International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:16 No:02 16

Microencapsulation of Green tea Extracts and its

Effects on the Physico-Chemical and Functional
Properties of Mango Drinks
Zokti James, A1. Badlishah Sham Baharin1*, Abdulkarim S. M.2, Faridah Abas2,
1
Department of Food Technology, 2 Department of Food Science, Faculty of food Science and technology, University Putra, Malaysia
UPM Serdang 43400 Selangor Malaysia
1 Zokti James, A (zoktii2002@yahoo.com).
2* Badlishah Sham Baharin (badli@upm.edu.my)
3 Abdulkarim S. M. (karimsabo@upm.edu.my)
4 Faridah Abas (faridah_abas@upm.edu.my
Abstract-- Green tea polyphenols have been reported to possess among functional foods because of the health promoting
many biological properties. Inspite of the many potential benefits characteristics. Functional foods plays the role of prevention
of green tea extracts, their sensitivity to high temperature, pH because of the ability to reduce those factors that compromise
and oxygen is a major disadvantage towards its effective good health (Shibamoto, Kanazawa, & Shahidi, 2008).Green
utilization in the food industry. Green tea leaves from Cameron tea catechin has been incorporated into various foods
highlands Malaysia were extracted using SFE. To improve the
including, bread, cakes biscuits, yoghurts , meat pastries etc.
stability, the green tea extracts was encapsulated by spray-drying
using different carrier materials including maltodextrin (MD), (Anandharamakrishnan, 2014; Yilmaz, 2006). However, green
gum arabic (GA) and chitosan (CTS) and their combination at tea catechins polyphenols have been reported to be susceptible
different ratios. Encapsulation efficiency, total phenolic content to degradation/ epimerization at certain temperature and pH of
and antioxidant capacity were determined and were found to be the environment which reduce the efficacy of the polyphenols
in the range of 71.41 - 88.04%, 19.32 - 24.90 (g100GAE), and 29. (Lun, Kwok, Huang, & Chen, 2003; Peters, Green, Janle, &
52 -38.05% respectively. Further analysis of moisture content, Ferruzzi, 2010). Green tea polyphenols are astringent, bitter
water activity, hygroscopicity and bulk density of the and acrid in taste which sometimes poses problem of
microparticles were carried out and the results ranged from; acceptability by the consumer (Narukawa et al., 2011). The
3
2.31 – 5.11%, 0.28 – 0.36, 3.22 – 4.71% and 0.22 – 0.28g/cm ,
therapeutic value of green tea presents an opportunity to
respectively. The ability of the microparticles to swell in SGF and
SIF was determined at 142.00 -188.63% and 207.55 -231.77% positively affect the outcome of risk of disease globally.
respectively. The microparticles with the best catechin efficiency, However, reports showed that the daily consumption of tea
total phenolic content and antioxidant activity were selected and beverage and bioavailability of native catechin monomers is
incorporated into mango drink and its effects on the less than 2% (Henning et al., 2004; Henning, choo, 2008).
physicochemical and functional properties of the product Effective administration of tea catechin polyphenols in the
evaluated during storage. The pH, total solid and viscosity of the body may require the use of appropriate vehicles to achieve
catechin microparticles supplemented range from 5.15 – 4.38, the required dose. Moreover, it is not certain if individual can
12.70 – 14.20%, and 12.55 -13.99(cps) respectively. The stability consume sufficient quantity of tea infusion that will provide
of green tea bioactives in the mango drink showed degradation
the anticipated health benefits in the conventional traditional
rate in the range of 16.47 – 29.72% compared to the non-
encapsulated powder (46.46%). The green tea extracts beverage. It is reported that a health conscious consumer may
microparticles show good degree of stability in the mango drink need to drink 10 cups (2000mL) of green tea in Japan to
and the antioxidant capacity of the mango drink was significantly maintain good health. If 200mL contains 100mg of tea
( p ≥ 0.05) different compared to the non-encapsulated powder catechin, therefore 10 cups of tea is equivalent to 1g of tea
at the end of the storage period. catechins (Hara, 2011). If we then go by the Japanese
Index Term-- Green tea catechins; Microencapsulation, recommendation the volumes of tea drinks appears too much.
Functional food; Mango drink; Stability The burdens of consumption of high water can be reduced by
simply turning the liquid into powder and then incorporate
1.0. INTRODUCTION into other foods which appear to be the current trend in the
Tea is an infusion of Camellia sinensis only second to water in food industry.
term of consumption globally. Green tea is a product from
non-fermented tea leaves as compared to black or oolong teas. Microencapsulation technology has been used as a successful
Epidemiological data have shown that consumption of tea has delivery tool in the pharmaceutical and food industries to
an inverse relation with reduced risk of certain chronic and protect and deliver heath ingredients to the consumers
degenerative diseases including certain forms of cancers, (Gouin, 2004; Taylor, Mozafari, Khosravi-darani, & Borazan,
cardiovascular diseases, diabetes, obesity, Alzheimer disease, 2008). Microencapsulation has enable formulators of
weight loss etc. (Basu & Lucas, 2007; Khan & Mukhtar, functional food ingredients to protect the stability and bring
2007). The major constituents of green tea catechin sustained release of polyphenols ( Lee, Ganesan, & Kwak,
polyphenols include catechin (C), epicatechin (EC), 2013; Lee et al., 2013; Oliveira, Santana, & Ré, 2006; Tang et
epigallocatechin (EGC), epigallocatechin gallate (EGCGC), al., 2013).
epicatechin gallate (ECG) and gallic acid. Tea has been listed
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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:16 No:02
Spray drying is an established technique that has been used for
microencapsulation of bioactive compounds (Gharsallaoui,
