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Entitled Optimizing the microwave-assisted extraction of phenolic antioxidants from apple
pomace and microencapsulation in cyclodextrins
For the degree of Master of Science
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Tameshia Ballard
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Peter Hirst
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Title of Thesis/Dissertation:
Optimizing the microwave-assisted extraction of phenolic antioxidants from apple pomace
and microencapsulation in cyclodextrins
Master of Science
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Vaishnavi Chandrasekar
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*Located at http://www.purdue.edu/policies/pages/teach_res_outreach/viii_3_1.html
OPTIMIZING THE MICROWAVE-ASSISTED EXTRACTION OF PHENOLIC
ANTIOXIDANTS FROM APPLE POMACE AND MICROENCAPSULATION IN
CYCLODEXTRINS
A Thesis
Submitted to the Faculty
of
Purdue University
by
Vaishnavi Chandrasekar
In Partial Fulfillment of the
Requirements for the Degree
of
Master of Science
August 2010
Purdue University
West Lafayette, Indiana
UMI Number: 1489654
All rights reserved
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a note will indicate the deletion.
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ii
For Amma and Appa
iii
ACKNOWLEDGMENTS
I would like to thank my advisor Dr. Tameshia Ballard for being my mentor and
friend, and guiding me through the graduate program. I enjoyed working with her and
learnt a lot in the process! I would also like to thank Dr. Fernanda San-Martin and Dr.
Peter Hirst for serving on my committee, and for all their help and advice.
I am grateful to all the faculty and staff in Food Science for their help over the
past two years. A special thanks to Dr. Nivens for his guidance in various analytical
techniques. Also to Dr. Liceaga for permitting me to use of her lab equipment whenever I
needed to. I am also thankful to Dr. Campanella and Bhavesh Patel for helping me with
the DSC.
I thank my office mates Rei and Mandar for their support and friendship during
these last two years. I am also eternally grateful to my ‘mafia’ friends in Food Science…
Preetha, Deepak, Shivangi and Azalenah for all their support and help, and the endless
cups of coffee that provided motivation during my day!
Finally, I am forever grateful to my family for their support and strength in all my
endeavors. My parents, for the love that keeps me positive everyday; my older sister
Preethi for her spontaneity and generosity; and my twin sister Vaidehi for being there
always.
iv
TABLE OF CONTENTS
Page
LIST OF TABLES……………………………………………………………………..…vi
LIST OF FIGURES…………………………………………………………………..….vii
ABSTRACT .................................................................................................................. ix
CHAPTER 1
INTRODUCTION ................................................................................1
Hypothesis ...................................................................................................................3
Objectives....................................................................................................................3
Significance and Rationale...........................................................................................4
Thesis organization ......................................................................................................4
References ...................................................................................................................6
CHAPTER 2
LITERATURE REVIEW .....................................................................8
2.1 Antioxidants ..........................................................................................................8
2.2 Apple polyphenols ...............................................................................................11
2.3 Extraction techniques ...........................................................................................13
2.3.1 Solvent extraction..........................................................................................13
2.3.2 Supercritical fluid extraction (SFE) ...............................................................14
2.3.3 Accelerated solvent extraction (ASE) ............................................................16
2.3.4 Microwave assisted extraction (MAE) ...........................................................17
2.4 Quantification of phenolic compounds .................................................................21
2.5 Estimation of antioxidant activity.........................................................................22
2.5.1 ORAC assay ..................................................................................................23
2.5.2 DPPH Assay .................................................................................................24
2.5.3 FRAP Assay ..................................................................................................25
2.5.4 TEAC Assay .................................................................................................26
2.6 Encapsulation of polyphenols...............................................................................26
2.6.1 Wall materials ...............................................................................................27
2.6.2 Analysis of inclusion complexes....................................................................32
2.7 Summary .............................................................................................................34
References .................................................................................................................35
v
Page
CHAPTER 3
OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION OF
PHENOLIC ANTIOXIDANTS FROM APPLE POMACE ........................................... 41
Abstract .....................................................................................................................42
Introduction ...............................................................................................................43
Methods and Materials...............................................................................................46
Results and Discussion ..............................................................................................50
Conclusion ................................................................................................................58
References .................................................................................................................75
CHAPTER 4
ENCAPSULATION OF POLYPHENOL EXTRACTS FROM APPLE
POMACE IN CYCLODEXTRINS ................................................................................77
Abstract .....................................................................................................................78
Introduction ...............................................................................................................79
Methods and materials ...............................................................................................81
Results and discussion ...............................................................................................85
Conclusion ................................................................................................................89
References .................................................................................................................96
CHAPTER 5
CONCLUSIONS ................................................................................98
vi
LIST OF TABLES
Table
Page
Table 3.1 Coded values and corresponding actual values of the optimization parameters
......................................................................................................................68
Table 3.2 Experimental design for extraction of phenolic compounds by MAE in terms of
coded values ..................................................................................................69
Table 3.3 Response surface models fitted based on TPC for different apple cultivars and
solvent systems ..............................................................................................70
Table 3.4 Response surface models fitted based on DPPH activity for different apple
cultivars and solvent systems .........................................................................71
Table 3.5 Optimum conditions for the extraction of phenolic antioxidant compounds by
MAE based on both TPC and DPPH activity .................................................72
Table 3.6 Predicted and experimental values of TPC and DPPH activity for Red
Delicious apple pomace extracted under optimal conditions for 60% ethanol .73
Table 3.7 HPLC quantification of major apple polyphenols from optimized MAE extracts
for each apple cultivar and solvent system ..................................................... 74
Table 4.1 HPLC quantification of apple polyphenols from Red Delicious apple pomace
extract............................................................................................................95
vii
LIST OF FIGURES
Figure
Page
Figure 2.1 Common polyphenols in apple ...................................................................... 12
Figure 2.2 Structure of Cyclodextrin molecule ............................................................... 30
Figure 2.3 Phase solubility diagram (adapted from Higuchi & Connors, 1965) .............. 32
Figure 3.1 Response surface of Red Delicious apple pomace extracted with 70% acetone
- Effect of extraction time and microwave power on TPC ............................. 59
Figure 3.2 Response surface of apple pomace extracts with 70% acetone - Effect of
solvent volume and power on TPC ............................................................... 60
Figure 3.3 Effect of solvent volume on TPC of Winesap apple pomace extracted with
70% acetone ................................................................................................. 61
Figure 3.4 Effect of solvent volume on DPPH inhibition percentage of Jonathan apple
pomace extracted with 70% acetone ............................................................. 62
Figure 3.5 Interaction plot of Red Delicious apple pomace extracted with 60% ethanol Effect of microwave power and time on TPC ............................................... 63
Figure 3.6 Response surface of Jonathan apple pomace extracted with 60% ethanol Effect of solvent volume and power on DPPH inhibition percentage ............ 64
Figure 3.7 Response surface of the effect of solvent volume and power on both TPC and
DPPH inhibition percentage (desirability) for Red Delicious apple pomace
extracted with 60% ethanol .......................................................................... 65
Figure 3.8 Response surface of the effect of solvent volume and power on both TPC and
DPPH inhibition percentage (desirability) for Red Delicious apple pomace
extracted with 70% acetone .......................................................................... 66
Figure 3.9 HPLC chromatogram at 280 nm of Red Delicious apple pomace extracted with
60% ethanol under optimum conditions ........................................................ 67
viii
Figure
Page
Figure 4.1 Complex formation study of apple extract with (A) α-CD (B) β-CD and (C) 2hydroxypropyl-β-CD .................................................................................... 90
Figure 4.2 DSC thermograms of pure apple pomace extract and inclusion complexes
under oxidative conditions ............................................................................ 93
Figure 4.3 Phase solubility study of apple pomace extract with α-, β, and γ-CD at 30 °C
(n=2) ............................................................................................................94
ix
ABSTRACT
Chandrasekar, Vaishnavi, M.S. Purdue University, August, 2010. Optimizing the
microwave-assisted extraction of phenolic antioxidants from apple pomace and
microencapsulation in cyclodextrins. Major Professor: Tameshia Ballard.
Apple pomace is the skin, seeds and flesh leftover after juice extraction, and is
rich in polyphenols that have demonstrated high antioxidant activity both in vivo and in
vitro. Despite this fact, apple pomace remains an underutilized agricultural waste product.
To add value and increase the commercial applicability of the pomace, efficient methods
for the extraction of phenolics from apple pomace are needed. Traditional solvent
extraction is both solvent and time intensive, leading to the consideration of alternate
extraction techniques such as microwave-assisted extraction (MAE). MAE of phenolic
compounds from pomace of four apple cultivars (Red Delicious, Golden Delicious,
Winesap and Jonathan) was optimized for various extraction parameters (microwave
power, extraction time, solvent volume to sample ratio and solvent type) using response
surface methodology. Optimum conditions were based on maximizing two responses:
total polyphenol content (TPC) and antioxidant activity as measured by the percent
inhibition (%IP) of the DPPH free radical. Response surface models were developed by
regression, taking into consideration significant model parameters (p<0.05). Each solvent
system showed similar optimum extraction conditions independent of apple cultivar. Red
Delicious pomace variety had the highest TPC (15.8 mg GAE/g DW) and DPPH radical
x
scavenging activity (94.4 % inhibition) obtained under the optimum conditions of 735 W
and 149 s, with 10.4 mL of 60% ethanol and 5.65 mL of 70% acetone, respectively.
Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin were some of the
major phenolic compounds identified by HPLC in the extracts.
Stabilization of the extracted phenolics obtained under optimized conditions was
carried out by encapsulation in α-, β-, and 2-hydroxypropyl-β-cyclodextrin. Inclusion
complex formation was confirmed by differential scanning calorimetry for all the three
CD types. Complexes were also found to be stable to oxidation up to temperatures of 250
°C. Phase solubility studies indicated an A L type diagram and established the formation
of a 1:1 complex of the extract with β-CD, with a moderate binding constant (K c = 1.02
mg GAE/g extract -1). The overall results of this research indicate that MAE is an
efficient method for extraction of valuable antioxidant compounds from apple pomace.
Further, their encapsulation in β-CD results in the formation of stable inclusion
complexes with enhanced solubility for potential use as functional food ingredients.
Keywords: apple pomace, microwave-assisted extraction, encapsulation, cyclodextrin,
inclusion complex
1
CHAPTER 1. INTRODUCTION
Polyphenols are a diverse group of compounds that are present in plants, and are
synthesized as secondary metabolites. They are known to possess very good antioxidant
activity (1). Currently the industry makes use of a number of synthetic antioxidants such
as BHA, BHT and TBHQ to combat the problem of lipid oxidation, and enhance the
shelf-life of lipids and lipid-containing foods. Due to increasing consumer demand for
healthier, all natural products, industry’s reliance on these synthetic compounds must be
minimized. Thus, the challenge for the food industry moving forward is to create foods
containing more natural ingredients while maintaining sensory quality. The use of these
naturally occurring antioxidant polyphenols in place of synthetic antioxidants in foods is
a potential area worth investigating.
Several million tons of apples are processed as juice or cider, generating hundreds
of tons of apple pomace as a waste stream globally (2). Polyphenols from apple pomace
have been extensively studied, and are known to possess high antioxidant and
antiproliferative activities and effectively inhibit lipid oxidation (3). The major phenolic
compounds present in apple include catechin, epicatechin, quercetin glycosides and
phloridzin (4). The type and amount of phenolic compounds present varies widely
2
depending on the apple cultivar, maturity and environmental growth conditions (3).
Polyphenols have conventionally been extracted from the apple matrix by solid-liquid
extraction using various organic solvents including ethanol, methanol, acetone, or
mixtures of these with water (5-6). However, conventional extraction techniques
consume large amounts of solvent, and are also time intensive. As a result, alternate
extraction techniques such as ultrasound-assisted extraction (7), sub-critical carbondioxide extraction (8) and pressurized liquid extraction (9) have been studied to improve
the extraction of phenolic compounds from apples.
Microwave-assisted extraction (MAE) is a relatively new extraction technique
that utilizes microwaves for heating up the sample and solvent system, rapidly and
homogenously. The principle of heating using microwaves is based on the direct action of
the electromagnetic radiation on the molecules through ionic conduction and dipole
rotation, resulting in heating (10). Polar solvents with high dielectric constants tend to
heat up more rapidly with microwaves than non-polar solvents with lower dielectric
constants (11). Thus, polar solvents or non-polar solvents diluted with water are
frequently employed solvent systems with MAE. MAE has been increasingly used for the
rapid extraction of bioactive compounds from plant sources (12-14). Compared to solvent
extraction, it has been shown to reduce solvent requirements and extraction time (12, 1415). However, the extraction of phenolic compounds from apple pomace using MAE has
not been studied and optimized.
As phenolic compounds are antioxidants, they are highly prone to oxidation, and
degrade in the presence of heat and light (16). This makes them difficult to store and
3
transport. Furthermore, many of these compounds are hydrophobic in nature, and
therefore, demonstrate limited solubility in water. Encapsulation of phenolic antioxidants
in suitable wall materials may enhance their functionality. Cyclodextrins, or modified
starch molecules, are commonly used wall materials for the encapsulation of hydrophobic
compounds (17). They are cyclic molecules that consist of a relatively hydrophobic
cavity which may form inclusion complexes with the phenolic compounds. Several
hydroxyl groups are present on the outer surface, which enhances the water solubility of
the inclusion complex. Modified cyclodextrins with higher water solubility have also
been developed by the addition of polar side chains. Therefore, the stability and
functionality of the extracted polyphenols may be improved by their encapsulation in
native or modified cyclodextrins.
Hypothesis
This work is based on the hypothesis that microwave-assisted extraction may be
used as a rapid and efficient technique to increase the yield of valuable natural
antioxidant compounds from apple pomace. Additionally, the water solubility and
oxidative stability of phenolic compounds present in the pomace extracts will be
increased by encapsulation in cyclodextrins.
Objectives
1. Optimize the microwave-assisted extraction of phenolic antioxidants from apple
pomace using response surface methodology.
4
2. Encapsulate apple pomace extract in cyclodextrins and characterize their
complex formation, oxidative stability and solubility using DSC and phase
solubility studies.
