Volatile Compounds in Fresh and Processed Oriental Mushrooms

VOLATILE COMPOUNDS IN FRESH AND PROCESSED ORIENTAL MUSHROOMS

A thesis in the fulfilment of the requirements for the degree of

Doctor of Philosophy

Lina Ashmore

B. Sci Hons (Food Science and Technology, UNSW, Australia)

B. Sci (Nutrition, University of Sydney, Australia)

School of Chemical Engineering

UNSW

Sydney, NSW, Australia

March 2014

ORIGINALITY STATEMENT

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed ……………………………………………......

14 March 2014 Date ……………………………………………......

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only).

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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14 March 2014 Date ……………………………………………......

AUTHENTICITY STATEMENT

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

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14 March 2014 Date ……………………………………………......

ACKNOWLEDGEMENT

I would like to thank my supervisor Dr. George Srzednicki and my co-supervisor Dr. John D. Craske for their constant support and constructive guidance, endless encouragement throughout my PhD studies. The transmission of their precious and huge scientific knowledge about the aroma world accumulated during several years of research was of a considerable help for the development of this work.

Avery special thanks for Dr. Noel Arrold from Li-Sun Exotic Mushrooms Ltd for his kindness and prompt supply of the exotic mushrooms.

I would like to thank Camillo Taraborelli and Dr. Victor Wong who trained and assisted me in using equipment in the laboratory and provided help with instruments. Moreover, I wish to thank my friends from the school of Chemical Sciences for making this journey interesting.

A special thanks to my colleague and friend Wiyanto Bie for his help and the pleasant work atmosphere throughout this entire journey and Dr. Veronica Chandra Hioe for her help and support.

I would like to thank my dear friend Shereene for her friendship and for her unconditional support during the difficult times.

At last, but not least, my heartfelt acknowledgements must go to my parents and siblings for their support and patience. I would have not been at this point without their encouragement and understanding.

ABSTRACT

Consumption of mushrooms is as old as the civilisation of people all over the world. The aroma is typical and special for each of the species of edible mushrooms. Despite their high consumption, only few studies were concerned with their aroma. This research aims at characterising the quality of the fresh and processed oriental mushrooms based on their aroma profile. As the volatile compounds (VC) are produced via metabolic and non- metabolic pathways, non-aggressive yet powerful techniques are needed to investigate their chemical composition. Their volatile profile has a potential to be used as quality control instrument useful for the industry. Ambient temperature vacuum distillation was successfully optimised to extract VC from fresh, boiled under reflux, dried at different temperatures (40, 50, 60, and 70 °C), and dried and boiled under reflux mushrooms samples. Four oriental mushroom species, Agrocybe aegerita (chestnut), Flammulina velutipes (enoki), Pleurotus ostreatus (oyster), and Lentinus edodes (shiitake) were studied. VC were identified by gas chromatography-mass spectrometry (GC-MS) using solid phase micro-extraction (SPME).

A storage study of 10 days was also conducted in order to detect changes in VC in the different mushroom species in order to monitor their deterioration. The main C8 compounds present in all the studied mushrooms were decreasing over time while aldehydes ‘content was increasing. Furthermore, sulphur compounds in shiitake contributing to the overall deterioration of the aroma of fresh mushrooms were also increasing.

The quality of dried materials depends on the physical and chemical changes occurring as a function of the processing conditions. Mushroom samples were dried at different temperatures to determine the most suitable one for aroma development. As for shiitake, 40 °C was chosen as the most suitable drying temperature while for chestnut 60 °C and for enoki and oyster 50 °C was the most appropriate. Some compounds such as limonene, furfuryl alcohol, 2-undecanone and some conjugated dienal in chestnut and oyster, pyrazine in enoki mushrooms, and lenthionine in shiitake were only generated via heat. Rehydration temperature played an important role in the liberation of VC. In general the maximum release of alcohols, aldehydes, pyrazine and conjugated diene from the dried material was higher at 100 °C rehydration temperature compared to 25 °C. The concentration of the main C8 compounds in chestnut, oyster mushrooms was decreasing when the dried samples were boiled under reflux as opposed to freshly boiled under reflux. In contrast, their concentrations

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in enoki and shiitake samples did not vary significantly. The qualitative and quantitative composition of the VC can be used as quality indicator and highlight the intensity of the processes in the studied mushrooms in the course of processing and storage. The knowledge of aroma profiles can assist in understanding of the mechanisms of the volatiles formation, generating desirable aromas or spoiling the product. Furthermore, the minimisation of degradation and deterioration of mushrooms during thermal processes requires the knowledge of the kinetics of degradation and process conditions. The outcome of this study is expected to provide a contribution to the science and also to the mushroom and food industry interested in formation and retention of pleasant aroma in fresh and processed mushrooms.

TABLE OF CONTENTS

ACKNOWLEDGEMENT ...... II ABSTRACT ...... III TABLE OF CONTENTS ...... V ABBREVIATIONS ...... VIII LIST OF FIGURES ...... IX LIST OF TABLES ...... XV 1. INTRODUCTION ...... 1

1.1 PROBLEM STATEMENT ...... 1 1.2 BACKGROUND ...... 1 1.3 AIMS ...... 2 1.3.1. Optimisation of vacuum distillation ...... 2 1.3.2. Identification of volatile compounds by Gas chromatography-Mass spectrometry (GC-MS) using solid phase micro-extraction (SPME) ...... 3 1.3.3. Detection of spoilage ...... 3 1.3.4. Optimisation of drying conditions ...... 3

2. LITERATURE REVIEW ...... 4

2.1 BACKGROUND ...... 4 2.2 IMPORTANCE OF VITAMIN D IN MUSHROOMS ...... 5 2.3 ORIENTAL MUSHROOMS: CHARACTERISTICS, NUTRITIONAL AND MEDICINAL PROPERTIES AND CULTIVATION...... 5 2.3.1 Agrocybe aegerita (Chestnut) ...... 6 2.3.2 Flammulina velutipes (Enoki) ...... 7 2.3.3 Pleurotus ostreatus (Oyster) ...... 8 2.3.4 Lentinus edodes (Shiitake) ...... 10 2.4 MUSHROOM AGEING ...... 13 2.5 AROMA QUALITY ...... 14 2.6 VOLATILE AROMAS IN MUSHROOMS ...... 17 2.6.1 Eight-carbon VC and the fungal aroma...... 20 2.6.2 Aroma Production Mechanism ...... 20 2.6.2.1 Oxidation step ...... 20 2.6.2.2 Cleavage of the hydroperoxide compounds ...... 20 2.6.3 Mechanism of sulphur compounds formation ...... 22 2.7 METHODS OF AROMA ANALYSIS ...... 23 2.7.1 Isolation ...... 23 2.7.1.1 Direct solvent extraction ...... 23 2.7.1.2 Subcritical water extraction (SPW) ...... 24 2.7.1.3 Supercritical carbon dioxide extraction ...... 24 2.7.1.4 Distillation ...... 25 2.7.1.4.1 Direct distillation ...... 25 2.7.1.4.2 Steam distillation (direct versus indirect) ...... 25 2.7.1.4.3 Vacuum steam distillation ...... 26 2.7.1.4.4 Simultaneous steam distillation/extraction ...... 27 2.7.1.5 Headspace techniques ...... 27 2.7.1.5.1 Static headspace ...... 28 2.7.1.5.2 Dynamic headspace (purge and trap) ...... 29

2.7.1.5.3 Headspace solid-phase microextraction (HS-SPME) ...... 30 2.7.2 Concentration...... 31 2.7.3 Gas chromatographic analysis (GC) ...... 31 2.7.4 Gas chromatography-olfactometry (GC-O) ...... 33 2.7.4.1 Dilution analysis ...... 34 2.7.4.2 Time-intensity method ...... 34 2.7.4.3 Detection frequency method ...... 34 2.7.4.4 Posterior intensity method ...... 35 2.8 RELATIONSHIP BETWEEN GC-O AND SENSORY DATA ...... 35 2.9 MUSHROOMS PRESERVATION ...... 35 2.9.1 Vacuum Cooling ...... 36 2.9.1.1 Application in the agri-food industry ...... 36 2.9.2 Modified atmosphere packaging (MAP) ...... 37 2.9.3 Drying and aroma compounds ...... 39 2.9.4 Drying using heat pump dryers ...... 39 2.9.5 Freeze-drying (lyophilisation) ...... 41 2.9.6 Vacuum drying ...... 42 2.9.7 Tunnel drying ...... 42 2.10 CONCLUSION ...... 43

3. MATERIALS AND METHODS ...... 44

3.1 MUSHROOM SAMPLES ...... 44 3.1.1 Mushrooms species ...... 44 3.1.2 Mushrooms collection ...... 44 3.2 Detection of spoilage ...... 44 3.3 DRYING ...... 44 3.4 TREATMENTS ...... 45 3.5 AMBIENT TEMPERATURE VACUUM DISTILLATION ...... 46 3.6 CONCENTRATION USING SPME ...... 46 3.7 IDENTIFICATION AND QUANTIFICATION OF VOLATILES COMPOUNDS USING GC-MS ...... 47 3.8 STATISTICAL ANALYSIS ...... 47

4. RESULTS AND DISCUSSION ...... 48

4.1 VC IN AGROCYBE AEGERITA (CHESTNUT MUSHROOMS) ...... 48 4.1.1. Fresh and stored chestnut mushrooms ...... 48 4.1.2 Drying curves ...... 59 4.1.3 Dried (D) chestnut mushrooms ...... 60 4.1.4 Dried and boiled under reflux (DBR) chestnut mushrooms ...... 72 4.1.5 Comparison between treatments ...... 85 4.2 VC IN FLAMMULINA VELUTIPES (ENOKI MUSHROOMS) ...... 100 4.2.1 Fresh and stored enoki mushrooms ...... 100 4.2.2 Drying curves ...... 107 4.2.3 Dried (D) enoki mushrooms ...... 108 4.2.4 Dried and boiled under reflux (DBR) enoki mushrooms ...... 116 4.2.5 Comparison between treatments ...... 126 4.3 VC IN PLEUROTUS OSTREATUS (OYSTER MUSHROOMS) ...... 138 4.3.1 Fresh and stored oyster mushrooms ...... 138 4.3.2 Drying curves ...... 147 4.3.3 Dried (D) oyster mushrooms ...... 148 4.3.4 Dried and boiled under reflux (DBR) oyster mushrooms ...... 157

4.3.5 Comparison between treatments ...... 167 4.4 VC IN LENTINUS EDODES (SHIITAKE MUSHROOMS) ...... 177 4.4.1 Fresh and stored shiitake mushrooms ...... 177 4.4.2 Drying curves ...... 184 4.4.3 Dried (D) shiitake mushrooms ...... 185 4.4.4 Dried and boiled under reflux (DBR) shiitake mushrooms ...... 196 4.4.5 Comparison between treatments ...... 206 4.5 COMPARISON BETWEEN SPECIES ...... 214

5. CONCLUSIONS AND RECOMMENDATIONS ...... 218 REFERENCES ...... 220 APPENDIX A. STANDARD ADDITION ...... 244 APPENDIX B. TIC AND MASS SPECTRA ...... 246 APPENDIX C1. PUBLICATION 1-IFR 20(3):1211-1214 (2013) ...... 260 APPENDIX C2. PUBLICATION 2-IFR 21(1):263-268 (2014)...... 264 APPENDIX C3. PUBLICATION 3-PROCEEDINGS OF I.C OF FABE 2013 ...... 270

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ABBREVIATIONS a.a. Amino acid

AEDA Aroma extraction dilution analysis

ANOVA Analysis of variance

BR Boiled under reflux

CHARM Hedonic aroma response method

D Dried

DBR Dried and boiled under reflux

F Fresh

FD Dilution factor

GABA Gamma aminobutyric acid

GC-MS Gas chromatography mass spectrometer

GC-O Gas chromatography-olfactometry

M Moisture content (%db)

MAP Modified atmosphere packaging

MR Moisture ratio (decimal)

PDMS/CARB/DVB Polydimethylsiloxane/Carboxen/Divinylbenzene

SPME Solid phase micro-extraction

VC Volatile compounds

Subscripts e equilibrium i initial

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LIST OF FIGURES

FIGURE 2.1: CHESTNUT MUSHROOMS (SOURCE: LI-SUN EXOTIC MUSHROOMS)...... 6 FIGURE 2.2: ENOKI MUSHROOMS (SOURCE: LI-SUN EXOTIC MUSHROOMS)...... 8 FIGURE 2.3: OYSTER MUSHROOMS (SOURCE: LI-SUN EXOTIC MUSHROOMS)...... 9 FIGURE 2.4: SHIITAKE MUSHROOMS (SOURCE: LI-SUN EXOTIC MUSHROOMS)...... 11 FIGURE 2.5: CULTIVATION PROCESS OF THE STUDIED MUSHROOMS. (SOURCE: LI-SUN EXOTIC MUSHROOMS, 2010)...... 12 FIGURE 2.6: A DENDROGRAM FOR ODOURS CLASSIFICATION...... 16 FIGURE 2.7: MECHANISM OF 1-OCTEN-3-OL FORMATION ...... 21 FIGURE 2.8: AROMA FORMATION MECHANISM ...... 22 FIGURE 4.1: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN C8 COMPOUNDS EXTRACTED FROM CHESTNUT MUSHROOMS OVER A 10 DAYS STORAGE PERIOD...... 54 FIGURE 4.2: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS DURING THE STORAGE OF CHESTNUT MUSHROOMS OVER A 10 DAYS STORAGE PERIOD...... 55 FIGURE 4.3: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES IN STORED CHESTNUT MUSHROOMS OVER A 10 DAYS STORAGE PERIOD...... 57 FIGURE 4.4: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC EXTRACTED FROM STORED CHESTNUT MUSHROOMS DURING 10 DAYS STORAGE PERIOD...... 58 FIGURE 4.5: MOISTURE RATIO OF CHESTNUT MUSHROOMS AT DIFFERENT DRYING TEMPERATURES VERSUS TIME ...... 60 FIGURE 4.6: VACUUM PACKED CHESTNUT MUSHROOMS DRIED AT 40 °C (LEFT) AND 50 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 67 FIGURE 4.7: VACUUM PACKED CHESTNUT MUSHROOMS DRIED AT 60 °C (LEFT) AND 70 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 67 FIGURE 4.8: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN C8 COMPOUNDS EXTRACTED FROM DRIED CHESTNUT AT DIFFERENT TEMPERATURES...... 68 FIGURE 4.9: NORMALISED RELATIVE CONCENTRATIONS OF ALDEHYDES EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 69 FIGURE 4.10: NORMALISED RELATIVE CONCENTRATIONS OF ALCOHOLS AND KETONES EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 70 FIGURE 4.11: NORMALISED RELATIVE CONCENTRATIONS OF OTHER SELECTED VC EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 71 FIGURE 4.12: NORMALISED RELATIVE CONCENTRATIONS OF C8 VOLATILE COMPOUNDS EXTRACTED FROM DRIED AND BOILED CHESTNUT MUSHROOMS...... 81

FIGURE 4.13: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES VC EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES FOLLOWED BY BOILING UNDER REFLUX...... 82 FIGURE 4.14: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES THEN BOILED UNDER REFLUX...... 83 FIGURE 4.15: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOL (GROUP 1) EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES FOLLOWED BY BOILING UNDER REFLUX...... 84 FIGURE 4.16: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 2) EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES FOLLOWED BY BOILING UNDER REFLUX...... 85 FIGURE 4.17: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF MAIN C8 COMPOUNDS EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS ...... 93 FIGURE 4.18: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF ALDEHYDES VC EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS...... 94 FIGURE 4.19: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF CONJUGATED ALDEHYDES VC EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS...... 95 FIGURE 4.20: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS VC EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS...... 96 FIGURE 4.21: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF KETONES AND SATURATED ALCOHOLS EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS...... 97 FIGURE 4.22: COMPARISON BETWEEN THE NORMALISED RELATIVE CONCENTRATIONS OF VC (GROUP 1) EXTRACTED FROM FRESH (F), BOILED UNDER REFLUX (BR), DRIED AT 60 °C (D 60 °C), AND DRIED AT 60 °C THEN BOILED UNDER REFLUX (DBR 60 °C) CHESTNUT MUSHROOMS...... 98 FIGURE 4.23: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 1) EXTRACTED FROM ENOKI MUSHROOMS DURING 10 DAYS OF STORAGE PERIOD...... 104 FIGURE 4.24: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 2) DETECTED IN ENOKI MUSHROOMS DURING 10 DAYS OF STORAGE PERIOD...... 105 FIGURE 4.25: NORMALISED RELATIVE CONCENTRATION OF 2-ETHYL-1-HEXANOL EXTRACTED FROM ENOKI MUSHROOMS DURING 10 DAYS OF STORAGE PERIOD...... 106

FIGURE 4.26: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 3) EXTRACTED FROM ENOKI MUSHROOMS DURING 10 DAYS OF STORAGE PERIOD...... 106 FIGURE 4.27: MOISTURE RATIOS OF ENOKI MUSHROOMS AT DIFFERENT DRYING TEMPERATURES VERSUS TIME ...... 108 FIGURE 4.28: VACUUM PACKED ENOKI MUSHROOMS DRIED AT 40 °C (LEFT) AND 50 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 114 FIGURE 4.29: VACUUM PACKED ENOKI MUSHROOMS DRIED AT 60 °C (LEFT) AND 70 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 114 FIGURE 4.30: NORMALISED RELATIVE CONCENTRATIONS MAIN ALCOHOLS AND KETONES EXTRACTED FROM DRIED ENOKI MUSHROOMS AT DIFFERENT TEMPERATURES...... 115 FIGURE 4.31: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC EXTRACTED FROM ENOKI MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 116 FIGURE 4.32: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS AND KETONES EXTRACTED FROM ENOKI MUSHROOMS DRIED AT DIFFERENT TEMPERATURES FOLLOWED BY BOILING UNDER REFLUX...... 124 FIGURE 4.33: NORMALISED RELATIVE CONCENTRATION OF SELECTED NEWLY FORMED VC EXTRACTED FROM ENOKI MUSHROOMS DRIED AT DIFFERENT TEMPERATURES FOLLOWED BY BOILING UNDER REFLUX...... 125 FIGURE 4.34: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN C8 COMPOUNDS IDENTIFIED IN ENOKI MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 133 FIGURE 4.35: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS AND KETONES VC (GROUP 1) IDENTIFIED IN ENOKI MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 134 FIGURE 4.36: SELECTED VC (GROUP 2) EXTRACTED FROM ENOKI MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 135 FIGURE 4.37: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 1) EXTRACTED FROM OYSTER MUSHROOMS DURING 10 DAYS STORAGE PERIOD...... 143 FIGURE 4.38: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 2) EXTRACTED FROM OYSTER MUSHROOMS DURING 10 DAYS STORAGE PERIOD...... 144 FIGURE 4.39: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 3) EXTRACTED FROM OYSTER MUSHROOMS DURING 10 DAYS STORAGE...... 145 FIGURE 4.40: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES VC (GROUP 4) EXTRACTED FROM OYSTER MUSHROOMS DURING 10 DAYS STORAGE PERIOD...... 146 FIGURE 4.41: MOISTURE RATIOS OF OYSTER MUSHROOMS AT DIFFERENT DRYING TEMPERATURES VERSUS TIME...... 148 FIGURE 4.42: VACUUM PACKED OYSTER MUSHROOMS DRIED AT 40 °C (LEFT) AND 50 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 153 FIGURE 4.43: VACUUM PACKED OYSTER MUSHROOMS DRIED AT 60 °C (LEFT) AND 70 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 153

FIGURE 4.44: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN C8 VC EXTRACTED FROM DRIED OYSTER MUSHROOMS...... 154 FIGURE 4.45: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS AND KETONES VC EXTRACTED FROM DRIED OYSTER MUSHROOMS...... 155 FIGURE 4.46: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES VC EXTRACTED FROM DRIED OYSTER MUSHROOMS...... 155 FIGURE 4.47: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 1) EXTRACTED FROM DRIED OYSTER MUSHROOMS...... 156 FIGURE 4.48: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS VCS EXTRACTED FROM DRIED AND BOILED UNDER REFLUX OYSTER MUSHROOMS...... 162 FIGURE 4.49: NORMALISED RELATIVE CONCENTRATIONS OF KETONES VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX OYSTER MUSHROOMS...... 163 FIGURE 4.50: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX OYSTER MUSHROOMS...... 164 FIGURE 4.51: NORMALISED RELATIVE CONCENTRATIONS OF OTHER VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX OYSTER MUSHROOMS...... 165 FIGURE 4.52: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS VC IN OYSTER MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 173 FIGURE 4.53: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALDEHYDES VC IN OYSTER MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 174 FIGURE 4.54: NORMALISED RELATIVE CONCENTRATIONS OF OTHER SELECTED VC IN OYSTER MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 175 FIGURE 4.55: BEHAVIOUR OF 1-OCTEN-3-OL AND 1-OCTEN-3-ONE IN OYSTER MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 176 FIGURE 4.56: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 1) AND THEIR BEHAVIOUR OVER 10 DAY STORAGE PERIOD...... 182 FIGURE 4.57: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED MAIN C8 COMPOUNDS AND THEIR BEHAVIOUR OVER 10 DAY STORAGE PERIOD...... 183 FIGURE 4.58: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC (GROUP 2) AND THEIR BEHAVIOUR OVER 10 DAY STORAGE PERIOD...... 184 FIGURE 4.59: MOISTURE RATIOS OF SHIITAKE MUSHROOMS AT DIFFERENT DRYING TEMPERATURES VERSUS TIME...... 185 FIGURE 4.60: VACUUM PACKED SHIITAKE MUSHROOMS DRIED AT 40 °C (LEFT) AND 50 °C (RIGHT) (AUTHOR PHOTOGRAPH)...... 192 FIGURE 4.61: VACUUM PACKED SHIITAKE MUSHROOMS DRIED AT 60 °C (RIGHT) AND 70 °C (LEFT) (AUTHOR PHOTOGRAPH)...... 192 FIGURE 4.62: NORMALISED RELATIVE CONCENTRATION OF THE MAIN C8 IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 193 FIGURE 4.63: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN SULPHUR COMPOUNDS IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 194

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FIGURE 4.64: NORMALISED RELATIVE CONCENTRATION OF ALDEHYDES VC IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 195 FIGURE 4.65: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED ALCOHOLS AND KETONES IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 196 FIGURE 4.66: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN C8 VC IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES AND BOILED UNDER REFLUX (DBR)...... 202 FIGURE 4.67: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN SULPHUR COMPOUNDS IN SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES AND BOILED UNDER REFLUX (DBR)...... 203 FIGURE 4.68: CHANGES IN THE NORMALISED RELATIVE CONCENTRATIONS OF ALDEHYDES COMPOUNDS IN DRIED THEN BOILED UNDER REFLUX (DBR) SHIITAKE AT DIFFERENT TEMPERATURES...... 205 FIGURE 4.69: CHANGES IN THE NORMALISED RELATIVE CONCENTRATION OF ALDEHYDES COMPOUNDS IN DRIED THEN BOILED UNDER REFLUX (DBR) SHIITAKE AT DIFFERENT TEMPERATURES...... 206 FIGURE 4.70: NORMALISED RELATIVE CONCENTRATIONS OF MAIN C8 COMPOUNDS IN SHIITAKE MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 211 FIGURE 4.71: NORMALISED RELATIVE CONCENTRATIONS OF THE MAIN SULPHUR COMPOUNDS IN SHIITAKE MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 212 FIGURE 4.72: NORMALISED RELATIVE CONCENTRATIONS OF SELECTED VC IN SHIITAKE MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 213 FIGURE 7.1: STANDARD ADDITION CURVE OF BENZALDEHYDE...... 244 FIGURE 7.2.: STANDARD ADDITION CURVE OF 3-OCTANONE ...... 244 FIGURE 7.3: STANDARD ADDITION CURVE OF NONANAL...... 245 FIGURE 7.4: TIC OF FRESH CHESTNUT MUSHROOMS AT D0...... 246 FIGURE 7.5: TIC OF FRESH CHESTNUT MUSHROOMS BOILED UNDER REFLUX...... 246 FIGURE 7.6: TIC OF DRIED CHESTNUT MUSHROOMS AT 60 °C...... 247 FIGURE 7.7: TIC OF DRIED (60 °C) AND BOILED UNDER REFLUX CHESTNUT MUSHROOMS...... 247 FIGURE 7.8: TIC OF FRESH ENOKI MUSHROOMS AT D0...... 248 FIGURE 7.9: TIC OF FRESH ENOKI MUSHROOMS BOILED UNDER REFLUX...... 248 FIGURE 7.10: TIC OF DRIED ENOKI MUSHROOMS AT 50 °C...... 249 FIGURE 7.11: TIC OF DRIED (50 °C) AND BOILED UNDER REFLUX ENOKI MUSHROOMS.... 249 FIGURE 7.12: TIC OF FRESH OYSTER MUSHROOMS AT D0...... 250 FIGURE 7.13: TIC OF FRESH OYSTER MUSHROOMS BOILED UNDER REFLUX...... 250 FIGURE 7.14: TIC OF DRIED OYSTER MUSHROOMS AT 50 °C...... 251 FIGURE 7.15: TIC OF DRIED (50 °C) AND BOILED UNDER REFLUX OYSTER MUSHROOMS. 251 FIGURE 7.16: TIC OF FRESH SHIITAKE MUSHROOMS AT D0...... 252

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FIGURE 7.17: TIC OF FRESH SHIITAKE MUSHROOMS BOILED UNDER REFLUX...... 252 FIGURE 7.18: TIC OF DRIED SHIITAKE MUSHROOMS AT 40 °C...... 253 FIGURE 7.19: TIC OF DRIED (40 °C) AND BOILED UNDER REFLUX SHIITAKE MUSHROOMS...... 253 FIGURE 7.20: MASS SPECTRA OF 1-OCTEN-3-OL...... 254 FIGURE 7.21: MASS SPECTRA OF 3-OCTANOL...... 254 FIGURE 7.22: MASS SPECTRA OF 3-OCTANONE...... 254 FIGURE 7.23: MASS SPECTRA OF 2-OCTEN-1-OL...... 255 FIGURE 7.24: MASS SPECTRA OF 2-UNDECANONE...... 255 FIGURE 7.25: MASS SPECTRA OF BENZALDEHYDE...... 255 FIGURE 7.26: MASS SPECTRA OF (E,E)-2,4-HEPTADIENAL...... 256 FIGURE 7.27: MASS SPECTRA OF PHENYLACETALDEHYDE...... 256 FIGURE 7.28: MASS SPECTRA OF LIMONENE...... 256 FIGURE 7.29: MASS SPECTRA OF N-HEXANAL...... 257 FIGURE 7.30: MASS SPECTRA OF N-OCTANOL...... 257 FIGURE 7.31: MASS SPECTRA OF NONANOL...... 257 FIGURE 7.32: MASS SPECTRA OF 1,2,4-TRITHIOLANE...... 258 FIGURE 7.33: MASS SPECTRA OF 2,3,5,6-TETRATHIAHEPTANE...... 258 FIGURE 7.34: MASS SPECTRA OF DIMETHYL DISULFIDE...... 258 FIGURE 7.35: MASS SPECTRA OF LENTHIONINE...... 259 FIGURE 7.36: MASS SPECTRA OF METHYL METHYLTHIOMETHYL DISULFIDE...... 259 FIGURE 7.37: MASS SPECTRA OF DILL ETHER...... 259

xiv

LIST OF TABLES

TABLE 4.1: CONCENTRATIONS OF VC EXTRACTED FROM CHESTNUT MUSHROOMS DURING 10 DAYS OF STORAGE AT 4 °C...... 52 TABLE 4.2: CONCENTRATIONS OF VC EXTRACTED FROM CHESTNUT MUSHROOMS DRIED AT DIFFERENT TEMPERATURES ...... 64 TABLE 4.3: CONCENTRATIONS OF VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX CHESTNUT MUSHROOMS...... 75 TABLE 4.4: COMPARISON BETWEEN CONCENTRATIONS OF VC IN CHESTNUT SAMPLES SUBJECTED TO VARIOUS TREATMENTS...... 89 TABLE 4.5: CONCENTRATIONS OF VC EXTRACTED FROM ENOKI MUSHROOMS DURING 10 DAYS STORAGE...... 102 TABLE 4.6: CONCENTRATIONS OF VC EXTRACTED FROM ENOKI MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 112 TABLE 4.7: CONCENTRATIONS OF VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX ENOKI MUSHROOMS...... 121 TABLE 4.8: COMPARISON BETWEEN CONCENTRATIONS OF VC IN ENOKI SAMPLES SUBJECTED TO VARIOUS TREATMENTS...... 130 TABLE 4.9: CONCENTRATIONS OF VC EXTRACTED FROM OYSTER MUSHROOMS DURING 10 DAYS STORAGE PERIOD...... 139 TABLE 4.10: CONCENTRATIONS OF VC EXTRACTED FROM OYSTER MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 150 TABLE 4.11: CONCENTRATIONS OF VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX OYSTER MUSHROOMS...... 160 TABLE 4.12: COMPARISON BETWEEN CONCENTRATIONS OF VC IN OYSTER MUSHROOMS SUBJECTED TO DIFFERENT TREATMENTS...... 171 TABLE 4.13: CONCENTRATIONS OF VC EXTRACTED FROM SHIITAKE MUSHROOMS DURING 10 DAYS STORAGE...... 179 TABLE 4.14: CONCENTRATIONS OF VC EXTRACTED FROM SHIITAKE MUSHROOMS DRIED AT DIFFERENT TEMPERATURES...... 188 TABLE 4.15: CONCENTRATIONS OF VC EXTRACTED FROM DRIED AND BOILED UNDER REFLUX SHIITAKE MUSHROOMS...... 200 TABLE 4.16: COMPARISON BETWEEN CONCENTRATIONS OF SELECTED VC IN SHIITAKE SAMPLES SUBJECTED TO DIFFERENT TREATMENTS...... 209 TABLE 7.1: RAW DATA FOR THE STANDARD ADDITION CURVE OF BENZALDEHYDE...... 244 TABLE 7.2: RAW DATA FOR THE STANDARD ADDITION CURVE OF 3-OCTANONE...... 245 TABLE 7.3: RAW DATA FOR THE STANDARD ADDITION CURVE OF NONANAL...... 245

1. INTRODUCTION

1.1 Problem statement

To characterise the quality of oriental mushrooms on the basis of their aroma profile.

1.2 Background

Since ancient times, mushrooms have been considered as a special type of food. The Greeks thought mushrooms gave power for warriors in the battlefield, the Pharaohs valued them as a delicacy, the Romans on the other hand considered them as a “food of the Gods” offered only on exceptional events, the Chinese viewed them as “elixir of life”, and the Mexican-Indians used them for medicinal purposes as well as hallucinogenic substances in religious ceremonies and in witchcraft (Miles and Chang, 2004).

Mushrooms have been widely consumed as a food or food ingredient because of their highly desirable flavour, aroma, and nutritional properties. They are source of proteins providing essential amino acids; they also contain carbohydrates, are a moderate source of crude fibre and ash and have low energetic value. The trends towards “functional foods” is causing reassessments of volatiles for effects going beyond the sensory properties such as antioxidative (Vuotto et al., 2000), antitumour (Sheu et al., 2007) and antiatherosclerotic effects (Guillamond et al., 2010).

Storage stability is an important factor in extending the shelf life of mushrooms by preventing off-flavour formation as well as discolouration and reduction in the nutrition value. Fresh mushrooms have a short shelf life therefore it is crucial to market them soon after harvest or to preserve them using processes such as cooling and drying. Drying is effective in preserving mushrooms as it removes enough water to inactivate the enzymes and micro- organisms. Drying is a simultaneous mass and heat transfer process where water is removed by diffusion from inside the food product to the air-food interface and from the interface to the air stream by convection. Some fresh mushrooms exhibit a slight odour, but upon drying or crushing, a new characteristic aroma gradually develops. Two main routes of VCs formation can be differentiated: biosynthesis of compounds via genetically determined pathways (Takeoka et al., 1995) and thermally induced reactions (Parliment et al., 1989).

Many mushrooms species have been poorly characterized and their aroma profile depends on proper processing conditions such as optimization of drying conditions. These conditions

have a major effect on the volatile profile as intense and pleasant aroma is of interest for the consumer and hence the industry as a tool for quality monitoring. The study of aroma compounds as well as their importance and release from the food matrix are crucial for the characterisation of a food. Nevertheless, despite their high consumption, only few studies are concerned with aroma.

The extraction technique is very important, as the compounds extracted are to be a true representation of the typical mushroom odour. In previous studies, simultaneous distillation- extraction (SDE), solid phase micro-extraction (SPME), and solvent extraction were used separately to analyse volatiles in mushrooms. However, the former technique is associated with the formation of secondary flavours as a result of degradation caused by lipid oxidation (Barcarolo et al., 1996) as well as products resulting from browning reactions (Werkhoff et al., 1998) and compounds obtained via heating are mixed with the desired components. Werkhoff et al. (1998) argued about the possibility of thermally induced artefacts yielding a falsified aroma and concluded that SDE produces more representative aromas of the food sample if it operates under vacuum. In the comparison of SPME with solvent extraction, the latter was found to be a more powerful extraction technique (Ewen, 2004).

Therefore, as volatile compounds are produced via metabolic and non-metabolic pathways, non-aggressive techniques are needed to investigate the chemical composition of the volatiles. The most appropriate techniques are such that result in the detection of as many compounds as possible.

1.3 Aims

In order to fill the information gap and characterise fresh and dried oriental mushrooms based on their aroma profile, the specific objectives of the present study are as follows:

1.3.1. Optimisation of vacuum distillation

Vacuum distillation will be used as the main extraction technique since the method of isolation determines the quality and yield of the extracts. Previously vacuum distillation performed on yellow passion fruit yielded flavour volatiles representing the typical aroma of the yellow passion fruit. Furthermore, this technique allowed the identification of compounds in yellow passion fruits that have never been reported before (Werkhoff et al., 1998). This technique was also used in a study to compare the volatile constituents in cold-pressed

bergamot oil and a volatile oil and it was concluded that extracts from vacuum distillation were of superior quality (Belsito et al., 2007).

1.3.2. Identification of volatile compounds by Gas chromatography-Mass spectrometry

(GC-MS) using solid phase micro-extraction (SPME)

The volatiles condensed in the cold traps are subject to extraction via SPME followed by GC/MS identification. SPME is sensitive, cost efficient and becomes a very efficient instrument when used in conjunction with gas chromatography-mass spectrometry analysis, allowing the concentration of the volatiles in a solvent-free manner.

1.3.3. Detection of spoilage

Mushrooms are perishable foods with a short shelf life of 1-3 days at room temperature. Their high moisture content as well as their complex matrix makes them susceptible to spoilage and quality deterioration (Manzi et al., 2004). The chemicals responsible for off odours result from chemical reactions occurring within the food matrix itself (e.g., lipid oxidation, enzymatic action). No type of food or food ingredient is immune to off-odour development. Some of the volatiles formed during ageing may be perceived as off-odours leading to rapid quality deterioration, therefore a need for an early detection is crucial for quality control.

1.3.4. Optimisation of drying conditions

The quality of dried foods depends on the physical and chemical changes occurring during processing and storage. These changes affect the physical structure and hence texture, appearance, and rehydratability. There is a difference between the volatile profile of fresh and dried mushrooms as losses of some volatile compounds may be due to the inactivation of volatiles forming enzymes and loss of precursors as new by products may be introduced by autoxidation and Maillard reactions.

2. LITERATURE REVIEW

2.1 Background

Mushrooms as defined by Chang and Miles (1992) are “a macrofungus with a distinctive fruiting body which can be either hypogeous or epigeous, large enough to be seen with the naked eye and to be picked by hand”. Mushrooms have been widely consumed as a food or food ingredients in many cultures and not only as a part of the normal diet, but also as a delicacy because they have a highly desirable taste and aroma (Mau et al., 1997; Nollet, 2009). They are the edible species of a large group of fungi belonging to division Basidiomycota in order Agaricales. Edible mushrooms refer to fungal species that are harvested either wild or cultivated. The number of mushroom species on Earth is estimated at 140,000, of which around 10% are known. Among them, ~50% are edible, more than 2000 are safe, and around 700 species possess pharmacological properties (Chang 1999; Wasser and Weis 1999; Reshetnikov et al., 2001). The most widely cultivated mushroom in the world is the button mushroom (A. bisporus) which gained their commercial value due to the flavour (Kües and Liu 2000). Edible mushrooms are characterised by short-shelf life (3-4 days at ambient temperature) due to the post-harvest changes. These changes are due to their high moisture content as well as to the high activity of enzymes such as protease or polyphenol oxidase, responsible for protein and sugar decrease and for a browning reaction during storage (Manzi, 2004).

Attributes such as butyrace- (butter-like), delicat- (delicious), olid- (ambrosial), suav- (sweet) or nidoros- (pungent) are referred to as aroma descriptors. Distinctive odours have long been and are still used as taxonomic markers for mushroom species (Larsen and Frisvad, 1995). The pleasant aromas along with the nutritional and medicinal properties have triggered the world-wide industrial production of various edible fungi. The investigation and identification of aroma compounds as well as determination of their relevance and release from the food matrix are of extreme importance for the characterisation of a food.

This review focuses on the importance of mushrooms and on their volatile compounds. It evaluates numerous methods used to isolate volatile compounds from other

constituents such as water, carbohydrates, lipids, vitamins, minerals etc. These methods are for extraction and identification of the isolated compounds. Processing methods, such as drying that impact on the volatile profile are also discussed.

2.2 Importance of vitamin D in mushrooms

Vitamin D plays an important role in calcium metabolism and bone mineralisation. It is the generic name of a closely related group of vitamins exhibiting similar biological activity of cholecalciferol (vitamin D3). Fungi are known as a good source of phytosterols which reduce cholesterol absorption and hence lower plasma cholesterol and low-density lipoprotein cholesterol (Shin et al., 2003). The most abundant phytosterol in mushrooms is ergosterol which, when exposed to UV light, undergoes photolysis to yield a variety of photoirradiation products mainly the previtamin D2, tachysterol, and lumisterol. The previtamin D2 then undergoes spontaneous thermal rearrangement to vitamin D2 (Mattila et al., 1994). Although most mushrooms contain ergosterol, only chanterelles have been reported to contain significant amounts of natural vitamin D2, ergocalciferol (Mattila et al., 1994; Rangel-Castro et al., 2002). Teichmann et al., (2007), in their investigations to study the effect of UV irradiation on sterol and vitamin D2 in cultivated and wild mushrooms confirmed that mushrooms exposure to UV-C irradiation (254 nm) could greatly increase vitamin D2 after harvest but UV-A (366 nm) showed rather modest vitamin D2-producing capability. Jasinghe and Perera (2005) reported that shiitake mushrooms contained a remarkably high concentration of ergosterol and its distribution varied in different parts of the mushroom tissue where the concentration in the gills was about twice higher than that of the outer layer of cap. Furthermore, it was suggested that optimum irradiation temperature and moisture content of around 70-80% maximises the yield of vitamin D2.

2.3 Oriental mushrooms: Characteristics, nutritional and medicinal properties and cultivation.

Mushrooms have been valued by human kinds for millions of years because of their nutritional and medicinal values. The following sections discuss the functional properties of the mushrooms studied in this dissertation as well as their cultivations.

2.3.1 Agrocybe aegerita (Chestnut)

Chestnut, currently classified in Agaricomycetes in family of Strophariaceae, is widely distributed in Korea, Japan, Europe and Africa. A. aegerita is fibrous with a peculiar fragrance compared to other mushroom species (Lo and Cheung, 2005). It is an edible mushroom with a fruity-wine flavour (Courtecuisse and Duhem, 1994). This mushroom is nutritionally important as it contains high moisture content (~90%), is a source of carbohydrates and fibre, it has also low energetic value and is rich in vitamins like vitamin B and minerals like sodium, potassium, phosphorus, and selenium (Ghorai et al., 2009). It is one of the popular mushrooms known to possess anti-fungal and antitumour properties. It may also assist in the alleviation of inflammatory conditions as well as reducing the incidence of stomach and breast cancer (Diyabalanage et al., 2008). That is because it has strong antioxidant effect and free radical scavenging ability which are correlated with its high level of phenolic compounds (Lo and Cheung, 2005).

As for its growth parameters, the spawn run does not require any light and has an incubation temperature of 21 to 27 °C with a %RH 95-100 and CO2 levels higher than 20000 ppm that lasts for 20 to 28 days. The primordial formation necessitates an initial temperature of 10 to 16 °C with a %RH 95-100 and CO2 levels less than 2000 ppm for a shorter period of time (7-14 days) and 500-1000 lux while the development of the fruiting body requires an incubation temperature of 13-18 °C and %RH 90-95 and CO2 levels less than 2000 ppm for 5 to 6 days. Its crop cycle involves two flushes with 10-14 days apart (Li-Sun Exotic mushrooms, 2010).

Figure 2.1: Chestnut mushrooms (Source: Li-Sun Exotic Mushrooms).

The cultivation of chestnut mushrooms is divided into several stages as shown in Figure 2.5. The first stage involves the receipt and mixing of raw materials (saw dust, rice bran,

pollard, lime, water, and gypsum) for the substrate production. The mixture is then filled into autoclave bags, sterilised for 2.15 h at 121 °C, cooled down, then inoculated with a pure culture of mushrooms (in this case chestnut ). It is then followed by an incubation stage at 25 °C then transferred to the tunnel for cropping, harvesting, packing and finally delivery (Li-Sun exotic mushrooms, 2010).

2.3.2 Flammulina velutipes (Enoki)

Enoki mushrooms, also known as winter mushroom and Enokitake is characterised for its abnormal feature of small caps and long stipes (Stamets, 2000). It requires low temperature for growth and therefore it is known as “low temperature mushroom”. Its cultivation using a sawdust mix in bottles started in Japan in 1928 and 1970 in Taiwan where the sawdust base is mixed with water and rice bran using polypropylene bottles to which the spawn is added. Once the mycelium is grown, the bottles are placed in the dark at 10-12 °C in order to stimulate the formation of the fruiting body primordial. Upon stems formation, the temperature is reduced to 3-5 °C. When the stems reach 2 to 3 cm, a cylinder of wax is placed around the bottle to provide stem support while preventing the formation of the cap and promoting the elongation of the stem. From a nutritional point of view, enoki mushrooms have high moisture content (~89%) and the dry mass includes carbohydrates (58%), protein (27.5%), fat (7%), and ash (7.4%) (Ko et al., 2007). It is also a source of amino acids with the most abundant compounds being lysine and glycine, the former being an essential limiting amino acid in most vegetable proteins. According to Chiang et al., (2006), enoki is a good source of the free amino acid gamma-aminobutyric acid (GABA) which functions as a neurotransmitter. It is also reported that this mushroom contains compounds, such as glucans and heteropolysaccharides involved in the prevention of hypercholesterolemia and cancer (Yoshioka et al., 1973; Leung et al., 1997) as well as inducing antibody production by modulation of TH-cell differentiation and function (Ghorai et al., 2009).

As for growth parameters, the spawn run does not require any light for a period of 14 to 18 days at an incubation temperature ranging between 21 and 24 °C, %RH 95-100 and with CO2 levels higher than 5000 ppm. Meanwhile, primordial formation necessitates a lower incubation temperature (7-10 °C) lasting for 3 to 5 days with %RH 95-100 and

CO2 levels between 2000 and 4000 ppm using light(100-200 lux). As for the development of the fruiting body, it requires a higher temperature (10-16 °C), longer

time (5-8 days), the same C02 levels and light requirements compared to the primordial formation but with lower relative humidity (%RH 90-95). Its cropping cycle consists of 2 to 3 flushes with 10-12 days apart (Li-Sun Exotic Mushrooms, 2010). Its cultivation method is similar to that of chestnut mushrooms discussed in 2.3.1 and is shown in Figure 2.5.

Figure 2.2: Enoki mushrooms (Source: Li-Sun exotic mushrooms).

2.3.3 Pleurotus ostreatus (Oyster)

Oyster mushroom (Pleurotus ostreatus) is one of the most widely cultivated mushrooms (Bano and Rajarathnam, 1986). Pleurotus species belongs to phylum Basidiomycota that produces oyster shaped mushrooms and accordingly they have been called oyster mushrooms. They can be white, blue or pink comprising about 40 species (Zadrazil, 1978). It is regarded as a delicacy due to its characteristic texture and pleasant flavour. P. ostreatus mushrooms have a high moisture content of ~90 % , are a good source of carbohydrates and proteins, low in fat generally the unsaturated are predominant over the saturated fatty acids, and are a source of minerals (potassium, phosphorous, magnesium) and vitamins (folic acid, thiamine, riboflavin, vitamin C). They also contain ergosterol which can be converted to vitamin D under ultraviolet irradiation (Bano and Rajarathnam, 1988). The main compounds responsible for the aroma of fresh, stored and thermally processed mushrooms are a series of aliphatic alcohols and ketones known as C8 compounds: 1-octen-3-ol, 2-octen-1-ol, 3-octanol, 1-octen-3-one, and 3-octanone. These compounds are formed by the oxidation of linoleic acid by the

enzymes lypoxygenase and hydroperoxide lyase (Tressl et al., 1982; Chen and Wu, 1984; Mau et al., 1992; Assaf et al., 1997).

As for the growth parameters, the spawn run has an incubation temperature of 24 °C with a relative humidity ranging between 85 and 95 % and CO2 levels between 5000 and 20000 ppm lasting for a period of 12 to 21 days. Meanwhile, the primrodia formation requires a lower incubation temperature (10-15.6 °C), lower CO2 levels (less than 1000 ppm), a higher relative humidity (95 to 100 %) and a shorter period of time (3 to 5 days) compared to the spawn run. Furthermore light is an important component in this stage (1000 to 2000 lux). As for the fruiting body development, it has the same CO2 levels and light requirements as the previous stage, however, it needs a higher incubation temperature (10 to 21 °C), a lower relative humidity (85 to 90 %) that continues for 4 to 7 days. Its cropping cycle involves 3 to 4 flush cycles, 7 to 14 days apart over 45 to 55 days (Source: Li-Sun Exotic Mushrooms).

Figure 2.3: Oyster mushrooms (Source: Li-Sun Exotic Mushrooms).

The cultivation of oyster mushrooms involves the receipt and mixing of raw materials and chemicals (benlate, cotton seed hulls, rice bran, pollard, lime, water, and gypsum) which are then filled into trays for a pasteurisation process that lasts 10 h at 65 °C, cooled down then a pure culture of oyster mushroom is added, incubated then transferred to the tunnel for cropping, harvesting, packing, and delivery.

2.3.4 Lentinus edodes (Shiitake)

Shiitake mushroom, also known as Lentinus edodes, is the second most common mushroom in the world. It was introduced into Japan during the 15th century then its production was improved by inoculating the logs with the mushroom spawn. It is now cultivated throughout Europe, America, Australia, New Zealand, Thailand, and most significantly in China (more than 500.000 tonnes/year). The use of the plastic-bag culture techniques caused an increase in its production worldwide. It has high moisture content (88-92%), low energetic value and contains proteins (26% of dry weight), lipids (mainly linoleic acid), carbohydrate, fibre and minerals, vitamins B-1, B-2, C, and ergosterol, the D provitamin. Besides its nutritive content, several compounds isolated displayed immunomodulatory, hypocholesterolemic, and antimicrobial properties (Chang, 1996). The action of ß-D-glucan “Lentinan”, an antitumor polysaccharide, is due to the activation of the host’s immune functions where it binds to the surface layer of lymphocyte or specific serum protein, activating macrophage, T-cells, NK cells, and increases the production of interleukins (IL-1, IL-2) and interferons related to the activation of effector cells (Hamuro and Chihara, 1985). Lentinus edodes among other mushrooms have been consumed for years in Korea, China, Japan and those practices form the basis of scientific studies of fungal medicinal activities, especially in the field of stomach, prostate and lung cancer (Ying et al., 1987, Hobbs, 1995 and 2000, Wasser and Weis 1997 and 1999). Heat-treated shiitake have cancer-preventive properties. They contain thiazolidine-4-carboxilic acid that blocks the formation of carcinogenic N- nitroso compounds from nitrates (which exist in vegetables and meat) that is produced in dried and heated mushrooms (Wasser and Weis, 1997; Hobbs, 2000; Stamets, 2000). The amount of nutrients and bioactive compounds differ in various strains and are affected by the substrate, fruiting bodies, and methods of cultivation (Wasser, 2005). Other studies carried by Bianco (1981) showed that L. edodes was effective against Staphylococcus aureus and Bacillus subtilis. It also has antithrombotic properties (Hokama and Hokama, 1981).

The growth parameters of shiitake mushrooms in this study are divided into four stages.

Spawn run with the incubation temperature 21-27 °C, %RH 95-100, with C02 level above 10000 ppm and light requirements of 50 to 100 lux for a duration of 35-70 days. Primordia formation necessitates an initial temperature formation of 10-16 °C followed by 16-21 °C, %RH 95-100, with CO2 level less than 1000 ppm and light requirements 10

of 500-2000 lux at 370-420 nm. The third stage is fruiting body development where the initial temperature is 16-18 °C which is then increased to reach 21-27 °C, %RH 60-80 with CO2 level less than 1000 ppm and light requirements of 500-2000 lux at 370-420 nm. The final stage is the cropping cycle which is performed every 2-3 weeks for 8 to 12 (16) weeks (Li-Sun Exotic Mushrooms). Its cultivation method is similar to that of chestnut mushrooms discussed in 2.3.1 and is shown in Figure 2.5.

Figure 2.4: Shiitake mushrooms (Source: Li-Sun Exotic Mushrooms).

Figure 2.5: Cultivation process of the studied mushrooms. (Source: Li-Sun Exotic Mushrooms, 2010).

2.4 Mushroom ageing

The economic importance of mushrooms quality requires the understanding of the biochemical and physiological mechanisms that determine quality.

Senescence is a genetically regulated oxidative process involving degradation of the cellular structures and enzymes as well as mobilisation of the by-products to other parts of the plant. This leads to an increase in lipid peroxidation and membrane leakiness caused by the generation of reactive oxygen species that takes place in plant tissues during senescence (Thompson et al., 1987).

In mushrooms, macroscopic post-harvest changes manifested by the elongation of the stipe, opening of the cap, growth of gills, and production of spores result from changes at the cellular level, breakdown, and synthesis (Braaksma et al., 1998). Post-harvest senescence in many horticultural commodities is accompanied by alteration in the cell membranes hence disruption of the barriers between compartments and loss of cell turgescence (Mazliak, 1987). In mushrooms, these changes cause softening of tissues as a result of changes in membrane permeability. Moreover, in mushrooms, softening or loss of firmness during post-harvest life is ascribed to changes in membrane permeability (Beelman et al., 1987). Tao et al., (2007) suggested that membrane permeability is directly related to storage conditions.

Braaksma et al., (1998) also showed that postharvest development of mushrooms is a tissue specific process such as cell growth by vacuolar expansion in the pileus tissue, while most of the cells in this tissue disappear. In the gills, the development is via cell divisions, while in the stipe, no growth in the cellular area is seen. Such observation correlates well with the hypothesis that protein breakdown in mushrooms (Burton, 1988) occurs only in stipe and pileus tissue. This is in line with observations of Donker and Braaksma (1997) that showed that changes in amino acid pools after harvest are tissue specific.

Nevertheless, lipoxygenases, the enzymes catalysing the direct conversion of the polyunsaturated fatty acids to give hydroperoxide products which are intermediate compounds in the volatile formation mechanism, play an important role in the breakdown of membrane lipids induced by senescence (Del Rio et al., 1998).

Mushroom discoloration is caused by the oxidation of phenolic compounds catalysed by tyrosinase, and the subsequent chemical reactions that convert the oxidised products into the brown polymer, melanin. Tyrosinase can be activated by proteolytic action. Also, proteases activity are enhanced during sporophores senescence, which is a harvest-induced event and this increase may be the reason for the discoloration and changes in texture as chitin synthase can also be proteolically activated. Burton et al., (1993) have suggested that phenolic compounds and tyrosinase are located in different compartments of the cell and that during bruising or post-harvest ageing, the compartments barriers may breakdown allowing the two components to mix giving the enzyme a greater access to its substrate and the browning reactions to occur. Although the beneficial effect of the increased proteolytic activity is likely to be nutritional, as it results in the release of the amino acids needed for the maturation of sporophores, undesirable secondary effects may occur such as tyrosinase activity (Hammond & Nichols, 1976).

2.5 Aroma quality

The interaction of taste, odour, and textural perception provides an overall sensation referred to as “flavour”. Flavour results from compounds that are divided into two groups, those responsible for taste and those responsible for odours known as aroma substances. Aroma substances are volatile organic compounds perceived by the odour receptor sites of the nose. They consist of a highly diversified classes of compounds, some of them being highly reactive and occurring in very low concentrations. They can be used as an objective guide for assessing the quality of raw or processed food (Belitz et al., 2009). Aroma compounds are usually divided into two categories: pleasant which include balsamic, almond, anise-like odours, floral or fruity, and unpleasant that include sulphurous, rubbery, alliaceous, ammoniacal, and putrid (Gilbert, 1934).

Techniques to measure aroma quality revolve around three criteria, their ability to (i) detect very small differences, (ii) produce indices with properties of interval-scale measurement for metric comparison with molecular properties, and (iii) avoid subjective comparison. Chastrette (1997) argues that the characterisation of odour quality depends on the basis behind odour classification, i.e. whether (i) it focuses upon global properties such as boiling point, molar volume, shape, (ii) electrostatic variables,

and (iii) geometric variables (distances within molecules). A typical dendogram for odour classification and characterisation is shown in Figure 2.1.

Fungal aroma is a typical and special trait of each edible mushroom species. Research on the quality of the cultivated fungal species is as crucial as the research conducted on agronomic techniques to obtain mushrooms with nutrition and medicinal importance, providing biological material for industrial purposes (Liu et al., 2004). Relevant targets are the pharmaceutical, diet supplements, and food industries aiming at obtaining new aromas, flavours, and nutritional values (Omarini et al., 2010).

Figure 2.6: A dendrogram for odours classification. Adapted from Wise et al., 2000.

Volatile aromas, nutrients and hence quality are greatly affected by growth media which is vital given the importance of flavour for consumer acceptance. As mentioned earlier, substrates for mushroom cultivation are mixtures of different agro-industrial wastes, such as wheat, rice, paddy straw, sawdust) and nitrogen supplements (wheat bran, soybean hull, millet). These substrates affect the chemical composition and hence the volatile profile, as well as the nutritional value of the cultivated mushrooms (Bonatti et al., 2004). Shashirekha et al. (2005) in their study on the effects of supplementing rice straw with growth substrate on the analytical characteristics of mushrooms showed that the volatile profile is influenced by the substrate composition and he reported a 35% increase in the total lipids. Nevertheless, fatty acids play a major role in the aroma contributing to the odour and flavour precursors via enzymatic oxidation, decarboxylation, or esterification thus highlighting their importance in the synthesis and generation of volatile compounds (Wu et al., 2005). Furthermore, Callac and Guinberteau (2005) identified a relationship between the ratio of volatile compounds and phylogenetic closeness indicating the potential importance of volatiles chemicals as taxonomic markers.

The same volatile metabolites contributing to the pleasant aroma in some foods like mushrooms for example, can be responsible for the off-odours in others such as 1- octen-3-one in dairy (Kinderlerer, 1989).

Other studies correlating aroma and quality were done by Hagenmaier (2002) and Tietel et al. (2010) who showed that storage was deleterious to the flavour of mandarins with an increase in off-flavour being indicative of the loss in flavour quality. Obenland et al. (2011) revealed that flavour loss was due to changes in aroma volatiles as a result of storage.

2.6 Volatile aromas in mushrooms

The aroma components of flavour result from a mixture of organic molecules exhibiting a wide range of chemical structures characterised by different physical and chemical properties such as volatility and polarity. Each volatile compound is characterised by its own aroma ranging from mushroom-like for 1-octen-3-ol to sweet and fruity for the ketone 3-octanone (see Table 2.1). Aroma compounds share one characteristic, namely

volatility. However, this attribute presents a challenge for analysts as there is no single isolation method giving a true representation of the aroma profile (Taylor, 2010).

Table 2.1: Aroma descriptor of some volatile compounds found in mushrooms.

Compounds Aroma descriptors Alcohols 1-octanol Fruity-flowery, orange, waxy, sweet 3-methyl-1-butanol Alcoholic, cheese, pungent 1-octen-3-ol Mushroom, butter, resinous

Ketones 1-octen-3-one Boiled mushrooms, fungal, metallic 3-octanone Fruity, sweet, floral, lavender, sweet ester 1-phenyl-1,2-ethanedione Camphorated

Aldehydes Hexanal Freshly cut grass Nonanal Fatty, floral Benzaldehyde Almond, sweet, phenolic

Pyrazines Pyrazine Dried mushroom

Terpene geraniol Rose, sweet Linalool Light, sweet, citrus Nerol Sweet, floral, slightly bitter

Lactones 4-hexanolide Fragrant 4-octanolide Coconut 4-nonanolide Fruity Adopted from Jong and Birmingham (1993).

Different mushroom species have different volatile compounds (VC) profile. For instance, Suillus bellini are a rich species of alcohols which are considered as the main odorants of the mushroom-like aroma. Other species such as Tricholomopsis rutilans and Fistulina hepatica which contain high ester percentages correspond to those that presented low percentage of non-volatile acids. Tricholoma equestre and Suillus luteus contain high levels of acetaldehyde well known for its almond-like aroma (Guedes de Pinho et al., 2008). Octen-3-ol, by far the major component in many mushroom species, is a minor volatile constituent in Marasmius alliaceus (Rapior et al., 1997).

Volatile norisoprenoids, such as ß-ionone, 6-methyl-5-hepten-2-one, trans- geranylacetone, and (E,E)-farnesylacetone are newly identified in mushrooms. These ketones are known as oxidative by-products or degradation products from carotenoids. Suillus bellini, Suillus granulatus, and Suillus luteus contain high levels of these compounds, which can be markers of this mushroom genus. Eugenol, lactones, indole, 2-piperidone, sterols, and nicotinamide content differ between mushroom species. Suillus granulatans, Boletus edulis, and Tricholoma equestre have high amounts of sterols whereas Suillus bellini, Amanita rubescens and Tricholomopsis rutilans have high levels of nicotinamide (Guedes de Pinho et al., 2008).

Methional, which according to Nagodawithana, (1995) could be formed by Strecker degradation of methionine is the most potent aroma-active compound in cooked pine- mushrooms (Cho et al., 2007). Nevertheless, 2-acetylthiazole was found only in cooked pine-mushrooms, suggesting that this compound might be formed due to heat exposure. Nagodawithana, (1995) argued that this compound might be formed in pine-mushrooms by condensation of Maillard intermediates followed by cyclisation.

Raw or dried Shiitake is used in Japanese and Chinese dishes because of its highly desirable taste and aroma (Mizuno, 1995). The characteristic volatile compounds are gradually generated via the drying process (Chyau and Wu, 1990). Two types of compounds constitute the VOC profile of shiitake, the eight-carbon compounds being the major flavour compounds in fresh mushroom and sulphur-containing characteristic compounds in dried shiitake (Chyau and Wu, 1990, Chang et al., 1991). The formation of C8- compounds results from enzymic activities during rupture and or drying of the tissue. They are formed enzymatically from linoleic acid.

Shiitake is dried for two reasons namely preservation and development of the specific shiitake aroma. VC in shiitake differ between fresh samples and those subjected to different heat treatments. The compound 1-octen-3-ol was found as the main volatile in fresh shiitake (74.7% peak area of total volatiles) while 1,2,4-trithiolane was reported in dried shiitake (66.3%) (Hong et al., 1988).

Mushroom quality is determined by its organoleptic characteristics of which the aroma is the most critical. Several researchers have investigated the VC present in mushrooms with nearly 150 different volatile compounds representing a wide range of chemical

classes including but not limited to alcohols, carbonyls, acids, esters, sulphurous compounds, that were identified in diverse mushroom species (Maga, 1981; MacLeod and Panchasara, 1983; Chen and Ho, 1986; Beltran-Garcia et al., 1997; Rapior et al., 1997; Cho et al., 2008). Chemical composition of mushrooms is closely linked to species, cultivating conditions and processing (Mau et al., 1997; Díaz et al., 2002; and Wu et al., 2005.

2.6.1 Eight-carbon VC and the fungal aroma

Eight-carbon VC are characteristic of the fungal aroma accounting for 44.3%-97.6% of the total volatile fraction depending on the extraction method used. Several researchers have shown the importance of a specific VC, 1-octen-3-ol, concentration being the most important determining the resulting aroma as discussed by Díaz et al., 2002, Dijkstra & Wiken 1976, and Pyysalo (1976).

Each VC is characterised by its own aroma ranging from mushroom-like for 1-octen-3- ol to sweet and fruity for the ketone 3-octanone (see Table 2.1).

2.6.2 Aroma Production Mechanism

The biosynthetic pathway of the 8 carbon VC formation in fungi is controversial. Fungi, like plants, use their fatty acids as a substrate to produce volatile compounds. These lipids are first oxidised and then cleaved to produce the short-chain VC. Linoleic acid is the precursor to 8-C VC formation in fungi, acting as substrate (Combet et al., 2006).

2.6.2.1 Oxidation step

Two proposed biochemical routes lead to the formation of 1-octen-3-ol either by autoxidation of linoleic acid and or by enzyme-catalysed oxidation followed by cleavage. The latter pathway was suggested to be the most likely route through the action of a lipoxygenase (a non-heme iron-containing dioxygenase), then the subsequent cleavage of the intermediate fatty acid hydroperoxide by a hydroperoxide lyase in 2 different pathways (Tressl et al., 1982). Combet et al. (2006) suggested that both enzymes are present in plants and play a vital role in short-chain volatile synthesis.

2.6.2.2 Cleavage of the hydroperoxide compounds

Hydroperoxide compounds have a short half-life, behaving as a precursor to a range of cytochrome P450 enzymes including the hydroperoxide lyase, which breaks the 20

hydroperoxide molecules into short-chain volatile compounds. The lyase enzymes operate in two cleavage modes, either homolytic or heterolytic, producing different by- products:

a) The first cleavage mechanism, called heterolytic is found in most plants: the enzyme breaks the hydroperoxide between the unsaturated carbon and the carbon holding the hydroperoxide group. (Combet et al., 2006).

b) The second mechanism is called homolytic and entails the cleavage of the hydroperoxide between the carbon attached to the hydroperoxide group and the saturated carbon. This mechanism has been observed in algae, grass, and mushrooms (Berger et al., 1986; Vick and Zimmerman, 1989). Figure 2.2 illustrates the mechanism of 1-octen-3-ol formation.

Figure 2.7: Mechanism of 1-octen-3-ol formation Source: Combet et al. (2006).

2.6.3 Mechanism of sulphur compounds formation

The specific aroma of Shiitake that is formed when dried shiitake is soaked in warm water or when chopped raw shiitake is left unrefrigerated is due to 1,2,3,5,6- pentathiepane known as lenthionine, a sulphur-containing VC generated enzymatically from its precursor lentinic acid in dry mushrooms (Morita & Kobayshi, 1966, Yasumoto et al., 1971a,b) (Figure 2.3). It can also be heat-decomposed to produce hydrogen sulphide (Wada et al., 1967). OHO O O OO O O O SSSS N OH lentinic Acid H NH2

glutamyltransferase - glutamic acid

OHO O O OO O des-glutamyl lentinic acid SSSS NH2

C-S lyase - pyruvic acid, NH 3

O O O O SSSS OH

Thiosulfinate

- acetaldehyde, formaldehyde

dithiirane SS

S S S S S S S

S S S S SS SS

1,2,3,5,6-pentathiepane 1,2,4,6-tetrathiepane hexathiepane lenthionine Figure 2.8: Aroma formation mechanism adopted from Mizuno (1995).

2.7 Methods of Aroma Analysis

A general pathway is followed by analysts when analysing aroma compounds. The steps are as follows:

1. Isolation

2. Concentration

3. Gas chromatography analysis

4. Gas chromatography-olfactrometry

2.7.1 Isolation

During VC isolation, a gentle yet powerful extraction technique is needed to prevent any possible changes caused by enzymatic reactions (hydrolysis of esters, oxidation of fatty acids and hydrogenation of aldehydes) and non-enzymatic reaction such as hydrolysis of glycosides, reduction of disulfides from the Maillard reaction, and reaction of amines, thiols, and aldehydes in the aroma concentrate (Belitz et al., 2009).

