Investigation and Optimization of Methods for the Extraction of Antioxidants and Polyphenols from

126 Pages·2017·1.9 MB·English

by  Andrea Dinardo

Investigation and Optimization of Methods for the Extraction of Antioxidants
and Polyphenols from Yellow European Plums (Prunus Domestica L.)

By
Andrea Di Nardo

A Thesis
presented to
The University of Guelph

In partial fulfillment of the requirements
for the Degree of
Master of Applied Science
in
Biological Engineering

Guelph, Ontario, Canada
© Andrea Di Nardo, May 2018
ABSTRACT
Investigation and Optimization of Methods for the Extraction of Antioxidants and
Polyphenols from Yellow European Plums (Prunus Domestica L.)
Andrea DiNardo Advisor:
University of Guelph, 2018 Ashutosh Singh, PhD
Yellow European Plums (YEPs) contain health promoting compounds which may be extracted for
use in functional food and nutraceutical industries. Conventional extraction methods include heat
reflux extraction (HRE) and Soxhlet extraction, but often result in reduced extraction yields due
to high temperature requirements and long extraction durations. Microwave-assisted extraction
(MAE) is one method which can mitigate these drawbacks because it allows rapid and
homogenous heating the biomatrix. The objective of this thesis is to determine the effects of
solvent and solvent concentration, time, and temperature during MAE in comparison to other
extraction methods. Concentration of phenolic compounds, specifically ascorbic acid, chlorogenic
acid, neochlorogenic acid, were quantified using a Folin-Ciocalteau method and HPLC analysis.
Antioxidant scavenging of free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Fe3+-
tripyridyltraizine (FRAP) were used to determine antioxidant capacity of samples. It was observed
that MAE yielded larger concentrations of bioactive compounds in comparison to other extraction
methods.
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ACKNOWLEDGEMENTS
This project was made possible by the generous support from the Natural Sciences and
Engineering Research Council of Canada, the Arrell Food Institute at the University of Guelph
and Food from Thought Initiative, the Ontario Ministry of Agriculture Food and Rural Affairs,
and the support of the Ontario Tender Fruit Marketing Board and the Niagara Peninsula Fruit and
Vegetable Growers Association.
To say that this thesis was completed as a personal achievement would be an understatement.
The completion of this work was done so as a collaborative effort by the incredible people who
surround me. I would first and foremost like to thank my advisor, Dr. Ashutosh Singh. I will never
be able to put into words just how thankful I am for all the guidance and trust you have given me.
The skills and knowledge I have gained under your leadership are invaluable and I will be forever
grateful to have been one of your graduate students.
Thank you to Dr. Jayasankar Subramanian for all your generosity and encouragement in all of
my projects and endeavours. Thank you to Dr. Manickavasagan Annamalai for serving as on my
advisory committee and for all your support. I would also like to thank Dr. Erica Pensini for being
a listening ear during my writer’s block and for serving on my defense committee.
I would like to extend gratitude to the staff here at the School of Engineering. Phil Watson,
Jeff Madge, and Jacqueline Fountain for technical support, and Jaqueline Floyd, Jenn McCreary,
and Jeanne Castellanos for administrative support. Thank you to Jose Varghese from SCP Sciences
for your support and guidance in installation, training, and troubleshooting of the microwave-
digestion unit.
The students from the Food Research Lab, who have become family, were an integral part
of my success. Thank you Harjeet, Rudy, Kelsie, Abhjeet, Ramandeep, Arshdeep, and Arvind.
From our coffee dates to our road trips and dance parties, you all helped keep me sane during this
process. To my friends Haleigh and Sarah, thank you for listening to me through all my rants and
always making me laugh.
Finally, I would like to thank my family. To my parents, Carlo and Elena, for continuously
supporting me in all my endeavours. I attribute all my success to your unwavering support. To my
siblings Daniela and Luca, you are constant sources of inspiration to me and always manage to put
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a smile on my face. And finally, to my fiancée, Gregory. Thank you for standing by me during my
many years of study. You have continuously encouraged me to pursue my dreams, no matter what
the cost, and I can never truly thank you enough. This thesis is dedicated to all of you.
v