Significance and Rationale
Several million tons of apple pomace is generated globally as an industrial waste
stream from apple cider or juice production. The extraction of phenolic antioxidant
compounds from this waste stream could prove to be beneficial in terms of generating
value-added food ingredients. Polyphenols have traditionally been extracted by solidliquid extraction, and conditions for maximizing yields have been optimized. However,
conventional solvent extraction is both time and solvent intensive. The use of alternative
extraction techniques, in this case MAE, may prove to be more practical. Once removed
from their native plant matrix, polyphenols are unstable and prone to oxidation in the
presence of heat and light. In addition, several of these phenolics have very low solubility
in water, which limits their applications in the food industry. Encapsulation of these
compounds in cyclodextrins may enhance their stability and improve their solubility,
thereby increasing overall commercial applicability.
Thesis organization
This thesis consists of five chapters. Following the introduction is the second
chapter consisting of a concise literature review. The third and fourth chapters outline the
work carried out under the two objectives stated above, and have been written in
manuscript format to be submitted to peer-reviewed journals. The third chapter focuses
on optimizing MAE of phenolic compounds from apple pomace. The fourth chapter
5
details the encapsulation of the polyphenol extracts in cyclodextrins that were obtained in
the MAE study. The final chapter summarizes the overall conclusions, and outlines some
possible future work that may be carried out.
6
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Dapkevicius, A.; Venskutonis, R.; van Beek, T. A.; Linssen, J. P. H., Antioxidant
activity of extracts obtained by different isolation procedures from some
aromatic herbs grown in Lithuania. J Sci Food Agr 1998, 77, (1), 140-146.
Vendruscolo, F.; Albuquerque, P. M.; Streit, F.; Esposito, E.; Ninow, J. L.,
Apple Pomace: A Versatile Substrate for Biotechnological Applications. Critical
Reviews in Biotechnology 2008, 28, (1), 1-12.
Boyer, J.; Liu, R. H., Apple phytochemicals and their health benefits. Nutr J
2004, 3, 5.
Lu, Y.; Foo, L. Y., Identification and quantification of major polyphenols in
apple pomace. Food Chemistry 1997, 59, (2), 187-194.
Foo, L. Y.; Lu, Y., Isolation and identification of procyanidins in apple pomace.
Food chemistry 1999, 64, (4), 511-514.
Garcia, Y. D.; Valles, B. S.; Lobo, A. P., Phenolic and antioxidant composition
of by-products from the cider industry: Apple pomace. Food Chemistry 2009,
117, (4), 731-738.
Lee, K. W.; Kim, Y. J.; Kim, D. O.; Lee, H. J.; Lee, C. Y., Major phenolics in
apple and their contribution to the total antioxidant capacity. Journal of
Agricultural and Food Chemistry 2003, 51, (22), 6516-6520.
Adil, I. H.; Çetin, H. I.; Yener, M. E.; BayIndIrlI, A., Subcritical (carbon
dioxide + ethanol) extraction of polyphenols from apple and peach pomaces, and
determination of the antioxidant activities of the extracts. The Journal of
Supercritical Fluids 2007, 43, (1), 55-63.
Alonso-Salces, R. M.; Korta, E.; Barranco, A.; Berrueta, L. A.; Gallo, B.;
Vicente, F., Pressurized liquid extraction for the determination of polyphenols in
apple. Journal of Chromatography A 2001, 933, (1-2), 37-43.
Eskilsson, C. S.; Bjorklund, E., Analytical-scale microwave-assisted extraction.
Journal of Chromatography A 2000, 902, (1), 227-250.
Wang, L.; Weller, C. L., Recent advances in extraction of nutraceuticals from
plants. Trends in Food Science & Technology 2006, 17, (6), 300-312.
Hong, N.; Yaylayan, V.; Raghavan, G.; Paré, J.; Bélanger, J., Microwaveassisted extraction of phenolic compounds from grape seed. Natural Product
Research 2001, 15, (3), 197-204.
Pan, X.; Niu, G.; Liu, H., Microwave-assisted extraction of tea polyphenols and
tea caffeine from green tea leaves. Chemical Engineering and Processing 2003,
42, (2), 129-133.
Kaufmann, B.; Christen, P., Recent extraction techniques for natural products:
microwave-assisted extraction and pressurised solvent extraction. Phytochemical
analysis 2002, 13, (2), 105-113.
Proestos, C.; Komaitis, M., Application of microwave-assisted extraction to the
fast extraction of plant phenolic compounds. LWT-Food Science and Technology
2008, 41, (4), 652-659.
7
16.
17.
Cheynier, V., Polyphenols in foods are more complex than often thought. Am J
Clin Nutr 2005, 81, (1), 223S-229.
Szejtli, J., Introduction and General Overview of Cyclodextrin Chemistry.
Chemical Reviews 1998, 98, (5), 1743-1754.
8
CHAPTER 2. LITERATURE REVIEW
2.1 Antioxidants
Lipids are a major component of foods, and are classified in very basic terms as
fats (lipids that are solid at room temperature) and oils (lipids that are liquid at room
temperature). They contribute to the sensory and flavor profile of various foods, and may
also serve certain functional properties within the food system such as emulsification.
Lipid oxidation is a major problem in the food industry, resulting in loss of nutritional
and sensory qualities and producing undesirable flavors, color and toxins (1). Food
manufacturers currently incorporate synthetic antioxidant compounds such as butylated
hydroxy toluene (BHT), butylated hydroxy anisole (BHA), propyl gallate (PG) and
tertiary-butyl hydroxy quinone (TBHQ) into food products to slow the rate of lipid
oxidation and enhance shelf life. These synthetic antioxidants have proven to be effective
in inhibiting lipid oxidation, but have shown toxicological effects, particularly in higher
concentrations (2). Structurally, all these compounds closely resemble naturally occurring
polyphenols. They all contain one or more hydroxyl groups with hydrogen donating
potential, attached to a modified benzene ring which provides resonance stabilization to
the radical form. As today’s consumers are demanding more all natural food products,
9
the addition of naturally derived polyphenols from plant sources may be a good
alternative to replace their synthetic counterparts.
Polyphenols are a wide group of compounds that are found in a variety of plant
tissues. They are secondary plant metabolites and are synthesized during normal plant
growth as well as during conditions of stress (3). These phenolic compounds contribute to
the organoleptic and sensory properties of plant foods in terms of enhancing their
astringency and bitterness. Polyphenols are a ubiquitous group of compounds that can be
classified into several types based on their chemical structure. Flavonoids are the largest
class of polyphenolic compounds with greater than 5000 compounds identified to date,
and they can be further classified into several subclasses such as flavones, flavonols,
flavanols and anthocyanidins. Other important groups of polyphenolic compounds
include phenolic acids, hydroxycinnamic acids, coumarins, stilbenes and lignans. Bravo
et al. (4) summarizes the major classes of naturally occurring polyphenolic compounds
based on their chemical structure.
Interest in phenolic compounds has been rapidly increasing owing to their roles
as effective antioxidants and scavengers of free radicals (5-6). Rice-Evans et al. (7)
investigated the structure of several naturally occurring phenolic compounds and how
they function as terminators of free radicals and chelators of metal ions that are capable
of catalyzing lipid peroxidation. Phenolics have several hydroxyl groups which are
capable of donating a hydrogen atom to free radicals to form phenoxy radical
intermediates. These intermediate radicals are relatively stable due to resonance. As a
result, a new chain reaction is not easily initiated. Due to their antioxidant properties,
10
there is good potential to extract and utilize these phenolic compounds as replacers of
synthetic antioxidants. The advantage of this approach would be to produce food
products with a clean label with no synthetic preservatives. Consumer demand for
products containing natural ingredients has been rapidly increasing, and nearly 76% of
consumers would choose a product containing ‘all natural’ ingredients rather than
synthetic compounds (Mintel, 2009).
A large amount of these natural antioxidant compounds have been found in
agricultural and industrial by-products. Grape pomace has been studied extensively and is
known to be rich in catechin, epicatechin and quercetin glycosides (8-9). Other
agricultural by-products that have been studied include citrus seeds and peels (10), carrot
pulp waste (11), spent tea leaves (12), cacao by-products (13) and soy-bean molasses
(14). All these studies showed that as a general rule, the natural phenolics extracted from
the plant matrix had comparable antioxidant activity to that of synthetic antioxidants, and
could be used as an alternative to the synthetic ones in foods. In some cases, the natural
extracts showed a reduced antioxidant potential when compared to synthetic antioxidants.
However, they could still be used advantageously as maximum levels established for
synthetic food additives need not be applicable to naturally occurring compounds (15).
A substantial amount of waste is generated from apple processing. Nearly 25 to
30% of the total world production of apples is estimated to be industrially processed into
products such as cider and juice, and this results in generation of several million tons of
apple pomace globally (9). Apple pomace is the skin, seeds and flesh leftover after juice
11
extraction, and is rich in several phenolic compounds (8). This waste stream thus has
excellent potential to be used as a source of valuable antioxidant byproducts.
2.2 Apple polyphenols
Consumption of apples has been related to several health benefits. A large number
of epidemiological and animal studies have shown a link between consumption of apples
and decreased risk of cancer and cardiovascular disease (16). Apple polyphenols have
been extensively studied and are found to contain high antioxidant and anti-proliferative
activity and effectively inhibit lipid oxidation (16). These health benefits have mainly
been attributed to phytochemicals including phenolic antioxidants present in apples. The
major polyphenols identified in apples are catechin, epicatechin, quercetin and quercetin
glycosides, phloridzin, caffeic acid and chlorogenic acid (Fig 2.1). Lee et al. (17)
quantified some of the major polyphenols in six cultivars of apples. The average
concentrations in mg/100 g fruit were: epicatechin 8.65; phloretin glycosides 5.59;
chlorogenic acid 9.02; procyanidin B 9.35; quercetin glycosides 13.2 and vitamin C 12.8.
The total phenols content varies widely depending on the apple cultivar, state of
maturation, availability of growth nutrients, etc.
12
Figure 2.1 Common polyphenols in apple
Several studies indicate a higher concentration of polyphenols in the apple peel
compared to the rest of the fruit (16). Quercetin glycosides are known to be exclusively
present in the peel. Some of the other polyphenols typically present in the peel include
procyanidins, catechin, epicatechin, phloridzin and chlorogenic acid. However, higher
concentrations of chlorogenic acid have been reported in the apple flesh compared to the
peel (18). Several animal studies were conducted to study and compare the beneficial
health effects of apple peel and flesh phenolic compounds. In vivo studies indicate that
apple peels were better able to inhibit cancer cell proliferation, had greater plasma
antioxidant capacity and showed greater inhibition of lipid peroxidation in rats (19).
A number of studies have reported the beneficial effects of individual apple
polyphenols in relation to reducing the risk of cancer and cardiovascular disease. Boyer
and Liu (16) have reviewed these studies comprehensively. However, the synergistic
effect of all the polyphenols present in apples should be considered rather than the effect
13
of individual compounds. Thus, there is very good potential to use phenolic extracts from
apples as a bioactive ingredient in foods.
2.3 Extraction techniques
Studies on extraction of these compounds from plant matrices have traditionally
involved solvent extraction. These classical extraction methods are time consuming,
require large volumes of solvent and often generate heat, and consequently cannot be
used for the extraction of thermolabile compounds, rendering these techniques
impractical for commercial large scale extraction. Therefore, other more rapid and
efficient extraction techniques are being explored such as supercritical fluid extraction,
accelerated solvent extraction and microwave assisted extraction. Descriptions of these
extraction methods will be given in the following sections as a means of comparison.
2.3.1 Solvent extraction
Solvent extraction involves addition of a solvent or a mixture of solvents to the
plant matrix for subsequent extraction of the desired analyte. The solvent diffuses into
the plant matrix and the analyte is then solubilized in the solvent. The solvent along with
the analyte can then be physically separated from the plant matrix using various filtration
and centrifugation methods. The particle size of the plant matrix is thus an important
factor. The smaller the particles, the easier it is for the solvent to diffuse into the matrix
and extract the analyte. The important solvent characteristics that govern the solubility of
phenolic compounds are polarity, degree of polymerization, interaction of phenolics with
other food components and formation of insoluble complexes (4). Common solvents
14
employed for the extraction of polyphenols are methanol, ethanol, acetone, water, ethyl
acetate, and to a lesser extent propanol, dimethylformamide, and their combinations (20).
Existing classical techniques used for extracting phytochemicals from plants include
soxhlet, hydrodistillation and maceration with an alcohol-water mixture or hot fat (21).
Traditionally, solvent extraction coupled with the use of heat and/or agitation has been
performed using a soxhlet extraction unit.
Extraction of phenolics from apple pomace has widely been carried out using
aqueous 70% acetone (9, 22-23). Foo and Loo (9) characterized the procyanidins in apple
pomace after extraction of about 200 g freeze-dried pomace using 70% aqueous acetone
(3 x 500 ml) at room temperature. The total phenols content of their extract was 7.24
mg/g DW. Quercetin glycosides accounted for more than half (4.24 mg/g DW) of the
total phenols present followed by phloridzin and its oxidative products.
2.3.2 Supercritical fluid extraction (SFE)
SFE with carbon dioxide is a non-thermal approach to extracting thermolabile
compounds, and has therefore been successfully used in extraction of phenolics. SFE
makes use of a supercritical fluid to extract the analyte of interest from the plant matrix.
A supercritical fluid is any substance which is held above its critical temperature and
pressure, and exhibits the characteristics of both a liquid and a gas. Such a fluid has the
advantage of lower viscosity and higher diffusivity. This increases the rate of mass
transfer and diffusion, thereby decreasing the extraction time from hours (for typical
solvent extraction) to a few minutes. The solvation power depends on the temperature
and pressure. Hence, selectivity for particular analytes can be increased by manipulating
15
these parameters. The extracted solutes, now dissolved in the supercritical gas, can then
be separated by depressurization. There is no residual solvent in the extract, as in the case
with other extraction techniques. Further, only small volumes of sample and solvents are
required. Supercritical fluid extracts have reported higher antioxidant power compared to
extracts obtained by solid-liquid or soxhlet extraction (24).
