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Membrane Performance and Potential Separation of Cytokinins During Ultrafiltration

of Skimmed Coconut Milk

Article  in  Journal of Computational and Theoretical Nanoscience · December 2013

DOI: 10.1166/asl.2013.5202

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 RESEARCH ARTICLE
Copyright © 2013 American Scientific Publishers Advanced Science Letters
All rights reserved
Printed in the United States of America
Vol. 19, 3620–3624, 2013

Membrane Performance and Potential Separation

of Cytokinins During Ultrafiltration of
Skimmed Coconut Milk
Ng Ching Yin, Abdul Wahab Mohammad∗ , and Ng Law Yong
Faculty of Engineering and Built Environment, Department of Chemical and Process Engineering,
Universiti Kebangsaan Malaysia, UKM Selangor 43600, Malaysia

Coconut milk is a milky-white perishable liquid. It is enriched with complex minerals, vitamins and value-added
bioproducts such as kinetin and zeatin, which are well-known as cell division promoters and commonly used in
medicinal, pharmaceutical and nutraceutical application. In this study, the ultrafiltration (UF) process was used
to separate kinetin and zeatin from skimmed coconut milk. The presence of kinetin and zeatin was confirmed
by high performance liquid chromatography (HPLC) analysis. The ultrafiltration membranes used were 30-kDa
polysulfone (PSf) and 30-kDa polyvinylidene fluoride (PVDF) membranes. Factors and mechanisms affecting
the flux declines were studied and postulated. The lower normalized flux was exhibited by PVDF membrane
during UF process. This showed that the extent of membrane fouling occurred on PVDF membrane compared to
PSF membrane. PVDFDelivered
membranesby Publishing
were shown to be Technology to: Ching
more hydrophobic Yinones
than PSf Ng by contact angle tests.
Morphological studies of IP:
both14.1.210.204
membranes were On: Fri, 30
carried out May 2014 07:36:15
by scanning electron microscopy (SEM). Fouling
layers formed on the membranesCopyright: American
were confirmed using Scientific
SEM. Publishers
Keywords: Ultrafiltration, Aqueous Coconut Milk, Kinetin, Zeatin, Morphological Study.

1. INTRODUCTION
Coconut milk is a milky-white oil-in-water emulsion and a com-
plex biological fluid, typically comprising fat, protein, carbohy-
drates and minerals, the major components being water and fat.
Coconut (Cocos nucifera L.), is a source of oil that is impor-
tant for commercial products all around the world. Coconut oil is
notable and famed for its short chain fatty acids, which is well-
recognized as a healthier oil supply compared to other crop oils.1
The content of coconut milk carries traces of minerals such as
potassium and sodium, as well as growth compounds known as
cytokinin. Cytokinin is a compound that is mainly composed of
kinetin, zeatin and N6 -benzyladenine. This substance has been
widely applied to crops as growth enhancers because it could
stimulates cell division in tissues and regulates plant growth and
development.2 In view of the fact that these substances are capa-
ble of stimulating cell division, they have the potential to be used
for medicinal purposes and skin care applications. Cytokinin has
been reported to have adverse effects on human cancerous cell
growth and enhances new cell development.3
Ultrafiltration (UF) is a pressure-driven membrane separation
process in which the membrane fractionates the components of a
liquid with respect to their sizes and structures. The pore sizes of
∗
Author to whom correspondence should be addressed.
the UF membrane is within the range of 0.01–0.1 m. With the
advances in membrane technology, membranes have also been
employed in the processing of high-valued bio-products.
Approximately 60 to 70% of coconut milk is composed
of fat. This fat content can be extracted and converted into
coconut oil. When the fat content is extracted from coconut
milk, the skimmed coconut milk still contains valuable materials
(11S globulin-typed protein, lauric acid, glycine, lysine and so
forth). Therefore, as part of the value enhancement of coconut
milk, we processed skimmed coconut milk by UF to separate
cytokinin contents. In previous study, solvent extraction and solid
phase extraction methods were used to extract and purify the
cytokinins.4 The objectives of this paper were to investigate the
performances of the UF process and determine the possibility of
separating and concentrating cytokinin in skimmed coconut milk
by UF.
2. REAGENTS AND MATERIALS
The phytohormone standards, kinetin and zeatin as shown
in Figure 1, were purchased from Sigma-Aldrich (Steinheim,
Germany). All the standards were dissolved in HPLC-grade
methanol with a concentration ranging from 10 to 500 M
and stored at 0 to 4  C for HPLC analysis. Purchased chemi-
cals were: methanol and formic acid (HPLC grade) from Tedia