Breheret et al., 1997 and Jeleń, (2003) showed that the fungal volatile profile is highly dependent on the extraction method selected. Larsen and Frisvad, (1995), when comparing methods for the collection of volatile chemical markers, noted that steam distillation extraction was producing volatile profiles dominated by the lipid degradation products, 1-octen-3-ol and 3-octanone, probably not a true representation of the organism volatile profile. There is a wide range of isolation and extraction techniques each presenting advantages as well as some drawbacks.

2.7.1.1 Direct solvent extraction

This method is known to be the simplest and most efficient isolation technique for aroma compounds based on the ability to utilise solubility as a basis for aroma isolation. The sample is added to a separating funnel and mixed with a solvent. Subsequently, the solvent extract is collected from the separating funnel, and dried with anhydrous salt then concentrated for GC analysis (Taylor, 2010). However, this technique doesn’t work for samples that contain lipids and hence, lipids must be separated from the solvent extract before further analysis. This additional step adds the probability of making errors, such as loss of volatiles or artefact contamination as well as increasing

the time for analysis. According to Taylor (2010), the aroma profile obtained via solvent extraction represents the relative solubility of various aroma constituents in the organic and aqueous phase. Several researchers have studied the advantages of the solvent extraction technique and found it simple and useful when combined with chromatography providing adequate chemical description of the mushrooms species studied (Rapior et al., 1997; Guedes De Pinho et al., 2008). However, it is limited by the chemical properties of the compounds studied (Taylor, 2010).

2.7.1.2 Subcritical water extraction (SPW)

This is an extraction technique, recently emerged to replace traditional extraction methods. It is carried out using hot water (100 to 374 °C), at high pressure (usually from 10 to 60 bar) to maintain water in the liquid state. Subcritical extraction has been widely used to extract different compounds from vegetables. For instance, Ibanez et al., (2003) investigated the extraction of antioxidant compounds of rosemary by SWE over a wide range of temperatures where water temperature greatly affected the extraction yield which increased at higher temperatures. Basile et al., (1998) extracted aroma from rosemary using SPE while Kubátová et al., (2001a) used it to characterise aroma in peppermint. This technique provides a number of advantages over traditional extraction techniques (organic solvents, solid-liquid extraction) mainly by having a shorter extraction times, higher quality, lower costs of the extracting agent, as well as being environmentally friendly. These advantages have been verified for the SWE of several plants such as laurel (Fernández-Perez, 2000), fennel (Gamiz-Garcıa and Luque de Castro, 2000), oregano (Soto Ayala and Luque de Castro, 2001) and kava (Kubátová, et al., 2001b).

2.7.1.3 Supercritical carbon dioxide extraction

The supercritical CO2 extraction is commonly used for the extraction of volatiles because of the non-toxic, non-flammable, readily available, high purity and low cost properties of CO2. Recently there has been an increased interest in supercritical and subcritical extraction using carbon dioxide as a solvent. Reverchon and Marco (2006) reviewed the applications of supercritical CO2 extraction in food processing, pharmaceuticals and nutraceuticals. Some compounds show little to moderate solubility in CO2 and hence the addition of solvents such as ethanol (Dobbs et al., 1986; Ke et al.,

1996) has been proposed to improve the solubility of polar compounds since CO2 is a 24

poor extractor for polar substances. Naik et al. (1989) and Tuan and Ilangantileke

(1997) have compared the advantages of liquid CO2 over the supercritical CO2 extraction of aroma compounds. Liquid CO2 was found to possess interesting properties for aroma and flavour compounds with medium molecular weight in addition to the low extraction temperature. The inert extraction atmosphere provides an advantage for the recovery of volatile and thermally labile compounds. Kubátová et al., (2001a), compared water-supercritical fluid extraction and CO2-supercritical fluid extraction with hydrodistillation for the extraction of essential oils form savory Satureja hortensis and peppermint (Mentha piperita) and concluded that the former method was highly selective for polar ozygenated flavour compounds when compared to CO2 supercritical extraction or hydrodistillation. Danh et al. (2010) used ethanol-modified supercritical

CO2 at 190 bar, 50 °C and 15 % volume ethanol to obtain the highest oil yield (5.9%) from vetiver grass, over three times and nearly double that of hydrodistillation and SCE with pure CO2.

2.7.1.4 Distillation

One of the most used isolation techniques involves distillation followed by solvent extraction with the main advantages being separation of volatile components from the non-volatiles, simplicity of the apparatus, and reproducibility.

2.7.1.4.1 Direct distillation

The sample is placed in a round bottom flask and mixed with water where it can be heated with continuous stirring. However, it presents some problems such as scorching (caused by excess heat), bumping (if the sample contains particulates), and foaming. These problems can be resolved by stirring and addition of antifoaming agents (DC polydimethyl siloxanes). However, these silicones may end up in the distillate (Marsili, 1997).

2.7.1.4.2 Steam distillation (direct versus indirect)

The sample may be distilled at atmospheric pressure followed by solvent extraction. Being simple and straightforward procedures, they are still used alone or combined with other techniques for the characterisation of flavours and aromas. Saritas et al. (2001) isolated the essential oils from aromatic lichens of various genera (Mnium and Plagiomnium) by hydrodistillation of fresh and dried plant parts and were able to

identify several VC including two unreported sesquiterpenes. Alternatively, a modified Likens-Nickerson extractor can be used allowing the introduction of steam into the system. However, the recoveries for lipid systems are not as good as for aqueous samples. The indirect application of steam to the distillation presents some advantages over the direct method such as speed and lesser degradation of material. The steam could be generated by heating the round bottom flask with a mantle or using a steam generator. The steam and volatiles are then condensed in a series of cooled traps such as ice water and dry ice/acetone or methanol (Marsili, 1997).

2.7.1.4.3 Vacuum steam distillation

Previously referred to as cryogenic trapping, this technique was first introduced by Joulain (1986) as a method for trapping VC emitted by Wisteria sinensis flowers. This technique is used when there is a possibility of material decomposition. It operates under high vacuum where an inert gas is incorporated into the system and a series of cooled traps are used to protect the pump from the water vapour and the sample form pump oil vapours. This distillation can also operate under high vacuum for samples containing a large amount of lipids, using the process of vaporising the flavour from a heated thin film of the oil under high vacuum. The distillate is then collected in a series of cooled traps of liquid nitrogen (Marsili, 1997). Werkhoff and Guntert (1996) used vacuum distillation to extract volatiles from Bourbon vanilla beans where they identified 16 new ester compounds and concluded that this method allowed the identification of esters which were important contributors to the overall sensation of natural vanilla flavour. However they did not specify the pressure and temperature at which the extractions were carried on. Werkhoff et al., (1998) also isolated flavour extracts of yellow passion fruit using different techniques and concluded that the vacuum headspace procedure (using a pressure of 1-10 mbar) resulted in the most powerful isolation technique yielding flavour volatiles representing the typical aroma of the yellow passion fruit. Furthermore, this technique allowed the identification of compounds in yellow passion fruits that have never been reported before. This technique was also used in a study to compare the volatile constituents in cold-pressed bergamot oil and a volatile oil and it was concluded that extracts from vacuum distillation were of superior quality (Belsito et al. 2007). Pennarun et al. (2001) compared hydrodistillation with a 600 Pa vacuum and high vacuum degassing and cold finger molecular distillation and concluded that hydrodistillation was the best method 26

the extract VC from oysters Crassostrea gigas. Ashmore et al. (2013) used ambient temperature vacuum distillation to extract VC from A. bisporus and concluded that this technique gave the most representative aroma of the fresh/raw mushrooms.

2.7.1.4.4 Simultaneous steam distillation/extraction

This technique combines steam distillation at atmospheric pressure and simultaneous extraction with organic solvents. This method involves dispersing the sample in water using a 500 mL to 5 L capacity flask with heat incorporated using a heated mantle or heated oil bath with stirrer if solids are present. The distillation process is performed for 1-3 hours, after which the system is cooled and the solvent from the central extracting tube is combined with that of the solvent flask. The solvent is dried using sodium sulphate then concentrated (Parliment, 1997). Marsili, (1997) discussed the advantages associated with this technique as; 1- a single operation removes all the aroma volatiles and concentrates them, 2- a small volume of solvent is required hence minimising artefact formation, 3-the percentage recovery is usually high, and 4- the system may operate under reduced pressure hence reducing thermal decomposition. However its major limitations are formation of secondary flavours as a result of degradation caused by lipids oxidation (Barcarolo and Casson, 1997) as well as products resulting from browning reactions (Werkhoff et al., 1998) and those obtained via heating are mixed with the desired components. Werkhoff et al. (1998) argued about the possibility of thermally induced artefacts yielding falsified aroma and concluded that this technique produces more representative aromas of the food sample if it operates under vacuum. Siegmund et al., (1997) found that 5,6-dihydro-2,4,6-trimethyl-4H-1,3,5-dithiazine, usually pointed to as an important aroma-active compound in cooked and cured meat aromas, is an artefact formed during the distillations performed using the Lickens-Nickerson SDE method.

2.7.1.5 Headspace techniques

A distinguishing attribute of aroma compounds is that in order to be noticeable by the olfactory system, they must be in the gas phase at specific concentration. The total volatile content represents the volatile composition in a food. However, it is often difficult to relate this profile to the volatile profile expressed when foods are consumed. The headspace techniques, coupled to gas chromatography-mass spectrometry (GC/MS) are practical and can be used on different kinds of samples whether solid, liquid or 27

gaseous. VC can be isolated and analysed using two techniques each having different requirements concerning instrumentation namely the static and dynamic (purge and trap) headspace techniques (Van Ruth, 2001).

2.7.1.5.1 Static headspace

This is based on the principle that aliquots of the gas phase are withdrawn from samples in a closed static headspace (closed bottle) after the equilibrium between the sample matrix and the gas phase above it has been reached. Then the aliquots are fed to the GC/MS system via a syringe. The position of the equilibrium depends on several parameters, such as temperature and sample matrix, therefore the matrix can be standardised via addition of Na2SO4 or Na2 CO3, internal standardisation, or the multiple headspace extraction procedure (MHE) (Hubschmann, 2001).

The main advantage of this technique is its ability to analyse a sample for low molecular weight (LMW) compounds without the presence of a solvent peak. This is important since many samples that are analysed are being assayed for residual solvent content (Marsili, 1997). It also allows for automation and is useful for sample screening preparations. This technique is sensitive, cheap, and involves simple sample preparation. It is mostly used in quality control situations where only major components are of interest (Taylor, 2010).

This technique is coupled with few drawbacks which limits its usage. For instance, the fact that only a portion of the headspace is analysed as determined by the headspace vials filling, causes the instrument to be insensitive for the determination of analytes with low concentrations in the original sample material. Although increasing the temperature increases the volatility of compounds, most static headspace instruments can only heat up to 150 °C (Marsili, 1997). Furthermore, the analysis at low temperatures limits the usefulness of this technique for the analysis of analytes with higher boiling points (Wampler, 1997).

Headspace filling should be performed under an inert gas atmosphere. Upon injection from headspace vials, air can regularly get into the GC system affecting column sensitivity (Hubschmann, 2001).

2.7.1.5.2 Dynamic headspace (purge and trap)

This method involves the continuous transfer of analytes from the sample matrix in the headspace phase which is performed by purging the sample with an inert gas, such as nitrogen or helium stripping the aroma compounds from the sample (Taylor, 2010). The inert gas serves to separate the volatile compounds from the matrix. The continuous stripping prevents the thermodynamic equilibrium between the sample and its vapour phases allowing efficient extraction. The inert gas is then supplied directly over or through the thermostatically controlled sample for a sufficient period of time to ensure the extraction of the majority of the volatiles (Barcarolo and Casson, 1997).

This process can be divided into two phases: (a) extraction and entrapment of VC; (b) desorption and transfer to the GC column (Barcarolo and Casson, 1997). During the trapping phase, the carrier gas travels to the trap where the volatiles are retained while the carrier gas is expelled. The trapping phase may involve adsorption onto a high surface area sorbent material or cold trapping by condensing or freezing the analyte in the trap (Marsili, 1997), However, there are some considerations to be taken when performing the trapping technique, such as chemical nature of the compounds and their thermal stability: sorption and desorption characteristics of the sorbent, breakthrough volume of the analytes on the sorbent, and the presence of contaminating materials (Wampler, 1997).

In a second step, the stripped organic VC are adsorbed on a porous material in short columns (Pillonel et al., 2002). The analytes can be desorbed by heating the trap only enough to revolatilise it without causing thermal degradation. However, care must be taken with the choice of sorbent as some may collect unwanted materials (Wampler, 1997). Breheret et al. (1997) noted the mushroom VC extracted and compared them with the solvent extraction technique and concluded that the headspace analysis identified compounds that were detectable by the human nose, whereas the former method allowed only general identification of VC. Werkhoff et al. (1998) evaluated the use of the dynamic headspace technique for VC extraction and concluded that this method isolated mainly the highly volatile compounds therefore failing to isolate some of the volatiles responsible for the true aroma of the yellow passion fruits. However, when the simultaneous distillation extraction was compared to the above technique, the former technique was characterised by the loss of the highly volatile compounds

(Werkhoff et al., 1998). This technique was applied successfully where Jiang and Kubota (2001) used the above technique to collect the VC in the leaves of a Japanese pepper.

Dynamic headspace offers many advantages of the static headspace technique. These include the absence of solvent peak and analysis of the analytes of interest (volatiles). Moreover, as the volume of sample can be controlled, the sensitivity of the dynamic headspace is increased allowing extraction of VC present at the part per billion. Nevertheless, using a dry purge step and a moisture control system, the water vapour can be eliminated from the GC/MS system. In addition, because of the sorbent selectivity and affinity, specific analytes can be collected, while the unwanted compounds can be discarded (Hübschmann, 2001).

This technique presents some limitations manifested by the complexity of the instrument and hence cost. For instance, the purge vessels must be sanitised cautiously, or economical single-use vessels made from polymer materials should be used (Hübschmann, 2001). Moreover, it is time consuming as several stages are performed per sample compared to the static sampling.

2.7.1.5.3 Headspace solid-phase microextraction (HS-SPME)

It is an adsorption/desorption technique developed by Arthur and Pawliszyn, (1990). It is made up of a short fibre of fused silica, coated with a stationary phase (often made of polydimethyl siloxane, carboxen), in a syringe-like device. When VC have reached equilibrium in the headspace of the sample to analyse, the fibre is inserted in the chamber. Analytes are then separated between the stationary-phase coating and the gas phase and adsorb/absorb on the fibre (Combet et al., 2006) After a period of time, the fibre is drawn back and removed to the gas chromatograph injection port, where the volatiles are thermally desorbed from the fibre and cold trapped on the head of the capillary column (Zhang and Pawliszyn, 1993; Yang and Peppard, 1994). This method is sensitive, cost efficient, fast and becomes a very efficient instrument when used in conjunction with gas chromatography-mass spectrometry analysis allowing the collection of fragrances from live plants with minimum disturbance of the specimen. Furthermore it allows the concentration of the volatiles in a solvent-free manner (Zhang and Pawliszyn, 1993; Yang and Peppard, 1994).

Several researchers have used HS-SPME as a main extraction method. For instance, Vereen et al. (2000) employed it to study the VC released by Fraser Firs, Augusto et al. (2000) in tropical Brazilian fruit, Wu and Wang, (2000) in fresh and processed shiitake mushroom. Zini et al. (2001) studied VC in Eucalyptus citriodora trees, Jeleń, (2006) in food taints and off-flavors, Lopez et al. (2007) in sulphur compounds in wines, Guedes De Pinho et al. (2008) in wild edible mushrooms and Jeleń and Szczurek (2010) in wine. However, when HS-SPME was compared to dichloromethane solvent extraction, solid-sample injection and Tenax thermal desorption, it was found that SPME is not the most powerful extraction technique (Ewen, 2004).

2.7.2 Concentration Concentration is the next step following extraction. It is performed by drying the solvent over sodium sulphate or magnesium sulphate and then carefully concentrating it on a steam bath using a Vigreux column (Wampler, 1997).

2.7.3 Gas chromatographic analysis (GC) Gas chromatography is used to separate the aroma compounds based on the differences in their affinities for the stationary phase. This means that compounds with a higher affinity for the stationary phase will have a longer residence time compared to compounds with lower affinity to the stationary phase. GC is usually coupled to a mass spectrometer (MS) enabling both identification and quantification of the eluted compounds. As a result, a chromatogram is generated showing a series of peaks, each peak will correspond to one aroma compound (Skoog et al., 1998).

The basic components of the Gas Chromatography unit are:

a) A carrier gas and control system

The carrier gas which is characterised by its optimal flow rate is provided from a high pressure gas cylinder and the choice depends upon the detector type. The most common gas used is helium for mass spectrometer and hydrogen for flame ionisation detector. b) A sample introduction system

Modern gas chromatography units usually come with an automatic sampling unit, through which a specific volume of sample is injected into the unit in a very short space 31

of time. In the past, with the old chromatography units, peak broadening occurred because of the time taken in the injection. An important consideration is that high temperatures should be maintained to make sure that there is no residue left in the system. There are two main methods of injection are used in analyses. These methods are known as split and splitless injections (Skoog et al., 1998).

Split injection

As the name suggests, this method involves only a small volume of the sample that enters the column and hence the column resolution is increased. However, split injection lacks the sensitivity to detect compounds present as small traces.

Splitless injection

This method is used in the area of trace analysis for analytes with narrow boiling point range which are not thermally labile. In this technique the split valve is closed, and the entire sample passes onto the column. Furthermore, as low temperatures are used, the analysis of heat sensitive compounds can be performed. c) A thermally controlled chromatographic column

The gas chromatography unit uses an open tubular or capillary column, where the sample is introduced in it is entirely without heat. The main disadvantage of a capillary column is its low capacity; however, sensitive detectors can decrease the need for high capacity columns. d) A detector

Many different detection techniques exist that can be linked to the output of the gas chromatography unit: 1) The flame ionisation detector, 2) The electrical conductivity detector, 3) The mass spectrometer. Each type of detector gives different types of selectivity.

The most popular detector to be connected to the gas chromatography unit is the mass spectrometer. This is due to its high sensitivity, a short response time that is independent of the flow rate, a temperature range from room temperature to at least 400 °C, and its great stability and reproducibility (Skoog et al., 1998).

Detectors can be classified into concentration dependent detectors and mass flow dependent detectors. The signal from the first is linked to the concentration of the solute in the detector and does not destroy the sample. Meanwhile, the signal from the second is dependent on the rate at which analytes enter the detector and unlike the former, it destroys the sample (Sheffield Hallam University, 1998). e) Recording system

It is necessary to record the data generated by the detector in a retrievable format. This can be in either computer memory or on a paper. Werkhoff et al. (1998) showed that the chromatograms of extracts obtained using the vacuum headspace technique contained high boiling components which may play an important role in the characterisation of the “tropical and very pleasant flavour” of yellow passion fruits.

2.7.4 Gas chromatography-olfactometry (GC-O)

Food flavour is defined as the sensation arising from the integration of signals generated as a result of sensing smell, taste, and irritating stimuli from the food or beverage (d'Acampora Zellner et al., 2008).

The introduction of GC-O in analytical aroma research allowed the differentiation of a wide range of odour-active and non-odour-active VC, related to their concentrations in the matrix under investigation. It is a unique analytical technique which associates the resolution power of capillary GC with the selectivity and sensitivity of the human nose. The gas-chromatography-olfactometry (GC-O) technique is a powerful and highly reproducible tool that is useful in linking the analytical results to the sensory perception of odours. In this technique, some of the separated odours are sent to a humidified sniffing port. The analyst can then place a descriptor on the volatile compound if it can be detected as an aroma compound (Cazes and Scott, 2003). One problem is that noses vary widely in their sensitivity and ability to discriminate odours. GC-O has been largely employed in combination with sophisticated methods which are classified in four categories: dilution, time-intensity, detection frequency, and posterior intensity methods.

2.7.4.1 Dilution analysis

The most widely used method is based on successive dilutions of an aroma extract until no odour can be detected by the panellists. This procedure can be performed either by combined hedonic aroma response method (CHARM) or aroma extraction dilution analysis (AEDA). In the former, the samples are presented to the panellists in an increasing dilution order and a dilution factor (FD) where the impact of an odour-active compound is determined. The results are then presented in the form of an aromagram where FD value is plotted against the retention index. In contrast, in the latter, the dilutions are presented in a random order to avoid bias and the panellists record the start and the end of each detected aroma. The results are also presented in the form of an aromagram where the duration of the odour is plotted against the dilution value (dilution value is analogous to the FD value in AEDA). AEDA is the most frequently used method to estimate the sensory contribution of single aroma compounds in mushrooms, cheese flavour, roasted beans and brewed coffee volatiles (Grosch et al., 2001) However, AEDA is limited by some drawbacks in such a way that it does not take into consideration the potential losses of odorants during the extraction procedure or the synergistic and suppressive effects of distinct compounds in a food matrix. With regards to CHARM, limitation can be observed in quantification analyses which require replication of the experiments by at least three trained assessors (Grosch et al., 2001; Iwabuchi et al., 2001)

2.7.4.2 Time-intensity method

This is based on the immediate recording of the intensity of the odour as a function of time. Although it can be performed in a single run, it requires trained assessors (Grosch et al., 2001; Iwabuchi et al., 2001).

2.7.4.3 Detection frequency method

This method uses the number of evaluators detecting aroma compound in the GC system’s effluent as a measure of its intensity. This GC-O does not require trained panellists and the results obtained are limited by the scale of measurement (Grosch et al., 2001; Iwabuchi et al., 2001).

2.7.4.4 Posterior intensity method

This method measures the intensity of aromatic compound and its scoring on a previously determined scale. The results may correlate well with the detection frequency results and to a lesser extent with the dilution analysis method (Grosch et al., 2001; Iwabuchi et al., 2001).

2.8 Relationship between GC-O and sensory data

The aroma of foodstuff is determined by the quality, the concentration of volatile compounds and their availability to the sensory system as a function of time (Pionnier et al., 2004). Information on the sensory attributes of a foodstuff can be provided by establishing their descriptive profiles with sensory descriptive analysis, such as a quantitative descriptive analysis (QDA) technique, which categorises and illustrates both qualitative and quantitative aspects of a foodstuff. The qualitative component comprises the descriptive terms, called “attributes”, which define the sensory profile of the samples, and the quantitative component measures the degree or intensity of each perceived attribute (Meilgaard et al., 1987; McTigue et al., 1989). Multiple attempts have been made to correlate the information obtained from sensory studies and the molecules exhibiting the typical taste of edible mushrooms (Kasuga et al., 1999; Harada et al., 2004). Cho et al., (2007) also investigated the combination of GC-O with sensory evaluation in pine mushrooms and showed that such combination is a powerful way to characterise the aroma of foodstuff and the use of multivariate statistical methods can be effective at revealing the relationship between data from instrumental and sensory analyses.

2.9 Mushrooms Preservation

Fresh foods are viewed as a matrix containing carbohydrates, proteins, lipids, water and water soluble components. These compounds are highly labile in fresh foods making them sensitive to chemical or physical damage. This molecular lability is decreased during the drying process as water is removed, thus concentrating the dissolved components (Nijhuis et al., 1998).

Fresh mushrooms are perishable and can be preserved only if they are subjected to postharvest technological treatments, such as drying and freezing. There are numerous quality indicators for mushrooms such as whiteness, cap development, stipe elongation, 35

number of ripe spores, texture, respiration rate, mannitol content, weight loss, and microbial deterioration (Gormley, 1975; Bartley et al., 1991; Lopez-Briones et al., 1992). Therefore, proper processing of mushrooms is crucial in order to maintain the quality of mushrooms needed for commercialisation (Gothandapani et al., 1997). 2.9.1 Vacuum Cooling

The vacuum cooling technique is a rapid evaporative cooling method extensively used for cooling some agricultural and food products. During vacuum cooling, the food commodities are loaded in a closed chamber and a vacuum is applied to evacuate air from the chamber (Zheng and Sun, 2004; Sun and Zheng, 2006). The evaporation is then achieved by reducing the pressure to the point where boiling of water can be evaporated at a lower temperature (Tambunan et al., 1994).

The vacuum cooling technique presents several advantages; for instance, as vacuum cooling is achieved through water evaporation occurring on the surface or inside the produce, the latter will have a uniform internal temperature distribution i.e. the temperature could be reduced at the same rate whether on top, centre or bottom (Malpas, 1972). Besides, the cooling rate depends on the porosity rather than the product dimensions making this technique an advantageous cooling method for large piles of produce (McDonald and Sun 2001). Furthermore, this technique is more hygienic since air goes only into the vacuum chamber at the end of the process unless the vacuum chamber is opened to release vacuum. However, vacuum cooling presents some drawbacks manifested by its limited applicability to moist and porous produce since the cooling is achieved through water evaporation. Also it necessitates that the amount of water loss should not cause significant deterioration to product quality (Sun and Zheng, 2006).

2.9.1.1 Application in the agri-food industry

The quality of fresh produce, such as cut flowers, fruits and vegetables begins to deteriorate upon harvesting and continues to decline quickly thereafter due to physiological changes of respiration, transpiration, and biosynthesis (Anon, 1981, Brosnan and Sun, 2001). Brosnan and Sun, (2001 and 2003) and Sun (1999) argued that vacuum cooling was effective in extending the shelf-life of cut daffodil and cut lily flowers. Artes and Martinez (1994, 1996), also demonstrated that vacuum cooling of lettuce followed by cold storage at 1 °C extended its shelf life for up to 14 days 36

compared to lettuce stored at ambient temperature with a shelf life of 3-5 days. Several studies were conducted on other fruits and vegetables varieties including broccoli (Sun, 1999), spinach (Sun, 2000), Chinese leaves (Sun, 2000), eggplants (Hayakawa et al., 1983), cucumber (Hayakawa et al., 1983), carrot (Hayakawa et al., 1983), peppers (Sherman and Allen, 1983), turnips (Ishii and Shinbori, 1988), strawberries (Anon, 1981), blackcurrants (Anon, 1981) and melons (Chambroy and Flanzy, 1980). Overall results show that vacuum cooling is an effective and efficient preservation method for these products.

As for mushrooms, their porous structure as well as their high moisture content made the vacuum cooling feasible and efficient hence prolonging their shelf life by 24 h after 102 h of storage (Burton et al., 1987 and Frost et al., 1989). Frost et al. (1989) investigated the effect of vacuum cooling on the quality of mushrooms stored at 5 °C after cooling and found no significant difference in product quality when comparing vacuum and conventional cooling. However, if mushrooms were slightly deteriorated prior to cooling, vacuum cooling applied will worsen the quality by enhancing the enzymatic browning of mushroom caps when compared with conventional cooling methods if these produce are stored at 1 °C for 8-10 days. On the other hand, Wang and Sun (2001) showed that vacuum cooling of mushrooms cause a 3.6% of weight loss which was higher than 2% for air blast chilling. However, Sun (1999) argued that during storage, vacuum cooled mushrooms experienced less water loss than air blast cooled ones which helped to compensate cooling loss and demonstrated that pre-wetting mushrooms prior to cooling is an effective method to increase product yield. 2.9.2 Modified atmosphere packaging (MAP)

Modified atmosphere packaging can be defined as an atmosphere with altered air composition in order to create a suitable atmosphere surrounding the product in an attempt to decrease its deterioration rate and hence extend its shelf life (Phillips, 1996;

Farber et al., 2003). Such atmospheres are richer in CO2 and poorer in O2 than air to potentially reduce respiration decay and physiological changes. Modified atmosphere packaging has been successfully applied to extend postharvest storage of Agaricus sp. and Pleurotus sp. mushrooms (Lopez-Briones et al., 1993; Villaescusa and Gil, 2003; Ares et al., 2007). MAP is simple, economical, and effective. Several studies have been carried out to determine the optimum conditions for MAP. Sveine et al. (1967) reported that 0.1% O2 37

and 5% CO2 were optimum conditions for prolonging shelf-life of mushrooms. Meanwhile, Roy et al. (1995) in his investigations to determine the proper concentrations of gases reported that 6% O2 was effective in decreasing cap development.

A low concentration of O2 has many advantages mainly decreasing the respiration rate, retarding cap development, reducing aerobic deterioration and weight loss, reducing tyrosinase activity and thus decreasing enzymatic browning (Kim et al., 2006). However, off-odours may develop due to fermentative metabolism under anaerobic conditions. Burton et al., (1987) suggested that the O2 level inside packages should be in the range of 3-4% to prevent anaerobic respiration. Kim et al. (2006) in their study on the influence of MAP on sensory quality of shiitake mushrooms also recommended that

O2 concentration remain above 5% in order to avoid off-odour generation. Tao et al. (2007), in their study on the effects of different storage conditions on the chemical and physical properties of mushrooms, showed that the degree of browning of the mushrooms stored under MAP increased slowly compared to those stored under cooling room conditions suggesting that MAP storage can efficiently extend the shelf life of the mushrooms. Furthermore, they found that mushrooms stored under MAP experienced a weight loss below 1% whereas those stored under cooling room conditions showed a weight loss between 10.12% and 14.78%. On the other hand, Lopez Briones et al. (1992) argued that the total mesophilic flora of mushrooms increased at 10 °C after four days of storage in various modified atmospheres. Furhtermore, Varoquaux et al. (1999) showed that MAP does not reduce respiration rate or metabolite consumption in mushrooms. Moreover, it has been demonstrated that CO2, at partial pressures between 5 to 20 kPa, prevented the opening of the cap, but induced both internal and external yellowing of the carpophores (Lopez Briones et al., 1993). An alternative method for prepacked mushrooms involves overwrapping with stretchable polyethylene or polyvinylchloride and partial heat sealing at the bottom of the package with a hot plate, in such a way that oxygen and carbon dioxide partial pressures within the overwrapped punnets can equilibrate with ambient atmospheres (Varoquaux et al., 1999). Mahajan et al. (2008) found that a low RH increased moisture loss in mushrooms causing shrinkage and quality deterioration. However, high RH caused a thin layer of 38

moisture to persist on caps and thus supported the growth of Pseudomonas tolaasii causing browning or yellowing of sporophore surface known as bacterial blotch as reported by Barber and Summerfield (1990) and Burton (1991). 2.9.3 Drying and aroma compounds

Mushrooms are a delicate type of produce owing to their high moisture content. Therefore they require adequate postharvest storage in order to maintain their quality. Lowering the temperature reduces respiration and transpiration rate, delays senescence, prevents wilting and shrivelling thus extending their shelf life (Burton and Twyning, 1989; Beit-Halachmy and Mannheim, 1992). Drying is an effective technique for the preservation of mushrooms and other food commodities. As water is removed, there is a fractional loss of the VC, increase in other compounds as well as changes in the enzymatic activity. During drying, Maillard reactions between sugars and amino acids may result in the formation of new compounds including VOC whose entity forms a specific aroma and flavour of the dry product (Misharina et al., 2009b).

Raw mushrooms contain a number of reactive compounds and hence any processing (cooking, drying) is accompanied by the formation of new VC resulting from various chemical reactions. During thermal processes, chemical reactions occur between amino acids and sugars resulting in the synthesis of new compounds including volatile compounds responsible for the aroma and taste of products. For instance, fresh Shiitake mushroom exhibits a slight odour but, upon drying, a characteristic sulphurous aroma gradually develops (Yasumoto et al., 1976). These compounds include but are not limited to 1,2,4-Trithiolane (C2H4S3), 1,2,4,6-tetrathiepane (C3H6S4), 1,2,3,4,5,6- hexathiepane (CH2S6), and lenthionine (Chen and Ho, 1986). Others, such as pine mushrooms are characterised by methional and 2-acetylthiazole formation due to exposure to heat (Rapior et al., 1997). Aromatic aldehydes were previously reported in various species of dried mushrooms. Aliphatic alcohols, aldehydes, and ketones are formed during the oxidation and decomposition of polyunsaturated fatty acids present in mushrooms by the activity of lipoxigenase and hydroperoxidase (Misharina et al., 2009b).

2.9.4 Drying using heat pump dryers

Drying is the most traditional method used for foods to be used as ingredients in sauces and soups. During the initial stage of drying, the process of moisture removal is fast 39

then becomes slower with increase in drying time. Van Arsdel (1973) argued that the surface tension was created as water is removed from the food surface causing shrinking. Furthermore, as more water is withdrawn, these structures are deformed or folded hence occupying a smaller volume resulting in shrinkage of the whole food product.

The heat pump dryer works by allowing hot and dry air to move through the food commodity to remove the moisture. The latter is then subjected to partial condensation by the effect of cooled airstream in the evaporator of the heat pump. The cool air is then reheated in the condenser of the heat pump and recycled back through the product. Such dryers are known to be energy efficient and have the ability to operate independently of outside ambient weather conditions, unlike solar dryers (Jon and Kiang, 2008). Mason (1994) found that ginger dried in a heat pump dryer retained 26% of its gingerol, the principal volatile flavour component responsible for its pungency compared to only 20% retention for rotary-dried commercial samples.

According to Saravacos et al. (1988), the rate of loss of volatiles depends on the concentration with the greatest losses occurring during the early stages of drying, when the initial concentration of the volatile component in the drying medium is low. Furthermore, as heat pump drying is performed in a sealed chamber, any volatilised compound remains in the chamber, and the partial pressure of that volatile compound progressively increases, delaying further volatilisation from the product.

Dried products include biomaterials (Alves-Filho and Strommen, 1996), mushrooms (Wongwises et al., 1997), sawn rubber, wood and bananas (Prasertsan and Saen- saby,1998) fruits (Soponronnarit et al., 1998), onions, carrots, potatoes and sweet potatoes, paddy seed (Fatouh et al., 1998), seed (Soponronnarit et al., 2000), and wet wool (Oktay, 2003). Mangkoltriluk et al. (2005) argued that increasing the temperature decreased the amount of volatile compounds in parsley leaves. This is in accordance with Sunthonvit et al., (2007) who showed that nectarines dried at low temperature using heat pump drier had the highest number and concentration of volatiles mainly lactones, terpenoids, esters, and aldehydes.

2.9.5 Freeze-drying (lyophilisation)

Lyophilisation enhances the textural properties of dried food samples such as strawberries or sliced mushrooms. Such low temperature processing maximises the aroma retention in food as water is removed by sublimation (Kompany and Rene, 1995).

Sublimation is the conversion of ice to water vapour without melting into liquid water hence protecting the physical structure of food. It operates under specific conditions (temperature below 0 °C and pressures below 614 Pa) where the frozen water in the sample causes ice sublimation by exposing it to low vapour pressure of water vapour (Baker, 1997). Water continues to be removed until the sample reaches moisture content of 5% However, such process is very expensive and time consuming taking more than 8 hours (Nollet, 2009).

Sometimes dehydration methods result in low quality dried products manifested by a change in the appearance and also in altering the natural balance between the flavour and colour. The loss of VC during drying is common and could be due to the inactivation of volatiles forming enzymes and their precursors. Freeman and Whenman (1975) in their study of drying fresh onions using hot air and freeze drying showed that the freeze drying resulted in a 45% reduction in the enzymes activity compared to 90% decrease using hot-air drying. In the case of mushrooms, Chang (1982) explained the brown discolouration of freeze-dried mushrooms caused by the reduction in the activity of polyphenoloxidase. Kompany and Rene (1995), in their study to determine the kinetics to retain 1-octen 3-ol, the most important aroma compound of fresh mushrooms, demonstrated that the concentration of VC declines quickly in the early stages of drying then stops at a certain moisture level. This finding was also confirmed by Martinez-Soto et al. (2001) who reported that the rapidly falling drying rate period is controlled by diffusion mechanism. Martínez-Soto et al. (2001) showed that the freeze- dried mushrooms were characterised by physical appearance similar to that of the fresh mushrooms. However, this drying method has the limitation of being very expensive costing three to five times more per weight of water removed than other drying techniques (Nollet, 2004).

2.9.6 Vacuum drying

This method is known as the standard and most accurate drying method for moisture analysis in foods. The vacuum drying heats foods up to 98-102 °C at low pressures of 3-13 kPa removing water rapidly, hence decreasing the drying time (Park and Bell, 2004).

Vacuum ovens can be connected to a vacuum line having a front door sealed with vacuum grease. During the drying process, dry air moves into the oven, which is crucial to the drying process, hence increases the efficiency of the vacuum drying. The drying conditions are set at 100 °C for up to 6 hours at a pressure of 3-13 kPa (Park and Bell, 2004).

As mushrooms are very sensitive to temperature, choosing the drying method can be the key to a successful operation and hence to better quality. Giri and Prasad (2007) argued that vacuum-drying of mushroom is faster than conventional hot-air drying and the dried mushrooms were a more porous dehydrated product, which rehydrated more quickly and more completely than the air dried product.

Artnaseaw et al. (2010) argued that drying at higher temperatures yields greater colour degradation of Jinda chili compared to drying at lower temperatures. This was explained by the fact that enzymatic browning reactions of Jinda chili are accelerated by temperature. Increase in colour degradation with an increase in drying temperature has also been reported by Ren and Chen (1998), Sacilik and Unal (2005) and Sharma and Prasad (2001). 2.9.7 Tunnel drying

This continuous type of dryer is built in the form of a long tunnel. It is provided with inlet and outlet of air with heating coils incorporated in the tunnel. The material to be dried is filled in trays and trucks loaded with these trays move progressively through the tunnel in contact with a current of hot gas to evaporate the moisture. The air flow in a tunnel dry can be concurrent, counter-current or a combination of both. The wet material enters at one end and dried product leaves at the other end (Gavhane, 2008).

The effects of some parameters related to the product quality and drying conditions such as product thickness, drying air temperature and time, relative humidity were investigated by many researchers (Henderson, 1974; Ozdemir and Devres, 1999; Yaldız 42

and Ertekin, 2001). Pal and Chakraverty (1997) showed that drying using a thin layer dryer takes place at a constant rate period for a short time, followed by the falling rate period. As the drying temperature is increased, the total drying time is reduced, and the rate of drying increased. They also confirmed that the relationship between moisture content and drying time is exponential and is in decreasing order. In terms of assessing the quality of dried mushrooms, treated samples (steam blanching followed by citric acid pre-treatment before drying) result in more shrinking of product with light discoloration compared to the untreated samples.

2.10 Conclusion

Mushrooms, the fruiting bodies of fungi, are appreciated not only for their texture and flavour but also for their chemical and nutritional characteristics. As various physiological and morphological changes occur after harvest, mushrooms need to be preserved to maintain their quality. Volatiles compounds are produced via metabolic and non-metabolic pathways and are greatly affected by the extraction methods as well as any postharvest treatment such as drying and freezing. The most appropriate extraction techniques are such that result in the detection of as many compounds as possible.

3. Materials and Methods

3.1 Mushroom samples

3.1.1 Mushrooms species

Four mushroom species were studied which included chestnut (Agrocybe aegerita) which were brown in colour open cap almost flat, enoki (Flammulina velutipes) which were clusters of long white stems with small caps, oyster (Pleurotus ostreatus) which were oyster-shaped cap, white in colour with short stems found in clusters, and shiitake (Lentinus edodes) which had dark brown caps. The mushrooms harvested were from the first flush.

Preliminary work on button mushrooms (Agaricus bisporus) purchased from local supermarket in Sydney, Australia, has been published in International Journal of Food Research (Appendix C).

3.1.2 Mushrooms collection

Mushrooms were hand-picked from Li-Sun Exotic Mushrooms (Bowral NSW, Australia) placed in eskies then transported back to the University of New South Wales Australia within 2 h after picking. Mushrooms were then weighed into 50 g (for extraction purpose) and 1 kg batches (for drying purpose), packed in plastic bags (PVC, polyvinyl chloride film) and placed in an incubator set at 4 °C until needed.

3.2 Detection of spoilage

The quality was assessed by extracting the VC using ambient temperature vacuum distillation followed by HS-SPME coupled to GC-MS. The analysis was carried on day 0 (D0) (collection date), day 1 (D1), day 2 (D2), day 3 (D3), day 4 (D4), day 7 (D7), and day 10 (D10).

3.3 Drying

The drying procedures were carried out using a laboratory-scale tunnel cabinet at 40, 50, 60, and 70 °C (±1) with 20 % RH. The dryer ran without the sample for 30 min to set the desired drying conditions before each drying experiment. Sample (1 kg) of whole mushrooms was uniformly spread on a metal tray in a single layer. The system comprised a fan, heating unit, steam injection unit, balance and drying chamber. The 44

tray with mushroom sample was placed on a metal frame inside the drying chamber. The metal frame was resting on the tray of a top loading electronic balance (PL6001-S, Mettler Toledo International Inc.) with a sensitivity of 0.1 mg located below the drying chamber. The balance was connected to a computer and the weights were recorded at 1 min interval. The temperature was measured using t-type thermocouples while relative humidity (RH) was measured using duct sensor (QF M65, Siemens). These measurements were recorded by a data logger (DATATAKER DT50) at 5 minutes interval during the whole drying process. The drying experiments were performed in duplicate. The moisture content was determined according to AOAC (1995). Each drying run lasted until the weight of the samples reached a constant value. The moisture content of samples was expressed on dry basis (mass of water/mass of dry solids). The changes in moisture content of samples during drying were expressed as moisture ratio (MR) as shown in equation 3.1

MR (3.1)

Where MR moisture ratio

M moisture content at a given time

Me equilibrium moisture content

Mi initial moisture content

3.4 Treatments a) Fresh mushrooms (F) were cleaned, blended to a puree and fed into a 2 L round bottom flask with water for ambient temperature vacuum distillation. b) Boiled under reflux mushrooms (BR): Same as a) and then boiled under reflux for 30 min c) Dried mushrooms (D): The mushrooms were cleaned, dried, and then subjected to ambient temperature vacuum distillation. d) Dried then boiled under reflux (DBR): The dried mushrooms were boiled under reflux then subjected to ambient temperature vacuum distillation.

3.5 Ambient temperature vacuum distillation

Ambient temperature vacuum distillation was performed for 4 h at 30 Pa on each of the mushroom samples treated as described in section 3.4. The vacuum distillation was carried out according to the method published by Ashmore et al. (2013) and Ashmore et al. (2014) (Appendix C).

3.6 Concentration using SPME

An SPME holder (Supelco, PA, USA) was used in the second stage of the experiment. SPME was performed on the combined content of cold traps to extract the VOC using a fused silica fibre coated with 50/30 µm layer of divinylbenzene/ carboxen/ polydimethylsiloxane (DVB/CAR/PDMS) (Supelco). The fibre was conditioned following the manufacturer’s instructions prior to its use and was thermally cleaned between analyses by injecting it to the GC injection port at 250 °C for 15 min. The sample vial was placed inside a water bath and was allowed to condition for the equilibrium time (10 min) without the fibre. After the equilibrium time, the fibre was introduced into the vial and was exposed to the headspace of the sample during the required extraction time (1.5 h). After extraction, the fibre was inserted into the injection port of the GC for thermal desorption of the analytes.

This fiber was chosen for the following reasons:

1- Previous studies have shown that it gives the most complete screening of the volatile profile in mushrooms (Silva Ferreira and Guedes de Pinho, 2003, Guedes de Pinho, et al. 2008.). 2- A DVB /CAR/ PDMS extract different types of analytes at the same time and have a higher efficiency for extracting odour compounds from water (Mani 1999). 3- The use of a mixed phase DVB/CAR/PDMS showed excellent sensitivity and reproducibility for the S-compounds studied as suggested by Herszage and Ebeler (2011). 4- SPME extractions were performed at room temperature (25+/-1 °C). That is because increasing the temperature increases the oxidation rate even though the volatility of analytes is increased (Mestres et al., 2002).

3.7 Identification and quantification of volatiles compounds using GC-MS

The SPME was injected in the splitless mode into the GC-MS and left for 0.5 min allowing the volatiles to desorb. GC-MS analysis was performed using Agilent 19091S- 433 instrument equipped with a DB-5ms column (30 m length * 0.25 mm i.d. * 0.25 µm film thickness). The carrier gas used was helium at a constant flow rate of 1.1 mL/min. The injector temperature was 250 °C. The oven temperature was initially held at 30 °C for 5 min, and then raised to 250 °C at a rate of 3 °C/min held at 250 °C for 25 min. All the chromatogram peaks were identified by means of the mass spectral library using a PC, then confirmed by comparison with the retention time of the authentic reference (Wiley 275, Hewlett-Packard Company, Palo Alto, CA.). For quantitation, the standard addition method was used where absolute ethanol (10.00 g) and mixture of standards (1- octen-3-ol, 3-octanol, benzaldehyde and nonanal) were prepared at 500 ppm. A volume of the mixture (5 µL) was added progressively to 1 mL mushroom extract placed in water bath set at 30 °C +/-1 °C. The calibration curve obtained by plotting GC peak area versus different concentrations of the standards was plotted and the samples were quantified by calculation of each peak area from the calibration line.

3.8 Statistical analysis

Statistical analysis was performed using the softwares Origin 8.6 and Graph pad Prism 5.0. Experiments were performed in triplicate and results were expressed as the mean value +/- standard deviation (SD) of 3 replicates. Analysis of variance (ANOVA) was used to compare the fresh and thermally treated samples and to determine the effect of storage time on the VCs and hence the quality of mushrooms. Tukey’s test was used to determine the significant difference at 95% confidence interval.

4. RESULTS AND DISCUSSION

4.1 VC in Agrocybe aegerita (Chestnut mushrooms)

4.1.1. Fresh and stored chestnut mushrooms

Chestnut mushrooms were stored at 4 °C for 10 days (see section 3.1.2) and the VC were extracted to determine how they were changing with storage time. The concentrations of the identified VC are shown in Table 4.1. The highest concentrations of the main C8 compounds were observed at D0. For 1-octen-3-one, its concentration was found to be 1.40×10-1±1.55×10-2 ppm at D0 which significantly decreased at D1 to 7.06×10-2±9.38×10-3 ppm, at D2 to 3.41×10-2±2.74×10-3 ppm, and at D3 to 7.08× 10-3±3.25×10-4 ppm. No significant change was observed between D3 and D10 reaching a minimum concentration of 2.66×10-3± 6.33×10-4 ppm at D10. As for 1-octen-3-ol, it had the highest concentration at D0 (1.20×100±5.97×10-2 ppm) which significantly decreased at D1 to reach 4.63×10-1±4.03×10-2 ppm) followed by a significant decrease at D3 (2.94×10-1±2.24×10-2 ppm) and D7 (1.16×10-1±2.17×10-2 ppm). The concentration of 3-octanone at D0 was found to be 1.81×100± 7.90×10-2 ppm which also significantly decreased at D1 to reach 7.24×10-1±5.21×10-2 ppm. Further storage significantly decreased its concentration at D2 to 4.27×10-1±6.55×10-2 ppm and at D3 to 1.25×10-1±3.47×10-3 ppm with no significant decrease between from D3 to D10. The concentration of 3-octanol significantly decreased from D0 (3.49×100 ±2.57×10-1 ppm) until D3 (1.01×100±9.75×10-2 ppm). Its lowest concentration was found at D10 (4.54×10-1±2.05×10-2 ppm) which was not significantly different from D4 (7.82×10-1 ±5.01×10-2 ppm) and D7 (5.52×10-1±1.94×10-2 ppm). As for 2-octen-1-ol, its concentration showed a significant decrease at D2 (5.33×10-1±2.46×10-2 ppm) followed by a significant decrease at D4 (3.62×10-1±2.48×10-2 ppm) and D7 (2.42×10-1 ±3.56×10-2 ppm). The concentration of n-octanol significantly decreased from D0 (2.20×10-1±1.11×10-2 ppm) to D3 (1.24×10-1±5.74×10-3 ppm) then at D7, a further significant decrease in its concentration was found where it reached 6.31×10-2±4.70× 10-3 ppm with no further considerable changes occurred at D10.

As for alcohols, 3-methyl-1-butanol had its concentration insignificantly increased from D0 (2.15×10-3±2.72×10-4ppm) to D3 (6.62×10-3±4.37×10-4 ppm). At D4, a significant change was found where its concentration reached 1.26×10-2±2.33×10-3 ppm which kept

increasing at D7 to reach 2.31×10-2±3.53×10-3 ppm and at D10 to reach 3.43×10-2 ±2.77×10-3 ppm. Meanwhile, 2-methyl-1-butanol had its lowest concentration at D0 (3.95×10-3±3.18×10-4 ppm) which significantly increased to 6.04×10-3±2.88×10-4 ppm at D1, and then 8.64×10-3±3.13×10-4 ppm at D3. No significant difference was found between D2 and D3 for this compound then, at D4, it reached a maximum concentration of 1.06×10-2±6.86×10-4 ppm then it decreased significantly again at D7 (9.03×10-3 ±5.63×10-4 ppm) which was not significantly different from D10. As for 1-pentanol, it had its highest concentration at D1 (1.13×10-3±1.11×10-4 ppm) which was not significantly different form D0 (1.05×10-3±1.11×10-4 ppm) but significantly different from D2 (8.93×10-4±4.54×10-5 ppm) with no significant difference found between D2, D3, D4, and D7. Interestingly, its concentration increased again at D10 (1.16×10-3 ±7.49×10-5 ppm). As for 1-hexanol, its highest concentration was detected at D0 (2.16×10-2 ±1.87×10-3 ppm) which significantly decreased at D3 (1.58×10-2±2.06×10-3 ppm) and reached a minimum of 7.48×10-3±5.08×10-4 ppm at D10. The concentration of 1-nonanol insignificantly increased from D0 to D1 (3.00×10-3±5.09×10-4 vs. 3.52×10-3 ±4.23×10-4 ppm) then significantly decreased at D10 to reach 1.08×10-3±1.48×10-4 ppm. As for 3,5,5-trimethyl-1-hexanol, its highest concentration was observed at D1 (1.81×10-3±2.99×10-4 ppm) which significantly decreased at D3 (9.72×10-4±3.10×10-5 ppm) and D7 (3.90×10-4±3.27×10-5 ppm) to reach a minimum of 2.89×10-4±2.08×10-5 ppm at D10. The concentration of 2-hepten-1-ol significantly decreased from D0 (3.57×10-3±2.87×10-4 ppm) to D1 (2.13×10-3±1.07×10-4 ppm), D2 (1.45×10-3±2.75×10-4 ppm), and D3 (8.58×10-4±2.70×10-5 ppm). No significant change was observed from D3 onwards and the lowest concentration was detected at D10 (4.30×10-4±4.60×10-5 ppm). As for phenyl ethyl alcohol, its concentration was the highest at D0 (1.58×10-2 ±1.89×10-3 ppm) which decreased at D2. At D3, its concentration significantly decreased to reach 1.11×10-2±6.81×10-4 ppm and kept decreasing until it reached a minimum of 6.28×10-3±4.73×10-4 ppm at D10 significantly different from the remaining storage days. No significant difference was found between D1, D2, D3, D4, and D7. Meanwhile, n-octanol had its maximum concentration at D0 (2.20×10-1±1.11×10-2 ppm) which then significantly decreased until D7 to reach 6.31×10-2±4.70×10-3 ppm which was not significantly different from D10. As for 1-phenylethanone, its concentration significantly decreased from D0 (1.70×10-3±2.28×10-4 ppm) to D1 (8.92×10-4±3.96× 10-5 ppm) then significantly decreased to reach 5.84×10-4±3.01×10-5 ppm at D3. Further

storage did not have a major impact on the concentration of this compound. The concentration of 2-ethyl 1-hexanol increased significantly form D0 (4.64×10-3 ±9.05×10-4 ppm) to D2 (5.62×10-2±2.24×10-3 ppm). At D7, its concentration significantly increased to reach 9.79×10-2± 6.07×10-3 ppm which was not significantly different from D10 (1.12×10-1±1.50×10-2 ppm). As for 1,8-cineole, its concentration insignificantly decreased from D0 to D1 (1.24×10-3±7.55×10-5 vs. 9.35×10-4±4.87×10-5 ppm). However, its concentration significantly increased to reach 2.69×10-3±3.44×10-4 at D7 and 6.47×10-3±2.03×10-4 ppm at D10. As for 2,6-bis(1,1-dimethylethyl)phenol, its concentration did not change significantly throughout the storage study with a maximum concentration of 2.42×10-2±2.52×10-3 ppm at D10 and a lowest concentration of 1.89×10-2±1.91×10-3 ppm at D1.

As for aldehydes, hexanal had its concentration increased with time with a minimum of 4.07×10-3±1.72×10-4 ppm at D0 which marginally increased at D1 (5.15×10-3±4.02×10-4 ppm) and reached a maximum of 1.22×10-2±5.45×10-4 ppm at D10. The concentration of benzaldehyde increased significantly from D0 (1.05×10-3 ±7.93×10-5 ppm) to D2 (2.70×10-3±1.14×10-4 ppm). At D4, its concentration significantly increased to reach 4.94×10-3±5.97×10-4 ppm and kept increasing significantly at D7 to reach 7.84×10-3 ±6.72×10-4 ppm and at D10, it reached a maximum of 1.06×10-2±5.81×10-4 ppm. As for (E)-2-octenal, its highest concentration was detected at D0 (7.70×10-3±4.52×10-4 ppm) which then significantly decreased at D1 to reach 1.55×10-3±2.69×10-4 ppm. Its lowest concentration was found at D10 (1.10×10-3±1.45×10-4 ppm) which was not significantly different from D1, D2, D3, and D4. The concentration of nonanal did not significantly change from D0 (1.81×10-3±2.47×10-4 ppm) to D3 (2.68×10-3±1.20×10-4 ppm). However a significant increase was observed at D7 (8.82×10-3±4.32×10-4 ppm and D10 (9.85×10-3±5.06×10-4 ppm). As for decanal, its concentration insignificantly increased from D0 (8.51×10-4±6.85×10-5) to D3 (1.89×10-3±5.24×10-4 ppm) then compared to D0, its concentration significantly increased at D4 (2.46×10-3±2.39×10-4 ppm). At D7 and D10, a significant increase was found (6.07×10-3±4.55×10-4 ppm and 8.34×10-3 ±6.94×10-4 ppm respectively).

Other compounds identified include (E,Z)-2,6-nonadienal which significantly decreased from D0 (1.55×10-3±1.74×10-4 ppm) to D1 (1.10×10-3±9.83×10-5 ppm) and D2 (8.50×10-4± 2.81×10-5 ppm) with no significant change between D2 and D3 and then

compared to D2, it significantly decreased at D4 to reach 5.45×10-4±5.52×10-5 ppm which was not significantly different from D3. At D10, it reached a minimum of 2.01×10-4±2.46×10-5 ppm which was significantly different from the rest of the storage time but not from D7. As for (Z)-1-ethyl-2-methylcyclohexane, its highest concentration was found at D0 (2.26×10-3±3.41×10-4 ppm) which was not significantly different from D1 but significantly different from D2 (1.18×10-3±1.63×10-4 ppm) and D4 (6.96×10-4±3.70× 10-5 ppm). No significant difference was found between D3, D4, D7, and D10 for this compound. Meanwhile, 3-heptanone had its highest concentration at D2 (1.63×10-3±3.26×10-4ppm) which was not significantly different from D1 but significantly higher than D3 (8.79×10-4±4.15×10-5 ppm) and D4 (4.11×10-4±3.26×10-5 ppm) and further storage did not significantly affect the concentration of this compound. As for 1-(2-furyl)-3-methyl-3-butene-1,2-diol, it highest concentration was found at D0 (2.45×10-3±4.87×10-4 ppm) which insignificantly decreased during storage until D2, then compared to D0, a significant decrease was found at D3 (9.53×10-4±2.37×10-5 ppm) and further storage did not significantly affect the concentration of this compound.

Table 4.1: Concentrations of VC extracted from chestnut mushrooms during 10 days of storage at 4 °C. Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD ppm SD ppm SD ppm SD (ppm) ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) 3-methyl-1- butanol 2.15×10-3 d 2.72×10-4 2.58×10-3 d 1.10×10-4 3.45×10-3 d 3.34×10-4 6.62×10-3 d 4.37×10-4 1.26×10-2 c 2.33×10-3 2.31×10-2 b 3.53×10-3 3.43×10-2 a 2.77×10-3 2-methyl-1- butanol 3.95×10-3 d 3.18×10-4 6.04×10-3 c 2.88×10-4 8.64×10-3 b 3.13×10-4 9.67×10-3 ab 3.29×10-4 1.06×10-2 a 6.86×10-4 9.03×10-3 b 5.63×10-4 9.11×10-3 b 7.80×10-4 1-pentanol 1.05×10-3 ab 1.11×10-4 1.13×10-3 a 1.11×10-4 8.93×10-4 bc 4.54×10-5 7.88×10-4 c 5.75×10-5 7.47×10-4 c 3.57×10-5 8.45×10-4 bc 4.97×10-5 1.16×10-3 a 7.49×10-5 Hexanal 4.07×10-3 c 1.72×10-4 5.15×10-3 bc 4.02×10-4 5.80×10-3 c 4.41×10-4 6.15×10-3 bc 3.98×10-4 7.10×10-3 abc 2.36×10-4 1.03×10-2 ab 2.53×10-4 1.22×10-2 a 5.45×10-4 1-hexanol 2.16×10-2 a 1.87×10-3 1.83×10-2 ab 2.50×10-3 1.64×10-2 ab 1.24×10-3 1.58×10-2 bc 2.06×10-3 1.41×10-2 bc 2.69×10-3 1.10×10-2 cd 1.47×10-3 7.48×10-3 d 5.08×10-4 6.84×10-4 3-heptanone 6.68×10-4 bc 6.16×10-5 1.39×10-3 a 1.53×10-4 1.63×10-3 a 3.26×10-4 8.79×10-4 b 4.15×10-5 4.11×10-4 c 3.26×10-5 5.05×10-4 bc 4.44×10-5 bc 2.14×10-4 Benzaldehyde 1.05×10-3 f 7.93×10-5 1.72×10-3 ef 1.44×10-4 2.70×10-3 de 1.14×10-4 3.32×10-3 d 4.97×10-4 4.94×10-3 c 5.97×10-4 7.84×10-3 b 6.72×10-4 1.06×10-2 a 5.81×10-4 2-hepten-1-ol 3.57×10-3 a 2.87×10-4 2.13×10-3 b 1.07×10-4 1.45×10-3 c 2.75×10-4 8.58×10-4 d 2.70×10-5 6.96×10-4 d 6.31×10-5 5.49×10-4 d 2.70×10-5 4.30×10-4 d 4.60×10-5 1-octen-3-one 1.40×10-1 a 1.55×10-2 7.06×10-2 b 9.38×10-3 3.41×10-2 c 2.74×10-3 7.08×10-3 d 3.25×10-4 5.46×10-3 d 5.95×10-4 4.56×10-3 d 5.22×10-4 2.66×10-3 d 6.33×10-4 1-octen-3-ol 1.20×100 a 5.97×10-2 4.63×10-1 b 4.03×10-2 3.86×10-1 b 2.37×10-2 2.94×10-1 c 2.24×10-2 2.19×10-1 c 1.68×10-2 1.16×10-1 d 2.17×10-2 7.39×10-2 d 5.60×10-3 3-octanone 1.81×100 a 7.90×10-2 7.24×10-1 b 5.21×10-2 4.27×10-1 c 6.55×10-2 1.25×10-1 d 3.47×10-3 7.32×10-2 d 2.97×10-3 6.13×10-3 d 2.89×10-4 6.17×10-3 d 3.12×10-4 3-octanol 3.49×100 a 2.57×10-1 2.25×100 b 4.17×10-1 1.63×100 c 2.09×10-1 1.01×100 d 9.75×10-2 7.82×10-1 d 5.01×10-2 5.52×10-1 d 1.94×10-2 4.54×10-1 d 2.05×10-2 1,8-cineole 1.24×10-3 c 7.55×10-5 9.35×10-4 cd 4.87×10-5 5.53×10-4 de 6.79×10-5 4.50×10-4 e 3.44×10-5 5.53×10-4 de 6.79×10-5 2.69×10-3 b 3.44×10-4 6.47×10-3 a 2.03×10-4 (Z)-1-ethyl-2- methylcycloh exane 2.26×10-3 a 3.41×10-4 2.19×10-3 a 1.35×10-4 1.18×10-3 b 1.63×10-4 8.43×10-4 bc 6.23×10-5 6.96×10-4 c 3.70×10-5 5.40×10-4 c 4.63×10-5 4.34×10-4 c 2.53×10-5 2-ethyl-1- hexanol 4.64×10-3 d 9.05×10-4 3.00×10-2 c 2.50×10-3 5.62×10-2 b 2.24×10-3 6.79×10-2 b 4.45×10-3 7.09×10-2 b 8.66×10-3 9.79×10-2 a 6.07×10-3 1.12×10-1 a 1.50×10-2 1-(2-furyl)-3- methyl-3- butene-1,2- diol 2.45×10-3 a 4.87×10-4 1.67×10-3 ab 2.71×10-4 1.65×10-3 ab 5.46×10-4 9.53×10-4 bc 2.37×10-5 6.84×10-4 c 4.57×10-5 3.54×10-4 c 2.62×10-5 2.00×10-4 c 1.96×10-5 1-phenyl 1.70×10-3 a 2.28×10-4 8.92×10-4 b 3.96×10-5 7.40×10-4 bc 2.46×10-5 5.84×10-4 cd 3.01×10-5 5.34×10-4 cd 3.71×10-5 4.74×10-4 d 2.62×10-5 4.10×10-4 d 2.69×10-5

Table 4.1 continued Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD ppm SD ppm SD ppm SD (ppm) ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) ethanone (E)-2-octenal 7.70×10-3 a 4.52×10-4 1.55×10-3 bc 2.69×10-4 1.59×10-3 bc 3.37×10-4 1.68×10-3 bc 2.78×10-4 1.27×10-3 bc 2.25×10-4 1.94×10-3 b 2.12×10-4 1.10×10-3 c 1.45×10-4 2-octen-1-ol 6.89×10-1 a 7.28×10-2 6.27×10-1 ab 2.15×10-2 5.33×10-1 bc 2.46×10-2 4.36×10-1 cd 1.79×10-2 3.62×10-1 d 2.48×10-2 2.42×10-1 e 3.56×10-2 1.98×10-1 e 1.63×10-2 n-octanol 2.20×10-1 a 1.11×10-2 1.94×10-1 a 1.33×10-2 1.58×10-1 a 3.55×10-3 1.24×10-1 b 5.74×10-3 9.34×10-2 b 3.24×10-3 6.31×10-2 c 4.70×10-3 4.25×10-2 c 5.93×10-3 Nonanal 1.81×10-3 d 2.47×10-4 2.05×10-3 d 3.73×10-4 2.28×10-3 cd 2.90×10-4 2.68×10-3 cd 1.20×10-4 3.11×10-3 c 1.43×10-4 8.82×10-3 b 4.32×10-4 9.85×10-3 a 5.06×10-4 Phenyl ethyl aclohol 1.58×10-2 a 1.89×10-3 1.40×10-2 ab 1.32×10-3 1.25×10-2 ab 1.18×10-3 1.11×10-2 b 6.81×10-4 1.13×10-2 b 1.09×10-3 1.24×10-2 ab 2.03×10-3 6.28×10-3 c 4.73×10-4 (E,Z)-2,6- nonadienal 1.55×10-3 a 1.74×10-4 1.10×10-3 b 9.83×10-5 8.50×10-4 c 2.81×10-5 7.51×10-4 cd 3.23×10-5 5.45×10-4 de 5.52×10-5 3.70×10-4 ef 4.36×10-5 2.01×10-4 f 2.46×10-5 1-nonanol 3.00×10-3 ab 5.09×10-4 3.52×10-3 a 4.23×10-4 2.65×10-3 abc 3.58×10-4 2.48×10-3 abc 8.70×10-4 2.21×10-3 bcd 3.33×10-4 1.47×10-3 cd 1.68×10-4 1.08×10-3 d 1.48×10-4 3,5,5- trimethyl-1- hexanol 1.54×10-3 ab 2.26×10-4 1.81×10-3 a 2.99×10-4 1.26×10-3 bc 9.89×10-5 9.72×10-4 cd 3.10×10-5 7.85×10-4 de 3.06×10-5 3.90×10-4 ef 3.27×10-5 2.89×10-4 f 2.08×10-5

Decanal 8.51×10-4 d 6.85×10-5 1.18×10-3 d 1.69×10-4 1.61×10-3 cd 1.81×10-4 1.89×10-3 cd 5.24×10-4 2.46×10-3 c 2.39×10-4 6.07×10-3 b 4.55×10-4 8.34×10-3 a 6.94×10-4 2,6-bis(1,1- dimethylethyl )phenol 1.96×10-2 a 2.02×10-3 1.89×10-2 a 1.91×10-3 2.18×10-2 a 2.46×10-3 2.28×10-2 a 2.08×10-3 2.38×10-2 a 3.63×10-3 2.15×10-2 a 2.08×10-3 2.42×10-2 a 2.52×10-3 *Standard deviation. Analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

The concentrations of the identified VC were normalised against the fresh samples to determine how the VC were changing with storage time. Figure 4.1 shows the behaviour of the main C8 compounds as they all decreased with storage time. As for1- octen-3-one, it significantly decreased from D0 to D3. At D4, the concentration slightly decreased with no significant difference found between D3, D4, D7, and D10. Meanwhile, 1-octen-3-ol had its normalised relative concentration significantly decreased from D0 to D1, with an insignificant change occurring at D2 then, it significantly decreased at D3 (no change was observed between D3 and D4) and continued to significantly decrease at D7 with no significant change at D10.

