The Investigation of 2-Acetyl-1-pyrroline Formation in Fragrant and Non-fragrant Rice during Cooking Thesis submitted for degree of Doctor of Philosophy Department of Food and Nutritional Science Xuan Wei Supervisors: Dr J. Stephen Elmore, Prof. Lisa Methven March 2019 Declaration I confirm that this is my own work and the use of all material from other sources has been properly and fully acknowledged. Xuan WEI March 2019 Abstract 2-Acetyl-1-pyrroline (2-AP) is a popcorn-like aroma compound with low odour detection threshold. This compound is regarded as the key contributor of fragrant rice aroma. 2-AP can be detected in fragrant rice, whereas the content of this compound in non-fragrant rice is too low to be detected. Levels of 2-AP in raw fragrant rice can be affected by abiotic stress and growth conditions, such as climate and soil conditions. Whereas it was originally thought that this compound could not be enhanced post-harvest, including cooking. One previous study in our research group demonstrated that 2-AP could be increased in fragrant rice after baking at 180 °C for 20 min. Hence, the objective of this project was to evaluate the mechanism of 2-AP formation in rice during cooking. 2-AP content was evaluated in fragrant and non-fragrant rice prepared by different cooking methods. The results of sensory evaluation on boiled milled rice confirmed that 2-AP and its popcorn-like aroma is the most important factor to differentiate fragrant and non-fragrant rice. Popcorn-like aroma was also noticed in boiled non-fragrant rice and the results from GC- olfactometry confirmed the presence of 2-AP in raw non-fragrant rice. Levels of 2-AP increased on baking milled rice (without water addition) at 180 °C. As the high level of lipids and polyphenols in rice bran might affect 2-AP formation during baking, 2-AP was not observed in rice bran after baking. When milled rice was baked at a range of temperatures, formation of 2-AP in fragrant and non- fragrant rice showed different trends; maximum 2-AP formation was observed in fragrant rice at around 100 °C, whereas 2-AP formation in non-fragrant rice only took place when the baking temperature was higher than 140 °C. Addition of intermediate compounds confirmed that the 2- AP formation at 180 °C occurred through Maillard reaction; the addition of external 1-pyrroline and methylglyoxal was shown to reduce 2-AP generation yield at 100 °C. These results suggested that formation of 2-AP in both fragrant and non-fragrant rice is driven by Maillard reaction at 180 °C baking. However, 2-AP generation in fragrant rice during baking at 100 °C may follow another reaction pathway and this pathway is sensitive to 1-pyrroline and methylglyoxal. Further research should focus on the effect of biosynthesis on 2-AP generation at 100 °C baking. i Acknowledgement I would like to express my deepest gratitude to my supervisors, Dr J. Stephen Elmore and Prof. Lisa Methven. I would never finish my PhD and this thesis without their guidance, supervision, encouragement and patience. I would like to thank Dr Jane Parker and Dr Dimitris Balagiannis who offered me support and advice on my whole PhD research, and thank Andy Dodson, who offered me the training and the technical support on GC-MS analysis. Also, I would like to thank Giract, the Bursary for the 1st year PhD students in Flavour Research. I am extremely grateful to them for honouring me a scholarship that supported me to start my first year PhD research. I would like to express my sincere acknowledgement to Prof. Don Mottram and British Society of Flavourists, who offered me to attend the 15th Flavourist Course, where I gained valuable knowledge on flavour chemistry and comprehensive understanding on this field. I would like to thank Dr Dody. D. Handoko. As my PhD project is based on his research, he is the person who led me into fragrant rice research field. Also, I would like to thank the Indonesia Centre of Rice Research, the institute that Dr Handoko works for, to provide me Sintanur and Ciherang rice samples. Last but not least, I would like to express my special gratitude to my parents, my entire family and my girlfriend Sherrie, for their loving, supporting, understanding, and encouraging on my PhD and entire life. ii Table of Contents Abstract ........................................................................................................................................... i Acknowledgement ..........................................................................................................................ii List of Figures ............................................................................................................................ viii List of Tables .................................................................................................................................. x List of Abbreviations .................................................................................................................... xi Chapter 1. Introduction ................................................................................................................ 1 Chapter 2. Evaluation of 2-acetyl-1-pyrroline in foods, with an emphasis on rice flavour .... 5 2.1. Introduction ....................................................................................................................... 6 2.2. Food Sources of 2-Acetyl-1-pyrroline .............................................................................. 6 2.2.1. Rice ......................................................................................................................... 7 2.2.2. Pandan .................................................................................................................... 9 2.2.3. Cereal products ..................................................................................................... 10 2.2.4. Other foods ........................................................................................................... 11 2.2.5. 2-Acetyl-1-pyrroline as a flavouring .................................................................... 12 2.3. Biological Formation of 2-Acetyl-1-pyrroline ................................................................ 13 2.3.1. Fragrant rice ......................................................................................................... 13 2.3.2. Formation of 2-acetyl-1-pyrroline by microorganisms ........................................ 19 2.4. Formation of 2-Acetyl-1-pyrroline through the Maillard Reaction ................................ 21 2.6. Stability and Stabilisation of 2-Acetyl-1-pyrroline ......................................................... 31 2.7. Extraction and Instrumental Analysis of 2-Acetyl-1-pyrroline ....................................... 33 2.7.1. Solvent-based extraction techniques ..................................................................... 33 2.7.2. Headspace techniques ........................................................................................... 36 2.7.3. Gas chromatography-mass spectrometry ............................................................. 38 2.7.4. Quantification of 2-acetyl-1-pyrroline .................................................................. 39 iii 2.8. Sensory Evaluation of 2-Acetyl-1-pyrroline ................................................................... 41 2.9. Conclusions ..................................................................................................................... 43 Chapter 3. Methods development of 2-acetyl-1-pyrroline extraction and quantification .... 44 3.1. Introduction ..................................................................................................................... 45 3.2. Materials and Methods .................................................................................................... 47 3.2.1. Plant materials and chemicals .............................................................................. 47 3.2.2. Rice sample preparation ....................................................................................... 47 3.2.3. 2-Acetyl-1-pyrroline extraction from rice samples ............................................... 47 3.2.3.1. Solid phase-extraction ................................................................................ 47 3.2.3.2. Headspace solid phase-microextraction .................................................... 48 3.3. Results and discussions ................................................................................................... 50 3.3.1. Optimisation of GC-MS analysis .......................................................................... 50 3.3.1.1. Separation of 2-AP ..................................................................................... 50 3.3.1.2. Determination of limit of quantification (LOQ) for 2-AP and establishment of calibration curve ................................................................................................. 53 3.3.2. Optimisation of extraction techniques for 2-AP in rice ........................................ 54 3.3.2.1. Solid phase-extraction ................................................................................ 54 3.3.2.2. Solid phase-microextraction ....................................................................... 55 4.3.2.3. The effect of the chosen extraction techniques on 2-acetyl-1-pyrroline analysis ...................................................................................................................