Content uploaded by Adolf K Awua
Author content
All content in this area was uploaded by Adolf K Awua
Content may be subject to copyright.
Available via license: CC BY 2.0
Content may be subject to copyright.
Available via license: CC BY 2.0
Content may be subject to copyright.
RESEARCH ARTICLE Open Access
Exploring the influence of sterilisation and
storage on some physicochemical properties of
coconut (Cocos nucifera L.) water
Adolf K Awua
*
, Edna D Doe and Rebecca Agyare
Abstract
Background: Fresh coconut (Cocos nucifera L) water is a clear, sterile, colourless, slightly acidic and naturally
flavoured drink, mostly consumed in tropical areas. It is a rich source of nutrients and has been used for medical
purposes. This study was designed to investigate changes in selected characteristics of coconut water after
autoclaving, gamma irradiation and storage. Also, the study was designed for assessing the possibility of measuring
the growth of bacterial in fresh, stored or sterilised coconut water using turbidity measurements (at wavelengths
between 600 nm and 800 nm) or by dry biomass determinations.
Results: Portions of coconut water aseptically extracted from the matured fruit, (average pH of 6.33 ± 0.17) were
either stored at 4°C, autoclaved at 121°C for 20 min., or irradiated with gamma rays at 5 kGy. Subsequent changes
in selected characteristics were determined. Autoclaving, gamma irradiation and long term storage of coconut
water at 4°C resulted both in the development of a pale to intense yellow colour and changes in turbidity. After
storage, the dry matter content of fresh, autoclaved and irradiated coconut water by 52.0%, 23.5% and 5.0%
respectively. There were also significant differences in the UV spectra before and after sterilisation and during the
storage of the coconut water. Although changes in total carbohydrates were observed, they were not significant (p
> 0.05).
Conclusions: The enormous differences in the characteristics before and after storage suggests that the use of
turbidity and dry biomass measurements for measuring the growth of bacteria in fresh, autoclaved and gamma
irradiated coconut water before storage is practicable without any possibility of interference by the innate turbidity,
colour and dry matter of the coconut water. However, this is not practicable after storing the coconut waters at 4°
C, since there were increases in the turbidity and dry matter of the coconut water to levels that will mask the
turbidity of a growing bacteria culture.
Background
Coconut (Cocos nucifera L)water,alsoreferredtoas
coconut juice, is a refreshing natural drink common and
mostly consumed in the tropical regions of the world
[1,2]. It is a clear, colourless, sweet, naturally flavoured
slightly acidic drink, with reported pH ranging between
4.2 and 6.0 [3,4].
Over six decades of research has shown that coconut
water contains proteins, fats, and is rich in carbohy-
drates and nutritionally important elements (potassium
being the most abundant) [3-7]. It is also a rich source
of essential amino acids (lysine, histidine, tyrosine and
tryptophan), fatty acids, glucose, fructose, cellulose,
sucrose, and organic acids such as tartaric, citric and
malic acids [7-10].
Coconut water’s rich enzyme systems include very
effective and selective reductase [1], polyphenol oxidase
(PPO) and peroxidase (POD). These are involved in its
development of a brownish colour when it is exposed to
air for a long time [11]. Based on its content and proper-
ties, coconut water has been used in the treatment of
child and adult diarrhoea, and gastroenteritis as well as
for urinary stone dissolution, short-term intravenous
hydration and protecting against gastrointestinal tract
infections [12].
* Correspondence: a_awua@yahoo.com
Cellular and Clinical Research Centre, Radiological and Medical Sciences
Research Institute, Ghana Atomic Energy Commission, Accra, Ghana
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
© 2011 Awua et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
While still in an undamaged fruit, coconut water
remains sterile and stable, but it may become unstable
when extracted from the fruit and stored for a few days
at 4°C. In Ghana, it is commonly available in the coastal
areas and mostly consumed fresh and directly from the
fruit. However, the conduct of its vendors and consumers
exposes it to a high risk of contamination with bacteria,
specifically coliforms.
Assessing the risk of bacteria infections through the
consumption of coconut water is made a difficulty since
there are limited reports that show the survival and
growth of pathogenic bacteria in coconut water. Consid-
ering the risk of bacteria contamination of coconut water
in Ghana, the possibilities of survival and growth of bac-
teria in coconut water and the potential for the use of
coconut water as a bacterial growth media in resource-
limited countries/laboratory like ours, we initiated this
study. We studied some characteristics of coconut water
obtained from locally grown crop. We also investigated
how these change with autoclaving, gamma irradiation
and storage. We report here, changes in colour, turbidity,
dry matter, pH and UV absorbance as well as the influ-
ence of temperature on the visible light absorbance of
coconut (Cocos nucifera L) water after autoclaving,
gamma irradiation and storage at 4°C. An assessment of
the possibility of measuring bacterial growth by measur-
ing the turbidity and dry biomass of bacteria cultures in
fresh, stored or sterilised coconut water was also
presented.