Carbon dioxide is often used in SFE because of its low critical values (T c =
31.1°C, P c = 72.8 atm). Carbon dioxide is also readily available in a highly pure form and
is inexpensive and non-toxic. Pure CO 2 , however, is not suitable for the extraction of the
more polar phenolic compounds. Therefore, a modifier such as EtOH or MeOH must be
used to increase solubility of these compounds in the supercritical fluid. The important
parameters involved in the extraction of polyphenols by this technique include
supercritical fluid carbon dioxide density, type of organic modifier used, modifier
percentage, extraction temperature and trap type. SFE is widely used for extraction of
plant phenolics owing to the absence of both heat and light during the extraction process,
which can reduce the incidence of degenerative reactions that may occur when using
other extraction techniques. A recent review on the applications of SFE in food analysis
and natural products analysis has been given by Mendiola et al. (25). Subcritical (carbon
dioxide and ethanol) extraction of polyphenols from apple pomace was optimized by Adil
et al. (26) using various pressures, temperatures, ethanol concentrations and extraction
times. Maximum extraction yields were obtained at 20% ethanol concentration, 60 °C at
60 MPa for 40 minutes. The total polyphenols content and DPPH free radical scavenging
of the extracts under these conditions was 0.47 mg gallic acid equivalents (GAE)/g fresh
16
weight of sample (1.566 mg GAE/g DW) and 3.3 mg DPPH/mg sample. The total
phenols content of this extract was low compared to extracts obtained previously from
solvent extraction techniques, probably because of limited solubility of the specific apple
polyphenols in carbon dioxide. Therefore, other extractions techniques need to be
considered for this purpose.
2.3.3 Accelerated solvent extraction (ASE)
ASE (also known as Pressurized Solvent Extraction or Pressurized Fluid
Extraction) is a solid-liquid extraction process which is performed at elevated
temperatures and pressures. The elevated pressure increases the boiling point such that
the solvent remains in a liquid state at elevated temperatures. The increased pressures
also tend to “push” the solvent into the matrix pores, thereby making the analyte more
accessible. This is a type of pressurized solvent extraction similar to SFE, and is usually
considered as an alternative to SFE for the extraction of polar compounds (27). This
technique is rapid (compared to classical extraction methods) and can be used with any
kind of solvent or solvent mixtures (with the exception of solvents which have flash
points at low temperatures and corrosive acids and bases).
Most of the applications for ASE are in the area of environmental research for the
extraction of pollutants from environmental matrices. Extensive work has been done in
the extraction of polycyclic aromatic hydrocarbons (PAH) and polychlorinated biphenyls
(PCB) as well as other pollutants. These have been summarized by GiergielewiczMoajska et al. (28) . Kaufmann and Christen (29) reviewed recent developments in
17
accelerated solvent extraction of natural products. Some of these include xanthones and
flavonones, curcuminoids, flavonolignans, terpenes and saponins.
The use of ASE in the extraction of bioactive compounds from apple pomace is
limited. Alonso-Salces et al. (30) optimized a pressurized liquid extraction of apple
polyphenols from Golden Delicious apple pomace by varying the temperature, solvent
composition, pressure and extraction time. The optimum extraction efficiency was
obtained at 5 minutes extraction time, 40 °C using 100% methanol at 1000 psi. The total
phenols obtained under these conditions as measured by reverse phase HPLC was 7.566
mg/g DW, which is comparable to yields obtained using solvent extraction techniques.
However, considering the capital cost of the instrumentation and relatively high solvent
volumes required, consideration of other extraction techniques may prove to be valuable.
2.3.4 Microwave assisted extraction (MAE)
Principle and Mechanism:
Microwaves are electromagnetic radiation with frequencies ranging from 0.3 to
300 GHz. Microwave assisted extraction has been used increasingly for extraction of
phenolics and other compounds. It has a number of advantages compared to other
extraction techniques, such as increased yield, shorter extraction time and use of less
solvent. MAE delivers energy rapidly to a total volume of solvent and solid plant matrix
with subsequent heating of the solvent and solid matrix, efficiently and homogenously.
The principle of heating using microwaves is based on the direct effect of the waves on
molecules by ionic conduction and dipole rotation. The ionic conduction generates heat
due to the resistance of the medium to ion flow. Dipole rotation also causes heat
18
generation as the polar molecules try to line up with the electric field and collide with
each other.
MAE involves first choosing a solvent in which the target analyte is soluble. The
polarity of the solvent is important as solvents with high dielectric constants (example
water) can absorb more microwave energy. Usually polar solvents are considered better
than non-polar ones (31) due to their high dielectric constants which results in rapid
heating on application of microwaves. On the other hand, several papers have described
the ‘broken cell-wall theory’ according to which microwave-transparent solvents with
lower dielectric constants, and thus lower microwave absorbing capacity, are better than
microwave-absorbing ones. This is because in a microwave-transparent solvent (e.g.
hexane), the microwaves are absorbed by the plant matrix alone. This matrix may have
some amount of water which gets heated up, disrupts the cellular structure and releases
the desired components into the surrounding solvent medium (32). This type of MAE is
also called a ‘cold-extraction’.
Parameters influencing MAE
There are a number of parameters that influence the microwave extraction
process. The commonly studied ones include choice of solvent, solvent volume,
extraction temperature and time, and matrix characteristics. Eskilsson et al. (33)
discusses each of these parameters in detail and are summarized below:
Choice of Solvent: The important characteristics that must be considered while choosing a
solvent are its microwave-absorbing properties, interaction of the solvent with the matrix,
19
and analyte solubility (34). The solvent must also be capable of selective solute
extraction. Common solvents employed are acetone, methanol, ethanol, hexane and
water, or various mixtures of these (35).
Solvent Volume: The volume of the solvent required varies depending on the sample, but
is usually within 10 – 30 ml (33). Very low volumes are required in some cases. For
example, phenol and methyl phenol extracted from soils required very small amounts of
solvent, with an optimum volume of 10 ml for a 5 gm sample size (36). The
solids/solvent ratio and solvent concentration are also important parameters that must be
taken into account. Shahidi and Naczk (37) found that changing the sample/solvent ratio
from 1:5 to 1:10 significantly increased the extraction yields of condensed tannins and
total phenolics from commercial canola meals. However, other studies have shown that
increasing the solvent volume may reduce the extraction yield after a certain value. Chee
et al. (38) reported this effect while extracting polycyclic aromatic hydrocarbons (PAH’s)
from sediment. In that study, it was found that 30 ml of solvent gave higher recoveries
than 45 ml. Kovaks et al. (39) also found on extracting free amino-acids from various
foodstuffs, that higher yield was obtained when the extraction was performed at lower
solvent volumes. The solids/solvent ratio must thus be obtained experimentally and varies
with each sample matrix.
Temperature: The influence of temperature seems to depend on the matrix. Some studies
have shown that the temperature is not a very significant factor (40) while others have
shown significant variation in extraction yields at different temperatures (41). Higher
temperatures, when used for long periods of time, result in degradation of thermolabile
20
phenolic compounds. Kiss et al. (42) studied the extraction of carotenoids from paprika
using MAE. The extractions were optimized to below 60 °C, as higher temperatures lead
to degradation and reduced yield.
Extraction Time: Extraction times are relatively short (typically 30 seconds to 20
minutes) in MAE as compared to other conventional extraction techniques (43). About
four minutes of irradiation was sufficient to extract the polyphenols from various
aromatic plants of Greek origin (44).
Matrix Characteristics: Some studies report that MAE is not matrix dependent, and
similar extraction yields are obtained irrespective of the nature of the matrix (45).
However, many other papers report that the matrix can have a profound effect on the
extraction yield. Lopez-Avila et al. (41) performed PAH (Polycyclic Aromatic
Hydrocarbon) extraction of 6 different matrices using the same procedure and obtained
recovery rates ranging from 57% to 99% for different matrices. The matrix moisture
content is an important parameter that can affect the extraction efficiencies, depending on
the nature of the solvent used. Samples with high water content can be easily extracted
using relatively non-polar solvents; while dry sample matrices are preferred for extraction
with more polar solvents.
Microwaves have been used increasingly for the fast extraction of a variety of
compounds. A number of scientific publications report the use of MAE for persistant
organic pollutants (POPs) (41, 46-49). A large number of pesticides have also been
extracted using MAE such as organochlorine pesticides (OCP) and OPP’s (36, 50-51). In
21
addition, microwaves have been used for the extraction of several biological compounds
such as essential oils from the leaves of rosemary and peppermint (52), taxanes from
taxus biomass (53) and extraction of ergosterol and total fatty acids from fungal hyphae
and spores, mushrooms, filtered air, artificially contaminated corn, naturally
contaminated grain dust and soil (54). Extraction of phenolic compounds from tea leaves
have been studied recently (55). Phenolic compounds have also been extracted from
grape seed (56) using MAE.
MAE is increasingly being used as an alternative to traditional extraction methods
for the removal of phenolics from plant tissues, as it significantly reduces extraction time
and solvent consumption while generating higher extraction yields (43, 55). Limited
studies have investigated the potential of MAE in the extraction of bioactive compounds
from plant waste streams such as apple pomace. The extraction of pectin from dried apple
pomace powder was optimized by Wang et al. (57), using HCl buffer (pH 1.01) at 499.4
W microwave power, 20.8 minutes extraction time and 0.069 solid/liquid ratio. However,
the extraction of polyphenols from apple pomace using MAE has not been previously
reported, and could be a potential tool for rapid and efficient extraction.
2.4 Quantification of phenolic compounds
Once the phenolic compounds are extracted from their plant source, they
are typically characterized and quantified by spectrophotometric and chromatographic
methods. The Folin-ciocalteu assay is the most widely used analytical method to estimate
the total polyphenol content in a sample. The phosphomolybdic acid in the Folin reagent
22
reacts with the phenols in alkaline solution to give a blue colored complex which is then
read spectrophotometrically at 760 nm. The phenolic content is commonly expressed as
percent gallic acid equivalents (GAE) per gram of sample. However, reducing substances
like sugars and ascorbic acid may also react with the Folin reagent and cause interference
and inaccurate estimation, which is one of the main drawbacks of the assay.
HPLC is the most common quantitative analytical tool used to study polyphenols,
such as anthocyanins, procyanidins, flavonoids, phenolic acids, etc. Several reviews on
the application of HPLC for the study of plant phenolics have been published. A typical
separation system involves a reverse phase HPLC (RP-HPLC) comprising a C-18 or C-30
stationary phase. Isocratic elution has been proved sufficient in some separations.
However, gradient elution is more commonly applied and results in better separation. A
number of different mobile phases have been employed, but a typical separation system
uses a binary solvent system comprising an aqueous component and a less polar organic
solvent such as acetonitrile. The common detectors used for food phenolics include UVVis, photodiode array (DAD) and UV-fluorescence detectors, although the use of various
other detectors has also been reported. Structural characterization of phenolics is usually
done using HPLC coupled with mass spectrometry (58).
2.5 Estimation of antioxidant activity
Polyphenols are known to be effective antioxidants and scavengers of free
radicals (7). There are a number of methods to estimate antioxidant capacity of phenolics.
Based on the mechanism of the reaction involved, assays are often classified as HAT
23
(hydrogen atom transfer) or SET (single electron transfer). HAT based assays measure
the ability of an antioxidant to quench free radicals by the donation of a hydrogen atom,
and some examples of this type include oxygen radical absorbance capacity (ORAC) and
total radical trapping antioxidant parameter (TRAP). SET based assays measure the
ability of the antioxidant molecule to reduce free radicals, metals or carbonyls by the
transfer of an electron. Examples of the SET type of antioxidant assays include diphenyl
picryl-hydrazyl (DPPH), trolox equivalence antioxidant capacity (TEAC), ferric ion
reducing antioxidant power (FRAP) as well as the Folin-ciocalteu total phenols assay
(59). Unfortunately, these different assays used to evaluate antioxidant potential vary
depending on the testing conditions, medium, type of substrate and reaction period. This
complicates our ability to effectively compare values of antioxidant effectiveness of plant
extracts from literature. Further, most of these assays occur under controlled conditions
which do not simulate physiological conditions, so understanding the antioxidant
potential of the test substrate in food and biological systems becomes complicated.
However, despite these drawbacks, these methods are still used for reporting antioxidant
potential because they are rapid, inexpensive and easy to use. Further, simple
comparisons of plant extracts obtained between different experimental treatments can be
successfully carried out with these types of assays due to their good within-run and
between-day reproducibility (59).
2.5.1 ORAC assay
The ORAC measures the antioxidant inhibition of peroxyl radical induced
oxidations by hydrogen atom transfer (60). The peroxyl radical reacts with a fluorescent
24
probe to form a non-fluorescent product, which is then quantified by fluorescence (59).
Antioxidants delay degradation of the probe by intercepting free radicals generated by
AAPH (2,2’-azobis(2-aminopropane) dihydrochloride), thereby causing a delay in
fluorescence decay. The reduced rate and amount of product formed over time can then
be monitored to estimate the antioxidant capacity, which is reported as micromoles of
Trolox equivalents per liter or gram of sample. Trolox is a water-soluble form of vitamin
E and is used as the antioxidant standard. The values are determined by measuring the
area under the curve.
ORAC is generally preferred over the other antioxidant estimation methods
because it models the reaction of antioxidants with peroxyl radicals as occurs in the
human body. Many food companies now include ORAC values on their product labels.
This assay may be easily automated and several improvements in the design of the
instrumentation and fluorescent probe (commonly fluorescein) have been reported (6061). The fluorescent markers used in the assay are very sensitive. However, their use
requires fluorometers for detection, which are rather expensive and not routinely
available in laboratories. Another disadvantage of this technique is the relatively long
analysis time (approximately 1 hour). Further, the ORAC reaction is temperature
sensitive and small temperature differences in the external wells of the microplate can
decrease the reproducibility of the assay (60).
2.5.2 DPPH Assay
DPPH is a stable nitrogen-centered free radical that can be used to estimate
antioxidant activity. This assay is based mainly on electron transfer reaction, rather than
25
hydrogen atom abstraction (59). The odd electron in DPPH gives a strong absorption
maximum at 517 nm and is purple in color. When the odd electron of the DPPH radical
becomes paired with a hydrogen from a free radical scavenging antioxidant to form
reduced DPPH –H, the molar absorptivity of the DPPH radical reduces from 9660 to
1640 and its color changes from purple to yellow (62). The antioxidant assay is based on
spectroscopic measurement of the loss of color at 517 nm after reaction with the test
compounds. This assay is extremely rapid and simple, requiring only a UV-Vis
spectrophotometer. However, there are a number of drawbacks with the DPPH that may
result in inaccurate interpretation of the antioxidant capacity. Certain compounds may
interfere by absorbing at the wavelength of measurement. Carotenoids, for example, have
been found to interfere (63). Further, DPPH is a very stable free radical unlike the highly
reactive radicals which are responsible for lipid oxidation in foods. Many antioxidants
show different activities with DPPH and with highly reactive radical species, leading to
either over- or under-estimation of the radical scavenging potential of the extract. Despite
these limitations, DPPH is one of the most widely used methods for reporting the
antioxidant potential of plant extracts because it is simple, rapid and relatively
inexpensive.