Roudaut, Chambin, Voilley, & Saurel, 2007) Spray drying has
been exploited by food industries because of the following
advantages: It is viewed as an already established technology,
has the ability to produce large amount of micocapsules, there
exists many approved shell materials for use or application,
can produce particles sizes of different variety, and is use for
food ingredients that are heat sensitive(Gharsallaoui et al.,
2007; Sansone et al., 2011).
Maltodextrin is one of the wall materials that is commonly use
in the industry for encapsulation of functional food ingredients
because of its unique characteristics (Cai, Luo, Sun, & Corke,
2004; “Gustavo , Barbosa- Canovas, Enrique Ortega-Rivas,
Pablo Juliano and, Hong Yan (2005).
Gum Arabic is a coating material derived from two species of
Acacia tree, Acacia sayel and Acacia Senegal. Gum Arabic is
primarily used as a stabilizer in food industry. It is a good
emulsifier with film forming properties and low viscosity in
aqueous solution which help spray-drying (Krishnan,
Kshirsagar, & Singhal, 2005; Wandrey, Bartkowiak, &
Harding, 2010). Gum arabic is edible completely and
resistance to a number of physicochemical conditions such as
acidic conditions when compared to others which is why is
good for microencapsulation green tea beverage being more
stable in acidic condition (de Vos, Faas, Spasojevic, &
Sikkema, 2010). Chitosan is a non- toxic biopolymer that is
biodegradable and biocompatible with many potentials for
use in biotechnological applications. Because of the cationic
and reactive functional groups chitosan is used in controlled
released formulation in pharmaceutical industries (Dudhani &
Kosaraju, 2010; Zhang & Kosaraju, 2007). Chitosan has
proved to be valuable in wine and juice industry. Recently
polyphenolic extracts from olive leaf was encapsulated by
spry drying using chitosan coating with high loading
efficiency compared to particle made from extrusion (Dudhani
& Kosaraju, 2010; Kosaraju, D’ath, & Lawrence, 2006). In
this study catechin is expected to serve as the main health
promoting agent, however, because chitosan has shown some
beneficial health properties its inclusion as a carrier material
will be of relevance. Encapsulation of catechin extracts using
maltodextrin and gum Arabic along with chitosan is a novel
approach.
One of the approaches to increase the bioavailability of
catechin it to administer tea in combination with fruit juices
(Ferruzzi, 2010; Henning et al., 2004, 2005; Henning SM,
choo JJ et al., 2008). Mango is one of the most popular
tropical fruit commonly called ‘king of fruits’ especially in
Asia. Very many high quality mango clones are found in
Malaysia. A major portion of mango is consumed as fresh
fruit locally. Mango possesses high nutritional potential
because of the relatively high β- carotene contents (pro-
vitamin V) and vitamin C apart from other antioxidant
compounds (Ribeiro, Barbosa, Queiroz, Knödler, & Schieber,
2008).
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Malaysia is blessed with variety of fruit and herbs including
tea (Arifullah, Vikram, Chiruvella, Shaik, & Abdullah Ripain,
2014). An attempt has been made to incorporate a Malaysian
local herb- mascotek (Ficusdeltoidea); known to possess high
antioxidant properties into mango drink in order to create
different way of enjoying the freshness and goodness of
mango (Halim-hasserihalim@gmail.com, 2014). One of the
objectives of this study is to produce an enriched mango drink
by incorporating encapsulated green tea extract powder into
mango drink and to evaluate the stability of the catechin
compounds during storage. Tea is produced in Malaysia;
therefore the outcome of this study will serve as an incentive
to tea producers and consumers of tea in Malaysia.
2.0 MATERIAL AND METHODS
2.1. Materials
Fresh tea leaves were obtained from Cameron highlands
Malaysia. Mango drink was purchased from the pilot
processing plant at the faculty of food Science University
Putra Malaysia. Maltodextrin (DE 10- 16), gum arabic,
chitosan were purchased from Scinfield chemicals (Malaysia),
and other chemicals were of analytical grade.
2.2. Microencapsulation of green tea
Green tea extracts was obtained through supercritical carbon
dioxide extraction following the method of Ghoreishi &
Heidari, (2013).
2.3. Preparation of Carbohydrate microcapsules
To find an appropriate formulation for wall material for the
encapsulation of green tea extracts obtained from the
supercritical fluid technique, a simplex centroid mixture of
experimental design based on previous studies (Vaidya,
Bhosale, & Singhal, 2006) was used. A blend of 40g (20%
w/v), commercial maltodextrin (MD), gum Arabic (GA) and
chitosan (CTS) were dispersed in distilled in water. Individual
wall materials were dissolved individually at (60-40oC) with
constant magnetic stirring for 30 minutes to give a final
volumes of 200ml with a total solid of between 18 -21 (oBrix).
Chitosan was separately dissolved in 5% acetic acid before
adding under magnetic stirrer and mixed properly. Two grams
(5% based on wall material used) of supercritical fluid extract
powder of green tea was added to the mixture. The mixture
was homogenized using a mini homogenizer (Model type-SP-
8 Malaysia) for 5minutes at 8000-9500 rpm until complete
dispersion was achieved. The slurry of the water, carrier
material and catechin extracts was spray dried in Buchi, 290
mini sprays dryer (Buchi- Switzerland) equipped with 0.7mm
diameter nozzle at an outlet temperature of 150±5oC, the inlet
temperature was determined by the outlet temperature. The
feed flow rate was 15ml/min. The microparticles prepared
were collected using the glass collecting chamber. The
microparticles powder were filled in aluminium pouches
immediately, sealed and kept in desiccator to cool and to
prevent absorption of moisture until further studies.
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2.4. HPLC analysis of catechins
The five major catechin phenolic compounds present in green
tea were analysed by HPLC using the method of Institute
