Production of antimicrobials and antioxidants from filamentous fungi A thesis submitted to the National University of Ireland for the degree of Doctor of Philosophy August 2014 By Helen A. Smith, B.Sc. Department of Biology Faculty of Science and Engineering Head of Department: Prof. Paul Moynagh Research Supervisors: Dr. Richard Murphy Table of Contents List of Tables viii List of Figures xi Declaration of Authorship xiii Dedication xiv Acknowledgements xv Abstract xvi Abbreviations xvii 1. INTRODUCTION 1 1.1 Filamentous fungi of therapeutic significance 1 1.1.1 History of therapeutic filamentous fungi 1 1.1.2 Fungal diversity 3 1.1.3 Structure and composition of fungal cells 5 1.1.3.1 Architecture of the cell wall 6 1.1.4 Nutritional requirement, metabolism and development 9 1.2 Secondary metabolites of filamentous fungi 11 1.2.1 Antibiotic production 12 1.2.2 Immunosuppressants 13 1.2.3 Statins 13 1.2.4 Alkaloids 14 1.2.5 Terpenes 14 1.2.6 Polyketides 15 1.2.6.1 Pigment production 16 1.2.6.2 Citrinin 17 1.2.7 Lipids 18 1.2.8 Other bioactive metabolites from filamentous fungi 18 1.3 Biomedicinal applications of filamentous fungi 20 1.3.1 Bioactive compounds 22 1.3.2 Antimicrobial compounds 24 1.3.2.1 Antibacterial activity 25 ii 1.3.2.2 Current antimicrobial strategies 27 1.3.2.3 Antifungal activity 29 1.3.2.4 Antiviral activity 30 1.3.3 Antitumor activity 31 1.3.3.1 Fungal polysaccharides and immune function 31 1.3.4 Antihypertensive activity 35 1.3.5 Concluding remarks 36 1.4 Fungi as a source of natural antioxidants 37 1.4.1 Oxidative stress 37 1.4.2 Natural antioxidants 39 1.4.3 Origin of antioxidants 41 1.4.4 Determination of antioxidant capacity 42 1.4.5 Bioactivity of fungal antioxidant compounds 44 1.5 Biomass generation from filamentous fungi 45 1.5.1 Biomass production using submerged cultivation processes 46 1.5.2 Factors that affect biomass generation and bioactive compound production in SLF 47 1.5.2.1 Culture medium 48 1.5.2.2 pH 48 1.5.2.3 Temperature 49 1.5.2.4 Agitation and inoculation density 50 1.5.3 Production of bioactive compounds and secondary metabolites by SLF 51 1.5.4 Extraction and purification of compounds from fungi 53 1.6 Application of fungi as a natural functional food source 56 1.6.1 Functional food 56 1.6.2 Current production status 57 1.6.3 Natural product industry 58 1.6.4 The future of drug discovery with fungi 59 1.7 Overview 60 iii 1.8 Project Objectives 62 2. MATERIALS AND METHODS 63 2.1 Materials 63 2.1.1 Chemicals, solvents and other reagents 63 2.2 Methods 66 2.2.1 Growth and maintenance of fungi 66 2.2.1.1 Storage and solid cultivation of fungi 66 2.2.1.2 Submerged liquid fermentation conditions 66 2.2.1.3 Separation of mycelial biomass 67 2.2.2 Effect of fermentation conditions on biomass production 67 2.2.2.1 Enhancement of medium composition for biomass generation 67 2.2.2.2 Carbohydrate supplementation 67 2.2.3 Compositional analysis of the fungal cell wall 68 2.2.3.1 Acid hydrolysis and sample preparation 68 2.2.3.2 Compositional analysis by high performance liquid chromatography 68 2.2.3.3 Chitin analysis 68 2.2.4 Preparation of fungal extracts 69 2.2.4.1 Hot water extraction 69 2.2.4.2 Methanol extraction 69 2.2.5 Antimicrobial activity 70 2.2.5.1 Inoculum preparation and storage 70 2.2.5.2 Bacterial adhesion assay 70 2.2.5.3 Agar well diffusion 71 2.2.5.4 Visibility of zones of inhibition (ZOI) 72 2.2.5.5 Antiyeast activity 72 2.2.6 Antioxidant activity 73 2.2.6.1 β-carotene bleaching assay 73 2.2.6.2 ABTS radical cation decolourisation assay 74 2.2.6.3 DPPH radical scavenging ability 74 2.2.6.4 Determination of antioxidant activity using a reducing power assay 75 iv 2.2.6.5 Cupric ion reducing antioxidant capacity (CUPRAC) 75 2.2.6.6 Metal ion chelating ability 76 2.2.7 Determination of antioxidant compounds 76 2.2.7.1 Total phenolic content 76 2.2.7.2 Total flavonoid content 76 2.2.7.3 Condensed tannin determination with vanillin-HCL 77 2.2.8 Phytochemistry 77 2.2.8.1 Thin layer chromatography 77 2.2.8.2 Identification of constituents 78 2.2.8.3 Bioautography 79 2.2.8.4 Antioxidant screening of compounds using bioautography 79 2.2.8.5 Extraction of compounds from TLC plates 79 2.2.9 Component extraction and identification 80 2.2.9.1 Solid phase extraction 80 2.2.9.2 UPLC-DAD analysis of phenolic compounds 80 2.2.9.3 Lipid analysis using liquid chromatography mass spectrometry (LC/MS) 81 2.2.9.4 Lipid extraction prior to gas chromatography mass spectrometry (GC/MS) 83 2.2.9.5 Fatty acid identification and quantification from different extracts using GC/MS 83 2.2.10 Statistical Analysis 84 2.2.11 Safety Information 84 3. BIOMASS GENERATION USING SUBMERGED LIQUID CULTURE 85 3.1 Screening of filamentous fungi for optimal culture conditions 87 3.1.1 Effect of