Extraction and assessment of antioxidant and antimicrobial activities of phenolics from cocoa pod husk (theobroma cacao l )

MINISTRY OF EDUCATION AND TRAINING
NHA TRANG UNIVERSITY

UWIHAYE FESTUS

EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND
ANTIMICROBIAL ACTIVITIES OF PHENOLICS FROM
COCOA POD HUSK (Theobroma cacao L.)

MASTER THESIS

KHANH HOA – 2020


MINISTRY OF EDUCATION AND TRAINING
NHA TRANG UNIVERSITY

UWIHAYE FESTUS

EXTRACTION AND ASSESSMENT OF ANTIOXIDANT AND
ANTIMICROBIAL ACTIVITIES OF PHENOLICS FROM
COCOA POD HUSK (Theobroma cacao L.)
MASTER THESIS

Major

Food Technology

Topic allocation decision

192/QD-DHNT on 03/03/2020

Decision in establishing the Committee

899/QD-DHNT on 04/09/2020

Defense date

19/09/2020

Supervisors
1. Dr. Nguyen Van Tang (Principal supervisor)
2. Dr. Tran Thi My Hanh (Co-supervisor)
Chairman
Assoc. Prof. Dr. Huynh Nguyen Duy Bao
Department of Graduate Studies

KHANH HOA – 2020


UNDERTAKING
I declare that the thesis entitled “Extraction and assessment of antioxidant and
antimicrobial activities of phenolics from cocoa pod husk (Theobroma cacao L.)”
is my own work. The work has not been presented elsewhere for assessment until the
time this thesis is submitted.
3rd September, 2020

Uwihaye Festus

iii

ACKNOWLEDGEMENTS
Many thanks and praises to God Almighty that he has been with me throughout
my study period in Vietnam.
Firstly, I would highly acknowledge Dr. Nguyen Van Tang, my principal
supervisor, and Dr. Tran Thi My Hanh, my co-supervisor for their help and guidance,
they gave me an amazing experience. I also would like to thank the financial support
for my Master thesis through the research project funded by the Ministry of Education
and Training, Vietnam entitled “Extraction of some bioactive compounds from cocoa
pod husk for potential application in the functional foods”.
Secondly, I also wish to appreciate my country, Rwanda through the Ministry of
Labour as it accepted me to join in Master program at the Nha Trang University,
Vietnam.
Thirdly, I am grateful to the supports from VLIR, Graduate Studies Department,
and Faculty of Food Technology, Nha Trang University. At large, I can not forget the
Nha Trang University’s entire teaching staff and my classmates for providing me with
a good and favorable working environment.
Lastly but not least, my special acknowledgment goes to my wife, Ugwaneza
Grace and my children, Jayden, Joanna and Jordan for their undeniable love,
encouragement, patience and understanding.
3rd September, 2020

Uwihaye Festus

iv


TABLE OF CONTENTS
UNDERTAKING ........................................................................................................ iii
ACKNOWLEDGEMENTS ......................................................................................... iv

TABLE OF CONTENTS...............................................................................................v
LIST OF ABBREVIATIONS ...................................................................................... ix
LIST OF TABLES.........................................................................................................x
LIST OF FIGURES ..................................................................................................... xi
CHAPTER 1 INTRODUCTION ...................................................................................1
1.1 PROBLEM STATEMENT ......................................................................................3
1.2 RESEARCH SIGNIFICANCE ................................................................................4
1.3 RESEARCH AIMS .................................................................................................4
1.3.1 Overall aim ...........................................................................................................4
1.3.2 Specific aims.........................................................................................................4
CHAPTER 2 REVIEW OF THE LITERATURE ..........................................................5
2.1 COCOA AND COCOA BY-PRODUCTS AS WASTES AND VALUE
ADDITION....................................................................................................................5
2.1.1 Husk of cocoa pod ................................................................................................5
2.1.2 Cocoa bioactive compounds with emphasis on phenolics .....................................6
2.1.3 CPH applications and phenolic compounds ..........................................................9
2.2 IMPACT OF PROCESSING METHODS ON PHENOLIC COMPOUNDS AND
ANTIOXIDATION POTENTIAL OF CPH ................................................................12
2.3

FRACTIONATION,

PURIFICATION,

CHARACTERIZATION

AND

QUANTIFICATION OF PHENOLIC COMPOUNDS ...............................................16
2.4 EVALUATION OF ANTIOXIDANTION POTENTIAL OF PHENOLICS ........17

2.5 COCOA PHENOLIC COMPOUNDS AND BIOLOGICAL ACTIVITIES ..........18
CHAPTER 3 MATERIALS AND METHODS ...........................................................20
3.1 EXPERIMENTAL PROCEDURE ........................................................................20
v