Method
Extraction of Coconut Water
Three matured green coconut fruits, with no visible
damage, were obtained from the local market (not more
than 48 hour after harvest). Parts of the mesocarp and
endocarp were removed to expose the endosperm, with-
out damage. After surface sterilisation with 70% ethanol
and exposure to UV light (240 nm) in a Biosafety Class II
Cabinet (Clean Air Systems Chennai, India), the endo-
sperm was cut-out with a sterile surgical blade and the
water transferred to a sterile Ducan bottle. After estimat-
ing the volumes, portions were stored at 4°C and -20°C.
The pHs of the fresh coconut water, the coconut water
after two weeks and one month of storage at 4°C were
determined (HANNA pH 211, Microprocessor pH meter,
Sigma-Aldrich, St. Louis USA). The sterility of each coco-
nut water extract and stored coconut water was tested by
inoculating nutrient agar plates and 10 mL Luria Bertani
broth (Sigma-Aldrich, St. Louis USA) with 150 μlof each
and incubating at 37°C for 24 hours.
Sterilisation of Coconut Water
About 200 mL of fresh coconut water was autoclaved at
121°C and 1.5 bars for 20 minutes (NAPCO model
9000-D, Pegasus Sci. Inc., Rockville USA). Another 250
mL was radiated with 5 kGy of gamma radiation (with a
dose rate of 10 kGy/h at 10 ± 0.5°C. Their pHs were
determined and portions stored at -20°C until further
use and at 4°C for further study.
During storage, the samples were intermittently trans-
ferred to room temperature (about 25°C), for not more
than 30 minutes in a day, once every week for one
month, to simulate the normal situation that may apply
in the use of stored liquid bacteria media.
Absorbance Spectra
The absorbance of the fresh, autoclaved and radiated
coconut water within a wavelength range of 200 nm and
600 nm were measured (UV-VIS 1210 Spectrophot-
ometer, Shimadzu Corp., Columbia MD, USA), with
repeats after two weeks and one month of storage at 4°C.
All determinations were performed in duplicates. Tem-
perature related absorbance spectra for stored fresh coco-
nut water were obtained by measuring it absorbance
(between 300 nm and 540 nm) after it has been main-
tained at a temperature (ranging from 35°C to 99.8°C) for
10 minutes.
Dry Matter Content
The amounts of dry matter in fresh, autoclaved and
radiated coconut water were determined before and after
one month of storage at 4°C. An aliquot of 1 mL of each
was centrifuged at 5000 rpm (Eppendorf centrifuge 5415
C) for 15 minutes. The pellets were air dried at 60°C
(GFL -7601 Hybridisation Incubator, GFL mbH, Burgwe-
del, Germany) and weighed repeatedly until a constant
weight was obtained (Sartorius CP64, Data Weighing
Systems, Grove IL, USA). All determinations were per-
formed in triplicates.
Total Carbohydrate
Total carbohydrate was determined according to the
Anthron’s method as reported by Eklöf et al., [13].
Statistical Analysis
MS. Excel microcomputer software (Microsoft Corpora-
tion) was used to obtain descriptive statistics (averages,
standard error etc) and percentage changes (increases
and decreases) in measured parameters. The student t-
test was used to analyse for statistical significance in the
differences in dry matter and the linear regression model
was used to analyse the relationship between abhorrence
and temperature.
Results
Coconut Water and Related Changes
The absence of colonies on nutrient agar plate and the
lack of change in the turbidity of LB broth inoculated
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 2 of 9
with coconut water indicate that the extraction process
was aseptically performed and the coconut water was
sterile. The average volume of water obtained ranged
between 250.0 and 300.0 mL (average of 266.7 ± 16.7
mL) with an average pH of 6.33 ± 0.17. The thickness
of the endosperm ranged between 0.5 cm and 0.6 cm
(average of 0.53 ± 0.03 cm).
After the gamma irradiation (at 5 kGy) and autoclaving,
a pale and intense yellow colour was observed respec-
tively (Figure 1). However, gamma irradiated, autoclaved
and fresh coconut water both recorded a wavelength of
maximum absorbance (l
max
) of 385 nm (Figure 2A).
Relative to the fresh coconut water (FCW), a stable and
an increase in turbidity was observed for the autoclaved
coconut water (ACW) and irradiated coconut water
(RCW) respectively. These differences in turbidity were
confirmed by the visible spectrum (Figure 2A) at wave-
lengths between 580 nm and 600 nm; where the absor-
bance of the autoclaved coconut water was just below
that of the fresh coconut water, and both were lower
than that of the irradiated coconut water. I.e. ACW =
FCW < RCW.
