Biochemical and Nutraceutical Analysis of Wild and Commercial Mushrooms By Sumaira Sharif M.Phil. (UAF) A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY FACULTY OF SCIENCES UNIVERSITY OF AGRICULTURE, FAISALABAD PAKISTAN 2016 DECLARATION I hereby declare that the contents of the thesis, “Biochemical and Nutraceutical Analysis of Wild and Commercial Mushrooms” are product of my own research and no part has been copied from any published source (except the references, standard mathematical or genetic models/equations/formulae/protocols etc). I further declare that this work has not been submitted for award of any diploma/degree. The University may take action if the information provided is found inaccurate at any stage. Sumaira Sharif 2002-ag-727 The Controller of Examinations, University of Agriculture, Faisalabad. ―We, the Supervisory Committee, certify that the contents and form of thesis submitted by Ms. Sumaira Sharif, Regd. No. 2002-ag-727 have been found satisfactory and recommend that it be processed for evaluation, by the External Examiner(s) for the award of degree‖. Supervisory Committee 1. Chairman __________________________ Dr. Muhammad Shahid 2. Member __________________________ Prof. Dr. Munir Ahmad Sheikh 3. Member __________________________ Prof. Dr. Sajjad-ur-Rahman DEDICATED To My loving and caring Abu Ammi and G Who always provide me compassion, unparalleled and unconditional love and support ACKNOWLEDGEMENTS In the name of Allah, the merciful, the beneficent Words are bound and knowledge is limited to praise Allah Subhanahu WA Taala, the omnipotent, the beneficent and merciful. It is by His grace and mercy alone, that I have come so far and achieved so much. Peace and blessings be upon Holy Prophet Muhammad (SAW), the everlasting source of guidance and knowledge for humanity. With genuine humanity, I acknowledge your aid, God. Please bless this work with your acceptance. I have a pleasure to ensure my sincere gratitude and deepest thanks to Dr. Muhammad Shahid, whose stimulating supervision, guidance and support made this work possible. I am grateful to him for holding me to a high standard thus enriching me with the skills and motivations to refine my research approach. I heartily thank him very much for his valuable help and kindness. I express my gratitude to Dr. Connie M. Weaver, who guided me during my research work in PURDUE University, West Lafayette, USA. Her support, encouragement, inspiring attitude and enthusiasm were impressive. The support, help and company of the whole group are appreciated. I wish to thank Prof. Dr. Munir A. Sheikh, (Ex. Dean Faculty of Sciences) Department of Biochemistry and Prof. Dr. Sajjad-UR-Rahman, Institute of Microbiology University of Agriculture, Faisalabad, for their guidance, encouragement, and help throughout my PhD studies. I am also thankful to my genius friends and fellows, Saira Mohsin, Mohsin Bhai, Dr. Ali Raza, Aisha, Dr. Sumia Akram, Nazia Kanwal, Dr. Sammar Abbas, Salman Bhai, Dr. Muhammad Mushtaq, Dr. Abid Ali, Ali bhai and shamshad Sb and especially to Dr. Asia Atta, Ghulam Mustafa and Dr. Mazhar Abbas for their love providing amenities and friendship. Many thanks are due to my nephews M. Faateh, M. Mahad, M. Haad, M. Ateeb and my nice‘s Wardah, Irha, Maryam and Abeeha for giving me so much joy and happiness. Special thanks to my uncle Iqbal Hussain Qureshi for guidance at each and every fraction of my life. I owe immense feelings of love and thanks for my affectionate and kind Mother and Father, asserts of my life whose prayers will never die whose love will never mitigate, as their prayers are always behind my each success. Thanks to my Brothers, Imran Babar and Farhan Ahmad and Sisters Humaira, Nadia, Guria and Nazia for their care and support. In last but not least I pay my cordial thanks to the special one who lives in my mind and soul, who is nearest, deepest and dearest to me for being there in time of need, for continuous support and encouragement for the fulfillment of my study. Sumaira Sharif TABLE OF CONTENTS Sr. No. TITLE Page No. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 2.1 Selected commercial and wild mushrooms 5 2.1.1 Pleurotus ostreatus 5 2.1.1.1 Scientific classification 5 2.1.1.2 Distribution, nutritional and medicinal uses 5 2.1.2 Lentinus edodes 6 2.1.2.1 Scientific classification 6 2.1.2.2 Distribution, nutritional and medicinal uses 6 2.1.3 Hericium erinaceus 7 2.1.3.1 Scientific classification 7 2.1.3.2 Distribution, nutritional and medicinal uses 7 2.1.4 Volvariella volvacea 8 2.1.4.1 Scientific classification 8 2.1.4.2 Distribution, nutritional and medicinal uses 8 2.1.5 Ganoderma lucidum 9 2.1.5.1 Scientific classification 9 2.1.5.2 Distribution, nutritional and medicinal uses 9 2.2 Nutritional analysis of mushrooms 11 2.2.1 Proximate composition 11 2.2.2 Protein contents 11 i 2.2.3 Carbohydrates and fiber contents 12 2.2.4 Fat