YEAST SMELL LIKE WHAT THEY EAT: ANALYSIS of VOLATILE ORGANIC COMPOUNDS of Malassezia Furfur in GROWTH MEDIA SUPPLEMENTED WITH
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! YEAST SMELL LIKE WHAT THEY EAT: ANALYSIS OF VOLATILE ORGANIC COMPOUNDS OF Malassezia furfur IN GROWTH MEDIA SUPPLEMENTED WITH DIFFERENT LIPIDS " THESIS PRESENTED AS PARTIAL REQUIREMENT TO OBTAIN THE TITLE AS MASTER OF SCIENCE IN CHEMISTRY Presented by: MABEL CRISTINA GONZÁLEZ MONTOYA Advisor CHIARA CARAZZONE, Ph.D. Co-advisor ADRIANA M. CELIS, Ph.D." ! CHEMISTRY DEPARTMENT UNIVERSIDAD DE LOS ANDES DECEMBER 2018 BOGOTÁ D.C. ! ! !2 CONTENT 1. ABSTRACT................................................................................................................................... 6 2. ACKNOWLEDGEMENTS ............................................................................................................7 3. INTRODUCTION TO THE GENERAL TOPIC............................................................................. 8 4. PROBLEM STATEMENT AND JUSTIFICATION OF THE RESEARCH PROJECT................... 9 5. OBJECTIVES OF THE STUDY.................................................................................................. 10 5.1. General objective............................................................................................................... 10 5.2. Specific objectives............................................................................................................. 10 6. THEORETICAL FRAMEWORK................................................................................................. 10 6.1. Microbial and fungal VOCs............................................................................................... 10 6.2. Methodologies used for analyse Microbial Volatile Organic Compounds (mVOCs)... 13 6.3. Statistical techniques used for volatile profiling............................................................ 15 6.3.1. Univariate analysis (UVA)........................................................................................ 16 6.3.2. Multivariate analysis (MVA)..................................................................................... 16 7. MATERIALS AND METHODS.................................................................................................... 19 7.1. Chemicals........................................................................................................................... 19 7.2. Media preparation............................................................................................................. 19 7.3. Yeast growth...................................................................................................................... 23 7.4. VOCs Extraction................................................................................................................ 23 7.5. Chromatographic analysis and annotation..................................................................... 24 7.6. Data analysis...................................................................................................................... 24 8. RESULTS AND DISCUSSION.................................................................................................... 25 8.1. Profiling of VOCs from M. furfur...................................................................................... 25 8.2.VOCs profile differentiation in four growth media in exponential and stationary phase 33 8.3. VOCs profile comparison in Agilent GC-MS with preliminary analysis in Thermo GC- MS 43 9. OUTPUT OF THE STUDY.......................................................................................................... 45 10. CONCLUSIONS......................................................................................................................... 45 11. BIBLIOGRAPHY......................................................................................................................... 45 12. APPENDIX.................................................................................................................................. 52 ! ! !3 TABLES INDEX Table 1. Some fungal VOCs their structures, functions and odors. Information of this Table has been reproduced from Morath et al., (2012). ...........................................................................................12 Table 2. Summary of some statistical techniques used in MVA. Information of this Table has been reproduced and combined from Eliasson et al., (2011), Putri & Fukusaki (2014) and Paliy & Shankar (2016). 