for Nutraceutical Advancement for determination of total
catechins and gallic acids in green tea (INA Method 111.002)
as used by (Perati, Borba, Mohindra, & Rohrer, 2011) with
minor modifications was followed., A reversed-phase C18
column (MetaChem PolarisTM Amide C18, 5µm, 4.6x250mm);
UV-Vis diode array detector, and a binary solvent system
containing acidified water( 0.1% orthophosphoric acid -
solvent A) and a polar organic solvent ( acetonitrile -solvent
B). Optimized gradient elution order was programmed as
follows: 0 min 96% A: 4% B; 12 min 85% A: 15% B; 22 min
75% A: 25% B; 24 min 85% A: 15% B; 30 min 85% A: 15%
B and 35 min 96% A: 4% B. The flow rate and injection
volume were set at 1mL/min and 10µL respectively with a
post run of 5minutes.The column temperature was 35% while
the wave length was at 280nm using UV detectors. Five stock
solutions of the various catechin standards were prepared by
dissolving 1mg of the individual standards catechins in 1.0mL
solvent to form a 1000 parts per million (ppm) concentrations
and stored in the refrigerator at 40C.
A specified amount of each solution was taken and the 5
aliquots were mixed and diluted to give a wide range of
standard mixtures. The concentration of each catechin in the
green tea extract was determined quantitatively based on the
chromatographic data of the standard mixture. The catechin
components were quantified using the calibration curves of the
standards obtained (Fig.7a).
2.5. Determination Microencapsulation yield
The method of (Robert et al., (2010) with some modification
was adopted for the determination microencapsulation
efficiency..
Total catechin of both surface and entrapped core material was
determined. A 200mg of green tea catechin encapsulated
microparticles was weighed accurately into a test-tube and
2mL of 50:8:42 (v/v/v) of methanol: acetic acid: water was
added. The dispersion was agitated using vortex for 1 min and
ultra-sonicated for 20 min. The supernatant was centrifuged at
9500rpm for 10 min and then filtered using syringe filter
0.45µm. To determine the surface catechin polyphenol 200
mg of the microparticles was treated with a mixture of 2mL
ethanol and methanol (1:1) it was vortexed for 1minutes and
was then filtered using 0.2µm Millipore syringe filters. The
amount of total catechin, TPC and DPPH were quantified. The
surface bioactive compound (SBC) percentage and the
microencapsulation efficiency (ME) of the micro-particles
were calculated according to the equation;
SBC = surface bioactive compounds Theoretical total
bioactive x100 (1)
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The microencapsulation efficiency (ME) = 100 – SBC (%)
(2)
2.6. Determination of degree of swelling of microparticles
The methods of Dudhani & Kosaraju, (2010); Oliveira,
Santana, & Re, (2005) with some modification was adopted.
Pre-weight catechin microparticles (200mg) were placed into
a previously soaked visking tubing dialysis bag (2 inf.
Diameter 14.3/ 26 mm pore size 25 angstroms WMCO 1200 –
16000 Dalton, UK) were aseptically placed in to a beaker
containing 150ml simulated gastric and intestinal fluid (pH
2.3 and 7.4 respectively). The content was stirred continuously
at 50rpm and was allowed to swell for 120 minutes during
which the swelling samples were periodically weighed at
interval (0, 20, 40, 60, 80, 100, and 120). The percentage
degree of swelling of the catechin microparticles was
gravimetrically determined using equation below:
Percentage (%) swelling = (Wt – WO) / WO x100
Where Wt represents the weight of swollen sample at the
stipulated time in the simulated fluid and the initial weight of
microparticles before swelling is WO.
2.7. Determination of Physicochemical properties of food
system (mango juice)
The following physicochemical properties of the mango drink
were determined including; pH, total solid, viscosity, TPC,
DPPH and total catechins contents.
To determine the stability of catechin in in mango drink,
various concentrations of the microcapsules (0.5, 1.0, and 2.0
%) containing catechin concentration in the range of 40.65 to
63.42µg/g were added into the mango juice in triplicates and
was mixed properly for 5 minutes at room temperature to
represent day 0. Four batches representing week, 1, 2, 3, and 4
were prepared and was stored at 4oC in a refrigerator. A 1mL
Sample from mixture of catechin microcapsules and mongo
drink was diluted with an equal volume of 70% methanol (v/v)
and was centrifuged at 4500rpm for 10 minutes. The
supernatant was collected using 0.2µm syringe micro-filters
and the amount of catechin released from the microparticles
was analysed for TPC, DPPH and total catechin (TC) using
UV- Vis spectrophometer and HPLC methods respectively.
All samples were prepared in triplicates.
2.7.1 Determination of pH
The pH of the juice both blank and the one that catechin
microparticles powder has been added was determined by pH
meter (pH 700 EU TECH, Instruments) at 7days intervals for
thirty days.
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2.7.2 Determination of Viscosity
Measurement of viscosity were done using a rheometer (
physical – Rheolab) with spindle No. 2 at 1550 s-1 shear rate
over a period of 1200 second at temperature of between 23-
250C and the viscosity means was recorded over the period.
All samples were measured in triplicates.
o
2.7.3. Determination of soluble solids ( Brix) of feed
A digital handheld refractometer (32 Atago Co, Ltd Tokyo
Japan) was used to determine the soluble solids (%). The
refractometer was cleaned properly and calibrated using
deionized distilled water (0 oBrix) before measurement was
carried out. To measure the soluble solids oBrix a drop of
mango was added to the refractometer at room temperature
(24oC) before taking the reading. Measurement was carried in
triplicate.
2.7.4 Determination of total phenolics compounds (TPC)
The amount of total phenol in the microparticles was
estimated colorimetrically following the methods used by
International Organization for standardization ISO14502-