1-octen-3-one 120 1-octen-3-ol 3-octanone 3-octanol 100 2-octen-1-ol n-octanol 80

Normalised relative concentration Normalised relative 0

0 2 4 6 8 10 Day

Figure 4.1: Normalised relative concentrations of the main C8 compounds extracted from chestnut mushrooms over a 10 days storage period.

The normalised relative concentration of 3-octanone significantly decreased from D0 until D3 and no further significant change was observed after that. As for 3-octanol, its normalised relative concentration significantly decreased from D0 to D3, and further storage did not significantly affect the normalised relative concentration of this compound while the normalised relative concentration of 2-octen-1-ol significantly decreased at D2, D4 and D7 but there was no significant change between D7 and D10. As for n-octanol, its normalised relative concentration significantly decreased from D0 to D7. No significant changes found D7 and D10.

Mau et al. (1992) and Wurzenberger and Grosch (1984), found that 1-octen-3-ol in A. bisporus decreased rapidly during the first 3 days, then dropped again through D9. These findings are in accordance with the results in this study where the level of 1- octen-3-ol significantly decreased from D0 to D1 and kept decreasing until D10.

Rapior et al. (1997) reported 3-octanone (fungal-fruity aroma) as the major VC and 1- octen-3-ol (fungal odour) as a minor compound in A. aegerita. These results were not in accordance with those reported by Takama et al. (1979) who found 1-octen-3-ol as the main VC in A. aegerita. The result of this study suggested that 3-octanol was the major VC during the storage study. Furthermore, Mau et al. (1992) showed that the content of 1-octen-3-ol in A. bisporus increased with each flush up to the third one then decreased in the fourth and the fifth, and finally drastically decreased at the sixth flush. As the mushrooms studied in this dissertation are collected from the first flush, it could be a reason why 1-octen-3-ol was not the major VC found.

Alcohols behaved differently from each other as shown in Figure 4.2. As for 1-hexanol, its highest normalised relative concentration was observed at D0 and it significantly decreased at D3. No significant difference was observed between D0, D1, and D2 and between D3, D4, and D7, and between D7 and D10.

1-hexanol 1-nonanol 3,5,5-trimethyl 1-hexanol 140 2-hepten-1-ol Phenyl ethyl aclohol 120

100

20 Normalised relative concentration relative Normalised

0 0 2 4 6 8 10 Day

Figure 4.2: Normalised relative concentrations of selected alcohols during the storage of chestnut mushrooms over a 10 days storage period.

Meanwhile, 1-nonanol had its highest normalised relative concentration at D1 which was not significantly different from D0, D2, and D3 but significantly different from D4, D7, and D10. However, no significant difference was found between D4, D7, and D10. The compound 3,5,5-trimethyl-1-hexanol had its highest normalised relative concentration at D1, slightly higher than D0 and then decreased significantly at D2. No significant change was observed between D2 and D3, D3 and D4, D4 and D7, and between D7 and D10. The normalised relative concentration of 2-hepten-1-ol significantly decreased from D0 to D3 and further storage did not have any effect. The normalised relative concentration of phenyl ethyl alcohol decreased with storage time with no significant change observed between D0, D1 and D2, and between D1, D2, D3, D4, and D7. However, a significant decrease was found at D10.

The aldehydes’ behaviour is shown in Figure 4.3. The normalised relative concentration of hexanal was the lowest at D0 and the highest at D10. No significant difference was observed between D0, D1, D2, D3, and D4 and between D2, D4, D7 and D10. The normalised relative concentration of benzaldehyde also increased with time with a minimum relative concentration observed at D0 which significantly increased from D2 onwards. No significant difference was observed between D0 and D1, and between D1 and D2. As for (E)-2-octenal, it showed a maximum normalised relative concentration at D0 which significantly decreased at D1. Further storage did not have a significant effect on this compound.

Hexanal 1200 Benzaldehyde (E)-2-octenal Nonanal 1000 Decanal

800

600

400

200 Normalisedrelativeconcentration 0

0 2 4 6 8 10 Day

Figure 4.3: Normalised relative concentrations of selected aldehydes in stored chestnut mushrooms over a 10 days storage period.

The normalised relative concentration of nonanal did not significantly change with storage from D0 until D3 and no significant change was observed between D2, D3, and D4. However, at D7, a significant increase was found which continued until D10. As for decanal, its normalised relative concentration was the lowest at D0 with a marginal increase observed until D3. From D4 onwards, a significant increase was found. No significant difference was detected between D2, D3, and D4. Meanwhile, (E,Z)-2,6- nonadienal had a maximum normalised relative concentration at D0 which then decreased significantly until D2. No significant difference was found between D2 and D3, D3 and D4, between D4 and D7 and between D7 and D10.

The normalised relative concentrations of selected VC are shown in Figure 4.4. The compound 3-methyl-1-butanol had its normalised relative concentration marginally increasing from D0 to D3 (no significant change between D0, D1, D2, and D3) then it significantly increased from D4 onwards. The normalised relative concentration of 2- ethyl-1-hexanol significantly increased from D0 to D2. No significant change was observed between D2, D3, and D4. At D7, a significant increase in the normalised relative concentration was detected which was not significantly different from D10. As for 1,8-cineole, its normalised relative concentration marginally decreased from D0 to

D1 and no significant change was found between D1 and D2 and between D2, D3, and D4. However, a significant increase was observed at D7 and D10.

3-methyl-1- butanol 2-ethyl-1- hexanol 1,8-cineole

2700

2400

2100

1800

1500

1200

900

600

300 Normalised relative concentration 0

0 2 4 6 8 10 Day

Figure 4.4: Normalised relative concentrations of selected VC extracted from stored chestnut mushrooms over a 10 days storage period.

These results suggest that the highest quality of the chestnut mushrooms is observed at D0 and their VC content was changing from D1 especially for the main C8 compounds. Meanwhile aldehydes’ content (benzaldehyde, decanal, hexanal, nonanal) was increasing with the storage time while some alcohol compounds (such as 1,8-cineole, 2- ethyl-1-hexanol and 3,5,5-trimethyl-1-hexanol) showed no significant difference between D3 and D4. Therefore it can be assumed that chestnut mushrooms are a delicate produce highly perishable with a quality that deteriorates rapidly therefore storage up to D4 would be the most suitable storage time. At D7, the mushrooms were still containing a significant amount of VC contributing to the pleasant aroma however the mushrooms were losing some of their important VC.

Mushrooms are living organisms where continued respiration and other metabolic reactions are still occurring after harvesting. However, the exact biochemical pathway of how these VC are evolving is still unknown and it requires further investigation which was not the aim of this study. Possible explanations for the changes in the VC profile could be due to biochemical changes in mushrooms during storage, microbial

changes or due to the nature of the packaging film (PVC film) where oxygen, carbon dioxide or water transmission rate may have affected the VC profile.

The compounds 2-methyl-1-butanol, 1-octen-3-one, 1-octen-3-ol, 3-octanone, 3- octanol, 2-octen-1-ol, n-octanol, (E)-2-octenal, and phenyl ethyl alcohol were previously reported by Rapior et al. (1997). In this study, a series of aldehydes (hexanal, nonanal and decanal), alcohols (3-methyl-1-butanol, pentanol, hexanol, 2-ethyl hexanol, 2-hepten-1-ol, 3,5,5-trimethyl-1-hexanol, and nonanol) as well as 1,8 cineole, 1-ethyl- 2-methyl-cis cyclohexane, 1-phenyl ethanone, 2,6-nonadienal, 3-butene-1,2-diol,1-(2- furanyl)-3-methyl, and 2,6-bis(1,1-dimethyl)phenol were also identified.

In this study, the concentrations of hexanal were increasing during storage which were not in accordance with the results of Zhang et al. (2010, 2009) who reported a decreased in hexanal concentration during the ripening of peach and kiwi fruit and correlated it to a decline in lypoxygenase and hydroperoxide lyase gene expression. Nonanal, characterised by a citrus-like or fatty aroma was previously reported as a potential contributor to avocado flavour during maturity (Obenland et al., 2012).

The compound 1,8-cineole noted for imparting a peppermint aroma was previously reported as an aroma contributor to Elder flower drink (Jørgensen et al., 2000).

4.1.2 Drying curves

The moisture ratios versus time of chestnut mushrooms dried at different temperatures are presented in Figure 4.5. Drying was faster at higher temperatures compared to lower temperatures (40 °C). The drying process can be divided into two stages where during the initial stage of drying, there was a rapid moisture removal from the mushroom samples, which decreased later with the increase in drying time. Stage 1 is due to the fact that moisture is removed from the food sample into the interface between the surface of the food and the drying air in the dryer while stage 2 involved the removal of moisture from the interior of the sample and is dependent on the rate of diffusion within the sample. At 40 °C, the mushrooms were dried for 16 h and the final MC reached was 4.5%. At 50 °C, the drying time was 15 h and the final MC was 2.8% while at 60 °C, the mushrooms were dried for 9 h with a final MC of 3.3% and at 70 °C, the mushrooms were dried for 8 h with a final MC of 1.5%.

1.0 0.9 0.8 0.7 0.6 40 °C 0.5 50 °C 0.4 60 °C

Moistureratio 0.3 70 °C 0.2 0.1 0.0 0 100 200 300 400 500 600 Time (min)

Figure 4.5: Moisture ratio of chestnut mushrooms at different drying temperatures versus time

4.1.3 Dried (D) chestnut mushrooms

Based on lack of published studies, it appears that volatile compounds in thermally processed chestnut mushrooms have not been studied so far. In this study, chestnut mushrooms were subjected to 4 different drying conditions namely 40, 50, 60, and 70 °C and the concentrations of the identified compounds are shown in Table 4.2. As for the main C8 VC, at 40 °C, 3-octanone was the major C8 compound (4.65× 10-1±2.37×10-2 ppm) followed by 3-octanol (1.19×10-1±1.95×10-2 ppm), 1-octen-3-ol (4.01×10-2±5.02×10-3 ppm), n-octanol (2.90×10-2±5.57×10-3 ppm), 2-octen-1-ol (1.41×10-2±3.29×10-3 ppm), 2-octanone (8.38×10-3±4.06×10-4 ppm), and 1-octen-3-one (8.84×10-4±7.51×10-5 ppm). At 50 °C, 3-octanone was still the major C8 compound with a concentration of 3.57×10-1±4.07×10-2 ppm which was not significantly different from that found at 40 °C, followed by 3-octanol which had a significantly lower concentration compared to 40 °C (5.99×10-2±7.58×10-3 ppm), then 1-octen-3-ol which had a concentration slightly higher compared to 40 °C (5.04×10-2 ±7.79×10-3 ppm), n- octanol with an insignificant change compared to 40 °C (2.72×10-2±2.22×10-3 ppm), 2- octen-1-ol (9.63×10-3±2.66×10-4 ppm), and 1-octen-3-one (1.01×10-3±6.92×10-5 ppm) both with insignificant changes compared to 40°C. At 60 °C, 3-octanone remained the major VC with a maximum concentration of 7.90×10-1±7.41×10-2 ppm followed by 1- octen-3-ol which showed a significant increase in its concentration reaching a maximum of 1.35×10-1±6.75×10-3 ppm with no significant change compared to 70 °C. As for 3- octanol, its concentration significantly decreased at 70 °C compared to 60 °C (1.06× 60

10-2±7.73×10-4 vs. 4.38×10-2±9.40×10-4 ppm) while 2-octanone had its highest concentration at 60 °C (1.28×10-2±6.35×10-4 ppm) significantly different from that obtained at 70 °C. The compounds 2-octen-1-ol and n-octanol had their maximum concentration at 60 °C (1.83×10-2±8.70×10-4ppm and 4.04×10-2±2.14×10-3 ppm respectively).

As for aldehydes, several compounds had their maximum concentration at 70 °C and they include 3-methyl butanal (1.69×10-2±4.45×10-3 ppm) and 2-methyl butanal (6.05× 10-3±5.32×10-4 ppm) with their concentrations significantly different from those found at 40, 50 and 60 °C, hexanal with a maximum of 2.46×10-2±7.94×10-4 ppm significantly different from that found at 60 °C (1.44×10-2±1.38×10-3 ppm) and with the lowest concentration retrieved at 50°C (7.96×10-3±3.78×10-4 ppm), nonanal with a maximum concentration of 8.33×10-3±3.28×10-4 ppm and which had the lowest concentration at 50 °C (2.68×10-3±2.77×10-4 ppm), benzaldehyde (2.10×10-1±3.91×10-2 ppm) which showed no significant difference between drying at 40, 50 , and 60 °C, (E)-2-octenal with a highest concentration of 4.98×10-3±9.66×10-4 ppm and a lowest of 1.42×10-3 ±2.08×10-4 at 50 °C, (E,E)-2,4-decadienal with a maximum of 6.57×10-3±3.10×10-4 ppm at 70 °C and the lowest at 60 °C (1.18×10-3±2.08×10-4 ppm) which was not significantly different from that found in 50 °C, and phenylacetaldehyde having a maximum concentration of 2.05×10-1±1.13×10-2 ppm and the lowest at 40 °C (4.83×10-3 ±5.52×10-4 ppm) not significantly different from 40 °C. Decanal on the other hand, had its highest concentration at 60 °C (3.84×10-3±2.57×10-4 ppm) which was significantly different from the lowest concentration detected at 50 °C (8.76×10-4±3.57×10-5 ppm). As for (E)-2-hexenal, its maximum concentration was found at 60 °C (1.95×10-3 ±1.38×10-4 ppm) which was significantly different from that found at 70 °C (4.89×10-4 ±2.85×10-5 ppm) but not significantly different from that found at 40 and 50 °C.

As for the remaining alcohols, 3-methyl-1-butanol, its highest concentration was found at 60 °C (4.32×10-2±4.10×10-3 ppm) which was not significantly different from 50 °C (4.07×10-2±3.66×10-3 ppm) but significantly different from its lowest concentration found in 70 °C samples (2.44×10-2±2.48×10-3 ppm). Meanwhile, 2-methyl-1-butanol also had its highest concentration at 60 °C (1.62×10-2±3.97×10-3 ppm), significantly different from that found in 70 °C samples but not significantly different from 40 and 50 °C samples. As for 1-pentanol, it had its maximum concentration at 50 °C (5.10×

10-3±3.58×10-4 ppm) significantly different D 40, 60, and 70 °C samples. The compound 1-hexanol had a maximum concentration of 1.50×10-1 ±2.34×10-2 ppm at 50 °C insignificantly different from that found at 40 °C or 60 °C but significantly different from its lowest concentration at 70 °C (8.04×10-2±1.89×10-3 ppm). As for 2- ethyl-1-hexanol, its highest concentration found was at 60 °C (7.88×10-3±1.52×10-4 ppm) which was not significantly different from 70 °C while it had the lowest concentration at 50 °C (6.20×10-3±8.17×10-4 ppm). Meanwhile 3-ethyl-5-hexen-3-ol had its maximum concentration at 60 °C (6.69×10-3±5.37×10-4 ppm) significantly different from that found in 40 and 70 °C samples but not significantly different from 50 °C. Phenyl ethyl alcohol had its highest concentration at 60 °C (9.49×10-2±3.22×10-3 ppm) and its lowest at 50 °C (5.53×10-2±1.67×10-3 ppm) which was comparable to that found in 40 °C samples (5.60×10-2±7.64×10-3 ppm). The compound 1-heptanol showed a maximum concentration at 60 °C (1.21×10-2±1.31×10-3 ppm), significantly different from its lowest concentration found in 70 °C samples (4.13×10-3±1.48×10-4 ppm). As for 1-nonanol, it showed no significant difference between 40, 50, 60, and 70 °C with the lowest concentration found was in 70 °C samples (1.09×10-2±6.47×10-4ppm). Meanwhile, 1-methyl-2-cyclohexen-1-ol had its highest concentration at 40 °C (3.04×10-3±4.97×10-4 ppm) which was significantly different from 50 °C (4.34×10-4 ±6.34×10-5 ppm) and 60 °C (1.03×10-3±1.05×10-4 ppm). It was not detected at 70 °C. The maximum concentration of benzyl alcohol was found at 50 °C (3.28×10-3±1.57× 10-4 ppm) which was significantly different from that obtained at 70 °C (7.89×10-4 ±8.95×10-5 ppm) but not significantly different from that found at 40 °C. As for 1,8- cineole, it had its highest concentration at 40 °C (1.40×10-3±2.82×10-4 ppm) which was significantly different from that found in 50 °C samples (8.40×10-4±3.54×10-5 ppm) but insignificantly different from 60 and 70 °C. Finally, 2,6-bis(1,1-dimethylethyl) phenol, its highest concentration was found at 70 °C (7.80×10-3±7.47×10-4 ppm) which was not significantly different from 40 and 60 °C but significantly different from 50 °C (4.57×10-3±8.77×10-4 ppm).

As for ketones, 3-heptanone was not detected at 40 °C but it had its maximum concentration at 70 °C (5.97×10-3±2.53×10-4 ppm) significantly higher than that found at 50 °C (1.42×10-3±5.53×10-5 ppm) and 60 °C (1.62×10-3±3.36×10-4 ppm). The compound 2-methylcyclohexanone had its highest concentration at 70 °C (3.73×10-3 ±3.52×10-4 ppm), significantly different from the other treatments with the lowest 62

concentration being found at 50 °C (7.61×10-4±3.93×10-5 ppm). The compound 2- undecanone had its maximum concentration at 60 °C (3.96×10-3±1.71×10-4 significantly different from its lowest concentration found at 70 °C. No significant difference was found between drying at 40 and 70 °C for this compound. As for 5-methyl-5-hexen-2- one, its highest concentration was found at 50 °C (2.59×10-3 ±3.49×10-4 ppm) which was significantly different from its lowest concentration found at 40 °C.

Other compounds identified include ethyl acetate which had its maximum concentration at 40 °C (4.82×10-4±2.49×10-5 ppm), significantly different from that found at 50 °C; however, it was not detected at 60 and 70 °C, p-tolylthioamide where different drying temperatures had no significant effect on its concentrations with the lowest concentration being found at 60 °C (1.57×10-3±2.22×10-4 ppm). As for 1,4-dimethyl-1- ethyl cyclohexane, its highest concentration was found at 40 °C (1.26×10-3 ±4.94×10-5 ppm) significantly different from that found in 50 °C samples. This compound was not detected at 60 and 70 °C. The compounds tetrahydro-3-methyl-4-methylene furan and 1-(2-furyl)-3-methyl-3-butene-1,2-diol had their highest concentrations at 40 °C (1.34×10-3±3.67×10-4 ppm and 3.66×10-3±2.88× 10-4 ppm respectively), significantly different from those found at the remaining drying temperatures.

Limonene had a maximum concentration of 5.36×10-3±5.84×10-4 ppm at 70 °C, significantly different from those found in 40, 50 and 60 °C samples. As for thiofuran, its highest concentration found was 4.09×10-3±3.25×10-4 ppm at 60 °C which was not significantly different from 50 °C samples but significantly different from its lowest concentration found at 70 °C (1.89×10-3±7.11× 10-4 ppm). As for 1,9-nonanediol, its maximum concentration was found at 50 °C (7.73×10-3±6.96×10-4 ppm) being significantly different from its lowest concentration detected at 70 °C (8.98×10-4 ±2.85×10-5 ppm). Finally, propanoic acid,2-methyl-1-(1,1-dimethylethyl)-2-methyl-1,3- propanediyl ester had its maximum concentration at 60 °C (2.77×10-3±2.90×10-4 ppm), significantly different from that found in 40, 50, and 70 °C.

Table 4.2: Concentrations of VC extracted from chestnut mushrooms dried at different temperatures D 40 °C D 50 °C D 60 °C D 70 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Ethyl acetate 4.82×10-4 a 2.49×10-5 2.28×10-4 b 3.31×10-5 ND ND ND ND

3-methyl butanal 7.44×10-4 b 2.40×10-5 1.40×10-3 b 2.75×10-4 2.75×10-3 b 3.01×10-4 1.69×10-2 a 4.45×10-3

2-methyl butanal 4.14×10-4 c 3.55×10-5 7.79×10-4 c 4.91×10-5 1.72×10-3 b 2.98×10-4 6.05×10-3 a 5.32×10-4

3-methyl-1-butanol 2.76×10-2 b 1.11×10-3 4.07×10-2 a 3.66×10-3 4.32×10-2 a 4.10×10-3 2.44×10-2 b 2.48×10-3

2-methyl-1-butanol 1.11×10-2 ab 6.20×10-4 1.56×10-2 ab 3.62×10-3 1.62×10-2 a 3.97×10-3 8.71×10-3 b 3.21×10-4

1-pentanol 2.91×10-3 b 1.64×10-4 5.10×10-3 a 3.58×10-4 2.50×10-3 b 4.10×10-4 2.63×10-3 b 1.43×10-4

Hexanal 8.71×10-3 c 5.58×10-4 7.96×10-3 c 3.78×10-4 1.44×10-2 b 1.38×10-3 2.46×10-2 a 7.94×10-4

1-hexanol 1.35×10-1 a 2.79×10-2 1.50×10-1 a 2.34×10-2 1.22×10-1 ab 1.77×10-2 8.04×10-2 b 1.89×10-3

3-heptanone ND ND 1.42×10-3 b 5.53×10-5 1.62×10-3 b 3.36×10-4 5.97×10-3 a 2.53×10-4

P-tolylthioamide 1.94×10-3 a 4.89×10-4 1.72×10-3 a 6.17×10-4 1.57×10-3 a 2.22×10-4 1.63×10-3 a 2.74×10-4

2-methylcyclohexanone 4.97×10-4 c 2.91×10-5 7.61×10-4 c 3.93×10-5 2.06×10-3 b 1.64×10-4 3.73×10-3 a 3.52×10-4

Benzaldehyde 6.09×10-3 b 3.80×10-4 1.14×10-2 b 7.71×10-4 4.22×10-2 b 8.19×10-3 2.10×10-1 a 3.91×10-2

1-heptanol 6.53×10-3 c 4.05×10-4 9.30×10-3 b 5.27×10-4 1.21×10-2 a 1.31×10-3 4.13×10-3 d 1.48×10-4

1-octen-3-one 8.84×10-4 c 7.51×10-5 1.01×10-3 c 6.92×10-5 2.62×10-3 b 7.26×10-4 5.45×10-3 a 5.02×10-4

1-octen-3-ol 4.01×10-2 b 5.02×10-3 5.04×10-2 b 7.79×10-3 1.35×10-1 a 6.75×10-3 1.22×10-1 a 6.63×10-3

3-octanone 4.65×10-1 b 2.37×10-2 3.57×10-1 b 4.07×10-2 7.90×10-1 a 7.41×10-2 1.62×10-1 c 2.03×10-2

Table 4.2 continued D 40 °C D 50 °C D 60 °C D 70 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-octanone 8.38×10-3 b 4.06×10-4 7.29×10-3 b 5.46×10-4 1.28×10-2 a 6.35×10-4 8.52×10-3 b 4.27×10-4

3-octanol 1.19×10-1 a 1.95×10-2 5.99×10-2 b 7.58×10-3 4.38×10-2 b 9.40×10-4 1.06×10-2 c 7.73×10-4

1,4-dimethyl-1-ethylcyclohexane 1.26×10-3 a 4.94×10-5 3.93×10-4 b 3.69×10-5 ND ND ND ND

Limonene 1.41×10-3 b 1.45×10-4 7.64×10-4 b 4.40×10-5 1.35×10-3 b 2.30×10-4 5.36×10-3 a 5.84×10-4

Tetrahydro-3-methyl-4-methylene,furan 1.34×10-3 a 3.67×10-4 5.41×10-4 b 5.44×10-5 ND ND ND ND

1.09×10-3 1,8-cineole 1.40×10-3 a 2.82×10-4 8.40×10-4 b 3.54×10-5 1.04×10-3 ab 1.87×10-4 5.09×10-5 ab

1-(2-furyl)-3-methyl-3-butene-1,2-diol 3.66×10-3 a 2.88×10-4 1.44×10-3 b 2.83×10-4 2.28×10-3 b 4.49×10-4 2.24×10-3 b 5.63×10-4

7.56×10-3 2-ethyl-1-hexanol 6.61×10-3 bc 1.40×10-4 6.20×10-3 c 8.17×10-4 7.88×10-3 a 1.52×10-4 1.18×10-4 ab

Phenylacetaldehyde 4.83×10-3 c 5.52×10-4 8.13×10-3 c 6.11×10-4 2.70×10-2 b 3.05×10-3 2.05×10-1 a 1.13×10-2

Benzyl alcohol 2.89×10-3 a 4.88×10-4 3.28×10-3 a 1.57×10-4 1.95×10-3 b 2.71×10-4 7.89×10-4 c 8.95×10-5

Thiofuran 2.38×10-3 bc 2.78×10-4 3.08×10-3 ab 3.18×10-4 4.09×10-3 a 3.25×10-4 1.89×10-3 c 7.11×10-4

1.86×10-3 5-methyl-5-hexen2-one 1.51×10-3 b 5.35×10-4 2.59×10-3 a 3.49×10-4 1.97×10-3 ab 3.37×10-4 2.56×10-4 ab

1-methyl-2-cyclohexen-1-ol 3.04×10-3 a 4.97×10-4 4.34×10-4 b 6.34×10-5 1.03×10-3 b 1.05×10-4 ND ND

(E)-2-octenal 1.46×10-3 b 4.59×10-4 1.42×10-3 b 2.08×10-4 4.84×10-3 b 1.51×10-4 4.98×10-3 a 9.66×10-4

2-octen-1-ol 1.41×10-2 ab 3.29×10-3 9.63×10-3 b 2.66×10-4 1.83×10-2 a 8.70×10-4 1.21×10-2 b 8.59×10-4

Table 4.2 continued D 40 °C D 50 °C D 60 °C D 70 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) n-octanol 2.90×10-2 b 5.57×10-3 2.72×10-2 b 2.22×10-3 4.04×10-2 a 2.14×10-3 1.50×10-2 c 1.17×10-3

Nonanal 4.14×10-3 c 5.06×10-4 2.68×10-3 d 2.77×10-4 6.33×10-3 b 4.04×10-4 8.33×10-3 a 3.28×10-4

Phenyl ethyl alcohol 5.60×10-2 c 7.64×10-3 5.53×10-2 c 1.67×10-3 9.49×10-2 a 3.22×10-3 7.67×10-2 b 5.51×10-3

(E)-2-hexenal 1.35×10-3 a 2.91×10-4 1.44×10-3 a 3.22×10-4 1.95×10-3 a 1.38×10-4 4.89×10-4 b 2.85×10-5

1,9-nonanediol 4.20×10-3 b 5.66×10-4 7.73×10-3 a 6.96×10-4 2.24×10-3 c 3.28×10-4 8.98×10-4 d 2.85×10-5

1-nonanol 1.15×10-2 a 2.01×10-3 1.82×10-2 a 6.44×10-3 1.78×10-2 a 1.12×10-3 1.09×10-2 a 7.53×10-5

3-ethyl-5-hexen-3-ol 3.66×10-3 b 5.79×10-4 4.74×10-3 a 2.75×10-4 6.69×10-3 a 5.37×10-4 1.50×10-3 c 6.47×10-4

Decanal 1.78×10-3 c 5.39×10-4 8.76×10-4 d 3.57×10-5 3.84×10-3 a 2.57×10-4 2.89×10-3 b 1.49×10-4

2-undecanone 2.52×10-3 b 3.84×10-4 3.96×10-3 a 1.71×10-4 1.67×10-2 a 5.21×10-3 2.15×10-3 b 1.61×10-4

(E,E)-2,4-Decadienal 1.88×10-3 b 2.20×10-4 1.37×10-3 b 2.24×10-4 1.18×10-3 b 2.08×10-4 6.57×10-3 a 2.36×10-4

2,6-bis(1,1-dimethylethyl)phenol 6.36×10-3 a 1.50×10-4 4.57×10-3 b 8.77×10-4 7.77×10-3 a 6.48×10-4 7.80×10-3 a 3.10×10-4 propanoic acid,2-methyl-1-(1,1-dimethylethyl)-2- 4.64×10-4 b 1.98×10-5 6.88×10-4 b 1.04×10-4 2.77×10-3 a 2.90×10-4 8.03×10-4 b 7.47×10-4 methyl-1,3-propanediyl ester

* Standard deviation, analyses are per 1 ml extract.

Means with the same letter within a row are not significantly different (p < 0.05).

Chestnut mushrooms dried at different temperatures are shown in figure 4.6 and 4.7 below. At lower temperature (40 and 50 °C) the mushrooms were lighter in colour compared to higher drying temperatures (60 and 70 °C).

Figure 4.6: Vacuum packed chestnut mushrooms dried at 40 °C (Left) and 50 °C (Right) (Author photograph).

Figure 4.7: Vacuum packed chestnut mushrooms dried at 60 °C (Left) and 70 °C (Right) (Author photograph).

The normalised relative concentrations of the main C8 compounds identified in chestnut mushrooms dried at different temperatures are shown in Figure 4.8.

1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalisedrelative concentration 0.0

ol l ol ne no nol -one 3- -1- a n- ano ta n e t c oct en-3 t t oc -oc 3-o n- oc 1- 3 2-octanone 2-octe 1- Compound Figure 4.8: Normalised relative concentrations of the main C8 compounds extracted from dried chestnut at different temperatures.

The compound 1-octen-3-one had its highest normalised relative concentration at 70 °C which was significantly different from that obtained at 40, 50 and 60 °C. However, no major change occurred between drying at 40 and 50 °C. As for 1-octen-3-ol, it had its maximum normalised relative concentration at 60 °C which was not significantly different from 70 °C but significantly different from 40 and 50 °C. Meanwhile, 3- octanone and 2-octanone had their maximum relative concentrations at 60 °C, significantly different from that obtained at 40, 50 and 70 °C. As for 3-octanol, drying at 40 °C resulted in the maximum retention of this compound which was significantly different from that obtained at 50, 60 and 70 °C. As for 2-octen-1-ol, the highest normalised concentration was found at 60 °C which was significantly different from 50 and 70 °C but not significantly different from 40 °C while for n-octanol, drying at 60 °C resulted in the maximum retention of this compound which was significantly different from that found in 40, 50 and 70 °C samples. However, no significant difference was found between 40 and 50 °C.

The normalised relative concentrations of aldehydes extracted from chestnut mushrooms are shown in Figure 4.9. At 70 °C, 3-methyl butanal, hexanal, benzaldehyde, nonanal, (E,E)-2,4-decadienal, and phenylacetaldehyde had their highest normalised relative concentration significantly different from those obtained at lower

temperatures. On the other hand, (E)-2-hexenal and decanal had their maximum normalised relative concentrations at 60 °C which were significantly different from those found in 70 °C samples.

1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2

Normalisedrelative concentration 0.0

al l al al al l n na n en ta xa ona bu He Decan cadiena N 2-hex e - d etaldehyde thyl e Benzaledhyde (E) 2,4- 3-m Compound (E,E)- Phenylac Figure 4.9: Normalised relative concentrations of aldehydes extracted from chestnut mushrooms dried at different temperatures. The normalised relative concentrations of alcohols and ketones extracted from dried chestnut are summarised in Figure 4.10. There was no significant difference between drying at 50 and 60 °C for 3-methyl-1-butanol and drying at 40 and 70 °C had significantly resulted in a significantly lower normalised relative concentration for this compound. As for 1-hexanol, no significant difference was found between 40, 50, and 60 °C and had its highest normalised relative concentration at 60 °C which was significantly different from 70 °C. As for 3-heptanone, drying at 70 °C resulted in the maximum normalised relative concentration which was significantly different from drying at lower temperatures. No significant change was observed between 50 and 60 °C and drying at 40 °C resulted in the loss of this compound. As for phenyl ethyl alcohol, its highest normalised concentration was found at 60 °C with no significant difference between 40 and 50 °C. As for 2-undecanone, its maximum normalised relative concentration was found at 60 °C which was significantly different from 40, 50, and 70 °C samples. As for 5-methyl-5-hexen2-one, its highest normalised relative concentration was found at 50 °C which was not significantly different from that found at 60 and 70 °C but significantly different from that obtained at 40 °C while 1-methyl-2-

cyclohexen-1-ol had its maximum normalised relative concentration at 40 °C which was significantly different from the ones found in D 50, 60 and 70 °C samples.

D 40 °C 1.2 D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalised relative concentration

0.0

l l l ol o ne o ne ne o n an o oh o -o 1- ta x an lc an 2 n- bu he pt a c en e 1- 1- e yl de x ex l- -h th n he oh hy 3 le -u 5- cl et y 2 l- cy m en hy 2- 3- Ph et l- m hy 5- et Compound m 1- Figure 4.10: Normalised relative concentrations of alcohols and ketones extracted from chestnut mushrooms dried at different temperatures.

The normalised relative concentrations of other selected VC extracted from chestnut dried at different temperatures are shown in Figure 4.11. Limonene had its highest normalised relative concentration at 70 °C which was significantly different from those obtained at lower drying temperatures while 1,8-cineole had its maximum normalised relative concentration at 40 °C which was significantly different from that found at 50 °C but not significantly different from that obtained at 60 and 70 °C. Benzyl alcohol had its maximum normalised relative concentration at 50 °C which was significantly different from those obtained at 60 and 70 °C but not significantly different from 40 °C. As for thiofuran, it had its highest normalised relative concentration at 60 °C which was not significantly different from that found at 50 °C but significantly different from 40 and 70 °C while 1,9-nonanediol had its maximum concentration at 50 °C which was significantly different form 40, 60 and 70 °C. As for 3-ethyl-5-hexen-3-ol, no significant difference was found between the normalised relative concentration at 50 and 60 °C but its highest normalised concentration was observed at 60 °C being significantly different from those obtained at 40 and 70 °C. The compound 2-ethyl-1- hexanol had its highest normalised relative concentration at 60 °C which was not

significantly different from 70 °C samples but significantly different from 40 and 50 °C samples.

1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2

Normalisedrelative concentration 0.0

e n ol ol en ole a i n n e a o in fur x c o aned m i n he o 1- Li 1,8- Th n ,9- yl- Benzyl alcohol 1 eth 2- Compound 3-ethyl-5-hexen-3-ol Figure 4.11: Normalised relative concentrations of other selected VC extracted from chestnut mushrooms dried at different temperatures.

Ho et al. (1989) showed that the majority of heterocyclic compounds are formed via Maillard reactions and that hydrolytic and pyrolytic degradation of food components (such as sugars and amino acids) as well as lipid oxidation contribute to the formation of heterocyclic compounds such as thiofuran also known as thiophene. This compound is said to be formed in the reaction of 2,4-decadienal and cysteine. Ho et al. (1989) also concluded that heterocyclic compounds containing nitrogen or sulphur compounds are characterised by a a strong sensory attributes at low concentrations and that 2,4- decadienal and hexanal can react with Maillard reaction intermediates to form heterocyclic compounds. Boelens et al. (1971) reported that thiophenes are commonly found in vegetables such as onions. Drying was faster at higher temperatures (70 °C) compared to lower temperatures (40 °C and different drying temperatures had different effects on the concentrations of different VC. Even compounds belonging to the same chemical class behaved differently. However, the rehydration temperature should be taken into consideration when choosing the most suitable drying temperature for chestnut mushrooms as it plays a vital role in the liberation of VC from the dried material.

4.1.4 Dried and boiled under reflux (DBR) chestnut mushrooms Chestnut samples dried at different temperatures and then boiled under reflux were again extracted using ambient temperature vacuum distillation followed by concentration and analysis using SPME coupled to GC-MS. Their concentrations were calculated using the standard addition method and the results are shown in Table 4.3. In DBR 40 °C samples, 3-octanone was the main C8 VC identified (9.08×10-2±4.07× 10-3 ppm) and its concentration significantly increased to 1.67×10-1±8.27×10-3 in DBR 50 °C samples and to 1.19×10-1±8.75×10-3 in DBR 60 °C samples and to 1.14×10-2 ±6.16×10-4 in DBR 70 °C samples. It was followed by 1-octen-3-ol with a concentration of 6.18×10-2±5.19×10-3 in DBR 40 °C samples which did not significantly increase when compared to DBR 50 °C samples but was significantly higher in DBR 60 °C (1.16×10-1±1.25×10-2 ppm) samples. As for 3-octanol, its maximum concentration was found in DBR 40 °C samples (3.13×10-2±5.56×10-3 ppm) then it significantly decreased in DBR 50 °C to reach 1.46×10-2±3.28×10-3 ppm followed by a further decrease in DBR 70 °C samples (5.96×10-3±3.68×10-4 ppm). As for 2-octen-1-ol, its concentration was found to be 1.15×10-2±1.01×10-3 ppm in DBR 40 °C samples which was significantly different from DBR 60 °C (1.44×10-2±9.67×10-4 ppm) and 70 °C samples (5.87×10-3±5.94×10-4 ppm). Meanwhile, n-octanol had a concentration of 1.45× 10-2±1.18×10-3 ppm in DBR 40 °C samples which was significantly lower than that found in DBR 60 °C (2.19×10-2±9.57×10-4 ppm). Its lowest concentration was found in DBR 70 °C samples (9.65×10-3±3.70×10-4 which was significantly lower than that found in DBR 40 and 60 °C samples. The saturated ketone, 1-octen-3-one had its highest concentration in DBR 50 °C samples (6.10×10-2±1.08×10-3 ppm) and the lowest in DBR 70 °C samples (8.66×10-4±2.16×10-5 ppm) both being significantly different from DBR 40 and 60 °C samples. The highest concentration of 2-octanone was found in DBR 60 °C samples (6.99×10-3±2.18×10-4 ppm) which was significantly different from DBR 50 °C samples (5.69×10-3±4.43×10-4 ppm) but not significantly different from DBR 40 °C samples. In DBR 70 °C samples, 2-octanone was not detected. Isooctanol had its highest concentration in DBR 60 °C samples (2.19×10-3±1.63×10-4 ppm) and the lowest in DBR 40 °C samples (4.91×10-4 ±5.50×10-5 ppm). As for the remaining alcohols, 3-methyl-1-butanol had a maximum concentration of 3.73×10-2±1.54×10-3 ppm in DBR 60 °C samples and the lowest in DBR 70 °C samples (7.35×10-3±2.02×10-4 ppm) while 2-methyl-1-butanol also had its highest concentration 72

in DBR 60 °C samples (8.40×10-3±3.59×10-4 ppm) which was significantly different from DBR 40 and 50 °C samples, and was not detected in DBR 70 °C samples. As for 1-pentanol, it had its highest concentration in DBR 50 °C (4.14×10-3±8.03×10-4 ppm) which was not significantly different from DBR 60 °C samples (2.86×10-3±8.82×10-4 ppm) but significantly different from DBR 40 and 70 °C samples. As for 1-hexanol, it had a much lower concentration in DBR 50 °C samples (8.61×10-2±2.32×10-3 ppm) compared to the aldehyde hexanal (2.41×10-1±3.68×10-2 ppm) which could be explained by the oxidation of alcohol to give aldehyde. The compound 2-hepten-1-ol had its maximum concentration in DBR 70 °C (7.95×10-3±4.99×10-4 ppm) and its lowest in DBR 50 °C samples (3.28×10-4±3.92×10-5 ppm). As for 1-acetylimidazole, its highest concentration was found in DBR 60 °C samples (1.57×10-2±1.80×10-3 ppm) and its lowest in DBR 70 °C samples (1.17×10-3±1.35×10-4 ppm) while 2,5-diformylthiophene had a maximum concentration of 6.83×10-3±5.81×10-4 ppm in DBR 60 °C samples and a minimum concentration of 2.71×10-4±2.21×10-5 ppm in DBR 50 °C samples which was not significantly different from DBR 70 °C samples. Furfuryl alcohol had the maximum concentration in DBR 70 °C samples (4.90×10-3±1.96×10-4 ppm) which was significantly different from the other treatments. No significant difference between DBR 50 and 60 °C samples was found for 2-ethyl-1-hexanol (6.51×10-3±1.05×10-4 vs. 6.11×10-3±3.80×10-4 ppm) while phenyl ethyl alcohol had its highest concentration in DBR 40 °C samples (5.97×10-2±3.71×10-3 ppm) and the lowest in DBR 70 °C samples (1.35×10-3±2.34×10-4 ppm). As for 1-nonanol, it had its maximum concentration in DBR 50 °C (8.60×10-3±3.16×10-4 ppm) which was not significantly different from DBR 60 °C samples (8.42×10-3±5.32×10-4 ppm) but significantly different from those found in DBR 40 and 70 °C samples. As for the ketones, the concentration 3-heptanone showed no significant difference between DBR 50 and 60 °C samples (1.21×10-2±1.73×10-3 vs. 1.38×10-2±9.69×10-4 ppm) while the concentration of 2-methylcyclohexanone showed no significant difference between DBR 40 and 50 °C samples (4.34×10-4±3.91×10-5 vs. 4.01×10-4 ±2.04×10-5 ppm). The compound 2-undecanone had its highest concentration in DBR 60 °C samples (2.71×10-2±1.77×10-3 ppm), significantly different from DBR 40, 50, and 70 °C samples while 1-phenylethanone showed no significant difference in concentrations between DBR 40 °C (1.75×10-3±3.41×10-4 ppm), 60 °C (1.50×10-3 ±7.43×10-5 ppm) and 70 °C samples (1.35×10-3±1.34×10-4 ppm).

Chen and Wu (1984) have shown that a reductase system in A. bisporus may reduce l- octen-3-one to l-octen-3-ol as well as reduce the double bond of l-octen-3-one to form 3-octanone. The compounds (E,E)-2,4-decadienal and (E,E)-2,4-nonadienal identified in dried and boiled under reflux chestnut were also identified in the aroma of baked Jewel sweetpotato and were characterised by having a watermelon and cooked starch aromas respectively. They are derived from the decomposition of linoleic or linolenic acid and have been previously found in potato chips, cooked rice, and wheat bread (Baltes & Song, 1994). As 2-pentyl furan, described as having a floral note, could also be formed from 2,4-decadienal and was identified in boiled potato aroma (Nursten and Sheen, 1974) and baked Jewel potatoes (Wang & Kay, 2000). These findings suggest that these compounds are the results of thermal reactions.

Table 4.3: Concentrations of VC extracted from dried and boiled under reflux chestnut mushrooms. Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

3-methyl butanal 8.01×10-3 c 5.79×10-4 1.90×10-2 ab 2.11×10-3 2.39×10-2 a 6.07×10-3 1.27×10-2 bc 2.56×10-3

2-methyl butanal 4.56×10-3 d 1.62×10-4 1.66×10-2 b 1.94×10-3 2.20×10-2 a 1.36×10-3 1.31×10-2 c 8.35×10-4

3-methyl-1-butanol 1.95×10-2 b 2.10×10-3 2.26×10-2 b 1.99×10-3 3.73×10-2 a 1.54×10-3 7.35×10-3 c 2.02×10-4

2-methyl-1-butanol 6.64×10-3 b 6.78×10-4 6.46×10-3 b 3.67×10-4 8.40×10-3 a 3.59×10-4 ND ND

Pyridine 1.28×10-3 bc 6.71×10-4 1.87×10-3 b 9.71×10-5 3.61×10-3 a 3.04×10-4 7.13×10-4 c 3.65×10-5

1-pentanol 2.29×10-3 b 5.38×10-5 4.14×10-3 a 8.03×10-4 2.86×10-3 ab 8.82×10-4 1.84×10-3 b 1.45×10-4

Hexanal 1.12×10-1 b 5.98×10-3 2.41×10-1 a 3.68×10-2 1.61×10-1 b 1.66×10-2 4.87×10-2 c 5.76×10-3

1-hexanol 6.35×10-2 b 5.40×10-3 8.61×10-2 a 2.32×10-3 7.15×10-2 b 4.83×10-3 1.47×10-2 c 3.02×10-3

3-heptanone 5.57×10-3 b 4.36×10-4 1.21×10-2 a 1.73×10-3 1.38×10-2 a 9.69×10-4 7.40×10-3 b 4.98×10-4

Heptanal 2.71×10-3 c 4.27×10-5 7.61×10-3 a 5.48×10-4 4.43×10-3 b 3.40×10-4 1.78×10-3 d 8.63×10-5

P-tolythioamide 1.95×10-3 a 1.99×10-4 1.62×10-4 b 3.54×10-5 2.58×10-3 a 3.75×10-4 2.42×10-3 a 2.40×10-4

2-methyl-1,6-heptadiene 2.23×10-4 c 2.18×10-5 6.12×10-4 a 2.70×10-5 4.53×10-4 b 4.20×10-5 2.34×10-4 c 4.04×10-5

2-methylcyclohexanone 4.34×10-4 a 3.91×10-5 4.01×10-4 a 2.04×10-5 3.13×10-4 b 3.65×10-5 1.67×10-4 c 3.14×10-5

Benzaldehyde 2.66×10-2 d 1.34×10-3 9.39×10-2 c 5.79×10-3 1.59×10-1 a 1.28×10-2 1.32×10-1 b 1.67×10-3

2-hepten-1-ol 4.15×10-4 b 7.42×10-5 3.28×10-4 b 3.92×10-5 8.34×10-4 b 2.88×10-5 7.95×10-3 a 4.99×10-4

1-acetylimidazole 8.37×10-3 b 3.46×10-4 5.90×10-3 b 8.49×10-4 1.57×10-2 a 1.80×10-3 1.17×10-3 c 1.35×10-4

Table 4.3 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

1-octen-3-one 5.58×10-3 c 3.32×10-4 6.10×10-2 a 1.08×10-3 1.95×10-2 b 1.54×10-3 8.66×10-4 d 2.16×10-5

1-octen-3-ol 6.18×10-2 b 5.19×10-3 6.83×10-2 b 2.62×10-3 1.16×10-1 a 1.25×10-2 6.54×10-2 b 3.27×10-3

3-octanone 9.08×10-2 c 4.07×10-3 1.67×10-1 a 8.27×10-3 1.19×10-1 b 8.75×10-3 1.14×10-2 d 6.16×10-4

2-pentyl furan 9.58×10-3 a 7.74×10-4 1.11×10-2 a 1.36×10-3 4.08×10-3 c 6.82×10-4 4.56×10-3 b 6.94×10-4

2-octanone 6.11×10-3 ab 6.41×10-4 5.69×10-3 b 4.43×10-4 6.99×10-3 a 2.18×10-4 ND ND

3-octanol 3.13×10-2 a 5.56×10-3 1.46×10-2 b 3.28×10-3 9.65×10-3 bc 7.33×10-4 5.96×10-3 c 3.68×10-4

2,2,4,6,6-pentamethyl-3-heptene 1.52×10-2 a 2.43×10-3 6.22×10-3 b 6.61×10-4 7.23×10-3 b 2.69×10-4 7.43×10-3 b 6.48×10-4

Limonene 8.54×10-5 b 1.90×10-5 2.73×10-4 b 8.87×10-5 3.86×10-4 b 3.04×10-5 9.19×10-3 a 3.68×10-4

Furfuryl alcohol 1.67×10-3 b 1.60×10-4 3.98×10-4 c 2.93×10-5 7.16×10-4 c 6.41×10-5 4.90×10-3 a 1.96×10-4

1-(2-furyl)-3-methyl-3-butene-1,2-diol 7.84×10-3 a 5.20×10-4 4.02×10-4 c 3.01×10-4 4.70×10-3 b 4.01×10-4 3.69×10-3 b 1.05×10-3

(Z)-3-ethyl-2-methyl-1,3-hexadiene, 3.13×10-3 b 2.03×10-4 7.38×10-3 a 3.62×10-4 6.55×10-3 a 3.97×10-4 ND ND

2-ethyl-1-hexanol 3.08×10-3 b 3.36×10-4 6.51×10-3 a 1.05×10-4 6.11×10-3 a 3.80×10-4 2.73×10-3 b 1.94×10-4

Phenylacetaldehyde 8.04×10-3 c 9.95×10-5 2.08×10-2 b 3.82×10-3 4.04×10-2 a 5.03×10-3 1.16×10-2 c 3.00×10-4

(E,E)-2,4-hexadienal 6.56×10-3 a 3.02×10-4 1.28×10-3 c 1.40×10-4 2.77×10-3 b 3.97×10-4 3.05×10-3 b 1.50×10-4

(E)-2-octenal 1.06×10-2 b 1.62×10-4 1.69×10-2 a 1.44×10-3 1.43×10-2 a 1.96×10-3 7.12×10-3 c 2.89×10-4

1-phenylethanone 1.75×10-3 a 3.41×10-4 6.58×10-4 b 2.02×10-5 1.50×10-3 a 7.43×10-5 1.35×10-3 a 1.34×10-4

2-octen-1-ol 1.15×10-2 b 1.01×10-3 1.27×10-2 ab 6.02×10-4 1.44×10-2 a 9.67×10-4 5.87×10-3 c 5.94×10-4

Table 4.3 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

n-octanol 1.45×10-2 b 1.18×10-3 1.40×10-2 bc 3.36×10-3 2.19×10-2 a 9.57×10-4 9.65×10-3 c 3.70×10-4

Nonanal 8.69×10-3 bc 2.45×10-4 1.07×10-2 ab 7.75×10-4 1.18×10-2 a 1.25×10-3 7.31×10-3 c 4.84×10-4

Phenyl ethyl alcohol 5.97×10-2 a 3.71×10-3 4.62×10-2 b 4.93×10-3 4.34×10-2 b 4.45×10-3 1.35×10-3 c 2.34×10-4

Isooctanol 4.91×10-4 c 5.50×10-5 1.47×10-3 b 3.94×10-4 2.19×10-3 a 1.63×10-4 8.24×10-4 c 2.97×10-5

(E,E)-2,4-octadienal 1.02×10-3 b 2.36×10-5 3.22×10-3 a 1.99×10-4 9.74×10-4 b 4.63×10-5 4.41×10-4 c 2.15×10-5

(E)-2-nonenal 3.29×10-3 b 2.31×10-4 5.27×10-3 a 1.57×10-4 5.06×10-3 a 2.19×10-4 4.83×10-3 c 2.66×10-4

1-nonanol 3.50×10-3 b 1.81×10-4 8.60×10-3 a 3.16×10-4 8.42×10-3 a 5.32×10-4 6.35×10-4 c 5.49×10-5

2,5-diformylthiophene 3.77×10-3 b 3.13×10-4 2.71×10-4 c 2.21×10-5 6.83×10-3 a 5.81×10-4 4.34×10-4 c 3.87×10-5

Decanal 2.07×10-3 ab 8.47×10-4 1.89×10-3 b 1.68×10-4 1.95×10-3 ab 9.80×10-5 3.12×10-3 a 3.01×10-4

(E,E)-2,4-nonadienal 1.53×10-3 c 3.51×10-4 3.27×10-3 b 2.21×10-4 4.53×10-3 a 5.41×10-4 4.34×10-4 d 3.80×10-5

Alpha-ethylidene-benzeneacetaldehyde, 1.43×10-3 b 6.61×10-5 1.41×10-3 b 7.30×10-5 2.90×10-3 a 1.68×10-4 2.79×10-3 a 1.50×10-4

2-undecanone 6.19×10-3 c 6.60×10-4 1.02×10-2 b 5.25×10-4 2.71×10-2 a 1.77×10-3 2.89×10-3 d 1.91×10-4

(E,E)-2,4-decadienal 3.55×10-2 b 2.41×10-3 4.96×10-2 b 6.46×10-3 1.21×10-1 a 1.05×10-2 1.45×10-3 c 1.90×10-4

2,6-bis(1,1-dimethylethyl)phenol 1.64×10-3 d 5.38×10-4 1.72×10-2 a 6.78×10-4 4.68×10-3 b 3.76×10-4 3.31×10-3 c 2.47×10-4

3,5,5-trimethyl-2-hexene 1.38×10-2 a 4.24×10-4 1.41×10-3 b 5.80×10-5 3.49×10-4 c 2.98×10-5 1.59×10-4 c 2.48×10-5

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

As for aldehydes, the main compound found in DBR 40 °C samples was hexanal with a concentration of 1.12×10-1±5.98×10-3 ppm followed by benzaldehyde (2.66×10-2 ±1.34×10-3 ppm), (E,E)-2,4-decadienal (3.55×10-2±2.41×10-3 ppm), (E)-2-octenal (1.06×10-2±1.62×10-4ppm), nonanal (8.69×10-3±2.45×10-4 ppm), phenylacetaldehyde (8.04×10-3±9.95×10-5 ppm), 3-methyl butanal (8.01×10-3±5.79×10-4 ppm), (E,E)-2,4- hexadienal (6.56×10-3±3.02×10-4 ppm), 2-methyl butanal (4.56×10-3±1.62×10-4 ppm), (E)-2-nonenal (3.29×10-3±2.31×10-4 ppm), heptanal (2.71×10-3±4.27×10-5 ppm), decanal (2.07×10-3±8.47×10-4 ppm), (E,E)-2,4-nonadienal (1.53×10-3±3.51×10-4), alpha- ethylidene-benzeneacetaldehyde (1.43×10-3±6.61×10-5 ppm),and (E,E)-2,4-octadienal (1.02×10-3±2.36×10-5 ppm). In DBR 50 °C samples, hexanal was also the main aldehyde found with a concentration of 2.41×10-1±3.68×10-2 significantly different from the other treatments where further increase in temperature significantly reduced its concentration reaching 1.61×10-1±1.66×10-2 ppm in DBR 60 °C samples and 4.87×10-2 ±5.76×10-3 ppm in DBR 70 °C samples. As for 2-methyl butanal, its highest concentration was found in DBR 60 °C samples (2.20×10-2±1.36×10-3 ppm) which was significantly different from DBR 50 °C (1.66×10-2±1.94×10-3 ppm) and DBR 70 °C (1.31×10-2±8.35×10-4 ppm) samples. Heptanal had its maximum concentration at 50 °C (7.61×10-3±5.48×10-4 ppm) followed by 60 °C (4.43×10-3±3.40×10-4 ppm being significantly different from 50 °C) and reaching a minimum of 1.78×10-3±8.63×10-5 in DBR 70 °C samples. Benzaldehyde had its concentration significantly increased in DBR 50 °C samples to reach 9.39×10-2±5.79×10-3 ppm and had its highest concentration in DBR 60 °C samples (1.59×10-1±1.28×10-2 ppm) which was also significantly different from that found in DBR 70 °C samples (1.32×10-1±1.67×10-3 ppm). Meanwhile, phenylacetaldehyde had a maximum concentration of 4.04×10-2 ±5.03×10-3 ppm in DBR 60 °C samples which was significantly different from DBR 40, 50, and 70 °C samples. As for (E,E)-2,4-decadienal it had a concentration of 4.96×10-2 ±6.46×10-3 ppm in DBR 50 °C samples which was significantly different from the maximum concentration found in DBR 60 °C samples (1.21×10-1±1.05×10-2 ppm). In DBR 70 °C samples, much of this compound was lost (1.45×10-3±1.90×10-4 ppm). Meanwhile, 3-methyl butanal had its maximum concentration in DBR 60 °C samples (2.39×10-2±6.07×10-3 ppm) which was significantly different from DBR 40 °C (8.01×10-3±5.79×10-4 ppm) and 70 °C samples (1.27×10-2±2.56×10-3 ppm). As for nonanal, no significant difference was found between the concentrations in DBR 50 °C

(1.07×10-2±7.75×10-4 ppm) and DBR 60 °C samples (1.18×10-2±1.25×10-3 ppm) with that found in DBR 60 °C samples being the highest concentration for that compound. No significant difference was found between DBR 50 and 60 °C for (E)-2-octenal (1.69×10-2±1.44×10-3 vs. 1.43×10-2±1.96×10-3 ppm) with that found in DBR 50 °C samples being the highest concentration detected. The volatile (E,E)-2,4-octadienal had its maximum concentration in DBR 50 °C samples (3.22×10-3±1.99×10-4 ppm) being significantly different from DBR 40, 60 and 70 °C samples while (E,E)-2,4-hexadienal had its highest concentration in DBR 40 °C samples (6.56×10-3±3.02×10-4ppm) followed by that found in DBR 60 °C (2.77×10-3±3.97×10-4 ppm) which was not significantly different from DBR 70 °C samples (3.05×10-3±1.50×10-4 ppm). As for (E)-2-nonenal, it had its maximum concentration in DBR 50 °C (5.27×10-3±1.57×10-4 ppm) which was not significantly different from DBR 60 °C but significantly different from its lowest concentration found in DBR 70 °C samples (4.83×10-3±2.66×10-4 ppm). Meanwhile, decanal had its highest concentration in DBR 70 °C (3.12×10-3±3.01×10-4 ppm) which was not significantly different from DBR 40 and 60 °C samples. As for (E,E)-2,4-nonadienal, it had its maximum concentration in DBR 60 °C (4.53×10-3 ±5.41×10-4 ppm) which was significantly different from DBR 40, 50, 70 °C with lowest concentration being found in DBR 70 °C samples (4.34×10-4±3.80×10-5 ppm). The compound alpha-ethylidene-benzeneacetaldehyde showed no significant difference between DBR 60 and 70 °C (2.90×10-3±1.68×10-4 vs. 2.79×10-3±1.50×10-4 ppm).

Other compounds identified include 2-methyl-1,6-heptadiene which had its highest concentration found in DBR 50 °C samples (6.12×10-4±2.70×10-5 ppm) and the lowest in DBR 40 °C samples (2.23×10-4±2.18×10-5 ppm) which was not significantly different from that found in DBR 70 °C samples. The highest concentration of 2-pentyl furan was found in DBR 50 °C samples (1.11×10-2±1.36×10-3 ppm) which was not significantly different from that found in DBR 40 °C samples (9.58×10-3±7.74×10-4 ppm) while in DBR 60 °C samples, losses of this compound occurred with a minimum concentration of 4.08×10-3±6.82×10-4 ppm. As for limonene, its highest concentration was found in DBR 70 °C samples (9.19×10-3±3.68×10-4 ppm), significantly different from DBR 40, 50, and 60 °C samples, with no significant changes occurred between DBR 40, 50 and 60 °C samples. The compound (Z)-3-ethyl-2-methyl-1,3-hexadiene was not detected in DBR 70 °C samples and its highest concentration was found in DBR 50 °C samples (7.38×10-3±3.62×10-4 ppm), not significantly different from DBR 60 °C samples. As for 79

p-tolylthioamide, it had its maximum concentration in DBR 60 °C samples (2.58×10-3 ±3.75×10-4 ppm) which was not significantly different from DBR 40 and 70 °C samples. The compound 2,2,4,6,6-pentamethyl-3-heptene had its highest concentration in DBR 40 °C (1.52×10-2±2.43×10-3 ppm) which was significantly different from DBR 50, 60, 70 °C. As for 1-(2-furyl)-3-methyl-3-butene-1,2-diol, its highest concentration was found in DBR 40 °C samples (7.84×10-3±5.20×10-4 ppm) and its lowest in DBR 50 °C samples (4.02×10-4±3.01×10-4 ppm). Pyridine was also identified and had its highest concentration in DBR 60 °C samples (3.61×10-3±3.04×10-4 ppm) which was significantly different from its lowest concentration found in DBR 70 °C samples (7.13×10-4±3.65×10-5 ppm). As for 3,5,5-trimethyl-2-hexene, it had its highest concentration in DBR 40 °C (1.38×10-2±4.24×10-4 ppm) and its lowest concentration in DBR 70 °C samples (1.59×10-4±2.48×10-5 ppm) which was not significantly different from DBR 60 °C. Finally, 2,6-bis(1,1-dimethylethyl)phenol had its highest concentration in DBR 50 °C (1.72×10-2±6.78×10-4 ppm) significantly different from its lowest concentration found in DBR 40 °C samples (1.64×10-3±5.38×10-4 ppm).

The normalised relative concentrations of the main C8 compounds are shown in Figure 4.12. The normalised relative concentration of 1-octen-3-one and 3-octanone were significantly different at the different temperatures being the highest when the mushrooms were dried at 50 °C then boiled under reflux. As for 1-octen-3-ol, its highest normalised relative concentration was observed in DBR 60 °C samples. As for 2- octanone, its highest normalised relative concentration in DBR 60 °C samples which was not significantly different from DBR 40 °C but significantly different from DBR 50 °C samples. However, 2-octanone could not be detected in DBR 70 °C samples. As for 3-octanol, its highest normalised relative concentration was detected in DBR 40 °C which was significantly different from DBR 50 °C. No significant change was observed between 50 and 60 °C, and between 60 and 70 °C. The normalised relative concentration of 2-octen-1-ol was the highest at DBR 60 °C which was not significantly different from DBR 50 °C samples but significantly different from DBR 40 and 70 °C samples. As for n-octanol, its highest normalised relative concentration was also observed in DBR 60 °C samples which was significantly different from 40, 50, and 70 °C.

DBR 40 °C 1.2 DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2

Normalised relativeconcentration 0.0

l o l ol - ne no o -1-ol ta n ten-3 -oc c -octanone 3 n-octan 1-o 3-octan 2 2-octe 1-octen-3-one Compound

Figure 4.12: Normalised relative concentrations of C8 volatile compounds extracted from dried and boiled chestnut mushrooms.

The normalised relative concentrations of selected aldehydes are shown in Figure 4.13. The compound 3-methyl butanal had its highest value at DBR 60 °C which was not significantly different from 50 °C but significantly different from 40 and 70 °C. As for benzaldehyde and phenylacetaldehyde, their normalised relative concentrations were the highest at DBR 60 °C and were significantly different from DBR 40, 50 and 70 °C samples. As for hexanal, it had its highest normalised relative concentration at DBR 50 °C which was significantly different from the other treatments. Nonanal had its highest normalised relative concentration at DBR 60 °C which was not significantly different from DBR 50 °C but significantly different from DBR 40 and 70 °C. As for (E)-2-octenal, it had its highest normalised relative concentration at DBR 50 °C which was not significantly different from DBR 60 °C samples but significantly different from DBR 40 and 70 °C samples.

1.4 DBR 40 °C 1.2 DBR 50 °C DBR 60 °C DBR 70 °C 1.0

0.8

0.6

0.4

0.2 Normalisedrelative concentration

l al l e na an ena tanal n u exa oct H No - ldehyd ta Benzaldehyde (E)-2 ace 3-methyl b Compound Phenyl

Figure 4.13: Normalised relative concentrations of selected aldehydes VC extracted from chestnut mushrooms dried at different temperatures followed by boiling under reflux.

The normalised relative concentrations of selected conjugated dienes and dienals are shown in Figure 4.14. The compounds 2-methyl-1,6-heptadiene and (E,E)-2,4- octadienal had their maximum normalised relative concentrations at DBR 50 °C being significantly different when compared to DBR 40, 60, and 70 °C samples. As for (Z)- 3- ethyl-2-methyl-1,3-hexadiene, its highest normalised relative concentration was found at DBR 50 °C which was not significantly different from DBR 60 °C but significantly different from DBR 40 °C and could not be detected in DBR 70 °C samples. The maximum normalised relative concentration of (E,E)-2,4-hexadienal was observed in DBR 40 °C samples which was significantly different from the other treatments. No significant difference was found between DBR 60 and 70 °C samples for this compound. As for (E,E)-2,4-nonadienal, its highest normalised relative concentration was found at DBR 60 °C samples being significantly different from DBR 40, 50 and 70 °C. The normalised relative concentration of (E,E)-2,4-decadienal was the maximum in DBR 60 °C samples which was significantly different DBR 40, 50 and 70 °C. No significant difference was found between DBR 40 and 50 °C.

1.2 DBR 40 °C DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

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0.0 Normalisedrelative concentration

l ne ne al na al al die die ien ie ien ien ta xa ad ad ad ad ep he ex oct on ec 6-h ,3- -h ,4- n -d -1, l-1 2,4 -2 2,4 2,4 yl hy )- ,E) )- )- th et ,E (E ,E ,E me -m (E (E (E 2- l-2 hy -et )-3 Compound (Z

Figure 4.14: Normalised relative concentrations of selected VC extracted from chestnut mushrooms dried at different temperatures then boiled under reflux.