The visible light spectra of all three samples of coconut
water (fresh, autoclaved and irradiated) before storage
was presented as Figure 2A. After two weeks of storage,
fresh coconut water developed a pale yellow colour,
increased in turbidity (Figure 1) and opened with a puff
sound. Its average increase in absorbance across the
range of wavelengths (Figure 2B) was also extensive, (by
about 500%). However, a slight increase in turbidity with
no change in the intensity of the pale yellow colour and a
mild increase in average absorbance (about 25%) were
observed for irradiated coconut water (Figure 2B).
On the other hand, there were changes in the turbid-
ity and the intensity of the yellow colour of autoclaved
coconut water (Figure 1D and 1E). The related spectra
indicated an increase in absorbance by about 108%
between 560 nm to 600 nm and a decrease by 41%
between 280 nm and 500 nm respectively (Figure 2B).
After four weeks of storage, the absorbance of the auto-
claved coconut water was restored to almost what it was
before storage, while the absorbance of the irradiated
coconut water did not show any appraisable change. On
the other hand, the absorbance of fresh coconut water
further increased by about 44% and became intense yellow
(Figure 2B and 2C). However, the increase in the intensity
of the yellow colour was masked by the increase in turbid-
ity; the true colour was observed after centrifugation to
pellet the suspended matter.
In summary, the autoclaved coconut water remained
relatively a clear medium while the fresh and irradiated
coconut water increased in turbidity during storage. For
the fresh and irradiated coconut water, sediments were
observed at the base of the containing bottles by the 4
th
week of storage. It was also observed that the cloudiness/
turbidity of various stored coconut water samples masked
their yellow colour. It should be noted that absorbance
between 600 nm and 700 nm has been used extensively
for measuring the growth (increase in cells) of bacteria in
liquid cultures; as such a cloudy solution/medium will
most likely cover-up little changes in turbidity of a grow-
ing bacteria culture, particularly at its lag phase.
A B C DE
Figure 1 Pictures of coconut water before and after sterilisation and storage at 4°C.(A). Fresh coconut water after extraction. (B). Fresh
coconut water after two weeks of storage at 4°C. (C). Irradiated coconut water after four weeks of storage at 4°C (no significant difference to
the observation before and after two weeks of storage). (D). Autoclaved mixture of coconut water before storage. (E). Autoclaved coconut water
after two weeks of storage. For B, C and E, the increase in turbidity/cloudiness of the water masked the intensity of the yellow colour; however,
it was observable after centrifugation.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 3 of 9
Changes in Dry Matter
The changes in turbidity observed were further studied
by determining the dry matter (mg/mL) of the coconut
water samples before and after the one month storage
period (Figure 3 and table 1). The dry matter content of
three different samples of fresh coconut water did not
differ significantly (p > 0.05) with each other. After
autoclaving, the dry matter reduced by 19.1% (not sig-
nificant, p>0.05) but increased by 14.3% after gamma
irradiation (not significant, p > 0.05). After four weeks
of storage at 4°C, fresh, autoclaved and irradiated coco-
nut water, increased in dry matter by 52.0%, 23.5% and
5.0% respectively. Regarding the combined effect of
autoclaving and storage, and irradiation and storage, it
was observed that the latter resulted in a 20% net
increase in dry matter while the former resulted in no
net change in dry matter within the period of storage.
Changes in UV Spectra
In comparison to the UV spectrum of fresh coconut
water stored for two weeks, a dramatic reduction in
absorbance (69.5% to 72.0%) was observed across all
wavelengths for the spectrum of autoclaved coconut
water stored for the same period. However, the pattern
of both spectra was not so different (Figure 4A and 4B).
With respect to gamma irradiated coconut water stored
for the same period, UV absorbance at wavelengths less
than 260 nm were extensively reduced while those at
1.80
2.00 Fresh CW
Autoclaved CW 1.80
2.00
g
e
AB
1.20
1.40
1.60
o
re storage
Radiated CW
1.20
1.40
1.60
w
eeks of stora
g
0.60
0.80
1.00
sorbance bef
o
0.60
0.80
1.00
b
ance after 2
w
0.00
0.20
0.40
Ab
0.00
0.20
0.40
Absor
b
260 360 460 560 660
Wavelenght (nm)
260
Wavel
Fresh CW
Autoclaved CW 1.80
2.00
e
Fresh CW
Autocl ved CW
C
Radiated CW
1.20
1.40
1.60
o
nth of storag
e
Radiated CW
0.60
0.80
1.00
a
nce after 1 m
o
0.00
0.20
0.40
Absorb
a
460 660
enght (nm)
260 460 660
wavelenght (nm)
Figure 2 Visible-light spectra of coconut water before and after sterilisation and storage at 4°C.(A). After autoclaving at 121°C and 15 psi
for 20 minutes and gamma irradiation at 5 kGy, absorbance of fresh, autoclaved and irradiated coconut water within the visible light region
were measured. (B). After two weeks of storing the fresh, autoclaved and gamma irradiated coconut water at 4°C, their absorbance were
measured at 20 nm interval within the visible light region. (C). After one month in storage at 4°C the absorbance measurements were repeated
as in B, These were all plots of the average of three absorbance readings at each wavelength. Absorbance readings at wavelengths from 320 nm
to 340 nm were very unstable and as such were excluded from the plot.