18 Table 3. Chemical composition of mDixon medium. .........................................................................20 Table 4. Chemical composition of oleic acid (OA), palmitic acid (PA) and OA+PA media. The com- ponents of the minimal medium (MM) are listed in Materials and Methods. .....................................21 Table 5. Volatiles ´ profiles of M. furfur in four media and two growth phases. .................................28 Table 6. Significant statistical univariate parameters found on 17 VOCs from M. furfur and com- pounds highlighted by PCA and PLS-DA. ........................................................................................34 ! ! !4 FIGURES INDEX Figure 1. Main metabolic pathways for mVOCs production. This figure has been reproduced from Schmidt et al., (2015). Volatiles are depicted in coloured dashed rectangles indicating different chemical classes. Representative examples are given per class: alcohols (e.g. ethanol), aldehydes (e.g. benzaldehyde), alkanes (e.g. undecane), alkenes (1-undecene), aromatic compounds (e.g. 2- phenylethanol), esters (e.g. 2-phenylethyl ester), fatty acids (e.g. butyric acid), isoprene, lactic acid, lactones (e.g. gammabutyrolactone), methylketones (e.g. acetone), monoterpenes (e.g. farnesol), nitrogen compounds (e.g. benzonitrile), sesquiterpenes (e.g. pinene) and sulphur compounds (e.g., dimethyl disulphide). .........................................................................................................................11 Figure 2. Headspace (HS) extraction techniques used for sampling mVOCs. Information of this Fig- ure has been reproduced from Agrawal (2004). ...............................................................................13 Figure 3. A graphical representation of the different analytical approaches and informatics tech- niques employed in metabolomics studies. A standard experimental design in metabolomics in- cludes sample preparation and data acquisition using for instance LC–MS, GC–MS, NMR or FT-IR. Once the data have been generated, several types of univariate statistical and multivariate algo- rithms such as discriminant analysis, classification trees and machine learning can be applied to reduce the dimensionality of the data and facilitate biological interpretation, this could also improve the design of subsequent or future experiments. Information of this Figure has been reproduced from Gromski et al., (2015). ..............................................................................................................15 Figure 4. Growth curves of M. furfur in mDixon, oleic acid (OA), palmitic acid (PA) and OA+PA medium. 23 Figure 5. Chromatograms of M. furfur in exponential and stationary phases grown in four media with different FA content. De = Dixon in Exponential phase, Ds = Dixon in Stationary phase, Oe = Oleic acid in Exponential phase, Os = Oleic acid in Stationary phase, OPe = Oleic acid + Palmitic acid in Exponential phase, OPs = Oleic acid + Palmitic acid in Stationary phase, Pe = Palmitic acid in Exponential phase, Ps = Palmitic acid in Stationary phase. ..........................................................26 Figure 6. Pirate-plots representing univariate statistical differences in the absolute peak areas of 17 VOCs in which the grouping variable Medium-Phase significantly affects the emission (P < 0.05 in ANOVA and Kruskal-Wallis test). Variation of data is represented by Bayesian High-Density Intervals after running 10000 iterations. Bars show median values for each treatment. The first line of plots is y-scaled from 0 to 16x107, in the following line y-axis were scaled from 0 to 1.2x107, while the oth- ers two lines are scaled from 0 to 1.2x106. ......................................................................................35 Figure 7. Heat-map of Hellinger transformed and Pareto-scaled peak areas from 61 tentative anno- tated volatiles found in M. furfur analysis using HS-SPME/GC-MS. Dendrograms represent similari- ties between samples (objects) and VOCs (variables) using Euclidean distances as a measure. Clus- ters of Medium-phase samples were colored according to the four growth media, while the com- pounds were colored according to the chemical group. Compounds with * represent VOCs report- ed previously in Fungi in the mVOC 2.0 Database. ...........................................................................37 Figure 8. 3-dimensional PCA of the volatiles’ profiles of M. furfur grown in eight different experi- mental treatments. Ellipses in this plot are confidence intervals of 95% using normal-distribution. 39 Figure 9. a) PLS-DA of volatiles’ profiles of M. furfur grown in eight different experimental treat- ments. Ellipses in this plot are confidence intervals of 95% using t-distribution. b) VIP-Plot showing the more important VOC from M. furfur differentiated between four medium and two growth phas- ! ! !5 es. The VIP score of each compound was calculated as a weighted sum of the squared correlations between the PLS-DA components and the original variables. .........................................................42 Figure 10. 3-dimensional PCA of the volatiles’ profiles