2.5.3 FRAP Assay
The FRAP assay is another assay which is based on singlet electron transfer. This
method has been widely used to evaluate the antioxidant capacity of several botanical
extracts (64-65). The FRAP assay measures the reduction of ferric 2,4,6-tripyridyl-striazine (TPTZ) to a colored (blue) product and this can be monitored
26
spectrophotometrically at 595 nm. The FRAP assay is simple, rapid and inexpensive and
does not require any special equipment. However, the results may fluctuate widely
depending on the analysis time. Fast reacting phenolics must be analyzed with short
reaction times while polyphenols that react slowly require longer reaction times for
detection (59).
2.5.4 TEAC Assay
The TEAC assay utilizes both electron transfer and hydrogen atom transfer to
estimate the antioxidant capacity. This method measures a long life radical cation ABTS+
which are formed by the oxidation of ABTS by oxidants. There is a decrease in color
which is measured spectrophotometrically for a fixed time to determine the antioxidant
activity of the test compound. This assay is rapid and simple. ABTS reacts over a wide
range of pH and is soluble in both aqueous and organic solvents.
2.6 Encapsulation of polyphenols
Phenolic compounds may easily be destroyed by light, oxygen and other factors
once removed from the plant matrix. This makes them difficult to store and transport. In
addition, several of these compounds are highly hydrophobic and insoluble in aqueous
systems. Encapsulation of these compounds using some wall material with high stability
and good solubility characteristics would help in overcoming these problems. This would
enable the use of these systems in oil-in-water emulsions, etc.
27
Encapsulation is a technique by which a material or a mixture of materials is
entrapped within another material or system. It is commonly used to stabilize the
entrapped compound, and to provide protection from environmental or subsequent harsh
processing conditions. In several cases, encapsulation enables the incorporation of the
compound in systems where it is insoluble in the native state. Encapsulation has
traditionally been used extensively in the pharmaceutical industry for the incorporation of
various drugs and other ingredients within a capsule. This was later extended to food
processing and has several applications. A number of components have been
encapsulated in the food industry, such as vitamins, minerals, amino-acids, flavors,
colorants, enzymes, sweeteners and antioxidants (66).
The substance that is encapsulated is often referred to as the core material, while
the coating material is called the shell, wall, carrier or encapsulant (67). The process of
encapsulation consists of two essential steps: the first is the formation of an emulsion
between the core material and the wall material. The second is drying or cooling of the
emulsion to obtain microcapsules. These microparticles may be obtained by chemical
processes, such as coacervation, co-crystallization, molecular inclusion and interfacial
polymerization, or they may be obtained by mechanical processes such as spray-drying
(most common), spray chilling/cooling, extrusion and fluidized bed. Madene et al. (67)
has reviewed the theory of each of these techniques in detail.
2.6.1 Wall materials
There are a number of wall materials available for use in encapsulation. A suitable
wall material must protect the core material from factors that may cause its degradation,
28
prevent premature interaction between the core material and other ingredients, limit
volatile losses and allow a controlled release of the core material under desired conditions
(66). There are several sources of wall materials permitted for use in food ingredients
such as proteins (milk or whey proteins, gelatin, etc.), natural gums (gum arabic,
alginates, carragenans, etc.), maltodextrins with different dextrose equivalence, waxes
and their blends. The right choice of a wall material can be determined experimentally by
forming and evaluating the microcapsules for encapsulation efficiency, stability under
different storage conditions, degree of protection provided to the core material and by
surface observation using scanning microscopy (68).
Gums
Acacia gum or gum Arabic has good emulsification properties and has been used
extensively for food emulsification (69). However, it is very expensive compared to other
available wall materials. Bixin (a carotenoid obtained from the seed of Bixa orellano)
was encapsulated with gum arabic and maltodextrin. The core material was found to be
more stable when trapped in gum Arabic, compared to maltodextrin + Tween 80 (70).
Zhang et al. (71) studied various microencapsulation methods of procyanidins extracted
from grape seeds using gum Arabic and maltodextrin as the wall material. Spray-drying
was used to prepare the microcapsules, with an efficiency of 88.84%.
Proteins
Proteins such as milk or whey protein and gelatin also have good functional
properties and possess good binding properties for flavor compounds. Whey proteins
29
have been successfully used for encapsulating anhydrous milk fat by spray drying, with
an encapsulation yield greater than 90% (72). Gelatin is a water-soluble wall forming
material that has wide applications in the food industry (73).
Carbohydrate based wall materials
Several carbohydrate compounds have been used for incorporation of ingredients.
Maltodextrins were found to be effective in protecting the carotenoids of paprika
oleoresin (74). Modified starches in which the starch molecule has been chemically
treated with hydrophobic groups such as octenyl side chains to increase their affinity for
hydrophobic flavor compounds, have widely been used as encapsulating materials for
flavor compounds (75).
Pectin is another polymer capable of producing stable emulsions at low
concentrations of around 1-2% (75). Alginates are gum like material obtained from
brown seaweed, and contains a number of free carboxylic acid groups which react with
divalent cations, mainly calcium to form stable gels (76). They are widely employed in
drug delivery systems. Deladino et al. (77) studied the encapsulation of natural
antioxidant compounds extracted from yerba mate (Ilex paraguariensis) using two
different systems, calcium alginate and calcium alginate-chitosan.
Cyclodextrins
Modified starches have very good functionality to be used as wall materials.
Cyclodextrins (CD) are enzymatically modified starch molecules obtained by the action
of cyclodextrin glucosyltransferase on starch. The starch molecule is cleaved by the
30
enzyme and the ends join together to form a circular molecule with six, seven or eight
glucose residues linked by α 1,4 glucosidic bonds, to form α-cyclodextrin, βcyclodextrin and γ-cyclodextrin, respectively (Fig 2.2 a). CDs can form inclusion
complexes with a variety of hydrophobic compounds, by entrapping the entire
compound, or at least part of the compound, in the apolar cavity. The complex formation
relates to its structure (Fig 2.2 b). The hydroxyl groups of the CD molecule are located on
the wider edge of the ring, resulting in a hydrophilic outer shell suitable for solubility in
aqueous food systems. The apolar carbon and hydrogen atoms and the ether-like oxygen
atoms are present in the inner surface to form the relatively hydrophobic cavity (78).
(a)
(b)
Figure 2.2 Structure of Cyclodextrin molecule (a) top view of α, β and γ-CD (b) 3 -d
surface view of β-CD
31
The important factors associated with complex formation with a guest molecule
are steric and thermodynamic effects (78). The size of the hydrophobic cavity must be
suitable with the guest molecule. In addition, the reaction must be driven forward by the
displacement of water molecules from the cavity, and formation of hydrophobic
interactions between the guest and the CD. This results in a thermodynamically favorable
reaction. Studies have shown that addition of excess water to the system tends to shift the
equilibrium to the left, resulting in dissociation of the guest-CD complex, and release of
the guest molecule (78).
β-CD is the most widely used CD for encapsulation purposes as it is more
available and lower priced compared to the other two parent CDs. However, it has lower
water solubility (1.85% w/v at 25°C) compared to α-CD (14.5% w/v) and γ-CD (23.2%
w/v) (78). However, the number of glucopyranosyl residues, and thus the cavity diameter
of β-CD is more suitable for the entrapment of a variety of molecules. Beta-cyclodextrins
have been commonly used for the encapsulation of phenolic compounds such as
resveratrol (79). Lucas-Abellan et al. (79) studied the encapsulation of quercetin and
myricetin flavonols in beta-cyclodextrins in acidic medium.
Derivatives of β-CD have been produced by chemical modification of the parent
molecule by aminations, etherifications and esterifications in order to produce wall
materials with improved solubility and stability characteristics (78). 2-hydroxypropyl-βCD is an example of a modified CD with improved solubility characteristics, and has
been used as an encapsulant for a variety of food ingredients.
32
2.6.2 Analysis of inclusion complexes
Phase solubility study
Enhancing the water solubility of hydrophobic compounds is one of the major
advantages of encapsulation. The most widely applied analytical method used to study
the aqueous solubility of inclusion complexes formed by cyclodextrins is the phase
solubility study developed by Higuchi and Connors (80). This method has been applied to
analyze the solubility of several ligand-drug complexes, and is widely used in the
pharmaceutical industry. Usually, an excess amount of the drug molecule is added to
aqueous solutions containing the ligand (cyclodextrin) at increasing ligand
concentrations. Inclusion complexes are allowed to form by stirring at constant
temperature. This may take anywhere between 48 hours to 1 week (78). Following this,
samples are filtered and the water soluble inclusion complexes are analyzed for
quantification of the drug molecule. Phase solubility diagrams are then constructed by
plotting the ligand (cyclodextrin) concentration on the x-axis and the drug concentration
on the y-axis (80). A typical phase-solubility curve is shown in Fig 2.3.
Figure 2.3 Phase solubility diagram (adapted from Higuchi & Connors, 1965)
33
Curves obtained are classified mainly into 2 categories, the formation of inclusion
complexes with good water solubility (A) and the formation of inclusion complexes with
poor water solubility (B). The A L type curve indicates a linear increase of drug solubility
with cyclodextrin concentration, and is indicative of a 1:1 binding of the drugcyclodextrin complex (78). AP and A N are representative of positively deviating and
negatively deviating isotherms respectively. B S and B I are representative of poorly
soluble and insoluble complexes. This method allows a qualitative assessment of
complex formation, and may also be used to derive stability constants between the drug
and ligand.
Differential Scanning Calorimetry (DSC)
DSC is a thermal analysis tool that measures the amount of energy required to
increase the temperature of the sample and reference, as a function of temperature. DSC
has been widely used for the verification of complex formation, by comparing the
thermograms of typically four types of samples: pure drug molecule, pure ligand
(cyclodextrin), the inclusion complex and a physical mixture of the ligand and drug,
prepared at the same ratio as for the inclusion complex. The disappearance of thermal
peaks associated with the pure drug or ligand, in the thermogram of the inclusion
complex is usually indicative of successful complex formation (81-82).
DSC studies carried out under an oxygen atmosphere have also been used for
checking the oxidative stability of the complex. The complex and pure drug compound
are placed in DSC pans with a hole on their lid, and the experiment is carried out under
an oxygen atmosphere. Degradation peaks are usually visible at low temperatures for the
34
pure drug molecule, while they are absent or shifted to the right in the thermogram of the
inclusion complex (81, 83).
2.7 Summary
Polyphenols are a ubiquitous group of plant compounds that have high
antioxidant activity, and have been shown to inhibit lipid oxidation. They have been
extracted from their native plant matrix traditionally by solvent extraction. However,
research has shown that conventional solid-liquid extraction consumes large amounts of
solvent and is also time intensive. Alternate extraction techniques have been discussed,
with emphasis on microwave-assisted extraction. Furthermore, studies indicate the poor
functionality of polyphenols in their native state due to low oxidative stability and poor
water solubility. Encapsulation of these compounds may improve their stability and
solubility for use in various food systems. Cyclodextrins or modified starch compounds
have been widely used in the pharmaceutical industry and may prove useful in
encapsulating polyphenolic plant extracts for widespread food applications.
35
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41
CHAPTER 3. OPTIMIZATION OF MICROWAVE-ASSISTED EXTRACTION
OF PHENOLIC ANTIOXIDANTS FROM APPLE POMACE†
Vaishnavi Chandrasekar1, Tameshia S. Ballard1*, Fernanda San-Martín1 and Peter Hirst2
1
Department of Food Science, Purdue University, West Lafayette, IN 47907
2
Department of Horticulture and Landscape Architecture, Purdue University, West
Lafayette, IN 47907
*
Correspondence should be sent to:
Tameshia S. Ballard, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN
47907
Phone: (765) 496-6545 Fax: (765) 494-7953 Email: tballard@purdue.edu
†
Submitted to J. Agricultural and Food Chemistry
42
Abstract
Microwave-assisted extraction (MAE) was used to extract phenolic antioxidants from
apple pomace of four cider apple varieties (Red Delicious, Golden Delicious, Winesap
and Jonathan). Response surface methodology was used to optimize solvent type (70%
acetone, 60% methanol and 60% ethanol), microwave power (100-900 W), solvent
volume to sample ratio (4-12 ml/g dry pomace) and extraction time (30-180 s) for each
pomace variety. Optimum conditions were based on total polyphenol content (TPC) and
antioxidant activity as measured by inhibition percentage (IP) of the 2,2-diphenyl-1picrylhydrazyl (DPPH) free-radical. Red Delicious pomace had the highest TPC (15.8 mg
GAE/g DW) and DPPH radical scavenging activity (94.4%) obtained under the optimum
extraction conditions. Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin
were some of the major phenolic compounds identified in the extracts. MAE was found
to be an effective method of extracting valuable antioxidant compounds from apple
pomace.
Keywords: polyphenols, apple pomace, microwave-assisted extraction, total polyphenol
content, DPPH, response surface methodology
43
Introduction
Waste generated from apple processing is substantial. It is estimated that about 25
to 30% of the total world production of apples is processed into cider, juice and other
products, which results in several million tons of apple pomace being generated globally
(1). Apple pomace consists of the peels, seeds, stems and flesh left over from juice or
cider processing and is primarily being treated as an agricultural waste material, with no
real value to processors. The pomace is currently being composted or used as animal
feed, although this application is limited due to its low protein content (2). However,
apple pomace is rich in phenolic antioxidants including epicatechin, caffeic acid,
phloridzin, quercetin glycosides and quercetrin (3). Phenolic extracts from apples have
shown high in vitro and in vivo antioxidant and antiproliferative activity (4).
Consumption of apples has been associated with reduced risk of cardiovascular disease
(5), lung cancer (6) and stroke (7), and this has largely been attributed to the presence of
polyphenols in the fruit, many of which remain in the pomace after juice extraction. The
relative quantities and types of phenolics present vary widely depending mainly on the
apple cultivar and other factors such as state of maturation and availability of growth
nutrients (8).