3.2 MATERIAL AND CHEMICALS .........................................................................21
3.2.1 Fresh cocoa pod husk ..........................................................................................21
3.2.2 Analytical reagents .............................................................................................21
3.3 EXPERIMENTAL METHODS .............................................................................21
3.3.1 Preparation of powder from crude CPH extract ..................................................21
3.3.2 Fractionation of crude extract from CPH by column chromatography ...............22
3.3.3 Preparation of solutions from crude CPH extract and derived fractions for
evaluation of phenolic content, antioxidation potential and antimicrobial activity ......23
3.3.4 Phenolic compounds identification in CPH extract.............................................23
3.3.5 Analysis of physicochemical properties of powdered extract from CPH ............25
3.3.6 Analysis of bioactive compounds .......................................................................26
3.3.7 Evaluation of biological activities .......................................................................27
3.3.8 Statistical analysis ...............................................................................................29
CHAPTER 4 RESULTS AND DISCUSSIONS ..........................................................30
4.1 PHYSICOCHEMICAL PROPERTIES OF CRUDE CPH POWDER ...................30
4.2 IDENTIFICATION OF PHENOLIC COMPOUNDS BY THIN-LAYER
CHROMATOGRAPHY ..............................................................................................32
4.3

IDENTIFICATION

OF

PHENOLIC

COMPOUNDS

BY

FTIR

SPECTROSCOPY .......................................................................................................33
4.4 IDENTIFICATION OF PHENOLIC COMPOUNDS BY HPLC ..........................35
4.5 BIOACTIVE COMPOUNDS OF DERIVED CPH FRACTIONS ........................42
4.5.1 Total phenolic content (TPC) ..............................................................................42
4.5.2 Total flavonoids content (TFC)...........................................................................44
4.5.3 Saponins .............................................................................................................45
4.6 ANTIOXIDANT ACTIVITIES OF CPH EXTRACT AND DERIVED
FRACTIONS ...............................................................................................................46
4.6.1 DPPH radical scavenging capacity (DRSC) .......................................................46
4.6.2 Cupric reducing antioxidant capacity (CUPRAC) ..............................................47
vi


4.6.3 Ferric reducing antioxidant power (FRAP) .........................................................48
4.7 ANTIMICROBIAL ACTIVITY OF CRUDE CPH EXTRACT ............................49
CHAPTER 5 CONCLUSIONS AND FUTURE PERSPECTIVES .............................52
5.1 CONCLUSIONS ...................................................................................................52
5.2 FUTURE PERSPECTIVES ...................................................................................52
REFERENCES................................................................................................................ I

vii

LIST OF SYMBOLS
g

: Gram

min

: Minute

mL

: Millilitre

mm

: Millimetre

nm

: Nanometre

s

: Second

W

: Watt



: Micro

viii


LIST OF ABBREVIATIONS
CBS

: Cocoa bean shells

CE

: Catechin equivalents

CPH

: Cocoa pod husk

CUPRAC

: Cupric reducing antioxidant capacity

DPPH

: 2,2-diphenyl-1-picrylhydrazyl

DRSC

: DPPH radical scavenging capacity

EE

: Escin equivalents

EtOH

: Ethanol

FRAP

: Ferric reducing antioxidant power

FTIR

: Fourier-transform infrared spectroscopy

GAE

: Gallic acid equivalents

HPLC

: High performance liquid chromatography

MAE

: Microwave assisted extraction

MeOH

: Methanol

MIC

: Minimum inhibition concentration

ORAC

: Oxygen radical absorbance capacity

RE

: Rutin equivalents

SC

: Saponins content

SFE

: Supercritical fluid extraction

TAA

: Total antioxidant activity

TE

: Trolox equivalents

TFC

: Total flavonoid content

TLC

: Thin layer chromatography

TPC

: Total phenolic content

WSI

: Water soluble index

ix


LIST OF TABLES
Table 2.1. Total phenolic content (TPC) of cocoa by-products ................................... 10
Table 2.2. MAE characteristics .................................................................................... 15
Table 4.1. Physiochemical properties of crude CPH powder ....................................... 31
Table 4.2. Bioactive compounds of crude extract and fractions from CPH ................. 46
Table 4.3. Antioxidant capacity of crude extract and fractions from CPH................... 49
Table 4.4. Antimicrobial activity of phenolic-enriched powder from CPH ................. 51