08
1
1.2
1.4
1.6
1.8
2
a
tter (mg/mL)
0
0.2
0.4
0.6
0
.
8
Dry m
a
Figure 3 Dry matter content of coconut water before and after
sterilisations and storage at 4°C. Aliquots (1 mL) of the different
samples of coconut water were centrifuged and the supernatant
discarded (this was done in triplicates). The pellets were dried at 60°
C and weighed every 30 minutes until a constant weight was
obtained (Sartorius CP64 weighing balance). The averages of three
weights for each sample were plotted with the standard error
indicated as error bars. The large standard errors (error bars)
observed for the irradiated and fresh coconut water after storage
were as a result of the fact that the sediments settles to the base of
the bottle very fast when mixed before measuring out the needed
volume for analysis. FCW2, FCW3, FCW4, = are different samples of
fresh coconut water; SFCW3 = stored fresh coconut water sample;
ACW2 = autoclaved coconut water sample; SACW2 = stored
autoclaved coconut water sample; RCW = irradiated coconut water;
SRCW4 = stored irradiated coconut water sample.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 4 of 9
wavelengths greater than 260 nm changed marginally,
resulting in a drastic change in the pattern of the spec-
tra (Figure 4A and 4C).
After one month of storage, the overall UV absor-
bance of fresh coconut water reduced by an average of
30%, resulting in a slight change in the pattern of the
spectrum (Figure 4A). For autoclaved coconut water,
large reductions (between 80% and 76%) in the overall
absorbance were observed but with a resultant marginal
change in the pattern of the UV-spectrum (Figure 4B).
With regards to irradiated coconut water, stored for the
same period, a reduction (between 30% and 40%) and a
significant change in the pattern of the UV spectrum
was observed (Figure 4C).
Changes in pH
An average pH of 6.33 ± 0.17 was recorded for the fresh
coconut water, however, a reduction by 1.5 units was
observed after autoclaving and gamma irradiation (table 2).
After one month of storage, the pH of the fresh coconut
water reduced by an average 2.3 pH units while those of
autoclaved and irradiated coconut water, did not change. A
mixture of samples of fresh coconut water studied also
recorded similar observations.
Temperature and Colour Change
There was an increase in the absorbance of stored fresh
coconut water, (at 4°C for two weeks) as its temperature
increased between 35°C and 99.8°C (Figure 5A and 5B)
with a l
max
at 385 nm. An observable larger increase in
the intensity of the yellow colour was seen at tempera-
tures 95°C or higher.
Changes in Total Carbohydrate
Although autoclaving, irradiation and storage resulted in
the reduction of the total carbohydrate content of coco-
nut water by about 18.9%, 6.2% and 6.9% respectively
(Figure 6), the influence of the two sterilisation methods
on the total carbohydrate content of coconut water was
not significant (p > 0.05).
Table 1 Changes in dry matter content of coconut water
due to sterilisations and storage
Samples % Change in dry matter
After treatment Stored Combination
Fresh coconut water –52.0 –
Autoclaved coconut water -19.1 23.5 0.0
Radiated coconut water 14.3 5.0 20.0
The coconut water was autoclaved at 121°C and 15psi for 20 minutes or
Irradiated at 5 kGy. All storage was at 4°C for 1 month.
0.70
0.75
a
ge
0.70
0.75
a
ge
0.20
0.25
r
age
AB C
0.55
0.60
0.65
of RCW after stor
a
2 weeks
1 month
0.60
0.65
of FCW after stor
a
2 weeks
1 month
010
0.15
of ACW after sto
r
2 weeks
1 month
0.45
0.50
Absorbance
0.50
0.55
Absorbance
0.05
0
.
10
Absorbance
0.40
180 230 280 330
Wavelenght (nm)
0.45
180 230 280 330
Wavelenght (nm)
0.00
180 230 280 330
Wavelenght (nm)
Figure 4 UV spectra of fresh, autoclaved and irradiated coconut water after storage for two weeks and one month. After two weeks
and one month of storage of; (A). Fresh coconut water, (B). Autoclaved coconut water (121°C and 15 psi for 20 minutes) and (C). Gamma
irradiation (5 kGy) coconut water the UV absorbance between 200 nm and 300 nm were measured at an interval of 20 nm in triplicates. These
plots are of the average of three absorbance readings at each wavelength. FCW = Fresh coconut water. ACW = Autoclaved coconut water. RCW
= Irradiated coconut water.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 5 of 9
Discussion
The observed sterility of the extracted, sterilised and
stored coconut water imply the changes observed for
the fresh, stored and sterilised coconut water were not
due to contaminating microbes as was the case in the
study by Chowdhury et al., [14] who also studied coco-
nut fruits obtained from the local market, with no data
on the age of maturity.