1(2005) and Robert et al., (2010) adopted.
2.7.5. Antioxidant activity determination
Antioxidant activity was determined by the catechins capacity
to scavenge stable DPPH radicals following the method of
(Rutz et al., 2013) with slight modification. Briefly, 1mL of
mango drink was extracted using ethanol (70% v/v), 100µLof
the supernatant was added to 3.9mL of ethanolic solution of
0.1 mM DPPH. The mixture was incubated at room
temperature (25oC) in the dark for 30 minutes. Sample
absorbance (AS sample) was measured using
spectrophotometer (Thermo Fisher Scientific-GENESY 10S
UV.VIS, Malaysia) at 517nm against absorbance of ethanol
blank (AB). Samples were determined in triplicate. The
radical scavenging activity was calculated using the formula:
% Inhibition = [(AB – AS /AB)] x 100.
Where: AB is absorbance of blank; AS is the absorbance of the
sample of green tea catechin microparticles.
2.7.6. Formulation of green tea catechin/ mango juice
Drink system
Bottled mango drink packaged in 100mL volumes prepared by
the Department of Food technology, Faculty of food science
and Technology University Putra for commercial purpose
were obtained in June, 2015. The drink was prepared by
diluting the concentrate to a ratio of 1:3 concentrate to water.
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The formulated drink contains the following per 100ml:
energy -33kcal, carbohydrate -8.1g, protein -0.1g, fat-0.0g,
total sugar- 8.0g, vitamin C -1.1mg, and fibre -0.1g. Batches
of supplemented mango drink samples were prepared by
incorporating powdered green tea microparticles into a pre-
weighed mango drinks before storage at 4oC for 4weeks.
3.0. RESULTS AND DISCUSSION
3.1. Microencapsulation Efficiency (ME)
Microencapsulation efficiency is carried with the sole aim to
determine the loss of core material before, during and after
processing microparticles. It is the percentage of entrapped
catechin over the total catechin in the system. The efficiency
microencapsulation and total catechin compounds are
presented in Figure1 and Table3.1. The encapsulation
efficiency of the total catechin as well as the summary of TPC
and DPPH scavenging activity of the spray dried
microparticles. The encapsulation efficiency was in the range
of 71.41 -88.04%, whereas the total catechin contents was in
the range of 7046.70 -8687.90mg/100g. The formulation with
100 per cent gum Arabic produced microparticles with highest
encapsulation efficiency (88.04%). The results of total
phenolic compounds and antioxidant capacity are presented in
Fig.2 and 3. The total phenolic compounds of the
microparticles were in the range of 24.90 -19.32 g/100gGAE
whereas the antioxidant capacity was in the range 29.52 -
38...05 -71.99%. However, the formulation with 25:74:1
(maltodextrin: gum arabic: chitosan) gave the lowest catechin
microparticles encapsulation efficiency (71.99%) but
produced the highest total polyphenolic compounds (24.74g/
100g GAE) and DPPH activity of 38.05% respectively. This
can be attributed to the combined antioxidant effects of gum
arabic, chitosan and green tea catechin. Antoxidative effect of
chitosan has been reported (Dudhani & Kosaraju, 2010;
Muzzarelli, n.d.; Shahidi,Arachi, 1999). Encapsulation
efficiency can be affected by the nature of wall materials and
the that will in turn influences the antioxidant activity
(Ezhilarasi, Indrani, Jena, & Anandharamakrishnan, 2013).
The physicochemical properties of the spry dried
microparticles were evaluated shown in (Table3.2). The
moisture content, water activity, degree of hygroscopicity,
bulk density, and tap density were in the range of 2.31-4.78%,
0.36-0.28, 3.22 -4.94%, 0.20 -0.28g/cm3 and 0.25g/cm3
respectively. These properties showed that the powders were
of better quality characteristics (Şahin Nadeem, Torun, &
Özdemir, 2011). Table 3.3 shows the five major catechin
compounds that were isolated by HPLC in the spray dried
green tea microparticles. The major catechin compounds
associated with antioxidant properties of catechin are C, EC,
EGC, EGCG, GCG and ECG. The results showed significant
(p ≤ 0.05) variation among elements within a column. The
result of the percentage individual catechin compounds
isolated in this study reflects previous studies by other
researchers on similar subject (Vuong, Golding, Nguyen, &
Roach, 2013; Vuong, Q.v., Nguyen, V., Golding J.B., Roach,
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Vuong, Golding, Nguyen, & Roach, 2010). Similarly, the
results of the antioxidant properties of green tea catechin
obtained after microencapsulation was also comparable to
similar works reported by other researchers (Bakowska-
Barczak & Kolodziejczyk, 2011; Peres et al., 2011). This
study supports the assertion that the antioxidant properties and
the efficacy of green tea catechins is attributed to the presence
catechin epimers and that encapsulation by spray drying using
carbohydrate polymers as carriers can be used to retain the
important catechin epimers after spray drying and storage.
Previous reports indicated good encapsulation efficiency of
catechin compounds when maltodextrins and gum arabic were
used as carrier materials for spray drying phenolic compounds
(Davidov-pardo & Arozarena, 2013; Taylor, Vaidya, Bhosale,
& Singhal, 2007; Zhang & Kosaraju, 2007).The low retention
of catechin added chitosan in this study could be a result of
emulsifying capacity in the feed solution and the interaction
that might have taken place between the core materials and
chitosan (Dudhani & Kosaraju, 2010; B. F. Oliveira et al.,
2006).Although the intention of this work was to use catechin
antioxidant as the main therapeutic material, because chitosan
has been reported to have health beneficial affects it very
possible that utilizing chitosan to deliver catechin may be of
added health benefits to the consumer. This justifies the
relevance considering chitosan-based food delivery system.
3.2. Swelling studies of microparticles
It was anticipated that the release of microparticles may occur
by swelling and degradation. Since the particles were prepared