The normalised relative concentrations of selected alcohols (group 1) are shown in Figure 4.15. The compounds 3-methyl-1-butanol and 1-nonanol had their highest value at DBR 60 °C significantly different from DBR 40 and 70 °C samples. The normalised relative concentrations of 2-ethyl-1-hexanol and 1-nonanol showed no significant difference between DBR 50 and 60 °C samples while 2-hepten-1-ol showed the highest normalised relative concentration in DBR 70 °C samples being significantly different from the other treatments. Isooctanol had its highest normalised relative concentration in DBR 60 °C samples which was significantly different from the other treatments. Phenyl ethyl alcohol exhibited the highest normalised relative concentration at DBR 40 °C which was significantly different from DBR 50, 60, and 70 °C samples but no significant difference was found between 50 and 60 °C.

1.2 DBR 40 °C DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

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Normalisedrelativeconcentration 0.0

l ol l ol ol n -o nol tano ona lcoh ten-1 n a 1-bu p - - l 1-hexa e Isooctan 1 yl h h hy 2- et t m 2-e Compound 3- Phenyl ethyl

Figure 4.15: Normalised relative concentrations of selected alcohol (group 1) extracted from chestnut mushrooms dried at different temperatures followed by boiling under reflux.

The normalised relative concentrations of selected VC (group 2) are shown in Figure 4.16. Pyridine had its maximum normalised relative concentration in DBR 60 °C samples which was significantly different from DBR 40, 50 and 70 °C samples, however, no significant difference was found between DBR 40 and 50 °C samples, and between DBR 40 and 70 °C samples. As for 2-pentyl furan, its normalised relative concentration was the highest in DBR 50 °C samples which was not significantly different from DBR 40 °C samples but significantly different from DBR 60 and 70 °C samples. The compounds limonene and furfuryl alcohol had their highest normalised relative concentrations in DBR 70 °C samples being significantly different from the other treatments. As for 3-heptanone, its maximum normalised relative concentration was found in DBR 60 °C samples which was not significantly different DBR 50 °C samples but significantly different from DBR 40 and 70 °C samples. Meanwhile, 2- undecanone had its highest normalised relative concentration in DBR 60 °C samples which was significantly different from the other treatments.

1.2 DBR 40 °C 1.0 DBR 50 °C DBR 60 °C DBR 70 °C 0.8

0.6

0.4

0.2 Normalisedrelativeconcentration 0.0

e n e l e e in a n ho n n d r ne o o o ri fu o lc an an y yl m a pt ec P t i yl e d en L r -h n p fu 3 -u 2- r 2 Fu Compound

Figure 4.16: Normalised relative concentrations of selected VC (group 2) extracted from chestnut mushrooms dried at different temperatures followed by boiling under reflux.

4.1.5 Comparison between treatments

Taking into consideration the amount of VC retained, it appears that 60 °C was the most suitable drying temperature for chestnut mushrooms. Statistical analysis including ANOVA and Tukey tests were used to show any significant changes between the different drying temperatures. The concentrations of the volatiles identified are shown in Table 4.4. The concentrations varied with different treatments. The main C8 compound identified in F samples was 3-octanol with a concentration of 3.49×100±2.57×10-1 ppm followed by 3-octanone with a concentration of 1.81×100±7.90×10-2 ppm, 1-octen-3-ol with 1.20×100±5.97×10-2 ppm, 2-octen-1-ol with 6.89×10-1±7.28×10-2 ppm, n-octanol with 2.20×10-1±1.11×10-2 ppm, and 1-octen-3-one with 1.40×10-1±1.55×10-2 ppm. When the samples were freshly boiled under reflux, the concentrations of these compounds significantly decreased which could be due to evaporation or oxidation. The compound 3-octanol remained the major VC with a concentration of 1.87×100±2.29×100 ppm followed by 3-octanone (4.57×10-1±7.08× 10-3 ppm), 1-octen-3-ol (3.26×10-1±4.18×10-2 ppm), 2-octen-1-ol (1.50×10-1±2.35×10-2 ppm), n-octanol (7.00×10-2±9.22×10-3 ppm), and 1-octen-3-one (3.34×10-2±3.73×10-3 ppm). Drying at 60 °C also significantly decreased the concentration of 3-octanol to

4.38×10-2±9.40×10-4 which was not significantly different from DBR 60 °C samples (9.65×10-3±7.33×10-4 ppm) while 3-octanone had a significantly higher concentration in D 60 °C samples compared to BR samples, however, in DBR 60 °C samples, its concentration was significantly reduced to 1.19×10-1±8.75×10-3 ppm. As for 1-octen-3- ol, its concentration also significantly decreased upon drying to 1.35×10-1±6.75×10-3 ppm which was not significantly different from DBR 60 °C samples (1.16×10-1 ±1.25×10-2 ppm). The compound 1-octen-3-one also experienced a significant decrease in its concentration in D 60 °C samples (2.62×10-3±7.26×10-4 ppm) and it was not significantly different from that found in DBR 60 °C samples. As for n-octanol, its concentration significantly decreased to 4.04×10-2±2.14×10-3 ppm then slightly decreased in DBR samples (2.19×10-2±9.57×10-4 ppm). The concentration of 2-octen-1- ol significantly deceased in D 60 °C samples, and boiling under reflux of the dried samples caused further significant losses in this compound reaching a minimum concentration of 1.44×10-2±9.67×10-4 ppm. Meanwhile, 2-octanone was only detected in the dried samples with a concentration of 1.28×10-2±6.35×10-4 in D 60 °C samples and then significantly decreased when the dried chestnut mushrooms were boiled under reflux (6.99×10-3±2.18×10-4 ppm).

As for aldehydes, (E)-2-octenal was the main aldehyde in F samples with a concentration of 7.70×10-3±4.52×10-4 which was not significantly different from BR and D 60 ° C samples but significantly lower than DBR 60 °C samples (1.43×10-2 ±1.12×10-3 ppm). Hexanal was the second most abundant aldehyde in F samples with a concentration of 4.07×10-3 ±1.72×10-4 ppm which was not significantly different from D 60 °C samples but significantly lower than those found in BR (7.06×10-2±5.39×10-3 ppm) and DBR 60 °C samples (1.61×10-1±1.66×10-2 ppm). Nonanal was also identified with an initial concentration of 1.81×10-3±2.47×10-4 ppm which significantly increased in BR samples (5.86×10-3±2.83×10-4 ppm). Its highest concentration was reached in DBR 60 °C (1.08×10-2±1.25×10-3) which was significantly different from D 60 °C samples. Benzaldehyde had its lowest concentration in F samples (1.05×10-3±7.93×10-5 ppm) which was not significantly different from BR samples (1.08×10-2±4.98×10-4 ppm) while its highest concentration was in DBR 60 °C samples (1.59×10-1±1.28×10-2 ppm). The compounds 3-methyl butanal and 2-methyl butanal had their highest concentration in DBR 60 °C samples (2.39×10-2±6.07×10-3 ppm and 2.20×10-2 ±1.36×10-3 ppm respectively) and were significantly different from BR, and D 60 °C 86

samples. Both compounds were not detected in F samples. Meanwhile the following compounds were only detected in DBR samples: heptanal (of 4.43×10-3±3.40×10-4 ppm), (E,E)-2,4-octadienal (9.74×10-4±4.63×10-5 ppm, (E,E)-2,4-nonadienal (4.53×10-3 ±5.41×10-4 ppm), and α-ethylidene-benzeneacetaldehyde (2.90×10-3±1.68×10-4 ppm). Phenylacetaldehyde, being absent in F and BR samples, was found in D 60 °C samples with a concentration of 2.70×10-2±3.05×10-3 ppm) significantly lower than that determined in DBR 60 °C samples (4.04×10-2±5.03×10-3 ppm). As for (E)-2-hexenal, its highest concentration was detected in DBR 60 °C (5.27×10-3±8.19×10-4 ppm) which was significantly different from BR (1.48×10-3±3.76×10-4 ppm) and D 60 °C samples (1.95×10-3±1.38×10-4 ppm). Decanal had its highest concentration in D 60 °C samples (3.84×10-3±2.57×10-4ppm) which was significantly different from F, BR, and DBR 60 °C samples and had the lowest concentration in F samples (8.51×10-4±6.85×10-5 ppm) which was not significantly different from BR samples (1.38×10-3±4.44×10-4 ppm). As for (E,Z)-2,6-nonadienal, it was only found in F samples with a concentration of 1.55×10-3±1.74×10-4 ppm. Finally, (E,E)-decadienal had its highest concentration in DBR 60 °C (1.21×10-1±1.05×10-2 ppm) which was significantly different from BR and D 60 °C samples. This compound was not detected in F samples.

As for the ketones, 3-heptanone had its highest concentration in DBR 60 °C samples (1.38×10-2±9.69×10-4 ppm), significantly higher than in the other treatments and had the lowest concentration in F samples (6.68×10-4±6.16×10-5 ppm). As for 5-methyl-5- hexen-2-one, being undetected in F and DBR samples, it had its highest concentration in D 60 °C (1.97×10-3±3.37×10-4 ppm) which was significantly higher than that found in BR samples (4.56×10-4±2.33×10-5 ppm). The compound 2-methyl cyclohexanone was also not detected in F samples and had its highest concentration in D 60 °C samples (2.06×10-3±1.64×10-4 ppm) which was significantly higher than those found in BR and DBR 60 °C samples. As for 1-phenylethanone, its highest concentration was found in F samples (1.70×10-3±2.28×10-4 ppm) which was not significantly different from DBR 60 °C (1.50×10-3±7.43×10-5 ppm). However, it was not detected in BR and D 60 °C samples. Meanwhile, 2-undecanone had its highest concentration at DBR 60 °C (2.71×10-2± 1.77×10-3ppm),significantly different from BR and D 60 °C and was not detected in F samples.

Among the alcohols identified was 3-methyl-1-butanol which had its highest concentration in D 60 °C (4.32×10-2±4.10×10-3 ppm) then it significantly decreased in DBR 60 °C samples (3.73×10-2±1.54×10-3 ppm), 2-methyl-1-butanol which also had its highest concentration in D 60 °C samples (1.62×10-2±3.97×10-3 ppm) and was significantly different from its lowest concentration found in F samples (3.95× 10-3±3.18×10-4 ppm). However, it was not detected in BR samples. As for 1-pentanol, it had its maximum concentration in DBR 60 °C (3.61×10-3±3.04×10-4 ppm) significantly different from the other treatments and the lowest concentration in F samples (1.05× 10-3±1.11×10-4 ppm). The concentration of 1-hexanol was the highest in D 60 °C samples (1.22×10-1±1.77×10-2 ppm) and the lowest in F samples (2.16×10-2±1.87×10-3 ppm). As for 2-hepten-1-ol, its highest concentration was found in D 60 °C (1.21×10-2 ±1.31×10-3 ppm) which was significantly higher than its lowest concentration found in DBR 60 °C samples (8.34×10-4±2.88×10-5 ppm). No significant difference in the concentration was found between F and D 60 °C samples for 1,8-cineole (1.24×10-3 ±7.55×10-5 and 1.04×10-3±1.87×10-4 ppm respectively) and was not detected in BR and DBR 60 °C samples while furfuryl alcohol was only detected in DBR 60 °C (7.16×10-4 ±6.41×10-5 ppm).

Meanwhile, some compounds were only detected in D 60 °C and include benzyl alcohol (1.95×10-3±2.71×10-4 ppm), 1,9-nonanediol (2.24×10-3±3.28×10-4 ppm) and 3-ethyl-5- hexen-3-ol (6.69×10-3±5.37×10-4 ppm). As for 2-ethyl hexanol, it had its highest concentration in DBR 60 °C samples (4.04×10-2±5.03×10-3 ppm) which was significantly different from that found in D 60 °C samples (7.88×10-3±1.52×10-4 ppm) while the lowest concentration was found in F samples (4.64×10-3±9.05×10-4 ppm). Phenyl ethyl alcohol had its highest concentration in D 60 °C (9.49×10-2±3.22×10-3 ppm) samples which was significantly higher than the other treatments. The concentration of 3,5,5-trimethyl-1-hexanol showed no significant difference between F and BR samples (1.54×10-3±2.26×10-4 ppm and 1.94×10-3±2.90×10-5 ppm respectively) and was not detected in D 60 °C and DBR 60 °C. Meanwhile, 2-nonen-1-ol was only identified in BR samples (3.96×10-3±4.40×10-4 ppm) and 1-nonanol had its highest concentration in D 60 °C samples (1.78×10-2±1.12×10-3 ppm) which was significantly different from F, BR and DBR 60 °C.

Table 4.4: Comparison between concentrations of VC in chestnut samples subjected to various treatments. F BR D 60 °C DBR 60 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

3-methyl butanal ND ND 1.64×10-3 b 2.50×10-4 2.75×10-3 b 3.01×10-4 2.39×10-2 a 6.07×10-3 2-methyl butanal ND ND 1.25×10-3 b 1.14×10-4 1.72×10-3 b 2.98×10-4 2.20×10-2 a 1.36×10-3 3-methyl-1-butanol 2.15×10-3 c 2.72×10-4 1.49×10-3 c 3.34×10-4 4.32×10-2 a 4.10×10-3 3.73×10-2 b 1.54×10-3 2-methyl-1-butanol 3.95×10-3 bc 3.18×10-4 ND ND 1.62×10-2 a 3.97×10-3 8.40×10-3 b 3.59×10-4 Pyridine ND ND 1.36×10-3 b 2.27×10-4 ND ND 3.23×10-3 a 2.59×10-4 1-pentanol 1.05×10-3 c 1.11×10-4 1.52×10-3 c 1.68×10-4 2.50×10-3 b 4.10×10-4 3.61×10-3 a 3.04×10-4 Hexanal 4.07×10-3 c 1.72×10-4 7.06×10-2 b 5.39×10-3 1.44×10-2 c 1.38×10-3 1.61×10-1 a 1.66×10-2 1-hexanol 2.16×10-2 c 1.87×10-3 2.29×10-2 c 1.27×10-3 1.22×10-1 a 1.77×10-2 7.15×10-2 b 4.83×10-3 3-heptanone 6.68×10-4 c 6.16×10-5 9.57×10-3 b 4.14×10-4 1.62×10-3 c 3.36×10-4 1.38×10-2 a 9.69×10-4 Heptanal ND ND ND ND ND ND 4.43×10-3 a 3.40×10-4 P-tolylthioamide ND ND ND ND 1.57×10-3 b 2.22×10-4 2.58×10-3 a 3.75×10-4 2-methyl-1,6-heptadiene ND ND ND ND ND ND 4.53×10-4 a 4.20×10-5 2-methylcyclohexanone ND ND 7.89×10-4 b 4.98×10-5 2.06×10-3 a 1.64×10-4 3.13×10-4 c 3.65×10-5 Benzaldehyde 1.05×10-3 c 7.93×10-5 1.08×10-2 c 4.98×10-4 4.22×10-2 b 8.19×10-3 1.59×10-1 a 1.28×10-2 2-hepten-1-ol 3.57×10-3 b 2.87×10-4 1.36×10-3 c 2.24×10-4 1.21×10-2 a 1.31×10-3 8.34×10-4 c 2.88×10-5 1-acetylimidazole ND ND ND ND ND ND 1.57×10-2 a 1.80×10-3 1-octen-3-one 1.40×10-1 a 1.55×10-2 3.34×10-2 b 3.73×10-3 2.62×10-3 c 7.26×10-4 1.95×10-2 bc 1.54×10-3 1-octen-3-ol 1.20×100 a 5.97×10-2 3.26×10-1 b 4.18×10-2 1.35×10-1 c 6.75×10-3 1.16×10-1 c 1.25×10-2 3-octanone 1.81×100 a 7.90×10-2 4.57×10-1 c 7.08×10-3 7.90×10-1 b 7.41×10-2 1.19×10-1 d 8.75×10-3 2-pentyl furan ND ND ND ND ND ND 4.08×10-3 a 6.82×10-4 2-octanone ND ND ND ND 1.28×10-2 a 6.35×10-4 6.99×10-3 b 2.18×10-4 3-octanol 3.49×100 a 2.57×10-1 1.87×100 b 2.29×100 4.38×10-2 c 9.40×10-4 9.65×10-3 c 7.33×10-4 1,8 cineole 1.24×10-3 a 7.55×10-5 ND ND 1.04×10-3 a 1.87×10-4 ND ND 89

Table 4.4 continued F BR D 60 °C DBR 60 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) 2,2,4,6,6-pentamethyl-3-heptene ND ND 5.61×10-3 c 2.76×10-4 8.53×10-3 a 5.74×10-4 7.23×10-3 b 2.69×10-4 Limonene ND ND 1.26×10-2 a 3.17×10-3 1.35×10-3 b 2.30×10-4 3.86×10-4 b 3.04×10-5 Furfuryl alcohol ND ND ND ND ND ND 7.16×10-4 a 6.41×10-5 1-(2-furyl)-3-methyl-3-butene-1,2- diol 2.45×10-3 b 4.87×10-4 ND ND 2.28×10-3 b 4.49×10-4 4.70×10-3 a 4.01×10-4 3-ethyl,2-methyl-1,3-hexadiene ND ND 3.98×10-3 b 1.93×10-4 ND ND 6.55×10-3 a 3.97×10-4 5-methyl-5-hexen-2-one ND ND 4.56×10-4 b 2.33×10-5 1.97×10-3 a 3.37×10-4 ND ND 2-ethyl-1-hexanol 4.64×10-3 c 9.05×10-4 1.23×10-2 b 1.42×10-3 7.88×10-3 b 1.52×10-4 4.04×10-2 a 5.03×10-3 Phenylacetaldehyde ND ND ND ND 2.70×10-2 b 3.05×10-3 4.04×10-2 a 5.03×10-3 Benzyl alcohol ND ND ND ND 1.95×10-3 a 2.71×10-4 ND ND Thiofuran ND ND ND ND 4.09×10-3 a 3.25×10-4 ND ND (E)-2-octenal 7.70×10-3 b 4.52×10-4 7.91×10-3 b 6.26×10-4 4.84×10-3 b 1.51×10-4 1.43×10-2 a 1.12×10-3 1-phenylethanone 1.70×10-3 a 2.28×10-4 ND ND ND ND 1.50×10-3 a 7.43×10-5 2-octen-1-ol 6.89×10-1 a 7.28×10-2 1.50×10-1 b 2.35×10-2 1.83×10-2 c 8.70×10-4 1.44×10-2 d 9.67×10-4 n-octanol 2.20×10-1 a 1.11×10-2 7.00×10-2 b 9.22×10-3 4.04×10-2 c 2.14×10-3 2.19×10-2 c 9.57×10-4 Nonanal 1.81×10-3 c 2.47×10-4 5.86×10-3 b 2.83×10-4 6.33×10-3 b 4.04×10-4 1.08×10-2 a 1.25×10-3 Phenyl ethyl alcohol 1.58×10-2 c 1.89×10-3 7.18×10-3 d 7.15×10-4 9.49×10-2 a 3.22×10-3 4.34×10-2 b 4.45×10-3 (E,Z)-2,6-nonadienal 1.55×10-3 a 1.74×10-4 ND ND ND ND ND ND (E,E)-2,4-octadienal ND ND ND ND ND ND 9.74×10-4 a 4.63×10-5 (E)-2-hexenal ND ND 1.48×10-3 b 3.76×10-4 1.95×10-3 b 1.38×10-4 5.27×10-3 a 8.19×10-4 1,9-nonanediol ND ND ND ND 2.24×10-3 a 3.28×10-4 ND ND 1-nonanol 3.00×10-3 b 5.09×10-4 1.44×10-3 b 2.50×10-4 1.78×10-2 a 1.12×10-3 8.42×10-3 b 5.32×10-4 3-ethyl-5-hexen-3-ol ND ND ND ND 6.69×10-3 a 5.37×10-4 ND ND

Table 4.4 continued F BR D 60 °C DBR 60 °C Compound ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) 2,5-diformylthiophene ND ND ND ND ND ND 6.83×10-3 a 5.81×10-4 3,5,5-trimethyl-1-hexanol 1.54×10-3 a 2.26×10-4 1.94×10-3 a 2.90×10-5 ND ND ND ND 2-nonen-1-ol ND ND 3.96×10-3 a 4.40×10-4 ND ND ND ND Decanal 8.51×10-4 c 6.85×10-5 1.38×10-3 bc 4.44×10-4 3.84×10-3 a 2.57×10-4 1.95×10-3 b 9.80×10-5 (E,E)-2,4-nonadienal ND ND ND ND ND ND 4.53×10-3 a 5.41×10-4 Alpha-ethylidene- benzylacetaldehyde ND ND ND ND ND ND 2.90×10-3 a 1.68×10-4 2-undecanone ND ND 9.42×10-4 c 2.94×10-5 1.67×10-2 b 5.21×10-3 2.71×10-2 a 1.77×10-3 (E,E)-2,4-decadienal ND ND 5.66×10-3 b 2.37×10-4 1.18×10-3 b 2.08×10-4 1.21×10-1 a 1.05×10-2 2,6-bis(1,1-dimethylethyl)phenol 1.96×10-2 a 2.02×10-3 2.23×10-2 a 2.86×10-3 7.77×10-3 a 6.48×10-4 4.68×10-3 b 3.76×10-4 Propanoic acid,2-methyl-1-(1,1- dimethylethyl)-2-methyl-1,3- propanediyl ester ND ND ND ND 2.77×10-3 a 2.90×10-4 ND ND * Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

Other compound identified were pyridine which was not detected in F and D 60 °C and had its highest concentration in DBR 60 °C (3.23×10-3±2.59×10-4 ppm) significantly higher than in BR samples (1.36×10-3±2.27×10-4 ppm).

The following compounds were only detected in DBR 60 °C and include 2-methyl-1,6-heptadiene (4.53×10-4±4.20×10-5 ppm), 1-acetylimidazole (1.57×10-2±1.80×10-3 ppm), 2-pentyl furan (4.08×10-3±6.82×10-4 ppm), and 2,5-diformylthiophene (6.83×10-3±5.81×10-4 ppm).

As for thiofuran, it was only found in D 60 °C (4.09×10-3±3.25×10-4 ppm). Meanwhile, 3-ethyl-2- methyl-1,3-hexadiene had its maximum concentration in DBR 60 °C samples (6.55×10-3 ±3.97×10-4 ppm) which was significantly lower than in BR samples (3.98×10-3±1.93×10-4 ppm). As for limonene, which was not detected in F samples, had its highest concentration in BR samples (1.26×10-2±3.17×10-3 ppm) which then significantly decreased in D 60 °C and DBR 60 °C samples. The compound p-tolylthioamide had its maximum concentration in DBR 60 °C samples (2.58×10-3±3.75×10-4 ppm) significantly different from that found in D 60 °C samples (1.57×10-3±2.22×10-4 ppm). As for 2,2,4,6,6-pentamethyl-3-heptene, its maximum concentration was found in D 60 °C (8.53×10-3±5.74×10-4 ppm) which was significantly different from BR samples (5.61×10-3±2.76×10-4 ppm) and DBR 60 °C samples (7.23×10-3±2.69×10-4 ppm). Meanwhile, 1-(2-furyl)-3-methyl-3-butene-1,2-diol was not detected in BR samples but had its maximum concentration in DBR 60 °C (4.70×10-3±4.01×10-4 ppm) which was significantly different from F and D 60 °C samples. The compound 2,6-bis(1,1-dimethylethyl)phenol had its highest concentration in BR samples (2.23×10-2±2.86×10-3 ppm) which was not significantly different from F and D 60 °C samples but significantly different than DBR 60 °C (4.68×10-3 ±3.76×10-4 ppm). The ester compound identified was propanoic acid, 2-methyl-1-(1,1- dimethylethyl)-2-methyl-1,3-propanediyl ester was detected and was only found in D 60 °C with a concentration of (2.77×10-3±2.90×10-4 ppm).

The concentrations of the main C8 compounds are shown in Figure 4.17. The concentrations of the identified compounds were normalised against the fresh concentrations. The compounds 1- octen-3-one, 1-octen-3-ol, 3-octanone, 3-octanol, 2-octen-1-ol, n-octanol had their highest normalised concentrations in fresh chestnut samples being significantly different from the other treatments. As for 2-octanone, it was not detected in F and BR samples probably because it was absent and only generated upon extended period of heat treatment such as drying or its level was well below the detection limit of the instrument. It had its highest normalised relative

concentration in D 60 °C samples which was significantly different from DBR 60 °C samples probably due to losses occurring during boiling under reflux.

1.2 F BR 1.0 D 60 °C DBR 60 °C 0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

e e l l l n ne n -o -3-ol o ano ano n an ano t t e t ten-1-o oc oct oc c - cten-3 - 3-oc n 1-oct 3- 2 1-o 2--o Compound

Figure 4.17: Comparison between the normalised relative concentrations of main C8 compounds extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms

The aldehydes’ behaviour in all four treatments was studied and their normalised relative concentrations are shown in Figure 4.18. The compounds phenylacetaldehyde, 3-methyl butanal, benzaldehyde, (E)-2-hexenal, hexanal, (E)-2-octenal, and nonanal had their highest normalised concentrations when the dried chestnut samples were boiled under reflux and were significantly different from the other treatments. One possible explanation is that these compounds in chestnut mushrooms need boiling water as a rehydration for a higher release of VC in dried samples.

Several compounds identified in fresh and thermally processed chestnut mushrooms were previously been reported as aroma contributors to the Elder flower drink and include linalool which imparts a flowery sweet aroma, (E)-2-octenal (with green grass-like aroma), 1-octen-3-one and 1-octen-3-ol (with a mushroom-like aroma) and limonene (characterised by a citrus, orange note) (Jørgensen et al., 2000).

1.4 F BR 1.2 D 60 °C DBR 60 °C 1.0

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration

0.0

de al de al al al al y an y en an en an eh t eh x x ct n ld bu ld he e o o a l a 2- H 2- N et hy nz )- )- ac et e E (E yl m B ( en 3- Ph Compound

Figure 4.18: Comparison between the normalised relative concentrations of aldehydes VC extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms.

The normalised relative concentrations of the conjugated dienals are shown in Figure 4.19 where (E,E)-2,4 octadienal and (E,E)-2,4-nonadienal had the highest amount in DBR 60 °C samples and were only detected when the dried chestnut samples were boiled under reflux. As for (E,Z)-2,6- nonadienal, it was only found in F samples, while (E,E)-2,4-decadienal had its maximum normalised relative concentration in DBR 60 °C significantly different from those found in BR and D 60 °C samples.

1.2 F BR 1.0 D 60 °C DBR 60 °C

0.8

0.6

0.4

0.2 Normalised relative concentration 0.0

al al al al ien ien ien ien ad ad ad ad on ct on ec -n -o n -d ,6 2,4 ,4 ,4 )-2 )- )-2 )-2 ,Z ,E ,E ,E (E (E (E (E Compound

Figure 4.19: Comparison between the normalised relative concentrations of conjugated aldehydes VC extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms.

The normalised relative concentrations of selected alcohols extracted chestnut mushrooms subjected to different treatments are shown in Figure 4.20. The compounds 3-methyl-1-butanol, 1- hexanol, benzyl alcohol, phenyl ethyl alcohol and 1-nonanol had their maximum normalised relative concentrations when the mushrooms samples were subjected to D 60 °C treatment significantly different from the other treatments. Meanwhile, 2-ethyl-1-hexanol had its highest normalised relative concentration in DBR 60 °C samples which was significantly different from the other treatments. As for 3,5,5-trimethyl-1-hexanol, it was not detected in D 60 °C and DBR 60 °C samples but had its highest normalised relative concentration in BR samples which was not significantly different from that found in F samples.

1.2 F BR D 60 °C 1.0 DBR 60 °C

0.8

0.6

0.4

0.2

Normalised relative concentration 0.0

l l l l l l l no no no ho ho no no a a a o lo a a ut ex ex lc c on ex -b -h -h l a l a -n -h -1 1 -1 zy hy 1 -1 yl yl n et yl th th Be l th e -e y e -m 2 en im 3 tr Ph 5- 5, Compound 3,

Figure 4.20: Comparison between the normalised relative concentrations of selected alcohols VC extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms.

The normalised relative concentrations of selected ketones and saturated alcohols are shown in Figure 4.21. The compounds 3-heptanone and 2-undecanone had their highest normalised relative concentrations when the chestnut samples were dried then boiled under reflux significantly different from F, BR, and D 60 °C samples. Meanwhile, 2-methylcyclohexanone and 2-hepten-1- ol had their highest normalised values in D 60 °C samples which were significantly different from the other treatments.

1.2 F BR 1.0 D 60 °C DBR 60 °C 0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

e e l l e on on 1-o 1-o on an an n- n- an pt ex te ne ec he h ep o d 3- clo -h 2-n un cy 2 2- yl eth Compound 2-m

Figure 4.21: Comparison between the normalised relative concentrations of ketones and saturated alcohols extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms.

The normalised relative concentration of selected VC (group 1) extracted from chestnut mushrooms subjected to different treatments are shown in Figure 4.22. Compounds which had their maximum normalised relative concentration in DBR 60 °C significantly different from the other treatments include pyridine, 2-pentyl furan, furfuryl alcohol, and 3-ethyl-2-methyl-1,3- hexadiene. As for 1,8-cineole, it was absent or not detected in BR and DBR 60 °C but had its highest normalised relative concentration in F samples which was not significantly different from D 60 °C samples. As for limonene, being absent in F samples, it had its highest normalised relative concentration in BR samples that was significantly different from that found in D 60 °C and DBR 60 °C samples while thiofuran was only detected in D 60 °C. Finally, 1-phenylethanone had its highest normalised relative concentration in F samples which was not significantly different from DBR 60 °C. However, it was not detected in BR and D 60 °C samples.

1.4

1.2 F BR D 60 °C 1.0 DBR 60 °C

0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

e n le e ol n e e in ra eo en h ra on en rid fu in on lco fu an di y yl -c m a io th xa P nt ,8 Li yl Th le he pe 1 ur ny 3- 2- rf e -1, Fu -ph yl 1 eth -m l-2 Compound thy 3-e

Figure 4.22: Comparison between the normalised relative concentrations of VC (group 1) extracted from fresh (F), boiled under reflux (BR), dried at 60 °C (D 60 °C), and dried at 60 °C then boiled under reflux (DBR 60 °C) chestnut mushrooms.

Rapior et al. (1997) also reported (E)-2-nonenal, limonene, (E,E)-2,4-decadienal, and 2- undecanone in steam distillate extracts from chestnut mushrooms (A. aegerita). These compounds were not detected in the storage study of fresh chestnut (section 4.1.1) but rather found when the mushrooms were heat processed. Therefore it can be suggested that ambient temperature vacuum distillation gave the most representative extracts of the original fresh material.

Several compounds identified in chestnut mushrooms have also been found in the ripe fruit of Morinda citrifolia using steam distillation-extraction and SPME and include 3-methyl butanal, 2- methyl butanal, benzaldehyde, benzyl alcohol, ethyl acetate, and limonene (Wei et al., 2011).

The rehydration temperature plays an important role in the liberation of VC from the dried samples. Some aldehydes such as benzaldehyde, (E)-2-octenal, nonanal, n-hexanal, heptanal, phenylacetaldehyde, (E)-2-hexenal, (E,E)-2,4-nonadienal, (E,E)-2,4-octadienal, and (E,E)- decadienal, as well as other compounds such 1-phenylethanone, 3-ethyl-2-methyl-1,3-hexadiene, had their highest amount liberated when the mushrooms were boiled under reflux (rehydration temperature 100 °C) compared to some alcohols that were in favour of 25 °C as a rehydration temperature (dried samples subjected to ambient temperature vacuum distillation) and include 3- methyl-1-butanol, 2-methyl-1-butanol, 1-hexanol, 2-hepten-1-ol, 1,8-cineole, benzyl alcohol, 1,9-

nonanediol, phenyl ethyl alcohol, and 1-nonanol. Martinez-Soto et al. (2001) argued that rehydration of P. ostreatus was much faster at 94 °C than at 25 °C.

In a series of aldehydes (C5-C10), the odour threshold reaches a minimum with octanal. In the case of saturated aldehydes, an E-configurated double bond at C2 increases the odour threshold in the case of alkenals 5:1 to 8:1 compared to their corresponding alkanals except for (E)-2-nonenal which possesses an odour threshold 17.5 times lower than that of nonanal. The odour threshold of the E-isomer exceeds that of the Z-isomer (Belitz et al., 2009). As for the formation of VC in mushrooms, several mechanisms were studied. Lipoxygenases enzymes are commonly found in plants where they may be involved in a number of aspects in plants physiology including but not limited to growth and development, senescence or responses to wounding (Vick and Zimmerman 1987). The fatty acid linoleic acid is involved in the formation of 1-octen-3-ol via oxidation by the action of lipoxygnease and hydroperoxidase lyase then a part of 1-octen-3-ol is oxidised using atmospheric oxygen to yield 1-octen-3-one. In fruits and vegetables, linoleic acid is subject to oxidative degradation by lipoxygenases and hydroperoxide lyases to yield aldehydes such as hexanal, (E)-2-hexenal, (E)-2-octenal. Furthermore a part of the aldehydes is then converted enzymatically to its corresponding alcohols. These compounds are also detected in mushrooms. A possible explanation could be that a special/different type of lypoxygenase and hydroperoxidase lyases is involved in the mechanism of aldehydes formation.

A possible explanation for the formation of 3-methyl butanal and 2-methyl butanal is by enzymatic transamination or oxidative deamination of the amino acids leucine and isoleucine where the amino acids are converted to α-keto acids then to the corresponding aldehydes by decarboxylation. Aldehdyes are derived from lipid degradation as reported by Schwab et al. (2008) and Obenland et al. (2012), in his study on VC in “Hass” avocado, identified hexanal, (E)- 2-hexenal, and 2,4-hexadienal described as having a green or grassy aroma, were associated with the ripening of the fruit. Snyder et al. (1985) identified hexanal and 2,4-decadienal as oxidation products from linoleic acid in extruded and baked zein and corn samples while Huang et al. (1987) identified 2-octanone among the thermal degradation products of corn oil. Phenylacetaldehyde formed via Strecker degradation of phenylalanine is characterised by a perfume aroma note and is found in many flavours such as fruit, chocolate, and honey (Scarpellino and Soukup, 1993). In contrast, benzaldehyde has a strong nutty, almond odour though it may also originate from the same amino acid (Wang and Kays, 2000).

4.2 VC in Flammulina velutipes (Enoki mushrooms)

4.2.1 Fresh and stored enoki mushrooms

Based on lack of published studies, it appears that the changes in the VC of enoki mushrooms during storage have not been studied so far. The storage trial over 10 days for enoki samples was also carried out (see section 3.1.2). Table 4.5 shows the concentration of VC on different days. At D0, the main VC was 3-octanone (4.54×10-1±1.96×10-2 ppm) followed by 3-octanol (3.35×10-1 ±4.67×10-2 ppm), 2,2,4,6,6-pentamethyl-3-heptene (8.30×10-2±2.15×10-3 ppm), 1-octen-3-ol (6.29×10-2±2.99×10-3 ppm), limonene (4.94×10-2±4.66×10-3 ppm), 1H-indol-5-ol (3.58×10-2 ±1.10×10-3 ppm), 2-ethyl hexanal (2.93×10-2±2.69×10-3 ppm) and 2,6-bis (1,1- dimethylethyl)phenol (1.53×10-2±1.32×10-3 ppm).

As for the C8 compounds, 1-octen-3-ol, its concentration significantly decreased at D1 (2.73×10-2 ±1.59×10-3 ppm) reaching the lowest concentration at D10 with a concentration of 8.51×10-4 ±3.66×10-5 ppm. Meanwhile, 3-octanone had its concentration decreased significantly over time with a maximum of 4.54×10-1±1.96×10-2 ppm at D0 and a minimum of 1.39×10-2 ±1.92×10-3 ppm at D10. The compound 3-octanol had its concentration significantly decreasing at D1 (2.61×10-1 ±3.63×10-2 ppm) and D2 (1.65×10-1±2.27×10-2 ppm). No significant difference was found between D2 and D3 but compared to D0, a significant decrease occurred at D4 and D7 (6.82×10-2 ±6.42×10-3 and 2.70×10-2±2.80×10-3 pm respectively). No significant difference was observed between D7 and D10.

As for the remaining alcohols, 3-methyl-1-butanol, it had its lowest concentration at D0 (1.20×10-3 ±2.05×10-4 ppm), not significantly different from D1, then it significantly increased at D2 to reach 2.29×10-3±2.06×10-4 ppm. At D4, it reached 3.38×10-3±1.69×10-4 ppm which was not significantly different from D7 but significantly different from its maximum concentration at D10 (7.66×10-3 ±3.16×10-4 ppm). Meanwhile, 2-methyl-1-butanol reached its maximum concentration at D7 (7.66×10-3±3.16×10-4 ppm), significantly different from the remaining days then it decreased at D10 to reach a minimum of 1.88×10-3 ±2.78×10-4 ppm. The compound 1H-indole-5-ol had a maximum concentration of 6.67×10-2±1.50×10-3 ppm at D10, statistically different from the remaining storage days. As for 2-ethyl-1-hexanol, its lowest concentration was found at D0 (9.23×10-3±7.97×10-4 ppm) which then significantly increased at D1 to reach 1.34×10-1±6.74×10-3 ppm with a further increase observed at D4 (1.96×10-1±2.33×10-2 ppm). The maximum concentration reached was 3.36×10-1±2.48×10-2 ppm at D10.

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As for aldehydes, hexanal had its concentration increased over time starting from 5.11×10-4 ±2.25×10-5 ppm at D0 and reaching 1.60×10-3±4.10×10-4ppm at D10, significantly different from the remaining storage days. Meanwhile, 3-methyl butanal significantly increased from D0 to D3 (1.12×10-3±2.25×10-4 vs. 2.57×10-3±3.39×10-4 ppm), then at D7, a significant increase was found where its concentration reached 4.11×10-3±1.86×10-4 ppm followed by a further significant increase at D10 (5.14×10-3±2.98×10-4 ppm) . As for 2-ethyl hexanal, its highest concentration was at D0 (2.93×10-2±2.69×10-3 ppm), not significantly different from D1 but significantly different from the remaining days where it reached a minimum of 1.53×10-3±3.74×10-4 ppm at D10. Octanal had its maximum concentration at D0 (8.04×10-3±2.12×10-4 ppm) which significantly decreased at D1 and D2 followed by a significant decrease at D4 (8.28×10-4±4.08×10-5 ppm) with no significant change occurred between D4, D7, and D10. As for nonanal, its concentration significantly increased from D0 (1.94×10-3±2.36×10-4 ppm) to D3 (3.39×10-3±7.40×10-4 ppm) until it reached a maximum of 6.44×10-3±2.48×10-4 ppm at D10. The compound decanal had its lowest concentration at D0 (1.97×10-3±2.60×10-4 ppm) which significantly increased at D2 (3.63×10-3±2.24×10-4 ppm), D4 (5.58×10-3±2.65×10-4 ppm) until D10 (9.14×10-3±2.23×10-4 ppm).

Other compounds identified include ethyl acetate which had its minimum concentration at D0 (1.41×10-3±1.52×10-4 ppm) then it marginally increased until D3. At D4, it significantly increased almost doubling to 2.98×10-3±6.04×10-4 ppm reaching its maximum at D10 (5.33×10-3 ±3.89×10-4 ppm), not statistically different from D7. Limonene had its highest concentration at D0 (4.94×10-2 ±4.66×10-3 ppm) which then significantly decreased at D1 (3.41×10-2±3.95×10-3 ppm) reaching a minimum of 1.95×10-3±2.89×10-4 ppm at D10, not significantly different from D7. Meanwhile, 2,2,4,6,6-pentamethyl-3-heptene had its maximum concentration at D0, not significantly different from D1 but significantly different from D2, D3, D4, D7,and D10. The concentration of dill ether increased with the storage time starting with 2.35×10-3±4.79×10-4 ppm at D0 followed by a significant increase at D4 (8.71×10-3±3.13×10-4 ppm) and reaching 2.97×10-2±4.11×10-3 ppm at D10. Dihydrocarveol had its highest concentration at D0 (2.29×10-3±2.77×10-4 ppm) which then significantly decreased at D2 (1.60×10-3±2.11×10-4ppm), D4 (1.04×10-3±2.48×10-4 ppm) until D10 (3.48×10-4±3.41×10-5 ppm). As for 1,3-Di-tert-butylbenzene, its concentration significantly increased from D0 (5.25×10-4±8.53×10-5 ppm) to D3 (1.78×10-3±5.14×10-4 ppm). Further storage also increased its concentration with a maximum of 3.84×10-3±4.67×10-4 ppm at D10. Meanwhile, 2,6-bis(1,1-dimethylethyl)phenol had its lowest concentration at D0 which significantly increased at D2 and reached a maximum of 4.74×10-2±4.10×10-3 ppm at D10.

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Table 4.5: Concentrations of VC extracted from enoki mushrooms during 10 days storage. Compound D0 D1 D2 D3 D4 D7 D10

ppm SD* ppm SD ppm SD ppm SD ppm SD ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Ethyl acetate 1.41×10-3 c 1.52×10-4 1.51×10-3 c 2.61×10-4 1.93×10-3 bc 2.50×10-4 2.46×10-3 bc 3.15×10-4 2.98×10-3 b 6.04×10-4 4.59×10-3 a 5.74×10-4 5.33×10-3 a 3.89×10-4

3-methyl butanal 1.12×10-3 d 2.25×10-4 1.45×10-3 d 3.54×10-4 1.60×10-3 d 2.34×10-4 2.57×10-3 c 3.39×10-4 3.15×10-3 c 3.50×10-4 4.11×10-3 b 1.86×10-4 5.14×10-3 a 2.98×10-4

3-methyl-1- butanol 1.20×10-3 e 2.05×10-4 1.77×10-3 de 1.34×10-4 2.29×10-3 cd 2.06×10-4 2.72×10-3 bc 2.80×10-4 3.38×10-3 b 1.69×10-4 5.01×10-3 b 3.17×10-4 7.66×10-3 a 3.16×10-4

2-methyl-1- butanol 5.59×10-3 b 2.57×10-4 4.83×10-3 bc 4.06×10-4 4.40×10-3 c 2.76×10-4 3.85×10-3 cd 4.51×10-4 3.13×10-3 d 1.65×10-4 7.66×10-3 a 3.16×10-4 1.88×10-3 e 2.78×10-4

Hexanal 5.11×10-4 b 2.25×10-5 6.51×10-4 b 1.70×10-5 7.00×10-4 b 3.33×10-5 7.25×10-4 b 3.28×10-5 7.87×10-4 b 3.04×10-5 8.61×10-4 b 2.33×10-5 1.60×10-3 a 4.10×10-4

1H-indol-5-ol 3.58×10-2 d 1.10×10-3 4.66×10-2 c 2.98×10-3 4.73×10-2 c 4.78×10-3 4.43×10-2 c 1.06×10-3 4.90×10-2 c 2.60×10-3 5.76×10-2 b 3.05×10-3 6.67×10-2 a 1.50×10-3

2-ethyl- hexanal 2.93×10-2 a 2.69×10-3 2.28×10-2 ab 4.44×10-3 1.98×10-2 b 2.27×10-3 1.70×10-2 b 3.04×10-3 7.72×10-3 c 4.51×10-4 3.70×10-3 c 5.41×10-4 1.53×10-3 c 3.74×10-4

1-octen-3-ol 6.29×10-2 a 2.99×10-3 2.73×10-2 b 1.59×10-3 1.12×10-2 c 5.24×10-4 8.55×10-3 cd 2.94×10-4 6.57×10-3 de 3.43×10-4 3.57×10-3 ef 1.90×10-4 8.51×10-4 f 3.66×10-5

3-octanone 4.54×10-1 a 1.96×10-2 4.05×10-1 a 2.74×10-2 3.21×10-1 b 3.55×10-2 2.79×10-1 b 8.12×10-3 1.33×10-1 c 4.15×10-2 6.41×10-2 d 2.96×10-3 1.39×10-2 d 1.92×10-3

2,2,4,6,6- pentamethyl- 3-heptene 8.30×10-2 a 2.15×10-3 7.19×10-2 a 4.34×10-3 2.15×10-2 b 3.06×10-2 3.62×10-3 b 3.83×10-4 3.26×10-3 b 2.84×10-4 2.69×10-3 b 2.98×10-4 2.29×10-3 b 3.09×10-4

3-octanol 3.35×10-1 a 4.67×10-2 2.61×10-1 b 3.63×10-2 1.65×10-1 c 2.27×10-2 1.03×10-1 cd 7.82×10-3 6.82×10-2 de 6.42×10-3 2.70×10-2 e 2.80×10-3 1.97×10-3 e 3.29×10-4

Octanal 8.04×10-3 a 2.12×10-4 6.22×10-3 b 3.71×10-4 4.16×10-3 c 5.70×10-4 3.80×10-3 c 3.08×10-4 8.28×10-4 d 4.08×10-5 5.14×10-4 d 2.96×10-5 3.89×10-4 d 2.05×10-5

Limonene 4.94×10-2 a 4.66×10-3 3.41×10-2 b 3.95×10-3 2.99×10-2 bc 3.29×10-3 2.24×10-2 cd 2.94×10-3 1.61×10-2 de 2.08×10-3 8.15×10-3 ef 5.39×10-4 1.95×10-3 f 2.89×10-4

2-ethyl-1- hexanol 9.23×10-3 e 7.97×10-4 1.34×10-1 d 6.74×10-3 1.63×10-1 cd 1.62×10-2 1.85×10-1 cd 1.18×10-2 1.96×10-1 c 2.33×10-2 2.79×10-1 b 3.32×10-2 3.36×10-1 a 2.48×10-2

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Table 4.5 continued Compound D0 D1 D2 D3 D4 D7 D10

ppm SD* ppm SD ppm SD ppm SD ppm SD ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Nonanal 1.94×10-3 d 2.36×10-4 2.25×10-3 d 3.77×10-4 2.71×10-3 cd 1.83×10-4 3.39×10-3 c 7.40×10-4 4.59×10-3 b 2.88×10-4 5.04×10-3 b 3.00×10-4 6.44×10-3 a 2.48×10-4

Dill ether 2.35×10-3 d 4.79×10-4 3.30×10-3 cd 4.50×10-4 4.42×10-3 cd 2.39×10-4 5.73×10-3 cd 4.78×10-4 8.71×10-3 c 3.13×10-4 1.67×10-2 b 3.01×10-3 2.97×10-2 a 4.11×10-3

Dihydro- carveol 2.29×10-3 a 2.77×10-4 1.76×10-3 ab 2.42×10-4 1.60×10-3 b 2.11×10-4 1.55×10-3 bc 1.75×10-4 1.04×10-3 cd 2.48×10-4 6.93×10-4 de 2.91×10-5 3.48×10-4 e 3.41×10-5

Decanal 1.97×10-3 e 2.60×10-4 2.65×10-3 e 3.11×10-4 3.63×10-3 d 2.24×10-4 4.31×10-3 d 3.10×10-4 5.58×10-3 c 2.65×10-4 7.80×10-3 b 2.53×10-4 9.14×10-3 a 2.23×10-4

1,3 di-tert- butylbenzene 5.25×10-4 e 8.53×10-5 9.36×10-4 e 6.47×10-5 1.13×10-3 de 9.35×10-5 1.78×10-3 cd 5.14×10-4 2.47×10-3 bc 2.06×10-4 3.04×10-3 b 1.06×10-4 3.84×10-3 a 4.67×10-4

2,6-bis(1,1- dimethylethyl )phenol 1.53×10-2 e 1.32×10-3 2.17×10-2 de 6.90×10-4 2.33×10-2 d 1.17×10-3 3.05×10-2 c 1.42×10-3 3.31×10-2 bc 2.74×10-3 3.94×10-2 b 3.47×10-3 4.74×10-2 a 4.10×10-3

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The normalised relative concentrations of selected VC (group 1) are shown in Figure 4.23. The compound 2-ethyl hexanal showed minor fluctuations, with significant changes occurring at D4. Meanwhile, octanal, described as having fatty aroma descriptor (Gürbüz et al., 2006), had its highest normalised relative concentration at D0 which then significantly decreased at D1 and D2 followed by a significant decrease from D4 onwards. As for 1-octen-3-ol, having a mushroom-like aroma (Cho et al., 2008), had its normalised relative concentration significantly decreasing at D1 with further storage decreasing its amount. Zhang et al., (2008) reported a decrease in the peak area of 1-octen-3-ol during maturity of straw mushrooms, which is consistent with the findings in this study. Meanwhile, 3-octanone had its normalised relative concentration significantly decreasing from D0 to D2 and kept decreasing until D10. The compound 3-octanol, which is characterised by a mushroom-like, buttery aroma (Cho et al., 2008), had its normalised relative concentration significantly decreasing at D1 and D2 with no major changes occurring at D3. However at D7, a significant difference was observed and no significant change was observed between D7 and D10.

1-octen-3-ol 3-octanone 3-octanol 120 Octanal 2-ethyl hexanal 100

Normalised relative concentration 0

0 2 4 6 8 10 Day

Figure 4.23: Normalised relative concentrations of selected VC (group 1) extracted from enoki mushrooms during 10 days of storage period.

The normalised relative concentrations of selected VC (group 2) identified in stored enoki mushrooms are shown in Figure 4.24. Limonene, characterised by a citrus-like aroma (Belitz et al., 2009), had a maximum relative concentration at D0 which then significantly decreased at D3 reaching the lowest value at D10. The normalised relative concentration of dill ether increased

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with the storage time peaking at D10. The naturally occurring dill ether has the typical odour of dill oil which has an odour perception threshold 25 ppb in water (Vokk et al., 2011). Dihydrocarveol possessing a minty organoleptic property (Flavors and fragrances SAFC, 2013) had the highest concentration at D0 which then significantly decreased at D2 and D7. The normalised relative concentration of hexanal slightly increased with time but a significant increase was observed at D10. As for 1H-indol-5-ol, it was fluctuating during storage reaching the highest amount at D10 significantly different from the other storage days. The sharp increase in the normalised concentration of hexanal is obvious. A possible explanation could be due to increase in natural variability of this compound with increasing storage time.

Limonene Hexanal 400 1H-indol-5-ol Dill ether Dihydrocarveol

300

200

100

0 Normalised relative concentration

0 2 4 6 8 10 Day

Figure 4.24: Normalised relative concentrations of selected VC (group 2) detected in enoki mushrooms during 10 days of storage period.

The compound 2-ethyl-1-hexanol, known for its rose-like aroma (Cho et al., 2008), had its minimum relative concentration at D0 which then significantly increased with storage peaking at D10 (Figure 4.25).

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2-ethyl-1-hexanol 7000

6000

5000

4000

3000

2000

1000 Normalised relative concentration

0 2 4 6 8 10 Day

Figure 4.25: Normalised relative concentration of 2-ethyl-1-hexanol extracted from enoki mushrooms during 10 days of storage period.

The normalised relative concentrations of selected VC (group 3) extracted from stored enoki mushrooms are shown in Figure 4.26. The normalised relative concentrations of decanal, ethyl acetate, 3-methyl butanal and 1,3-di-tert-butyl benzene were increasing with storage with a significant increase observed at D4 compared to D0.

Decanal Ethyl acetate 3-methyl butanal 900 1,3-di-tert-butyl benzene

800

700

600

500

400

300

200

100 Normalisedrelativeconcentration 0

0 2 4 6 8 10 Day

Figure 4.26: Normalised relative concentrations of selected VC (group 3) extracted from enoki mushrooms during 10 days of storage period.

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Possible explanations for the presence of aldehydes in enoki mushrooms could be found in several studies. The compound 3-methyl butanal, characterised by a malty aroma (Belitz et al., 2009), is a volatile Strecker aldehyde formed from the reaction of α-amino acid (leucine) and α-dicarbonyl compound according to Vernin and Parkanyi (1982). Octanal which is described as having fatty aroma descriptor (Gürbüz et al., 2006) is a VC from oxidation of the unsaturated fatty acids (oleic and linoleic acid) while nonanal (with citrusy or floral aroma) and decanal (having sweet waxy, orange aroma descriptor) (Gürbüz et al., 2006) from the oxidation of oleic acid and hexanal which has green-like odour (Belitz et al., 2009) from the oxidation of linoleic acid and arachidonic acid (Badings, 1970).

Mushrooms are living organisms where continued respiration and other metabolic reactions are still occurring after harvesting. However, the exact biochemical pathway of how these VC are evolving is still unknown and it requires further investigation which was not the aim of this study Possible explanations for the changes in the VC profile could be due to biochemical changes in mushrooms during storage, microbial changes or due to the nature of the packaging film (PVC film) where oxygen, carbon dioxide or water transmission rate may have affected the VC profile.

It can be concluded that at D4, some VC start to significantly increase (aldehydes such as decanal, 3-methyl butanal, and hexanal and the alcohol 2-ethyl 1-hexanol) or decrease (alcohols such as 1- octen-3-ol, 3-octanol, 1-indol-5-ol and ketones such as 3-octanone) and hence the quality of enoki mushrooms is affected.

4.2.2 Drying curves

The moisture ratios versus time of enoki samples dried at different temperatures are presented in Figure 4.27. As expected, drying is faster at higher temperatures compared to lower temperatures (40 °C). The drying curves as shown in Figure 4.27 can be divided into two stages. Stage one is characterised by a fast removal of moisture and stage two which has a slower rate of moisture removal. Stage 1 is due to the fact that water is removed from the food sample into interface between the surface of the food and the drying air in the tunnel dryer while stage 2 deals with removing the moisture from the interior of the sample and is subjected to the diffusion process within the sample. The mushrooms were allowed to dry until they reached a constant weight. At 40 °C, the mushrooms were dried for 18.5 h and the final MC reached was 2.1%. At 50 °C, the drying time was 17 h and the final MC was 1.1% while at 60 °C, the mushrooms were dried for

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10 h with a final MC of 3.2% and at 70 °C, the mushrooms were dried for 7 h with a final MC of 2.2%.

It can be seen that drying at 50 and 60 °C resulted in a very close moisture ratio curves and the effect of temperature on VC will be discussed in the following sections.

Figure 4.27: Moisture ratios of enoki mushrooms at different drying temperatures versus time.

4.2.3 Dried (D) enoki mushrooms

The effect of different drying temperatures on VC was studied. Compounds belonging to the same chemical class behaved differently under the same conditions. The concentrations of studied compounds are shown in Table 4.6. The compound 3-methyl-1-butanol was the major VC found at 40 °C with a concentration of 7.86×10-2±1.67×10-3 ppm which decreased significantly when the drying temperature reached 70 °C (3.06×10-2±3.40×10-3 ppm) followed by 2-methyl-1-butanol (4.53×10-2±7.28×10-3 ppm) which also had its lowest concentration at 70 °C (1.83×10-2±1.60× 10-3 ppm). Following is 3-octanone with a concentration of 2.43×10-2±1.33×10-3 ppm which increased significantly when the samples were dried at 60 °C (5.22×10-2±3.40×10-3 ppm) followed by 2,6-bis(1,1-dimethylethyl)phenol with a maximum concentration of 2.01×10-2±5.09×10-3 ppm at 50 °C which was not significantly different from those determined at the remaining drying temperatures, then 1H-indol-5-ol (1.32×10-2±4.88×10-3 ppm) which increased with increasing

temperature to 50 °C (2.46×10-2±4.38×10-3 ppm) but decreased when the drying temperature reached 60 °C (6.91×10-3±2.07×10-4 ppm) and 70 °C (7.69×10-3±6.77×10-4 ppm).

As for the main C8 compounds, increasing the temperature from 40 °C to 50 °C did not have a major impact on the concentration of 1-octen-3-ol (5.67×10-3±1.59× 10-3 vs. 6.39×10-3±1.56×10-3 ppm). However, at 60 °C most of this compound was lost (1.27×10-3±2.33×10-4 and decreased even further at 70 °C (6.95×10-4 ±8.18×10-5 ppm). The highest concentration of 1-octen-3-one was found at 40 °C (2.97×10-3±4.80×10-4ppm) and then it decreased dramatically when the drying temperature reached 50 °C (6.27×10-4±1.34×10-4 ppm). A further increase in temperature from 60 to 70 °C did not have a major impact on the retention of the latter compound (6.80×10-4±1.13×10-4 vs. 7.05×10-4±1.79×10-5 ppm). Meanwhile, 3-octanone had its highest concentration at 60 °C (5.22×10-2±3.40×10-3 ppm) significantly different from 40, 50, and 70 °C samples and had its lowest concentration at 70 °C (9.86×10-3±1.66×10-4 ppm). As for 3-octanol, increasing the temperature from 40 to 60 and 70 °C significantly decreased the concentration of this compound (6.97×10-3±1.23×10-3 vs. 4.59×10-3±6.17×10-4 vs. 4.19×10-3±8.15×10-4 ppm) while n-octanol had the lowest concentration at 70 °C where most of the compound was lost due to heat (1.12×10-3 ±1.67×10-4 ppm), being significantly different from 40 and 50 °C samples but no significantly different from 60 °C samples.

As for the terpenes, the highest concentration was detected at 60 °C. Increasing the temperature from 40 °C to 60 °C significantly increased the concentration of limonene (1.22×10-3±4.04×10-4 vs. 2.53×10-3±2.44×10-4 ppm) and linalool (7.95×10-4±1.15×10-4 vs. 1.64×10-3±2.51×10-4 ppm). At 70 °C, both compounds had significantly lower concentrations.

As for aldehydes, 3-methyl butanal and 2-methyl butanal showed no significant difference between the different drying conditions both with the maximum concentrations were found at 70 °C (6.78×10-3±2.51×10-3 and 8.08×10-3±3.41×10-3 ppm respectively). Hexanal had its highest concentration at 40 °C (6.98×10-3±5.95×10-4 ppm) which was significantly different from 50, 60, and 70 °C samples and had its lowest at 70 °C (7.58×10-4±1.12×10-4 ppm) while decanal showed no significant difference between the different drying conditions with the highest concentration was found at 50 °C (2.83×10-3±1.77×10-3 ppm) and the lowest at 60 °C (1.11×10-3±1.94×10-4 ppm). As for nonanal, its concentration was not significantly affected by the different drying temperatures with the highest concentration being found at 50 °C (4.55×10-3±1.71×10-4 ppm),

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unlike benzaldehyde which had its highest concentration at 70 °C (4.07×10-3±3.63×10-4 ppm), significantly different from its lowest concentration found at 40 °C (6.68×10-4±2.64×10-5 ppm).

As for ketones, the concentration of 5-methyl-3-hexen-2-one was not affected by the different dying conditions with the highest concentration was found at 60 °C (1.20×10-3±1.79×10-4 ppm). As for 2,7-octandione, it had its maximum concentration at 70 °C (2.59×10-3±3.10×10-4 ppm) which was not significantly different from 60 °C samples but significantly different from 40 and 50 °C samples (1.65×10-3±2.42×10-4 ppm). Meanwhile, 2-undecanone had the highest concentration at 50 °C (3.30×10-3±1.16×10-3 ppm) significantly different from that found in 40 °C samples. As for 10-dimethyl-5,9-undecadien-2-one, it had its maximum concentration at 60 °C (1.10×10-2±2.73×10-3 ppm) which was not significantly different from 50 °C samples, but significantly different from 40 °C (1.14×10-3±1.45×10-4 ppm) and 70 °C samples (2.66×10-3 ±5.47×10-4 ppm).

Other alcohol compounds identified include 1-pentanol which had its highest concentration at 50 °C (2.33×10-3±3.79×10-4 ppm) being not significantly different from 40 °C samples but significantly different from 60 °C samples (1.22×10-3±1.32×10-4 ppm) and 70 °C samples (1.33×10-3±1.37×10-4 ppm), 1-hexanol which also had its highest concentration at 50 °C (1.79× 10-2±2.47×10-3 ppm) and significantly different from 40, 60, and 70 °C samples. As for 1- heptanol, no significant differences in the concentrations were found between 40, 50, and 70 °C with a maximum concentration of 3.65×10-3±8.81×10-4 ppm at 40 °C. The compound 2-ethyl-1- hexanol had its concentration affected by the temperature increase and decreased significantly when the drying was carried at 70 °C compared to 40 °C (2.82×10-3±4.33×10-4 vs. 5.91×10-3 ±1.33×10-3 ppm). Phenyl ethyl alcohol showed no significant difference between the different drying conditions with its maximum concentration found was at 50 °C (7.77×10-3±1.12×10-2 ppm) while 2-cyclohexen-1-ol had its maximum concentration at 40 °C (1.27×10-3±2.79×10-4 ppm), significantly different from 70 °C samples (6.74×10-4±3.08×10-5 ppm) but not significantly different from 50 and 60 °C samples. Meanwhile, the concentration of 1-undecanol did not significantly vary between the different drying conditions with the maximum concentration found was at 40 °C (9.24×10-4±1.97×10-4 ppm) while 1-nonanol had its maximum concentration at 50 °C (2.07×10-2±1.66×10-3 ppm), significantly different from that found at 40, 60, and 70 °C. As for 2,6-bis(1,1-dimethylethyl)phenol, it showed no significant difference between the different drying conditions with its highest concentration found at 50 °C (2.01×10-2±5.09×10-3 ppm).

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Other compounds identified include ethyl acetate which had its highest concentration was found at 50 °C (5.07×10-3±1.14×10-3 ppm), significantly different from those found at 40, 60, and 70 °C. As for 2,2,4,6,6-pentamethyl-3-heptene, it had a maximum concentration of 9.71×10-3 ±1.12×10-3 ppm 40 °C, not significantly different from 50 °C samples but significantly different from 60 °C (1.55×10-3±5.04×10-4 ppm) and 70 °C samples (5.75×10-3±1.19×10-3 ppm). Meanwhile, 1-(2- furyl)-3-methyl-3-butene-1,2-diol which showed no significant difference between the different drying conditions. As for 1,3-di-tert butylbenzene, its highest concentration was found at 60 °C (2.21×10-3±7.14×10-5 ppm) significantly different form 40, 50, and 70 °C samples.