Although the maturity of the fruit could not have been
determined accurately, the thickness of the endosperm
were within the range (between 4 and 10 mm) reported for
12 month old matured coconut fruit by Santoso, et al., [8].
The volumes of coconut water (266.7 ± 16.7 mL) were
lower than those reported (350 mL to 500 mL) by Camp-
bell-Falck et al., [7] and Santoso, et al., [8]. These variation
in volume of coconut water, as well as variations in enzyme
activity, pH and nutritive values have been reported to be
due to differences in plant variety, locality of cultivation
and the age of maturity (before and after eighth month),
[15,16].
The observed differences in colour and turbidity (Figure
1) were confirmed in the visible spectra (Figures 2A-C).
When exposed to oxygen, polyphenol oxidases catalyse the
hydroxylation of monophenols, such as in tyrosine (present
in coconut water), to O-diphenols; these are further oxi-
dised to O-quinones and semiquinone, which polymerised
to different extents, leading to the yellow, orange or brown
colours [11,16-18]. The added heating to 60°C or above
enhances the activity of the enzymes (PPO and POD),
which is reduced/lost at temperatures above 90°C [11].
Such colour changes (due to PPO and POD) have been
reported for Mueller-Hinton broth after autoclaving [19].
The change in colour after gamma irradiation is
explainable on similar bases, since irradiation with
gamma rays (ionizing radiations) leads to the formation
of hydroxyl phenal radicals (phenol side group of tyro-
sine and gallic acid) [20] which then serves as the pre-
cursor for the coloured O-quinones.
Table 2 pH variations in coconut water before and after
sterilisation and storage
Sample Initial
pH
Magnitude of pH decrease after
Autoclaving Irradiation Storage
Fresh coconut water 6.0-6.5 1.5 1.5 2.0
Mix coconut water 6.5 1.5 –2.5
Autoclaved mix coconut
water
5.0 ––0.0
Autoclaved coconut
water
5.0 ––0.0
Radiated coconut water 4.5 ––0.0
The coconut water was autoclaved at 121°C and 15psi for 20 minutes or
Irradiated at 5 kGy. All storage was at 4°C for 1 month.
1.2
1.4
A
06
0.8
1.0
a
nce of fresh CW
35.0 oC
45.0 oC
55.0 oC
65.0 oC
0.2
0.4
0
.
6
Absorb
a
75.0 oC
85.0 oC
95.0 oC
99.8 oC
0.0
250 300 350 400 450 500 550 600
Wavelenght (nm)
y = 0.0066x + 0.4075
R² = 0.8689
100
1.20
1.40
B
0.60
0.80
1
.
00
Absorbance
y = 0.0023x - 0.0039
R² = 0.9293
0.00
0.20
0.40
30 40 50 60 70 80 90 100 110
Temperature (oC)
385 nm 540 nm
Figure 5 Visible light spectrum and temperature profile of fresh coconut water stored for two weeks. Duplicates of 10 mL of fresh
coconut water were incubated at 35°C with an increase in temperature by 10°C every 10 minutes. At the end of each incubation period,
absorbances were measured at wavelengths intervals of 20 nm starting from 280 nm to 540 nm, (A). presents the visible light spectrum at each
incubation temperature. (B). presents the regression of the absorbance at the wavelength of maximum absorbance (l
max
, 385 nm) and
temperature as well as that between the absorbance at 540 nm and temperature.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 6 of 9
The visible light spectra after two weeks of storage
(Figure 2B) also indicate changes in colour and turbidity
of fresh, autoclaved and irradiated coconut water. Simi-
lar observations were made by Puchakawimol et al., [18]
where fresh coconut water became orange-yellow in col-
our and increased in the saturation of colour on the
later days of storage at room temperature. Also,
Chowdhury et al., [14] showed that after one month of
storage at 0°C, fresh coconut water changed in colour
and flavour, increased in pH and turbidity with the for-
mation of gas in addition to being contaminated with
fungi.
These observations suggest that the reactions of poly-
phenol oxidase and peroxidase, and the formation of the
insoluble matter continued during storage at 4°C. The
decrease and subsequent increase in colour intensity of
autoclaved coconut water after 2 weeks and one month
respectively, suggests that polyphenol oxidase and peroxi-
dase (which catalyse reversibly, both oxidation and reduc-
tion reactions [11,18,16,21]) in response to the reduction
and increase in oxygen (due to storage in an air tight con-
tainer for two weeks, and after opening the air tight con-
tainer respectively) converted the chromophore and its
precursors reversibly. However, the continuous increase in
the turbidity and absorbance above 540 nm (Figure 2B
and 5), suggests that the reactions forming the insoluble
matter and colour were still occurring and that the
enzymes involved where also heat resistant.