x

LIST OF FIGURES

Figure 2.1. Classification of dietary phenolic compounds in foods and their general
chemical structures. ....................................................................................................... 7
Figure 2.2. The cocoa fruit structures1and wastes2. Source:......................................... 11
Figure 2.3. Scheme of MAE apparatus. ....................................................................... 15
Figure 2.4. Strategic steps for treating material from plant by targeting phenolics. ..... 17
Figure 3.1. Overall experimental procedure for extraction and assessment of biological
activity of phenolics from CPH ................................................................................... 20
Figure 4.1. TLC of phenolic-enriched extract from cocoa pod husk using 30%
methanol as solvent...................................................................................................... 33
Figure 4.2. FTIR spectrum of phenolic-enriched extract from CPH ............................ 34
Figure 4.5. HPLC chromatograms at 272 nm of mixed standards (A and B) and 8
fractions (C to J)... ....................................................................................................... 39
Figure 4.6. HPLC chromatograms at 272 nm of 6 re-fractionated fractions (A to F).. 41

xi


ABSTRACT
The main objectives of this research were to extract phenolic compounds from
the CPH using already optimized conditions as well as evaluate bioactive compounds
and biological functions of obtained extracts and fractions. Microwave-assisted
extraction (MAE) was used for extraction under optimum condition (50 mL/g, 600 W,
30 min, and 5 s/min) and water as a solvent. The CPH extract fractionation was done
by column chromatography (CC). Qualitative analysis of phenolic compounds was
done using TLC, FTIR, and HPLC. Quantitative determination for total phenolics
(TPC), total flavonoids (TFC) and total saponins (SC) shown in milligram of gallic
acid, catechin and escin equivalents per gram of dried CPH, respectively, together with

antioxidant abilities were analyzed by colorimetric assays coupled with UV
spectrophotometry. The results showed that there was a significant difference
(p<0.05), with higher bioactive compounds in crude CPH extract, 14.96  0.86 for
TPC, 119.17  18.63 for TFC and 451.58  44.09 for SC, compared to the content of
CPH extract fractions. The antioxidation analysis results showed that there was a
significant difference (p<0.05), with CPH extract having higher antioxidation
capacities, DRSC, CUPRAC and FRAP with values of 54.12  0.19, 66.12  1.94 and
76.17  5.00 mg TE/g dry extract, respectively, than values in CPH extract fractions.
FTIR and HPLC analysis indicated that the fractions from the crude CPH extract
contained

some

major

phytochemical

compounds

including

theobromine,

theophylline, and (-)-epigallocatechin gallate. The crude CPH extract antimicrobial
activity was evaluated on two bacterial strains (Bacillus subtilis and Escherichia coli)
and one fungal strain (Candida albicans) by use of agar diffusion method. The extract
showed some traces and 1 mm diameter of inhibition zones against test bacterial
strains and fungal strain, respectively. Hence, this study underline the probable
exploitation of CPH extract as natural source of antioxidant and antibacterial which
could have considerable and valuable use in different domains including food,

cosmetic sector, and pharmacy, to mention few.
Keywords: Cocoa pod husk, microwave-assisted extraction, bioactive
compounds, phenolic compounds, biological activities, antioxidant, antimicrobial.

xii


CHAPTER 1 INTRODUCTION
The cocoa tree (Theobroma cacao L.) is said to be have been first found in
Amazon region of South America, and now its farming has been expanded and vast
plantations are localized in both tropical and subtropical regions. Africa high percentage
of cocoa production share (68%), followed by Asia (17%) and Latin America (15%) [1].
In 2013, 3.9 million tons of cocoa bean, worth around $12 billion, global production was
achieved [2,3]. In general, cocoa market place increase by 3% each year and 40 to 50
million people worldwide benefit from cocoa farming opportunities [1]. It was reported
that not more than 450g of chocolate are obtained from 400 cocoa beans [1], which
means that 10 tons of wet cocoa pod husk (CPH) come from 1 ton of dry beans [4].
Cocoa producing countries generate a lot of by-products considered as waste
[3] , and they are not used but discarded. Considering fresh weight, a great percentage
of fresh cocoa pod is considered to be waste [2,5]. That portion covers cocoa shell
(8.15 ± 0.78%), cocoa pod husk (70.15 ± 0.67%) and cocoa bean pulp (21.70 ± 0.20%)
[6]. Not only the bad smell, but also cocoa pod husk left in the field of cocoa
plantation is associated with environmental problems and disease propagation like
black pod rot [4] and with ches' bloom [7]. Around 6.4 (1.5 times cocoa beans) [2] and
55 million tons (13 times cocoa beans) [8] of cocoa pod shell and cocoa pod husk are
world annual production, respectively. There is a need to exploit those cocoa byproducts. The interest in cocoa by-products comes from their availability and high
phenolic content [1]. Phenolics from cocoa, the same as those of other plants, have
been part of various studies as bioactive compounds [9], and were proven to have
properties which are important to the health like antioxidants,....[10].
In the current trend, the health benefits of phenolic compounds has been topic

of discussion and research [11]. Epidemiological studies have reported that phenolics
are probable candidates for prevention of chronic diseases [5]. Farahmandfar et al.[12]
reported that the phenomenon through which antioxidant ability happens is by redox
reactions that inhibit highly reactive molecules effects. As it was reported, Abdul
Karim et al.[13] has shown that phenolic compounds delayed skin aging and protected