Extraction of phenolics from apple pomace has been carried out typically by
solvent extraction. Foo and Lu (1) obtained a phenolic extract with 70% aqueous acetone
(1500 ml/200 g) that contained 7.24 mg GAE/g dry apple pomace and mainly consisted
of quercetrin glycosides. Adil et al. (9) optimized the subcritical (carbon dioxide and
ethanol) extraction of phenolics from apple pomace at 20% ethanol concentration, 60°C
44
at 60 MPa for 40 min. The total phenols content and DPPH free radical scavenging
activity of the extracts obtained was 1.57 mg GAE/g dry pomace and 3.3 mg DPPH/mg
sample, respectively. The low values were attributed to limited solubility of apple
phenolics in carbon dioxide. Alonso-Salces et al. (10) optimized the pressurized liquid
extraction of apple polyphenols from Golden Delicious apple pomace. Total phenols
content of the extracts was 7.57 mg/g dry sample obtained under the optimum conditions
of 5 min extraction time, 40°C using 100% methanol at 1000 psi. Considering the capital
cost of instrumentation needed for these extraction methods and the relatively high
solvent volumes required, consideration of other extraction techniques may be helpful.
Microwave-assisted extraction (MAE) is a relatively new extraction technique
that delivers microwave energy rapidly to a total volume of solvent and solid plant
matrix. This results in subsequent heating of the solvent and solid matrix, efficiently and
homogenously (11). The principle of heating using microwaves is based on the direct
effect of the waves on molecules by ionic conduction and dipole rotation. The plant
matrix may contain some water which is rapidly heated by microwaves, disrupting the
cellular structure and releasing the desired components into the surrounding medium
(12). There are a number of parameters that influence the microwave extraction process.
Those most commonly studied include choice of solvent, solvent volume, extraction
temperature and time, and matrix characteristics (13).
MAE is increasingly being used as an alternative to traditional extraction methods
for the removal of phenolics from plant tissues, as it significantly reduces extraction time
and solvent consumption while generating higher extraction yields. Ballard et al. (14)
45
found that MAE increased the extraction yield of phenolic antioxidants from peanut skins
using less solvent and in a fraction of the time required for traditional solid-liquid
extraction. Limited studies have investigated the potential of MAE in the extraction of
bioactive compounds from plant waste streams such as apple pomace. The extraction of
pectin from dried apple pomace powder was optimized by Wang et al. (15) using 499 W
microwave power, 20.8 minutes extraction time and 0.069 solid:liquid ratio. However,
the extraction of polyphenols from apple pomace using MAE has not been previously
reported and could be a potential tool for rapid and efficient extraction.
The influence of several factors on a desired response can be determined using
response surface methodology (RSM) which was first described by Box and Wilson (16).
This method requires fewer experimental runs while still producing statistically
acceptable results (17). It is widely used to evaluate the effect of various factors and their
interactions on the response variable, and to optimize multiple responses simultaneously.
The central composite design (CCD) is the most commonly used experimental design in
this method, and has been used in optimization studies for a number of applications
including the extraction of bioactive compounds from plant sources (18-20).
The objective of this work was to optimize and compare the extraction of
phenolic antioxidants from pomace of four common apple cider varieties (Red Delicious,
Golden Delicious, Winesap and Jonathan) using MAE. Response surface methodology
was used to optimize solvent type, microwave power, solvent volume to sample ratio and
extraction time for each pomace variety. Optimum conditions were based on total
46
polyphenol content (TPC) and demonstrated antioxidant activity as measured by
inhibition percentage (IP) of the DPPH free-radical.
Methods and Materials
Chemicals
Catechin, epicatechin, procyanidin B2, caffeic acid, chlorogenic acid, phloridzin,
hydrocaffeic acid, hydrocoumaric acid, quercetin and quercetrin were purchased from
Sigma Aldrich Chemical Co. (St. Louis, MO). 2,2-Diphenyl-1-Picrylhydrazyl (DPPH),
Folin-Ciocalteu reagent, sodium carbonate anhydrous and 3,4,5-trihydroxybenzoic acid
(gallic acid) were obtained from VWR International (Seattle, WA). Analytical grade
methanol, ethanol and acetone, and HPLC grade acetic acid and water were obtained
from Mallinckrodt Baker Inc. (Phillipsburg, NJ).
Preparation of samples
Frozen pomace from four cider apple varieties (Red Delicious, Golden Delicious,
Winesap and Jonathan) was provided by Beirsdorfer Orchard (Guilford, IN). Pomace
samples were placed in Ziploc freezer bags, flushed with nitrogen gas and stored at -20
°C until use. Samples were lyophilized for 48 hr. in a freeze dryer (Virtis Co., Gardiner,
NY), ground to less than 1 mm particle size using a bench top coffee grinder (Hamilton
Beach brand, Washington, NC) and used for extraction.
47
Microwave-assisted extraction
Extractions were carried out in a Mars Xpress microwave extraction system
(CEM Corp., Matthews, NC). One gram of ground apple pomace was accurately weighed
and quantitatively transferred to teflon-lined extraction vessels. The appropriate volume
of solvent was added, a stir-bar was inserted and the vessel was closed and exposed to
microwaves for the designated time period. The extraction vessels were allowed to cool
to room temperature after extraction. Extracts were then transferred to 15 ml centrifuge
tubes and centrifuged at 1050 rpm for 20 min. in a Dynac centrifuge (Clay Adams,
Division of Becton Dickinson and Co., Parsippany, NJ). The supernatant was then
transferred to glass centrifuge tubes, flushed with nitrogen gas and stored at -20°C until
analysis. Extracted samples were stored no longer than 2 weeks prior to analysis. Two
replications were carried out for each treatment.
Total phenols determination
The total polyphenols content (TPC) of the extracts was determined using the
Folin-Ciocalteu assay as outlined by Spanos & Wrolstad (21). Gallic acid standards
ranging from 0.1 to 0.5 g/L were prepared. 0.1 ml of the sample and standards were taken
in 15 ml test tubes. 3 ml of 0.2 N Folin-Ciocalteu reagent (Sigma-Aldrich Co., St. Louis,
MO) was added to each tube and mixed on a vortex mixer. 2 ml of 9% (w/v) sodium
carbonate solution in water was added to each tube, and again mixed using a vortex
mixer. Absorbance was read after 2 hr. at 765nm using DU720 spectrophotometer
(Beckman Coulter Inc., Fullerton, CA). The phenolic content of the extracts were
expressed in mg gallic acid equivalents (GAE)/g dry weight (DW) of apple pomace.
48
DPPH radical scavenging assay
The free radical scavenging activity was determined spectrophotometrically using
a modified version of the DPPH assay reported by Garcia et al. (22). Extracted samples
were evaporated to dryness under vacuum, and reconstituted in same volumes of
methanol prior to analysis. 40 µL of sample, appropriately diluted in methanol, was
added to 1.45 mL DPPH solution (40 mg/L in methanol). The solution was mixed using a
vortex mixer and allowed to stand in the dark for 30 minutes. Absorbance of the samples
was read at 517 nm using a DU720 spectrophotometer (Beckman Coulter Inc., Fullerton,
CA). The decrease in absorbance was calculated in comparison to a blank sample (40 µL
methanol and 1.45 mL DPPH) and results were expressed as inhibition percentage (IP):
IP (%) = 1 - (A e/A b ), where A e = absorbance of sample extract and A b = absorbance of
blank.
HPLC analysis
The major phenolic compounds in the extracts were analyzed by reversed phase
HPLC based on the method used by Suarez et al. (23). An Agilent 1200 Series HPLC
(Santa Clara, CA) system with a diode array detector was used. An Xterra C-18 column
(3.9 x 100 mm, I.D. 3.5 µm; Waters Corp.) was utilized and the column temperature was
equilibrated to 25 °C. The binary mobile phase consisted of solvent A: 2% aqueous acetic
acid and solvent B: 100% methanol. The gradient elution was performed as follows: a
linear step from 0 to 45% of solvent B in 55 min, an isocratic step of 15 min and finally
another linear step from 45 to 55% of solvent B in 10 min. Flow rate of the mobile phase
49
was set at 0.8 mL/min. Injection volume was 10 µL. Identification of phenolics in the
extracts was achieved by comparing their spectra and retention times with those of
externally injected standards, when available. Absorbance was measured at 313 nm for
detection of hydroxycinnamic acids, 355 nm for flavonol glycosides and 280 nm for the
rest of phenolic compounds (benzoic and 3-phenylpropionic acids, flavanols,
procyanidins and dihydrochalcones).
Experimental design and statistical analysis
Response surface methodology with a central composite design was used to
optimize the extraction of phenolic antioxidant compounds from each of the four apple
pomace varieties and three solvent systems. Design Expert 7.1.6 (Stat-Ease Inc.,
Minneapolis, MN) was used for experimental design and statistical analysis. The effects
of three independent variables: solvent volume to sample ratio (A), microwave power (B)
and extraction time (C) on the two measured responses (TPC and IP) were studied. The
coded values and corresponding actual values of these independent variables are shown
in Table 3.1. The variables were studied at five levels: -1.7, -1.0, 0.0, 1.0, 1.7 (with α =
1.7), with four replicates at the center, giving rise to 18 experimental points (Table 3.2).
The data was analyzed to fit the quadratic model given below:
Y = β 0 + Σ β i X i + Σ β ii X i 2 + ΣΣ β ij X i X j
whereY is the predicted response; X i and X j are independent variables; β 0 is a constant ; β i,
β ii and β ij are the coefficients for the linear, quadratic and interaction terms respectively.
Models were generated through least-squares regression analysis. Three-dimensional
50
response surface and contour plots were generated by the software by keeping one
variable at the optimal value and varying the other two. The models were evaluated based
on their F statistic, p value and R2 value generated by analysis of variance (ANOVA).
Significance of model terms was established at the 5% probability level (p < 0.05).
Significance of difference test was carried out using SAS statistical software package
(SAS Version 9.2, SAS Institute Inc., NC).
Results and Discussion
The concentrations of solvents used and the range of extraction parameters chosen
in this study were based on preliminary experiments (data not shown). Response surface
models were fitted based on the TPC and DPPH radical scavenging activity for each
apple cultivar and solvent system.
Model fitting
Response surface models were fitted using regression, taking into consideration
only significant factors (p < 0.05). Model assumptions of normality, constant variance
and independence of observations were checked. Transformation of the data was carried
out in some cases to obtain a better model fit, where the response variable is related to the
model parameters as a power function instead of the customary linear model. ANOVA of
the regression equations were performed. The predicted models in terms of coded units,
along with their F and p statistics and R2 values are shown in Table 3.3 (based on TPC)
51
and Table 3. 4 (based on DPPH IP). The models obtained using the three different solvent
systems are discussed in detail.
Acetone extraction
All models for acetone extracts based on TPC were found to be significant (p <
0.05) with R2 values of 0.90, 0.87, 0.92 and 0.85 for Red Delicious, Golden Delicious,
Jonathan and Winesap apple pomace varieties, respectively. This indicated that the
models could be used to predict the response variables.
Solvent volume, microwave power and time were found to be significant model
parameters. Fig. 3.1 shows the effect of microwave power and extraction time on the
TPC of Red Delicious pomace. Increase in microwave power from 265 W to 735 W and
extraction time from 61 s to 149 s produced higher yields of total phenols in the extract.
However, it was found that further increases in microwave power and extraction time
lead to superheating of the solvent, which exceeded pressure limits of the microwave
extraction unit. Due to this constraint, a larger range of these parameters were not
studied and optimizations were carried out within this range. This was true for all
subsequent extractions.
The effect of solvent volume and power on the TPC of pomace extracts can be
seen in Fig. 3.2. In Fig. 3.2 (a), solvent volume of 5.6 ml/g DW and microwave power of
735 W yielded the maximum predicted TPC of 0.46 mg GAE/ml (2.6 mg GAE/g dry
pomace) for Golden Delicious apple pomace variety. Similar patterns were observed for
Winesap and Red Delicious pomace samples extracted with 70% acetone. In general,
52
smaller solvent volumes produced higher extraction yields. For the Winesap pomace
extraction, it was found that as the solvent volume was reduced from 10.3 to 5.6 ml, the
TPC increased by 54.2% from 0.59 to 0.91 mg GAE/g DW (Fig. 3.3). This is in
contradiction to what is typically observed during conventional extraction methods where
higher solvent volumes generally produce increased yields of phenolic compounds.
However, there have been reports of the opposite trend occurring using MAE, where
higher solvent volumes have been shown to decrease extraction yield in some instances.
For example, it was found that lower solvent volumes lead to higher yields of pectin from
apple pomace using MAE (15). The same phenomenon was observed when MAE was
used to extract polycyclic aromatic hydrocarbons (PAHs) from sediment using
acetone:hexane (1:1) as the extraction solvent (24) and for the MAE of felodipine tablets
(25). It is not currently known why lower solvent volumes have lead to higher extraction
yields when using MAE and this phenomenon warrants further investigation.
Response surface models for acetonic extracts fitted based on the DPPH radical
scavenging activity were also found to be significant (p < 0.05) with R2 values of 0.95,
0.93, 0.82 and 0.66, respectively for Red Delicious, Golden Delicious, Jonathan and
Winesap pomace samples. The solvent volume to sample ratio significantly impacted the
extraction of phenolics with high DPPH radical scavenging activity for all the apple
pomace varieties. When extracting Jonathan pomace with 70% acetone, there was an
18.5% increase in the inhibition percentage from 73.09 to 86.62, as the solvent volume
was decreased from 10.3 to 5.6 ml/g DW (Fig. 3.4). Thus, it appears that MAE of apple
53
pomace using 70% acetone requires lower solvent volumes for maximum TPC and DPPH
activity.
Ethanolic extraction
The models for ethanolic extraction were also found to be significant (p < 0.05)
and were adequate for predicting the response variables. The factors, microwave power
and extraction time, had a significant effect on the models based on TPC. Higher
microwave power and extraction time produced maximum TPC values in all the apple
pomace samples. The interaction plot between microwave power and time for Red
Delicious apple pomace extracted with 60% ethanol illustrates the effect of power and
time on TPC (Fig. 3.5). Even though solvent volume did not seem to be significant, its
interaction effects were fairly significant (p < 0.1), and as a result, higher solvent
volumes were found to be more effective in obtaining extracts with higher total phenols.
Solvent volume was the only parameter that had a significant effect on the DPPH
inhibition percentage in Jonathan ethanolic extracts (Fig. 3.6). Decreasing the solvent
volume resulted in higher DPPH radical scavenging activity in the extract.