as food ingredient, release of catechin was characterized in
condition mimicking digestion in both the gastric (pH 2.3) and
intestinal (pH7.4) conditions. The microparticles of green tea
from the formulated wall materials demonstrated swelling of
142.00 – 188.65% over a period of 120 minutes in the SGF (
Fig.3.4a&b); whereas the swelling index in simulated
intestinal (SIF) was in the range 207.55 -231.77%
(Fig.3.5a&b). There was a significant difference (p ˂ 0.05) in
swelling index of microcapsules containing chitosan on SGF
and SIF when compare to others without chitosan blend.
Swelling of microparticles containing high ratio of
maltodextrin reached equilibrium within the first 40 minutes,
whereas blends of maltodextrin /gum arabic / chitosan reached
swelling equilibrium within 60- 100 minutes. The pH of the
medium played a significant (P ˂ 0.05) role in the swelling
capacity of catechin loaded microparticles. All the
microparticles swell less in the SGF except those containing
chitosan blends compared to those incubated in SIF
conditions. There are very limited reports on pH dependent
swelling index and stability of catechin microparticles in
related studies(Dudhani & Kosaraju, 2010). Reports on insulin
pH dependent swelling and chitosan nanoparticles shows that
chitosan swells more in the gastric pH, compared to the
intestinal pH (Dudhani & Kosaraju, 2010, Shu &Zhu,2002).
In this study the swelling pattern of the microparticles can be
attributed to the high swelling of chitosan and its ability to
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20
uncoil the carbohydrate molecules to an extended structure
with higher molecular weight because of the pH controlled
electrostatic interaction between anions and chitosan film
Agarwal, V., & Mishra, (1999) reported similar behaviour in
chitosan containing spray dried particles. Although there are
few on pH motivated swellings, higher swelling in the case
of chitosan blend microparticles may be attributed protonation
of the amino group of chitosan when pH decreases ( Oliveira,
Santana, & Ré, 2006). The degree of swelling observed may
have been caused by factors such particles size and catechin
wall materials interaction.
3.3. Physicochemical analysis of mango drink
3.3.1. Effects of storage on pH, total solid ( oBrix) and
viscosity
Fig3.6a shows the mean values of changes in pH of mango
/catechin supplemented drink stored at 4±1 oC for 30 days. It
was observed that when the concentration of catechin
microcapsules increased from 0.5 to 2.0% the pH value of
the drink increased from 5.15 to 5.17 except in the case of
free (non-encapsulated) green tea extract where the pH
decreased significantly ( p ≤ 0.05) to 5.09. However, it was
generally observed that there was a reduction in the pH values
(5.15 to 5. 12) at day - 0 depending on the carrier materials.
In this study it was observed that as the storage period
increases the pH values decreases generally so that by the
third week storage period there was no significant ( p ≤
0.05) difference among the groups including the control
sample. Except for the free (non-encapsulated) green tea
extract which shows lower pH at the end of the storage period.
It has been reported that the normal pH range of mango fruit
drink is in the range 4.5 -5.0 (Vasquez- Calcedo et al.; (2002).
It was observed that as the period of storage increased
irrespective of the concentration of the microparticles the pH
values decreases significantly (p ≤ 0.05) for both the control
and test samples. However, pH value of the control samples
and that of free showed significantly (p ≤ 0.05) difference
from that catechin microcapsules incorporated samples. The
pH of the supplemented mango drink was generally within the
normal range after the storage period. The addition of catechin
microcapsules in the mango drink did significantly affect the
pH of the mango drink.
3.3.2 Total soluble solid (TS)
The result of the total soluble solid (oBrix) as was monitored
during the storage period at 4oC is shown in Fig3.6b. It was
observed that the value of the total soluble solid ( oBrix) did
not show significant (p ≥ 0.05) difference between the control
mango drink and the catechin incorporated mango at 0 day.
However, it was observed that as the concentration of the
incorporated microparticles in the drink increased from 0.5 –
2.0% the value of the oBrix also increased significantly (p ≤
0.05). As the period of incubation increased; the value oBrix
also increased and finally stabilizes at the end of the forth
week. The introduction of the microparticles did not
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significantly (p ≤ 0.05) affect the total solid of the mango
drink since value of the oBrix was within the normal range 10-
16% which considered normal (Vasquez- Calcedo et al.;
2002).
3.3.2 Viscosities
The analysis of viscosity is a simple but rapid method by
which we determine the thoroughness of the rate of mixing of
the ingredients. It also enables us to assess how the level of
solids, degree emulsification and hydrolysis can impact the
quality and consistency of the product. When the viscosity is
increases it tends to maintain the stability of the product by
keeping the insoluble ingredients in suspension and preserving
the homogeneity of the solutions (Rossman, 2009).
The viscosity value of the mango drink supplement as
examined during the storage period is presented in Fig3.6c.
The result shows that the viscosity was not significantly (p ≤
0.05) different when the microparticles were introduced
during the first two weeks of the storage period (0, 1 & 2), but
sample without the microparticles (non-encapsulated green
tea extracts and control) were significantly (p ≤ 0.05)
different. At the end of the storage period the viscosity values
increased significantly (p ≤ 0.05) for the entire sample
irrespective of the concentration and composition of the
microparticles.
3.3.4. Effect of time and temperature on the Stability
catechin in mango drink stored for 30 days at 4 oC
Fig3.7 a, b &c shows the representative chromatograms of