The increase in colour and turbidity of irradiated coco-
nut water for two weeks with no significant increase after
an additional two weeks needs further investigation for a
clearer understanding, since it is generally accepted that
the activity of POD and PPO are not significantly affected
by low dose (about 5 kGy) gamma radiations [22]. How-
ever, Frylinck et al., [23] detected a partial inactivation of
these enzymes in mango fruit after low dose gamma irra-
diation. Therefore, we ask, is it that in this study of coco-
nut water only small quantities of phenal radicals were
produced after irradiation? Or is it that the activities of
these enzymes were greatly reduced or lost after irradia-
tion? A study that will measure the activity of these
enzymes and estimate the changes in the concentration
of radical in fresh and irradiated coconut water before
and at regular time intervals during storage may help to
throw more light on this.
The results of the dry matter (Figure 3 and table 1) cor-
related with the changes in the absorption spectra
between the wavelength 560 nm and 600 nm (Figure 2A-
C), as an indication of turbidity. Autoclaving resulted in a
decrease in dry matter by 1.1%, seen as a lower absor-
bance of autoclaved coconut water compared to that of
fresh coconut water (Figure 2A). Conversely, irradiation
resulted in an increase in dry matter (Figure 2A) and an
associated increase in absorbance in that same range.
Similar correlations were observed after storage. (Figure
2B and 2C; table 1).
The results presented as Figure4demonstratethe
changes in UV absorption of coconut water due to a com-
bination of sterilisation and storage. Since coconut water
is a mixture and/or a solution of various biomolecules,
including aromatic amino acids (tyrosine), gallic acids, fla-
vour giving aromatic hydrocarbons, biphenal products of
the enzymes POD and PPO and others as reported by
Yong et al., 2009 [24], the significance of the changes in
UV absorption in relation to its biochemical and nutri-
tional components cannot be evaluated with this data.
However, these changes provide evidence for the occur-
rence of both physical and biochemical reactions (to dif-
ferent extents) after sterilisation and during storage at 4°C.
The interactions between biomolecules have been shown
as one of the physical processes by which the UV spectra
of a molecule may be changed [25]. On the other hand,
the reformation of aromatic system via proton and elec-
tron rearrangement or perturbations as a result of expo-
sure to radiations, are some of the chemical process that
influence and change absorption in the near UV region
[26].
The reduction in pH, as was observed after autoclaving,
irradiation and storage (table 2) was a result of the forma-
tion of the phenol radicals and oxides, leading to the for-
mation of the yellow colour. Stored fresh coconut water
experienced the greatest change in pH probably because
its POD and PPO activities were not reduced (as was
expected after autoclaving and gamma irradiation) and
therefore contributed to a higher release of protons.
80 0
100.0
120.0
140.0
160.0
180.0
200.0
bohydrate(mg/mL)
0.0
20.0
40.0
60.0
80
.
0
ACW
TotalCar
Beforetreatment
SFCW RCW4
Aftertreatment
Figure 6 Total carbohydrate content of fresh and sterilised
coconut water. The total carbohydrate was determined by the
Anthrone’s method. The plots are of triplicate determination with
the standard error indicated as the error bars. SFCW, = Stored fresh
coconut water; ACW = autoclaved coconut water; RCW = irradiated
coconut water. ‘Before treatment’and ‘After treatment’imply before
and after, autoclaving for ACW, storage for SFWC and radiation for
RCW.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 7 of 9
The temperature profile (Figure 5A and 5B) indicates
that the changes in absorbance at 385 nm (indicative of
colour) and 540 nm were linearly related to an increase in
the temperature of stored coconut water (R
2
of 0.87 and
0.93 respectively, p < 0.05).Thesedataindicatethatthe
reactions leading to the formation of the yellow colour
were increasingly activated by the increase in temperature
up to 99.8°C.
The reduction in the total carbohydrate of fresh coco-
nut water during storage (Figure 6) was the result of
reactions that use sugars and other simple carbohydrates
in the formation of the insoluble matter, (the base sub-
stance of the coconut endosperm) which resulted in the
increase in turbidity (Figure 1). Autoclaving, with its
associated high temperature and pressure, is known to
reduce nutritional value of media through the activation
of a myriad of reaction involving amino acids, sugars,
proteins and other carbohydrates [27,28]. This, most
likely resulted in the reduction of its total carbohydrate
(Figure 6). Gamma irradiation leads to the formation of
reactive radicals with little potential of being involved in
reactions such as those associated with sugars, as such, a
marginal reduction in total carbohydrate was observed
for Irradiated coconut water.