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Table 4.6: Concentrations of VC extracted from enoki mushrooms dried at different temperatures. Compound D 40 °C D 50 °C D 60 °C D 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Ethyl acetate 2.71×10-3 b 6.87×10-4 5.07×10-3 a 1.14×10-3 1.20×10-3 bc 4.93×10-5 3.45×10-4 c 3.70×10-5

3-methyl butanal 5.43×10-3 a 3.37×10-4 3.37×10-3 a 3.12×10-4 4.39×10-3 a 1.41×10-3 6.78×10-3 a 2.51×10-3

2-methyl butanal 3.99×10-3 a 1.98×10-4 3.76×10-3 a 3.97×10-4 5.17×10-3 a 1.98×10-3 8.08×10-3 a 3.41×10-3

3-methyl-1-butanol 7.86×10-2 ab 1.67×10-3 6.15×10-2 b 1.06×10-2 7.87×10-2 a 6.50×10-3 3.06×10-2 c 3.40×10-3

2-methyl-1-butanol 4.53×10-2 a 7.28×10-3 3.62×10-2 a 6.53×10-3 5.20×10-2 a 9.19×10-3 1.83×10-2 b 1.60×10-3

1-pentanol 2.11×10-3 a 3.46×10-4 2.33×10-3 a 3.79×10-4 1.22×10-3 b 1.32×10-4 1.33×10-3 b 1.37×10-4

Hexanal 6.98×10-3 a 5.95×10-4 8.34×10-4 b 1.86×10-4 7.98×10-4 b 1.43×10-4 7.58×10-4 b 1.12×10-4

1-hexanol 1.23×10-2 b 2.60×10-3 1.79×10-2 a 2.47×10-3 5.19×10-3 c 8.15×10-4 4.86×10-3 c 4.71×10-4

1H-indole-5-ol 1.32×10-2 b 4.88×10-3 2.46×10-2 a 4.38×10-3 6.91×10-3 b 2.07×10-4 7.69×10-3 b 6.77×10-4

Benzaldehyde 6.68×10-4 c 2.64×10-5 1.74×10-3 b 5.26×10-4 1.83×10-3 b 3.09×10-4 4.07×10-3 a 3.63×10-4

1-heptanol 3.65×10-3 a 8.81×10-4 3.44×10-3 a 3.73×10-4 1.28×10-3 b 9.19×10-4 1.96×10-3 ab 2.54×10-4

1-octen-3-one 2.97×10-3 a 4.80×10-4 6.27×10-4 b 1.34×10-4 6.80×10-4 b 1.13×10-4 7.05×10-4 b 1.79×10-5

1-octen-3ol 5.67×10-3 a 1.59×10-3 6.39×10-3 a 1.56×10-3 1.27×10-3 b 2.33×10-4 6.95×10-4 b 8.18×10-5

3-octanone 2.43×10-2 b 1.33×10-3 1.50×10-2 c 1.20×10-3 5.22×10-2 a 3.40×10-3 9.86×10-3 d 1.66×10-4

3-octanol 6.97×10-3 a 1.23×10-3 5.05×10-3 ab 6.67×10-4 4.59×10-3 b 6.17×10-4 4.19×10-3 b 8.15×10-4

2,2,4,6,6-pentamethyl-3-heptene 9.71×10-3 a 1.12×10-3 7.71×10-3 ab 1.13×10-3 1.55×10-3 c 5.04×10-4 5.75×10-3 b 1.19×10-3

5-methyl-3-hexen-2-one 1.10×10-3 a 2.19×10-4 8.11×10-4 a 3.33×10-5 1.20×10-3 a 1.79×10-4 7.16×10-4 a 7.91×10-5

Limonene 1.22×10-3 bc 4.04×10-4 1.57×10-3 b 1.25×10-4 2.53×10-3 a 2.44×10-4 7.26×10-4 c 6.53×10-5

1-(2-furyl)-3-methyl-3-butene-1,2-diol 3.24×10-3 a 3.84×10-4 2.13×10-3 a 6.96×10-4 2.56×10-3 a 9.53×10-4 1.71×10-3 a 4.75×10-4

2-ethyl-1-hexanol 5.91×10-3 a 1.33×10-3 5.78×10-3 ab 3.30×10-4 4.42×10-3 ab 1.03×10-3 2.82×10-3 b 4.33×10-4

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Table 4.6 continued Compound D 40 °C D 50 °C D 60 °C D 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2,7-octandione 7.04×10-4 c 8.60×10-5 1.65×10-3 b 2.42×10-4 2.39×10-3 a 2.86×10-4 2.59×10-3 a 3.10×10-4

n-octanol 4.57×10-3 ab 1.29×10-3 5.39×10-3 a 4.57×10-4 2.82×10-3 bc 5.72×10-4 1.12×10-3 c 1.67×10-4

Linalool 7.95×10-4 b 1.15×10-4 7.60×10-4 b 9.27×10-5 1.64×10-3 a 2.51×10-4 7.10×10-4 b 8.67×10-5

Nonanal 3.06×10-3 a 6.87×10-4 4.55×10-3 a 1.71×10-4 3.50×10-3 a 5.57×10-4 3.69×10-3 a 2.40×10-4

Phenyl ethyl alcohol 6.12×10-4 a 8.66×10-5 7.77×10-3 a 1.12×10-2 5.70×10-4 a 6.62×10-5 7.34×10-4 a 9.15×10-5

2-cyclohexen-1-ol 1.27×10-3 a 2.79×10-4 1.10×10-3 ab 9.95×10-5 1.25×10-3 a 2.52×10-4 6.74×10-4 b 3.08×10-5

1-undecanol 9.24×10-4 a 1.97×10-4 7.69×10-4 a 1.05×10-4 6.33×10-4 a 7.20×10-5 7.07×10-4 a 6.97×10-5

1-nonanol 1.10×10-2 b 3.13×10-3 2.07×10-2 a 1.66×10-3 1.32×10-3 c 2.70×10-4 1.52×10-3 c 1.45×10-4

Decanal 1.93×10-3 a 5.74×10-4 2.83×10-3 a 1.77×10-3 1.11×10-3 a 1.94×10-4 2.12×10-3 a 2.38×10-4

1,3-di-tert butylbenzene 1.01×10-3 b 5.15×10-4 6.18×10-4 b 7.23×10-5 2.21×10-3 a 7.14×10-5 8.54×10-4 b 1.25×10-4

2-undecanone 1.15×10-3 b 1.87×10-4 3.30×10-3 a 1.16×10-4 1.80×10-3 ab 5.37×10-4 1.51×10-3 ab 2.24×10-4

6-10-dimethyl-5,9-undecadien-2-one 1.14×10-3 b 1.45×10-4 1.03×10-2 a 4.39×10-4 1.10×10-2 a 2.73×10-3 2.66×10-3 b 5.47×10-4

2,6-bis(1,1-dimethylethyl)phenol 1.58×10-2 a 7.00×10-3 2.01×10-2 a 5.09×10-3 1.21×10-2 a 1.69×10-3 1.41×10-2 a 5.20×10-3

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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Enoki mushrooms dried at different temperatures are shown in figure 4.28 and 4.29 below. At lower temperature (40 and 50 °C) the mushrooms were lighter in colour compared to higher drying temperatures (60 and 70 °C).

Figure 4.28: Vacuum packed enoki mushrooms dried at 40 °C (Left) and 50 °C (Right) (Author photograph).

Figure 4.29: Vacuum packed enoki mushrooms dried at 60 °C (Left) and 70 °C (Right) (Author photograph).

The normalised relative concentrations of the main alcohols and ketones found in enoki mushrooms dried at the different temperatures are shown in Figure 4.30. The relative

concentrations of VC have been normalised to the highest concentration found under each condition. The highest amount of 1-octen-3-one was found when enoki samples were dried at 40 °C being significantly different when compared to 50, 60 and 70 °C. The compounds 1-octen- 3-ol, 3-octanol, 2-ethyl-1-hexanol, and n-octanol showed no significant difference when comparing their normalised relative concentration at 40 °C and 50 °C. As for 3-octanone, it had the highest amount at 60 °C significantly different when compared to the other dried samples while 2-undecanone had its maximum relative concentration at 50 °C which was significantly different from 40 °C samples but not significantly different from that found in 60 and 70 °C samples.

1.4 D 40 °C D 50 °C 1.2 D 60 °C D 70 °C 1.0

0.8

0.6

0.4

Normalisedrelative concentration 0.2

0.0 e l e l l l e n -o n no no no n -o -3 no ta a ta no -3 en ta c ex c a en ct c -o h -o ec ct o -o 3 1- n d -o 1- 3 l- un 1 hy 2- et 2- Compound

Figure 4.30: Normalised relative concentrations main alcohols and ketones extracted from dried enoki mushrooms at different temperatures.

Different classes of compounds such as aldehydes, ketones, alcohols, and terpenes differed in their behaviours as shown in Figure 4.31. The normalised relative concentration of nonanal showed no significant difference when different drying temperatures were compared. In contrast, benzaldehyde showed the maximum normalised relative concentration at 70 °C being significantly different from the other drying temperatures.

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D 40 °C 1.2 D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalisedconcentrationrelative 0.0

l l l a o ne -o de ne an lo e 5 y -o n na on l- eh 2 o i m do ld n- N L i in a e L - nz ex 1H e -h B l-3 hy et Compound m 5-

Figure 4.31: Normalised relative concentrations of selected VC extracted from enoki mushrooms dried at different temperatures.

The highest normalised relative concentrations for terpenes (linalool and limonene) were found at 60 °C. As for 1H-indol-5-ol, it had the maximum normalised relative concentration when samples were dried at 50 °C while 5-methyl-3-hexen-2-one showed no significant difference between the different drying temperatures.

4.2.4 Dried and boiled under reflux (DBR) enoki mushrooms

Boiling the dried enoki mushrooms under reflux resulted in an increase in the concentrations of some VC as well as the release of some new ones. The concentrations of the dried and boiled under reflux enoki mushrooms are shown in Table 4.7. Phenylacetaldehyde was found to be the major VC in DBR 40 °C samples (5.43×10-2±7.84×10-3 ppm) followed by 2-methyl butanal (4.03×10-2±8.49×10-3 ppm), 3-methyl butanal (3.40×10-2±7.35×10-3 ppm), benzaldehyde (1.71×10-2±3.00×10-3 ppm), 2,2,4,6,6-pentamethyl-3-heptene (1.68×10-2±1.75×10-3ppm), hexanal (1.62×10-2±4.42×10-4ppm), 2,6-bis (1,1-dimethylethyl)phenol (1.43×10-2±2.96×10-3 ppm), dill ether (1.27×10-2±1.38×10-3 ppm), and 1H-indol-5-ol (1.22×10-2±2.15×10-3 ppm).

As for the C8 compounds, 1-octen-3-one had its highest in DBR 50 °C samples (6.66×10-3 ±6.09×10-4 ppm) significantly different from its lowest concentration found in DBR 60 °C samples (6.48×10-4±9.79×10-5 ppm) with no significant difference found between DBR 40 °C and

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70 °C samples. As for 3-octanone, it had a concentration of 4.35×10-3±6.20×10-4 ppm in DBR 40 °C samples which was not significantly different compared to DBR 50 °C and 60 °C samples but significantly different from that found in DBR 70 °C samples (1.93×10-3±4.21×10-4ppm). The concentration of 1-octen-3-ol increased significantly to almost double when the temperature increased from 40 °C to 50 °C (4.19×10-3±1.053×10-3 vs. 8.36×10-3±7.61×10-4 ppm) in DBR 40 and 50 °C samples, but again further increase in temperatures resulted in a significant loss of this compound in DBR 60 and 70 °C samples (2.66×10-3±4.28×10-4 and 2.37×10-3±6.64×10-4 ppm respectively). A significant increase in the concentration of 3-octanol was observed when the drying temperature increased peaking in DBR 60 °C samples (5.25×10-3±5.24×10-4 ppm) then decreased again in DBR 70 °C samples (1.00×10-3±3.07×10-4 ppm) with no significant difference found between DBR 40, 50, and 70 °C samples. As for n-octanol, its highest concentration was found in DBR 50 °C (5.22×10-3±4.48×10-4 ppm) significantly different from its lowest concentration found in DBR 60 °C (7.17×10-4±1.53×10-4 ppm). No significant difference was found between DBR 40 and 70 °C samples.

As for the remaining alcohols, 3-methyl-1-butanol had its maximum concentration in DBR 50 °C (8.39×10-3±6.15×10-4 ppm) not significantly different from DBR 40 °C but significantly different form DBR 60 and 70 °C samples. As for 1-pentanol and 1-hexanol, they both had their highest concentrations in DBR 50 °C (5.98×10-3±8.95×10-4 ppm and 1.14×10-2±2.21×10-3 ppm respectively) significantly different from those found in DBR 40, 60, and 70 °C samples. Meanwhile, 1H-indol-5-ol and 2-ethyl-1-hexanol had their maximum concentrations in DBR 50 °C samples (1.57×10-2±7.20×10-3 ppm and 3.95×10-3±3.65×10-4ppm respectively), not significantly different from DBR 40 °C samples but significantly different from DBR 60 and 70 °C samples. As for 1-nonanol, it had its maximum concentration in DBR 70 °C samples (5.96×10-3±1.08×10-3 ppm) which was not significantly different from DBR 50 °C samples but significantly different from DBR 40 °C (2.89×10-3±8.23×10-4 ppm) and DBR 60 °C (7.46×10-4 ±1.29×10-4 ppm) while dihydrocarveol had its highest concentration in DBR 50 °C samples (2.15×10-3±5.37×10-4 ppm) which was significantly different form DBR 60 and 70 °C samples and was not detected in DBR 40 °C samples. Meanwhile, 2,6-bis(1,1-dimethylethyl)phenol showed no significant difference between DBR 40, 50 and 60 °C samples, but in DBR 70 °C, its concentration was significantly lower (7.24×10-3±1.80×10-4 ppm).

As for aldehydes, The compounds 3-methyl butanal and 2-methyl butanal had their concentrations peaking in DBR 40 °C samples (3.40×10-2±7.35×10-3 ppm and 4.03×10-2±8.49×

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10-3 ppm respectively) then significantly decreased in DBR 50 °C samples. Hexanal had its highest concentration in DBR 50 °C samples (2.37×10-2±1.70×10-3 ppm), significantly different from DBR 40 samples but not significantly different from DBR 60 and 70 °C samples, while heptanal had its highest concentration in DBR 70 °C samples (2.64×10-3±3.70×10-4 ppm), significantly from the remaining treatments. As for benzaldehyde, its concentration marginally increased when comparing DBR 40, 50, and 60 °C samples which were significantly lower than its maximum concentration found in DBR 70 °C samples (3.85×10-2±1.55×10-3 ppm). The concentration of phenylacetaldehyde slightly decreased when comparing DBR 40 and 50 °C samples (5.43×10-2±7.84×10-3 vs. 4.20×10-2±1.45×10-3 ppm) then significantly decreased in DBR 60 °C (2.30×10-2±2.81×10-3 ppm). No significant difference was observed between DBR 40 and 50 °C samples and between DBR 50 and 70 °C samples for this compound while decanal showed no significant difference between DBR 40 °C and 50 °C (2.95×10-3±1.62×10-4 and 5.68×10-3 ±1.33×10-3 ppm respectively), however, in DBR 60 °C samples, its concentration was significantly reduced to 1.11×10-3±4.86×10-4 ppm which was not significantly different from DBR 70 °C samples (1.68×10-3±6.06×10-4 ppm).

As for ketones, drying at 60 °C followed by boiling under reflux resulted in the maximum retention of 2-propanone (1.16×10-2±1.88×10-3 ppm) which was significantly different from DBR 40, 50, and 70 °C samples while 3-heptanone had its highest concentration in DBR 40 °C samples (2.53×10-3±2.87×10-4 ppm) which was significantly different from DBR 50, 60, and 70 °C samples. As for 2-hexanone, it showed no significant difference between DBR 50 and 60 °C samples while its lowest concentration was found in DBR 40 °C samples (2.31×10-3±3.94×10-4 ppm. Meanwhile, 1,2-cyclohexadione had an increase in its concentration as the temperature increased peaking in DBR 70 °C samples (5.89×10-3±3.81×10-4 ppm) significantly different form DBR 40, 50, and 60 °C samples. As for 2,7-octanedione, it had its maximum concentration in DBR 70 °C samples (3.06×10-3±1.19×10-4 ppm) which was not significantly different from DBR 50 °C samples but significantly different from its lowest concentration found in DBR 40 °C samples (2.10×10-3±6.75×10-5 ppm). Acetophenone had its highest concentration in DBR 40 °C samples (1.75×10-3±2.87×10-4 ppm) which was not significantly different from DBR 50 °C samples but significantly different from DBR 60 and 70 °C samples. Meanwhile 2-undecanone, its highest concentration was found in DBR 70 °C samples (5.02×10-3±8.21×10-4 ppm) which was significantly different form DBR 40, 50 and 60 °C samples. Meanwhile camphor showed no significant difference between DBR 40 and 50 °C samples (2.58×10-3±3.72×10-4 and 3.12×10-3

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±5.21×10-4 ppm respectively) but in DBR 60 °C samples, its concentration significantly decreased to reach a minimum of 9.91×10-4±8.74×10-5 ppm which was not significantly different from DBR 70 °C samples. As for 6,10-dimethyl-5,9-undecadien-2-one, no significant difference was found between the different treatments with its highest concentration was obtained in DBR 50 °C (1.23×10-3±4.51×10-4 ppm).

Aldehydes behaviour is explained in section 4.1.5 while the formation of 2-octanone is discussed in section 4.2.5. However, understanding the behaviour of some compounds requires further analysis as these information cannot be found in the literature.

As for pyrazine compounds, different drying temperatures did not have a major impact on the concentrations of 2,6-dimethylpyrazine and 3-ethyl-2,5-dimethylpyrazine where the highest concentration of the former compound was found in DBR 70 °C samples (2.56×10-3±3.44×10-4 ppm) while for the latter compound, it was found in DBR 40 °C samples (2.69×10-3±5.85×10-4 ppm). As for 2-ethyl-6-methylpyrazine, its highest concentration was found in DBR 60 °C (4.04×10-3±5.65×10-4 ppm) which was significantly different from DBR 40, 50, and 70 °C.

As for the terpene compounds, limonene showed no significant difference between the different treatments with the maximum concentration found was in DBR 40 °C (5.98×10-3±7.19×10-4 ppm) while linalool had the highest concentration in DBR 60 °C samples (5.98×10-3±7.19×10-4 ppm) which was significantly different compared to other drying temperatures. The formation of pyrazine compounds and the reason behind pyrazine and terpene compounds’ detection only in DBR can be found in section 4.2.5 where some compounds need higher rehyhdration temperature for their release from their dried material. Furthermore, Martinez-Soto et al. (2001) argued that rehydration of P. ostreatus was much faster at 94 °C than at 25 °C.

Other compounds identified include for 2-methyl furan with a maximum concentration of 1.73×10-3±2.61×10-4 ppm in DBR 60 °C samples, ethyl acetate with its highest concentration in found DBR 50 °C samples (3.86×10-3±7.32×10-4 ppm). The compound 2,3,4,5-tetrahydropyridine was also identified with a maximum concentration of 7.62×10-3±8.13×10-4 ppm in DBR 50 °C, significantly different from DBR 40, 60, and 70 °C samples. Meanwhile, 2,2,4,6,6-pentamethyl-3- heptene had its highest concentration in DBR 40 °C samples (1.68×10-2±1.75×10-3 ppm), significantly different form DBR 60 and 70 °C samples but not significantly different form DBR 40 °C samples. As for 2-ethyl-1,4-dimethylbenzene, it was not affected by the different drying conditions with its maximum concentration found in DBR 50 °C samples (1.43×10-3±5.11×10-4

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ppm). Meanwhile 1-(2-furyl)-3-methyl-3-butene-1,2-diol, its highest concentration was found in DBR 50 °C (6.64×10-3±1.20×10-4 ppm) which was not significantly different from DBR 40 °C samples but significantly different from DBR 60 °C (3.13×10-3±5.17×10-4 ppm) and DBR 70 °C samples (3.24×10-3±7.30×10-4 ppm). As for (E,E)-2,4-heptadienal, its maximum concentration was found in DBR 50 °C (2.32×10-3±3.25×10-4 ppm), significantly different from DBR 40,60, and 70 °C samples. Finally, dill ether, showed a maximum concentration in DBR 40 °C (2.58×10-3 ±3.72×10-4 ppm), significantly different from DBR 50, 60, and 70 °C samples.

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Table 4.7: Concentrations of VC extracted from dried and boiled under reflux enoki mushrooms. Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-propanone 5.63×10-3 b 1.03×10-3 2.51×10-3 b 7.11×10-4 1.16×10-2 a 1.88×10-3 5.55×10-3 b 1.41×10-3

2-methyl furan 7.99×10-4 b 3.47×10-5 1.01×10-3 b 2.14×10-4 1.73×10-3 a 2.61×10-4 9.00×10-4 b 3.44×10-5

Ethyl acetate 1.83×10-3 b 4.78×10-4 3.86×10-3 a 7.31×10-4 4.91×10-4 c 7.71×10-5 6.16×10-4 c 5.83×10-5

3-methyl butanal 3.40×10-2 a 7.35×10-3 1.02×10-2 b 5.06×10-3 2.62×10-2 a 8.01×10-4 2.03×10-2 ab 6.50×10-3

2-methyl butanal 4.03×10-2 a 8.49×10-3 1.29×10-2 b 3.12×10-4 2.18×10-2 b 6.51×10-4 1.59×10-2 b 5.70×10-3

3-methyl-1-butanol 6.45×10-3 ab 1.35×10-3 8.39×10-3 a 6.15×10-4 4.63×10-3 b 3.55×10-4 1.61×10-3 c 2.10×10-4

2-hexanone 2.31×10-3 c 3.94×10-4 7.04×10-3 a 6.34×10-4 5.05×10-3 ab 1.02×10-3 3.35×10-3 bc 9.05×10-4

1-pentanol 1.86×10-3 b 4.01×10-4 5.98×10-3 a 8.95×10-4 7.84×10-4 b 4.54×10-5 8.07×10-4 b 1.02×10-4

Hexanal 1.62×10-2 b 4.42×10-4 2.37×10-2 a 1.70×10-3 2.09×10-2 ab 9.45×10-4 2.03×10-2 ab 3.35×10-3

Heptanal 1.06×10-3 b 1.51×10-4 1.58×10-3 b 4.84×10-4 1.20×10-3 b 2.49×10-4 2.64×10-3 a 3.70×10-4

1-hexanol 5.12×10-3 b 7.53×10-4 1.14×10-2 a 2.21×10-3 1.90×10-3 bc 4.51×10-4 1.78×10-3 c 6.62×10-4

3-heptanone 2.53×10-3 a 2.87×10-4 1.18×10-3 b 1.52×10-4 1.01×10-3 b 1.68×10-4 7.19×10-4 b 2.94×10-5

2,6-dimethylpyrazine 1.96×10-3 a 3.37×10-4 2.40×10-3 a 5.28×10-4 1.77×10-3 a 1.73×10-4 2.56×10-3 a 3.44×10-4

1H-indol-5-ol 1.22×10-2 ab 2.15×10-3 1.57×10-2 a 7.20×10-3 4.44×10-3 b 4.24×10-4 4.71×10-3 b 6.77×10-4

1,2-cyclohexadione 4.67×10-4 b 1.21×10-4 5.03×10-4 b 6.15×10-5 6.77×10-4 b 2.85×10-5 5.89×10-3 a 3.81×10-4

Benzaldehyde 1.71×10-2 b 3.00×10-3 2.33×10-2 b 4.55×10-3 2.53×10-2 b 4.97×10-3 3.85×10-2 a 1.55×10-3

1-octen-3-one 4.20×10-3 b 1.19×10-3 6.66×10-3 a 6.09×10-4 6.48×10-4 c 9.79×10-5 3.36×10-3 b 6.59×10-4

1-octen-3-ol 4.19×10-3 b 1.05×10-3 8.36×10-3 a 7.61×10-4 2.66×10-3 b 4.28×10-4 2.37×10-3 b 6.64×10-4

3-octanone 4.35×10-3 a 6.20×10-4 3.96×10-3 a 6.16×10-4 4.33×10-3 a 1.70×10-4 1.93×10-3 b 4.21×10-4

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Table 4.7 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2,3,4,5-tetrahydropyridine 1.25×10-3 b 4.98×10-4 7.62×10-3 a 8.13×10-4 7.52×10-4 b 5.52×10-5 1.00×10-3 b 8.58×10-5

3-octanol 7.28×10-4 b 8.11×10-5 1.42×10-3 b 2.75×10-4 5.25×10-3 a 5.24×10-4 1.00×10-3 b 3.07×10-4

2-ethyl-6-methylpyrazine 1.46×10-3 b 4.75×10-4 1.70×10-3 b 4.79×10-4 4.04×10-3 a 5.65×10-4 2.58×10-3 b 3.61×10-4

2,2,4,6,6-pentamethyl-3-heptene 1.68×10-2 a 1.75×10-3 1.22×10-2 ab 2.84×10-3 7.34×10-3 b 1.01×10-3 8.24×10-3 b 1.65×10-3

(E,E)-2,4-heptadienal 1.39×10-3 b 2.82×10-4 2.32×10-3 a 3.25×10-4 1.33×10-3 b 2.81×10-4 1.06×10-3 b 3.57×10-5

2-ethyl-1,4-dimethylbenzene 1.05×10-3 a 4.82×10-4 1.43×10-3 a 5.11×10-4 7.86×10-4 a 4.56×10-5 6.99×10-4 a 1.17×10-4

Limonene 2.60×10-3 a 2.14×10-4 2.49×10-3 a 8.65×10-5 1.89×10-3 a 8.40×10-4 2.03×10-3 a 6.20×10-4

1-(2-furyl)-3-methyl-3-butene- 1,2-diol 5.73×10-3 a 5.82×10-4 6.64×10-3 a 1.20×10-4 3.13×10-3 b 5.17×10-4 3.24×10-3 b 7.30×10-4

2-ethyl-1-hexanol 3.52×10-3 a 2.82×10-4 3.95×10-3 a 3.65×10-4 1.65×10-3 b 2.36×10-4 1.63×10-3 b 6.59×10-4

Phenylacetaldehyde 5.43×10-2 a 7.84×10-3 4.20×10-2 ab 1.45×10-3 2.30×10-2 c 2.81×10-3 3.90×10-2 b 6.89×10-3

2,7-octanedione 2.10×10-3 b 6.75×10-5 2.48×10-3 ab 4.53×10-4 2.26×10-3 b 3.43×10-4 3.06×10-3 a 1.19×10-4

n-octanol 2.50×10-3 b 8.73×10-4 5.22×10-3 a 4.48×10-4 7.17×10-4 c 1.53×10-4 3.17×10-3 b 6.10×10-4

2,5-dimethyl-3-ethylpyrazine 2.69×10-3 a 5.85×10-4 2.50×10-3 a 8.93×10-4 2.06×10-3 a 5.29×10-4 1.54×10-3 a 3.14×10-4

Linalool 2.63×10-3 b 2.69×10-4 3.50×10-3 b 2.98×10-4 5.98×10-3 a 7.19×10-4 1.13×10-3 c 1.10×10-4

Nonanal 7.35×10-3 a 2.80×10-3 6.36×10-3 a 1.21×10-3 4.18×10-3 b 5.67×10-4 8.12×10-3 a 1.58×10-4

Camphor 2.58×10-3 a 3.72×10-4 3.12×10-3 a 5.21×10-4 9.91×10-4 b 8.74×10-5 6.61×10-4 b 5.65×10-5

Acetophenone 1.75×10-3 a 2.87×10-4 1.29×10-3 ab 2.83×10-4 7.64×10-4 bc 3.80×10-5 5.31×10-4 c 2.89×10-5

1-nonanol 2.89×10-3 b 8.23×10-4 5.08×10-3 a 8.44×10-4 7.46×10-4 c 1.29×10-4 5.96×10-3 a 1.08×10-3

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Table 4.7 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Dill ether 1.27×10-2 a 1.38×10-3 8.71×10-3 b 1.22×10-3 5.90×10-3 c 8.96×10-4 3.80×10-3 c 4.98×10-4

Dihydrocarveol ND ND 2.15×10-3 a 5.37×10-4 1.10×10-3 b 2.67×10-4 7.32×10-4 bc 1.41×10-4

Decanal 2.95×10-3 a 1.62×10-4 5.68×10-3 a 1.33×10-3 1.11×10-3 b 4.86×10-4 1.68×10-3 b 6.06×10-4

2-undecanone 2.53×10-3 b 4.57×10-4 2.88×10-3 b 7.29×10-4 1.94×10-3 b 3.31×10-4 5.02×10-3 a 8.21×10-4

6-10-dimethyl-5,9-undecadien-2- -4 -5 -3 -4 -4 -5 -4 -5 one 9.03×10 a 5.27×10 1.23×10 a 4.51×10 6.84×10 a 3.04×10 7.00×10 a 4.18×10

-2 -3 -2 -3 -2 -3 -3 -4 2,6-bis(1,1-dimethylethyl)phenol 1.43×10 ab 2.96×10 1.71×10 a 6.22×10 1.24×10 ab 1.56×10 7.24×10 b 1.80×10

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The normalised relative concentrations of selected alcohols and ketones are shown in Figure 4.32. Boiling under reflux of the samples dried at 50 °C resulted in the maximum release of 1-octen-3- one and 1-octen-3-ol, and n-octanol significantly different from DBR 40, 60, and 70 °C samples. The normalised relative concentration of 3-octanone did not change significantly when comparing the normalised values in DBR 40 °C, 50 °C, and 60 °C samples. As for 3-octanol, its highest normalised relative concentration was found in DBR 60 °C samples, significantly different from those determined at the remaining drying conditions and its lowest in DBR 40 °C samples while 2- ethyl-1-hexanol showed no significant difference between DBR 40 and 50 °C samples and between DBR 60 and 70 °C samples. As for 2-undecanone, its highest normalised relative concentration was found in DBR 70 °C significantly different from that found in DBR 40, 50, and 60 °C samples.

DBR 40 °C 1.4 DBR 50 °C DBR 60 °C DBR 70 °C 1.2

1.0

0.8

0.6

0.4

Normalisedrelativeconcentration 0.2

0.0 ne ol ne ol ol ol ne -o 3- o an an an o -3 n- an ct ct x an n te ct -o -o he ec te oc -o 3 n 1- d oc 1- 3 l- un 1- hy - Compound et 2 2-

Figure 4.32: Normalised relative concentrations of selected alcohols and ketones extracted from enoki mushrooms dried at different temperatures followed by boiling under reflux.

As for the newly formed VC (Figure 4.33), camphor, also known as 1,7,7-trimethylbicyclo[2.2.1] heptan-2-one, was only detected when the dried samples were boiled under reflux. Its highest normalised relative concentrations were found in DBR 50 °C samples which was not significantly different from 40 °C samples. These amounts were significantly different from DBR 60 °C and 70 °C samples. Dill ether, also known as 3,6-dimethyl-2,3,3 alpha, 4,5,7 alpha-hexahydro-1- benzofuran had the highest normalised value in DBR 40 °C samples. Meanwhile, drying at 60 °C

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followed by boiling under reflux was found to be the best temperature for the maximum retention of 2-methyl furan while DBR 50 °C was found to be the best drying temperature for (E,E)-2,4- heptadienal. DBR 70 °C was the optimum temperature for 1,2-cylcohexadione which was significantly different DBR 40, 50, and 60 °C samples. As for the pyrazine compounds, 2,6- dimethylpyrazine and 2,5-dimethyl-3-ethylpyrazine, no significant losses were found between DBR 40, 50, 60, and 70 °C samples.

1.4

1.2 DBR 40 °C DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2

Normalised relative concentration 0.0

n e e l r r e ra zin ion ena ho the zin l fu ra ad di mp ll e ra hy lpy ex pta Ca Di lpy et hy loh he hy -m et yc ,4- -et 2 im 2-c )-2 - 3 6-d 1, ,E yl 2, (E eth Compound im 5-d 2,

Figure 4.33: Normalised relative concentration of selected newly formed VC extracted from enoki mushrooms dried at different temperatures followed by boiling under reflux.

Linalool and limonene are known as antibacterial compounds as they have been found effective against food-borne pathogens Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Capmpylobacter jejuni (Chen et al., 2008; Sandasi et al., 2008). The naturally occurring dill ether showed the typical odour of dill oil which has an odour perception threshold 25 ppb in water (Vokk et al., 2011). As for 6,10-dimethyl, 5-9-undecadien-2-one, it is characterised by a green and rosy floral odour and fresh-floral with reported usage in baked goods, frozen dairy, gelatines and puddings. It was found in citronella oil, yellow passion fruit, peppermint, tomato, carrots, melon, parmesan cheese, figs, mango, red sage, nectarines, clams and pork (Burdock, 2004). This VC was also detected in freshly harvested and stored rice (Bryant and McClung 2011). Indoles are known for their fecal odour in high concentrations and floral odour in high dilution. Benzaldehyde, the main characteristic component of bitter almond oil is used in aroma compositions for its bitter

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almond odour. Camphor , a terpenoid also known as 1,7,7-trimethylbicyclo[2.2.1]-2-heptanone, (Chen et al., 2013) is the main components of oils obtained from the camphor tree Cinnamomum camphora, has a slight mint odour and is used mainly as a plasticizer and in perfuming industrial products (Bauer et al., 2001). Camphor also exhibits a wide range of biological activities such as antibacterial, antifungal, antimutagenic, antitussive, and insecticidal properties (Chen et al., 2013).

These results show that 50 °C was a suitable drying temperature aiming at preserving the VC while extending the shelf life of the product.

4.2.5 Comparison between treatments

The choice of a suitable drying temperature should be a compromise between (a) suitable condition for the maximum retention and preservation of VCs, (b) adequate moisture removal for the extension of the shelf life (c) savings in terms of energy usage. Therefore drying at 50 °C appears as the most suitable drying temperature. Drying at 50 °C resulted in the maximum retention of VC in enoki and thus it was chosen as the suitable temperature and was used to compare the 4 treatments: fresh (F), boiled under reflux (BR), dried (D), and dried and boiled under reflux (DBR). The choice of 50 °C as the most suitable drying temperature was based on the overall concentrations of VC of importance in enoki mushrooms such as the main C8 compounds, aldehydes, ketones, pyrazines and terpenes. Furthermore, the behaviour of VC when the dried samples were boiled under reflux was also taken into consideration as some of VC tend to form at higher rehydration temperature rather than ambient temperature rehydration such as 2-propanone, 2-methyl furan, 2-hexanone, 3-heptanone, heptanal, 2,6-dimethyl pyrazine, 2,3,4,5- tetrahydropyridine, 2-ethyl-6-methyl pyrazine, (E,E)-2,4-heptadienal, 2-ethyl-1,4- dimethylbenzene, phenylacetaldehyde, (E)-2-octenal, 2,5-dimethyl-3-ethyl pyrazine, and camphor (section 4.2.4). Statistical analysis such as ANOVA and Tukey tests were used to show any significant changes between the different drying temperatures (see table 4.6).

In terms of concentrations (Table 4.8), 1H-indol-5-ol was the major VC detected in (BR) samples (3.58×10-2±3.96×10-3 ppm) followed by 3-octanone (2.82×10-2±4.54×10-3 ppm), 2-ethyl-1- hexanol (2.66×10-2±2.93×10-3 ppm), 2,6-bis(1,1-dimethylethyl)phenol (2.14×10-2±4.49×10-4 ppm) and 3-octanol (1.18×10-2±1.51×10-3 ppm). Compared to (F) samples, some compounds were newly formed or released upon boiling under reflux and include but are not limited to 2-propanone (2.99×10-3±5.46×10-4 ppm), 3-heptanone (7.34×10-4±6.19×10-5 ppm), heptanal (7.98× 10-4±6.97×10-5 ppm), benzaldehyde (4.10×10-3±6.07×10-4 ppm), 1-octen-3-one, (7.91× 10-4±7.68×10-5ppm), 5-metyl-3-hexen-2-one (9.37×10-3±1.94×10-4 ppm), phenylacetaldehyde

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(5.05×10-3±5.72×10-4 ppm), (E)-2-octenal (1.15×10-3±1.01×10-4 ppm), 2-cyclohexene-1-one (6.04×10-4±3.39×10-5 ppm), linalool (1.03×10-3±1.48×10-4 ppm), and 2-undecanone (7.41× 10-4±4.23×10-5 ppm). Other compounds found in BR samples include 2-methyl butanal (2.19× 10-3±1.08×10-4 ppm), 1-pentanol (4.12×10-3±1.29×10-4 ppm), n-octanol (2.37×10-3±1.85×10-4 ppm) and 1-nonanol (7.28×10-4±2.08×10-5 ppm). Comparing the four treatments, 2-propanone was only found in BR samples (2.99×10-3±5.46×10-4 ppm) and DBR samples (2.51×10-3±7.11×10-4 ppm) with no significant difference between the 2 treatments, while 2-methyl furan was only found in DBR samples with a concentration of 1.01×10-3±2.14×10-4 ppm. Ethyl acetate had its maximum concentration in D 50 °C (5.07×10-3±1.14×10-3 ppm) samples which was not significantly different from DBR 50 °C samples (3.86×10-3±7.31×10-4 ppm) but significantly different from F (1.41×10-3±1.52×10-4 ppm) and BR (1.27×10-3±1.31×10-4 ppm) samples. The compounds 3-methyl butanal and 2-methyl butanal both had their highest concentration in DBR 50 °C samples (1.02×10-2±5.06×10-3 ppm and 1.29×10-2±3.12×10-4 ppm).

As for the alcohols, 3-methyl-1-butanol had its maximum concentration in D 50 °C samples (6.15×10-2±1.06×10-2 ppm) which was significantly different from F, BR and DBR 50 °C samples with a minimum concentration of 6.82×10-4±4.78×10-5 ppm found in BR samples. The compound 2-methyl-1-butanol had its highest concentration in D 50 °C samples (3.62×10-2 ±6.53×10-3 ppm), its lowest in BR samples (5.70×10-4±9.97×10-5 ppm) and was not detected in DBR samples. As for 1-pentanol, its highest concentration was found in DBR 50 °C samples (5.98×10-3±8.95×10-4 ppm) which was significantly different from its lowest concentration found at D 50 °C (2.33× 10-3±3.79×10-4 ppm) and was not detected in F samples. Meanwhile 2-hexanone was only found in DBR 50 °C with a concentration of 7.04×10-3±6.34×10-4 ppm. Some of the compounds had their maximum concentrations in DBR 50 °C samples which were significantly different from the other treatments and include hexanal (2.37×10-2±1.70×10-3 ppm), 3-heptanone (1.18× 10-3±1.52×10-4 ppm), heptanal (1.58×10-3±4.84×10-4 ppm), benzaldehyde (2.33×10-2±4.55×10-3 ppm), 1-octen-3-one (6.66×10-3±6.09×10-4 ppm), 1-(2-furyl)-3-methyl-3-butene-1,2-diol (6.64×10-3±1.20×10-4 ppm). As for 1-hexanol, it was not found in F and BR samples but its highest concentration was found in D 50 °C samples (1.79×10-2±2.47×10-3 ppm), not significantly different from DBR 50 °C samples while 1-octen-3-ol had its maximum concentration in F samples (5.84×10-2±6.43×10-3 ppm) which was significantly different from the other treatments and with a minimum concentration of 5.90×10-3±2.80×10-4 in BR samples. Meanwhile 2,6- bis(1,1-dimethylethyl)phenol showed no significant difference between the different treatments.

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As for the remaining ketones, 1-octen-3-one, being not detected in F samples, had its maximum concentration in DBR 50 °C (6.66×10-3±6.09×10-4 ppm) and its lowest in D 50 °C samples (6.27×10-4±1.34×10-4 ppm) which was not significantly different from BR samples (7.91× 10-4±7.68×10-5 ppm). Meanwhile 6-10-dimethyl-5,9-undecadien-2-one had its highest concentration in D 50 °C samples (1.03×10-2±4.39×10-4 ppm) which was significantly different DBR 50 °C samples (1.23×10-3±4.51×10-4 ppm).

Other compounds identified include octanal which had its highest concentration in F samples (7.66×10-3±2.12×10-4 ppm) and its lowest in BR samples (7.11×10-4±3.07×10-5 ppm). This compound was not found in D 50 °C and DBR 50 °C samples. As for 2,2,4,6,6-pentamethyl-3- heptene, its lowest concentration was found in BR samples (2.16×10-3±2.70×10-4 ppm) which was significantly different from the other treatments while limonene had its highest concentration in F samples (4.94×10-2±4.66×10-3 ppm) which was significantly different from BR, D 50 °C and DBR 50 °C samples. Meanwhile, 1,3-di-tert butyl benzene had its highest concentration in BR samples (6.67×10-4±4.72×10-5 ppm) which was not significantly different from that found in D 50 °C (6.18×10-4±7.23×10-5 ppm). As for nonanal, its highest concentration was found in DBR 50 °C (6.36×10-3±1.21×10-3 ppm) which was not significantly different from BR and D 50 °C but significantly different from F samples (1.93×10-3±2.36×10-4 ppm).

As for dill ether, its highest concentration was found in BR samples (9.08×10-3±7.18×10-4 ppm) which was not significantly different from DBR 50 °C samples (8.71×10-3±1.22×10-3 ppm) with its lowest concentration found in F samples (2.35×10-3±4.79×10-4 ppm) and was undetected in D 50 °C samples. Meanwhile, dihydrocarveol had its maximum concentration in F samples (7.21×10-3±3.52×10-4 ppm) which was significantly different from BR (2.06×10-3±7.05×10-4 ppm) and DBR samples (2.15×10-3±5.02×10-4 ppm). Decanal had its highest concentration in BR samples (5.56×10-3±4.45×10-4 ppm) significantly different from F samples but not significantly different from D 50 °C and DBR 50 °C samples while 1,3-di-tert butylbenzene showed no significant difference between BR and D 50 °C samples but was not identified in DBR 50 °C. Other compounds identified only in DBR 50 °C samples include 2,6-dimethyl pyrazine (2.40 ×10-3±5.28×10-4 ppm), 1,2-cyclohexadione (5.03×10-4±6.15×10-5 ppm), 2,3,4,5-tetrahydropyridine (7.62×10-3±8.13×10-4 ppm), 2-ethyl-6-methylpyrazine (1.70×10-3±4.79×10-4 ppm), (E,E)- 2,4-heptadienal (2.32×10-3±3.25×10-4 ppm), 2-ethyl-1,4-dimethylbenzene (1.43×10-3±5.11×10-4 ppm), 2,5-dimethyl-3-ethylpyrazine (2.50×10-3±8.93×10-4 ppm) and camphor (3.12×10-3 ±5.21×10-4 ppm).

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Huang et al., (2004) found reported that 3-methyl butanal is derived from the amino acid leucine. According to Muriel et al. (2004) and Chen et al. (2000), thermal processing speeds up lipid oxidation and degradation of alkenals such as (E)-2-nonenal, alkadienals (2,4 hexadienal), alcohols (hexanol) and furans. Pyrazine compounds such as, 2,6-dimethylpyrazine, 2,3,4,5- tetrahydropyridine, 2-ethyl-6-methylpyrazine, 2,5-dimethyl-3-ethylpyrazine were detected when the dried mushrooms were boiled under reflux, but when the samples were freshly boiled under reflux, the former compounds were absent. MacLeod and Panchasara (1983), in their studies on the volatiles in freshly cooked and dried and cooked A. bisporus also reported the formation of pyrazines only in the dried and cooked product.

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Table 4.8: Comparison between concentrations of VC in enoki samples subjected to various treatments. Compound F BR D 50 °C DBR 50 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-propanone ND ND 2.99×10-3 a 5.46×10-4 ND ND 2.51×10-3 a 7.11×10-4

2-methyl furan ND ND ND ND ND ND 1.01×10-3 a 2.14×10-4

Ethyl acetate 1.41×10-3 b 1.52×10-4 1.27×10-3 b 1.31×10-4 5.07×10-3 a 1.14×10-3 3.86×10-3 a 7.31×10-4

3-methyl butanal 1.12×10-3 b 2.25×10-4 2.48×10-3 b 8.23×10-4 3.37×10-3 b 3.12×10-4 1.02×10-2 a 5.06×10-3

2-methyl butanal ND ND 2.19×10-3 b 1.08×10-4 3.76×10-3 b 3.97×10-4 1.29×10-2 a 3.12×10-4

3-methyl-1-butanol 1.20×10-3 b 2.05×10-4 6.82×10-4 b 4.78×10-5 6.15×10-2 a 1.06×10-2 8.39×10-3 b 6.15×10-4

2-methyl-1-butanol 5.59×10-3 b 2.57×10-4 5.70×10-4 b 9.97×10-5 3.62×10-2 a 6.53×10-3 ND ND

1-pentanol ND ND 4.12×10-3 b 1.29×10-4 2.33×10-3 b 3.79×10-4 5.98×10-3 a 8.95×10-4

2-hexanone ND ND ND ND ND ND 7.04×10-3 a 6.34×10-4

Hexanal 5.11×10-4 c 2.25×10-5 5.86×10-3 b 8.03×10-4 8.34×10-4 c 1.86×10-4 2.37×10-2 a 1.70×10-3

3-heptanone ND ND 7.34×10-4 b 6.19×10-5 ND ND 1.18×10-3 a 1.52×10-4

Heptanal ND ND 7.98×10-4 b 6.97×10-5 ND ND 1.58×10-3 a 4.84×10-4

1-hexanol ND ND ND ND 1.79×10-2 a 2.47×10-3 1.14×10-2 a 2.21×10-3

2,6-dimethylpyrazine ND ND ND ND ND ND 2.40×10-3 a 5.28×10-4

1H-indol-5-ol 3.95×10-2 a 1.10×10-3 3.58×10-2 a 3.96×10-3 2.46×10-2 ab 4.38×10-3 1.57×10-2 b 7.20×10-3

1,2-cyclohexadione ND ND ND ND ND ND 5.03×10-4 a 6.15×10-5

Benzaldehyde ND ND 4.10×10-3 b 6.07×10-4 1.74×10-3 b 5.26×10-4 2.33×10-2 a 4.55×10-3

1-octen-3-one ND ND 7.91×10-4 b 7.68×10-5 6.27×10-4 b 1.34×10-4 6.66×10-3 a 6.09×10-4

1-octen-3-ol 5.84×10-2 a 6.43×10-3 5.90×10-3 b 2.80×10-4 6.39×10-3 b 1.56×10-3 8.36×10-3 b 7.61×10-4

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Table 4.8 continued Compound F BR D 50 °C DBR 50 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

3-octanone 4.38×10-1 a 4.47×10-2 2.82×10-2 b 4.54×10-3 1.50×10-2 b 1.50×10-3 3.96×10-3 b 6.16×10-4

2,3,4,5-tetrahydropyridine ND ND ND ND ND ND 7.62×10-3 a 8.13×10-4

5-metyl-3-hexen-2-one ND ND 9.37×10-3 a 1.94×10-4 8.11×10-4 b 3.33×10-5 ND ND

2-ethyl-6-methylpyrazine ND ND ND ND ND ND 1.70×10-3 a 4.79×10-4

3-octanol 3.35×10-1 a 4.67×10-2 1.18×10-2 b 1.51×10-3 5.05×10-3 b 6.67×10-4 1.42×10-3 c 2.75×10-4

Octanal 7.66×10-3 a 2.12×10-4 7.11×10-4 b 3.07×10-5 ND ND ND ND

2,2,4,6,6-pentamethyl,3- heptene 5.36×10-2 a 4.21×10-2 2.16×10-3 c 2.70×10-4 7.71×10-3 bc 1.13×10-3 1.22×10-2 b 2.84×10-3

(E,E)-2,4-heptadienal ND ND ND ND ND ND 2.32×10-3 a 3.25×10-4

2-ethyl-1,4-dimethylbenzene ND ND ND ND ND ND 1.43×10-3 a 5.11×10-4

Limonene 4.94×10-2 a 4.66×10-3 1.44×10-3 b 3.93×10-4 1.57×10-3 b 1.25×10-4 2.59×10-3 b 8.65×10-5

1-(2-furyl)-3-methyl-3-butene- 1,2-diol ND ND ND ND 2.13×10-3 b 6.96×10-4 6.64×10-3 a 1.20×10-4

2-ethyl-1-hexanol 9.23×10-3 b 7.97×10-4 2.66×10-2 a 2.93×10-3 5.78×10-3 b 3.30×10-4 3.95×10-3 b 3.65×10-4

Phenylacetaldehyde ND ND 5.05×10-3 b 5.72×10-4 ND ND 4.20×10-2 a 1.45×10-3

(E)-2-octenal ND ND 1.15×10-3 a 1.01×10-4 ND ND ND ND

2-cyclohexen-1-ol ND ND 6.04×10-4 b 3.39×10-5 1.10×10-3 a 9.96×10-5 ND ND n-octanol ND ND 2.37×10-3 b 1.85×10-4 5.39×10-3 a 4.57×10-4 5.22×10-3 a 4.48×10-4

2,5-dimethyl-3-ethylpyrazine ND ND ND ND ND ND 2.50×10-3 a 8.93×10-4

Linalool ND ND 1.03×10-3 b 1.48×10-4 1.20×10-3 b 8.40×10-4 3.50×10-3 a 2.98×10-4

Nonanal 1.93×10-3 b 2.36×10-4 5.25×10-3 a 6.47×10-4 4.55×10-3 a 1.71×10-4 6.36×10-3 a 1.21×10-3

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Table 4.8 continued Compound F BR D 50 °C DBR 50 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Camphor ND ND ND ND ND ND 3.12×10-3 a 5.21×10-4

1-nonanol ND ND 7.28×10-4 c 2.08×10-5 2.07×10-2 a 1.66×10-3 5.08×10-3 b 8.44×10-4

Dill ether 2.35×10-3 b 4.79×10-4 9.08×10-3 a 7.18×10-4 ND ND 8.71×10-3 a 1.22×10-3

Dihydrocarveol 7.21×10-3 a 3.52×10-4 2.06×10-3 b 7.05×10-4 ND ND 2.15×10-3 b 5.02×10-4

Decanal 1.97×10-3 b 2.60×10-4 5.56×10-3 a 4.45×10-4 2.83×10-3 ab 1.77×10-3 2.95×10-3 a 1.62×10-4

1,3-Di-tert butylbenzene 5.25×10-4 b 8.53×10-5 6.67×10-4 a 4.72×10-5 6.18×10-4 a 7.23×10-5 ND ND

2-undecanone ND ND 7.41×10-4 b 4.23×10-5 3.30×10-3 a 1.16×10-4 2.88×10-3 a 7.29×10-4

6-10-dimethyl-5,9-undecadien- 2-one ND ND ND ND 1.03×10-2 a 4.39×10-4 1.23×10-3 b 4.51×10-4

2,6-bis(1,1- dimethylethyl)phenol 1.53×10-2 a 1.32×10-3 2.14×10-2 a 4.49×10-4 2.01×10-2 a 5.09×10-3 1.71×10-2 a 6.22×10-3

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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In order to compare the effects of the treatments on VC, the concentrations were normalised against the highest value. As shown in Figure 4.34, 1-octen-3-one was not detected in F samples and had its highest normalised relative concentration in DBR 50 °C which was significantly different from BR and D 50 °C samples while 1-octen-3-ol, 3-octanone, and 3-octanol had their highest normalised concentration in F samples which were significantly different from the other treatments. Meanwhile, n-octanol had its maximum normalised value in D 50 °C samples which was not significantly different from DBR 50 °C but significantly different from BR samples and was not found in F samples.

F BR 1.2 D 50 °C DBR 50 °C

1.0

0.8

0.6

0.4

0.2 Normlaisedrelativeconcentration 0.0

e l e l l on 3-o on no no -3- n- an cta cta en cte ct -o -o ct -o 3-o 3 n 1-o 1 Compound

Figure 4.34: Normalised relative concentrations of the main C8 compounds identified in enoki mushrooms subjected to different treatments.

The normalised relative concentrations of selected alcohol and ketones VC (group 1) are shown in Figure 4.35. The compound 1-nonanol had its maximum normalised value in D 50 °C samples which was significantly different from its lowest value found in BR samples but was not identified in F samples. As for 2-cyclohexen-1-ol, its normalised relative concentration showed a significant difference between BR and D 50 °C samples; however, it was not identified in F and DBR 50 °C samples, while 2-ethyl-1-hexanol had its highest normalised relative concentration in BR samples which was significantly different from the remaining treatments. Meanwhile, the normalised relative concentration of 2-undecanone showed no significant difference between D 50 °C and

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DBR 50 ° C and was absent in F samples. As for 5-methyl-3-hexen-2-one, it was not found in F or DBR 50 °C samples but had its highest normalised concentration in BR samples which was significantly different from D 50 °C samples. As for 6,10-dimethyl-5,9-undecadien-2-one, it had the highest normalised relative concentration in D 50 °C samples which was significantly different from that found in DBR 50 °C samples. However, it was not found in F and BR samples.

1.4

F 1.2 BR D 50 °C 1.0 DBR 50 °C

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration

0.0

l l l no -o no ne ne ne na -1 xa no 2-o 2-o no xen he eca n- n- 1- he 1- d xe die lo yl- -un -he ca cyc eth 2 l-3 de 2- 2- ety -un -m 5,9 5 yl- eth Compound im 0-d 6-1

Figure 4.35: Normalised relative concentrations of selected alcohols and ketones VC (group 1) identified in enoki mushrooms subjected to different treatments.

The normalised relative concentrations of selected VC (group 2) identified in enoki subjected to different treatments are shown in Figure 4.36. The compound 1H-indol-5-ol showed no significant difference between F, BR, samples and D samples but its normalised relative concentration significantly decreased in DBR samples. The compounds benzaldehyde, phenylacetaldehyde, linalool, and hexanal had their maximum normalised relative concentration in DBR 60 °C samples which were significantly different from the other treatments. Meanwhile, limonene had its highest normalised concentration in F samples which was significantly different from the other treatments. Dill ether (3,6-dimethyl-2,3,3a,4,5,7a-hexahydro-1-benzofuran) was not detected when the

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samples were dried and had its highest normalised relative concentration in BR samples which was not significantly different from DBR 50 °C samples.

1.4 F BR 1.2 D 50 °C DBR 50 °C 1.0

0.8

0.6

0.4

0.2 Normalised relative concentration 0.0

ol e r e ol al e 5- yd he en lo n yd l- eh et on a xa eh do d ll in e d n al Di im L H al -i nz L et H e ac 1 B yl Compound en Ph

Figure 4.36: Selected VC (group 2) extracted from enoki mushrooms subjected to different treatments.

According to Muriel et al. (2004) and Chen et al. (2000), thermal processing speeds up lipid oxidation and degradation of alkenals such as (E)-2-nonenal, alkadienals (2,4 hexadienal), alcohols (hexanol) and furans (section 4.2.5).

Pyrazines have been widely reported in the literature. Qian and Reineccius, (2002) studied aroma compounds in Parmigiano-Reggiano cheese were carried and 2,6-dimethylpyrazine was found to have a nutty or chocolate aroma characteristics with a “very strong” aroma intensity, while 2,5- dimethyl-3-ethyl-pyrazine had a roasted or baked aroma descriptor and the intensity of the aroma was referred to as “strong”. Fan and Qian (2006) described 2-ethyl-6-methylpyrazine as having a nutty or roasted aroma.

Food systems that have been found to contain pyrazine compounds include beef products (Liebich et al., 1972; Watanabe and Sato, 1971a,b), cocoa products (Van Praag et al., 1968), coffee (Bondarovich et al., 1967), peanuts (Waller et al., 1971) popcorn (Walradt et al., 1970), potato products (Buttery et al., 1970, 1971), rye crisp bread (Von Sydow and Anjou, 1969), soy products (Manley and Fagerson, 1970), tomatoes (Ryder, 1969), peas (Murray et al., 1970), green bell peppers (Buttery et al., 1969a,b), sesame oil (Takei et al., 1969), dairy products (Ferretti and

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Flanagan, 1972), roasted pecans (Wang and Odell, 1972), roasted filberts (Sheldon et al., 1972) and chicken broth (Wilson and Katz, 1972).

Most of these compounds have been found in foods that were toasted or subjected to an extended heat treatment. Several theories on pyrazine formation were proposed. Dawes and Edwards (1966) concluded that pyrazines in heated foods were the results of condensation reactions between sugars and amino acids. Hough et al., (1952) postulated that at lower temperatures and over a prolonged period of times, pyrazines can result from the reaction of sugars and amino acids to form ditetrahydroxybutylpyrazine intermediate through condensation followed by rearrangement and cleavage to form alkylpyrazines and that, at higher temperatures, intermediate rearrangements and cleavage of sugars results in the formation of hydroxycarbonyl which condense with the nitrogen (from amino acid) to yield alkylpyrazines. Mason et al., (1966) also reported the formation of pyrazine compounds in roasted peanuts and concluded that the former compounds resulted from the reaction between amino acids and sugars. Van Praag et al., (1968) discussed the role of ammonia in pyrazine formation and concluded that the former compound was an intermediate. Koehler et al. (1969) studied the variables affecting pyrazine formation in a sugar- amino acid model system and concluded that pyrazine yield began at 100 °C and the yield increased as the temperature increased until the temperature reached 150 °C were the yield fluctuated and postulated that higher temperatures resulted in the destruction of these compounds after being formed. Other factors studied by Koehler et al. (1969) affecting the pyrazine yield were the type of amino acids (certain amino acids reacted to yield more pyrazines than others), the source of carbon (glucose, fructose, sucrose, arabinose) which reacted with asparagine where fructose yielded more pyrazines due to its greater ability to fragment. Reineccius et al., (1972) studied pyrazine compounds in cocoa beans and concluded that factors affecting the former compounds formations were time, temperature and the ratio of sugar to amino acid where ratio other than 1:1 decreased the yield of pyrazine formation. In the experiments with Flammulina velutipes in the current study, the work carried by Kim et al. (2009) showed that enoki contained asparagine among other amino acids and 5 sugar types (ribose, xylose, mannose, glucose, and trehalose) with fructose being absent. Furan compounds were also formed as a result of the Maillard reaction due to the thermal processing (Golovnya and Misharina, 1994).

As for benzaldehyde, and phenylacetaldehyde, possible explanation for their formation was discussed in chestnut mushrooms (section 4.1.5).

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The results of this study showed that some compounds were only identified when the samples were freshly boiled under reflux (BR) or when dried and boiled under reflux (DBR) which suggest that these compounds required heated or boiled water for their release and hence detection and identification. These compounds include 2-propanone, 2-methyl furan, 2-hexanone, 3-heptanone, heptanal, 2,6-dimethyl pyrazine, 2,3,4,5-tetrahydropyridine, 2-ethyl-6-methyl pyrazine, (E,E)-2,4-heptadienal, 2-ethyl-1,4-dimethylbenzene, phenylacetaldehyde, (E)-2-octenal, 2,5-dimethyl-3-ethyl pyrazine, and camphor. Furthermore, the rehydration temperature plays an important role in the release of VC as suggested by Martinez-Soto et al. (2001) who found the rehydration of mushrooms was much faster at 94 °C compared to 25 °C. Other compounds were not found in F samples but with thermal processing, these compounds contributed to the overall aroma of enoki samples and include 2-propanone, 2-methyl furan, 2-methyl butanal, 1-pentanol, 2-hexanone, 3-heptanone, Heptanal, 1-hexanol, 2,6-dimethyl pyrazine, 1,2-cyclohexadione, benzaldehyde, 1-octen-3-one, 2,3,4,5-tetrahydropyridine, 5-methyl-3-hexen-2-one, 2-ethyl-6- methyl pyrazine, (E,E)-2,4-heptadienal, 2-ethyl-1,4-dimethylbenzene, 1-(2-furyl)-3-methyl-3- butene-1,2-diol, phenylacetaldehyde, (E)-2-octenal, 2-cyclohexen-1-ol, n-octanol, 2,5-dimethyl,3- ethylpyrazine, linalool, camphor, 1-nonanol, 2-undecanone, and 6-10-dimethyl-5,9-undecadien-2- one.

The pyrazine compounds 2,6-dimethyl pyrazine and 2,5-dimethyl-3-ethylpyrazine were previously reported by Misharina et al. (2009b) in dried cepe mushrooms.

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4.3 VC in Pleurotus ostreatus (Oyster mushrooms)

4.3.1 Fresh and stored oyster mushrooms

VC were extracted and identified from fresh/raw oyster mushrooms stored over 10 days period (see section 3.1.2) and their concentrations were calculated and normalised against D0. The concentrations of VC identified during the storage study are shown in Table 4.9. It appears that 3- octanol was the major VC determined throughout the storage period. At D0, it had a concentration of 4.57×100±3.67×10-1 ppm followed by 1-octen-3-ol (2.16×100±2.92×10-1 ppm), 3-octanone (2.05×100±1.75×10-1 ppm), n-octanol (4.56×10-1±2.91×10-2 ppm), 2-octen-1-ol (2.21×10-1 ±2.72×10-2 ppm), 1-octen-3-one (4.30×10-2±3.39×10-3 ppm), ethyl octanoate (3.32×10-2±5.55×10-3 ppm), and octanal (2.77×10-2±3.79×10-3 ppm). At D1, 3-octanol was also the major VC detected but with a lower concentration (3.94×100±2.74×10-1 ppm) followed by 3-octanone (1.76×100±1.97×10-1 ppm), 1-octen-3-ol (1.23×100±1.59×10-1 ppm), and n-octanol (4.19×10-1 ±1.28×10-2 ppm). At D3, the concentration of 3-octanol (2.89×100±5.69×10-1 ppm) and 3- octanone (1.07×100±4.76×10-2 ppm) decreased significantly and further storage significantly reduced their respective concentrations compared to D0. No significant changes were observed for n-octanol between D0 and D2, but at D3, its concentration decreased almost by half compared to D0 (2.84×10-1±2.35×10-2 vs. 4.56×10-1±2.91×10-2 ppm). Further significant decrease was observed at D10 (1.67×10-1±1.88×10-2 ppm). As for 1-hexanol, its concentration fluctuated between D0 and D3, then at D4, its concentration decreased almost by half compared to D0 (2.55×10-3±1.22×10-4 vs. 4.49×10-3±6.08×10-4 ppm) with no significant changes occurred between D4 and D7 but the concentration significantly decreased at D10 (1.02×10-3±1.02×10-4 ppm). The concentration of 2-ethyl-1-hexanol was significantly increasing up until D2 with the lowest concentration found was at D0 (3.19×10-3±1.84×10-4 ppm) and the highest at D7 (1.83×10-1 ±2.44×10-2 ppm). As for 3-methyl-1-butanol, its concentration was increasing with time with a minimum of (1.82×10-3±1.08×10-4 ppm) at D0 followed by a significant increase at D7 (7.94×10-3 ±6.46×10-4 ppm), and D10 (1.84×10-2±3.54×10-3 ppm). The concentration of 2-methyl-1-butanol was fluctuating with time with its lowest concentration found at D1 (2.05×10-3±1.80×10-4 ppm) and a maximum concentration of 2.56×10-2±2.80×10-3 ppm at D10.