3620 Adv. Sci. Lett. Vol. 19, No. 12, 2013 1936-6612/2013/19/3620/005 doi:10.1166/asl.2013.5202
Adv. Sci. Lett. 19, 3620–3624, 2013 RESEARCH ARTICLE

weight cut-off of 30 kDa. The effective membrane area in this

experiment was 15.2 cm2 . The filtration processes were per-
formed using the Sterlitech stirred cell. The operating parameters
were: 2.4 bar of pressure, 400 rpm stirring rate and 50±05  C of
temperature. The permeate flux was measured periodically over
the course of the experiment for a duration of one hour.

5. CONTACT ANGLE ANALYSIS

Fig. 1. Chemical structure of the phytohormone compounds.5 6
(Fairfield, USA), triethylamine (TEA) (HPLC grade) from Merck
(Hohenbrunn, Germany). Whatman filter paper, 0.45 m, and
polypropylene (Mill, Maidstone, Kent, England) were used to
filter skimmed coconut milk before the UF process. TEA was
used to adjust the pH value of the buffer solution (0.1% formic
acid) to pH 3.2. The PSf and PVDF ultrafiltration flat sheet mem-
branes with a molecular weight cut-off of 30 kDa were obtained
from GE Osmonics (Minnetonka, MN). The other apparatuses
used were chemically-resistant stirred cell (model HP4750, Ster-
litech, Kent, USA) and hot plate (Wisestir MSH-20D, Daihan,
Seoul, Korea). Sodium hydroxide (NaOH) was obtained from
Tedia (Fairfield, USA) for particle-size distribution tests.
3. PREPARATION OF SKIMMED
Delivered by Publishing Technology
COCONUT MILK
IP: 14.1.210.204 On: Fri, 30
Initially, the purchased local coconut milk Copyright:
was filtered through
Americana Scientific
nylon sieve cloth to remove residues and large particles. A sim-
ple pasteurization (60  C for 4 min) process was conducted in
order to reduce the microbial load to 10%.7 To extract the fat,
centrifugation was conducted. There were three different phases
of solution obtained after the centrifugation: cream, aqueous and
solid phases. The feed for the ultrafiltration process was from the the wavelength was fixed at 265 nm.
aqueous phase, which was also called skimmed coconut milk. To
avoid and reduce bacterial contamination, skimmed coconut milk
solution was kept at 4  C or below and used within 5 days after
the solution was pre-filtered through a 0.45-m polypropylene
Whatman filter paper (to remove larger particles). Enrichment of
the valuable nutrients in coconut milk was proven and it was
showed in Table I.
The contact angle measurement device (KRÜSS GmbH Model
FM40mk2 Easy Drop with the SW4001 software and an accu-
racy of ±010 ) was used in contact angle analysis, with deion-
ized water at ambient temperature. The sessile drop method was
applied in this test.8
6. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC)
INSTRUMENTATION AND PROCEDURE
The analysis of phytohormones was done using a High per-
formance liquid chromatography (HPLC) system (Agilent Tech-
nologies 1200 Series, Santa Clara, USA). HPLC is an attractive
approach for polar and thermally reactive phytohormones. Con-
ventional UV has been widely applied in HPLC for separating
phytohormones in samples.9
Data were processed by the accompanying system software
(ChemStation for LC 3D Systems). Prior to HPLC analysis, the
samples were
filteredYin
to: Ching by aNg
0.45-m Whatman glass microfibre
filter. 10-l filtered samples were injected into a C18 reverse
May 2014 07:36:15
phase column (Zorbax SB-C18 100 Å, 150 mm in length, 2.1 mm
Publishers
in diameter, Agilent Technologies, Santa Clara, USA). The ini-
tial HPLC running condition was a methanol (0.1%)-formic acid
buffer (10:90, v/v). The column thermostat was set at 25  C. The
flow rate was 0.3 mL min−1 throughout the whole separation
process. To achieve high accuracy detection of phytohormones,
Table II shows the phytohormone or cytokinin (kinetin and
zeatin) standard response characteristics using HPLC analysis.
Meanwhile, Eq. (1) was used to calculate the rejection percent-
age of the solute after ultrafiltration, where Cp is the solute
Table II. Phytohormone standard response characteristics using
HPLC.