CONTRIBUTIONS OF AUTHORS
The following are the manuscripts prepared for publication:
i. DiNardo, A., Subramanian, J., and Singh, A. (2018). Investigation of Antioxidant Content
and Capacity in Yellow European Plums. International Journal of Fruit Science, 18(1): 99-
116.
ii. DiNardo, A., Subramanian, J., and Singh, A. (2018). Intensification of Phenolic Extraction
from Yellow European Plums by use of Conventional, Microwave-, and Ultrasound-
Assisted Extraction. Separation Science and Technology (Under Review).
iii. DiNardo, A., Brar, H.S., Subramanian, J., and Singh, A. (2018). Optimization of
Microwave-Assisted Digestion Parameters and Characterization of Phenolic Compounds in
Yellow European Plums. The Canadian Journal of Chemical Engineering (Accepted).
The work reported here was performed by Andrea DiNardo and supervised by Dr. Ashutosh
Singh of the School of Engineering, University of Guelph, Guelph. Ontario. The entire research
work was conducted in the Food Research Lab, School of Engineering, University of Guelph,
Guelph. Ontario.
Co-authors, Harjeet Singh Brar (M.A.Sc. Student, School of Engineering, University of
Guelph), Dr. Jayasankar Subramanian (Professor, Department of Plant Agriculture, University of
Guelph), and Dr. Ashutosh Singh (Associate Professor, School of Engineering, University of
Guelph), provided guidance and support in data analysis and in the development of the
manuscripts.
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Table of Contents
ACKNOWLEDGEMENTS ........................................................................................................... iii
CONTRIBUTIONS OF AUTHORS .............................................................................................. v
List of Figures ................................................................................................................................. x
List of Tables ................................................................................................................................ xii
List of Abbreviations ................................................................................................................... xiv
CHAPTER 1 INTRODUCTION .................................................................................................... 1
1.1 Hypothesis ............................................................................................................................. 2
1.2 Research Objectives .............................................................................................................. 2
1.2.1 Overall Objective ............................................................................................................ 2
1.2.2 Specific Objectives ......................................................................................................... 2
CHAPTER 2 LITERATURE REVIEW ......................................................................................... 3
2.1 Introduction ........................................................................................................................... 3
2.2 Food supplements and nutraceuticals .................................................................................... 4
2.2.1 Phenolic compounds ....................................................................................................... 5
2.3 Conventional methods of extraction ..................................................................................... 7
2.3.1 Heat reflux extraction (HRE) ......................................................................................... 8
2.3.2 Soxhlet extraction ........................................................................................................... 8
2.4 Novel Methods of Extraction ................................................................................................ 9
2.4.1 Ultrasound-assisted extraction (UAE) ............................................................................ 9
2.4.2 Supercritical fluid extraction (SFE) .............................................................................. 10
2.4.3 Pressurized fluid extraction (PFE) ................................................................................ 10
2.4.4 Microwave-assisted extraction (MAE) ......................................................................... 11
2.4 Extraction of polyphenols from fruits and vegetables ........................................................ 13
2.4.1 Apples ........................................................................................................................... 13
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2.4.2 Berries ........................................................................................................................... 14
2.4.3 Asparagus ..................................................................................................................... 15
2.4.5 Yellow European Plums (YEPs) .................................................................................. 15
2.4.7 Other food sources of phenolic compounds ................................................................. 16
Conclusion ................................................................................................................................. 16
References ................................................................................................................................. 17
CHAPTER 3 INVESTIGATION OF ANTIOXIDANT CONTENT AND CAPACITY IN
YELLOW EUROPEAN PLUMS ................................................................................................. 24
Abstract ..................................................................................................................................... 24
3.1 Introduction ......................................................................................................................... 24
3.2 Materials and Methods ........................................................................................................ 26
3.2.1 Fruits and chemicals ..................................................................................................... 26
3.2.2 Preparation of methanolic plum extracts ...................................................................... 26
3.2.3 Determination of total phenolic content ....................................................................... 27
3.2.4 Scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals ..................... 27
3.2.5 Determination of ferric reducing antioxidant potential (FRAP) .................................. 28
3.2.6 High performance liquid chromatography (HPLC) analysis ........................................ 28
3.2.7 Statistical analysis......................................................................................................... 30
3.3 Results and Discussion ........................................................................................................ 30
3.3.1 Determination of total phenolics .................................................................................. 30
3.3.2 HPLC analysis .............................................................................................................. 32
3.3.3 Determination of radical scavenging activity ............................................................... 34
3.3.4 Determination of ferric reducing antioxidant potential (FRAP) .................................. 35
3.3.5 Correlation between phenolic content and antioxidant activity ................................... 37
Conclusion ................................................................................................................................. 40
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References ................................................................................................................................. 40
CONNECTING TEXT ................................................................................................................. 45
CHAPTER 4 INTENSIFICATION OF PHENOLIC EXTRACTION FROM YELLOW
EUROPEAN PLUMS BY USE OF CONVENTIONAL, MICROWAVE-, AND
ULTRASOUND- ASSISTED EXTRACTION ............................................................................ 46
Abstract ..................................................................................................................................... 46
4.1 Introduction ......................................................................................................................... 46
4.2 Materials and Methods ........................................................................................................ 49
4.2.1 Fruits and chemicals ..................................................................................................... 49
4.2.2 Sample preparation ....................................................................................................... 49
4.2.3 Microwave extraction, ultrasound extraction, and heat reflux extraction .................... 49
4.2.4 Determination of total phenolic content ....................................................................... 51
4.2.5 Quantification of total phenolic content ....................................................................... 51
4.2.6 Antioxidant assays ........................................................................................................ 52
4.2.7 Statistical analysis......................................................................................................... 52
4.3 Results and discussion ......................................................................................................... 53
4.3.1 Heat reflux extraction (HRE) ....................................................................................... 53
4.3.2 Microwave-assisted extraction (MAE) ......................................................................... 57
4.3.3 Ultrasound-assisted extraction (UAE) .......................................................................... 60
4.3.4 Selection of a suitable PI approach............................................................................... 65
Conclusions ............................................................................................................................... 67
References ................................................................................................................................. 67
CONNECTING TEXT ................................................................................................................. 72
CHAPTER 5 OPTIMIZATION OF MICROWAVE-ASSISTED DIGESTION PARAMETERS
AND CHARACTERIZATION OF PHENOLIC COMPOUNDS IN YELLOW EUROPEAN
PLUMS ......................................................................................................................................... 73
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Abstract ..................................................................................................................................... 73
5.1 Introduction ......................................................................................................................... 73
5.2 Materials and Methods ........................................................................................................ 75
5.2.1 Fruits and chemicals ..................................................................................................... 75
5.2.2 Sample preparation ....................................................................................................... 75
5.2.3 Microwave-assisted digestion (MD) ............................................................................ 76
5.2.4 Determination of total phenolic content (TPC) ............................................................ 77
5.2.5 Quantification of phenolic compounds ......................................................................... 77
5.2.6 Antioxidant Assays ....................................................................................................... 78
5.2.7 Statistical analysis......................................................................................................... 79
5.3 Results and Discussion ........................................................................................................ 80
5.3.1 MD parameter selection................................................................................................ 80
5.3.2 MD process optimization.............................................................................................. 88
5.3.3 Comparison of MD with conventional extraction ........................................................ 99
Conclusions ............................................................................................................................. 100
References ............................................................................................................................... 100
CHAPTER 6 SUMMARY, CONCLUSION AND FUTURE WORK ...................................... 104
6.1 Summary and conclusion .................................................................................................. 104
6.2 Future work ....................................................................................................................... 105
Appendix ................................................................................................................................. 107
Supplementary Tables ......................................................................................................... 107
Supplementary Figures ........................................................................................................ 110
x