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Table 4.9: Concentrations of VC extracted from oyster mushrooms during 10 days storage period. Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD ppm SD ppm SD ppm SD ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

3-methyl 5.42×10-3 a 5.89×10-4 5.15×10-3 ab 2.25×10-4 4.78×10-3 abc 2.21×10-4 4.62×10-3 abc 2.75×10-4 4.10×10-3 bcd 6.20×10-4 3.79×10-3 cd 5.71×10-4 3.22×10-3 d 4.90×10-4 butanal

2-methyl 1.61×10-3 cd 1.85×10-4 9.01×10-4 d 4.15×10-5 2.39×10-3 bc 2.87×10-4 2.34×10-3 bc 4.38×10-4 2.18×10-3 bc 3.25×10-4 3.16×10-3 b 4.38×10-4 4.33×10-3 a 5.65×10-4 butanal 1,1-diethoxy 3.90×10-3 d 3.15×10-4 5.16×10-3 cd 5.33×10-5 6.49×10-3 cd 4.42×10-4 8.54×10-3 cd 3.79×10-4 1.00×10-2 c 9.74×10-4 1.64×10-2 b 3.43×10-3 3.00×10-2 a 4.06×10-3 ethane 3-methyl-1- 1.82×10-3 c 1.08×10-4 2.20×10-3 c 3.50×10-4 2.54×10-3 c 1.93×10-4 2.68×10-3 c 2.47×10-4 4.57×10-3 bc 5.80×10-4 7.94×10-3 b 6.46×10-4 1.84×10-2 a 3.54×10-3 butanol 2-methyl-1- 5.67×10-3 c 3.23×10-4 2.05×10-3 d 1.80×10-4 3.02×10-3 cd 1.31×10-4 4.01×10-3 cd 1.40×10-4 4.48×10-3 cd 6.38×10-4 1.61×10-2 b 1.22×10-3 2.56×10-2 a 2.80×10-3 butanol Hexanal 1.50×10-3 f 4.02×10-4 2.47×10-3 e 1.58×10-4 2.77×10-3 de 2.17×10-4 3.47×10-3 cd 3.41×10-4 4.30×10-3 c 2.49×10-4 5.63×10-3 b 1.69×10-4 7.27×10-3 a 6.38×10-4 butanoic acid, 3-methyl- 1.65×10-3 e 4.67×10-4 3.63×10-3 d 4.49×10-4 4.42×10-3 d 3.06×10-4 6.14×10-3 c 2.32×10-4 6.55×10-3 c 2.51×10-4 7.93×10-3 b 4.95×10-4 9.76×10-3 a 4.97×10-4 ,ethyl ester 1-hexanol 4.49×10-3 a 6.08×10-4 4.18×10-3 a 2.24×10-4 4.31×10-3 a 7.77×10-5 4.67×10-3 a 9.78×10-4 2.55×10-3 b 1.21×10-4 2.49×10-3 b 1.54×10-4 1.02×10-3 c 1.02×10-4 3-heptanone 1.01×10-3 b 9.14×10-5 3.46×10-4 b 8.27×10-5 8.03×10-4 b 9.22×10-5 1.08×10-3 b 1.94×10-4 6.49×10-4 b 3.64×10-5 7.03×10-4 b 7.15×10-5 1.79×10-2 a 1.52×10-3 Benzaldehyde 2.60×10-3 e 6.42×10-4 1.91×10-2 d 1.06×10-3 2.42×10-2 cd 7.83×10-4 3.00×10-2 bc 4.60×10-3 3.37×10-2 ab 3.51×10-3 3.90×10-2 a 1.69×10-3 3.96×10-2 a 2.67×10-3 1-octen-3-one 4.30×10-2 a 3.39×10-3 3.88×10-2 a 3.64×10-3 3.12×10-2 b 1.69×10-3 2.93×10-2 b 3.45×10-3 2.79×10-2 b 2.08×10-3 1.85×10-2 c 1.38×10-3 1.32×10-2 c 1.69×10-3 1-octen-3-ol 2.16×100 a 2.92×10-1 1.23×100 b 1.59×10-1 6.99×10-1 c 6.97×10-2 4.23×10-1 cd 3.88×10-2 1.16×10-1 d 1.41×10-2 7.74×10-2 de 5.07×10-3 4.59×10-2 e 4.78×10-3 3-octanone 2.05×100 a 1.75×10-1 1.76×100 a 1.97×10-1 1.34×100 b 2.41×10-1 1.07×100 bc 4.76×10-2 8.04×10-1 cd 9.44×10-2 5.76×10-1 d 8.27×10-2 3.94×10-1 d 5.88×10-2 3-octanol 4.57×100 a 3.67×10-1 3.94×100 ab 2.74×10-1 3.32×100 bc 2.43×10-1 2.89×100 cd 5.69×10-1 2.25×100 de 2.90×10-1 2.10×100 de 2.46×10-1 1.38×100 e 2.38×10-1 n-octanal 2.77×10-2 a 3.79×10-3 2.45×10-2 ab 2.33×10-3 2.07×10-2 bc 2.13×10-3 1.85×10-2 bcd 3.02×10-3 1.63×10-2 cd 1.59×10-3 1.53×10-2 cd 1.98×10-3 1.37×10-2 d 1.17×10-3 2-ethyl-1- 3.19×10-3 b 1.84×10-4 4.21×10-2 b 7.01×10-3 1.33×10-1 a 6.17×10-3 1.73×10-1 a 2.05×10-2 1.78×10-1 a 1.48×10-2 1.83×10-1 a 2.44×10-2 1.60×10-1 a 5.21×10-2 hexanol (E)-2-octenal 2.63×10-3 d 2.86×10-4 3.88×10-3 d 3.56×10-4 5.79×10-3 c 3.49×10-4 6.62×10-3 c 3.61×10-4 7.48×10-3 c 4.72×10-4 1.01×10-2 b 1.08×10-3 1.28×10-2 a 9.31×10-4 2-octen-1-ol 2.21×10-1 a 2.72×10-2 7.26×10-2 b 1.08×10-2 6.78×10-2 bc 2.98×10-3 4.07×10-2 bcd 7.13×10-3 3.67×10-2 cd 1.63×10-3 2.95×10-2 d 1.95×10-3 1.89×10-2 d 2.79×10-3 n-octanol 4.56×10-1 a 2.91×10-2 4.19×10-1 a 1.28×10-2 3.93×10-1 a 2.80×10-2 2.84×10-1 b 2.35×10-2 2.54×10-1 bc 2.69×10-2 2.05×10-1 cd 1.79×10-2 1.67×10-1 d 1.88×10-2 Nonanal 3.44×10-3 c 2.21×10-4 4.13×10-3 c 1.82×10-4 4.30×10-3 c 1.64×10-4 4.92×10-3 bc 2.20×10-4 5.38×10-3 bc 4.16×10-4 6.35×10-3 b 3.06×10-4 1.10×10-2 a 1.82×10-3

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Table 4.9 continued Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD ppm SD ppm SD ppm SD ppm SD ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) 2- ethylhexanoic 3.12×10-4 c 1.93×10-5 3.29×10-4 c 1.03×10-5 3.50×10-4 c 3.18×10-5 3.52×10-4 c 4.36×10-5 3.88×10-4 c 3.93×10-5 7.61×10-4 b 9.48×10-5 9.51×10-4 a 3.50×10-5 acid (E)-2-nonenal 2.59×10-3 c 3.13×10-4 7.97×10-3 b 8.10×10-4 1.13×10-2 b 1.17×10-3 1.17×10-2 b 1.20×10-3 1.19×10-2 ab 7.09×10-4 1.22×10-2 ab 6.96×10-4 1.62×10-2 a 2.00×10-3 Ethyl 3.32×10-2 a 5.55×10-3 2.68×10-2 ab 3.42×10-3 2.22×10-2 bc 2.66×10-3 1.76×10-2 cd 2.66×10-3 1.44×10-2 cde 1.67×10-3 1.08×10-2 de 1.25×10-3 8.96×10-3 e 9.70×10-4 octanoate Decanal 1.73×10-3 e 1.84×10-4 1.77×10-3 e 1.39×10-4 2.15×10-3 de 2.89×10-4 2.73×10-3 d 1.50×10-4 3.41×10-3 c 1.80×10-4 4.59×10-3 b 2.56×10-4 5.25×10-3 a 3.56×10-4 2,6-bis(1,1- dimethylethyl) 1.65×10-3 b 3.74×10-4 1.63×10-3 b 4.01×10-4 1.76×10-2 a 5.37×10-3 1.89×10-2 a 2.36×10-3 1.81×10-2 a 5.46×10-3 1.65×10-2 a 2.13×10-3 1.34×10-2 ab 8.62×10-3 phenol. * Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The concentration of ketones VC significantly decreased with storage. For instance, the concentration of 1-octen-3-one decreased with storage time from 4.30×10-2± 3.39×10-3 ppm at D0 to 3.12×10-2± 1.69×10-3 ppm at D2 reaching a minimum of 1.32×10-2±1.69×10-3 ppm at D10. No significant difference was found between D2 and D3, and D4. As for 3-heptanone, its concentration decreased from D0 to D1 (1.01×10-3±9.15×10-5 vs. 3.46×10-4±8.27×10-5 ppm) and D2 (8.03×10-4±9.22×10-5 ppm) but a significant increase was observed at D10 (1.79×10-2 ±1.52×10-3 ppm).

The concentration of the saturated alcohols was decreasing with increasing storage time. The compound 1-octen-3-ol had the highest concentration at D0 (2.16×10-0±2.92×10-1 ppm) which significantly decreased at D1 to 1.23×100±1.59×10-1 ppm and D2 to 6.99×10-1±6.97×10-2 ppm then reaching a minimum of 4.59×10-2±4.78×10-3 ppm at D10 which was significantly different from D0, D1, D2, D3, and D4. As for 2-octen-1-ol, its concentration significantly decreased from D0 to D1 (2.21×10-1±2.72×10-2 vs. 7.26×10-2±1.08×10-2 ppm) then significantly decreased at D4 (3.67×10-2±1.63×10-3 ppm) which was not significantly different from D3 and reached a minimum of 1.89×10-2±2.79×10-3 ppm at D10.

Aldehydes VC behaved differently in comparison to each other. For instance, the concentration of benzaldehyde increased significantly from D0 (2.60×10-3±6.42×10-4 ppm) to D1 (1.91×10-2± 1.06×10-3 ppm), and kept increasing until it reached 3.96×10-2±2.67×10-3 at D10. No significant difference was found between D4, D7 and D10. In contrast, the concentration of 3-methyl butanal was decreasing with storage with the highest amount found was at D0 (5.42×10-3±5.89×10-4 ppm) and the lowest at D10 (3.22×10-3±4.90×10-4 ppm) with no significant changes occurring between D4, D7 and D10 while the concentration of 2-methyl butanal was changing in the opposite direction with an initial concentration of 1.61×10-3±1.85×10-4 ppm at D0 then increased significantly at D7 (3.16×10-3±4.38×10-4 ppm) and reached a a maximum of 4.33×10-3±5.65×10-4 ppm at D10. Hexanal which is characterised by a cut-grass like aroma (Cho et al., 2007) had its concentration increasing with time with its minimum being at D0 (1.50×10-3±4.02×10-4 ppm) then it increased significantly at D3 (3.47×10-3±3.41×10-4 ppm) and kept increasing significantly compared to D0 until it reached a maximum of 7.27×10-3±6.38×10-4 ppm at D10. The concentration of octanal was slightly decreasing with time with the highest value found at D0 (2.77×10-2±3.80×10-3 ppm) and decreased almost by half at D10 (1.37×10-2±1.17×10-3 ppm). As for nonanal, its concentration was increasing with time having the lowest value at D0 (3.44×10-3 ±2.21×10-4 ppm) then increased to 5.38×10-3±4.16×10-4 ppm at D4 reaching its maximum concentration at D10 significantly higher compared to the other days (1.10×10-2±1.82×10-3 ppm).

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As for decanal, its concentration was increasing slowly with time but at D4, it significantly increased to reach 3.41×10-3±1.80×10-4 ppm and further storage significantly increased its concentration to reach 5.25×10-3±3.56×10-4 ppm at D10. The saturated aldehydes (E)-2-octenal and (E)-2-nonenal behaved similarly where their concentrations were increasing with time reaching a maximum amount at D10 with 1.28×10-2±9.31×10-4 ppm for (E)-2-octenal and 1.62×10-2±2.00×10-3 ppm for (E)-2-nonenal. No significant difference was found between D1, D2, D3, D4, and D7 and between D7 and D10 for (E) 2-nonenal but a significant change occurred between D7 and D10 for (E)-2-octenal.

The concentrations of esters and carboxylic acids were also changing during storage. The concentration of butanoic acid, 3-methyl-ethyl ester was almost doubled between D0 and D1 (1.65×10-3±4.67×10-4 vs. 3.63×10-3±4.49×10-4 ppm) and significantly increased with the storage time reaching 6.14×10-3±2.32×10-4 ppm at D3, 7.93×10-3±4.95×10-4 ppm at D7 and 9.76×10-3 ±4.97×10-4 ppm at D10. This compound was also detected in pine mushrooms study conducted by Cho et al., (2007) and having a pleasant odour note (sweet and floral). As for 2-ethylhexanoic acid, its concentration significantly increased from 3.12×10-4±1.93×10-5 ppm at D0 and to 7.61×10-4±9.48×10-5 ppm at D7, and to 9.51×10-4±3.50×10-5 ppm at D10. A significant decrease was found for ethyl octanoate at D3 compared to D0 (1.76×10-2±2.66×10-3 vs. 3.32×10-2 ±5.55×10-3 ppm) reaching a minimum concentration of 8.96×10-3±9.70×10-4 ppm at D10.

The concentration of 1,1-diethoxy ethane was increasing with storage with a minimum of 3.90×10-3±3.15×10-4 at D0 and a maximum of 3.00×10-2±4.06×10-3 ppm at D10. As for 2,6- bis(1,1-dimethylethyl)phenol, its concentration was slightly increasing with time with a minimum of 1.63×10-3±4.01×10-4 ppm at D1 and a maximum of 1.89×10-2±2.36×10-3 at D3.

These results suggest that at D7, the quality of oyster mushrooms decreased compared to D0 but the mushrooms still contained a considerable amount of VC contributing to the overall aroma of fresh oyster mushrooms.

The normalised relative concentrations of selected VC (group 1) are shown in Figure 4.37. The compound 3-heptanone did not show any major fluctuations from D0 to D7. However, at D10, a significant increase was observed while for benzaldehyde a significant increase appears when comparing D0 to the rest of the storage days increasing almost 7 times from D0 to D1 and 9.3 times from D0 to D2. No major fluctuations were seen between D2 and D3, D3 and D4, D4 and D7, D4, D7 and D10. The normalised relative concentration of 2-ethyl 1-hexanol increased 13 times from D0 to D1 and almost 3 times from D1 to D2. No significant change was observed after

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D2. As for 2,6-bis(1,1-dimethylethyl)phenol, it showed no significant difference between D0 and D1 but a exhibited a significant increase at D2 with no significant change afterwards.

2-ethyl-1-hexanol Benzaldehyde 2,6-bis(1,1-dimethylethyl) phenol 3-Heptanone 7000

6000

5000

4000

3000

2000

1000

Normalisedrelative concentration 0

0 2 4 6 8 10 Day

Figure 4.37: Normalised relative concentrations of selected VC (group 1) extracted from oyster mushrooms during 10 days storage period.

The compound 2-ethyl-1-hexanol appeared to be the major VC in fresh and stored oyster mushrooms. Possible explanations are that chemical and metabolic reactions occurring during postharvest resulted in such finding and nature variability of this compound with time.

The normalised relative concentration of selected alcohols and ketones (group 2) during storage are shown in Figure 4.38. The normalised relative concentration of 1-octen-3-ol, 3-octanone, 3- octanol, 2-octen-1-ol, n-octanol, and 1-octen-3-one decreased with the storage time. As for 1- octen-3-ol, it showed a significant decrease between D0 and D4 then no major fluctuations were observed between D4 and D7, then at D10, the normalised relative concentration was significantly decreased compared to D4. Meanwhile, 2-octen-1-ol showed a significant decrease between D0 and D1 with no major changes occurring between D1, D2 and D3 and no major decrease occurred from D3 onwards. The levels of 3-octanone and 3-octanol were decreasing throughout the storage with a significant decrease between D0 and D2, D0 and D3, and D0 and D4. No significant changes occurred from D4 onwards. As for n-octanol, no significant changes occurred between D0 and D2, but at D3, the normalised relative concentration significantly decreased followed by a further decrease at D7. Meanwhile, the normalised relative concentration of 1-octen-3-one

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significantly decreased from D0 to D2 with no significant change between D2, D3, and D4. A significant decrease occurred at D7 which was not significantly different from D10.

1-octen-3-ol 3-octanone 3-octanol 140 2-octen-1-ol n-octanol 120 1-octen-3-one

100

0 Normalised relative concentration relative Normalised

0 2 4 6 8 10 Day

Figure 4.38: Normalised relative concentrations of selected VC (group 2) extracted from oyster mushrooms during 10 days storage period.

The normalised relative concentrations of selected VC (group 3) are shown in (Figure 4.39). The normalised relative concentration of 1,1-diethoxy ethane (acetal) and butanoic acid, 3-methyl- ethyl ester were increasing throughout the storage trial with a significant increase observed at D10 almost 7 times higher than the original amount at D0 for 1,1-diethoxy ethane and almost 6 times higher than D0 for butanoic acid, 3-methyl-,ethyl ester. No major changes were observed for ethyl octanoate from D4 onwards while the normalised relative concentration of 2-ethylhexanoic acid was increasing with the storage time especially between D7 and D10.

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1,1-diethoxy ethane butanoic acid, 3-methyl-,ethyl ester Ethyl octanoate 2-ethylhexanoic acid 900

800

700

600

500

400

300

200

Normalised relative concentration 100

0 0 2 4 6 8 10 Day

Figure 4.39: Normalised relative concentrations of selected VC (group 3) extracted from oyster mushrooms during 10 days storage.

The normalised relative concentrations of selected aldehydes (group 4) are shown in Figure 4.40. Hexanal content was increasing and at D4, its normalised relative concentration was 2.8 times higher than that at D0 whereas at D10, it was 4.8 times higher than that at D0. The normalised relative concentration of (E)-2-octenal was also increasing with a significant increase at D2, D7 and D10. Nonanal was also changing with the storage time, increasing almost 3 times at D10 compared to D0 while decanal amount showed a small increase in the normalised relative concentration between D0 and D2 with an increase of 2.6 times at D7 compared to D0. The normalised relative concentration of 3-methyl butanal was the highest at D0 then it decreased significantly at D4. Further storage did not significantly decrease its normalised relative concentration. As for octanal, its level decreased significantly at D2 then at D10. Meanwhile, the normalised concentration of (E)-2-nonenal significantly increased at D1 followed by small increases until a significant increase was observed at D10.

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3-methyl butanal Hexanal Octanal (E)-2-octenal 700 Nonanal (E)-2-nonenal Decanal 600

500

400

300

200

100 Normalisedrelative concentration 0 0 2 4 6 8 10 Day

Figure 4.40: Normalised relative concentrations of selected aldehydes VC (group 4) extracted from oyster mushrooms during 10 days storage period.

The compounds 3-octanone, 3-octanol, 1-octen-3-ol and benzaldehyde were previously identified by Beltran-Garcia and his colleagues (1997) who studied VC extracted from oyster mushrooms and their antibacterial activities. They also found that benzaldehyde in the vacuum distillate and explained its formation as a “response to stress” suffered during vacuum with the degree of stress related to the amount of this compound. In contrast to the data reported by Nyegue et al. (2003) who reported 1-octen-3-one as the major VC in P. ostreatus, the major VC found by Tsai et al. (2009) was 1-octen-3-ol with octanal and (E)-2-octenal compounds being undetected. As for Kabbaj et al. (2002) who studied VC production in oyster mushrooms, concluded that 3-octanone was the major VC and was responsible for the fruity-lemon like odour of P. ostreatus followed by 3-octanol which had a sweet herbaceous odour then 1-octen-3-ol (mushroom alcohol) while 1- octen-3-one, 2-octen-1-ol, and n-octanol were absent in the fruiting body of the oyster mushrooms. Hong et al. (1986) and Jung and Hong (1991) studied VC in Korean P. ostreatus and found 1-octen-3-ol as the main C8 volatile followed by 1-octen-3-one and 3-octanol. However, in the present study, 3-octanol was the major VC identified followed by 3-octanone and 1-octen-3-ol.

Zhang et al. (2008) found that the content of 1-octen-3-ol and 3-octanone remained at nearly the same levels during maturity while Kabbaj et al. (2002) reported that the content of 1-octen-3-ol was increasing while 2-methylbutanal and 2-methyl-1-butanol decreased during three days storage of the oyster mushrooms which contradicted the results in this study which showed that as the mushrooms were aging, their C8 content decreased while 2-methyl butanal and 2-methyl-1-

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butanol content increased. Possible explanation for the increase or decrease in the amount of VC is discussed in section 4.2.1

Kabbaj et al. (2002) also studied VC of P. ostreatus mycelium grown on solid support, agar surface, and liquid medium and concluded that the aroma produced by the mycelium is directly related to the type of culture with the most similar VC with the fruit bodies are those grown on sugarcane bagasse impregnated with nutritive solution. Furthermore, he explained the differences between the aromatic spectra of the fruiting bodies and those of the mycelia grown on agar surface or liquid culture could be attributed to the mode of growth where in liquid culture, mycelial growth involved pellet formation and growth under stress caused by the low concentration of the dissolved oxygen while on agar surface and solid support, the growth is apical and similar to that in the fruiting bodies.

The compound 1-octen-3-ol has been found in several non-fungal sources such as black currants (Anderson and Von Sydow, 1964), cranberries (Anjou and Von Sydow 1967), and potatoes (Nursten and Sheen 1974; Dijkstra 1976). However, in dairy products, 1-octen-3-ol was identified as a mushroom off-flavour resulting from lipid oxidation (Stark and Forss 1962).

A possible explanation for the formation of 3-methyl butanal and 2-methyl butanal is by enzymatic transamination or oxidative deamination of the amino acids leucine and isoleucine as discussed in section 4.1.5. Kubo et al. (1993) found octanol as a flavour compound in mate tea which also had inhibitory effect on the proliferation of many organisms while Wang and Kays (2000) reported that benzaldehyde which has a strong nutty aroma may be originated from phenylalanine via Strecker degradation.

The obvious discrepancies in the described main volatiles of fruiting bodies between the different studies conducted might be attributed to specific strains, culture conditons as well as the extraction techniques.

4.3.2 Drying curves

The changes in moisture ratio of oyster mushrooms over time as a function of drying temperature are presented in Figure 4.37. As seen in this figure, all lines have two stages. The moisture ratio is rapidly reduced and then slowly decreasing with increasing drying time. It clearly appears that the drying temperature affects the total drying time. The rate of moisture loss is higher at higher temperatures (70 °C) and the total drying time is reduced with the increase in air temperature

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probably due to the fact that higher temperature of air had increased the temperature of the product, which accelerated the movement of moisture from the samples. The mushrooms were allowed to dry until they reached a constant weight. At 40 °C, the mushrooms were dried for 18 h and the final MC reached was 8.37%. At 50 °C, the drying time was 15 h and the final MC was 3.88% while at 60 °C, the mushrooms were dried for 11 h with a final MC of 1.91% and at 70 °C, the mushrooms were dried for 9 h reaching a final MC of 2.91%.

1.00 0.90 40 °C 50 °C 0.80 60 °C 0.70 70 °C 0.60 0.50 0.40 0.30 0.20

Moisture (MR) ratio Moisture 0.10 0.00 0 200 400 600 800 1000 1200 Time (min)

Figure 4.41: Moisture ratios of oyster mushrooms at different drying temperatures versus time.

4.3.3 Dried (D) oyster mushrooms

The concentrations of VC identified in oyster mushroom subjected to different drying treatments are shown in Table 4.10. In terms of concentration, 3-octanol was the major VC found at 40 °C (2.75×100±3.13×10-1 ppm) and 50 °C (2.86×100±4.67×10-1 ppm) followed by 3-octanone (1.92×100±1.25×10-1 ppm and 1.90×100±3.99×10-2 ppm respectively). At 60 and 70 °C it was the opposite where 3-octanone was the major VC with a significant increased concentration at 60 °C (2.35×100±2.91×10-1 ppm) and 70 °C (3.15×100±6.64×10-2 ppm) followed by 3-octanol with its concentration significantly decreased at 60 ° C (1.75×100±3.16×10-1ppm) and 70 °C (1.37×100± 3.05×10-1 ppm). As for 1-octen-3-ol, it had the highest concentration at 50 °C (6.43×10-1±5.16 ×10-2 ppm) which was significantly different from 60 °C (5.59×10-3±4.25 x10-4 ppm) but not

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significantly different from 70 °C (5.68×10-1±4.83×10-2 ppm). As for n-octanol, increasing the temperature decreased its concentration which peaked at 40 °C (2.56×10-1±4.29×10-2 ppm) then significantly decreased to reach a minimum of 7.96×10-3±7.12 ×10-4 ppm at 70 °C. Meanwhile, 2- octen-1-ol had the maximum concentration at 40 °C (2.30×10-2±5.56×10-3 ppm) which was significantly different from the lower concentration found in 60 °C (5.15×10-3±1.09×10-3 ppm) and 70 °C samples (8.08×10-3±3.08×10-4 ppm). Meanwhile, 2-ethyl-1-hexanol had the highest concentration at 50 °C (2.09×10-2±2.37×10-3 ppm) which was significantly different from the values determined for other drying temperatures and the lowest at 40 °C (3.93×10-3±5.52×10-4 ppm). No significant difference was found between 60 °C and 70 °C for 3-methyl-1-butanol (1.40×10-2±3.40×10-3 vs. 1.31×10-2±2.21 ×10-3 ppm) with the lowest concentration found was at 40 °C (2.20×10-3±5.36×10-5 ppm). Meanwhile, 2-undecanone had its highest concentration at 70 °C (2.42×10-2±1.45×10-3 ppm) being significantly different from that found at 40, 50, and 60 °C.

As for the aldehydes, octanal was the major VC at 40 °C (3.41×10-2±4.91×10-3 ppm) and 70 °C (4.22×10-2±2.34×10-3 ppm) followed by (E)-2-nonenal (3.22×10-2±8.78×10-3 ppm). At 50 °C, (E)- 2-nonenal was the major VC (4.03×10-2±1.34×10-3 ppm) being significantly different from that determined at the other drying temperatures followed by hexanal (3.36×10-2±2.22×10-3 ppm) and octanal (1.70×10-2±8.21×10-4 ppm). The compounds 3-methyl butanal and 2-methyl butanal both had their maximum concentrations at 50 °C (4.21×10-3±4.61×10-5 ppm and 5.88×10-3±5.58×10-4 ppm respectively) which were significantly different from 40 and 60 °C but not significantly different from 70 °C. Meanwhile, (E,E)-2,4-hexadienal had its highest concentration at 70 °C (1.32×10-2±3.42×10-3 ppm) which was significantly different from 40, 50, and 60 °C. As for hexanal, its maximum concentration was found at 60 °C (3.67×10-2±1.25×10-3 ppm) which was not significantly different from 50 °C but significantly different from that found at 40 and 70 °C. Phenylacetaldehyde had its maximum concentration at 70 °C (4.30×10-3±7.89×10-5 ppm) significantly different from 40, 50, and 60 °C with the lowest concentration reaching 8.21×10-4 ±9.55×10-5 ppm at 40 °C. As for (E)-2-octenal, it had its highest concentration at 70 °C (8.08× 10-3±3.08×10-4 ppm) which was significantly different from the lowest concentration found at 60 °C (1.68×10-3±4.35×10-4 ppm). Drying at 40 °C followed by rehydration at 25 °C resulted in the release of the minimum amount of benzaldehyde (1.95×10-3±1.70×10-4 ppm), nonanal (2.58×10-3±3.82×10-5 ppm), and decanal (1.68×10-3±3.07×10-4 ppm).

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Table 4.10: Concentrations of VC extracted from oyster mushrooms dried at different temperatures. Compound D 40 °C D 50 °C D 60 °C D 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

3-methyl butanal 1.17×10-3 c 5.35×10-5 4.21×10-3 a 4.61×10-5 2.90×10-3 b 7.44×10-4 4.04×10-3 ab 6.74×10-4

2-methyl butanal 5.36×10-4 c 9.82×10-5 5.88×10-3 a 5.58×10-4 3.07×10-3 b 4.88×10-4 4.18×10-3 ab 1.12×10-3

(E,E)-2,4-hexadienal 4.82×10-4 b 8.66×10-5 7.88×10-4 b 4.34×10-5 3.12×10-3 b 3.43×10-4 1.32×10-2 a 3.42×10-3

3-methyl-1-butanol 2.20×10-3 b 5.36×10-5 1.36×10-2 a 6.46×10-3 1.40×10-2 a 3.40×10-3 1.31×10-2 a 2.21×10-3

Hexanal 1.23×10-3 c 1.88×10-4 3.36×10-2 a 2.22×10-3 3.67×10-2 a 1.25×10-3 2.78×10-2 b 6.65×10-4

2,3,5-trimethylfuran ND ND ND ND 1.62×10-3 b 3.40×10-5 4.65×10-3 a 4.99×10-4

4-methyl-3-penten-2-one ND ND ND ND 2.47×10-3 a 1.20×10-4 2.19×10-3 a 1.82×10-4

1,2-dimethylbenzene 1.12×10-2 c 9.78×10-4 3.30×10-2 a 3.39×10-3 2.00×10-2 b 1.53×10-3 8.76×10-3 c 5.29×10-4

1-hexanol 1.06×10-2 b 1.04×10-3 2.44×10-2 a 1.16×10-3 1.43×10-2 b 1.10×10-3 1.29×10-2 b 5.05×10-3

3-heptanone 5.03×10-4 b 5.07×10-5 6.90×10-3 a 1.09×10-3 1.85×10-3 b 2.93×10-5 1.42×10-3 b 2.23×10-4

Benzaldehyde 1.95×10-3 c 1.70×10-4 4.18×10-3 b 2.95×10-4 4.71×10-3 b 7.36×10-4 1.44×10-2 a 1.48×10-3

1-octen-3-ol 1.10×10-2 b 1.19×10-3 6.43×10-1 a 5.16×10-2 5.59×10-3 b 4.25×10-4 5.68×10-1 a 4.83×10-2

3-octanone 1.92×100 c 1.25×10-1 1.90×100 c 3.99×10-2 2.35×100 b 2.91×10-1 3.15×100 a 6.64×10-2

3-octanol 2.75×100 a 3.13×10-1 2.86×100 a 4.67×10-1 1.75×100 b 3.16×10-1 1.37×100 b 3.05×10-1

Octanal 3.41×10-2 b 4.91×10-3 1.70×10-2 c 8.21×10-4 1.32×10-2 c 2.20×10-3 4.22×10-2 a 2.34×10-3

5-methyl-1,3-benzenediol 3.34×10-3 c 6.80×10-4 3.60×10-3 c 6.25×10-4 1.32×10-2 a 1.69×10-3 7.54×10-3 b 1.06×10-3

2-methoxy-6-methylpyrazine 6.06×10-4 c 2.75×10-5 4.57×10-3 b 3.33×10-4 7.57×10-3 a 1.33×10-3 8.49×10-3 a 9.04×10-4

Limonene 3.92×10-4 b 8.13×10-5 1.15×10-3 a 1.68×10-4 1.36×10-3 a 2.26×10-4 1.08×10-3 a 1.01×10-4

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Table 4.10 continued Compound D 40 °C D 50 °C D 60 °C D 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-ethyl-1-hexanol 3.93×10-3 b 5.52×10-4 2.09×10-2 a 2.37×10-3 6.04×10-3 b 6.73×10-4 4.65×10-3 b 6.21×10-4

Phenyacetaldehyde 8.21×10-4 c 9.55×10-5 1.21×10-3 c 1.10×10-4 2.73×10-3 b 4.38×10-4 4.30×10-3 a 7.89×10-5

(E)-2-octenal 3.18×10-3 b 2.96×10-4 2.29×10-3 bc 3.18×10-4 1.68×10-3 c 4.35×10-4 8.08×10-3 a 3.08×10-4

2-octen-1-ol 2.30×10-2 a 5.56×10-3 2.07×10-2 a 2.18×10-3 5.15×10-3 b 1.09×10-3 8.08×10-3 b 3.08×10-4

n-octanol 2.56×10-1 a 4.29×10-2 1.75×10-1 b 1.59×10-2 8.10×10-2 c 1.60×10-2 7.96×10-3 c 7.12×10-4

Nonanal 2.58×10-3 c 3.82×10-5 5.64×10-3 b 8.45×10-4 4.55×10-3 b 2.03×10-4 1.54×10-2 a 2.83×10-3

(E )-2-nonenal 3.22×10-2 ab 8.78×10-3 4.03×10-2 a 1.34×10-3 1.90×10-2 bc 3.99×10-3 1.54×10-2 c 2.34×10-3

Ethyl octanoate 2.50×10-3 b 2.93×10-4 4.95×10-3 b 7.90×10-4 1.39×10-2 a 1.98×10-3 1.54×10-2 a 2.34×10-3

Decanal 1.68×10-3 b 3.07×10-4 3.36×10-3 a 2.08×10-4 3.06×10-3 ab 9.14×10-4 2.72×10-3 ab 5.40×10-4

2-undecanone 1.09×10-3 c 4.85×10-5 4.36×10-3 b 1.95×10-4 2.79×10-3 bc 8.57×10-4 2.42×10-2 a 1.45×10-3

2,6-bis(1,1- dimethylethyl)phenol 1.92×10-2 a 2.66×10-3 3.51×10-2 a 2.91×10-3 1.76×10-2 a 2.60×10-3 2.42×10-2 a 1.45×10-3

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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Other compounds identified were 2,3,5-trimethylfuran which was absent at 40 and 50 °C and had the highest concentration at 70 °C (4.65×10-3±4.99×10-4 ppm) which was significantly different from that found at 60 °C (1.62×10-3±3.40×10-5 ppm), and 4-methyl-3-penten-2-one which was also not identified at 40 and 50 °C but had its maximum concentration at 60 °C (2.47×10-3 ±1.20×10-4 ppm) which was not significantly different from 70 °C. As for 1,2-dimethylbenzene, its highest concentration was found at 50 °C (3.30×10-2±3.39×10-3 ppm) which was significantly different from the other drying temperatures. Other compounds which also had their maximum concentrations at 50 °C include 1-hexanol (2.44×10-2±1.16×10-3 ppm) and 3-heptanone (6.90×10-3 ±1.09×10-3 ppm). As for 5-methyl-1,3-benzendiol, it had the highest concentration at 60 °C (1.32 ×10-2±1.69×10-3 ppm), significantly different from those found at 40, 50, and 70 °C with the lowest concentration reaching 3.34×10-3±6.80×10-4 ppm at 40 °C. As for 2-methoxy-6-methyl pyrazine, no significant difference was found between 60 °C (7.57×10-3±1.33×10-3 ppm) and 70 °C samples (8.49×10-3±9.04×10-4 ppm) but the concentration decreased significantly when compared to 40 °C (6.06×10-4±2.75×10-5 ppm) and 50 °C (4.57×10-3±3.33×10-4 ppm). When comparing the concentrations of limonene at 40 °C (3.92×10-4±8.13×10-5 ppm) and 60 ° C (1.36×10-3±2.26×10-4 ppm), a significant increase in its concentration was observed. As for ethyl octanoate, it had its highest concentration at 70 °C (1.54×10-2±2.34×10-3 ppm) and the lowest significantly different at 40 °C (2.50×10-3±2.93×10-4 ppm). Finally, 2,6-bis(1,1- dimethylethyl)phenol had its highest concentration at 50 °C (3.51×10-2±2.91×10-3 ppm) which was not significantly different from those found at 40, 60, and 70 °C.

The results show that some compounds are more abundant when the samples are dried at 40 °C while others exhibited the maximum retention at 50 °C and few compounds at 60 and 70 °C. The choice of a suitable drying temperature should be a compromise between (a) suitable condition for the maximum retention and preservation of VCs, (b) adequate moisture removal for the extension of the shelf life (c) savings in terms of energy usage. Therefore drying at 50 °C appears as the most suitable drying temperature.

Aldehydes formed by the peroxidation of unsaturated fatty acids are considered as important aroma contributor only when they are produced in foods in a concentration higher than their odour threshold concentration. Therefore, the aroma active compounds, including hexanal which has green-like aroma and (E) 2-nonenal characterized by stale or hay aroma (Chen et al., 2009) which seem to be the products from the peroxidation of linoleic acid, can produce the relatively high odour threshold. As a matter of fact, (E)-2-nonenal has an odour threshold 17.5 times lower than nonanal. In chiral compounds as well as cis/trans isomers such as C6 and C9 aldehydes with a

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double bond, the molecule geometry influences the odour intensity and quality (Belitz et al., 2009).

Oyster mushrooms dried at different temperatures are shown in figure 4.42 and 4.43 below. At lower temperature (40 and 50 °C) the mushrooms were lighter in colour compared to higher drying temperatures (60 and 70 °C).

Figure 4.42: Vacuum packed oyster mushrooms dried at 40 °C (Left) and 50 °C (Right) (Author photograph).

Figure 4.43: Vacuum packed oyster mushrooms dried at 60 °C (Left) and 70 °C (Right) (Author photograph).

The normalised relative concentrations of the main C8 compounds are shown in Figure 4.44. The highest normalised relative concentration of 1-octen-3-ol was at 50 °C which was not significantly different from 70 °C. Meanwhile, 3-octanol and 2-octen-1-ol showed no significant difference between drying at 40 and 50 °C, but at higher temperatures, their normalised relative concentrations was significantly decreased. Drying at 70 °C resulted in the maximum generation and retention of 3-octanone while n-octanol had the greatest amount at 40 °C which again decreased with increasing temperatures.

1.4 D 40 °C D 50 °C 1.2 D 60 °C D 70 °C 1.0

0.8

0.6

0.4

0.2 Normalisedrelative concentration

0.0

l l l l o ne o o o 3- o an 1- an n- an ct n- ct te ct -o te -o oc -o 3 oc n 1- 3 2- Compound

Figure 4.44: Normalised relative concentrations of the main C8 VC extracted from dried oyster mushrooms.

Drying at different temperatures resulted in alcohol and ketone VC behaving differently from each other with some favouring low drying temperatures, while other higher temperatures (Figure 4.45). Drying at 50 °C was the most suitable temperature for 3-heptanone which showed the highest amount at 50 °C being significantly different from the other drying temperatures while 2- undecanone had its maximum normalised relative concentration at 70 °C significantly different from the remaining drying temperatures. No significant difference was found for 4-methyl-3- penten-2-one when dried at 60 and 70 °C and no traces of this VC were found at 40 or 50 °C. As for 1-hexanol and 2-ethyl-1-hexanol, they had the highest normalised relative concentration at 50 °C, while 3-methyl-1-butanol had the highest amount at 60 °C which was not significantly different from 50 and 70 °C.

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1.4 D 40 °C D 50 °C 1.2 D 60 °C D 70 °C 1.0

0.8

0.6

0.4

0.2 Normalised relative concentration 0.0

l l ne ne ne ol o o no no -o an an an ta a -2 ex ex ut p ec en -h -h -b he nd nt 1 -1 -1 3- -u e yl yl 2 -p th th l-3 -e e hy 2 -m et Compound 3 m 4-

Figure 4.45: Normalised relative concentrations of selected alcohols and ketones VC extracted from dried oyster mushrooms.

Aldehydes VC also varied in their normalised relative concentration under different drying temperatures (Figure 4.46). Octanal, nonanal, (E)-2-octenal, benzaldehyde, (E,E)-2,4-hexadienal, and phenylacetaldehyde had their maximum relative concentration at 70 °C. As for (E)-2-nonenal, it had the highest normalised relative concentration at 50 °C which was not significantly different from that found at 40 °C but significantly different from D 60 and 70 °C samples.

D 40 °C 1.2 D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration 0.0

l l al l l l e l e na na n na na na yd na yd ta na ca xa cte e eh ie eh Oc o e e -o on ld ad ld N D H -2 -n a ex ta ) -2 z h ce (E E) Ben ,4 a ( )-2 ny ,E he Compound (E P

Figure 4.46: Normalised relative concentrations of selected aldehydes VC extracted from dried oyster mushrooms.

155

Meanwhile, the normalised relative concentration of decanal showed no significant difference between drying at 50, 60, and 70 °C; however, drying at 40 °C resulted in major losses of this compound. As for hexanal, drying at 60 °C resulted in the highest normalised concentration of this compound which was not significantly different from 50 °C but significantly different from 40 and 70 °C samples.

The normalised relative concentrations of selected VC (group 1) extracted from dried oyster are shown in Figure 4.47. The compound limonene had its highest normalised relative concentration at 60 °C which was not significantly different from D 50 and 70 °C samples but significantly different from D 40 °C samples. The compound ethyl octanoate showed no significant difference between D 60 and 70 °C samples and 2,3,5-trimethylfuran had its maximum normalised value at 70 °C which was significantly different from that found at 60 °C. However, this compound was not identified in D 40 and 50 °C samples. As for 5-methyl-1,3-benzendiol, its highest normalised relative concentration was found at 60 °C which was significantly different form the other treatments while 2-methoxy-6-methyl pyrazine showed no significant difference between D 60 and 70 °C samples and had its lowest value in D 40 °C samples.

1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalisedrelative concentration 0.0

e e n l e en at ra io in n no fu nd az mo ta yl ze yr Li oc th en l p yl e -b y th rim ,3 th E -t l-1 me ,5 hy 6- 2,3 et y- -m ox 5 eth Compound 2-m

Figure 4.47: Normalised relative concentrations of selected VC (group 1) extracted from dried oyster mushrooms.

Dijkstra (1976) reported that the concentration of 1-octen-3-ol in dried A. bisporus was much lower than in fresh mushrooms which was comparable to the findings in this study.

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The compounds benzaldehyde and phenylacetaldehyde, were previously identified as aroma compounds in a Japanese fermented vegetable product (Imai et al., 1991 and Kasahara et al., 1982) while hexanal was previously reported as an undesirable flavour volatile generated during the fermentation of cucumber (Zhou et al., 2000). All the compounds identified in this study contribute to the mushroom aroma of oyster mushrooms. The main C8 compounds as well as alcohols, ketones, pyrazine and furan compounds are key compounds in the dried mushrooms.

4.3.4 Dried and boiled under reflux (DBR) oyster mushrooms

Based on lack of published studies, it appears that VC in dried and boiled under reflux oyster mushrooms have not been studied before. The concentration of VC in dried and boiled under reflux oyster mushrooms is shown in Table 4.11. In DBR 40 °C samples, phenylacetaldehyde was the major VC with a relative concentration of 5.14×10-2±3.22×10-3 ppm followed by 3-methyl butanal (3.40×10-2±7.35×10-3 ppm), 2-methyl butanal (2.66×10-2±1.63×10-3 ppm), benzaldehyde (1.71×10-2±3.00×10-3 ppm), 3-octanol (1.68×10-2±1.75×10-3 ppm), hexanal (1.62×10-2±4.43×10-4 ppm), and 2,6-bis(1,1-dimethylethyl) phenol (1.53×10-2±1.18×10-3 ppm). In DBR 50 °C samples, phenylacetaldehyde was also the main VC detected but its concentration significantly decreased to 4.21×10-2±1.46×10-3 ppm followed by benzaldehyde which had a significant increase in its concentration compared to 40 °C (2.78×10-2±7.26×10-3 vs. 1.71×10-2±3.00×10-3 ppm), hexanal which also significantly increased compared to 40 °C (2.39×10-2±1.70×10-3 ppm), 3-octanol (1.50×10-2±1.78×10-3 ppm), 3-methyl butanal and 2-methyl butanal which showed a significant decrease in their concentrations (1.43×10-2±9.86×10-4 ppm and 1.17×10-2±1.24×10-3 ppm respectively). The concentration of 3-octanol was not significantly lower in DBR 50 °C samples (1.50×10-2±1.78×10-3 ppm) compared to DBR 40 °C samples while 3-heptanone showed a significant increase in its concentration when comparing DBR 40 and 50 °C samples (2.73×10-3 ±3.29×10-4 vs. 1.28×10-2±1.22×10-3 ppm). In DBR 60 °C samples, (E)-2-nonenal was the major VC with its concentration significantly different when compared to the other drying temperatures (3.25×10-2±3.85×10-3 ppm) followed by 3-methyl butanal with a concentration of 2.71×10-2 ±7.33×10-4 ppm significantly different from that found in DBR 50 °C samples, benzaldehyde (2.33×10-2±1.36×10-3 ppm) which was not significantly different when compared to DBR 50 °C samples followed by phenylacetaldehyde with a significant decrease in its concentration (2.30×10-2±2.82×10-3 ppm). As for 2-methyl butanal, it also showed a significant decrease in its concentration (2.09×10-2±1.58×10-3 ppm) in DBR 60 °C samples compared to DBR 40 °C samples. Hexanal showed not significant change when comparing DBR 50, 60, and 70 °C samples

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although its concentration marginally decreased in DBR 60 and 70 °C samples. As for 3-octanol, increasing the temperature to 60 °C and 70 °C followed by boiling under reflux significantly decreased its concentration (1.08×10-2±4.86×10-4 and 8.82×10-3±7.59×10-4 ppm respectively). Meanwhile, 2,6-bis(1,1-dimethylethyl)phenol had a non-significant decrease in its concentration in DBR 70 °C samples compared to DBR 60 °C samples (8.27×10-3±4.43×10-4vs.1.25×10-2 ±1.56×10-3 ppm). At 70 °C, phenylacetaldehyde was also the main VC with a concentration of 4.20×10-2±2.19×10-3 ppm followed by benzaldehyde (3.85×10-2±1.55×10-3 ppm) both being significantly different from those found in DBR 60 °C samples. As for 3-methyl butanal, 2-methyl butanal and hexanal, they showed no significant decrease in their concentrations in DBR 70 °C samples compared to DBR 60 °C samples. Meanwhile, 2-methyl furan had its maximum concentration in DBR 70 °C samples (1.08×10-3±7.45×10-5 ppm) which was not significantly different when compared to DBR 40, 50 and 60 °C samples. Octanal was not detected in DBR 40 °C samples which could be either because of its very low concentration that was below the detection limit of the instrument or the prolonged heating time followed by boiling under reflux for 30 min that caused this VC to be lost. However, it had its highest concentration in DBR 50 °C samples (1.91×10-3±8.98×10-5 ppm) significantly different from DBR 60 and 70 °C samples. As for heptanal, it had its highest concentration in DBR 70 °C samples (2.56×10-3±3.44×10-4ppm) which was not significantly different from DBR 40 and 50 °C and had its lowest concentration in DBR 60 °C samples (1.78×10-3±1.73×10-4 ppm) while (E,E)-2,4-decadienal had its maximum concentration in DBR 40 °C samples (4.62×10-3±6.13×10-4 ppm) significantly different from DBR 50, 60 and 70 °C samples and had its lowest concentration in DBR 50 °C samples (1.44×10-3 ±4.45×10-4 ppm). Nonanal had its highest concentration in DBR 70 °C samples (8.13×10-3 ±1.59×10-4 ppm) significantly different from DBR 50 and 60 °C samples and its lowest in DBR 60 °C samples (4.19×10-3±5.68×10-4 ppm) while decanal had its maximum concentration in DBR 50 °C samples (7.30×10-3±4.85×10-4 ppm), significantly different from the other drying temperatures and its lowest in DBR 60 °C samples (1.26×10-3±3.24×10-4 ppm).

Alcohols and ketones VC behaved differently. The concentration of 3-methyl-1-butanol was the maximum in DBR 40 °C samples (3.60×10-3±6.44×10-4 ppm) which was significantly different from DBR 50, 60, and 70 °C samples while 2-methyl-1-butanol had its maximum concentration in DBR 50 °C, significantly different from the remaining treatments. As for 1-hexanol, it had a maximum concentration of 5.25×10-3±5.54×10-4 in DBR 40 °C samples which was significantly different when compared to DBR 50 °C samples (1.28×10-3±5.32×10-4 ppm). Meanwhile, 1-octen- 3-one had its highest concentration in DBR 50 °C samples (6.87×10-3±9.56×10-4 ppm) which was

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significantly different from its lowest concentration found in DBR 40 °C samples (1.21×10-3 ±1.54×10-4 ppm). As for 1-octen-3-ol, it had its highest concentration in DBR 50 °C samples (8.03×10-3±2.62×10-4 ppm), significantly different from those found in DBR 60 °C (4.30×10-3 ±2.17×10-4 ppm) and 70 °C samples (4.29×10-3±1.89×10-4 ppm) and had its lowest concentration in DBR 40 °C samples (4.19×10-3±6.20×10-4 ppm). Cyclooctanol and 3-octanone had their highest concentrations in DBR 40 °C samples (3.64×10-3±3.43×10-4 and 8.05×10-3±4.88×10-4 ppm respectively) significantly different from the other treatments. As for 2-octanone, it reached a maximum of 2.63×10-3±1.83×10-4 ppm in DBR 60 °C samples, significantly different from DBR 40 and 50 °C samples but not significantly different from DBR 70 °C samples. Meanwhile, 2- ethyl-1-hexanol had its highest concentration in DBR 50 °C samples (6.65×10-3±1.20×10-4 ppm) significantly higher compared to the other treatments. The compound 3-heptanone had its maximum concentration in DBR 50 °C (1.28×10-2±1.22×10-3 ppm), significantly different form DBR 40, 60 and 70 °C samples. As for 1-nonanol, it had its highest concentration in DBR 70 °C (6.70×10-3±2.08×10-4ppm) significantly different from DBR 40, 50 and 60 °C, but had its lowest concentration in DBR 40 °C samples (2.89×10-3±8.23×10-4 ppm). As for 2-undecanone, DBR 70 °C resulted in the maximum retention of this compound with a concentration of 7.87×10-3 ±5.89×10-4 ppm significantly different from those found in DBR 40, 50, and 60 °C samples.

As for 2-methoxy-6-methyl pyrazine, it had its highest concentration in DBR 50 °C samples (3.33×10-3±3.26×10-4 ppm) which was significantly different from the other treatments. As for 3- ethyl-2-methyl-1,3-hexadiene, it had its maximum concentration in DBR 40 °C samples, significantly different from DBR 50 °C samples (5.73×10-3±5.82×10-4 vs. 2.59×10-3±8.65×10-5 ppm). There was no significant difference in the concentration of this compound between DBR 50, 60, and 70 °C samples. The treatment DBR 50 °C resulted in the maximum retention of (E)-2- octenal (5.91×10-3±5.66×10-4 ppm), 2-octen-1-ol (5.88×10-3±2.97×10-4 ppm), and n-octanol (7.89×10-3±3.91×10-4 ppm), decanal (7.30×10-3±4.85×10-4 ppm), all being significantly different from the other remaining treatments. The monoterpene linalool had its concentration almost doubled with every 10 °C increase in the temperature peaking in DBR 60 °C (8.34×10-3±6.57×10-4 ppm), however, in DBR 70 °C samples, much of this compound was lost (1.13×10-3±1.10×10-4 ppm). Meanwhile, 1-acetylimidazole had its maximum concentration in DBR 40 °C (5.43×10-3 ±4.54×10-4 ppm) which was significantly different from those found in DBR 50, 60, and 70 °C samples.

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Table 4.11: Concentrations of VC extracted from dried and boiled under reflux oyster mushrooms. Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-methyl furan 7.79×10-4 a 1.45×10-5 1.06×10-3 a 3.06×10-4 1.06×10-3 a 9.51×10-5 1.08×10-3 a 7.45×10-5

3-methyl butanal 3.40×10-2 a 7.35×10-3 1.43×10-2 c 9.86×10-4 2.71×10-2 ab 7.33×10-4 2.20×10-2 bc 2.55×10-3

2-methyl butanal 2.66×10-2 a 1.63×10-3 1.17×10-2 c 1.24×10-3 2.09×10-2 b 1.58×10-3 1.98×10-2 b 8.01×10-4

3-methyl-1-butanol 3.60×10-3 a 6.44×10-4 2.23×10-3 b 2.63×10-4 1.32×10-3 b 2.36×10-4 1.24×10-3 b 2.67×10-4

2-methyl-1-butanol 1.80×10-3 b 3.47×10-4 3.13×10-3 a 2.71×10-4 9.66×10-4 c 5.59×10-5 1.95×10-3 b 4.53×10-4

Hexanal 1.62×10-2 b 4.43×10-4 2.39×10-2 a 1.70×10-3 2.17×10-2 a 5.88×10-4 2.03×10-2 ab 3.35×10-3

1-hexanol 5.25×10-3 a 5.54×10-4 1.28×10-3 b 5.32×10-4 1.75×10-3 b 3.02×10-4 1.78×10-3 b 6.62×10-4

3-heptanone 2.73×10-3 b 3.29×10-4 1.28×10-2 a 1.22×10-3 1.04×10-3 c 1.31×10-4 7.48×10-4 c 2.55×10-5

Heptanal 1.90×10-3 ab 2.45×10-4 2.50×10-3 a 2.47×10-4 1.78×10-3 b 1.73×10-4 2.56×10-3 a 3.44×10-4

Benzaldehyde 1.71×10-2 c 3.00×10-3 2.78×10-2 b 7.26×10-3 2.33×10-2 bc 1.36×10-3 3.85×10-2 a 1.55×10-3

1-acetylimidazole 5.43×10-3 a 4.54×10-4 2.44×10-3 b 1.88×10-4 2.95×10-3 b 2.05×10-4 2.25×10-3 b 3.17×10-4

1-octen-3-one 1.21×10-3 c 1.54×10-4 6.87×10-3 a 9.56×10-4 2.81×10-3 b 2.79×10-4 3.26×10-3 b 2.04×10-4

1-octen-3-ol 4.19×10-3 b 6.20×10-4 8.03×10-3 a 2.62×10-4 4.30×10-3 b 2.17×10-4 4.29×10-3 b 1.89×10-4

Cyclooctanol 3.64×10-3 a 3.43×10-4 1.35×10-3 c 1.96×10-4 6.34×10-4 d 7.43×10-5 2.57×10-3 b 1.52×10-4

3-octanone 8.05×10-3 a 4.88×10-4 6.71×10-3 b 3.64×10-4 5.23×10-3 c 6.43×10-4 4.62×10-3 c 3.06×10-4

2-octanone 1.07×10-3 b 1.43×10-4 1.23×10-3 b 1.34×10-4 2.63×10-3 a 1.83×10-4 2.11×10-3 a 3.48×10-4

3-octanol 1.68×10-2 a 1.75×10-3 1.50×10-2 a 1.78×10-3 1.08×10-2 b 4.86×10-4 8.82×10-3 b 7.59×10-4

Octanal ND ND 1.91×10-3 a 8.98×10-5 9.67×10-4 b 4.16×10-5 1.00×10-3 b 6.50×10-5

2-methoxy-6-methylpyrazine 1.58×10-3 b 1.22×10-4 3.33×10-3 a 3.26×10-4 1.02×10-3 b 1.03×10-4 1.48×10-3 b 2.70×10-4

3-ethyl-2-methyl-1,3-hexadiene 5.73×10-3 a 5.82×10-4 2.59×10-3 b 8.65×10-5 2.44×10-3 b 1.36×10-4 2.13×10-3 b 3.19×10-4

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Table 4.11 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

2-ethyl-1-hexanol 3.52×10-3 b 2.82×10-4 6.65×10-3 a 1.20×10-4 1.65×10-3 c 2.37×10-4 3.54×10-3 b 4.24×10-4

Phenylacetaldehyde 5.14×10-2 a 3.22×10-3 4.21×10-2 b 1.46×10-3 2.30×10-2 c 2.82×10-3 4.20×10-2 b 2.19×10-3

(E)-2-octenal 2.77×10-3 b 1.44×10-4 5.91×10-3 a 5.66×10-4 2.09×10-3 b 1.70×10-4 2.52×10-3 b 2.10×10-4

2-octen-1-ol 2.12×10-3 c 3.24×10-4 5.88×10-3 a 2.97×10-4 1.80×10-3 c 1.52×10-4 3.17×10-3 b 5.20×10-4

N-octanol 2.29×10-3 b 2.88×10-4 7.89×10-3 a 3.91×10-4 8.64×10-4 c 5.27×10-5 1.44×10-3 bc 4.48×10-4

Linalool 2.63×10-3 c 2.69×10-4 4.68×10-3 b 4.13×10-4 8.34×10-3 a 6.57×10-4 1.13×10-3 d 1.10×10-4

Nonanal 7.02×10-3 a 1.87×10-4 5.78×10-3 b 1.99×10-4 4.19×10-3 c 5.68×10-4 8.13×10-3 a 1.59×10-4

(E)-2-nonenal 1.48×10-3 b 2.89×10-4 3.59×10-3 b 3.10×10-4 3.25×10-2 a 3.85×10-3 1.49×10-3 b 1.52×10-4

1-nonanol 2.89×10-3 c 8.23×10-4 4.93×10-3 b 5.95×10-4 4.59×10-3 b 4.87×10-4 6.70×10-3 a 2.08×10-4

Decanal 3.10×10-3 b 4.01×10-4 7.30×10-3 a 4.85×10-4 1.26×10-3 c 3.24×10-4 1.54×10-3 c 3.81×10-4

2-undecanone 2.76×10-3 b 8.10×10-4 2.74×10-3 b 5.23×10-4 2.46×10-3 b 1.86×10-4 7.87×10-3 a 5.89×10-4

(E,E)-2,4-decadienal 4.62×10-3 a 6.13×10-4 1.44×10-3 c 4.45×10-4 2.66×10-3 b 2.87×10-4 1.94×10-3 bc 2.48×10-4

2,6-bis(1,1-dimethylethyl) phenol 1.53×10-2 a 1.18×10-3 1.38×10-2 a 3.67×10-3 1.25×10-2 ab 1.56×10-3 8.27×10-3 b 4.43×10-4

* Standard deviation (SD), analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The normalised relative concentration of selected alcohols VC from dried and boiled under reflux oyster mushrooms are shown in Figure 4.48. The values are normalised against the highest values calculated. Boiling under reflux of the samples dried at 40 °C resulted in the maximum retention of 3-methyl-1-butanol, 1-hexanol, and cyclooctanol being significantly different from the other treatments. As for 1-octen-3-ol, it had the maximum normalised relative concentration in DBR 50 °C samples which was also significantly different from DBR 40, 60, and 70 °C samples. As for 2-ethyl-1-hexanol, 2-octen-1-ol, and n-octanol, they had their highest normalised relative concentration in DBR 50 °C and were significantly different from DBR 40, 60 and 70 °C samples. Unlike the above mentioned alcohols, 1-nonanol had the maximum normalised relative concentration in DBR 70 °C samples while 3-octanol had its highest normalised concentration in DBR 40 °C which was not significantly different from DBR 50 °C samples but significantly different from DBR 60 and 70 °C samples.

DBR 40 °C 1.2 DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration

0.0 l l l l l l l l l no no -o no no no -o no no ta a -3 ta ta a -1 ta a u ex en c c ex en c on -b h t oo -o h t -o n 1 1- oc cl 3 1- oc n 1- l- 1- y l- 2- hy C hy et et m 2- 3- Compound

Figure 4.48: Normalised relative concentrations of selected alcohols VCs extracted from dried and boiled under reflux oyster mushrooms.

The normalised relative concentrations of ketones VC extracted from dried oyster at different temperatures followed by the boiling under reflux are shown in Figure 4.49. The compounds 3- heptanone and 1-octen-3-one had their maximum normalised relative concentrations in DBR 50 °C samples which were significantly different form the other treatments. No significant

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difference was found between DBR 60 and 70 °C samples for both compounds. Meanwhile, 3- octanone had the highest normalised relative concentration in DBR 40 °C samples being significantly different from those found in DBR 50, 60 and 70 °C while 2-octanone showed no significant difference between DBR 60 and 70 °C samples. As for 2-undecanone, its highest normalised relative concentration was found in DBR 70 °C samples which was significantly different from those found in DBR 40, 50, and 60 °C samples.

DBR 40 °C 1.2 DBR 50 °C DBR 60 °C DBR 70 °C 1.0

0.8

0.6

0.4

0.2 Normalisedconcentration relative

0.0

e e e e e on on on on on an 3- an an an t n- ct ct c ep te -o -o de -h oc 3 2 un 3 1- 2- Compound

Figure 4.49: Normalised relative concentrations of ketones VC extracted from dried and boiled under reflux oyster mushrooms.

The normalised relative concentration of selected aldehydes VC are summarised in Figure 4.50. Hexanal had the highest normalised value in DBR 50 °C samples which was significantly different from DBR 40 °C samples but was not significantly different from DBR 60 and 70 °C samples. Octanal, being undetected in DBR 40 °C samples, had its highest normalised value in DBR 50 °C samples which was significantly different from DBR 60 and 70 °C. Decanal and (E)-2-octenal also had the maximum normalised relative concentration found in DBR 50 °C samples being significantly different from those found in DBR 40, 60, and 70 °C samples. Meanwhile, nonanal had its maximum normalised relative concentration in DBR 70 °C samples which was significantly different from those found in DBR 40, 50 and 60 °C samples. As for benzaldehyde, it also had its highest normalised value in DBR 70 °C samples and was significantly different from those found in DBR 40, 50, and 60 °C samples. As for (E)-2-nonenal, it was found to have the highest normalised relative concentration in DBR 60 °C samples which was significantly different from those found in DBR 40, 50 and 70 °C samples. Meanwhile, 3-methyl butanal and

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phenylacetaldehyde both had their maximum normalised relative concentrations in DBR 40 °C which were significantly different from DBR 50, and 70 °C samples.

1.4 DBR 40 °C 1.2 DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2 Normalisedconcentration relative 0.0

al al al al al al e al e n n n n n n yd n yd a ta a ca te ne h ta h ex c on e oc o de u de H O N D 2- -n al l b al )- )-2 nz hy et (E E e et ac ( B m yl 3- en Ph Compound

Figure 4.50: Normalised relative concentrations of selected aldehydes VC extracted from dried and boiled under reflux oyster mushrooms.

The normalised relative concentrations of other VC extracted from dried and boiled under reflux oyster mushroom are shown in Figure 4.51. The compound (E,E)-2,4-decadienal, 1- acetylimidazole, and 3-ethyl-2-methyl-1,3-hexadiene had their maximum normalised relative concentration in DBR 40 °C which were significantly different from those found in DBR 50, 60, and 70 °C samples. Linalool had its maximum normalised relative concentration in DBR 60 °C samples while 2-methoxy-6-methylpyrazine had its highest normalised relative concentration in DBR 50 °C samples which was significantly different form the other treatments. As for 2-methyl furan, there was no significant difference between DBR 40, 50, 60 or 70 °C samples as the normalised relative concentrations determined at these temperatures were not significantly different from each other.

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1.4

1.2 DBR 40 °C DBR 50 °C 1.0 DBR 60 °C DBR 70 °C

0.8

0.6

0.4

0.2 Normalisedconcentration relative 0.0 l n l le e e oo ra na o in en al u ie az az i in l f d id r ad L y ca m py ex th de li l -h e 4- ty hy ,3 -m 2, ce et -1 2 )- -a m yl ,E 1 6- th (E y- e ox -m th l-2 e hy -m et Compound 2 3-

Figure 4.51: Normalised relative concentrations of other VC extracted from dried and boiled under reflux oyster mushrooms.

The VC determined in oyster mushroom samples that had been dried (D) and dried and boiled under reflux (DBR) showed that the same compound behaved differently depending on the rehydration temperature. The former was at 25 °C when the ambient temperature vacuum distillation is carried out or at 100 °C upon boiling the dried samples under reflux followed by vacuum distillation at ambient temperature. Some compounds are generated when the rehydration temperature was 100 °C such as 2-methyl furan, 2-methyl-1-butanol, heptanal, 1-octen-3-one, cyclooctanol, 2-octanone, 3-ethyl-2-methyl-1,3-hexadiene, linalool, 1-nonanol, and (E,E)-2,4- decadienal. Other compounds from dried samples were only detected in dried oyster samples with 25 °C rehydration temperature such as (E,E)-2,4-hexadienal, 2,3,5-trimethylfuran, 4-methyl-3- penten-2-one, 5-methyl-1,3-benzendiol, limonene, and ethyl octanoate. These losses could be due to (a) the vaporisation of the VC when the samples were boiled under reflux for 30 min although it was assumed to be a closed system or (b) when the sample in the round bottom flask was left to cool for 5 min in order to prevent the cracking of the flask and to minimise the loss of VC if they are to be transferred to another flask for ambient temperature vacuum distillation or (c) losses occurred during the 4 h extraction using the vacuum distillation where some VC escaped the third trap and were captured in the molecular sieve used to protect the pump.

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Choosing 50 °C as the most appropriate drying temperature, comparison was made between the behaviour of similar compounds when they were rehydrated at 25 °C and 100 °C. Some compounds showed a higher concentration when the dried samples were boiled under reflux compared to dried samples rehydrated at 25 °C. These compounds include 3-methyl butanal (1.43×10-2±9.86×10-4 vs. 4.21×10-3±4.61×10-5 ppm), 2-methyl butanal (1.17×10-2±1.24×10-3 vs. 5.88×10-3±5.58×10-4 ppm), 3-heptanone (1.28×10-2±1.22×10-3 vs. 6.90×10-3±1.09×10-3 ppm), benzaldehyde (2.78×10-2±7.26×10-3 vs. 4.18×10-3±2.95×10-4 ppm), phenylacetaldehyde (4.21× 10-2±1.46×10-3 vs. 1.21×10-3±1.10×10-4 ppm), (E)-2-octenal (5.91×10-3±5.66×10-4 vs. 2.29× 10-3±3.18×10-4 ppm), nonanal (5.78×10-3 ±1.99×10-4 vs. 5.64×10-3±8.45×10-4 ppm), and decanal (7.30×10-3±4.85×10-4 vs. 3.36×10-3±2.08×10-4 ppm). Some compounds had a higher concentration when the samples were only dried before being subjected to ambient temperature vacuum distillation in comparison with samples that were dried and boiled under reflux. These compounds include 3-methyl-1-butanol (1.36×10-2±6.46×10-3 vs. 2.23×10-3±2.63×10-4 ppm), hexanal (3.36× 10-2±2.22×10-3 vs. 2.39×10-2±1.70×10-3 ppm), 1-hexanol (2.44×10-2±1.16x10-3 vs. 1.28× 10-3±5.32×10-4 ppm), 1-octen-3-ol (6.43×10-1±5.16×10-2 vs. 8.03×10-3±2.62×10-4 ppm), 3- octanone (1.90×100±3.99 x10-2 vs. 6.71×10-3±3.64×10-4 ppm), 3-octanol (2.86×100±4.67×10-1 vs. 1.50×10-2±1.78×10-3 ppm), octanal (1.70×10-2±8.21×10-4 vs. 1.91×10-3±8.98×10-5 ppm), 2-ethyl- 1-hexanol (2.09×10-2±2.37×10-3 vs. 6.65×10-3±1.20×10-4 ppm), 2-octen-1-ol (2.07×10-2±2.18× 10-3 vs. 5.88×10-3±2.97×10-4 ppm), n-octanol (1.75×10-1±1.59×10-2 vs. 7.89×10-3±3.91×10-4 ppm), (E)-2-nonenal (4.03×10-2±1.34×10-3 vs. 3.59×10-3±3.10×10-4 ppm), 2-undecanone (4.36× 10-3±1.95×10-4 vs. 2.74×10-3±5.23×10-4 ppm) and 2,6-bis(1,1-dimethylethyl)phenol (3.51×10-2 ±2.91 x10-3 vs. 1.38×10-2±3.67×10-3 ppm).

In this study, several compounds were identified and were not previously reported in dried and boiled under reflux oyster mushrooms and include 2-methyl furan, 2-methoxy-6-methyl pyrazine and 3-ethyl-2-methyl-1,3-hexadiene. The compounds benzaldehyde, linalool, and 2,4-decadienal have been also identified as compounds contributing to the aroma of baked “Jewel” sweet potatoes (Wang and Kays, 2000). A possible explanation for the presence of 2-octanone, (E)-2-octenal, and phenylacetaldehyde as well as the mechanism of 3-methyl butanal and 2-methyl butanal formation were discussed in section 4.1.5

Ohta et al. (1990) reported linalool as a key compound contributing to the aroma of the distilled sweet potato spirit (Kansho-shochu). He also found that in the raw storage roots, terpene alcohols are present primarily in the form of ß-glucoside conjugates which can be hydrolysed by ß- glucosidase and that the presence of linalool in cooked sweet potatoes may be liberated from non-

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volatile glycosides by enzyme hydrolysis and or heating. In this study, linalool was only identified when oyster samples were thermally processed suggesting that the exposure to high temperature allowed the liberation of this aroma-active compound.