1


it from UV radiations. Grillo et al.[14] showed that because of high fiber and
antioxidants content, the CPH could be used in food and beverages.
Cocoa bioactive compounds are in various categories which include phenolics,
flavonoids, saponins, and many others, and they affect some of the bioactivities [9]. It
was reported that the minimization of waste from cocoa industry by value addition
should be strategic [10]. By use of supercritical fluid extraction of cocoa hulls, a value
of 1.8% TPC was achieved [9], and another process at the optimum conditions using
MeOH for the extraction, 0.52 % yield was obtained [5].
Exploitation of cocoa by-products considered as waste can reduce
environmental pollution and improve the value for both cocoa farming and processing
industry [2]. Valadez-Carmona et al.[5] reported that cocoa waste have a considerable
phenolic content which make them attractive to production of value-added products.
Polyphenol content of 1.09g/100g dry weight was achieved from cocoa shell [6] and
4.6% soluble phenolics in CPH extraction [4]. Extraction of phenolics is very
important and the conditions during process can affect bioactive compounds and their
activities [15]. Md Yusof et al.[10] indicated that its crucial to properly choose an
extraction method as different techniques give different outcomes of bioactive
compounds. Various solvents extracted polyphenolics in the CPH, 8.48 mg GAE/g
total phenolic as the maximum value was obtained by water extraction [6]. The choice
of solvent is important as its physicochemical characteristics impact on amount and
type of phenolic compound [15]. More polyphenolic content, 2-3 times more, were

realized in cocoa bean husk by use of aqueous solvents, than using pure ones [16]. The
process of extracting of polyphenolics has been implemented traditionally way by use
of conventional methods, currently there is a need to adapt novel methods and one of
them is microwave-assisted extraction. Abbas Delazar et al. [17] reported that method
relies on microwaves heating during extraction.
By referring to the previous studies, extraction of phenols in the CPH by
microwave-assisted extraction (MAE) and water serving as extraction medium on
optimized conditions has not been reported.
There are a number of advantages associated with the use of MAE including
does not take long to get the yield, just little quantity of extraction medium is required,
2


very effective and cheap [17], and using water during extraction process is safe and
environmental friendly due to that it can produce clean and safe extracts [18].
In most cases, a traditional technique yields low amount of phenolics when compared
to modern technology [10].
The research main intentions are: (a) extracting and recovering of phenolic
compounds in the CPH using already optimized conditions, and (b) evaluating
bioactive functions of the extracted phenolics from the CPH.
1.1 PROBLEM STATEMENT
So many researches have been conducted and identified natural bioactive
compounds with different biological activities (prevention of oxidation, aging,
cancer,...) in different edible plants. Nevertheless, the use of normal foods to generate
other food components is seen as unprofessional which could also affect food prices,
and consumers will be required to incur more cost [15]. By referring to the trending
demand for maximum valorization of food waste [14], there was an involvement and
willingness to isolate natural bioactive compounds from unused and ignored plant
materials such as peels, leaves and pulps, and worldwide many industries have
embarked in drafting and implementing measures which can minimize process byproducts considered as waste [14]. Additionally, the use of synthetic additives has

been quite long under criticism due to probable undesirable side effects to human
health [19].
Grillo et al.[14] also reported that despite the publication number of patents and
research papers related to food waste value addition strategies, the targeted objective is
still far from being full achieved.
In the cocoa industry, chocolate production requires the use of fresh cocoa
beans, which are fermented and dried, while cocoa pod husk and shell account for high
percentage of cocoa fruit are discarded to the landfill. Every year, there is an increase
in production and processing of cocoa beans which correlate with an increase in waste
reading to the disposal of million tons of cacoa pod husks [4]. Chocolate market was
projected to increase at a 2.3% annual rate, and wastes generated by chocolate industry
were estimated at a 3.1% increase annual rate between 2014 and 2019 [14].