4. ULTRAFILTRATION (UF) Phytohormone standard

MEMBRANE PROCESS Kinetin Zeatin

Mean retention time
The ultrafiltration flat sheet membranes used were polysulfone Mina 26.8 18.4
(PSf) and polyvinylidene fluoride (PVDF) with a molecular RSD (%) 0.19 0.002
Peak areaa
Mean 7327.34 612.17
Table I. Content of nutrients of fresh and skimmed coconut milk. RSD (%) 1.7 0.5
Equation of calibration curve Y = 1604x − 7492 Y = 1224x − 5331
Nutrient content Fresh coconut milk Skimmed coconut milk R2 0.977 0.999
Protein, % (w/w) 2.29 2.45 Linear range (ppm) 2–100 2–100
Fat, % (w/w) 23.8 0.20 LOQ (M)c 1.7 3.47
Carbohydrate, % (w/w) 5.54 4.02
Note: a Data were measured with repeated injection (n = 5) of plant hormone standard at
Ash, % (w/w) 0.87 1.33
a concentration of 20 ppm each; b In the calibration equation, x represents concentration
Moisture, % (w/w) 67.5 92.0 of the analyte (ppm) and y the peak area (mAu · s); c LOQ was estimated based on
Energy, kcal/100 g 963 kJ 117.75 kJ S/N = 10.

3621
RESEARCH ARTICLE
concentration in permeates and Cf is the solute concentration in
the feed obtained through the HPLC analysis.
 
