic acid was weaker in flaxseed-flour sourdough bread when compared to wheat-flour sourdough bread. In addition, collaborative work in plants indicated synergistic antifungal effects of coriolic acid with other plant components. These indicate that the antifungal activities of HFA can be affected by the matrixes of food and plants synergistically or antagonistically. Due to the bacterial capacity to convert unsaturated fatty acids into HFA, the bacterial production of HFA and fatty acid metabolism in situ were also evaluated in a fermented sausage model with well-controlled microbiota (Chapter 7). HFAs were detected in the model sausages, but antifungal HFAs were not produced at levels that exert antifungal activity. This supports the compatibility of fermented sausage with the use of mold as ripening agents. Unpublished experimental data also documented that the antifungal activity of lactic acid bacteria in model dairy systems was not dependent on their ability to convert fatty acids to HFA. Results presented in this thesis contribute to understanding the structure-function relationship of HFAs, their mode of action, and their application in situ. This can further enable the development of lipid-based antifungal approaches suitable for application in food and agriculture, which in turn has the potential to reduce food waste and so help to address the increasing global food and agricultural demands. iii Preface This is an original work by Nuanyi Liang. Chapter 2 is a book chapter published as “Liang, N., and Curtis, J. M. (2020) ‘Conventional and Current Methods for the Analysis of Hydroxy Fatty Acids.’ In Lipidomics: Current and Emerging Techniques. Royal Society of Chemistry, Cambridge, UK (pp. 175- 222).” I performed literature review on the distribution of hydroxy fatty acids in nature and conventional and current methods for their analysis, and I wrote the chapter. Jonathan Curtis provided guidance during the writing and reviewed and revised several versions of the chapter. Chapter 3 is an experimental work published as “Chen, Y. Y., Liang, N. Y., Curtis, J. M., & Gänzle, M. G. (2016). Characterization of linoleate 10-hydratase of Lactobacillus plantarum and novel antifungal metabolites. Frontiers in Microbiology, 7, 1561.” I performed analysis of hydroxy fatty acids, and performed fermentation, extraction, isolation and antifungal activity assay of hydroxy fatty acids together with Yuan Yao Chen; other experiments described in the chapter were performed by Yuan Yao Chen. The manuscript was written by Yuan Yao Chen, and revised by me, Curtis, J. M. & Gänzle, M. G. This work was also published in Yuan Yao Chen’s PhD thesis, “Chen, Y. Membrane lipid homeostasis and stress resistance in Escherichia coli and Lactobacillus plantarum. University of Alberta, 2017.” Chapter 4 is an experimental work published as “Liang, N., Cai, P., Wu, D., Pan, Y., Curtis, J. M., & Gänzle, M. G. (2017). High-speed counter-current chromatography (HSCCC) purification of antifungal hydroxy unsaturated fatty acids from plant-seed oil and Lactobacillus cultures. Journal of Agricultural and Food Chemistry, 65(51), 11229-11236.” I performed the majority of lab work with support from Pengfei Cai and Datong Wu related to iv purification of hydroxy fatty acids via HSCCC. The manuscript was written by me and revised by all authors. Chapter 5 is an experimental work submitted as “Liang, N., Dacko, A., Tan. A. K., Xiang. S., Curtis, J. M., & Gänzle, M. G., An investigation into the relationship between the structures of monohydroxy unsaturated fatty acids and their antifungal activities.“ to Food Research International. I conducted the majority of the lab work and mentored Andrea Dacko, who assisted the extraction of hydroxy fatty acids and antifungal test of some of the hydroxy fatty acids, Sheng Xiang, who assisted with the measurement of LAUDAN assay and antifungal test of some of the hydroxy fatty acids, and Alexander Tan, who conducted the analysis of sterol content. The manuscript was written by me and revised by all the other authors. Chapter 6 is an experimental work published as “Quattrini, M.*, Liang, N.*, Fortina, M. G., Xiang, S., Curtis, J. M., & Gänzle, M. G. (2018). Exploiting synergies of sourdough and antifungal organic acids to delay fungal spoilage of bread. International Journal of Food Microbiology (*: Both authors contributed equally to the manuscript.), 302, 8-14.” I participated in the experimental design and performed the fungi-challenging test in sourdough bread with addition of hydroxy fatty acids, with the assistance of Sheng Xiang, and provided mentorship to Mattia Quattrini, who performed the other experiments. The manuscript was written by Mattia Quattrini and revised by me and other co-authors. Chapter 7 is currently prepared for submission to a peer-reviewed scientific journal. I conducted the identification and purification of hydroxy fatty acids, as well as the validation of LC-MS method and quantitation of fatty acids. Kaixing Tang and I performed the extraction of fatty acids from model sausages. The manuscript was written by me and revised by other co-authors. Statistical analysis was performed by Kaixing Tang with the assistance of Weilan Wang. The quantitation of hydroxy fatty acids has been published in Kaixing v Tang’s MSc thesis, “Tang, K. Effect of starter culture on accumulation of taste active amino acids, free fatty acids, and on survival of pathogenic Escherichia coli in dry fermented beef sausages. University of Alberta, 2018.” Appendix 1 is a manuscript prepared for submission. Lucie Nečasová assisted the separation of gangliosides. Yuan Yuan Zhao and Jonathan Curtis contributed to the experimental design. The manuscript was written by Nuanyi Liang and revised by other coauthors. vi This thesis is dedicated to my grandparents, Neng Liang (梁能) and Yuezhen Liang (梁月贞), and to all of the love and kindness I receive in life. vii Acknowledgements Thank you, Dr. Michael Gänzle and Dr. Jonathan Curtis, for the enormous support and guidance. Your dedication to research and teaching, as well as your genuine caring for students, are truly amazing. You are always patient in explaining every piece of knowledge, even the most trivial one; you are always there to help, even in the busiest days; and you are always proud of us, even for the smallest achievement. You encourage us to try new things and explore without the fear of failure. Your unconditional support is really the best thing I can ask for from supervisors. Thank you, my supervisory committee member, Dr. David Bressler for all of your help and advice. You have helped me grow as a scientist with a critical mind. Your outstanding work as both a scientist and an entrepreneur is truly inspiring. Thank you, Dr. Xingfang Li and Dr. Steve Labrie for being my examiners and providing incredible feedbacks. Thank you, Dr. Feral Temelli for being an excellent chair in such a special time. Thank you, Dr. Stephen Strelkov and Dr. Randall Weselake for being my candidacy examiners and helped me expand my knowledge in plant science and lipid science. Thank you, Dr. Lingyun Chen for chairing my candidacy exam. Thank you, all the collaborators, Dr. Yuanjiang Pan, Dr. Pengfei Cai, Dr. Datong Wu, Dr. Yuanyao Chen, Dr. Mattia Quattrini, Dr. Yuan Yuan Zhao, Kaixing Tang, Sabrina Strafella, Andrea Dacko, Alexander Tan, Sheng Xiang, Lucie Nečasová, Fangqin Hu, Azadeh Yasari, Dr. Stephen Strelkov and Dr. Nat Kav. I also want to thank Dr. Grigor Bantchev from United States Department of Agriculture. I feel very fortunate to work closely with incredibly intelligent, hardworking and dedicated scientists like you. Thank you, Mitacs, for your generous financial supports. Your awards for me have let me pursue my dream as a scientist.