catechin compounds obtained during HPLC analysis of
mango drink supplemented, Fig.7 (a) represent the
chromatogram of reference standard catechin compounds, (b)
chromatogram of green tea catechin analysis at week 0
storage, and (c) represents the chromatogram of catechins
analysis at the end of storage period (week 4). Fig3.8a, b &c
showed the stability of total catechin present in the
supplemented mango drink isolated by HPLC at different
concentration (0.5, 1.0 and 2.0%, whereas Fig3.9 illustrates
total percentage degradation rate of catechins. The
encapsulated catechin compounds were relatively stable at the
storage temperature (4oC) with degradation rate in the range
16.47 -29.72 %. However, the rate of degradation of catechin
compounds in the free catechin extract powder (control) was
higher than those of the encapsulated powder catechin
(45.26%) at the end of 4 weeks storage. The rate of release of
catechin in the mango drinks matrix increase proportionally
with the concentration of incorporated microparticles (2.0 ˃
1.0 ˃ 0.5). The higher the concentration of the catechin
microparticles the higher the level of catechin released. The
stability of catechin was affected by type of wall materials and
time (Fig3.9).
The effects of temperature, pH and food ingredients on the
stability catechin from green tea extracts have been
extensively studied (Chang, 2006; Friedman, , Levin, C. E.,
Choi, ., Lee, & Kozukue, 2009; Lun Su et al., 2003; ). In the
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21
report ascorbic acid one of the ingredients in mango is
reported to play dual roles as an antioxidant and pro-oxidant
(Hara, 2001; Lun Su et al., 2003). In this study the minimal
degradation in the microencapsulated green tea catechin may
be attributed to the combined effects of low temperature, and
pH. Other ingredients might have also played significant roles.
Previous study on epimerization reaction of EGCG, EGC,
ECG and EC, shows that epimerization will result in
corresponding dimmers like GCG, CG, GC respectively. We
observed that the peak area of GC a corresponding dimer from
EGCG sharply increased at the end of the 4th week storage
(Fig3.7c). This may have been caused by epimerization
reaction of other corresponding epimers. This finding is in
agreement with that of Chen et al.; (201), who reported
instances where catechin exhibited varying stability went it
was added to commercially sold soft drinks containing sucrose
or ascorbic acid. The high stability of catechins in mango juice
to water as observed in this study may be linked to the
chemical composition of the product.
3.3.5. Determination TPC
The amount of total phenolic content and antioxidant activity
of the supplemented mango drink as monitored during the
storage period of 4 weeks at 4oC is shown in Fig3.10a &b.
The polyphenols in green tea extracts is attributed to
catechins. The release rate of TPC and antioxidant was
proportional to increase in concentration of microparticles
incorporated (0.5, 1.0 and 2.0% w/v.) The TPC of the control
sample decreased by 20.0% against the TPC of the catechin
supplemented mango drink (3.01 -6.66%) [Fig3.10a]. There
was no significant (p ≤ 0.05) difference in the total phenolic
content mong the catechin microcapsules incorporated into the
supplemented mango drinks except for MD: GA: CTS
(25:74:1) which decreased by as much as 6.66%. This may
be attributed to synergy between catechins / mango drink
matrix, ingredients and other factors inherent in the mango
drink (Madhujith & Shahidi, 2006, Becker et al.; 2004).
Fig.3.10.b showed the evaluation of the DPPH radical
scavenging activity of the mango drink supplemented with
catechin microparticles stored at 4oC for 4 weeks. The
percentage stability of the radical scavenging activity was
between 79.92 - 96.17% thus less than 25% degradation rate.
The free catechin extracts (control) powder exhibited less
antioxidant activity compared to the encapsulated catechin
powder at the storage temperature. It has been reported that
the antioxidant properties of foods correlate with the presence
of selected compounds within the food system (Ramadan,
Sharanabasappa, Seetharam, Seshagiri, & Moersel, 2006). We
can infer that the antioxidant properties of the mango drinks
evaluated is a reflection of different endogenous antioxidant
and that of the green tea catechins which contributed to the
radical quenching activity or efficiency of the mango drink.
On the whole, the result of this study agrees with those found
in the literature on green tea antioxidant activities.
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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:16 No:02
CONCLUSION
At the end of this study we found out that encapsulated
supercritical fluid (Camellia sinensis) green tea catechin
extracts microparticles could be a suitable phenolic compound
for adding into mango drink. The encapsulated catechin
compounds were more stable in the supplemented mango
drinks in comparison with the non-encapsulated catechin
powder with improved functionality. The catechin extracts
did not affect the physicochemical properties of the mango
drink in terms of pH, total solid (oBrix), and viscosity of the
drinks. Among the four most important catechin monomers
EGCG and EGC showed higher degradation rate than EC and
ECG. This may be attributed to the high antioxidant
potentials due to the number of hydroxyl groups. Therefore
this study suggests that catechin microcapsules have the
potential to be used as a functional food ingredient for heath
drink.
ACKNOWLEDGEMENT
The authors will like to thank The University Putra Malaysia
for the financial support for the faculty of Food Science and
Technology. We are so grateful to Associate prof. Dr.
Badlishah S.B., who secure the grant secured the grant for the
research.
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Table3.1.
Total polyphenol and catechin of green tea microparticles and their entrapped efficiency
Wall material (%) TPC (g/100gGAE) DPPH (%) TC(mg/100g) Efficiency TC (%)