contents 13 2.2.5 Ash contents 13 2.3 Mineral contents of mushrooms 13 2.4 Amino acids composition of different mushrooms 14 2.5 Nutraceutical contents of mushrooms 17 2.5.1 Tocopherols 17 2.5.2 Alkaloids 18 2.5.3 Saponins and tannins 18 2.5.4 Carotenoids 18 2.5.5 Lycopene 19 2.5.6 Terpenoids 19 2.6 Antimicrobial potential of mushrooms 20 2.7 Antioxidant potential of mushrooms extracts 21 2.8 Anticancer potential of mushrooms 23 2.9 Lipid lowering effects of mushrooms 24 2.10 Phenolic acids profile of different mushrooms 25 2.11 Sugar contents of mushrooms 26 2.12 Fatty acids composition of mushrooms 26 2.13 Polysaccharides composition 28 3 MATERIALS AND METHODS 29 3.1 Standards, chemicals and reagents 29 3.2 Analytical instruments used in the research 30 3.3 Sample collection and preparation 31 ii 3.4 Selected mushrooms 32 3.5 Molecular identification of Ganoderma lucidum 32 3.5.1 Selection of fungi 32 3.5.2 Preparation of fungal mycelia 33 3.5.3 DNA isolation 33 3.5.3.1 Confirmation of isolated DNA by agarose gel electrophoresis 34 3.5.3.2 DNA quantification 34 3.5.4 Polymerase chain reaction (PCR) 35 3.5.4.1 Primers 35 3.5.4.2 Reaction mixture setup 35 3.5.4.3 Temperature cycling 35 3.5.4.4 Recovery of amplified gene from agarose gel 36 3.5.5 DNA Sequencing and Alignment Search (BLAST) 36 3.6 Proximate analysis of selected mushrooms 36 3.6.1 Estimation of crude fat 36 3.6.2 Estimation of crude protein 37 3.6.3 Estimation of crude fiber 37 3.6.4 Determination of ash contents 37 3.6.5 Determination of total carbohydrates 38 3.6.6 Total energy 38 3.7 Protein estimation by Bradford assay 38 3.8 Amino acids analysis 38 3.9 Minerals analysis 39 3.9.1 Instrumentation 39 3.10 Classical organic solvent extraction (COSE) 39 3.10.1 Extraction of selected mushrooms 40 3.11 Antimicrobial activity 40 iii 3.11.1 Antimicrobial activity by disc diffusion method 40 3.11.2 Minimum inhibition concentration (MIC) 41 3.11.3 Antibacterial activity by well diffusion method 41 3.11.4 Inhibition of microbial biofilm 42 3.12 Phenolic contents and antioxidant activity 42 3.12.1 Folin-Ciocalteu assay 42 3.12.2 Total flavonoid contents 43 3.12.3 DPPH scavenging activity assay 43 3.12.4 Reducing power 43 3.13 Brine shrimp lethality assay 44 3.13.1 Lethality concentration determination 44 Thrombolytic activities of selected mushrooms 3.14 44 extracts and fractions 3.14.1 Sample preparation 45 3.14.2 Collection of blood samples 45 3.14.3 Preparation of clot 45 3.14.4 Weight of clot before lysis 45 3.14.5 Addition of mushrooms extracts and fractions 45 3.14.6.1 Effect of concentration of sample 46 3.14.6.2 Effect of incubation time 46 3.14.6.3 Effect of the amount of sample 46 3.15 Anticancer potential of selected mushrooms 46 3.15.1 In vitro cell proliferation assay 46 α-Glucosidase and tyrosinase inhibition activities of 3.16 46 selected mushrooms 3.16.1 α-Glucosidase inhibition activity 46 3.16.2 Tyrosinase inhibition activity 47 iv 3.17 Phytochemical analysis 47 3.17.1 Qualitative analysis 47 3.17.1.1 Tests for alkaloids (Mayer‘s Test) 47 3.17.1.2 Test for detection of flavonoids (Alkaline reagent test) 47 3.17.1.3 Test for tannins 47 3.17.1.4 Test for saponins by froth test 48 3.17.2 Quantitative analysis 48 3.17.2.1 Alkaloid determination 48 3.17.2.2 Saponins determination 48 3.17.2.3 Flavonoids determination 48 3.17.2.4 Determination of tannins 49 3.17.2.5 Determination of β-carotenes 49 3.18 Analysis of phenolic acids by HPLC 49 3.18.1 Sample extraction and preparation 49 3.18.2 Instrumentation and chromatography 49 3.19 Tocopherols analysis of selected mushrooms 50 3.19.1 Sample preparation 50 3.19.2 HPLC analysis 51 3.20 Fatty acids analysis of selected mushrooms 52 3.20.1 Extraction 52 3.20.2 Instrumentation and chromatography 52 3.21 Determination of monosaccharide by alditol acetates 53 3.21.1 Hydrolysis 53 3.21.2 Reduction 53 3.21.3 O-Acetylation 53 3.22 Partially methylated alditol acetates (PMAA) 53 3.22.1 Methylation 53 3.22.2 Hydrolysis 54 v 3.22.3 Reduction 54 3.22.4 Acetylation 54 3.23 Glucan analysis of selected mushrooms 54 3.23.1 Extraction of crude polysaccharides 54 3.23.2 Purification procedures 55 3.23.3 Fractionation and purification 55 3.23.4 Phenol sulfuric acid assay 55 3.23.5 Characterization of glucans 56 3.23.6 Ultraviolet Visible Spectroscopy (UV/VIS) 56 3.23.7 Fourier Transformation Infrared Spectroscopy (FT-IR) 56 3.23.8 Scanning Electron Microscopy (SEM) 56 3.23.9 Preparation of silver nano-particles using polysaccharides 57 3.24 Statistical analysis 57 4 RESULTS AND DISCUSSION 58 4.1 Molecular Studies of Ganoderma lucidum 59 4.1.1 Isolation of genomic DNA 59 4.1.1.1 Quantification of isolated DNA 59 4.1.2 Polymerase Chain Reaction 59 4.1.3 Sequencing and BLAST results 60 Ganoderma lucidum internal transcribed spacer 1 (ITS), 4.1.4 60 partial sequence 4.2 Proximate analysis of selected mushrooms 62 4.2.1 Proximate composition of wild G.