3


Despite being considered to be waste, research has proven some of potential
uses and detection of bioactive compounds. Vriesmann et al.[4] has showed that a
4.6% soluble phenolics was found in cocoa pod husk, and 45.6-46.4 mg GAE of
soluble phenolics were reported by Abdul Karim et al.[20]. It was reported that cocoa
pod husk contains phenolic acid including caffeic acid [15]. It was also indicated that
cocoa pod husk has anti-caries activity [16].
The extraction and valorization of phenolic compounds from cocoa pod husk
which is renewable, sustainable and its availability is in huge quantity source and is of
great importance as it has many applications in different domains (food industry,
cosmetics, pharmacy,...). The proper usage of the cocoa pod husk could decrease their
environmental impact and problems, and contribute to the economic advantages
[4,16,21]. It is required to apply appropriate techniques, which can achieve the
intention of waste value addition, and this needs a shift from conventional methods.
These techniques are associated with so many drawbacks including being hard to

apply, a lot of time of getting for high yield, huge quantity of solvents, and sometimes
degradation of bioactive compounds and vaporization of other important components,
especially aromas and flavours. Non-conventional techniques are able to overcome the
mentioned limitations, hence fit for positive outcomes in terms of projected bioactive
compound quantity and quality.

1.2 RESEARCH SIGNIFICANCE
The outcomes of this research could probably be significant for the potential
different beneficial uses in food industry, pharmacy, cosmetics, etc. In addition, the
information obtained could be the base for further researches. It would also strengthen
valorization of the CPH, which will further reduce the environmental problems and
cocoa plantation diseases spread caused by its discard.
1.3 RESEARCH AIMS
1.3.1 Overall aim
The general intention of the research was to extract phenolic compounds in the CPH
using already optimized conditions and evaluate their biological functions in vitro.
1.3.2 Specific aims
i) To prepare and identify main phenolic compounds in the crude CPH extract
and derived fractions.
ii) To evaluate antioxidant and antimicrobial activities of the crude CPH
extract and derived fractions.
4


CHAPTER 2 REVIEW OF THE LITERATURE
There was a focus of some topics which included cocoa and cocoa wastes,
mainly the CPH, bioactive compounds with emphasis on phenolics, MAE as one of
methods for phenolics extraction, phenolics purification, and then phenolic biological
activities (antioxidant, antibacterial,..)
2.1 COCOA AND COCOA BY-PRODUCTS AS WASTES AND VALUE

ADDITION
2.1.1 Husk of cocoa pod
The fresh fruits of cocoa tree are in various shapes and colours, the tree is fixed
either on main branches or those of aside. Once cocoa is broken down and inner
content known as cocoa beans are removed, the remaining material consist of three
main parts which are cocoa mucilage, cocoa bean shell and cocoa pod husk (Figure
2.2) [22].
Since the CPH has been considered as one of the most important chocolate
processing cocoa pod derivative products ignored [3,4]. The CPH pose a serious
disposal problem, as per the estimations quantity of CPH related to the production of 1
ton of dry beans, are 10 times higher that of dry beans [4,14]. In fact, the CPH is not
used properly and could be handled as unwanted material to be discarded. In normal
case, the activity of removing cocoa seeds from cocoa pods is done directly in the field
and the CPH is not collected, so it decomposes and creates inappropriate conditions to
the environment.
Beyond production of bad smells, the CPH under decomposition in the field
could also spread cocoa tree diseases, and the most common known is black pod rot
[4,20,23,24]. The estimated production loss per year caused by black pod rot could be
of 30 up to 90%, whereas at the global level it ranges between 20 to 30% [25].
Almost 75% of fresh cocoa pod is covered by CPH mass [25], meaning also
that around 750 kg will be considered as material to discard [26,27], and it contains
11.88 g/kg tannins [27]. Even though, there still some issues on value addition of the
CPH, different ways of exploitation have been implemented and are targeting to
minimize the discard of cocoa by-products and this lead to the production of valuable
5


products, used in different domains such as food antioxidants, dietary fibers, animal
feed, etc.
2.1.2 Cocoa bioactive compounds with emphasis on phenolics

Bioactive compounds occur in some foods in little amount and are beyond the
normal nutritional role [28], whereas Rachmawaty et al.[29] defines bioactive
compounds just as food components in little quantity which could be vitally nutritional
or not, and could give important properties to life. Phenolic compounds in plants
(around 8000 types) [30,31] are secondary metabolites, often produced in plant
subsequent phase to growth. Shikimic acid and malonic acid pathways are channels for
phenolic compounds generation and this induce to define the terms phenolic and
polyphenol as all secondary natural metabolites which are end-products to shikimate
phenylpropanoids-flavonoids pathways by biogenetic [32].
For Vriesmann and de Oliveira Petkowicz, and Dias et al.[32,33], "phenolic"
or "polyphenol" as a chemical definition, is a chemical material which the hydroxyl
replacements attached to the benzene ring are one of esters, methyl ethers, glycosides
and others, and is called a phenol when one hydroxyl is replaced and polyphenols
when more than one are replaced. Lecumberri et al.[34] defines phenols as a big
group of chemicals that their hydroxyl moieties are attached on aromatic ring, and
numerous categories of these compounds exist. More than 4000 known phenolic
compounds are flavonoids, one of the main phenolics found in plants. Flavonoids
chemical structure consist of C15 which in positions of 6-3-6 carbon atoms. The
main classification of flavonoids comes from different substituents to C3 in the
middle of two aromatic rings [33] (Figure 2.1).