Different drying temperatures and hence different drying times led to differences in the amount of volatile compounds. As the mushrooms were dried until they reached a constant weight, the drying time played an important role in the formation/and loss of VC. At 40 °C, the mushrooms were dried for 18 h and the final MC reached was 8.37%. At 50 °C, the drying time was 15 h and the final MC was 3.88% while at 60 °C, the mushrooms were dried for 11 h with a final MC of 1.91% and at 70 °C, the mushrooms were dried for 9 h reaching a final MC of 2.91%. Nevertheless, the rehydration temperature temperature is also a crucial factor in the liberation of theses volatiles.

4.3.5 Comparison between treatments

Raw mushrooms contain a significant number of aroma-active compounds and therefore processing (boiling under reflux and drying) is accompanied by the appearance of new volatile substances resulting from various chemical reactions (e.g. lipid oxidation and degradation of alkenals) taking place during thermal processing.

The effects of various treatments on the concentration of VC in fresh oyster mushrooms are summarized in Table 4.12. Drying at 50 °C resulted in the maximum retention of VC in oyster mushrooms and thus it was chosen as the suitable temperature and was used to compare the 4 treatments: Fresh (F), boiled under reflux (BR), Dried (D 50 °C), and dried and boiled under reflux (DBR 50 °C).

Different treatments had different effects on the release of VC from mushrooms samples where compounds belonging to the same chemical class behaved differently to each other. Alcohols VC which had their maximum concentration in D 50 °C samples significantly different from the other treatments include 3-methyl-1-butanol (1.36×10-2±6.46×10-3 ppm), 1-hexanol (2.44× 10-2±1.16×10-3 ppm), 2-ethyl-1-hexanol (2.09×10-2±2.37×10-3 ppm), and 2,6-bis(1,1- dimethylethyl)phenol (3.51×10-2±2.91×10-3 ppm).

Aldehydes VC which had their maximum concentrations in DBR 50 °C samples significantly different from the other treatments include 3-methyl butanal (1.43×10-2±9.85×10-4 ppm), 2-methyl butanal (1.17×10-2±1.24×10-3 ppm), benzaldehyde (2.78×10-2±7.26×10-3 ppm),

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phenylacetaldehyde (4.21×10-2±1.46×10-3 ppm), (E)-2-octenal (5.91×10-3±5.66×10-4 ppm), nonanal (5.78×10-3±1.99×10-4 ppm), and decanal (7.30×10-3±4.85×10-4 ppm).

Other compounds identified include hexanal which had its maximum concentration in D 50 °C (3.35×10-2±2.22×10-3 ppm) which was not significantly different from BR samples (3.21× 10-2±4.50×10-3 ppm) but significantly different from the other treatments. As for 1-octen-3-one, it had its highest concentration in BR samples (5.01×10-1±1.70×10-2 ppm) which was significantly different from the remaining treatments while 1-octen-3-ol had its maximum concentration in F samples (2.16×100±2.92×10-1 ppm) which then significantly decreased in BR samples (3.84×10-1 ±3.45×10-2 ppm). No significant difference was found between BR and D 50 °C for this compound, however, the concentration was further reduced in DBR 50 °C compared to F and D 50 °C samples.

The following compounds had their maximum concentration in F samples and were significantly different from the other treatments and include 3-octanol (4.57×100±3.68×10-1 ppm), octanal (2.77×10-2±3.80×10-3 ppm), 2-octen-1-ol (2.21×10-1±2.72×10-2 ppm), n-octanol (4.56×10-1 ±2.91×10-2 ppm), and ethyl octanoate (3.32×10-2±5.55×10-3 ppm).

As for 3-octanone, it showed no significant difference between F and D 50 °C samples (2.06×100±1.75×10-1 and 1.90×100±3.99×10-2 ppm respectively) while butanoic acid,3- methyl,ethyl ester showed no significant difference between F and BR samples (1.65×10-3 ±4.67×10-4 and 2.69×10-3±2.76×10-4 ppm). The compound 2-methyl-1-butanol also had its highest concentration in F samples which was not significantly different from BR samples (5.67×10-3 ±3.23×10-4 and 4.90×10-3±7.50×10-5 ppm respectively). Meanwhile, 3-heptanone had its maximum concentration in DBR 50 °C samples (1.28×10-2±1.22×10-3 ppm) which was significantly different from the remaining treatments while 2-methoxy-6-methyl pyrazine was not identified in F and BR samples and had its highest concentration in D 50 °C (4.57×10-3±3.33×10-4 ppm) which was significantly different from DBR 50 °C samples. As for 2-ethyl hexanoic acid, being unidentified in D and DBR 50 °C samples had its highest concentration in BR samples (1.87×10-4±1.57×10-5 ppm) which was significantly different from F samples (3.12×10-4 ±1.93×10-5 ppm). The compound (E)-2-nonenal had its maximum concentration in D 50 °C samples (4.03×10-2±1.34×10-3 ppm) which was significantly different from the remaining treatments and had its lowest in BR samples (1.74×10-3±1.29×10-4 ppm). Meanwhile, 2- undecanone, being absent in F samples, had its maximum concentration in D 50 °C samples (4.36×10-3±1.95×10-4 ppm) significantly different from the other treatments.

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Some compounds were only found in the dried samples which were rehydrated at 25 °C and include (E,E)-2,4-hexadienal (7.88×10-4±4.34×10-5 ppm), 5-methyl-1,3-benzendiol (3.60× 10-3±6.25×10-4 ppm) and limonene (1.15×10-3±1.68×10-4 ppm), whereas other compounds were only identified in DBR 50 °C samples and include 2-methyl furan (1.06×10-3±3.06×10-4 ppm), heptanal (2.50×10-3±2.48×10-4 ppm), 1-acetylimidazole (2.44×10-3±1.88×10-4 ppm), cyclooctanol (1.35×10-3±1.96×10-4 ppm), 2-octanone (1.23×10-3±1.34×10-4 ppm), 3-ethyl-2-methyl-1,3- hexadiene (2.59×10-3±8.65×10-5 ppm), linalool (4.68×10-3±4.13×10-4 ppm), 1-nonanol (4.93×10-3 ±5.95×10-4 ppm), (E,E)-2,4-decadienal (1.44×10-3±4.45×10-4 ppm) where these concentrations were significantly different from those found in the remaining treatments.

Misharina et al. (2009a) identified the key substances in boiled oyster mushrooms and include 1- octen-3-one, 1-octen-3-ol, 2-octen-1-ol, and 3-octanone. He also reported that ketones are important contributors to mushrooms aroma with 1-octen-3-one having a low threshold concentration of odour which in water is 0.004-0.01ppm while 1-octen-3-ol is approximately 0.1 ppm i.e. 10-25 times higher and found that this alcohol has a much higher concentration than 1- octen-3-one which is in accordance with the results in this study when the mushrooms were fresh but upon boiling under reflux, the concentration of 1-octen-3-one was higher.

Picardi and Issenberg (1973) studied the changes in the amount of VC during thermal processing and found a significant increase of benzladehyde in the heating process, suggesting the formation of this aldehyde in response to thermal stress. Misharina et al. (2009b) studied the VC in dry cepe and oyster mushrooms and reported the main C8 compounds along with hexanal, benzaldehyde, (E)-2-hexenal, (E)-2-octenal, 1-nonanol, benzaldehyde, phenylacetaldehyde, and (E,E)-2,4- decadienal. However, ethyl acetate, 3-methyl butanal, 2-methyl butanal, 3-methyl-1-butanol, 2- methyl-1-butanol, 3-heptanone, 2-methylcyclohexanone, thiofuran, 2-octanone, 5-methyl-5- hexen-2-one, 1-methyl-2-cyclohexen-1-ol, 1,8-cineole, 2-undecanone, 1,9-nonanediol, cyclooctanol, 2,3,5-trimethylfuran, 5-methyl-1,3-benzenediol, 6-methyl-2-methoxypyrazine, 2- methyl-3-ethyl-1,3-hexadiene, and ethyl octanoate found in dried and dried and boiled under reflux oyster mushrooms carried in this study were not reported by Misharina et al. (2009b). It should be noted that the rehydration temperature plays a crucial role in the liberation and retention of VC from the dried samples as discussed in section 4.1.5.

Çağlarırmak (2007) in his work on the nutrients and the VC in Pleurotus species reported nonadecanoic acid, 9,12-octadecadien-1-ol, cis-linoleic acid methyl ester, 2-nitrocyclooctanone, hexadecadienoic acid, methyl ester where none of these compounds were identified in this study.

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Furthermore, none of the aldehydes were identified in Çağlarırmak’s work (2007) while the main C8 compounds were briefly mentioned. The discrepancies in the results could be due to composition of growth medium, growth conditions, genetic variations, storage, flush cycle, as well as drying and extraction techniques.

Pyysalo and Niskanen (1977) reported the aroma of boiled oyster mushrooms is rather “weak and less expressed” and did not contain any distinguishing compounds compared to other species. The compound 2,6-bis(1,1-dimethylethyl)phenol has not been reported in oyster mushrooms before. A similar compound belonging to the same chemical class known as 2,6-bis(1,1-dimethyethyl)-4- methylphenol has been reported as a volatile component of T.borchii Vitt and in truffle samples (Díaz et al., 2002).

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Table 4.12: Comparison between concentrations of VC in oyster mushrooms subjected to different treatments. Compound F BR D 50 °C DBR 50 °C ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) 2-methyl furan ND ND ND ND ND ND 1.06×10-3 a 3.06×10-4 3-methyl butanal 5.42×10-3 b 5.89×10-4 3.68×10-3 c 3.00×10-4 4.25×10-3 bc 4.60×10-5 1.43×10-2 a 9.85×10-4 2-methyl butanal 1.61×10-3 c 1.85×10-4 1.18×10-3 c 1.28×10-4 5.88×10-3 b 5.58×10-4 1.17×10-2 a 1.24×10-3 (E,E)-2.4-hexadienal ND ND ND ND 7.88×10-4 a 4.34×10-5 ND ND 3-methyl-1-butanol 1.81×10-3 b 1.07×10-4 6.92×10-4 c 3.15×10-5 1.36×10-2 a 6.46×10-3 2.23×10-3 b 2.63×10-4 2-methyl-1-butanol 5.67×10-3 a 3.23×10-4 4.90×10-3 a 7.50×10-5 ND ND 3.13×10-3 b 2.71×10-4 Hexanal 1.49×10-3 c 4.01×10-4 3.21×10-2 a 4.50×10-3 3.35×10-2 a 2.22×10-3 2.38×10-2 b 1.70×10-3 Butanoic acid,3-methyl,ethyl ester 1.65×10-3 a 4.67×10-4 2.69×10-3 a 2.76×10-4 ND ND ND ND 1-hexanol 4.48×10-3 b 6.07×10-4 6.36×10-3 b 4.18×10-4 2.44×10-2 a 1.16×10-3 1.28×10-3 c 5.32×10-4 3-heptanone 1.00×10-3 c 9.14×10-5 2.99×10-3 c 1.78×10-4 6.90×10-3 b 1.09×10-3 1.28×10-2 a 1.22×10-3 Heptanal ND ND ND ND ND ND 2.50×10-3 a 2.48×10-4 Benzaldehyde 2.59×10-3 b 6.41×10-4 7.11×10-3 b 3.00×10-4 4.18×10-3 b 2.95×10-4 2.78×10-2 a 7.26×10-3 1-acetylimidazole ND ND ND ND ND ND 2.44×10-3 a 1.88×10-4 1-octen-3-one 4.30×10-2 b 3.40×10-3 5.01×10-1 a 1.70×10-2 ND ND 6.87×10-3 c 9.56×10-4 1-octen-3-ol 2.16×100 a 2.92×10-1 3.84×10-1 bc 3.45×10-2 6.43×10-1 b 5.16×10-2 8.03×10-3 c 2.62×10-4 Cyclooctanol ND ND ND ND ND ND 1.35×10-3 a 1.96×10-4 3-octanone 2.06×100 a 1.75×10-1 1.64×10-1 b 1.75×10-2 1.90×100 a 3.99×10-2 6.71×10-3 b 3.64×10-4 2-octanone ND ND ND ND ND ND 1.23×10-3 a 1.34×10-4 3-octanol 4.57×100 a 3.68×10-1 1.30×100 c 1.72×10-1 2.86×100 b 4.67×10-1 1.50×10-2 d 1.78×10-3 Octanal 2.77×10-2 a 3.80×10-3 9.37×10-3 c 5.98×10-4 1.70×10-2 b 8.21×10-4 1.91×10-3 d 8.98×10-5 5-methyl-1,3-benzendiol ND ND ND ND 3.60×10-3 a 6.25×10-4 ND ND 2-methoxy-6-methylpyrazine ND ND ND ND 4.57×10-3 a 3.33×10-4 3.33×10-3 b 3.26×10-4 Limonene ND ND ND ND 1.15×10-3 a 1.68×10-4 ND ND 3-ethyl-2-methyl-1,3- hexadiene ND ND ND ND ND ND 2.59×10-3 a 8.65×10-5 2-ethyl-1-hexanol 3.19×10-3 c 1.84×10-4 7.42×10-3 b 4.91×10-4 2.09×10-2 a 2.37×10-3 6.65×10-3 b 1.20×10-4 Phenylacetaldehyde ND ND ND ND 1.21×10-3 b 1.10×10-4 4.21×10-2 a 1.46×10-3 (E)-2-octenal 2.63×10-3 b 2.86×10-4 2.52×10-3 b 3.10×10-4 2.29×10-3 b 3.18×10-4 5.91×10-3 a 5.66×10-4 2-octen-1-ol 2.21×10-1 a 2.72×10-2 5.84×10-2 b 3.91×10-3 2.07×10-2 c 2.18×10-3 5.88×10-3 c 2.97×10-4

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Table 4.12 continued Compound F BR D 50 °C DBR 50 °C ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) n-octanol 4.56×10-1 a 2.91×10-2 1.45×10-1 b 4.26×10-3 1.75×10-1 b 1.59×10-2 7.89×10-3 c 3.91×10-4 Linalool ND ND ND ND ND ND 4.68×10-3 a 4.13×10-4 Nonanal 3.44×10-3 b 2.21×10-4 3.18×10-3 b 4.23×10-4 5.64×10-3 a 8.45×10-4 5.78×10-3 a 1.99×10-4

2-ethyl hexanoic acid 3.12×10-4 b 1.93×10-5 1.87×10-4 a 1.57×10-5 ND ND ND ND (E)-2-nonenal 2.59×10-3 b 3.13×10-4 1.74×10-3 b 1.29×10-4 4.03×10-2 a 1.34×10-3 3.59×10-3 b 3.10×10-4 1-nonanol ND ND ND ND ND ND 4.93×10-3 a 5.95×10-4 Ethyl octanoate 3.32×10-2 a 5.55×10-3 6.27×10-4 b 1.01×10-4 4.95×10-3 b 7.90×10-4 ND ND Decanal 1.73×10-3 c 1.84×10-4 1.38×10-3 c 4.37×10-4 3.36×10-3 b 2.08×10-4 7.30×10-3 a 4.85×10-4 2-undecanone ND ND 1.26×10-3 b 2.19×10-4 4.36×10-3 a 1.95×10-4 2.74×10-3 b 5.23×10-4 (E,E)-2,4-decadienal ND ND ND ND ND ND 1.44×10-3 a 4.45×10-4 2,6-bis(1,1- dimethylethyl)phenol 1.65×10-3 d 3.74×10-4 2.80×10-2 b 2.28×10-3 3.51×10-2 a 2.91×10-3 1.38×10-2 c 3.67×10-3 * Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The comparison between different treatments was also aiming at establishing the differences in the behaviour of VC under different conditions used within a particular pattern. Alcohols VC extracted from fresh, boiled under reflux, dried (50 °C) and dried and boiled under reflux samples are compared in Figure 4.52. Again, the concentration of each compound was normalised against the values determined the fresh mushrooms in order to see how these compounds are changing after processing. The normalised relative concentration of 3-methyl-1-butanol was the highest in DBR 50 °C samples which was significantly different from that found in F, BR and DBR. As for 1-octen-3-ol, it had its maximum normalised relative concentration in F samples which was significantly different from the other treatments with no significant difference was found between BR and D samples for this compound but the normalised relative concentration in D and DBR samples was significantly different. This could be due to the rehydration temperature followed by some losses when the samples were boiled under reflux. As for 3-octanone, the highest normalised relative concentration was found in F samples which was not significantly different from D 50 °C samples. However, after boiling the dried samples under reflux, losses of this compound occurred which could be due to evaporation. As for 3-octanol, 2-octen-1-ol, and n-octanol, they had their maximum normalised relative concentration in F samples which were significantly different from those obtained in the other treatments.

1.2

F 1.0 BR D 50 °C 0.8 DBR 50 °C

0.6

0.4

0.2 Normalisedrelativeconcentration 0.0

l l none a ctanol -butanol octano octen-3-o 3- N-o l-1 1- 3-oct 2-octen-1-ol Compound 3-methy

Figure 4.52: Normalised relative concentrations of selected alcohols VC in oyster mushrooms subjected to different treatments.

Aldehydes’ behaviour was different to alcohols as shown in Figure 4.53. The normalised relative concentrations of 3-methyl butanal, (E)-2-octenal, and decanal were the highest in DBR 50 °C

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samples and were significantly different from the other treatments. Octanal had the highest normalised relative concentration in F samples while no significant difference was found for nonanal in D and DBR 50 °C which were significantly different from F and BR samples.

1.2 F BR 1.0 D 50 °C DBR 50 °C

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration 0.0

anal ct ctenal O -o Nonanal Decanal yl butanal h (E)-2 Compound 3-met

Figure 4.53: Normalised relative concentrations of selected aldehydes VC in oyster mushrooms subjected to different treatments.

The normalised relative concentrations of other VC extracted from different treatments are shown in Figure 4.54. The maximum relative concentrations of benzaldehyde and 3-heptanone were found in DBR 50 °C samples which were significantly different from the other treatments. Meanwhile, 1-octen-3-one had its highest normalised value in BR samples and was undetected in D 50 °C samples, while 1-hexanol and 2-ethyl-1-hexanol had their maximum relative concentrations in D 50 °C samples. No significant difference was found between the normalised relative concentrations found for 1-hexanol between F and BR samples while no significant difference was found between BR and DBR samples for 2-ethyl-1-hexanol.

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1.2 F BR 1.0 D 50 °C DBR 50 °C 0.8

0.6

0.4

0.2

0.0 Normalised relativeconcentration -0.2

one 3- ldehyde 1-hexanol enza 3-heptanone -octen- B 1 Ethyl octanoate Compound 2-ethyl-1-hexanol

Figure 4.54: Normalised relative concentrations of other selected VC in oyster mushrooms subjected to different treatments.

The compound 1-octen-3-one was found in fresh oyster mushrooms unlike in the case of A. bisporus where the latter compound was only found in cooked mushrooms. As for 1-octen-3-ol, it is formed from the oxidation of linoleic acid followed by a series of enzymatic reactions while 1- octen-3-one can be synthesised via the oxidation of 1-octen-3-ol. One possible explanation of the formation or the increase in its amount in mushrooms could be that in the presence of heat, 1- octen-3-ol gets oxidised to 1-octen-3-one and according to Figure 4.55, the normalised relative concentration of 1-octen-3-ol decreased upon boiling the samples under reflux while that of 1- octen-3-one increased. The normalised relative concentration of 1-octen-3-ol was the highest in F samples being significantly different from the other treatments. No significant difference was found between BR and D samples for this compound. As for 1-octen-3-one, the maximum normalised relative concentration was found in BR samples which was significantly different from the other treatments.

Maga (1981) also reported that the content of 1-octen-3-one increased during the boiling of champignons with the maximum amount found after 30 min of boiling and therefore, the difference between raw and boiled mushrooms could be in the increase of 1-octen-3-one and the decrease in 1-octen-3-ol since of 1-octen-3-one is a product of the oxidation of 1-octen-3-ol.

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F 4 BR D 50 °C DBR 50 °C

0 Normalisedrelativeconcentration

1-octen-3-ol 1-octen-3-one Compound

Figure 4.55: Behaviour of 1-octen-3-ol and 1-octen-3-one in oyster mushrooms subjected to different treatments.

The compounds 3-methyl butanal, 2-methyl butanal, 3-heptanone, butanoic acid, 3-methyl, ethyl ester, 2-ethyl hexanoic acid, ethyl octanoate, (E)-2-octenal, 2-ethyl-1-hexanol, (E)-2-nonenal and 2-undecanone were not reported by Misharina et al. (2009a). Ethyl octanoate has been previously identified in Fragaria chiloensis berries and was characterised as having sweet and fruity aroma while 2-ethyl 1-hexanol was described as having a green aroma (Prat et al., 2013).

Some of the compounds identified in oyster mushrooms were also reported in red raspberries by Klesk et al. (2004) and include 2-undecanone, which was described as having a floral or citrus note, hexanal (green fruity), 1-octen-3-one (mushroom-like), 1-hexanol (sweet, watermelon), octanol (herbal, floral), linalool (floral citrus), hexanal (apple-like) and nonanal (floral and fruity).

In this study, phenylacetaldehyde was not found in raw mushrooms but rather in dried and dried and boiled under reflux mushrooms. However, Zhang et al. (2008) identified this compound in raw mushrooms using steam distillation which involved heating the samples.

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4.4 VC in Lentinus edodes (Shiitake mushrooms)

4.4.1 Fresh and stored shiitake mushrooms

The VC behaviours of fresh shiitake samples stored over 10 days (see section 3.1.2) were also studied and are shown in Table 4.13. The compound 3-octanone was the major VC found at D0 with a concentration of 4.21×100±4.84×10-1 ppm followed by 3-octanol (1.39×100±1.21×10-1 ppm), 1-octen-3-ol (6.24×10-1±6.34×10-2 ppm), 2-octen-1-ol (5.04×10-1±1.73×10-2 ppm), n- octanol (2.22×10-1±3.79 ×10-2 ppm), dimethyl disulphide (1.17×10-1±1.20×10-2 ppm), 1-octen3- one (5.52×10-2±4.44×10-3 ppm), (3E)-1,3-octadiene (5.09×10-2±2.38×10-3 ppm) and dimethyl trisulfide (4.74×10-2±4.56×10-3 ppm). At D1, 3-octanone was also the main VC detected (4.12×100±8.31×10-1 ppm) followed by 3-octanol (1.21×100±4.14×10-1 ppm) with both concentrations decreasing and a similar pattern was also observed for D2 for these 2 compounds. At D3, the concentration of 3-octanone significantly decreased to reach 1.94×100±4.64×10-1 ppm and then it significantly decreased at D7 to 5.18×10-1±2.87×10-2 ppm. No significant change occurred between D7 and D10. As for 3-octanol, its concentration marginally decreased with storage and reached a minimum of 3.07×10-1±2.93×10-2 ppm at D10. No significant change occurred between D0, D1 and D2, and between D7 and D10. The compound 1-octen-3-ol had its highest concentration at D0 (6.24×10-1±6.34×10-2 ppm) then it significantly decreased to reach a minimum of 2.02×10-3±3.20×10-4 ppm at D10 while 1-octen-3-one had its highest concentration at D0 (5.52×10-2±4.44×10-3 ppm) which then significantly decreased at D1 to 3.67×10-3±4.85×10-4 ppm and further storage did not have any significant impact on its concentration. Meanwhile, 2- octen-1-ol showed no significant decrease between D0 and D2, however, at D3, its concentration significantly decreased to reach 1.03×10-1±2.58×10-2 ppm. No significant difference was found between D3 and D4, and between D7 and D10 reaching a minimum of 1.02×10-2±3.32×10-3 ppm at D10. As for n-octanol, no significant change was observed between D0 and D2 but at D3, its concentration significantly decreased to reach 1.20×10-1±3.88×10-2 ppm compared to D0 and reached a minimum of 2.11×10-2±3.31×10-3 ppm at D10. No significant difference was found between D3 and D4, and between D4 and D7. As for (3E)-1,3-octadiene, it had its highest concentration at D0 (5.09×10-2±2.38×10-3 ppm) then it significantly decreased at D1 (4.31×10-2 ±2.86×10-3 ppm), D2(3.72×10-2±2.50×10-3 ppm), and D3 (9.17×10-4±6.40×10-5 ppm). Further storage did not have a significant impact on the concentration of this compound. Dimethyl disulphide had its lowest concentration at D0 (1.17×10-1±1.20×10-2 ppm) which significantly increased at D3 (2.32×10-1±1.17×10-2 ppm) and reached a maximum of 6.82×10-1±3.99×10-2 ppm at D10 which was not significantly different from D7 (5.89×10-1±7.30×10-2 ppm). Dimethyl

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trisulfide also had its lowest concentration at D0 (4.74×10-2±4.56×10-3 ppm) which did not vary significantly at D1 and D2 but significantly increased at D4 (1.46×10-1±1.80×10-2 ppm) to reach a maximum of 3.84×10-1±4.39×10-2 at D10. Carbon disulphide also had its maximum concentration at D10 (1.07×10-2±6.40×10-3ppm) which was significantly different from the remaining storage days. As for 1-pentanol, it had its highest concentration at D2 (2.58×10-3±4.92×10-4 ppm) and its lowest at D10 (6.23×10-4±5.14×10-5 ppm). No significant difference was found between D0, D1 and D2 for 2-ethyl-1-hexanol but a significant increase was observed at D3 (5.78×10-2±3.11×10-3 ppm) and its concentration kept increasing until it reached a maximum of 1.46×10-1±2.73×10-2 ppm at D10. As for (E)-2-octenal, it had its highest concentration at D1 (6.88×10-3±3.67×10-4 ppm) which was not significantly different from D0 (6.09×10-3±1.29×10-4 ppm), however, at D2, its concentration significantly decreased to reach 2.05×10-3±3.77×10-4 ppm. No significant difference was found between D2, D3, D4, and D7. As for 1,2,4-trithiolane, it had the lowest concentration at D0 (9.15×10-3±5.06×10-4 ppm) which significantly increased at D2 (2.45×10-2 ±3.77×10-3 ppm) followed by a small increase until it reached a maximum of 6.14×10-2±6.23×10-3 ppm at D10, significantly different from the remaining storage days. The compound methyl (methylthio) methyl disulphide also had its minimum concentration at D0 (2.49×10-3±2.94×10-4 ppm) which significantly increased with storage until it reached a maximum of 3.39×10-1 ±1.79×10-2 ppm at D10. As for 1,4-dimethyltetrasulfide, its lowest concentration was also observed at D0 (1.41×10-3±3.15×10-4 ppm) with slight increases observed at D1 and D2. At D4, a significant increase was observed (2.70×10-2±1.93×10-3 ppm) and its concentration kept significantly increasing at D7 (4.28×10-2±1.70×10-3 ppm) and D10 (5.76×10-2±6.68×10-3 ppm). As for tris(methylthio) methane, no significant difference was found between D0, D1, D2, and D3 with the lowest concentration found was at D0 (7.20×10-4±5.06×10-5 ppm) but a significant increase was seen at D4 (3.14×10-3±1.47×10-4 ppm) and its concentration kept increasing until it reached 1.55×10-2±1.61×10-3 ppm at D10. Nonanal had its highest concentration at D3 (7.63×10-3 ±1.05×10-3 ppm) which was significantly different from the remaining storage days while its corresponding alcohol, 1-nonanol, had its highest concentration at D4 (4.10×10-3±4.93×10-4 ppm) also significantly different from the other storage days. As for decanal, its minimum concentration was observed at D0 (1.60×10-3±3.41×10-4 ppm) and the maximum at D10 (5.76×10-2±6.68×10-3 ppm) being significantly different from the remaining storage days. Meanwhile the concentration of 2,6-bis(1,1-dimethylethyl)phenol was not affected by storage with a minimum concentration of 2.84×10-3±3.35×10-4 ppm at D1 and a maximum concentration of 1.40×10-2±2.42×10-3 ppm at D4.

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Table 4.13: Concentrations of VC extracted from shiitake mushrooms during 10 days storage. Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD (ppm) ppm SD ppm SD ppm SD ppm SD (ppm) ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) Carbon 5.27×10-3 disulfide 1.39×10-3 b 2.97×10-4 1.46×10-3 b 2.83×10-4 1.24×10-3 b 3.62×10-4 1.70×10-3 b 7.40×10-4 2.28×10-3 b 6.40×10-4 ab 7.79×10-4 1.07×10-2 a 6.40×10-3 Dimethyl disulfide 1.17×10-1 d 1.20×10-2 1.57×10-1 cd 2.40×10-2 1.92×10-1 bcd 2.28×10-2 2.32×10-1 bc 1.17×10-2 2.77×10-1 b 1.75×10-2 5.89×10-1 a 7.30×10-2 6.82×10-1 a 3.99×10-2 1-Pentanol 1.15×10-3 bc 1.38×10-4 6.18×10-4 c 6.20×10-5 2.58×10-3 a 4.92×10-4 1.71×10-3 b 2.98×10-4 1.10×10-3 bc 4.39×10-4 2.81×10-3 a 2.04×10-4 6.23×10-4 c 5.14×10-5 (3E)-1,3- octadiene 5.09×10-2 a 2.38×10-3 4.31×10-2 b 2.86×10-3 3.72×10-2 c 2.50×10-3 9.17×10-4 d 6.40×10-5 1.53×10-3 d 1.65×10-4 2.05×10-3 d 3.13×10-4 1.60×10-3 d 3.16×10-4 Benzaldehyd e ND ND ND ND ND ND ND ND ND ND ND ND ND ND Dimethyl trisulfide 4.74×10-2 e 4.56×10-3 8.01×10-2 de 5.41×10-3 9.69×10-2 cde 9.56×10-3 1.12×10-1 cd 9.36×10-3 1.46×10-1 c 1.80×10-3 2.69×10-1 b 1.45×10-2 3.84×10-1 a 4.39×10-2 1-octen-3- one 5.52×10-2 a 4.44×10-3 3.67×10-3 b 4.85×10-4 1.60×10-3 b 2.34×10-4 1.68×10-3 b 2.28×10-4 1.50×10-3 b 2.10×10-4 1.28×10-3 b 1.93×10-4 7.75×10-4 b 1.16×10-4 5.30×10-2 1-octen-3-ol 6.24×10-1 a 6.34×10-2 3.62×10-1 b 3.77×10-2 2.65×10-1 c 3.69×10-2 1.16×10-1 d 1.90×10-2 9.65×10-2 d 6.97×10-3 de 4.58×10-3 2.02×10-3 e 3.20×10-4 3-octanone 4.21×100 a 4.84×10-1 4.12×100 a 8.31×10-1 5.16×100 a 1.04×100 1.94×100 b 4.64×10-1 4.22×10-1 b 2.54×10-2 5.18×10-1 b 2.87×10-2 5.35×10-1 b 4.44×10-2 3-octanol 1.39×100 a 1.21×10-1 1.21×100 ab 4.14×10-1 1.05×100 abc 1.52×10-1 7.74×10-1 bcd 1.25×10-1 6.38×10-1 cd 1.48×10-1 4.47×10-1 d 6.47×10-2 3.07×10-1 d 2.93×10-2 2-ethyl-1- hexanol 1.16×10-3 c 1.41×10-4 1.71×10-2 c 5.10×10-3 3.07×10-2 c 3.52×10-3 5.78×10-2 b 3.11×10-3 8.15×10-2 b 4.60×10-3 1.21×10-1 a 1.78×10-2 1.46×10-1 a 2.73×10-2 (E)-2-octenal 6.09×10-3 a 1.29×10-4 6.88×10-3 a 3.67×10-4 2.05×10-3 bc 3.77×10-4 1.10×10-3 c 4.41×10-4 1.49×10-3 c 7.23×10-4 1.36×10-3 c 4.74×10-4 2.83×10-3 b 4.51×10-4 2-octen-1-ol 5.04×10-1 a 1.73×10-2 4.04×10-1 a 8.67×10-2 4.06×10-1 a 5.38×10-2 1.03×10-1 bc 2.58×10-2 3.65×10-2 c 8.09×10-3 2.39×10-2 b 1.43×10-3 1.02×10-2 c 3.32×10-3 n-octanol 2.22×10-1 a 3.79×10-2 1.91×10-1 ab 3.56×10-2 1.74×10-1 ab 1.95×10-2 1.20×10-1 bc 3.88×10-2 6.15×10-2 cd 7.92×10-3 3.19×10-2 d 3.29×10-3 2.11×10-2 d 3.31×10-3 1,2,4- trithiolane 9.15×10-3 d 5.06×10-4 1.83×10-2 cd 5.59×10-3 2.45×10-2 b 3.77×10-3 2.93×10-2 bc 4.75×10-3 4.01×10-2 b 6.96×10-3 4.16×10-2 b 2.08×10-3 6.14×10-2 a 6.23×10-3

Nonanal 3.77×10-3 b 4.70×10-4 3.68×10-3 b 6.05×10-4 4.58×10-3 b 7.80×10-4 7.63×10-3 a 1.05×10-3 3.88×10-3 b 6.28×10-4 5.37×10-3 b 2.46×10-4 4.69×10-3 b 5.24×10-4 methyl(meth ylthio)methy l disulfide 2.49×10-3 e 2.94×10-4 5.45×10-2 d 4.45×10-3 8.34×10-2 c 6.20×10-3 9.79×10-2 c 8.85×10-3 1.05×10-1 c 1.00×10-2 1.34×10-1 b 1.24×10-2 3.39×10-1 a 1.79×10-2 nonanol 1.51×10-3 bc 2.45×10-4 8.43×10-4 c 6.95×10-5 1.07×10-3 c 5.41×10-5 9.03×10-4 c 2.05×10-5 4.10×10-3 a 4.93×10-4 2.29×10-3 b 4.31×10-4 3.46×10-3 a 2.98×10-4

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Table 4.13 continued Compound D0 D1 D2 D3 D4 D7 D10 ppm SD* ppm SD (ppm) ppm SD ppm SD ppm SD ppm SD (ppm) ppm SD (ppm) (ppm) (ppm) (ppm) (ppm) Decanal 1.60×10-3 d 3.41×10-4 2.17×10-3 cd 9.86×10-4 3.23×10-3 bcd 4.59×10-4 4.20×10-3 b 4.64×10-4 3.50×10-3 bc 7.94×10-4 4.47×10-3 b 1.85×10-4 6.61×10-3 a 5.96×10-4 1,4- dimethyltetra sulfide 1.41×10-3 e 3.15×10-4 2.39×10-3 e 2.77×10-4 4.23×10-3 de 6.62×10-4 1.04×10-2 d 8.86×10-4 2.70×10-2 c 1.93×10-3 4.28×10-2 b 1.70×10-3 5.76×10-2 a 6.68×10-3

Tris(methylt hio) methane 7.20×10-4 d 5.06×10-5 7.94×10-4 d 5.16×10-5 9.92×10-4 d 1.73×10-4 1.34×10-3 cd 1.32×10-4 3.14×10-3 bc 1.47×10-4 4.19×10-3 b 7.66×10-4 1.55×10-2 a 1.61×10-3 2,6-bis(1,1- dimethylethy l)phenol 4.70×10-3 a 4.80×10-4 2.84×10-3 a 3.35×10-4 1.28×10-2 a 1.76×10-2 1.35×10-2 a 2.91×10-3 1.40×10-2 a 2.42×10-3 1.02×10-2 a 5.63×10-3 9.44×10-3 a 2.69×10-4 * Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The normalised relative concentrations of selected VC (group 1) are shown in Figure 4.56. No significant difference was observed for carbon disulfide when comparing D0 to D7. At D10, its normalised relative concentration significantly increased but was not significantly different from D7. As for dimethyl disulfide, its normalised relative concentrations started to increase slightly throughout the storage trial with a significant increase starting at D3. At D7, its normalised relative concentration significantly increased until it reached the highest value at D10. Meanwhile, (3E)-1,3-octadiene had its maximum normalised relative concentration at D0 which significantly decreased until D3 with further storage not significantly affecting its content. Dimethyl trisulfide showed a significant increase at D3 and D7 and reached a maximum normalised relative concentration at D10 significantly different from the other storage days. As for 3-octanone, its normalised relative concentration was the highest at D2 which was not significantly different from D0 and D1, then at D3, it significantly decreased. Further storage did not significantly affect its content. Meanwhile, (E)-2-octenal had its highest normalised relative concentration at D1 which was not significantly different from D0. However, at D2, a significant decrease was found. As for 1,2,4-trithiolane, it had it lowest normalised relative concentration at D0 followed by a non- significant increase from D2 until D7. At D10, it reached its highest normalised value significantly different from the rest of the days. Meanwhile, decanal had its lowest normalised relative concentration at D0 which was not significantly different from D1 and D2. Compared to D0 and D1, its normalised relative concentration significantly increased at D3 and reached a maximum at D10, significantly different from the remaining storage days.

Based on these results, it can be concluded that shiitake mushrooms can last up to D4 where the important compounds such as the main C8 and a lower concentration of sulphur compounds contribute to the pleasant aroma of shiitake mushrooms. At D7, they can still be consumed; however, the VC profile at D7 is significantly different from the profile at D0 especially with the increase in sulphur content. The reasons for these changes are due to biochemical and microbial changes as well as the nature of the packaging film as already mentioned in section 4.2.1.

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800 Carbon disulfide 700 Dimethyl disulfide (3E)-1,3-octadiene 600 Dimethyl trisulfide 3-octanone concentration

(E)-2-octenal 500 1,2,4-trithiolane Decanal 400 relative

300

200 Normalised

100

0 0 2 4 6 8 10 Day

Figure 4.56: Normalised relative concentrations of selected VC (group 1) and their behaviour over 10 day storage period.

Wu and Yang (2000) found 1-octen-3-ol as the major VC in fresh crushed shiitake followed by 3- octanone. The amount of the former compound decreased with aging (after 3 h) while 3-octanone increased. Hong et al. (1988) also reported 1-octen-3-ol as the major VC in fresh shiitake. However, this study suggests that 3-octanone was the major VC detected. Given the fact that mushrooms were studied within 4 hour of harvesting and vacuum distillation was carried for 4 hours it can be safely suggested that 3-octanone is the major VC in fresh shiitake studied from the first flush compared to previous work with unknown flush cycle. Furthermore, Mau et al. (1992) argued that the content of 1-octen-3-ol changes during the crop cycle, peaking at the third flush in Agaricus bisporus. Benzaldehyde was not detected in fresh shiitake, but was present in the processed samples. One possible explanation is that its concentration is very low and below the detection limit of the instrument and that heating increased its amount.

The normalised relative concentrations of selected C8 compounds are shown in Figure 4.57. The compound 1-octen-3-ol had its highest normalised value at D0 which significantly decreased from D0 to D3. No significant difference was found between D3, D4, and D7 but a significant decrease was observed at D10. As for 1-octen-3-one, its highest normalised relative concentration was found at D0 then it decreased significantly at D1 and further storage did not significantly impact its content. Meanwhile, 3-octanol also showed the highest normalised relative concentration at D0 which significantly decreased at D3. No significant difference was found between D3 and D4, and between D4, D7, and D10. As for n-octanol, its highest normalised relative concentration was also

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found at D0, and then it slightly decreased until D2. No significant difference was found between D2 and D3, and between D3 and D4, and between D4, D7, and D10.

1-octen-3-ol 1-octen-3-one 160 3-octanol n-octanol 140

120

100

0 Normalisedrelativeconcentration -20 0 2 4 6 8 10 Day

Figure 4.57: Normalised relative concentrations of selected main C8 compounds and their behaviour over 10 day storage period.

The normalised relative concentrations of selected VC (group 2) are shown in Figure 4.58. Methyl (methylthio)methyl disulphide had its lowest normalised relative concentration at D0 which significantly increased at D1, D2 and D7 until it reached a maximum at D10. As for tris(methylthio) methane, its normalised relative concentration did not change significantly between D0 and D3, then at D4 (compared to D3), a small increase was found and continued until D7 with a significant increase occurred at D10. Meanwhile, 1,4-dimethyltetrasulfide showed no significant difference in its normalised relative concentration between D0 and D2 and between D2 and D3. However, at D4, its normalised content significantly increased and kept significantly increasing until D10. As for 2-ethyl-1-hexanol, it showed no significant difference between D0 and D2, then at D3 and D7, its normalised relative concentration significantly increased. No significant difference was found between D3 and D4, and between D7 and D10.

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2-ethyl-1-hexanol Methyl(methylthio) methyl disulfide Tris(methylthio) methane 1,4-dimethyltetrasulfide 16000

14000

12000

10000

8000

6000

4000

2000

0 Normalised relative concentration -2000 0 2 4 6 8 10 Day

Figure 4.58: Normalised relative concentrations of selected VC (group 2) and their behaviour over 10 day storage period.

Wu and Yang (2000) reported that 1-octen-3-ol decreased with ageing mushrooms at room temperature while dimethyl disulfide and dimethyl trisulfide increased. These findings are in accordance with the results of this study except that the mushrooms were stored at 4 °C until used.

4.4.2 Drying curves

Four different drying temperatures were used to determine the effect of drying time and temperature and the quality of the mushrooms in terms of their VC content. The moisture ratio of the samples as a function of drying time is presented in Figure 4.59 for 40, 50, 60 and 70 °C drying air temperatures. All lines have two stages: the moisture ratio rapidly declined then slowly decreased with increasing drying time. It can be also seen that drying temperature has a considerable effect on the total drying time. The highest rate of mushroom loss was observed at 70 °C and the lowest at 40 °C. The drying curves at 60 and 70 °C coincided. . The mushrooms were allowed to dry until they reached a constant weight. At 40 °C, the mushrooms were dried for 24 h and the final MC reached was 12.0%. At 50 °C, the drying time was 14 h and the final MC was 5.5% while at 60 °C, the mushrooms were dried for 12 h with a final MC of 4.4% and at 70 °C, the mushrooms were dried for 10 h reaching a final MC of 3.8%.

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1.00

0.90 40 °C 0.80 50 °C 0.70 60 °C 0.60

0.50 70 °C

0.40

MoistureRatio (MR) 0.30

0.20

0.10

0.00 0.00 400.00 800.00 1200.00 1600.00 Time (min)

Figure 4.59: Moisture ratios of shiitake mushrooms at different drying temperatures versus time.

4.4.3 Dried (D) shiitake mushrooms

Aromas of fresh and dried shiitake are significantly different as sensed by the nose and the instrument. The dried shiitake is known for its characteristic aroma being absent in fresh shiitake. For instance, 1-octen-3-ol, known as the mushroom alcohol gives a sweet, earthy odour with a sweet herbaceous taste and is referred to as mushroom alcohol (Chen and Wu, 1984) while 3- octanone offers a sweet, fruity cheese aroma with mushroom taste. Dimethyl trisulphide gives garlic and onion odour (Flavors and fragrances SAFC, 2013) while 1, 2, 4-trithiolane possesses boiled beef or onion aroma (Macleod 1994).

In terms of concentrations (Table 4.14), 3-octanol was the major C8 VC with a concentration of 1.04×100±5.08×10-2 ppm followed by 3-octanone (8.63×10-1±3.80×10-2ppm), 2-octen-1-ol (1.88×10-1±4.39×10-3 ppm) n-octanol (1.24×10-1±1.68×10-2 ppm), and 1-octen-3-ol (1.07×10-1 ±7.16×10-3 ppm) at 40 °C . At 50 and 70 °C, 1-octen-3-ol was the major VC among the C8 compounds followed by 3-octanone but at 60 °C, 3-octanone was the major VC with a concentration of 4.76×10-1±8.17×10-2 ppm. The concentration of 1-octen-3-ol was significantly different between 40 °C and 50 °C (1.07×10-1±7.16×10-3 vs. 1.57×100±7.30×10-2 ppm), 50 °C and 60 °C (1.57×100±7.30×10-2 vs. 1.39×10-1±2.34×10-2 ppm), and 50 °C and 70 °C samples (1.57×100±7.30×10-2 vs. 7.08×10-2±5.88×10-3 ppm) (p > 0.05) but was not significantly different

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between 40, 60 and 70 °C. The concentration of 3-octanone was significantly different in the samples dried at four different temperatures with the highest found was at 50 °C (1.50×100±6.98×10-2 ppm) and the lowest at 70 °C (4.88×10-2±3.62×10-2 ppm). As for 3-octanol, its concentration was significantly different in all the treatments with the highest concentration found was at 40 °C (p>0.05) with no significant difference found between drying at 60 and 70 °C (2.41×10-2±4.65×10-3ppm and 5.50×10-3±3.57×10-4ppm respectively). The concentration of n- octanol was also affected by the drying times and temperatures and was found to be not significantly different when comparing 40 °C and 50 °C samples (1.24×10-1±1.68×10-2 vs. 1.63×10-1± 2.27×10-2 ppm) and between 60 °C and 70 °C (1.40×10-2±1.05×10-3 vs. 4.70×10-3 ±4.37×10-4 ppm). As for 2-octen-1-ol, its highest concentration was observed at 40 °C (1.88×10-1 ±4.39×10-3 ppm) which was significantly different from that obtained at 50 °C (9.27×10-2 ±6.24×10-3 ppm), 60 °C (1.98×10-2±1.38×10-3 ppm) and 70 °C (5.51×10-3±3.73×10-4 ppm).

As for the sulphur compounds, dimethyl trisulfide was found to be peaking at 40 °C (1.19×100±1.66×10-1ppm) and not being significantly different from 50 °C, (1.35×100±6.32×10-2 ppm) ( p<0.05) but was then greatly reduced when the temperature increased to 60 °C (2.98×10-2± 1.22×10-3 ppm) and 70 °C (1.20×10-2±3.23×10-3ppm). Dimethyl disulphide was the second highest sulphur VC detected with the highest concentration at 40 °C (6.29×10-1±8.36×10-2 ppm) which then significantly decreased when the temperature reached 50 °C (2.55×10-1±3.09×10-2 ppm), and 60 °C (1.33×10-2±1.84×10-3 ppm). No significant difference was found between 60 ° C and 70 °C samples. The concentration of carbon disulphide was found to be the highest at 50 °C (1.71×10-1±1.09×10-2 ppm) which was significantly higher than that found at 40 °C (2.32×10-3± 5.57×10-4 ppm), 60 °C (1.40×10-2±3.42×10-3 ppm) and 70 °C (2.88×10-2±1.88×10-3 ppm). A 10 °C increase in temperature from 40 °C to 50 °C almost tripled the concentration of 1, 2, 4-trithiolane (1.07×10-2±1.63×10-3 ppm vs. 3.69×10-1±4.77×10-2 ppm). A 20 °C increase in temperatures from 40 to 60 °C had doubled the concentration of 1,2,4-trithiolane (1.07×10-2±1.63×10-3 ppm vs. 2.45×10-1±4.78×10-2 ppm). However, at 70 °C, its concentration decreased significantly compared to 50 and 60 °C (1.51×10-1±3.59×10-3 ppm). As for 1,3,5-trithiane, increasing the drying temperature from 40 to 50 °C did not significantly affect its concentration (2.19×10-3±7.97×10-4 vs. 1.56×10-2±7.08×10-4 ppm), however, at 60 °C, its concentration was significantly decreased to 4.76×10-4±6.63×10-5 ppm which was not significantly different from that found at 70 °C (1.70× 10-4±7.39×10-6 ppm). As for tris(methylthio) methane, 40 °C resulted in the maximum retention of this compound (3.97×10-3±4.55×10-4 ppm) which was significantly different from the other drying temperatures. The compound s-methyl methane thiosulphonate had its highest concentration at

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70 °C(6.96×10-3±7.12×10-4 ppm) which was not significantly different from 50 °C samples but significantly different from 40 °C (2.97×10-3±3.19×10-4 ppm) and 60 °C samples (4.76×10-3 ±3.62×10-4 ppm). As for methyl (methylthio) methyl disulfide, drying at 50 °C resulted in the highest concentration of this compound (1.72×10-1±3.37×10-3 ppm) which was significantly different from that found at 40 °C (1.17×10-3±1.56×10-4 ppm). Increasing the drying temperature to 60 °C and 70 °C significantly decreased its concentration (5.67×10-3±2.44×10-4 ppm and 2.23×10-3±8.79×10-5 ppm respectively). No significant difference between the 60 °C and 70 °C drying temperature was found for this compound. As for 1,4-dimethyltetrasulfide, its relative concentration significantly increased when the temperature increased from 40 °C to 50 °C (4.28×10-3±3.66×10-4 vs. 2.33×10-2±1.42×10-3ppm). However, a further increase in temperature from 50 °C to 60 °C significantly decreased its concentration (2.33×10-2±1.42×10-3ppm vs. 5.23×10-4±6.45×10-5 ppm). No significant difference was found between drying at 60 and 70 °C for this compound. The compound 2-(methylthio)acetic acid had its highest concentration at 40 °C (2.96×10-3±1.34×10-4 ppm) which was not significantly different from that determined at 60 °C (2.53×10-3±3.73×10-4 ppm) but significantly different from that obtained at 50 °C (2.05×10-3 ±9.16×10-4 ppm) and 70 °C (2.02×10-3±4.26×10-4 ppm). Meanwhile, 1, 2, 4, 5-tetrathiane had its highest concentration at 40 °C (4.74×10-2±5.05×10-3 ppm) which significantly decreased when the temperature was increased to 50 °C to reach a concentration of 1.30×10-2±2.27×10-3 ppm. At 60 °C, its concentration was significantly lower than that found at 40 °C but not significantly different from that determined at 70 °C (2.81×10-2±5.95×10-4 ppm). As for 1,2,4,6-tetrathiepane, 40 °C resulted in the maximum retention of this compound (1.24×10-1±2.49×10-2 ppm) which was significantly different from that found at 50 °C (4.24×10-2±6.05×10-3 ppm), 60 °C (3.60×10-2 ±2.44×10-3 ppm), and 70 °C (6.31×10-2±8.14×10-4 ppm). Lenthionine had its highest concentration at 70 °C (6.38×10-2±3.95×10-3 ppm) and its lowest at 40 °C and (3.93×10-3± 5.57×10-4 ppm) respectively. No difference was found between 50 °C and 60 °C (4.87×10-2 ±1.85×10-3 ppm and 4.71×10-2 ±6.49×10-3 ppm).

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Table 4.14: Concentrations of VC extracted from shiitake mushrooms dried at different temperatures. Compound D 40 °C D 50 °C D 60 °C D 70 °C ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) Propanal 2.59×10-3 bc 1.20×10-4 1.48×10-3 c 8.11×10-5 4.90×10-3 a 1.06×10-3 3.45×10-3 ab 3.34×10-4 Carbon disulfide 2.32×10-3 c 5.57×10-4 1.71×10-1 a 1.09×10-2 1.40×10-2 bc 3.42×10-3 2.88×10-2 b 1.88×10-3 3-methyl butanal 1.43×10-3 b 3.83×10-4 2.03×10-3 b 4.16×10-4 2.46×10-3 b 4.15×10-4 3.75×10-3 a 4.69×10-4 Pentanal 1.83×10-3 b 7.06×10-4 2.55×10-3 b 3.48×10-4 1.14×10-2 a 6.16×10-3 5.74×10-3 ab 8.31×10-4 Dimethyl disulfide 6.29×10-1 a 8.36×10-2 2.55×10-1 b 3.09×10-2 1.33×10-2 c 1.84×10-3 1.73×10-2 c 1.00×10-3 Hexanal 2.57×10-3 c 3.88×10-4 2.07×10-2 a 2.36×10-3 1.16×10-2 b 1.05×10-3 1.56×10-2 b 1.71×10-3 (3E)-1,3-octadiene 1.06×10-3 b 1.54×10-4 1.50×10-2 ab 1.43×10-2 2.13×10-2 a 4.05×10-3 1.56×10-2 ab 2.66×10-3 3-heptanone 4.67×10-4 c 2.43×10-5 1.02×10-3 b 5.06×10-5 1.45×10-3 b 2.15×10-4 3.53×10-3 a 3.58×10-4 3-(methylthio)-butanal 3.31×10-3 c 2.66×10-4 6.08×10-3 b 5.68×10-4 6.85×10-3 b 4.96×10-4 8.38×10-3 a 8.04×10-4 Benzaldehyde 7.83×10-3 b 1.57×10-3 1.14×10-2 b 2.03×10-3 7.04×10-2 a 7.99×10-3 7.34×10-2 a 4.18×10-3 Dimethyl trisulfide 1.19×100 a 1.66×10-1 1.35×100 a 6.32×10-2 2.98×10-2 b 1.22×10-3 1.20×10-2 b 3.23×10-3 S-methyl methane thiosulphonate 2.97×10-3 c 3.19×10-4 6.48×10-3 a 8.07×10-4 4.76×10-3 b 3.62×10-4 6.96×10-3 a 7.12×10-4 1-octen-3-ol 1.07×10-1 b 7.16×10-3 1.57×100 a 7.30×10-2 1.39×10-1 b 2.34×10-2 7.08×10-2 b 5.88×10-3 3-octanone 8.63×10-1 b 3.80×10-2 1.50×100 a 6.98×10-2 4.76×10-1 c 8.17×10-2 4.88×10-2 d 3.62×10-2 3-octanol 1.04×100 a 5.08×10-2 6.97×10-1 b 3.68×10-2 2.41×10-2 c 4.65×10-3 5.50×10-3 c 3.57×10-4 Octanal 6.41×10-3 b 7.79×10-4 6.01×10-3 b 5.48×10-4 1.24×10-2 a 1.35×10-3 1.47×10-2 a 1.73×10-3 Furfuryl alcohol 2.70×10-3 a 3.87×10-4 1.53×10-3 b 5.42×10-4 5.38×10-4 c 3.80×10-5 3.80×10-4 c 6.34×10-5 3.71×10-3 a 4.29×10-4 2.16×10-3 b 3.40×10-4 1.57×10-3 b 5.48×10-4 2.38×10-3 b 2.63×10-4 1-(2-furyl)-3-methyl-3-butene-1,2-diol 2-ethyl-1-hexanol 3.61×10-3 b 7.87×10-4 8.43×10-3 a 5.54×10-4 4.61×10-3 b 8.17×10-4 8.50×10-3 a 5.53×10-4 Phenylacetaldehyde 2.99×10-3 b 6.07×10-4 1.55×10-2 a 2.72×10-3 2.33×10-2 a 3.20×10-3 1.66×10-3 c 2.71×10-4 1-phenylethanone 2.88×10-3 a 1.18×10-4 2.03×10-3 ab 5.87×10-4 1.49×10-3 b 4.53×10-4 1.66×10-3 b 2.71×10-4 2-octen-1-ol 1.88×10-1 a 4.39×10-3 9.27×10-2 b 6.24×10-3 1.98×10-2 c 1.38×10-3 5.51×10-3 d 3.73×10-4 n-octanol 1.24×10-1 a 1.68×10-2 1.63×10-1 a 2.27×10-2 1.40×10-2 b 1.05×10-3 4.70×10-3 b 4.37×10-4 1,2,4-trithiolane 1.07×10-2 c 1.63×10-3 3.69×10-1 a 4.77×10-2 2.45×10-1 b 4.78×10-2 1.51×10-1 c 3.59×10-3 Nonanal 1.11×10-2 a 1.34×10-3 1.16×10-2 a 7.61×10-3 5.31×10-3 b 6.18×10-4 4.74×10-3 b 3.27×10-4

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Table 4.14 continued Compound D 40 °C D 50 °C D 60 °C D 70 °C ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm) 1,3,5-trithiane 2.19×10-3 a 7.97×10-4 1.56×10-2 a 7.08×10-4 4.76×10-4 b 6.63×10-5 1.70×10-4 b 7.39×10-6 Phenyl ethyl alcohol 2.64×10-3 b 1.59×10-4 5.07×10-3 a 4.82×10-4 3.09×10-3 b 7.34×10-4 3.08×10-3 b 4.01×10-4 Methyl(methylthio)methyl disulfide 1.17×10-3 c 1.56×10-4 1.72×10-1 a 3.37×10-3 5.67×10-3 b 2.44×10-4 2.23×10-3 b 8.79×10-5 Isooctanol 1.43×10-3 c 1.06×10-4 1.95×10-3 ab 2.84×10-4 1.52×10-3 bc 2.87×10-4 2.24×10-3 a 1.89×10-4 1-nonanol 1.18×10-2 c 1.75×10-3 1.74×10-2 bc 5.33×10-3 2.96×10-2 b 2.59×10-3 6.01×10-2 a 5.36×10-3 Decanal 1.75×10-2 a 1.27×10-3 7.03×10-3 b 6.92×10-4 3.41×10-3 c 3.86×10-4 3.84×10-3 c 3.27×10-4 1,4-dimethyltetrasulfide 4.28×10-3 b 3.66×10-4 2.33×10-2 a 1.42×10-3 5.23×10-4 c 6.45×10-5 2.64×10-4 c 7.41×10-6 Tris(methylthio),methane 3.97×10-3 a 4.55×10-4 2.19×10-3 b 7.43×10-4 1.47×10-3 b 2.29×10-4 1.40×10-3 b 1.64×10-4 2-(methylthio)acetic acid 2.96×10-3 a 1.34×10-4 2.05×10-3 b 9.16×10-4 2.53×10-3 ab 3.73×10-4 2.02×10-3 b 4.26×10-4 7.37×10-4 b 7.70×10-5 3.11×10-3 ab 8.61×10-4 4.24×10-3 a 5.48×10-4 4.62×10-3 a 3.47×10-4 2-formyl-1-isopentyl pyrrole 2-undecanone 6.82×10-3 a 5.34×10-4 1.67×10-3 c 7.48×10-4 5.34×10-3 b 1.97×10-4 5.66×10-3 ab 3.66×10-4 1,2,4,5-tetrathiane 4.74×10-2 a 5.05×10-3 1.30×10-2 c 2.27×10-3 2.91×10-2 b 5.40×10-3 2.81×10-2 b 5.95×10-4 2,4-dimethyl pyridine 1.32×10-1 a 2.69×10-2 1.05×10-1 a 1.03×10-2 5.40×10-3 b 7.43×10-4 4.40×10-3 b 3.97×10-4 1,2,4,6-tetrathiepane 1.24×10-1 a 2.49×10-2 4.24×10-2 b 6.05×10-3 3.60×10-2 b 2.44×10-3 6.31×10-2 b 8.14×10-3 2,6-bis(1,1-dimethyl)phenol 3.30×10-3 a 5.30×10-4 3.89×10-3 a 4.51×10-4 4.13×10-3 a 3.52×10-4 2.63×10-3 a 1.90×10-3 Lenthionine 3.93×10-3 c 5.57×10-4 4.87×10-2 b 1.85×10-3 4.71×10-2 b 6.49×10-3 6.38×10-2 a 3.95×10-3 * Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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As for aldehydes, propanal had its highest concentration at 60 °C (4.90×10-3±1.06×10-3 ppm) which was not significantly different from that determined at 70 °C (3.45×10-3±3.34×10-4 ppm), but significantly different from those found at 40 °C (2.59×10-3±1.20×10-4 ppm) and 50 °C (1.48×10-3±8.11×10-5 ppm). As for 3-methyl butanal, it had its highest concentration at 70 °C (3.75×10-3±4.69×10-4 ppm) which was significantly different from those obtained at 40 °C (1.43×10-3±3.83×10-4 ppm), 50 °C (2.03×10-3±4.16×10-4 ppm), and 60 °C (2.46×10-3±4.15×10-4 ppm) which were not significantly different when compared against each other. Pentanal had its maximum concentration at 60 °C (1.14×10-2±6.16×10-3 ppm) which was not significantly different from 70 °C but significantly higher than that determined at 40 °C (1.83×10-3±7.06×10-4 ppm). Hexanal had its highest concentration at 50 °C (2.07×10-2±2.36×10-3 ppm) which was significantly different from the lowest concentration determined at 40 °C (2.57×10-3±3.88×10-4 ppm). No significant difference was found between samples dried at 60 or 70 °C for this compound. Meanwhile, 3-(methylthio) butanal had its highest concentration at 70 °C (8.38×10-3±8.04×10-4 ppm) which was significantly higher than its lowest concentration found at 40 °C (3.31× 10-3±2.66×10-4 ppm). No significant difference was found between drying at 60 or 70 °C for benzaldehyde (7.04×10-2±7.99×10-3 vs. 7.34×10-2±4.18×10-3 ppm) with both concentrations being significantly higher than that determined at 40 °C (7.83×10-3±1.57×10-3 ppm) and 50 °C (1.14× 10-2±2.03×10-3 ppm). The behaviour of octanal was similar to that of benzaldehyde with its maximum concentration found at 70 °C (1.47×10-2±1.73×10-3 ppm). As for phenylacetaldehyde, its highest concentration found was at 60 °C (2.33×10-2±3.20×10-3 ppm) which was not significantly different from 50 °C but significantly higher than 40 °C (2.99×10-3±6.07×10-4 ppm) and 70 °C (1.66×10-3±2.71×10-4 ppm). As for nonanal, no significant difference was found between drying at 40 °C (1.11×10-2±1.34×10-3 ppm) or 50 °C (1.16×10-2±7.61×10-3 ppm), however, increasing the drying temperature to 60 and 70 °C significantly decreased its concentration (5.31×10-3±6.18×10-4 and 4.74×10-3±3.27×10-4 ppm respectively) . Meanwhile, decanal had its maximum concentration at 40 °C (1.75×10-2±1.27×10-3 ppm), significantly higher than the concentration found at 50, 60, and 70 °C.

As for the ketones, 3-heptanone had its highest concentration at 70 °C (3.53×10-3±3.58×10-4 ppm) significantly higher than its lowest concentration determined at 40 °C (4.67×10-4±2.43×10-5 ppm). Meanwhile, 2-undecanone showed no significant difference between drying at 40 or 70 °C and had its lowest concentration at 50 °C (1.67×10-3±7.48×10-4 ppm).

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The compound (3E)-1,3-octadiene had its maximum concentration at 60 °C (2.13×10-2±4.05×10-3 ppm) which was significantly different from that found at 40 °C but not significantly different from those found in 50 and 70 °C samples. As for 1-phenylethanone, no significant difference was found between drying at 40 and 50 °C (2.88×10-3±1.18×10-4 vs. 2.03×10-3±5.87×10-4 ppm). Furfuryl alcohol had its maximum concentration at 40 °C (2.70×10-3±3.87×10-4 ppm) and the lowest at 70 °C (3.80×10-4±6.34×10-5 ppm) which was not significantly different from that obtained at 60 °C. As for 2-ethyl-1-hexanol, no significant difference was found between drying at 50 or 70 °C (8.43×10-3±5.54×10-4 vs. 8.50×10-3±5.53×10-4ppm). However, drying at 40 or 60 °C resulted in a significantly lower concentration. Drying at 50 °C resulted in the maximum retention of phenyl ethyl alcohol (5.07×10-3±4.82×10-4 ppm) with no significant difference between 40, 60, and 70 °C while 2,4-dimethylpyridine had its maximum concentration at 40 °C (1.24×10-1 ±2.49×10-2 ppm) which was significantly different from 50, 60 and 70 °C. Isooctanol and 1- nonanol had their highest concentrations at 70 °C (2.24×10-3±1.89×10-4 and 6.01×10-2±5.36×10-3 ppm respectively) significantly different from those determined at the remaining drying temperatures. The compound 1-(2-furyl)-3-methyl-3-butene-1,2-diol had its maximum concentration at D 40 °C (3.71×10-3±4.29×10-4 ppm) which was significantly different from those determined at the other drying temperatures. Meanwhile, 2-formyl-1-isopentyl pyrrole had its highest concentration at 70 °C (4.62×10-3±3.47×10-4 ppm) which was not significantly different from 50 and 60 °C samples but significantly different from 40 °C samples. Finally, 2,6-bis(1,1- dimethylethy)phenol showed no significant difference between the concentrations of samples dried under different conditions with the maximum concentration found at D 60 °C (4.13×10-3 ±3.52×10-4 ppm).

Even though the drying curves of 60 and 70 °C were close, the concentrations of VC were different. Possiible explanation could be due to the fact that lower drying temperatures results in longer drying time causing more losses of VC due to evaporation as opposed to higher drying temperature and shorter drying time. Other possible explanation is that the formation or release of some compounds requires a specific temperature or range of temperautres. Nevertherless, the rehydration temperature plays a key role in the liberation of VC from the dried material.

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Shiitake mushrooms dried at different temperatures are shown in figure 4.60 and 4.61 below.

Figure 4.60: Vacuum packed shiitake mushrooms dried at 40 °C (Left) and 50 °C (Right) (Author photograph).

Figure 4.61: Vacuum packed shiitake mushrooms dried at 60 °C (Right) and 70 °C (Left) (Author photograph).