FREE GTE 28.19±0.30a 43.07±0.83a 9867.20±63.43a NA

MD: GA: CTS (25:74:1) 24.90±0.05b 38.05±1.27b 7046.70±53.11d 71.41±1.62g

MD: GA: CTS (0:99:1) 24.74±0.07 b 37.80±0.55c 7217.85±39.41cd 73.15±0.57g

MD: GA: CTS (50:49:1) 24.16±0.09b 36.92±0.57c 7117.25±52.93d 72.10±0.22g

MD: GA: CTS (99:0:1) 22.94±0.05c 35.05±0.04d 7313.57±36.56bcd 74.12±2.31g

MD: GA: CTS (75:24:1) 22.88±0.45c 34.96±0.55d 7722.22±46.72bcd 78.26±0.42f

MD: GA: CTS (0:100:0) 21.74±0.07d 33.22±0.39e 8687.90±51.12b 88.04±0.08b

MD: GA: CTS (100:0:0) 21.46±0.04d 32.79±0.12ef 8042.90±51.29bcd 81.51±0.15e

MD: GA: CTS (25:75:0) 21.32±0.04d 32.58±0.43ef 8404.88±51.75cd 85.18±1.63c

MD: GA: CTS (50:50:0) 21.10±0.06d 32.24±0.41f 8232.20±54.85d 83.43±0.27d

MD: GA: CTS (75:25:0) 19.32±0.14e 29.52±0.34g 8019.09±54.15c 81.27±0.13e

Results are mean ± SD of three determinations of catechin microparticles based on 5% carrier materials used. Means values in columns carrying the same superscript letters are not
significantly (p ≤ 0.05) different. NA –not applicable

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Table3.2.
Physicochemical properties of spray-dried green tea microparticles
Sample particle Moisture (%) Hygroscopicity (%) Water activity(aw) Bulk density(g/cm3) Tap density(g/cm3)

MD:GA:CTS(100:0:0) 3.16±0.06f 4.55±0.59c 0.28±0.01h 0.28±0.04a 0.35±0.06a

MD:GA:CTS(0:100:0) 4.58±0.04c 4.94±0.34b 0.26±0.03i 0.26±0.10b 0.34±0.08a

MD:GA:CTS(75:25:0) 2.49±0.06g 4.40±0.01cd 0.31±0.02f 0.23±0.01ef 0.34±0.08a

MD:GA;CTS(50:50:0) 3.36±0.06e 4.71±0.03b 0.34±0.01d 0.25±0.04bc 0.33±00ab

MD:GA:CTS(25:75:0) 2.31±0.01h 5.75±0.11ab 0.34±0.01d 0.25±0.00bcd 0.34±0,.08a

MD:GA:CTS(99:0:1) 4.33±0.05d 3.22±0.14e 0.36±0.10b 0.22±0.003f 0.29±0.00d

MD:GA:CTS(0:99:1) 5.11±0.06ab 3.94±0.10cde 0.25±0.05j 0.24±0.003cde 0.30±0.06cd

MD:GA:CTS(75:24:1) 3.33±0.04ef 3.46±0.16de 0.35±0.01c 0.23±0.00ef 0.31±0.00bc

MD:GA:CTS(50:49:1) 4.32±0.02d 3.96±0.02cde 0.32±0.10e 0.24±0.00d 0.30±0.06cd

MD:GA:CTS(25:74:1) 4.78±0.04b 3.34±0.04e 0.34±0.10d 0.20±0.00g 0.25±0.04e

Crude powder(BLK) 5.45±0.05a 6.15±0.23a 0.45±0.01a 0.16±0.10h 0.15±0.15f

Mean that do not share letters vertically are significantly different. Values are mean of three independent determinations