Cp
%Rejection = 1 − × 100 (1)
Cf
7. SCANNING ELECTRON MICROSCOPY
(SEM) IMAGING
SEM micrographs were obtained using Gemini Model SUPRA
55VP, ZEISS, with the magnification of 1000×, 3000× and
5000×. A thin gold layer was applied on samples by using a
gold sputter coater (Emscope SC 500, Ashford, Kent, UK) before
scanning.
8. PARTICLE-SIZE DISTRIBUTION TEST
Mastersizer 2000 (Malvern Instruments, Worcestershire, UK)
was used to measure particle sizes. The size range of particles
that could be read from the instrument was between 0.02 and
2000 m. Standard solution of kinetin and zeatin phytohormones
were prepared for this test.
9. RESULTS AND DISCUSSION
9.1. Membrane Characteristics
Membrane hydraulic permeability was carried out by using ultra-
pure water before brought to solution filtration process. Both
membranes achieved high regression coefficient; R2 = 0983
(PSf) and 0.986 (PVDF). Due to theDelivered by Publishing
contact angle measurement,Technology
fouled PVDF membrane (8073 ± 071) IP: 14.1.210.204
was On: Fri, 30
proved to be slightly
more hydrophobic than PSf membrane (7787 Copyright:
± 095). American Scientific
9.2. Normalized Flux
There was a significant difference between the flux behavior of
PSf and PVDF membranes, as shown in Figure 2. PVDF mem-
branes exhibited lower normalized fluxes throughout this study
compared to PSf ones. It is postulated that the lower normal-
ized fluxes of the PVDF membranes is caused by the higher
fouling rate or deposition rate of the foulants on the mem-
brane surface. The foulants deposited were consisted with broad
Fig. 2. Normalized fluxes for PSf and PVDF membranes within a duration
of 60 minutes.
3622
Adv. Sci. Lett. 19, 3620–3624, 2013
particle-size of constituents ranged from 1 nm to 20 m. This
observation is in good agreement with other studies postulating
that the hydrophobicity of the membrane surface could affect
the degree of adsorption of the foulants onto the membrane sur-
face (Shi et al. 2008). Thus, in this research work, PSf mem-
branes, with higher hydrophilicity, exhibited higher normalized
fluxes throughout the process and a lower degree of adsorption of
foulants onto the membranes. A greater degree of fouling on the
PVDF membrane surface was also observed through the SEM
images.
9.3. Morphological Study—Scanning Electron
Microscopy (SEM)
Figures 3(a)–(d) and 4(a)–(d) showed SEM images of the sur-
face and cross-section of PSf and PVDF membranes, respec-
tively, for both fresh and fouled membranes. These membrane
surfaces were deposited with different shaped particles or com-
pounds compared to the fresh membranes. Based on the images,
the PVDF membrane was apparently more porous than the PSf
one. Membrane fouling was believed to have been initiated soon
after a very short period of filtration time, which was further
proven by observing the sharp decline in the permeate flux as
shown in Figure 2. It was postulated that the concentration polar-
ization phenomenon occurred on membrane surfaces because a
layer of mixture was formed on the fouled surfaces of both mem-
branes, as shown in the scanned SEM pictures (Figs. 6 and 7).
The fouling layer that contributed to this concentration polariza-
tion probably consisted of coconut fats (creams remained after
the centrifugation process), proteins, suspended solids and other
to: Ching
small molecules suchYin
as Ng
vitamins, minerals, micelles and phy-
May 2014
tohormones. 07:36:15 polarization has been observed to be
Concentration
Publishers
more pronounced for porous membranes due to the higher con-
vective flow of the solutes to the membrane surface. This obser-
vation has been made and claimed by other researchers as well
(Carroll et al. 2000), and proven by the PVDF membrane surface
analysis. This explained the higher rate of solute deposition on
PVDF membranes with lower normalized fluxes. Based on the
contact angle measurement, surface hydrophobicity or the contact
angle value recorded for the PVDF membrane was higher after
the gel layer formation on membrane surfaces. Besides, the con-
tinuous coconut milk separation process might have contributed
to the fouling cake layer formation on membrane surfaces. This
Fig. 3. (a) Fresh PSf membrane with a magnification of 5000×; (b) fouled
PSf membrane with a magnification of 5000×; cross-section of (c) fresh PSf
membrane and (d) fouled PSf membrane with a magnification of 800×.
Adv. Sci. Lett. 19, 3620–3624, 2013
Fig. 4. (a) Fresh PVDF membrane with a magnification of 5000×; (b) fouled
PVDF membrane with a magnification of 5000×; cross-section of (a) fresh
PVDF membrane and (b) fouled PVDF membrane with a magnification of
3000×.
was due to the transportation and deposition of smaller sized
compounds on membrane surfaces, leading to the growth of the
fouling cake layer. Attachments of smaller sized compounds to
membrane surfaces were much easier than those of large com-
pounds, consistent with the results of other studies.10
The cross-sectional structures of fresh and used PSf and PVDF
(Figs. 3(c) and (d) and 4(c) and (d)) membranes were exactly
the same. Thus, we could conclude that the foulants did not pen-
etrate through the membrane pores. It was further proven from
the images captured that the finger-like pores of membranes were
not clogged by the foulants. These results suggested
Delivered that theTechnology
by Publishing
suspended solids, coconut proteins and IP: other small molecules
14.1.210.204 On: Fri, 30 May bound
played an important role in determining Copyright:
the surface fouling
Americanof Scientific
UF membranes.
9.4. Separation of Cytokinins (Kinetin and
Zeatin) by UF Membranes
Figure 5 shows the performance of PSf and PVDF membranes