6


Figure 2.1 Classification of dietary phenolic compounds in foods and their
general chemical structures. Source: Andrea and Cano[27]
Generally, the main objectives of the techniques applied when extracting
compounds with bioactivities among many others are: (a) to obtaining bioactive
compounds of target function in a plant under study, (b) increasing the efficacy of

techniques used in analysis, (c) boosting bioassay sensitivity by the increase the
interested compound concentration, (d) obtaining converted bioactive compounds
which are much applicable due to they are easy to sense and extract, and (e) providing
a stable technique which is not affected by changes in plant under study [35]. Cocoa
has been related to several health and technological benefits due to phenolic fraction in
its composition. Of those, phenolic compounds and procyanidins have been ranked the
most important subgroup under flavonoids classification [30].
Cocoa bioactive composition is made of various group of compounds and those
include phenolic acids, flavonoids, saponins and their derivatives and other small
7


fractions constituents [9]. Among many phenolic compounds identified in cocoa byproducts, procyanidins emerge as the most abundant and its content is functional of
where it orginated and production status [36]. Cyanidin-3-galactoside as well as
cyanidin-3-arabinoside as anthocyanins at a concentration of 0.02 to 0.4% dry mater
together with epicatechin and catechin as polyphenols at a level of 2 to 4% dry mater,
these compounds were detected in cocoa bean flour, which its fat content was
removed. The procyanidins content level obtained after extraction is various dependent
of some intrinsic and extrinsic factors during extraction process. Its probable to work
under maximum conditions which give higher percentage of end-product, and
methanol-water (8:2 v/v) was proposed to be suitable mixture in extracting coloring
chemicals in cocoa pod shell samples [2]. In comparative study done by Azila Abdul
Karim et al.[13] in Malaysia on antioxidant properties of cocoa pod husk and shell, it
was noticed that between them, cocoa pod shell was lower in antioxidation by DPPH
scavenging ability, but both were below antioxidation shown by gallic acid, a phenolic
used as standard, and the CPH was much higher in antioxidation by measured by
FRAP method than cocoa pod shell.
Different plants have shown to have extracts with bioactive functions. For
example, extracted compounds from P. amarus were possibly active in healing some
of the health ailment including hepatitis, plasmodia, inflammation, malaria,

diabetic...Those functional properties observed are much related to the biochemical
constituents in extracts [37]. Another plant, the artichoke (Cynara scolymus L.) has
phenolic compounds which have shown bioactivity through their antioxidation,
antifungal, and antimicrobial activities [38]. The presence of phenolic compounds with
biological activities, in Oxalis corniculta L. was also confirmed [39]. Cocoa and its
by-products have high content of phenolic compounds and many studies have been
conducted so far due to some properties of their phenolic content like antioxidant and
antiradical activities. Cocoa bean hull has pigments and bioactive compounds with
distinct biological activities, and this has made cocoa bean hull to be a more attractive
material for extraction. By use of the most common method for total phenolic content
evaluation using Folin Ciocalteu reagent, a 1.8% TPC in average was realized [9].
In general, it was reported that the extracts with high total phenolic contents
would be expected to exhibit much of oxidation and microbial inhibitions hence their
8


effects will be profound when used as food components [19]. In a study of evaluating
antioxidantion abilities of cocoa and its by-products, Martínez et al.[24] reported a
strong correlation (R2 > 0.95) was observed between TPC and all antioxidation activity
assays (DPPH, ABTS and FRAP). A study conducted on cocoa liquors to evaluate its
phenolic compounds level and their structure and antioxidation capability has realized
that antioxidation obtained was much related to the quantity of phenolics (coefficient
of determination was greater than 0.9), this allowed the suggestion that the dominant
constituents which contributed to the antioxidantion achieved were having a
characteristic of polyphenolic structure [31]. Tiburcio [40] also confirmed a very
corresponding ratio of phenolics level to antioxidation effect was observed. Sotelo et
al.[31] has reported that alteration of the quantity and the quality of the polyphenols in
the food matrix is a result of modulation of the polyphenolics production synthesis of
phenolic compounds. The unique chemical structure will determine specific
antioxidant capacity of each type of substance, and those alterations in the type of