Different drying temperatures and times affect the VC in different ways. Figure 4.62 shows the normalised relative concentration of the main C8 compounds. The maximum retention of 1-octen- 3-ol and 3-octanone was at 50 °C being significantly different when compared to the amount retained at 40, 60 and 70 °C. As for 3-octanol and 2-octen-1-ol, they had their maximum relative concentration at 40 °C and were significantly different when compared to 50, 60 and 70 °C (p>0.05). As for n-octanol, its highest normalised relative concentration was at 50 °C which was

not significantly different from that determined at 40 °C but significantly different from 60 and 70 °C. Meanwhile, isooctanol had its maximum normalised relative concentration at 70 °C which was not significantly different from 50 °C but significantly different from those found in 40 and 60 °C samples.

1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalised relative concentration 0.0

-ol ne ol -ol ol ol -3 no tan -1 tan tan ten ta oc ten oc oc c -oc 3- c n- so 1-o 3 2-o I Compound

Figure 4.62: Normalised relative concentration of the main C8 in shiitake mushrooms dried at different temperatures.

As for the sulphur compounds (Figure 4.63), drying at 50 °C resulted in the highest normalised relative concentration of carbon disulfide which was significantly different from the other drying temperatures while drying at 40 °C resulted in the maximum preservation of dimethyl disulfide and 1,2,4,5-tetrathiane . As for dimethyl trisulfide, no significant difference was found between drying at 40 or 50 °C but the normalised relative concentration significantly decreased when the drying temperature reached 60 or 70 °C. The compounds methyl(methylthio)methyl disulphide, 1,2,4 trithiolane and 1,4-dimethyltetrasulfide had their highest normalised relative concentrations at 50 °C which were significantly different from those determined at 40, 60, and 70 °C.

Drying at 60 °C and 70 °C was found to be harsh on the sulphur compounds except for lenthionine which showed the highest amount retained at 70 °C. When compared to 40 °C, it can be seen that most of the lenthionine is retained which could be explained by the fact that higher temperature is needed for the formation or release of this compound.

193

D 40 °C 1.2 D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

e e e e e e e e fid lfid lfid lan lfid lfid ian nin sul isu isu hio su su th hio di d tr rit ,di tra tra nt on yl yl -t yl lte -te Le rb eth eth ,2,4 eth hy 4,5 Ca im im 1 )m et ,2, D D hio dim 1 ylt ,4- eth 1 l(m thy Compound Me

Figure 4.63: Normalised relative concentrations of the main sulphur compounds in shiitake mushrooms dried at different temperatures.

The normalised relative concentrations of aldehydes are shown in Figure 4.64. Drying at 70 °C resulted in the maximum retention of 3-methyl butanal and 3-(methylthio) butanal which were significantly different from those obtained at the remaining drying conditions. The normalised relative concentration of benzaldehyde and octanal was also the highest at 70 °C but was not significantly different from that found at 60 °C but significantly different from those found at 40 and 50 °C. The normalised relative concentration of hexanal was the highest at 50 °C and significantly different from 40, 60 and 70 °C. Nonanal showed no significant difference between 40 and 50 °C but a significant loss is observed at 60 and 70 °C. As for decanal, its highest normalised relative concentration was observed at 40 °C while phenylacetaldehyde had its maximum normalised relative concentration at 60 °C which was not significantly different from that found at 50 °C but significantly higher than those found at 40 and 70 °C.

194

D 40 °C 1.4 D 50 °C D 60 °C 1.2 D 70 °C

1.0

0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

al al al de al de al al tan an an hy an hy an an bu ex but de ct de on ec yl H io) zal O tal N D eth lth en ace -m thy B nyl 3 me he 3-( P Compound

Figure 4.64: Normalised relative concentration of aldehydes VC in shiitake mushrooms dried at different temperatures.

The normalised relative concentrations of selected alcohols and ketones are shown in Figure 4.65. Drying at 70 °C resulted in the maximum retention of 3-heptanone and 1-nonanol being significantly different from the other drying temperatures while furfuryl alcohol had its maximum normalised relative concentration at 40 °C significantly different from that determined at 50, 60, and 70 °C. As for 2-ethyl-1-hexanol, no significant difference in the normalised relative concentration was found between drying at 50 and 70 °C, both being significantly higher when compared to 40 and 60 °C. Meanwhile, phenyl ethyl alcohol had its highest normalised relative concentration at 50 °C which was significantly higher than those obtained at 40, 60, and 70 °C. As for 2-undecanone, its maximum normalised relative concentration was found at 40 °C which was not significantly different from that found at 70 °C but significantly higher than those found at 50 and 60 °C.

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1.2 D 40 °C D 50 °C D 60 °C 1.0 D 70 °C

0.8

0.6

0.4

0.2 Normalisedrelativeconcentration 0.0 ne hol nol hol nol ne ano lco xa lco na ano ept l a -he l a -no ec -h ry l-1 thy 1 nd 3 rfu hty l e 2-u Fu 2-e eny Ph Compound

Figure 4.65: Normalised relative concentrations of selected alcohols and ketones in shiitake mushrooms dried at different temperatures.

Pyrroles are heterocyclic compounds formed in the Maillard reaction imparting sweet and corn- like to caramel-like aromas. Their mechanism of formation is similar to thiophenes which involves Maillard model systems comprising a sugar and a sulphur-containg amino acid (Boelens et al., 1971).

4.4.4 Dried and boiled under reflux (DBR) shiitake mushrooms

The effect of boiling under reflux of the shiitake samples dried under different conditions was also studied. One assumption was made that the boiling under reflux is carried in a closed system where the “cooked” shiitake do not lose any volatiles due to evaporation but are rather condensed back to the flask where the sample is being boiled. Furthermore, boiling under reflux of the dried samples is an imitation of the “cooking process” carried by the consumer, allowing to evaluate the effect of rehydration using boiling water and hence liberation of VC from the dried samples.

The concentrations of the VC identified are shown in Table 4.15. In DBR 40 °C samples, 1-octen- 3-ol was the major compound detected (2.11×100±9.85×10-2 ppm) then it significantly decreased with increasing temperature (1.06×100±4.93×10-2 ppm at 50 °C to 1.34×10-1±4.06×10-2ppm at 60 °C. No significant difference was found between DBR 60 and 70 °C samples. As for 1-octen-3- one, it had its highest concentration in DBR 40 °C samples (1.41×10-1±1.94×10-2 ppm) which significantly decreased with increasing temperatures (5.22×10-2±4.43×10-3 ppm at 50 °C, 2.83× 10-3±4.16×10-4 ppm at 60 °C, and 3.19×10-3±3.60×10-4ppm at 70 °C with no significant difference

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observed between DBR 60 and 70 °C samples). Meanwhile, 3-octanol showed no significant difference between DBR 40 and 50 °C (2.38×10-1±5.38× 10-2ppm vs. 2.06×10-1±2.93×10-2 ppm), but in DBR 60 °C samples, its concentration significantly decreased to reach 1.30×10-2±1.86×10-3 ppm and in DBR 70 °C samples, it showed an insignificant decrease in its concentration compared to DBR 60 °C. The compounds 2-octen-1-ol and n-octanol showed similar behaviour to 3-octanol with the highest concentration in DBR 40 °C (1.55×10-1±3.25×10-2 and 5.34×10-2±3.67×10-3 ppm respectively) not significantly different from DBR 50 °C but significantly different from DBR 70 °C (8.75×10-3±1.96×10-4 and 1.56×10-3±1.58×10-4 ppm respectively) which were not significantly different from those found in DBR 60 °C samples.

As for the sulphur compounds, carbon disulphide had its highest concentration in DBR 60 °C samples which was not significantly different from that found in DBR 50 °C (2.69×10-2±3.95×10-3 vs. 1.98×10-2 ±2.59×10-3 ppm). No significant difference was found between DBR 40 and 70 °C for this compound. Boiling under reflux of the dried shiitake at 50 °C significantly affected the dimethyl disulphide and dimethyl trisulphide content with their concentrations greatly decreasing (4.41×10-1±4.25×10-2 ppm in DBR 40 °C vs. 2.35×10-1±3.67×10-2 ppm in DBR 50 °C for the dimethyl disulphide and 4.70×10-1±3.98×10-2 ppm in DBR 40 °C vs. 3.13× 10-1±3.81×10-2 ppm in DBR 50 °C for dimethyl trisulphide). As for 1, 2, 4-trithiolane, its concentration significantly increased from 1.43×10-1±2.83×10-2 ppm in DBR 40 °C samples to 5.69×10-1±3.71×10-2 ppm in DBR 50 °C samples and its lowest concentration was found in DBR 70 °C samples (1.19×10-1 ±1.88×10-2 ppm), not significantly different from DBR 40 °C samples. Meanwhile, 1,3,5-trithiane had its maximum concentration in DBR 40 °C samples (5.68×10-3±3.65×10-4 ppm) which was significantly different from its lowest concentration found in DBR 70 °C samples (4.82×10-4 ±3.35×10-5 ppm). As for methyl(methylthio)methyl disulphide, no significant difference was found between DBR 40 and 50 °C (2.00×10-1±1.95×10-2 vs. 1.69×10-1±1.53×10-2 ppm), however increasing the drying temperature to 60 °C followed by boiling under reflux significantly decreased its concentration reaching a minimum of 2.94×10-2±2.03×10-3 in DBR 60 °C samples. The compounds 1,4-dimethyltetrasulfide, tris(methylthio) methane, and 2-(methylthio)acetic acid had their maximum concentrations in DBR 40 °C samples (1.46×10-2±3.08×10-3 ppm, 5.21×10-4 ±3.50×10-5 ppm, and 5.36×10-4±4.44×10-5 ppm respectively) which were significantly different from those found in DBR 50, 60, and 70 °C samples. Meanwhile, 1,2,4,5-tetrathiane had its highest concentration in DBR 60 °C samples which was not significantly different from DBR 70 °C but significantly different from DBR 40 °C samples (7.70×10-4±1.08×10-4 ppm) and DBR 50 °C samples (5.95×10-4±6.81×10-5 ppm). Meanwhile, 2,3,5,6-tetrathiaheptane had its maximum

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concentration in DBR 40 °C (2.11×10-3±7.40×10-5 ppm), significantly different from DBR 50 and 60 °C but not significantly different from DBR 70 °C. The compound 1,2,4,6-tetrathiepane had its highest concentration in DBR 50 °C samples (5.17×10-3±6.44×10-4 ppm), significantly lower than those found in DBR 40, 60, and 70 °C samples with the lowest concentration determined in DBR 40 °C (9.26×10-4±3.92×10-5 ppm). As for lenthionine, it had its minimum concentration of 5.39×10-4±2.91×10-5 ppm in DBR 70 °C samples which was significantly different from DBR 50 °C samples (1.10×10-3±1.77×10-4 ppm) but not significantly different from those found in DBR 40 °C samples (7.07×10-4±3.58×10-5 ppm) and DBR 60 °C samples (6.44×10-4±1.11×10-4 ppm).

As for aldehydes, 3-methyl butanal had its maximum concentration in DBR 70 °C samples (5.83×10-3±1.06×10-3 ppm) which was not significantly different from DBR 60 °C but significantly different from DBR 40 °C (1.96×10-3±3.51×10-4 ppm) and DBR 50 °C (1.03×10-3 ±7.97×10-4 ppm). Pentanal had its highest concentration in DBR 40 °C (2.08×10-2±1.37×10-3 ppm) which was significantly different form its lowest concentration found in DBR 50 °C samples (1.81×10-3±5.74×10-4 ppm). As for hexanal, no significant difference was found between DBR 40 and 50 °C samples (2.91×10-2±4.20×10-3 and 2.34×10-2±2.98×10-3 ppm) however, in DBR 60 and 70 °C samples, its concentration significantly decreased (6.41×10-3±9.60×10-4 ppm and 8.42× 10-3±4.52×10-4 ppm respectively). The compound 3-(methylthio)butanal had its maximum concentration in DBR 50 °C (1.80×10-2±3.28×10-3 ppm) which was significantly different from those determined at the remaining treatments. Meanwhile benzaldehyde had its highest concentration at DBR 70 °C (4.06×10-2±5.99×10-3 ppm) which was not significantly different from DBR 50 and 60 °C, but significantly different from its lowest content found in DBR 40 °C samples (2.04×10-2±4.55×10-3 ppm). As for octanal, no significant difference was found between DBR 40 and 50 °C, however its concentration significantly decreased in DBR 60 °C samples to reach a minimum of 2.48×10-3±2.20×10-4 ppm which was not significantly different from DBR 70 °C samples. Nonanal and decanal had their highest concentration at DBR 40 °C (7.78×10-3 ±3.96×10-4 and 9.29×10-3±4.95×10-4 ppm) and the lowest at DBR 50 °C (3.76×10-3±4.03×10-4 and 5.02×10-4± 1.57×10-5 ppm respectively) while the concentration of propanal showed no significant difference between the different drying temperatures with a maximum concentration of 2.94×10-3 ±8.62×10-4 in DBR 40 °C samples.

As for the remaining alcohols, furfuryl alcohol had its maximum concentration in DBR 40 °C samples (3.53×10-3±3.85×10-4 ppm) which was significantly different from that found in DBR 50, 60, and 70 °C samples, 2-ethyl-1-hexanol had its highest concentration in DBR 70 °C samples

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(4.22×10-3±3.27×10-4 ppm) which was not significantly different from DBR 40 °C samples but significantly different form its lowest concentration found in DBR 60 °C samples (2.47×10-3 ±1.21×10-4 ppm). Meanwhile, phenyl ethyl alcohol had its maximum concentration in DBR 60 °C samples (2.37×10-3±1.31×10-4 ppm) which was significantly different from that found in DBR 40 °C (5.81×10-4±5.64×10-5 ppm). No significant difference was found between DBR 50 and 70 °C samples. Finally, no significant difference was observed between DBR 40 and 50 °C samples for 1-nonanol which was significantly higher than its concentration found in DBR 60 and 70 °C samples (8.64×10-4±1.87×10-5 and 1.13×10-3±1.98×10-4 ppm).

Other compounds identified include 2-formyl-1-isopentyl pyrrole which had its highest concentration in DBR 70 °C (1.5×10-2±1.39×10-3 ppm) significantly different from the other drying temperatures and its lowest concentration found in DBR 40 °C (5.41×10-4±5.17×10-5 ppm). As for 2-undecanone, its highest concentration was found at DBR 70 °C (2.67×10-3±4.28×10-4 ppm) significantly different from the remaining samples with the lowest concentration determined in DBR 60 °C samples (6.91×10-4±8.33×10-5 ppm). As for 1-phenylethanone, its highest concentration was found in DBR 40 °C (5.12×10-3±4.26×10-4 ppm) which was significantly different from those found in DBR 50, 60, and 70 °C samples. The compound 2,4- dimethylpyridine had its maximum concentration in DBR 40 °C (6.01×10-3±3.85×10-4 ppm) significantly different from DBR 50, 60, and 70 °C samples. Finally, the concentration of 2,6- bis(1,1-dimethyl)phenol showed no significant difference in its concentration between DBR 40, 50, and 60 °C, however, it was significantly different form DBR 70 °C samples (2.36×10-3 ±2.42×10-3 ppm).

Wu and Wang (2000) reported that 1-octen-3-ol, 1-octen-3-one, 3-octanone and methyl (methylthio)methyl disulfide were present at a lower concentration when the rehydration temperature used was 40 °C compared to 70 °C. These findings are compatible with the results of this study as higher temperatures (boiling under reflux and hence rehydration temperature of 100 °C in this case) allowed a greater liberation of VC compared to 25 °C. Furthermore, 1-octen- 3-one was not detected in D 40 °C samples but rather in DBR 40 °C samples suggesting that either this compound is present in traces amount well below the detection limit of the instrument or higher temperature is needed for its maximum release from the dried material.

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Table 4.15: Concentrations of VC extracted from dried and boiled under reflux shiitake mushrooms. Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Propanal 2.94×10-3 a 8.62×10-4 2.25×10-3 a 2.41×10-4 1.95×10-3 a 2.49×10-4 2.10×10-3 a 3.07×10-4

Carbon disulfide 1.48×10-2 b 5.01×10-3 1.98×10-2 ab 2.59×10-3 2.69×10-2 a 3.95×10-3 1.16×10-2 b 3.81×10-3

3-methyl butanal 1.96×10-3 b 3.51×10-4 1.03×10-3 b 7.97×10-4 5.51×10-3 a 5.74×10-4 5.83×10-3 a 1.06×10-3

Pentanal 2.08×10-2 a 1.37×10-3 1.81×10-3 c 5.74×10-4 1.37×10-2 b 5.06×10-3 6.94×10-3 bc 6.73×10-4

Dimethyl disulfide 4.41×10-1 a 4.25×10-2 2.35×10-1 b 3.67×10-2 5.81×10-2 c 9.63×10-3 4.69×10-2 c 3.47×10-3

Hexanal 2.91×10-2 a 4.20×10-3 2.34×10-2 a 2.98×10-3 6.41×10-3 b 9.60×10-4 8.42×10-3 b 4.52×10-4

3-(methylthio)butanal 3.59×10-3 b 2.63×10-3 1.80×10-2 a 3.28×10-3 1.83×10-3 b 3.44×10-4 3.57×10-4 b 7.38×10-5

Benzaldehyde 2.04×10-2 b 4.55×10-3 3.33×10-2 a 3.89×10-3 3.36×10-2 a 4.46×10-3 4.06×10-2 a 5.99×10-3

Dimethyl trisulfide 4.70×10-1 a 3.98×10-2 3.13×10-1 b 3.81×10-2 4.26×10-2 c 3.03×10-3 2.53×10-2 c 5.60×10-3

1-octen-3-one 1.41×10-1 a 1.94×10-2 5.22×10-2 b 4.43×10-3 2.83×10-3 c 4.16×10-4 3.19×10-3 c 3.60×10-4

1-octen-3-ol 2.11×100 a 9.85×10-2 1.06×100 b 4.93×10-2 1.34×10-1 c 4.06×10-2 5.54×10-2 c 5.80×10-4

3-octanone 4.87×10-1 b 3.73×10-2 6.65×10-1 a 5.35×10-2 7.42×10-2 c 5.25×10-3 7.39×10-3 c 2.23×10-4

3-octanol 2.38×10-1 a 5.38×10-2 2.06×10-1 a 2.93×10-2 1.30×10-2 b 1.86×10-3 3.53×10-3 b 2.78×10-4

Octanal 1.47×10-2 a 3.75×10-3 1.61×10-2 a 3.96×10-3 2.48×10-3 b 2.20×10-4 5.93×10-3 b 4.71×10-4

Furfuryl alcohol 3.53×10-3 a 3.85×10-4 2.77×10-4 c 1.49×10-5 7.87×10-4 c 2.13×10-5 1.62×10-3 b 4.72×10-4

2-ehtyl,1-hexanol 3.75×10-3 ab 2.62×10-4 3.26×10-3 b 3.38×10-4 2.47×10-3 c 1.21×10-4 4.22×10-3 a 3.27×10-4

1-phenylethanone 5.12×10-3 a 4.26×10-4 1.80×10-3 b 2.68×10-4 1.46×10-3 b 4.10×10-4 2.34×10-3 b 3.00×10-4

2-octen-1-ol 1.55×10-1 a 3.25×10-2 1.53×10-1 a 2.78×10-2 1.22×10-2 b 1.67×10-3 8.75×10-3 b 1.96×10-4

n-octanol 5.34×10-2 a 3.67×10-3 5.18×10-2 a 3.08×10-3 1.46×10-3 b 1.37×10-4 1.56×10-3 b 1.58×10-4

1,2,4-trithiolane 1.43×10-1 c 2.83×10-2 5.69×10-1 a 3.71×10-2 4.72×10-1 b 3.77×10-2 1.19×10-1 c 1.88×10-2

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Table 4.15 continued Compound DBR 40 °C DBR 50 °C DBR 60 °C DBR 70 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Nonanal 7.78×10-3 a 3.96×10-4 3.76×10-3 c 4.03×10-4 5.40×10-3 b 5.53×10-4 5.39×10-3 b 3.19×10-4

1,3,5-trithiane 5.68×10-3 a 3.65×10-4 2.47×10-3 b 3.69×10-4 1.31×10-3 c 3.35×10-4 4.82×10-4 d 3.35×10-5

Phenyl ethyl alcohol 5.81×10-4 c 5.64×10-5 1.39×10-3 b 1.72×10-4 2.37×10-3 a 1.31×10-4 1.40×10-3 b 1.58×10-4

Methyl(methylthio)methyl disulfide 2.00×10-1 a 1.95×10-2 1.69×10-1 a 1.53×10-2 2.94×10-2 b 2.03×10-3 3.34×10-2 b 3.78×10-3

1-nonanol 2.02×10-3 a 2.87×10-4 1.89×10-3 a 3.75×10-4 8.64×10-4 b 1.87×10-5 1.13×10-3 b 1.98×10-4

Decanal 9.29×10-3 a 4.95×10-4 5.02×10-4 c 1.57×10-5 3.58×10-3 b 2.65×10-4 2.66×10-3 b 4.87×10-4

1,4-dimethyltetrasulfide 1.46×10-2 a 3.08×10-3 1.33×10-3 b 2.38×10-4 7.29×10-4 b 4.99×10-5 2.16×10-3 b 1.51×10-4

Tris(methylthio) methane 5.21×10-4 a 3.50×10-5 ND ND ND ND ND ND

2-(methylthio)acetic acid 5.36×10-4 a 4.44×10-5 ND ND ND ND ND ND

2-formyl-1-isopentyl pyrrole 5.41×10-4 c 5.17×10-5 5.71×10-4 c 5.20×10-5 3.30×10-3 b 2.70×10-4 1.15×10-2 a 1.39×10-3

1,2,4,5-tetrathiane 7.70×10-4 b 1.08×10-4 5.95×10-4 b 6.81×10-5 1.83×10-3 a 2.60×10-4 1.41×10-3 a 2.02×10-4

2-undecanone 1.13×10-3 b 1.07×10-4 5.83×10-4 b 2.95×10-5 6.91×10-4 b 8.33×10-5 2.67×10-3 a 4.28×10-4

2,3,5,6-tetrathiaheptane 2.11×10-3 a 7.40×10-5 9.05×10-4 b 1.46×10-4 1.20×10-3 b 1.34×10-4 1.80×10-3 a 3.67×10-4

2,4-dimethylpyridine 6.01×10-3 a 3.85×10-4 7.46×10-4 bc 5.84×10-5 3.41×10-4 c 3.00×10-5 9.96×10-4 b 1.37×10-4

1,2,4,6-tetrathiepane 9.26×10-4 c 3.92×10-5 5.17×10-3 a 6.44×10-4 2.14×10-3 b 1.10×10-4 2.83×10-3 b 3.84×10-4

2,6-bis(1,1-dimethyl)phenol 2.13×10-2 a 7.48×10-3 2.11×10-2 a 2.89×10-3 2.07×10-2 a 4.31×10-3 2.36×10-3 b 2.42×10-3

Lenthionine

7.07×10-4 b 3.58×10-5 1.10×10-3 a 1.77×10-4 6.44×10-4 b 1.11×10-4 5.39×10-4 b 2.91×10-5

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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The highest amount of 1-octen-3-ol was found in D 50 °C samples with 25 °C rehydration temperature, therefore, comparing that amount with its corresponding 100 °C rehydration temperature (drying followed by boiling under reflux) showed that the rehydration temperature plays a crucial role in the liberation of VC from the dried material. Furthermore, the saturated ketone, 1-octen-3-one, believed to be the product of oxidation of 1-octen-3-ol was only detected when the dried mushrooms were boiled under reflux which emphasized the importance of the rehydration in the release of aroma. As shown in Table 4.14, the increase in temperature and drying time resulted in a decrease in the concentration of this compound suggesting that 40 °C is the most suitable drying temperature for the preservation and maximization of 1-octen-3-one.

The normalised relative concentrations of the main C8 compounds are shown in Figure 4.66. DBR 40 °C resulted in the maximum retention of 1-octen-3-one and 1-octen-3-ol while DBR 50 °C resulted in the maximum preservation of 3-octanone. No significant difference in the normalised relative concentrations were found between DBR 40 and 50 °C for the compounds 3-octanol, 2- octen-1-ol, and n-octanol which were significantly different from those determined in DBR 60 and 70 °C samples.

DBR 40 °C 1.2 DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2 Normalised concentrationrelative 0.0

e l e l l l n -o n no -o no -o -3 no a -1 a -3 n a ct n ct n te ct -o te -o te oc -o 3 oc n oc 1- 3 2- 1- Compound

Figure 4.66: Normalised relative concentrations of the main C8 VC in shiitake mushrooms dried at different temperatures and boiled under reflux (DBR).

As for the sulphur compounds (Figure 4.67), carbon disulphide had its maximum normalised relative concentration at DBR 60 °C which was significantly different from that determined at

202

DBR 40 and 70 °C. Boiling under reflux of the shiitake dried at 40 °C resulted in the maximisation of dimethyl disulphide and dimethyl trisulphide, while in DBR 50 °C samples, 1,2,4 trithiolane and lenthionine had their highest normalised concentration. As for 1,2,4,5-tetrathiane, it had its highest normalised value in DBR 60 °C, being not significantly different from DBR 70 °C samples while methyl (methylthio) methyl disulphide had its maximum normalised relative concentration in samples dried at 40 °C which was not significantly different from DBR 50 °C samples but significantly different from that obtained at DBR 60 and 70 °C samples.

1.2 DBR 40 °C DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2

Normalisedrelativeconcentration 0.0

e e e e e e e fid fid fid lan fid ian in ul ul ul io ul th ion dis dis ris ith dis ra th n yl l t tr yl tet en bo th hy ,4- th 5- L ar e et ,2 e 4, C im im 1 )m ,2, D D hio 1 ylt th me yl( Compound eth M

Figure 4.67: Normalised relative concentrations of the main sulphur compounds in shiitake mushrooms dried at different temperatures and boiled under reflux (DBR).

Hong et al., (1988) also reported 1,2,4-trithiolane as the major VC found in dried shiitake. According to this study, dimethyl trisulphide was the major VC at 40 °C and 50° C, while at 60 °C and 70 °C, 1,2,4-trithiolane was found to have the highest concentration. When shiitake samples were boiled under reflux for 30 min, dimethyl disulphide and dimethyl trisulphide were the major VC found in dried shiitake at 40 °C, however, when the samples were dried at 50, 60 and 70 °C, 1,2,4-trithiolane was the major VC. One explanation could be that rehydration temperature plays an important role in the liberation of volatiles from the dried material. Another possible explanation is that boiling under reflux resulted in losses of the sulphur compounds studied except for 1,2,4-trithiolane and lenthionine where their relative concentration was rather increased.

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The following compounds 1,2,4-trithiolane, 1,2,4,5-tetrathiane, 1,2,4,6-tetrathiepane and lenthionine have been also reported in red algae (Chondria californica ) which suggest that the red algae and shiitake mushrooms have these compounds in common and could share the same mechanism of their formation (Wratten and Faulkener, 1976) while 1,2,4-trithiolane was reported as a VC in eggs (Gil and Macleod, 1981) as well as a product of the reaction between hydrogen sulphide and D-glucose (Sakaguchi and Shibamoto, 1978).

Methyl (methylthio)methyl disulfide has been reported as a compound in broccoli, cabbage, and cauliflower (Buttery et al., 1976) and also a contributor the typical flavour of several plants such as Hua gabonii (‘garlic plant”) (Jirovetz et al., 2002) and Marasmius alliaceus (Rapior et al., 1997). Its mechanism of formation as been reported by Kubec et al., (1998) where S-methyl cysteine and its sulfoxide are degraded to methyl (methylthio)methyl disulphide suggesting a combined enzymatic and thermal origin (including thermal degradation of products of methionine) of the later compound as a secondary by-product. Kubota et al., (1994) also suggested that naturally occurring amino acids can be a precursor for methyl (methylthio)methyl disulphide formed by C-S lyase-mediated enzymatic conversion without heat treatment. Its sensory properties are described as garlic-like, alliaceous, sulphury, and cooked cabbage-like (Rapior et al., 1997).

Lenthionine was detected in processed mushrooms only. This compound was first identified in dried shiitake by Morita and Kobayashi, (1966) and Wada et al. (1967). Ito et al. (1978) also concluded that lenthionine is formed enzymatically by soaking dried mushroom in water at neutral pH. Studies performed by Hiraide et al. (2010) showed that the amount of lenthionine produced by drying at 40 °C and 60 °C was very small. However, upon rehydration, the amount was relatively larger than that without rehydration. In this study, rehydration at 25 °C resulted in the maximum liberation of lenthionine compared to when the dried samples were boiled under reflux. The production of lenthionine in shiitake might be affected by physical damage on the cellular level and interfering with lenthionine synthesis initiation during the drying process.

The normalised relative concentration of aldehydes is shown in Figure 4.68. The compound 3- methyl butanal had its highest normalised value at DBR 70 °C samples which was not significantly different from DBR 60 °C but significantly different from DBR 40 and 50 °C. As for hexanal, its highest normalised value was found in DBR 40 °C samples which was not significantly different form DBR 50 °C samples but significantly different from DBR 60 and 70 °C while 3-(methylthio)butanal had its highest normalised concentration at DBR 50 °C significantly different from the other treatments. Meanwhile benzaldehyde showed no significant

204

difference between DBR 50, 60, and 70 °C while octanal showed no significant difference between DBR 40 and 50 °C. Nonanal and decanal had their maximum normalised relative concentrations in DBR 40 °C samples which were significantly different from the other treatments.

1.6 DBR 40 °C 1.4 DBR 50 °C DBR 60 °C 1.2 DBR 70 °C

1.0

0.8

0.6

0.4

0.2

0.0 Normalisedrelative concentration -0.2 al al al e al al al an an an yd an an an ut ex ut eh ct on ec l b H )b ald O N D hy hio nz et ylt Be 3-m th me Compound 3-(

Figure 4.68: Changes in the normalised relative concentrations of aldehydes compounds in dried then boiled under reflux (DBR) shiitake at different temperatures.

Other compounds that were isolated and identified are shown in Figure 4.69 and include furfuryl alcohol which had its maximum normalised relative concentration in DBR 40 °C samples significantly different the other treatments while 2-ethyl-1-hexanol had its maximum normalised relative concentration in DBR 70 °C samples which was not significantly different from that obtained in DBR 40 °C samples. Phenyl ethyl alcohol had its highest normalised value at DBR 60 °C samples significantly different form the other treatments while nonanol showed no significant difference between DBR 40 and 50 °C but higher temperatures followed by boiling under reflux significantly affected its normalised concentration. Finally, 2-undecanone had its maximum normalised relative concentration in DBR 70 °C samples which was significantly different from the other treatments.

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1.2 DBR 40 °C DBR 50 °C DBR 60 °C 1.0 DBR 70 °C

0.8

0.6

0.4

0.2 Normalised concentrationrelative 0.0

l l l l e ho no ho no on lco xa lco na an l a he a No ec y -1- yl d fur yl eth -un ur ht yl 2 F 2-e en Ph Compound

Figure 4.69: Changes in the normalised relative concentration of aldehydes compounds in dried then boiled under reflux (DBR) shiitake at different temperatures.

4.4.5 Comparison between treatments

The behaviour of VC in samples subjected to different treatments was compared. Drying at 40 °C was chosen as the most suitable drying temperature as it allowed the maximum preservation as well as formation of VC in shiitake samples. VC that were extracted from F, BR, D 40 °C and DBR 40 °C were identified and are shown in Table 4.16. As expected, some compounds showed an increase in their concentrations, other a decrease while new compounds were formed. It is also expected that compounds belonging to the same chemical class also behaved differently. In general, drying generated a higher concentration of sulphur compounds as opposed to freshly boiled under reflux samples. The choice of a suitable drying temperature should be a compromise between (a) suitable condition for the maximum retention and preservation of VCs, (b) adequate moisture removal for the extension of the shelf life (c) savings in terms of energy usage. Therefore drying at 50 °C appears as the most suitable drying temperature.

The main C8 compounds behaved differently in comparison to each other. For instance, 1-octen- 3-one had its highest concentration in DBR 40 °C (1.41×10-1±1.94×10-2 ppm), significantly different from its lowest concentration found in F samples with no significant difference found between F and BR samples (1.22×10-3±7.37×10-5 vs. 6.57×10-3±5.83×10-4 ppm). As for 1-octen-3- ol, its highest concentration was found in BR samples (3.04×100±1.27×100 ppm), not significantly different from DBR 40 °C samples but significantly different from F and D 40 °C samples.

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Meanwhile, 3-octanone had its maximum concentration in F samples (4.21×100±4.84×10-1 ppm) which decreased significantly during boiling under reflux (7.04×10-2±1.98×10-2 ppm) while 3- octanol had its highest concentration in D 40 °C (1.04×100±5.08×10-2 ppm), significantly higher than its lowest concentration found in BR samples (1.12×10-1±4.30×10-2 ppm). The maximum concentration of 2-octen-1-ol was found in BR samples (2.91×10-1±6.09×10-2 ppm), significantly different from the other treatments while n-octanol had its highest concentration in D 40 °C samples (1.88×10-1± 4.39×10-3 ppm) which was not significantly different from F samples but significantly different from BR samples (7.01×10-2±2.55×10-2 ppm).

As for the sulphur compounds, no significant difference was found between the concentrations of carbon disulphide in F, BR, and D 40 °C while the highest concentration was found at DBR 40 °C. As for dimethyl disulphide, no significant difference was found between F and BR samples (1.17×10-1±1.20×10-2 and 1.88×10-2±4.23×10-3 ppm respectively) while the highest concentration was found in D 40 °C samples. Dimethyl trisulfide also showed no significant difference between F and BR samples (4.74×10-2±4.56×10-3 and 2.86×10-2±5.24×10-3 ppm) with the highest concentration found in D 40 °C (1.19×100±1.66×10-1 ppm). Methyl(methylthio)methyl disulfide and 2,3,5,6-tetrathiaheptane both had their highest concentrations in D 40 °C (4.63×10-1±4.81× 10-2 and 1.32×10-1±2.69×10-2 ppm) significantly different from their concentrations in BR samples (4.02×10-2±1.98×10-2 and 3.33×10-3±1.27×10-3 ppm respectively). Meanwhile, 1,2,4- trithiolane and 1,4-dimethyltetrasulfide showed no significant difference between D 40 °C and DBR 40 °C and no significant difference between the lowest concentrations determined in F and BR samples (3.77×10-3±4.70×10-4 vs. 5.01×10-2±1.92×10-2 ppm for 1,2,4-trithiolane, and 1.27× 10-3±1.22×10-3 vs. 1.33×10-3±3.50×10-4 ppm for 1,4-dimethyltetrasulfide. As for tris(methylthio)methane, its highest concentration was found in F samples (5.44×10-3±3.39×10-4 ppm) significantly different from its lowest concentration found in BR samples (3.95× 10-4±8.90×10-5 ppm). No significant difference was found between BR and DBR samples (3.95×10-4±8.90×10-5 vs. 5.21×10-4±3.50×10-5 ppm). Finally, lenthionine was only detected in dried samples with the highest concentrations been found in DBR 40 °C (4.70×10-1±3.98×10-2 ppm).

As for aldehydes, benzaldehyde had its highest concentration in DBR 40 °C (2.04×10-2±4.55×10-3 ppm) which was significantly different from BR samples (1.95×10-3±7.79×10-4 ppm) and D 40 °C (7.83×10-3±1.57×10-3 ppm) and was not detected in F samples. As for (E)-2-octenal, its highest concentration was found in F samples (5.04×10-1±1.73×10-2 ppm) and was significantly different from BR samples (2.49×10-3±7.00×10-4 ppm), however, it was not detected in D 40 °C and DBR

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40 °C samples. Nonanal had its lowest counteraction in BR samples (6.37×10-3±3.82×10-4 ppm), not significantly different from DBR 40 °C (7.78×10-3±3.96×10-4 ppm) but significantly different from its highest concentration found in F samples (5.38×10-2±3.85×10-3 ppm). As for decanal, no significant difference was found between D 40 °C and DBR 40 °C (1.18×10-2±1.75×10-3 vs. 9.29×10-3±4.95×10-4 ppm) while the lowest concentration was found in BR samples (1.29×10-3 ±7.29×10-4 ppm).

As for the remaining alcohols, 1-pentanol showed no significant difference between F and BR samples (1.15×10-3±1.38×10-4 vs. 1.69×10-3±6.21×10-4 ppm respectively) while 2-ethyl-1-hexanol showed no significant difference between BR (2.51×10-3±1.18×10-3 ppm), D 40 °C (3.61× 10-3±7.87×10-4 ppm) and DBR 40 °C samples (3.75×10-3±2.62×10-4 ppm) while its lowest concentration was found in F samples (1.16×10-3±1.41×10-4 ppm) which was not significantly different from BR samples. As for 1-nonanol, it reached the maximum concentration of 1.55×10-2 ±5.90×10-3 ppm at D 40 °C and the lowest concentration of 1.35×10-3±7.32×10-4 ppm, not significantly different from F and DBR samples while 2,6-bis(1,1-dimethylethyl) phenol had its maximum concentration in DBR 40 °C samples (2.13×10-2±7.48×10-3 ppm) which was significantly different from the remaining treatments.

Other compounds identified include (3E)-1,3-octadiene with a maximum concentration of 5.09× 10-2±2.38×10-3 ppm in F samples and the lowest in D 40 °C (1.06×10-3±1.54×10-4 ppm). During boiling under reflux, its concentration significantly decreased to reach 6.56×10-3± 1.90×10-3 ppm and finally limonene which was only found in BR samples (5.36×10-4±8.29×10-5 ppm).

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Table 4.16: Comparison between concentrations of selected VC in shiitake samples subjected to different treatments. Compound F BR D 40 °C DBR 40 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

Carbon disulfide 1.39×10-3 b 2.97×10-4 3.69×10-3 b 1.28×10-3 2.32×10-3 b 5.57×10-4 1.48×10-2 a 2.01×10-3

Dimethyl disulfide 1.17×10-1 c 1.20×10-2 1.88×10-2 c 4.23×10-3 6.29×10-1 a 8.36×10-2 4.41×10-1 b 4.25×10-2

1-pentanol 1.15×10-3 a 1.38×10-4 1.69×10-3 a 6.21×10-4 ND ND ND ND

(3E)-1,3-octadiene 5.09×10-2 a 2.38×10-3 6.56×10-3 b 1.90×10-3 1.06×10-3 c 1.54×10-4 ND ND

Benzaldehyde ND ND 1.95×10-3 bc 7.79×10-4 7.83×10-3 b 1.57×10-3 2.04×10-2 a 4.55×10-3

Dimethyl trisulfide 4.74×10-2 c 4.56×10-3 2.86×10-2 c 5.24×10-3 1.19×100 a 1.66×10-1 4.70×10-1 b 3.98×10-2

1-octen-3-one 1.22×10-3 b 7.37×10-5 6.57×10-3 b 5.83×10-4 ND ND 1.41×10-1 a 1.94×10-2

1-octen-3-ol 6.24×10-1 bc 6.34×10-2 3.04×100 a 1.42×10-1 1.07×10-1 c 7.16×10-3 2.11×100 ab 9.85×10-2

3-octanone 4.21×100 a 4.84×10-1 7.04×10-2 c 1.98×10-2 8.63×10-1 b 3.80×10-2 4.87×10-1 bc 3.73×10-2

3-octanol 3.07×10-1 b 2.93×10-2 1.12×10-1 c 4.30×10-2 1.04×100 a 5.08×10-2 2.38×10-1 b 5.38×10-2

Limonene ND ND 5.36×10-4 a 8.29×10-5 ND ND ND ND

2-ethyl-1-hexanol 1.16×10-3 b 1.41×10-4 2.51×10-3 ab 1.18×10-4 3.61×10-3 a 7.87×10-4 3.75×10-3 a 2.62×10-4

(E)-2-octenal 5.04×10-1 a 1.73×10-2 2.49×10-3 b 7.00×10-4 ND ND ND ND

2-octen-1-ol 1.78×10-1 b 5.40×10-3 2.91×10-1 a 6.09×10-2 1.50×10-1 c 5.95×10-3 1.55×10-1 c 3.25×10-2

n-octanol 1.75×10-1 a 2.15×10-3 7.01×10-2 b 2.55×10-2 1.88×10-1 a 4.39×10-3 5.34×10-2 b 3.67×10-3

1,2,4-trithiolane 3.77×10-3 b 4.70×10-4 5.01×10-2 b 1.92×10-2 1.24×10-1 a 1.68×10-2 1.43×10-1 a 2.83×10-2

Nonanal 5.38×10-2 a 3.85×10-3 6.37×10-3 c 3.82×10-4 1.07×10-2 b 1.63×10-3 7.78×10-3 c 3.96×10-4

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Table 4.16 continued Compound F BR D 40 °C DBR 40 °C

ppm SD* (ppm) ppm SD (ppm) ppm SD (ppm) ppm SD (ppm)

methyl(methylthio)methyl disulfide 7.90×10-4 c 5.90×10-5 4.02×10-2 c 1.98×10-2 4.63×10-1 a 4.81×10-2 2.00×10-1 b 1.95×10-2

1-nonanol 1.51×10-3 b 2.45×10-4 1.35×10-3 b 7.32×10-4 1.55×10-2 a 2.91×10-3 2.02×10-3 b 2.87×10-4

Decanal 1.60×10-3 b 3.41×10-4 1.29×10-3 b 7.29×10-4 1.18×10-2 a 1.75×10-3 9.29×10-3 a 4.95×10-4

1,4-dimethyltetrasulfide 1.27×10-3 b 1.22×10-3 1.33×10-3 b 3.50×10-4 1.75×10-2 a 1.27×10-3 1.46×10-2 a 3.08×10-3

Tris(methylthio) methane 5.44×10-3 a 3.39×10-4 3.95×10-4 c 8.90×10-5 4.28×10-3 b 3.66×10-4 5.21×10-4 c 3.50×10-5

2,3,5,6-tetrathiaheptane 3.99×10-2 b 2.78×10-3 3.33×10-3 c 1.27×10-3 1.32×10-1 a 2.69×10-2 2.11×10-3 c 7.40×10-5

2,6-bis(1,1-dimethylethyl) phenol 4.70×10-3 b 4.80×10-4 6.28×10-3 b 1.08×10-3 3.30×10-3 b 5.30×10-4 2.13×10-2 a 7.48×10-3

Lenthionine ND ND ND ND 3.93×10-3 b 5.57×10-4 4.70×10-1 a 3.98×10-2

* Standard deviation, analyses are per 1 mL extract.

Means with the same letter within a row are not significantly different (p < 0.05).

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Comparison between fresh, boiled under reflux, dried at 40 °C followed by boiling under reflux was also established and the results are shown in Figure 4.70 where the values are normalised to the fresh state. As for 1-octen-3-one, it had the highest value when it was dried then boiled under reflux and was not detected in D 40 °C samples. The compounds 3-octanone and n-octanol had the highest amount when the mushrooms were fresh, but no significant difference was found for n- octanol when the fresh samples were compared to D 40 °C samples. As for 1-octen-3-ol, it had the highest normalised relative concentration in BR samples which was not significantly different from DBR 40 °C while for 3-octanol, the highest normalised relative concentration was found in D 40 °C samples. As for 2-octen-1-ol, it had its maximum normalised relative concentration in BR samples, significantly different from F, D 40 °C and DBR 40 °C samples.

1.6 F BR 1.4 D 40 °C DBR 40 °C 1.2

1.0

0.8

0.6

0.4

0.2 Normalisedrelative concentration 0.0

e ol e ol ol ol on 3- on n 1- n 3- n- an ta n- ta n- te ct oc te oc te c -o 3- c n- oc -o 3 -o 1- 1 2 Compound

Figure 4.70: Normalised relative concentrations of main C8 compounds in shiitake mushrooms subjected to different treatments.

As for the sulphur compounds (Figure 4.71), carbon disulphide and lenthionine had their highest normalised relative concentration in DBR 40 °C samples, significantly different from the other treatments. It can also be seen that lenthionine was only found when the shiitake samples were dried and dried then boiled under reflux. Meanwhile, dimethyl disulphide, dimethyl trisulfide and methyl(methylthio)methyl disulfide had their maximum normalised relative concentration in D 40 °C samples, significantly different from the other treatments while the normalised relative concentration of 1,2,4-trithiolane and 1,4-dimethyltetrasulfide showed no significant difference between D 40 °C and DBR 40 °C.

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1.4 F BR 1.2 D 40 °C DBR 40 °C 1.0

0.8

0.6

0.4

0.2 Normlaisedrelativeconcentration

0.0

e e e e e e e fid fid fid an fid fid in sul sul sul iol sul sul ion di di tri ith di ra nth on yl yl -tr yl tet Le rb eth eth 2,4 eth hyl Ca im im 1, )m et D D hio im ylt ,4-d eth 1 l(m thy Compound Me

Figure 4.71: Normalised relative concentrations of the main sulphur compounds in shiitake mushrooms subjected to different treatments.

The normalised relative concentrations of other selected VC extracted from shiitake mushrooms subjected to different treatments are shown in Figure 4.72. The compounds (3E)-1,3-octadiene, (E)-2-octenal, and nonanal had their highest normalised value in the F samples of mushrooms which was significantly higher when compared to other treatments while limonene was only present in BR samples which could be explained by either the fact that it was well below the detection limit of the instrument or that it was lost during drying. Meanwhile, no significant difference was found in the normalised relative concentration of 2-ethyl-1-hexanol and decanal when comparing D 40 °C and DBR 40 °C which were significantly higher compared to the other treatments. As for 1-nonanol, its highest content was found D 40 °C samples which was significantly higher compared to the other treatments with no significant difference found between F, BR, and DBR 40 °C.

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F 1.2 BR D 40 °C 1.0 DBR 40 °C

0.8

0.6

0.4

0.2 Normalised relative concentration

0.0

e e l l l l l n n no na na no na ie ne a te a a a ad o ex c on on ec ct im -h -o N -n D -o L -1 )-2 1 ,3 yl E -1 th ( E) -e (3 2 Compound

Figure 4.72: Normalised relative concentrations of selected VC in shiitake mushrooms subjected to different treatments.

The effects of storage and drying temperatures on the quality on shiitake were assessed. Some VC tend to increase with time, such as the sulphur compounds, resulting in off-odours and off-flavours in mushrooms, while the concentration of some other VC decreased. Different drying temperatures also affected the VC profile with drying at 40 °C appears to be the most suitable drying temperature. Different heat treatments had different effect on the VC content where lenthionine was only found in dried samples and the discussed sulphur compounds showed a higher concentration when the shiitake samples were dried and dried then boiled under reflux. Rehydrating temperature of the dried samples also played a vital role in the generation of aroma compounds. The overall aroma of shiitake seems likely to be a subtle balance of various functionalised compounds.

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4.5 COMPARISON BETWEEN SPECIES

Chestnut, enoki, oyster, and shiitake mushrooms were investigated for their VC content and were characterised on the basis of their aroma profile. Studies on the postharvest physiology of mushrooms to improve quality and shelf life are in accordance with the needs of consumers. The 4 mushroom species shared similar compounds such as some of the main C8 compounds (1-octen-3- ol, 3-octanone, and 3-octanol) as well as aldehydes (hexanal, nonanal, and decanal). They also contained distinctive compounds that allowed the differentiation between them. For instance, in fresh samples, A. aegerita had 3-octanol as the main C8 compound followed by 3-octanone while F. velutipes and L. edodes had 3-octanone as the major C8 compound followed by 3-octanol. Compared to the other species, P. ostreatus had 3-octanol as the major VC followed by 1-octen-3- ol which came as the second most abundant VC as opposed to the third place in A. aegerita and F. velutipes.

Fresh chestnut mushrooms contained 1,8-cineole, (E,Z)-2,6 nonadienal, and 3,5,5-trimethyl-1- hexanol which were only detected in this specie, while enoki mushrooms were characterised by the presence of several compounds that were absent in chestnut mushrooms such as the indole compound (1H-indol-5-ol) as well as limonene, dill ether, and dihydrocarveol. Fresh enoki mushrooms did not contain 1-octen-3-one, 2-octen-1-ol or n-octanol or benzaldehyde. Fresh oyster mushrooms were the only species containing butanoic acid, 3-methyl-ethyl ester, 2-ethyl hexanoic acid, ethyl octanoate and (E)-2-nonenal. Meanwhile, shiitake mushrooms were characterised by the presence of sulphur compounds that gave these mushrooms a distinctive aroma compared to the other species. These sulphur compounds each with a specific aroma descriptor such as carbon disulphide, dimethyl disulphide, and dimethyl trisulfide, contributed to the overall and balanced subtle aroma. Oyster and enoki mushrooms shared 3-methyl butanal, known for its malty aroma, which was absent in chestnut and shiitake mushrooms.

During storage, the concentration of the main C8 compounds in the four mushrooms species decreased with time while that of aldehydes and 2-ethyl-1-hexanol increased. Sulphur compounds identified in shiitake were also increasing during storage and hence contributing to the deterioration in quality and off-flavours formation. Benzaldehyde was not identified in fresh shiitake which could be due to its very low level well below the detection limit of the instrument.

Boiling under reflux of the fresh chestnut samples increased the concentration 3-methyl butanal, 2-methyl butanal (assuming they were present in the fresh state and in this case, their levels were below the detection limit of the instrument), as well the generation of pyridine, 2-methyl

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cyclohexanone, limonene, 3-ethyl-2-methyl-1,3-hexadiene, 5-methyl-5-hexen-2-one, (E)-2- hexenal, 2-nonen-1-ol, 2-undecanone, and (E,E)-2,4-decadienal.

As for enoki mushrooms, several compounds were formed after boiling under reflux and include 2-propanone, 1-pentanol, 3-heptanone, heptanal, benzaldehyde, 1-octen-3-one, 5-metyl-3-hexen- 2-one, phenylacetaldehyde, (E)-2-octenal, 2-cyclohexen-1-ol, n-octanol, linalool, 1-nonanol, and 2-undecanone. Other compounds showed an increase in their concentrations such as hexanal, nonanal, 2-ethyl-1-hexanol and dill ether while others exhibited a decrease in their concentrations such as 1-octen-3-ol, 3-octanone, 3-octanol, octanal and limonene.

Meanwhile boiling under reflux of oyster mushrooms had a limited impact on these mushroom species where an increase in the concentration of hexanal, 1-octen-3-one, and 2-ethyl hexanol was observed.

As for shiitake samples, boiling under reflux resulted in the loss of some of the main C8 compounds with their concentrations decreasing significantly such as 3-octanone, 3-octanol and n- octanol while other compounds such as 1-octen-3-ol and 2-octen-1-ol showed a significant increase upon boiling under reflux. Limonene was formed only when fresh shiitake samples were boiled under reflux. As for the sulphur compounds, no significant difference was found between fresh and boiled under reflux except for tris(methylthio) methane and 2,3,5,6-tetrathiaheptane where their concentrations significantly decreased upon heating.

The four mushroom species were subjected to the same drying conditions but each species was in favour of a different drying temperature probably due to their distinctive morphology that allowed the maximum retention of VC. The compounds identified in the following section include VC from D and DBR samples at the most suitable drying temperature. Drying at 70 °C proved to be harsh on VC for all four mushrooms species.

For chestnut samples, drying at 60 °C was found to be the most suitable for the maximum retention of VC which allowed the generation of VC that were not present in the fresh state or in some cases below the detection limit of the instrument such as 3-methyl butanal, 2-methyl butanal, pyridine, 2-methyl cyclohexanone, 2-pentyl furan, 2-octanone, limonene, furfuryl alcohol, (Z)-3- ethyl-2-methyl-1,3-hexadiene, 5-methyl-5-hexen-2-one, phenylacetaldehyde, benzyl alcohol, thiofuran, (E,E)-2,4-octadienal, (E)-2-hexenal, 1,9-nonanediol, (E,E)-2,4-nonadienal, alpha- ethylidene- benzeneacetaldehyde, 2-undecanone, (E,E)-2,4-decadienal and propanoic acid,2- methyl-1-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl ester.

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As for enoki mushrooms, drying at 50 °C resulted in the maximum retention of VC with the newly formed compounds include 2-propanone, 2-methyl furan, 2,6-dimethylpyrazine, 1,2- cyclohexadione, 2,3,4,5-tetrahydropyridine, 5-metyl-3-hepten-2-one, 2-ethyl-6-methylpyrazine, (E)-2,4-heptadienal, phenylacetaldehyde, (E)-2-octenal, 2-cyclohexen-1-ol, n-octanol, 2,5- dimethyl-3-ethylpyrazine, linalool, camphor, 2-undecanone, 2,6-dimethyl-3-propylpyrazine, and 6-10-dimethyl-5,9-undecadien-2-one.

The most suitable drying temperature for oyster mushrooms was found to be 50 °C where newly generated compounds identified include 2-methyl furan, (E,E)-2,4-hexadienal, heptanal, 2- methoxy-6-methylpyrazine, 2-methyl-3-ethyl-1,3-hexadiene, phenylacetaldehyde, 5-methyl-1,3- benzendiol, 2-octanone, (E,E)-2,4-decadienal, and 2-undecanone. In addition, an increase in the concentration of several compounds was observed such as 2-methyl butanal, hexanal, 1-hexanol, 3-heptanone, benzaldehyde, 2-ethyl-1-hexanol, nonanal, (E)-2-nonenal, and decanal. The compounds butanoic acid, 3-methyl-, ethyl ester and 2-ethyl hexanoic acid were not identified in the dried samples.

As for shiitake mushrooms, benzaldehyde and lenthionine were absent in the fresh samples with benzaldehyde already discussed above and lenthionine is only formed when the samples were dried. Drying resulted in the maximum retention and/or formation of sulphur compounds such as dimethyl disulphide, dimethyl trisulfide, 1,2,4-trithiolane, methyl(methylthio)methyl disulphide, 1,4-dimethyltetrasulfide and 2,3,5,6-tetrathiaheptane compared to the fresh state. Furthermore, the concentration of selected alcohols such as 2-ethyl-1-hexanol, 3-octanol, n-octanol, 1-nonanol was also increased during drying.

The rehydration temperature played a crucial role in the liberation of VC form the dried material in all four species. For chestnut, rehydration at 100 °C allowed the liberation of the following compounds pyridine; heptanal; 2-methyl-1,6-heptadiene; 1-acetylimidazole; 2-pentyl furan; furfuryl alcohol; 3-ethyl,2-methyl,1,3-hexadiene; (E,E)-2,4-octadienal; (E,E)-2,4-nonadienal, and alpha-ethylidene- benzeneacetaldehyde. Other compounds had their concentrations decreasing when the dried samples were boiled under reflux such as 2-methyl cyclohexanone, 2-hepten-1-ol, 3-octanone, 2-octanone, 2-octen-1-ol, phenyl ethyl alcohol, 1-nonanol, and decanal.

As for enoki mushrooms boiling under reflux of the dried samples also allowed the maximum liberation of some compounds such as 2-propanone, 2-methyl furan, 3-methyl butanal, 2-methyl butanal, 1-pentanol, 2-hexanone, hexanal, 3-heptanone, heptanal, 2,6-dimethylpyrazine, 1,2- cyclohexadione, benzaldehyde, 2,3,4,5-tetrahydropyridine, 2-ethyl-6-methylpyrazine, (E,E)-2,4-

216

heptadienal, phenylacetaldehhyde, 2,5-dimethyl-3-ethylpyrazine, linalool, nonanal, camphor, dill ether, decanal and 2-undecanone. Meanwhile other compounds experienced a decrease in their concentrations and include 3-methyl-1-butanol, 1-hexanol, 1-nonanol, dihydrocarveol and 6-10- dimethyl-5,9-undecadien-2-one. Other compounds were lost during or after boiling under reflux and these compounds include 2-methyl-1-butanol, 5-metyl-3-hexen-2-one and 2-cyclohexen-1-ol.

As for oyster mushrooms, some compounds needed 100 °C as a rehydration temperature and include 3-methyl butanal, 2-methyl butanal, benzaldehyde, 3-ethyl-2-methyl-1,3-hexadiene, (E)-2- octenal, linalool, 1-nonanol, decanal , and (E,E)-2,4-decadienal. As for the main C8 compounds, boiling under reflux of the dried samples resulted in further losses of VC compared to dried samples and to freshly boiled under reflux samples. Other compounds had a higher concentration with a rehydration temperature at 25 °C compared to DBR samples and include hexanal, 1- hexanol, 3-octanone, 3-octanol, octanal, (E)-2-nonenal, and 2-undecanone.

As for shiitake, 1-octen-3-one was detected after the dried samples were boiled under reflux. Other compounds had a higher concentration when the dried samples were boiled under reflux compared to when the dried samples were subjected directly to ambient temperature vacuum distillation (25 °C rehydration temperature). These compounds include carbon disulphide, dimethyl disulphide, benzaldehyde, 1-octen-3-ol, 3-octanone, and lenthionine. Other compounds which showed a lower concentration when the dried samples where boiled under reflux compared to only dried samples include dimethyl trisulfide, 3-octanol, n-octanol, nonanal, methyl(methylthio)methyl disulphide, 1-nonanol, tris(methylthio) methane, and 2,3,5,6- tetrathiaheptane.

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5. CONCLUSIONS AND RECOMMENDATIONS

A. aegerita, F. velutipes, P. ostreatus, and L. edodes mushrooms were investigated for the VC content and characterised based on their aroma profile.

Ambient temperature vacuum distillation was successfully optimised and used for the extraction of VC from the mushrooms species as it gave the highest quality and the most representative aroma of the original product.

Each mushroom species had a distinctive aroma profile that allowed it to be differentiated from the other species. Mushrooms are known for their high perishability due to their high moisture content as well as their complex matrix of carbohydrates, proteins, lipids, vitamins and minerals making them susceptible to spoilage and deterioration. Since only few studies have been concerned with the storage of mushrooms, the storage study conducted in this dissertation gave an input about the changes in the VC profile over time where the main C8 compounds present in all the studied mushrooms were decreasing over time while aldehydes’ content was increasing as well as the sulphur compounds in shiitake contributing to the overall deterioration of the aroma of mushrooms.

Drying also had a significant impact on the VC profile and hence the quality of the dried mushrooms is affected by the physical and chemical changes occurring during processing and storage. The rate of moisture loss for all mushroom species was higher at higher temperatures (70 °C) and the total drying time is reduced with the increase in air temperature due to the fact that higher temperature of air had increased the temperature of the product, which accelerated the removal of moisture from the samples. The choice of the most suitable drying temperature was (a) a compromise between retention of the already present VC, (b) the maximisation of the newly formed compounds, (c) economic point of view. As for shiitake, 40 °C was chosen as the most suitable drying temperature while chestnut required 60 °C and enoki and oyster 50 °C. Some compounds such as pyrazine in enoki mushrooms, and lenthionine in shiitake were only generated via heat.

Rehydration temperature played an important role in the liberation of VC. In general the maximum release of alcohols, aldehydes, pyrazine and conjugated dienal from the dried material was higher at 100 °C rehydration temperature compared to 25 °C. The concentrations of the main C8 compounds in chestnut, oyster mushrooms were decreasing when the dried samples were

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boiled under reflux as opposed to freshly boiled under reflux while with enoki and shiitake samples it did not significantly vary.

Future work could be investigation of the role of C8 compounds mainly 1-octen-3-ol in mushrooms metabolism and possible ways whereby they can be manipulated by growers to improve the aroma and flavour of their products. Furthermore, analysis of the best rehydration temperature for the liberation of VC from the dried material is of great importance to minimise losses during processing and retain the maximum amount of VC.

Finally, the volatile profile of the different species can be used for the development of an electronic nose enabling the detection, recognition and differentiation of simple or complex VC of different species.

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APPENDIX A. Standard Addition

4.00E+07

1.80E+09 3.00E+07 1.60E+09 y = 3E+08x + 3E+07 2.00E+07 R² = 0.9978 1.40E+09 1.00E+07 1.20E+09 0.00E+00 1.00E+09

Residual 0 2 4 6

Area 8.00E+08 -1.00E+07

6.00E+08 -2.00E+07 4.00E+08 -3.00E+07 2.00E+08 -4.00E+07 0.00E+00 Volume (mL) 0Volume 2(mL) 4 6 Figure 7.1: Standard addition curve of benzaldehyde.

Table 7.1: Raw data for the standard addition curve of benzaldehyde. Volume Area Residual Slope Intercept 0 7753203 -25008236 318122730.4 32761439 1 339966913 -10917256 2 705387823 36380923 3 1012972479 25842849 4 1313134099 7881738.4 5 1589195073 -34180018

1.80E+09 1.00E+08 y = 3E+08x + 1E+08 1.60E+09 8.00E+07 R² = 0.9417 1.40E+09 6.00E+07 1.20E+09 4.00E+07 1.00E+09 2.00E+07

Area 8.00E+08 0.00E+00

6.00E+08 Residual 0 2 4 6 4.00E+08 -2.00E+07 2.00E+08 -4.00E+07 0.00E+00 -6.00E+07 0 2 4 6 -8.00E+07 Volume (mL) Volume (mL)

Figure 7.2.: Standard addition curve of 3-octanone.

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Table 7.2: Raw data for the standard addition curve of 3-octanone. Volume Area Residual Slope Intercept 0 85965187 -50540395 312835323.2 136505582.3 1 429235306 -20105600 2 846907240 84731011 3 1147577346 72565794 4 1321646628 -66200247 5 1680231635 -20450563

1.60E+08 8.00E+06 y = 3E+07x + 8E+06 1.40E+08 R² = 0.9946 6.00E+06 1.20E+08 4.00E+06 1.00E+08 8.00E+07 2.00E+06 Area 6.00E+07 0.00E+00 Residual 4.00E+07 0 2 4 6 -2.00E+06 2.00E+07 0.00E+00 -4.00E+06 0 2 4 6 -6.00E+06 Volume (mL) Volume (mL)

Figure 7.3: Standard addition curve of nonanal.

Table 7.3: Raw data for the standard addition curve of nonanal. Volume Area Residual Slope Intercept 0 3963319 -3721380 27751690.69 7684698.952 1 42274627 6838237 2 62581586 -606494 3 88091475 -2848296 4 117461487 -1229975 5 148011060 1567908

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APPENDIX B. TIC and Mass Spectra

Figure 7.4: TIC of fresh chestnut mushrooms at D0.

Figure 7.5: TIC of fresh chestnut mushrooms boiled under reflux.

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Figure 7.6: TIC of dried chestnut mushrooms at 60 °C.

Figure 7.7: TIC of dried (60 °C) and boiled under reflux chestnut mushrooms.

Figure 7.8: TIC of fresh enoki mushrooms at D0.

Figure 7.9: TIC of fresh enoki mushrooms boiled under reflux.

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Figure 7.10: TIC of dried enoki mushrooms at 50 °C.

Figure 7.11: TIC of dried (50 °C) and boiled under reflux enoki mushrooms.

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Figure 7.12: TIC of fresh oyster mushrooms at D0.

Figure 7.13: TIC of fresh oyster mushrooms boiled under reflux.

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Figure 7.14: TIC of dried oyster mushrooms at 50 °C.

Figure 7.15: TIC of dried (50 °C) and boiled under reflux oyster mushrooms.

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Figure 7.16: TIC of fresh shiitake mushrooms at D0.

Figure 7.17: TIC of fresh shiitake mushrooms boiled under reflux.

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Figure 7.18: TIC of dried shiitake mushrooms at 40 °C.

Figure 7.19: TIC of dried (40 °C) and boiled under reflux shiitake mushrooms.

Figure 7.20: Mass spectra of 1-octen-3-ol.

Figure 7.21: Mass spectra of 3-octanol.

Figure 7.22: Mass spectra of 3-octanone.

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Figure 7.23: Mass spectra of 2-octen-1-ol.

Figure 7.24: Mass spectra of 2-undecanone.

Figure 7.25: Mass spectra of benzaldehyde.

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Figure 7.26: Mass spectra of (E,E)-2,4-heptadienal.

Figure 7.27: Mass spectra of phenylacetaldehyde.

Figure 7.28: Mass spectra of limonene.

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Figure 7.29: Mass spectra of n-hexanal.

Figure 7.30: Mass spectra of n-octanol.

Figure 7.31: Mass spectra of nonanol.

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Figure 7.32: Mass spectra of 1,2,4-trithiolane.

Figure 7.33: Mass spectra of 2,3,5,6-tetrathiaheptane.

Figure 7.34: Mass spectra of dimethyl disulfide.

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Figure 7.35: Mass spectra of lenthionine.

Figure 7.36: Mass spectra of methyl methylthiomethyl disulfide.

Figure 7.37: Mass spectra of dill ether.

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APPENDIX C1. Publication 1-IFR 20(3):1211-1214 (2013)

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APPENDIX C2. Publication 2-IFR 21(1):263-268 (2014).

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