growth media on fungal biomass and pH over time 87 3.1.2 Effect of nutrient supplementation on pH and biomass production 92 3.2 Effect of carbon supplementation on fungal cell wall composition 99 v 3.3 General Conclusions 107 4. ANTIMICROBIAL ACTIVITY OF FILAMENTOUS FUNGI 108 4.1 Pathogen binding capacity of fungal components 108 4.1.1 General discussion 116 4.2 Examination of antimicrobial activity 116 4.2.1 Screening of antimicrobial activity of filamentous fungi 117 4.2.2 Antiyeast activity 127 4.3 General Conclusions 128 5. ANTIOXIDANT ACTIVITY OF FUNGAL EXTRACTS 131 5.1 Extraction process and recovery 133 5.2 Assessment of antioxidant capacity for various fungal extracts 135 5.2.1 Antioxidant capacity by β-carotene/linoleic acid bleaching 135 5.2.2 Radical scavenging activity of various fungi as determined by ABTS+ and DPPH methods 139 5.2.2.1 DPPH radical scavenging ability of extracts 139 5.2.2.2 The ABTS+ radical scavenging activity assay 143 5.2.3 Antioxidant assessment by reduction of metal ions 144 5.2.3.1 The Ferric Reducing Antioxidant Power (FRAP) 145 5.2.3.2 Cupric ion reducing antioxidant capacity (CUPRAC) 150 5.2.4 Chelating ability against ferrous ions 154 5.2.5 Overview of the antioxidant capacity of fungal extracts 158 5.3 Antioxidant capacities of extracts in relation to total phenolics and other compounds 161 5.3.1 Total phenolic content of fungal extracts in relation to antioxidant capacity 163 vi 5.3.2 Relationship between antioxidant activities, as determined by ABTS+, FRAP and chelating assays, and total phenolics, flavonoids and condensed tannin content 169 5.4 General Conclusions 180 6. IDENTIFICATION OF ACTIVE FUNGAL CONSTITUENTS 183 6.1 Isolation of bioactive constituents using TLC 183 6.1.1 TLC method development 184 6.1.2 Screening of compound class using TLC 186 6.1.3 Preliminary isolation and identification of bioactive compounds 194 6.1.4 Bioactivity of isolated compounds from several filamentous fungi 200 6.1.5 General discussion 207 6.2 Bioactive compound analysis 208 6.2.1 UPLC-DAD analysis of phenolic compounds 208 6.2.2 Fatty acid analysis of active isolates separated using TLC 215 6.2.3 Fatty acid identification and quantification from different extracts using gas chromatography mass spectrometry (GC/MS) 234 6.3 General Conclusions 240 7. CONCLUSIONS 242 BIBLIOGRAPHY 251 vii List of Tables CHAPTER 1: INTRODUCTION Table 1.1 Some important medicinal mushrooms 3 Table 1.2 Biological activity of products of various filamentous fungi 21 Table 1.3 Antimicrobial activities of some filamentous fungi 26 CHAPTER 2: MATERIALS AND METHODS Table 2.1 UPLC-DAD solvent gradient levels 81 Table 2.2 List of fatty acid standards 82 CHAPTER 3: BIOMASS GENERATION USING SUBMERGED LIQUID CULTURE Table 3.1 Effect of growth media on fungal biomass and pH over time 88 Table 3.2 Overview of optimal conditions for generation of mycelial biomass 91 Table 3.3 Compositional analysis of fungal cell wall components following supplementation with various carbon sources 101 CHAPTER 4: ANTIMICROBIAL ACTIVITY OF FILAMENTOUS FUNGI Table 4.1 Detection times of bacterial growth t0.05 (h) for E. coli 10778 in microtitration plates coated with fungal cell wall components 110 Table 4.2 Detection times of bacterial growth t0.05 (h) for S. epidermidis 1798 in microtitration plates coated with fungal cell wall components 111 Table 4.3 Antibacterial activity of several filamentous fungi against Gram- negative bacteria in terms of zone of inhibition (mm) 120 Table 4.4 Antibacterial activity of several filamentous fungi against Gram- positive bacteria in terms of zone of inhibition (mm) 121 Table 4.5 Antiyeast activity of several filamentous fungi by agar well diffusion 127 viii CHAPTER 5: ANTIOXIDANT ACTIVITY OF FUNGAL EXTRACTS Table 5.1 Extraction recovery (%) from filtrate 133 Table 5.2 Antioxidant activity (%) of fungal extracts measured by β-carotene bleaching 136 -1 Table 5.3 Scavenging activity (EC50 mg mL ) of fungal extracts on DPPH radicals 141 -1 + Table 5.4 Scavenging activity (EC50 mg mL ) of fungal extracts on ABTS radicals 144 -1 Table 5.5 Reducing power (EC50 mg mL ) of fungal extracts 146 Table 5.6 CUPRAC (λ450nm) of selected fungal crude, hot water and methanol extracts 152 -1 Table 5.7 Chelating effect (EC50 mg mL ) of fungal extracts 155 -1 + Table 5.8 EC50 values (mg mL ) of fungal extracts in ABTS , reducing power and chelating ability assays 159 Table 5.9 Total phenolic content (mg GAE g-1) of various extracts of fungi 164 Table 5.10 Correlation between antioxidant activity and total phenolic content 166 Table 5.11 Total flavonoid content (mg QE g-1) of various extracts 170 Table 5.12 Correlation between antioxidant activitya and total flavonoid content 171 Table 5.13 Total condensed tannin content