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Table3. 3
Result showing individual catechin compounds obtained by high pressure liquid chromatography (HPLC) analysis
Wall material C EC EGC EGCG GCG ECG TC (mg/100g)
MD:GA:CTS(%)
100:00:0 73.78C 478.91abc 2330.33d 3963.67d 380.06bc 803.56abc 8042.90bcd
C abc c b cd
00:100:0 77.72 502.57 2449.99 4468.89 350.02 844.55abc 8687.90ab
C ab a ab ab
75:25:0 72.38 545.87 2681.22 4558.06 437.34 924.49a 8019.09bcd
C ab c c cde
50:50:0 73.78 528.23 2455.01 4173.52 338.62 680.86bc 8232..20bcd
bc a bc b def
25:75:0 69.90 578.99 2540.29 4371.71 308.72 532.48abc 8404.88bcd
c de e e efg
99:00:1 81.48 318.99 2138.29 3746.08 281.21 131.34c 7313.57bcd
c e f f efg
00:99:1 130.33 90.72d 1968.36 3480.93 286.64 767.14abc 7217.85cd
a bcd e e fg
75:24:1 132.33 431.77 2125.98 3669.09 275.51 835.00ab 7722..10cd
ab de f f g
50:49:1 98.62 358.35 1968.95 3405.78 235.06 706.34abc 7114.25d
bc e f f cd
25:74:1 86.95 296.19 1938.78 3336.93 363.28 745.78abc 7046.70d
bc a ab a a
GTE 90.03 574.78 2582.99 4704.55 423.22 651.91bc 9867.20a
Results are mean ± SD of three determinations of catechin microparticles based on 5% carrier materials used. Means values in columns carrying the

Same superscript letters are not significantly (p ≤ 0.05) different.

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Fig. 3.1. Encapsulation efficiency of total catechin entrapped. Values are means of three independent determination ± SD. Means carrying different letters are
significantly (p ≤ 0.05) different (Turkey’s multiple- range test

Fig. 3.2. total polyphenolic content green tea catechin microparticles entrapped using different wall material. Values are means of three independent determination
±SD.Values carrying different letters are significantly different (p ≤ 0.05) different.

Fig. 3.3. Antioxidant activity of green tea microparticles as determined by DPPH assay. Values are means of three independent determinations ± SD. Values
carrying different letters are significantly (p ≤ 0.5) different (Turkey’s multiples –range test).

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Fig. 3.4. (a) Effects of incubation on simulated gastrointestinal fluids (SGF pH 2.3) on the swelling behaviour of spray- dried microparticles. (MD-
maltodextrin, GA – gum arabic, CTS –chitosan).

Fig. 3.4(b). Degree of swelling of catechin microparticles in (SGF) Values are presented as means± SD. Values that carry different letters are significantly (p ≤
0.05) different (Turkey’s multiple –range test).

Fig. 3.5 (a). Effects of incubation on simulated intestinal fluids (SIF pH 7.4) on the swelling behaviour of spray- dried microparticles: (MD- maltodextrin, GA –
gum arabic, CTS –chitosan).

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Fig. 3.5 (b). Degree of swelling of catechin microparticles in SIF condition; values are presented as means ±SD. Values that carry different letters are
significantly (p ≤ 0.05) different (Turkey’s multiple –range test).

Fig. 3.6a. Change in pH of supplemented mango drink during stored at 4oC for 4 weeks

Fig. 3.6b.Change in total solid (oBrix) of supplemented mango drink during 4oC for 4 weeks

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Fig. 3.6c. Changes in viscosity of supplemented mango drink at during storage

Fig.7a. HPLC chromatogram of catechin standards

Fig. 6b Fig.7b.Representative HPLC chromatograms from Supplemented mango drink (week 0)

Fig. 3.7c. Representative HPLC chromatograms from Supplemented mango drink (wee 4)

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Fig. 3.8. Stability of catechin microparticles on catechin –supplemented mango drinks at (a) 0.5%, (b) 1.0 (c) 2.0% concentrations. Values are presented as the
means of three replicate determination ± SD.

Fig. 3.9. Percentage degradation of green catechin in Mango drink during 4 weeks storage at 4 oc. Values carrying different letters are significantly ( p ≥ 0.05)
different. Values are presented as means ± SD.
a

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Fig. 3.10. (a) Changes in total phenolic content of catechin microparticles released in catechin-mango drink stored at 4oc for 4weeks, (b) changes in antioxidant
activity of catechin- mango drink supplements at 4oC for 4 weeks. Values are presented as the means of three replicate determination ± SD.

165902-7373- IJBAS-IJENS @ April 2016 IJENS IJENS