Methanolic extraction
The methanolic models based on TPC were also found to be significant (p <
0.05). Inverse transformations of the data were required for ensuring the validity of
model assumptions and obtaining better fit. In this case, the response was inversely
related to the model parameters. Models based on the DPPH inhibition percentage were
found to be significant for Red Delicious, Golden Delicious and Winesap pomace
54
extracts. Trends similar to those obtained for ethanolic extracts, where increasing
microwave power and time lead to increased TPC were observed. In addition, a decrease
in solvent volume caused an increase in DPPH activity for all pomace varieties extracted
using 60% methanol. Extraction time was a significant factor in all the models. An
increase in the extraction time within the range tested in this study yielded extracts with
higher TPC and DPPH activity.
Model optimization and verification
The objective of the optimization was to achieve extraction conditions that yield
extracts containing high amounts of phenolic antioxidants and demonstrate high DPPH
radical scavenging activity. The TPC and DPPH inhibition percentage were closely
correlated (R2 >0.80) for all the extraction conditions. However, the ethanolic and
methanolic extracts that had high phenolic content exhibited lower radical scavenging
activity when compared to acetone extracts having lower TPC. This indicates the
specificity of certain polyphenols to be extracted under different conditions, and the
varying degrees of antioxidant capacities of individual compounds. The Design Expert
software was used to estimate the “sweet-spot” in which the optimization was based on
maximizing both TPC and DPPH activity simultaneously. The predicted optimum
conditions were referred to as “desirability” and are shown in Table 5.5. Each solvent
system had identical extraction conditions independent of the apple cultivar. A
microwave power of 735 W was observed as optimum for all samples and solvent
systems, indicating that phenolic compounds are not degraded by high temperatures in
this method as they are exposed for very short periods of time.
55
In general, Red Delicious pomace extracts contained higher amounts of total
phenols compared to the other cultivars (Table 3.5). The optimum conditions were
predicted at 10.3 ml solvent volume per gram dry sample extracted at 735 W power and
149 s extraction time. Due to equipment constraints that resulted in solvent superheating,
higher microwave powers and extraction times were not tested. The maximum DPPH
inhibition percentage of 94% was obtained using 5.65 ml of 70% acetone at 735 W and
149 s extraction time. The response surface plots optimized for both the TPC and DPPH
IP (desirability) for Red Delicious samples using acetone and ethanol solvent systems are
shown in Figs. 3.7 and 3.8.
To determine the accuracy of the model, Red Delicious pomace was extracted
under the predicted optimum conditions based on TPC and DPPH IP (Table 3.6).
Extractions were performed in triplicate and analyzed for their TPC and DPPH IP.
Extraction with 60% ethanol resulted in an experimental TPC value of 16.8 mg GAE/g
DW compared to the predicted value of 15.8 mg GAE/g DW, representing a 6.5%
increase over the predicted value. The radical scavenging activity was also higher by
4.1% (77.12) compared to the predicted value (74.1). A possible explanation for this
deviation could be that the optimal design points were located near the edge of the model,
which is typically less accurate than the model center. However, the experimental values
are still satisfactorily close (< 10%) to the predicted values. When extracting with 70%
acetone, smaller deviations from the predicted responses were observed, possibly due to
the fact that these models had better fit and R2 values.
56
HPLC characterization
The major phenolic compounds extracted under optimal conditions by MAE were
quantified by HPLC (Table 3.7). Extracts from all apple pomace varieties were found to
contain catechin (192.51-351.60 mg/kg), phloridzin (25.25-102.9 mg/kg), caffeic acid
(9.32-44.92 mg/kg), chlorogenic acid (22.56-38.40 mg/kg) and quercetrin (44.69-211.34
mg/kg). However, each solvent system showed a slightly different phenolic profile,
indicating the ability of different solvents to preferentially extract certain compounds.
Catechin and chlorogenic acid were found to be present exclusively in the methanolic
extracts, independent of pomace variety. Caffeic acid was also present in higher amounts
in the methanolic extracts as compared to acetone and ethanol in the four tested apple
cultivars. Procyanidin B2 was present in the methanolic extracts from Jonathan and
Winesap varieties. This selectivity can be attributed to solvent polarity. Methanol is the
most polar solvent and thus was capable of extracting the more polar phenolics and
hydroxycinnamic acids.
Acetonic extracts had higher amounts of phloridzin compared to the other solvent
systems, independent of pomace variety. This may be responsible for the high
antioxidant activity of these extracts. Lee et al. (4) found that phloretins were one of the
major contributors of antioxidant activity in apple pomace extracts along with quercetin.
The HPLC chromatogram of Red Delicious apple pomace extracted with 60% ethanol is
shown in Fig. 8. A significant peak (retention time 25.38 min) was obtained in the
chromatogram of all extracts, but remained unidentified due to lack of standards, and is
57
suspected to be a quercetin glycoside. High amounts of quercetin glycosides were
reported in gala apple pomace analyzed by HPLC (26).
Comparison with other extraction techniques
MAE was compared to traditional solid-liquid extraction for the ability to extract
polyphenols from apple pomace. Wijngaard and Brunton (27) optimized the solvent
extraction of polyphenols from an industrial blend of cider varieties. Optimum conditions
were obtained using 100 ml/g of 65% acetone extracted at 25 °C for 60 minutes, which
resulted in an extract containing 15.1 mg GAE/g DW. Slightly higher, although not
significantly different amounts of total phenols (15.8 mg GAE/g DW) were observed in
the Red Delicious pomace extracts in the current work using MAE, and this was verified
by experimental runs. It can be noted that though comparable amounts of total phenols
were extracted by both techniques, significantly shorter extraction times and solvent
volumes were required for MAE compared to the traditional solid-liquid extraction
method employed by Wijngaard and Brunton (27). The optimal extraction time for MAE
was predicted as 149 s versus 60 min for solid-liquid extraction, resulting in an
approximate 95% reduction in extraction time. This is consistent with other studies (2829) that have shown reduced extraction time of MAE compared with conventional
extraction methods as well as other relatively new extraction techniques (ultrasound
assisted extraction, supercritical fluid extraction, etc). In addition, MAE required
significantly lower solvent volumes (10.3 ml/g) compared to solvent extraction (100
ml/g).
58
Conclusion
MAE was found to be a viable alternative to traditional solvent extraction
techniques for the extraction of phenolic compounds from apple pomace. Phenolic
antioxidants from a variety of apple pomace cultivars were extracted under conditions of
varying extraction parameters (solvent volume to sample ratio, power and time) and
solvent systems. Red Delicious pomace variety had the highest TPC (15.8 mg GAE/g
DW) and DPPH radical scavenging activity (94.4%) compared to the other pomace
varieties. The optimal extraction conditions for this cultivar based on TPC were 10.4 mL
of 60% ethanol at 735 W and 149 s. Optimal conditions for DPPH radical scavenging
activity were 5.65 mL of 70% acetone at 735 W and 149 s. The predicted values were
verified experimentally and found to be satisfactory in predicting the response variables
(TPC and DPPH). Catechin, phloridzin, caffeic acid, chlorogenic acid and quercetrin
were some of the major polyphenols present in the extracts. Compared to traditional
solvent extraction, MAE produces extracts with comparable yields and significantly
shorter extraction times and lower solvent volumes. Thus, MAE can be used for the rapid
extraction of valuable antioxidants from industrial plant waste streams such as apple
pomace, thereby adding value to a waste material and increasing the potential
development of these extracts into functional food ingredients and natural food
preservatives.
59
Design-Expert® Software
total phenols
Design points below predicted value
0.928981
0.36367
0.69
Actual Factor
A: solvent volume = 8.00
0.635
total phenols (mg GAE/g DW)
X1 = B: power
X2 = C: time
0.58
0.525
735.29
0.47
617.65
500.00
149.12
127.06
B: power (W)
382.35
105.00
82.94
60.88
264.71
C: time (s)
Figure 3.1. Response surface of Red Delicious apple pomace extracted with 70% acetone
- Effect of extraction time and microwave power on TPC
60
Design-Expert® Software
Original Scale
total phenols
Design points below predicted value
0.749713
0.232543
0.46
Total phenols (mg GAE/g DW)
X1 = A: solvent volume
X2 = B: power
Actual Factor
C: time = 105.00
(a)
0.405
0.35
0.295
0.24
735.29
5.65
617.65
6.82
500.00
8.00
A: solvent volume (ml)
B: power (W)
382.35
9.18
10.35
264.71
Design-Expert® Software
Original Scale
total phenols
Design points below predicted value
0.749713
0.232543
Actual Factor
C: time = 105.00
(b)
0.46
Total phenols (mg GAE/g DW)
X1 = A: solvent volume
X2 = B: power
0.405
0.35
0.295
0.24
735.29
5.65
617.65
6.82
500.00
8.00
A: solvent volume (ml)
382.35
9.18
B: power (W)
10.35 264.71
Design-Expert® Software
total phenols
Design points below predicted value
1.1443
0.493787
0.96
Actual Factor
C: time = 105.00
0.855
total phenols (mg GAE/g DW)
X1 = A: solvent volume
X2 = B: power
(c)
0.75
0.645
735.29
0.54
617.65
500.00
5.64706
6.82353
B: power
382.35
8
9.17647
10.3529
264.71
A: solvent volume (ml)
Figure 3.2. Response surface of extracts with 70% acetone - Effect of solvent volume
and power on TPC on (a) Golden Delicious apple pomace (b) Red Delicious apple
pomace and (c) Winesap apple pomace
61
Figure 3.3. Effect of solvent volume on TPC of Winesap apple pomace extracted with
70% acetone
62
Figure 3.4. Effect of solvent volume on DPPH inhibition percentage of Jonathan apple
pomace extracted with 70% acetone
63
Figure 3.5. Interaction plot of Red Delicious apple pomace extracted with 60% ethanol Effect of microwave power and time on TPC
64
Design-Expert® Software
inhibition percentage
Design points above predicted value
Design points below predicted value
93.0412
62.0275
Actual Factor
C: time = 105.00
DPPH inhibition percentage (%)
X1 = A: solvent volume
X2 = B: power
91
86.75
82.5
78.25
74
735.29
617.65
5.64706
6.82353
500.00
8
382.35
9.17647
A: solvent volume (ml)
10.3529
B: power (W)
264.71
Figure 3.6. Response surface of Jonathan apple pomace extracted with 60% ethanol Effect of solvent volume and power on DPPH inhibition percentage
65
Design-Expert® Software
Desirability
Design points above predicted value
Design points below predicted value
1
0.700
0
0.525
Actual Factor
C: time = 149.12
0.350
Desirability
X1 = A: solvent volume
X2 = B: power
0.175
0.000
735.29
10.35
617.65
9.18
500.00
B: power (W)
8.00
382.35
6.82
264.71
A: solvent volume (ml)
5.65
Figure 3.7. Response surface of the effect of solvent volume and power on both TPC and
DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with
60% ethanol
66
Design-Expert® Software
Desirability
Design points below predicted value
1
0
0.940
X1 = A: solvent volume
X2 = B: power
0.705
Desirability
Actual Factor
C: time = 149.12
0.470
0.235
0.000
735.29
617.65
500.00
5.65
6.82
8.00
382.35
9.18
10.35
B: power (W)
264.71
A: solvent volume (ml)
Figure 3.8. Response surface of the effect of solvent volume and power on both TPC and
DPPH inhibition percentage (desirability) for Red Delicious apple pomace extracted with
70% acetone
67
Figure 3.9. HPLC chromatogram at 280 nm of Red Delicious apple pomace extracted
with 60% ethanol under optimum conditions
68
Table 3.1. Coded values and corresponding actual values of the optimization parameters
Code
-1
0
1
Solvent volume (A)
(ml/g DW)
5.6
8.0
10.3
Power (B)
(W)
264.7
500.0
735.3
Time (C)
(s)
61
105
149
69
Table 3.2. Experimental design for extraction of phenolic compounds by MAE in terms of coded
values
Experiment no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Solvent volume (A)
(ml/g DW)
-1
1
-1
1
-1
1
-1
1
0
0
0
0
-1.7
+1.7
0
0
0
0
Power (B)
(W)
+1
+1
+1
+1
-1
-1
-1
-1
0
0
0
0
0
0
0
0
-1.7
+1.7
Time (C)
(s)
-1
-1
+1
+1
-1
-1
+1
+1
0
0
0
0
0
0
-1.7
+1.7
0
0
70
Table 3.3. Response surface models fitted based on TPC for different apple cultivars and solvent
systems
Solvent
system
70%
Acetone
60% EtOH
60%
MeOH
a
Apple cultivar
Model for TPC (in coded units)a
F value
p value
R2
Red Delicious
total phenols = 0.52 - 0.13A + 0.048B +
0.054C + 0.048A2
17.14
< 0.0001
0.90
Golden Delicious
1/(total phenols) = 2.84 + 0.62A - 0.28B
- 0.35C - 0.31AB - 0.33AC
12.25
0.0003
0.87
Jonathan
total phenols = 0.73 - 0.19A + 0.042B +
0.052C + 0.054A2
27.75
< 0.0001
0.92
Winesap
total phenols = 0.69 - 0.16A + 0.052C +
0.053A2
17.72
< 0.0001
0.85
Red Delicious
total phenols = 0.63 + 0.17B + 0.17C
3.58
0.0323
0.66
Golden Delicious
1/(total phenols) = 2.69 + 0.43A - 0.50B
- 0.53C
7.55
0.0021
0.80
Jonathan
(total phenols)-1.5 = 1.70 + 0.50A - 0.38B
- 0.29C - 0.35AB
10.74
0.0004
0.82
Winesap
1/(total phenols) = 1.98 + 0.33A - 0.31B
- 0.37C
8.57
0.0018