Considering the changes observed thus far, it would be
convenient to measure the increase in bacteria cells
(growth) by measuring the dry biomass or turbidity of bac-
teria culture in fresh, autoclaved or gamma irradiated
coconut water with out storage. This is because prior to
storage, all of the three coconut water samples recorded
low turbidity, low dry matter (< 1.5 mg/mL) and a wave-
length of maximum absorbance of less than 600 nm.
These would not obscure small changes in absorbance/
turbidity of a growing bacteria culture, measured at wave-
lengths equal to or greater than 600 nm. However, for
both stored fresh and gamma irradiated coconut water,
the high and increasing turbidity and dry matter content
(> 1.5 mg/mL) during storage imply possible difficulties in
recording small changes during the measurements of tur-
bidity or dry biomass (particularly at the lag phase) of a
growing bacteria culture. The same possibility of inconsis-
tencies and non-reproducibility is expected with auto-
claved coconut water because of the decrease and
subsequent increase in its turbidity and dry matter despite
that these were low.
Conclusion
Sterilization of fresh coconut water by either autoclaving
or low dose gamma irradiation led to the development of
an intense or pale yellow colour respectively. Also asso-
ciated with these, were different extents of variations in
turbidity, dry matter and pH. The storage of fresh coconut
water at 4°C for both two and four weeks resulted in the
development of an intense yellow colour and a striking
change in turbidity, dry matter and pH.
The use of turbidity measurement for measuring the
growth of bacteria in fresh, autoclaved and gamma irra-
diated coconut water is practicable without any possibi-
lityofinterferencebytheverylowturbidityandbright
colours of these different coconut waters. However, this
may result in consistencies and, or non-reproducible
data if stored fresh, stored autoclaved or stored gamma
irradiated coconut water were used.
Abbreviations
FCW: Fresh coconut water; RCW: Irradiated coconut water; ACW: Autoclaved
coconut water; SFCW: Stored fresh coconut water; SRCW: Stored irradiated
coconut water; SACW: Stored autoclaved coconut water.
Acknowledgements
We are thankful to our senior colleagues for their useful suggestions and
support for this study. Specifically we are grateful to Mr. Oti Kwasi Gyamfi, CCRC
and Mr. David Bansa, NRC. We are also thankful to Miss. Margaret Dadzie and
Mr. Kofi Bedzera, Mr. Maxwell Ofori Appiah and Mr. Jonathan Okai Armah
(Gamma Irradiation Facility, GAEC), and Patricia Agyare for their assistance.
Authors’contributions
AKA conceived the study, and was involved with its design and the
performance of all laboratory analyses, statistical analyses and drafted the
manuscript. EDD participated in the design of the study and performance of
all laboratory analyses, RA was involved with the design, acquisition of
samples and participated in the performance of all the laboratory analyses
and the drafting of the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 1 July 2011 Accepted: 27 October 2011
Published: 27 October 2011
References
1. Fonseca AM, Monte FJQ, da Conceic M, de Oliveiraaão F: Coconut water
(Cocos nucifera L.) –A new biocatalyst system for organic synthesis.
Journal of Molecular Catalysis B: Enzymatic 2009, 57:78-82.
2. Walter EHM, Kabuki DY, Esper LMR, Sant’Ana AS, Kuaye AY: Modelling the
growth of Listeria monocytogenes in fresh green coconut (Cocos
nucifera L.) water. Food Microbiology 2009, 26:653-657.
3. Pradera ES, Fernandez E, Calderin O: Coconut water, a clinical and
experimental study. Amer J Dis Child 1942, 64:977-995.
4. DeSilva CC, Perera PM, Dias AP: Coconut water. Pediatria Int 1959, 9:225.
5. Olurin EO, Durowoju JEO, Bassir O: Intravenous coconut water therapy in
surgical practice. West Afr Med J 1972, 21:124-131.
6. Vigliar R, Sdepanian VL, Fagundes-Neto U: Biochemical profile of coconut
water from coconut palms planted in an inland region. J Pediatr (Rio J)
2006, 82:308-312.
7. Campbell-Falck D, Thomas T, Falck TM, Tutuo N, Clem K: The intravenous
use of coconut water. Am J Emerg Ied 2000, 18:108-111.
8. Santoso U, Kubo K, Ota T, Tadokoro T, Maekawa A: Nutrient composition
of kopyor coconuts (Cocos nucifera L.). Food Chemistry 1996, 51(2):299-304.
9. Jayalakshmy A, Arumughan C, Narayanan CS, Mathew AG: Changes in
chemical composition of coconut water during maturation. J Food Sci
Technol 1986, 23:203-207.
10. Pue AG, Riva W, Sundarao K, Kaluarin C, Singh K: Preliminary studies on
changes in the coconut water during maturation of the fruit Sci.Edited
by: New Guinea, M 1992, 2:81-84.