phenolic compounds could change the ability of inhibiting oxidation for the particular
food being analyzed.
2.1.3 CPH applications and phenolic compounds
The CPH value has many fields of applications. It has been reported to be the
main ingredient in many of value-added products (animal feed, soap...). Other
biotechnological opportunities for CPH value addition are its use to make fuels and as
functional ingredient in food [22].
The good proximate composition (protein around 6%, crude fat up to 10%, fiber
around 4%) of CPH has attracted the attempt to consider it as an important ingredient
to be incorporated in feed of animals [22], but theobromine presented at high
concentration has a detrimental effect on animals [3]. Though, the chemical
composition of the CPH is complex but one of chemicals contained is phenolic
compounds [41]. Due to the availability of cocoa pod husk, its important to exploit
them and generate compounds with potential functional activities to be used in food
industry and pharmacy [4].

9


Table 2.1 Total phenolic content (TPC) of cocoa by-products
Cocoa by-products
TPC (mg GAE/g)

shell of

husks of

Pulp

bean (%)

pod (%)

(g/100g DM)

197

46–57

104

Source: Haydeé et al.[22]

DM: Dry mater

As per reports from Lu et al., and Rachmawaty et al.[25,29], phenolic acids
content in the CPH was between 0.46 and 0.69 g GAE/g, whereas the TPC of fresh
CPH reported was around minimum of 2 and maximum of 3 mg GAE/g [25], and
45.6-46.4 mg GAE/g of soluble phenolics were reported by Abdul Karim et al.[20].
For Martínez et al.[24], a range of 2.07 and 3.65 mg GAE/g was established as the
TPC value of the CPH, whereas range of 16.40-23.01 mg GAE /g was realized by
Sotelo, Alvis and Arrázola [42].
Phenolic compounds were detected during the study of rheological properties of
pectins in the CPH determined under various treatments [43]. It was indicated that the
proportion of cocoa bean and cocoa pulp of fresh cocoa pod fruit was 23% and 26%
and that of the CPH was the more than the double of the two above [44]. In general, in
plants, polyphenols are found in most external parts [4,14,45]. The value addition of
cocoa by-products which are discarded, has been discovered by referencing on cocoa
beans [14] (Table 2.1).
A study conducted on polyphenolic compounds and their antioxidation in the

extracts from some plants has established higher concentration of polyphenols in fruit
peels than pulp, reaching up to twice the amount found in pulps of bananas and
tangerines, which also reflected in higher oxidation inhibition power [31]. The flesh of
kiwifruit was less in polyphenolics and flavonoids content than its pericarp, also the
content of peels and reflected biological functions including antioxidation and
bacterial inhibition were much more than that of the flesh [46]. Cocoa pod has got
three successive parts, the one out known as epicarp, the intermediate part known as
mesocarp, and the one inside known as endocarp [22]. Depending on the clones, at the
ripening, there is variation in thickness and color, green to red at the ripening stage.
Those changes could reflect the maturity stages and be associated with accumulation
of bioactive compounds at different levels [47].
10


Figure 2.2 The cocoa fruit structures1and wastes2. Source: Campos-Vega et al.[47]

A number of conditions can impact on cocoa phenolic compound composition
and those include environmental associated factors like where the cocoa originated,
growth stage, factors in production process like high or low temperature, use of
chemicals, and storage associated factors [22]. Procyanidins have been identified as
the most abundant polyphenol group in cocoa [48]. A variation in polyphenolic
compounds of cocoa derived products has been reported. Cocoa powder had a higher
range of TPC (0.33-6.5%) than dark chocolate (0.17-3.65%) [45], whereas in the
cocoa pod shell, range of polyphenolics was 1.3-1.8%, which include some of flavan3-ols, little quantity of anthocyanins and flavonols [14].
Yapo et al.[23] reported that during the CPH compounds extraction, the four
step sequential method was used whereby fractions in hot aqueous ethanol, cold
aqueous acetone and luke warm water were considered as soluble phenolics, and
fractions in hot acidified butanol as insoluble portion. From the study results, TPC
from husk of “fresh” cocoa pod has been realized to be > 6.9%, higher than the range
2.0 to 3.0% of both cocoa bean hull and kernel products under study, and which have

