VOLATILE COMPOUNDS IN FRESH AND PROCESSED ORIENTAL MUSHROOMS
A thesis in the fulfilment of the requirements for the degree of
Doctor of Philosophy
by
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 ……………………………………………......
COPYRIGHT STATEMENT
‘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.'
Signed ……………………………………………......
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.’
Signed ……………………………………………......
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.
ii
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
iii
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.
iv
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
v
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
vi
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
vii
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
viii
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
ix
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
x
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
xi
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
xii
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
xiii
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
xv
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
1
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
2
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.
3
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
4
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.
5
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 anti- tumour 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,
6
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
7
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
8
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.
9
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).
11
Figure 2.5: Cultivation process of the studied mushrooms. (Source: Li-Sun Exotic Mushrooms, 2010).
12
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).
13
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,
14
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).
15
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
17
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).
18
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
19
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).
21
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).
22
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
23
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
25
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) (Hu bschmann, 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 (Hu bschmann, 2001).
28
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
29
(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).
30
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).
32
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.
33
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).
34
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 post- harvest 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.
40
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).
41
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.
43
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