11. Matsui KN, Gut JAW, de Oliveira PV, Tadini CC: Inactivation kinetics of
polyphenol oxidase and peroxidase in green coconut water by
microwave processing. Journal of Food Engineering 2008, 88:169-176.
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 8 of 9
12. Mandal SM, Dey S, Mandal M, Sarkar S, Maria-Neto S, Franco OL:
Identification and structural insights of three novel antimicrobial
peptides isolated from green coconut water. Peptides 2009, 30:633-637.
13. Eklöf JS, Gullström M, Björk M, Asplund ME, Hammar L, Dahlgren A,
Öhman MC: The importance of grazing intensity and frequency for
physiological responses of the tropical sea grass. Thalassia hemprichii.
Aquatic Botany 2008, 89(3):337-340.
14. Chowdhury MGF, Rahman MM, Islam TAFM, Islam MS, Islam MS: Processing
and Preservation of Green Coconut Water. J Innov Dev Strategy 2009,
2(3):01-05.
15. Puchakawimol P, Jangchud K: Study on the quality changes of aromatic
coconut at different maturity. Proceedings of the 41st Kasetsart University
Annual Conference Bangkok, Thailand: (in Thai with English abstract) 2003,
166-174.
16. Duarte ACP, Coeiho MAZ, Leite SGF: Identification of peroxidase and
tyrosinase in green coconut water. Cien Tecnol Aliment 2002, 3(5):266-270.
17. Murasaki-Aliberti N, da C, Da Silva RMS, Gut JAW, Carmen C: Thermal
inactivation of polyphenoloxidase and peroxidase in green coconut
(Cocos nucifera) water. Inter J Food Sci and Techn 2009, 44(12):2662-2668.
18. Puchakawimol P, Jangchud K, Wuttijumnong P, Mahakarnjanakul W: Quality
of burnt aromatic coconut. Proceedings of the 43rd Kasetsart University
Annual Conference, Bangkok, Thailand (in Thai with English abstract) 2005,
586-593.
19. Galeazzi L, Groppa G, Giunta S: Mueller-Hinton Broth Undergoes Visible
Oxidative Color Changes in the Presence of Peroxidase and Hydrogen
Peroxide. J Clin Microbiol 1990, 28(9):2145-2147.
20. Maciel MI, Oliveira SL, Silva IP: Effects of different storage conditions on
preservation of coconut (Cocos nucifera) water. Journal of Food Processing
and Preservation 1992, 16:13-22.
21. Weng Z, Hendrickx M, Maesmans G, Tobback P: Immobilized peroxidase: a
potential bioindicator for evaluation of thermal processes. Journal of
Food Science 1991, 56(2):567-570.
22. Kim D, Song H, Lim S, Yun H, Chung J: Effects of gamma irradiation on
the radiation-resistant bacteria and polyphenol oxidase activity in fresh
kale juice. Radiation Physics and Chemistry 2007, 76(7):1213-1217.
23. Frylinck L, Dubery IA, Schabort JC: Biochemical changes involved in stress
response and ripening behavior of g-irradiated mango fruit.
Phytochemistry 1987, 26:684-686.
24. Yong JWH, Ge L, Ng YF, Tan SN: The Chemical Composition and
Biological Properties of Coconut (Cocos nucifera L.) Water. Molecules 2009,
14:5144-5164.
25. Nakamura Y, Tonomura Y, Hagihara B: Change in the Ultraviolet Spectrum
of Solubilized Ca
2+
-Dependent ATPase from Sarcoplasmic Reticulum due
to Binding with Ca
2+
Ions. J Biochem 1979, 86(2):443-446.
26. Nakano H, Tamai N, Nii M, Tsukamoto M, Abe N: Change in the UV-VIS
Absorbance of Amino Acids as a Result of Femtosecond Laser
Irradiation. Journal of Laser Micro/Nanoengineering 2007, 2(1):100.
27. Onyeike EN, Omubo-Dede TT: Effect of heat treatment on the proximate
composition, energy values, and levels of some toxicants in African yam
bean (Sphenostylis stenocarpa) seed varieties. [abstract]. Plant Foods Hum
Nutr 2002, 57(3-4):223-31.
28. Nutrition: decontamination of diets. In the Mouse in biomedical research..
Second edition. Edited by: Fox JG, Barthold SW, Davisson MT, Newcomer
CE, Quimby FW, Smith AL. California Academic Press; 2007:362-364.
doi:10.1186/1756-0500-4-451
Cite this article as: Awua et al.: Exploring the influence of sterilisation
and storage on some physicochemical properties of coconut (Cocos
nucifera L.) water. BMC Research Notes 2011 4:451. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Awua et al.BMC Research Notes 2011, 4:451
http://www.biomedcentral.com/1756-0500/4/451
Page 9 of 9