List of Figures
Figure 2.1. Number of published articles from 1997 to 2017 on nutraceuticals and functional foods
(Wed of Science Database, 2018) ................................................................................................... 5
Figure 2.2. Basic chemical structure of a flavonoid (Adopted from (Routray and Orsat, 2012)) . 6
Figure 2.3. Chemical structure of stilbene and lignin (Adopted from (Dai and Mumper, 2010)) . 7
Figure 2.4. Soxhlet extraction apparatus (Adopted from Singh and Orsat (2015)) ....................... 9
Figure 3.1. HPLC chromatogram of fresh plum methanolic extract. Ascorbic acid (peak 1), neo-
chlorogenic acid (peak 2), and chlorogenic acid (peak 3) ............................................................ 29
Figure 3.2. Correlation between total phenolic content and DPPH scavenging activity for freeze
dried genotypes (where ● = V98261, =V94021, =V91074, ▲=V98197, and =V95141) ... 38
Figure 3.3. Correlation between total phenolic content and FRAP activity for freeze dried
genotypes (where ● = V98261, =V94021, =V91074, ▲=V98197, and =V95141) ............ 38
Figure 3.4. Correlation between total phenolic content and DPPH scavenging activity for fresh
genotypes (where ● = V98261, =V94021, =V91074, ▲=V98197, and =V95141) ............ 39
Figure 3.5. Correlation between total phenolic content and FRAP activity for fresh genotypes
(where ● = V98261, =V94021, =V91074, ▲=V98197, and =V95141) ............................. 39
Figure 4.1. Response surface profile of the effect of power level (%) and time (min) on total
phenolic content of YEPs.............................................................................................................. 58
Figure 4.2. Response surface profile of the effect of power level (%) and time (min) on DPPH
scavenging activity (%) of YEPs .................................................................................................. 59
Figure 4.3. Response surface profile of the effect of power level (%) and time (min) on ferric
reducing antioxidant potential (FRAP) of YEPs .......................................................................... 60
Figure 5.1. Total phenolic content during MD when using A. ethanol, and B. methanol as
extraction solvents ........................................................................................................................ 83
Figure 5.2. DPPH scavenging activity during MD when using A. ethanol and B. methanol as
extraction solvents ........................................................................................................................ 84