0.65
Red Delicious
(total phenols)-1 = 2.18 + 0.33A - 0.39B
- 0.39C - 0.24C2
10.03
0.0006
0.85
Golden Delicious
1/(total phenols) = 3.96 + 0.65A - 0.66B
- 0.78C - 0.63AC - 0.52B2 - 0.48C2
7.45
0.0026
0.84
Jonathan
1/(total phenols) = 1.75 + 0.22A - 0.21B
- 0.20C
6.48
0.0040
0.78
Winesap
1/(total phenols) = 2.04 - 0.40B - 0.37C
7.06
0.0069
0.49
only includes significant model terms (p < 0.05)
71
Table 3.4. Response surface models fitted based on DPPH activity for different apple cultivars
and solvent systems
Solvent
system
70%
Acetone
60% EtOH
60%
MeOH
a
Apple cultivar
Model for DPPH (in coded units)a
F value
p value
R2
Red Delicious
inhibition percentage = 69.32 - 13.46A + 2.57
B + 4.09C + 2.62B2 + 2.31C2
46.99
< 0.0001
0.95
Golden Delicious
inhibition percentage = 48.61 - 12.12A + 3.52B
+ 5.13C + 4.23BC + 3.87A2
19.56
< 0.0001
0.93
Jonathan
inhibition percentage = 79.85 - 6.76A + 1.98C
34.42
< 0.0001
0.82
Winesap
inhibition percentage = 79.31 - 6.69A
31.10
< 0.0001
0.66
Red delicious
inhibition percentage = 62.64 - 7.69A + 4.89B
+ 6.31C
11.03
0.0004
0.82
Golden delicious
inhibition percentage = 32.74 + 0.98B + 4.18C
+ 8.86BC
3.51
0.0437
0.43
Jonathan
inhibition percentage = 82.23 - 8.15A
45.52
< 0.0001
0.74
Winesap
inhibition percentage = 46.80 - 5.29A + 6.09C
5.95
0.0078
0.56
Red Delicious
inhibition percentage = 50.12 + 9.64B + 8.44C
+ 5.61B2 + 6.68C2
7.33
0.0024
0.80
Golden Delicious
1/(inhibition percentage) = 0.031 + 4.031 x
10-003A - 4.584 x 10-003B - 6.062 x 10-003C 5.156 x 10-003AC
3.38
0.0421
0.51
Jonathan
not significant
Winesap
inhibition percentage = 62.12 + 7.38C
5.53
0.0159
0.42
only includes significant model terms (p < 0.05)
72
Table 3.5. Optimum conditions for the extraction of phenolic antioxidant compounds by MAE
based on both TPC and DPPH activity
Apple
cultivar
Solvent system
Solvent volume
(A)
(ml/g DW)
Power (B)
(W)
Time (C)
(s)
TPC
(mg GAE/g DW)
DPPH
Inhibition
Percentage (%)
Red
Delicious
70% Acetone
60% EtOH
60% MeOH
5.65
10.35
10.35
735.29
735.29
735.29
149.11
149.12
149.12
8.88
15.81
10.24
94.36
74.08
87.36
Golden
Delicious
70% Acetone
60% EtOH
60% MeOH
5.65
10.35
10.35
735.28
735.29
735.29
149.12
149.12
149.12
2.90
8.64
9.92
71.29
46.75
46.75
Jonathan
70% Acetone
60% EtOH
60% MeOH
5.65
10.35
8.00a
735.29
735.29
500a
149.12
149.12
105a
5.79
7.65
-
88.60
90.38
-
Winesap
70% Acetone
60% EtOH
60% MeOH
5.65
10.35
8.00a
735.29
735.29
735.29
149.12
149.12
149.12
5.67
5.83
6.28
88.60
62.20
75.47
a
term not significant in model
73
Table 3.6. Predicted and experimental values of TPC and DPPH activity for Red Delicious apple
pomace extracted under optimal conditions for 60% ethanol
Solvent
system
Optimum
conditions
60%
ethanol
Solvent volume
(ml/g DW)
10.4
Power (W)
735
Time (s)
149
Solvent volume
(ml/g DW)
70%
acetone
a
Predicted
yield
Experimental
yielda
TPC
(mg GAE/g DW)
15.8
16.8 ±0.97
DPPH activity (%)
74.1
77.1 ±3.44
5.65
TPC
(mg GAE/g DW)
8.88
9.30 ±0.65
Power (W)
735
DPPH activity (%)
94.4
93.7 ±4.20
Time (s)
149
Mean ± standard deviation (n=3)
Table.3.7. HPLC quantification of major apple polyphenols from optimized MAE extracts for each apple cultivar and solvent system
Apple cultivar
Concentration (mg/kg dry pomace)*
Solvent system
Catechin
Epicatechin
Procyanidin B2
Phloridzin
c
nd
nd
nd
nd
nd
nd
d
nd
nd
nd
nd
nd
nd
34.78
f
41.20
g
33.02
a
nd
nd
nd
136.24
nd
nd
b
nd
nd
nd
21.04
nd
nd
Red Delicious
60% MeOH
70% Acetone
60% EtOH
297.56
nd
nd
Golden Delicious
60% MeOH
70% Acetone
60% EtOH
192.51
nd
nd
Jonathan
60% MeOH
70% Acetone
60% EtOH
351.60
nd
nd
Winesap
60% MeOH
70% Acetone
60% EtOH
313.36
nd
nd
a
b
Gallic acid
Caffeic acid
52.78
b
82.59
h
25.25
d
nd
nd
b
189.09
32.71
f
10.00
d
27.63
c
38.40
nd
nd
a
80.83
f
46.33
i
59.20
g
nd
nd
a
378.81
35.40
f
9.32
e
24.63
b
37.16
nd
nd
a
125.03
d
68.76
c
119.65
c
nd
nd
c
23.08
33.89
f
9.77
b
34.67
b
38.00
nd
e
22.56
a
169.52
c
115.15
a
211.34
e
nd
nd
d
14.39
44.92
f
9.89
b
35.19
a
35.12
nd
nd
b
76.88
g
44.69
d
81.66
69.68
a
102.90
b
79.80
46.88
c
71.28
d
50.51
Chlorogenic acid
Quercetrin
d
Quercetin
nd
nd
nd
c
nd
nd
nd
b
nd
nd
124.30
e
nd
nd
nd
*
Data expressed as means; n=2
nd = not detected
Means with different letters represent significant difference within a column
74
75
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Lu, Y.; Foo, L. Y., Identification and quantification of major polyphenols in apple
pomace. Food Chemistry 1997, 59, (2), 187-194.
Lee, K. W.; Kim, Y. J.; Kim, D. O.; Lee, H. J.; Lee, C. Y., Major phenolics in
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Sesso, H. D.; Gaziano, J. M.; Liu, S.; Buring, J. E., Flavonoid intake and the risk
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Gillman, M. W.; Cupples, L. A.; Gagnon, D.; Posner, B. M.; Ellison, R. C.;
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77
CHAPTER 4. ENCAPSULATION OF POLYPHENOL EXTRACTS FROM
APPLE POMACE IN CYCLODEXTRINS
†
Vaishnavi Chandrasekar1, Tameshia S. Ballard1*, Fernanda San-Martín1 and Peter Hirst2
1
Department of Food Science, Purdue University, West Lafayette, IN 47907
2
Department of Horticulture and Landscape Architecture, Purdue University, West
Lafayette, IN 47907
*
Correspondence should be sent to:
Tameshia S. Ballard, 745 Agriculture Mall Drive, Purdue University, West Lafayette, IN
47907
Phone: (765) 496-6545 Fax: (765) 494-7953 Email: tballard@purdue.edu
†
Prepared for Submission to J. Agricultural and Food Chemistry
78
Abstract
Extracts of apple pomace are rich in polyphenols that have high antioxidant
activity. Encapsulation of Red Delicious apple pomace extracts obtained by microwaveassisted extraction was investigated in three types of cyclodextrins: α-cyclodextrin, βcyclodextrin and 2-hydroxypropyl-β-cyclodextrin. Quercitrin (84.5 mg/g dry weight of
apple pomace), phloridzin (82.2 mg/g) and caffeic acid (18.22 mg/g) were the major
phenolic compounds present in the acetone extracts as analyzed by HPLC. Inclusion
complexes of the extract with cyclodextrins (CDs) were formed by dissolution in aqueous
systems for 48 hours followed by freeze-drying to obtain dry complexes. Complex
formation was verified by DSC and confirmed for all the three CD types. Inclusion
complexes were found to be stable under oxidative conditions up to 250 °C. Phase
solubility studies indicated an A L type diagram indicating the formation of a 1:1 complex
of the extract with β-CD with a moderate binding constant (Kc = 1.02 mg GAE/g extract 1
). α-CD and 2-hydroxypropyl-β-CD did not prove to be effective in forming soluble
inclusion complexes from the current techniques employed. Encapsulation of the
polyphenol rich apple pomace extract in β-CD results in the formation of stable
complexes with enhanced solubility for use as functional food ingredients.
Keywords: apple pomace extract, polyphenols, encapsulation, cyclodextrin, DSC, phase
solubility
79
Introduction
Apple pomace is the waste stream (seeds, skin, flesh) generated from apple juice
or cider production. Currently it is used mainly as animal feed, although this application
is limited due to its low protein and vitamin content (1). Apple pomace is rich in several
polyphenols, such as quercetin glycosides, catechin, epicatechin, chlorogenic acid and
phloridzin (2). These polyphenols have been shown to exhibit high antioxidant and
radical scavenging activity in vitro (3-4), and may serve as potential replacers of
synthetic antioxidants in lipid-based foods that are prone to oxidation. Therefore, the
extraction of valuable natural antioxidant compounds from apple pomace may prove to
be economically beneficial. In spite of these potential benefits, the use of these
compounds as bioactive ingredients in foods is limited by their low water solubility and
poor oxidative stability.
Encapsulation of bioactive ingredients has been widely used in the food industry
to protect these compounds from oxidation, thereby increasing their stability. Further,
encapsulation in suitable wall materials may also serve to enhance the solubility, enabling
their use in aqueous food systems (5). Encapsulation is commonly carried out by simply
emulsifying the guest molecule with a suitable wall material in aqueous solution, after
which the water may be removed by freeze-drying or spray-drying to obtain dry inclusion
complexes (6). The use of cyclodextrins (CDs) as encapsulates in the food industry is
steadily increasing (7). In the past, CDs have been primarily used in pharmaceutical
applications for the encapsulation of hydrophobic drug molecules and stabilization of
light or oxygen-sensitive compounds (8). However, do to their broad range of protective
80
effects and generally recognized as safe (GRAS) status in the US, CDs are now finding
greater application in the food industry as well.
CDs are cyclic oligosaccharides that consist of 6, 7 or 8 glucose units linked
together by 1,4-α-glucosidic bonds, and are classified as α-CD, β-CD and λ-CD,
respectively. These molecules are chemically and physically stable, and are obtained by
the enzymatic modification of starch (9). They consist of a relatively hydrophobic cavity
in the center, with water soluble hydroxyl groups located on the outer surface. This
enables them to form inclusion complexes with hydrophobic guest molecules, while
increasing their solubility and bioavailability. The size of the CD cavity determines the
encapsulation efficiency of various phenolic compounds. Alpha-CD has the smallest
cavity of the three native CDs followed by β- and γ-CD. Modified CDs have been
developed with improved water solubility, such as 2-hydroxypropyl-β-CD (2-HP-β-CD).
Limited studies have looked at the use of CDs for encapsulation of whole plant extracts
containing a number of compounds, each likely having varying encapsulation efficiencies
with the different types of cyclodextrins. The encapsulation of complex mixtures such as
olive leaf extract (10) and propolis extract (6) have been achieved in β-CD.
Complex formation of inclusion compounds may be confirmed using several
techniques such as FTIR, NMR, SEM, X-ray diffraction and differential scanning
calorimetry (DSC) (11-12). DSC is a thermal analysis tool that is commonly employed
for confirming complex formation in the solid state, by comparing the thermogram of the
prepared inclusion complex with the guest molecule (6, 10-12). Disappearance of thermal
events associated with the guest molecule, in the thermogram of the inclusion complex is
81
indicative of complex formation (11). When carried out under an oxygen atmosphere,
DSC may also be used to study the oxidative stability of the inclusion complex (13).
In this work, encapsulation of the polyphenolic extract obtained from Red
Delicious apple pomace was studied using three types of cyclodextrins: α-cyclodextrin,
β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin. The main phenolic constituents in
the extract were analyzed by HPLC. Complex formation and oxidative stability were
analyzed by DSC. Further, the solubility of the inclusion complexes in water was
investigated using phase solubility studies.
Methods and materials
Reagents and Chemicals
α-CD, β-CD and 2-HP-β-CD, catechin, epicatechin, procyanidin B2, caffeic acid,
chlorogenic acid, phloridzin, hydrocaffeic acid, hydrocoumaric acid, quercetin and
quercetrin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO). Analytical
grade methanol, ethanol and acetone, and HPLC grade acetic acid and water were
obtained from Lilly Stores (West Lafayette, IN).
Preparation of apple pomace extract
Red Delicious apple pomace was provided by Biersdorfer Orchard (Guilford, IN).
The pomace was stored at -20 °C in nitrogen-flushed Ziploc freezer bags until use.
Samples were lyophilized for 48 hours in a freeze dryer (Virtis Co., Gardiner, NY) and
ground to less than 1 mm particle size. Microwave-assisted extraction was carried out in
82
a Mars Xpress microwave extraction system (CEM Corp., Matthews, NC) using the
optimized extraction conditions reported by Chandrasekar et al. (14). Briefly, apple
pomace was extracted with 5.6 ml of 70% acetone for 149 seconds at 735 W microwave
power. The extracts obtained were transferred to 15 ml centrifuge tubes and centrifuged
at 1050 rpm for 20 minutes in a Dynac centrifuge (Clay Adams, Division of Becton
Dickinson and Co., Parsippany, NJ). The supernatant was evaporated to remove the
organic solvent in a rotary vacuum evaporator. The aqueous extract was frozen and
lyophilized for 48 hours in a freeze-dryer (Virtis Co., Gardiner, NY) to obtain dry
polyphenol extract.
HPLC analysis
The polyphenol extract was resuspended in an equal volume of methanol and
analyzed by reverse-phase HPLC based on the method of Suarez et al.(15), using an
Agilent 1200 Series HPLC (Santa Clara, CA) with a diode array detector. A Waters C-18
column (3.9 x 100 mm, I.D. 3.5 µm) equilibrated at 25 °C was employed, and the
injection volumes was 10 µl. The mobile phase consisted of solvent A: 2% aqueous
acetic acid and solvent B: 100% methanol. The gradient elution first consisted of a linear
step from 0 to 45% of solvent B in 55 minutes, an isocratic step of 15 minutes, and finally
another linear step from 45 to 55% of solvent B in 10 minutes. The mobile phase flow
rate was set at 0.8 ml/min. Absorbance spectra was collected at 355 nm for detection of
flavonol glycosides, 313 nm for hydroxycinnamic acids and 280 nm for the other
phenolic compounds (procyanidins, dihydrochalcones, benzoic and 3-phenylpropionic
83
acids). External injected standards were used to identify and quantify some of the major
phenolic compounds present.