in terms of the rejection of kinetin and zeatin phytohormones
in solution (skimmed coconut milk) and standard solutions
(standard solutions refer to those solutions prepared by dissolving
kinetin in water or zeatin in water). It was found that the rejection
of zeatin (45%) from the solution (skimmed coconut milk) was
higher for both membranes compared to kinetin (25%). Although
Fig. 5. Rejection of kinetin and zeatin within solution and standard solution
by PSf (30-kDa) and PVDF (30-kDa) membranes.
RESEARCH ARTICLE
Fig. 6. Particle-size distributions of kinetin and zeatin phytohormones.
kinetin and zeatin molecules have roughly the same molecular
weight, which is 220 g mol−1 , both of them displayed different
rejections when tested using the membranes in this work. Thus,
it was postulated that the results obtained from this experimental
work were caused by some other factors (such as interactions
between the kinetin and zeatin molecules with other components
in the coconut milk which dependent on their molecules reactiv-
ity). The rejections of these two different phytohormones from
standard solution were significantly different to those from the
skimmed coconut milk solution. Zeatin has been recognized as
the most active phytohormone.11 Referring to the second carbon
of zeatin molecules (Fig. 1), there is an active and open double
bond in theto:zeatin
Ching Yin NgIt is believed that zeatin compounds
molecule.
actively 2014 to 07:36:15
the other compounds in the skimmed coconut
Publishers
milk to form bigger and more complex ones. This is worth
detailed investigation in the near future. Hence, the rejection of
zeatin was much higher than that of kinetin during the ultrafil-
tration process of skimmed coconut milk solution, although their
molecular sizes are relatively similar.
Figure 6 exhibits the particle-size distributions of kinetin and
zeatin in standard solution. The ranges of kinetin and zeatin
particle-sizes were between 0.05 and 0.40 m. Although kinetin
and zeatin have the same range of sizes, zeatin from skimmed
coconut milk was retained more than kinetin when the same
membranes were used for comparison. Fig. 5 showed the rejec-
tion of both cytokinins after UF process. As zeatin itself is an
active binder due to its molecular structure, it is not surprising to
find out that the rejection of zeatin was greater compared to that
of kinetin during the ultrafiltration process of skimmed coconut
milk. According to this result, it is interesting to find out whether
zeatin and kinetin can be permeated through the ultrafiltration
membrane successfully.
The produced kinetin and zeatin with anti-ageing and cell divi-
sion functions could be successfully applied in nutraceutical and
pharmaceutical fields in the near future. This paper could be
used as a preliminary study for the performance of polysulfone
(PSf) and polyvinylidene fluoride (PVDF) ultrafiltration mem-
branes and their potential in the separation of kinetin and zeatin
from skimmed coconut milk.
10. CONCLUSION
The finding indicated that the PSf membrane is more suitable for
the ultrafiltration of skimmed coconut milk solution as it is more
3623
 RESEARCH ARTICLE
hydrophilic than the PVDF membrane. Thus, the hydrophilicity
of the PSf membrane can be used to lower the membrane foul-
ing tendency. Existence of phytohormones (kinetin and zeatin)
in skimmed coconut milk was verified by HPLC. Concentra-
tions of kinetin in the feed, retentate, and permeate were deter-
mined to be less than 8 mg/ml. However, concentrations of
zeatin in the feed, retentate, and permeate were determined to
be less than 3 mg/ml. Separation of the phytohormones was suc-
cessfully achieved using the UF process with 30,000-MWCO
membranes. Most of the phytohormones was collected in the per-
meates, where the rejection percentage was less than 47%. The
types of membrane material used (PSf or PVDF) did not have
any significant or major effects on the result of phytohormone
separation. SEM images showed the formation of foulant layers,
which were believed to be caused by suspended solids, coconut
proteins and other small molecules. To obtain a higher purity
of phytohormones, nanofiltration of permeates from the UF pro-
cess has been suggested for future investigations and research
work.
Delivered by Publishing Technology to: Ching Yin Ng
IP: 14.1.210.204 On: Fri, 30 May 2014 07:36:15
Copyright: American Scientific Publishers
3624
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Adv. Sci. Lett. 19, 3620–3624, 2013
Acknowledgment: The authors wish to express their grati-
tude for the financial support from the 02-01-02-SF1021 Grants.
References and Notes
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2. L. Sáenz, L. H. Jones, C. Oropeza, D. VláčIl, and M. Strnad, Plant Growth
Regul. 39, 205 (2003).
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and P. Niemann, Phytochemistry 71, 1350 (2010).
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5. J. Barciszewski, F. Massino, and B. F. C. Clark, Int. J. Biol. Macromol. 40, 182
(2007).
6. J. W. H. Yong, L. Ge, Y. F. Ng, and S. N. Tan, J. Mol. 14, 5144 (2009).
7. R. Hagenmaier, Coconut Aqueous Processing, Publications, University of San
Carlos, Cebu City, Philippines (1980).
8. L. Palacio, J. I. Calvo, P. Prádanos, A. Hernández, P. Väisänen, and
M. Nyström, J. Membr. Sci. 152, 189 (1999).
9. J. Barciszewski, G. E. Siboska, B. O. Pedersen, B. F. C. Clark, and S. I. S.
Rattan, FEBS Lett. 393, 197 (1996).
10. J. Altmann and S. Ripperger, J. Membr. Sci. 124, 119 (1997).
11. A. Meyer, W. Rypniewski, M. Szymañski, W. Voelter, J. Barciszewski, and
C. Betzel, Biochim. Biophys. Acta, Proteins Proteomics 1784, 1590 (2008).
Received: 15 March 2012. Accepted: 27 April 2012.