undergone fermentation and roasting. The above results suggest that the CPH was rich
in proanthocyanidins compared to other by-products from fermentation and roasting
treatments. It was found that condensed tannins were lower in the CPH samples than
the other two cocoa by-products, and it could have been the results of chemical
changes, the most important being phenolics converted into insoluble products, when
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kernel was fermented. Also, the insoluble portion was increased by end-products of
Maillard reaction, which occurred in the presence of heat. Discovery for the outcomes
of oxidation inhibition ability evaluation that active radicals were scavenged by
soluble phenolics from the CPH compounds at the level of more than 85% compared
to the maximum scavenging ability of 68.0% realized by both kernel and hull
products. This showed that antioxidation measured by scavenging ability was higher in
the CPH products than in kernel and hull products. Additionally, the calculated EC50
of kernel and hull products with a maximum value of 55.0 g/g was two times higher
than that of CPH products. The same trend was also obtained by scavenging ability
evaluation by ABTS method. Abdul Karim et al.[20] reported that in two different
studies, TPC in the CPH varied between 45.6-46.4 mg GAE of soluble phenolics and
55.93 - 57.07 mg GAE per g.
2.2 IMPACT OF PROCESSING METHODS ON PHENOLIC COMPOUNDS
AND ANTIOXIDATION POTENTIAL OF CPH
Among some of extraction processes having an impact on the CPH phenolics
content and antioxidants activity have been published. Though, Abdul Karim et al.[20]
reported that the CPH total phenolic content with a maximum value of 57 mg GAE/g
dry mater but minimum value of TPC of around 4 mg GAE/g dry mater was realized
in function of sample origin and type of solvent. Campos-Vega, Nieto-Figueroa and
Oomah [47] found that much of TPC (3.5 mg GAE/g) was obtained in the CPH
samples from Cone region (Ecuador) when extracting the CPH by MeOH-acetone,
compared to 2.0 mg GAE/g realized by ethanolic extraction. The same trend was also

observed for its antioxidation capacity. The CPH extract was gained by two different
solvents by maceration method, in which 70% acetone was found to have extracted
phenolics content almost double times (around 95 mg GAE/g) as compared to ethanolwater (7:3 v/v) at 50 mg GAE/g) [41]. In addition, the milling method has been proven
to probably impact on the CPH phenolic content. When the CPH powder under oven
drying of less than one millimeter was treated by dry-milling, around 5% of phenolic
content by dry weight was achieved, but for those of less than one point seven
millimeter, this gave a 7% phenolic content when extracted [22].
The use of microwave as a novel method may protect phenolic content and
increase the extraction yield, but it should be noted that each method has its effect.
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Some of processing methods, which affected phenolics and antioxidants of CPH were
reported. It was found that in comparison to hot air and freeze-drying methods, drying
of CPH using microwave achieved higher TPC and other phenolic compounds. For
example, during the CPH extraction at the power of 595 W for 11.5 min, 3.4 higher
times TPC content was achieved.
Drying by use of microwave is recommended as it can improve on the
extraction of bioactive constituents [47]. Though, generally freeze drying has been
performed well than drying by air in terms of affecting extraction of phenolic
compounds, but its implementation should be done careful to avoid negative impacts
on composition of extracts, which could lead to the reduction of phenolic content [49].
In another report, it was shown that there was a considerable increase in TPC when the
CPH treated with acetic acid was sun-dried, compared to those were not treated. Acid
treatment was suggested to be important in inhibiting oxidation caused by PPO [50].
Furthermore, Daniel Oduro et al.[42] indicated that higher phenolic extracted by
ultrasound with maximum TPC of 23 mg GAE/g compared to the maximum TPC of
19 mg GAE/g obtained when extraction was done by agitation process.
The TPC of dry CPH extracted by ethanol solvent (5:5 v/v) reduced by 42%
when samples were autoclaved at 120oC but a 37% more TPC was achieved when the

CPH samples were fermented by a fungus strain (Rhizopus stolonifera NRRL 28169).
It is believed that fermentation has caused conversion of some molecules into new
products, which contributed to the increase of phenolic content [51]. Shalini [52]
found the same way that antioxidation ability was increased when the CPH was treated
by a different fungus strain (Rhizopus stolonifera LAU 07). However, Peralta-Jiménez
and Cizares-Macías [51] found that there was much of phenolic content in the
extract from the CPH without any treatment than those found in the CPH, which has
undergone fermentation and the latter has higher antioxidation ability measured by
scavenging capacity of DPPH and reduction of ORAC. The observed outcome is
assumed to be molecular changes of polyphenolics.
A study, where the CPH was treated by a fungus strain, more than 11 and 89%
decrease in phenolic content and tannin content was observed, respectively. The
decrease could have been the results of some compounds decomposition which were
then used by fungus [53]. Observed rise in phenolics of the CPH after fermentations is
due to the phenolics migration induced from the inner parts of cocoa pod, which is the
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