Preparation of inclusion complex
The inclusion complexes of the polyphenol extract were prepared with α-CD, βCD and 2-HP-β-CD according to the method of Mourtzinos et al. (10) with some
modification. 25 mg of polyphenol extract was dispersed in 10 ml of 9 mM aqueous
solutions of the cyclodextrins and mixed at 30 °C for 48 hours. The suspension was then
filtered through a 0.45 µm PVDF filter. The filtrate was frozen in liquid nitrogen and
lyophilized for 48 hours in a freeze dryer (Virtis Co., Gardiner, NY).
Preparation of physical mixture
Physical mixtures of the polyphenol extract with α-CD, β-CD and 2-HP-β-CD
were prepared by mixing the two components in the same weight ratio as the inclusion
complex, using a mortar and pestle for about 5 min until a homogeneous blend was
obtained.
Study of complex formation by DSC
Thermal analysis of the inclusion complexes was carried out using a Q2000 DSC
(TA Instruments, New Castle, DE) under a nitrogen atmosphere according to the method
of Mourtzinos et al.(10). Four types of samples were analyzed to compare their
thermograms; pure apple polyphenol extract, pure cyclodextrin (α, β and 2-HP-β),
physical mixtures and inclusion complexes. Calibration was carried out initially using
indium metal and the scan rate used was 10 °C/min from 30-230 °C. Samples were
84
weighed with ±0.01 mg accuracy and placed in hermetically sealed aluminum pans.
Experiments were conducted in duplicates for each sample.
Study of oxidative stability by DSC
Oxidative stability of the inclusion complexes was evaluated by thermal analysis
using DSC under an oxygen atmosphere, as described by Mourtzinos et al. (10) .
Inclusion complexes (with α, β and 2-HP-β-CDs) and pure polyphenol extract were
weighed at ±0.01 mg accuracy in aluminum pans and sealed with lids containing a
pinhole. Samples were heated from room temperature to 120 °C under an oxygen
atmosphere at a heating rate of 90 °C/min. After 1 minute at 120 °C for temperature
equilibration, the sample was heated up to 300 °C at a rate of 10 °C/min. DSC
experiments were conducted in duplicates.
Phase solubility study
Phase solubility study was conducted based on the method of Higuchi and
Connors (16). An excess amount of the polyphenol extract was added to increasing
concentrations of α-CD, β-CD and 2-HP-β-CD aqueous solutions (3-15 mM). The
inclusion complexes were allowed to form by continuous shaking for 48 hours at 30 °C.
The solutions were filtered through 0.45 µm PVDF filter. The filtrate was frozen and
lyophilized in a freeze dryer (Virtis Co., Gardiner, NY) to remove the water. The dried
inclusion complexes were then extracted with 100% methanol by sonication for 5
minutes. The extract obtained was filtered and the total phenols in the filtrate was
measured spectrophotometrically using the Folin-ciocalteu assay according to the method
85
of Spanos and Wrolstad (17). Gallic acid was used as the standard, and results were
expressed in terms of gallic acid equivalents (GAE)/g pomace extract. The experiments
were carried out in duplicate, and stability constants were calculated from the phasesolubility graph, using the Higuchi-Connors equation.
Results and discussion
HPLC analysis
Extracts obtained by microwave-assisted extraction of Red Delicious apple
pomace were analyzed by HPLC, and the phenolics present were identified based on the
retention times of the standards. The major polyphenols present include quercitrin,
phloridzin and caffeic acid, and their amounts are shown in Table 4.1. In addition, a
significant peak (retention time 25.38 min) was observed in the spectra measured at 280
nm, but remained unidentified due to lack of standards and is suspected to be a quercetin
glycoside. High amounts of quercetin glycosides were reported to be present in Gala
apple pomace extracts analyzed by HPLC (4).
Study of complex formation
The DSC results obtained under inert nitrogen atmosphere to verify the formation
of inclusion complexes between the apple polyphenol extract and the three types of CDs
studied are shown in Fig 4.1. Thermograms were obtained for four types of samples: pure
86
polyphenol extract, pure cyclodextrin, the physical mixture and the inclusion complex. In
Fig 4.1A, the pure polyphenol extract (a) shows an endothermic peak at about 160 °C,
and several endothermic peaks after 180 °C. These correspond to melting points of
caffeic acid (m.p. 195°C) and quercitrin (175°C), denoting degradation of the extract
constituents. Pure α-CD (b) showed an endothermic peak at 190 °C, possibly due to the
elimination of water. All these peaks are present in the physical mixture (c) as well.
However, the endothermic peaks associated with the pure polyphenol extract as well as
α-CD are absent in the thermogram of the inclusion complex (d), which is indicative of
successful inclusion of the guest molecule. Once the inclusion complex is formed, the
stability and melting characteristics of the resulting compound are altered, and as a result,
they show disappearance of thermal events associated with the initial guest molecule and
wall material.
Figure 4.1B shows the thermograms obtained for the β-CD samples. Again, pure
β-CD (b) shows endothermic peaks at 140 °C and 190 °C, possibly due to water
elimination from the sample. The peaks associated with the pure polyphenol extract and
pure β-CD are present in the physical mixture (c) but are absent in the thermogram of the
inclusion complex (d). Thus, complex formation between β-CD and polyphenol extract
may also be inferred from this result. β-CD has been shown to form complexes with other
types of complex extracts as well, such as the flavonoid rich St John’s wort extract
(Hypericum perforatum) (18), propolis ethanolic extract (6) and oleuropein rich olive leaf
extract (10).
87
Results obtained for 2-HP-β-CD are shown in figure 4.1C, and also shows
complex forming ability with the apple pomace extract. Pure 2-HP-β-CD shows an
endothermic peak at 220 °C, which is also present in the thermogram of the physical
mixture (c), with a slight shift to the left, indicating no complex formation by this
process. However, the DSC scan of the inclusion complex did not show any endothermic
peak in this temperature range, confirming the formation of a complex with the CD.
Thus, all the three types of CDs seem to have the ability to form a complex with the
polyphenolic extract obtained from apple pomace.
Study of oxidative stability
The thermograms of pure apple pomace extract and the inclusion complexes (with
α, β and 2-HP-β-CDs) under oxidative conditions are shown in figure 4.2. The pure
extract shows exothermic peaks at 185 °C and higher, which relates to the oxidation of
polyphenols in the extract. Phenolic compounds are sensitive to oxidation, and typically
degrade at temperatures over 100 °C, giving rise to various decomposition products (1920). Exothermic peaks were also present in the thermogram of the inclusion complexes
beyond 250 °C. This indicates the ability of the inclusion complexes obtained with all 3
types of CD’s to protect the pure extract against oxidation up to temperatures of 250 °C.
Phase solubility study
Phase solubility studies were carried out to investigate the dissolution of the
inclusion complexes formed using the three types of cyclodextrins in water, and the
results are shown in Fig. 4.3. Since apple pomace extract is a complex mixture of several
88
phenolic compounds, the results were described in terms of total phenols measured using
the Folin-ciocalteu assay, and expressed as mg GAE/g pure extract. This approach is
commonly employed when forming inclusion complexes with whole plant extracts (6) as
compared to encapsulation of a single compound. The solubility of the inclusion
complexes formed using β-CD was found to increase linearly (R2 = 0.9802) with increase
in cyclodextrin concentration. The slope of this line was estimated to be < 1 (0.0191)
indicating an A L type diagram and 1:1 binding between the guest molecule and wall
material (10). The stability constant for the extract-β-CD complex was calculated based
on the Higuchi-Connors equation (16) and was determined as K c = 1.02 (mg GAE/g
extract)-1. Several studies have confirmed the formation of soluble inclusion complexes
using β-CD with similar A L type phase solubility diagrams (6, 10, 12).
Inclusion complexes formed using α-CD and 2-HP-β-CD did not show any
increase in solubility, and the total phenols measured was found to remain constant and
low, indicative of a B I type phase solubility diagram. It is possible that these results are
due to the strong binding of the guest molecules within the cavities of α-CD and 2-HP-βCD, and thus inability to extract out the complexed polyphenols from the cavity.
However, another possibility is that these CDs have poor complex forming ability with
apple polyphenols resulting in very small amounts of phenolics being encapsulated. One
of the key factors for inclusion complex formation is steric effects, and the size of the
hydrophobic cavity plays an important role in forming the inclusion complex with guest
molecules. α-CD is smaller and can typically complex with low molecular weight guest
molecules, or compounds with aliphatic side chains (7), and is known to be less effective
89
than β-CD with regard to the formation of inclusion complexes with phenolic compounds
(21). DSC studies carried out under nitrogen atmosphere qualitatively indicate the
formation of an inclusion complex between the apple extract and α-CD/2-HP-β-CD, but
do not provide a quantitative estimation of encapsulation efficiencies. It is not possible to
come to a definite conclusion regarding this, and further studies are required in order to
identify the cause for this discrepancy.
Conclusion
Extracts obtained from Red Delicious apple pomace is rich in several polyphenols
that have shown high antioxidant activity. Some of the compounds identified by HPLC
include quercetrin, caffeic acid and phloridzin. The encapsulation of these compounds
was prepared by dissolution in three types of cyclodextrins: α-CD, β-CD and 2-HP-β-CD
followed by freeze-drying to obtain dry complexes. DSC studies verified the formation of
inclusion complexes between the apple polyphenol extract with all three types of
cyclodextrins. The formation of inclusion complexes was also found to be effective in
protecting the polyphenol extract from oxidation up to temperatures of 250 °C. Phase
solubility studies indicate a 1:1 binding of the polyphenol rich extract with β-CD, and
increasing water solubility of the complex with increase in CD concentration. Given the
antioxidant potential of these phenolic compounds, the solid inclusion complexes
obtained with β-CD may prove valuable for use as a stable and water-soluble food
ingredient.
90
Figure 4.1. Complex formation study of apple extract with (A) α-CD (B) β-CD and (C) 2hydroxypropyl-β-CD
4.1A. Thermograms of (a) pure apple pomace extract (b) pure α-CD (c) physical mixture
(d) inclusion complex
91
4.1B. Thermograms of (a) pure apple pomace extract (b) pure β-CD (c) physical mixture
(d) inclusion complex
92
4.1C. Thermograms of (a) pure apple pomace extract (b) pure 2-hydroxypropyl-β-CD (c)
physical mixture (d) inclusion complex
93
Figure 4.2. DSC thermograms of pure apple pomace extract and inclusion complexes
under oxidative conditions
94
Total phenols (mg GAE/g extract)
0.4
0.35
0.3
0.25
β-CD
0.2
α-CD
0.15
2-hydroxypropyl-β-CD
0.1
0.05
0
0
3
6
9
12
15
Cyclodextrin concentration (mM)
Figure 4.3. Phase solubility study of apple pomace extract with α-, β, and γ-CD at 30 °C
(n=2)
95
Table 4.1. HPLC quantification of apple polyphenols from Red Delicious apple pomace
extract
Polyphenol
Amount (mg/kg dry apple pomace)a
Quercitrin
46.33 ± 1.98
Phloridzin
82.59 ± 2.61
Caffeic acid
10.01 ± 0.23
Gallic acid
0.33 ± 0.08
Catechin
0.15 ± 0.21
a
expressed as mean ± S.D. (n=2)
96
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98
CHAPTER 5. CONCLUSIONS
Millions of apples are processed globally into juice, cider and other products,
generating large amounts of apple pomace as a waste stream. This waste stream is
severely under-utilized, and is currently being used as animal feed or compost. Apple
pomace is rich in polyphenols such as quercetin glycosides and hydroxycinnamic acids
that have very good antioxidant and radical scavenging activity. The use of these natural
phenolic extracts as replacers of synthetic antioxidants in foods may prove valuable from
a consumer standpoint. Removal of these compounds from the apple matrix has
conventionally been done by solid-liquid extraction, using mainly organic solvents in
combination with a small amount of water. However, these methods require extensive
amounts of solvent and extraction time, and consideration of alternate extraction
techniques may prove more efficient. Additionally, these compounds are hydrophobic
and prone to oxidation, limiting their use as stable food ingredients. Therefore, their
encapsulation in cyclodextrins (CDs) was also studied to enhance stability and water
solubility.
The microwave-assisted extraction of phenolic antioxidants was optimized from
apple pomace of four apple varieties (Red Delicious, Golden Delicious, Winesap and
99
Jonathan) using response surface methodology. Optimum extraction conditions were
based on the total polyphenol content (TPC), as well as the antioxidant activity measured
by the inhibition percentage (IP) of the DPPH free radical. The important parameters that
were optimized included solvent type (70% acetone, 60% methanol and 60% ethanol),
solvent volume to sample ratio (4-12 ml/g), extraction time (30 - 180 s) and microwave
power (100-900 W). Good correlation (R2>0.80) was obtained between TPC and DPPH
activity in all extracts. All solvent systems seemed to show identical extraction
conditions, independent of pomace variety. Red Delicious apple pomace extracts
contained highest TPC (15.8 mg GAE/g DW) and DPPH radical scavenging activity
(94.4 % inhibition) obtained under the optimum conditions of 735 W and 149 s, with 10.4
mL of 60% ethanol and 5.65 mL of 70% acetone, respectively.
Encapsulation of phenolic extracts obtained from Red Delicious apple pomace
was carried out using α-CD, β-CD and 2-hydroxypropyl-β-CD by dissolution in aqueous
solutions followed by freeze-drying to obtain dry inclusion complexes. Complex
formation was confirmed by DSC for all three CD types. The inclusion complexes
formed were also found to be stable and resistant to oxidation up to temperatures of 250
°C, compared to the pure apple pomace extract. Phase solubility studies were carried out
to study the water solubility of the complexes. A 1:1 binding of the polyphenol rich
extract with β-CD was observed, demonstrating increasing water solubility of the
complex with increase in CD concentration. However, complexes formed with α-CD and
2-hydroxypropyl-β-CD did not show increased water solubility. It is concluded that β-CD
100
inclusion complexes were effective in enhancing both stability and solubility of the
phenolic extracts, and serves as a potential food ingredient.
Future work
Recommended future work could involve extension of the MAE optimization
study to other potential waste streams that are rich in phenolic compounds.
In the encapsulation study, examining the mechanisms for dissolution of the
inclusion complexes and identification of the compounds encapsulated may be
worthwhile. This would help identify which phenolic compounds present in the extracts
are involved in binding with the hydrophobic cavity of the CD and forming inclusion
complexes. In addition, structural characterization of the guest-host complex using NMR
can be carried out. It is also suggested that release kinetics of the encapsulated guest
molecules be explored. Additionally, the application of these inclusion complexes as
antioxidants could be investigated in model food systems.
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