International Food Research Journal 22(2): 631-637 (2015)

Journal homepage: http://www.ifrj.upm.edu.my

Effectiveness of ascorbic acid and sodium metabisulfite as anti-browning

agent and antioxidant on green coconut water (Cocos nucifera) subjected to
elevated thermal processing
*
Tan, T. C., Cheng, L. H., Bhat, R., Rusul, G. and Easa, A. M.

Food Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800
USM, Penang, Malaysia

Article history Abstract

Received: 7 April 2014 Thermal processing of green coconut water (GCW) caused non-enzymic browning and
Received in revised form: development of rancidity. Effect of the addition of several combinations of ascorbic acid
9 September 2014 (AA) (0 to 100 ppm) and sodium metabisulfite (SMB) (0 to 30 ppm) on brown discolouration
Accepted: 12 September 2014
and rancidity of GCW during elevated thermal processing (121°C for 5 min at 15 psi) was
investigated. Addition of AA and/or SMB significantly (P<0.05) reduced brown discolouration
Keywords of processed GCW, with SMB being approximately 7 times more effective than AA. Rancidity
in GCW was significantly (P<0.05) reduced with the addition of SMB and/or AA, with SMB
Ascorbic acid being the most effective; approximately 35 times more effective than AA, respectively. SMB
Sodium metabisulfite can be recommended as an effective controller of browning and rancidity in thermally processed
Coconut water
GCW.
Thermal processing © All Rights Reserved

Introduction coconut water, ready-to-drink coconut water posed to

have great potential in beverage market.
Consumers living in tropic countries can easily To commercialise coconut water as a ready-to-
enjoy fresh coconut water straight from the coconuts. drink beverage, adequate heat treatment is important
However, for those living outside of the tropics, whole because coconut water is susceptible to microbial and
coconuts can be hard to find. Coconut water has been oxidative enzymatic spoilage (Campos et al., 1996;
dubbed “miracle water” for its numerous health- Tan et al., 2014). Thermal processing is one of the most
giving benefits. Besides being highly nutritious due widely used preservation method in food industries
to the presence of sugars, vitamins, minerals and because high temperature could provide inactivation
proteins (Santoso et al., 1996; Yong et al., 2009; Tan of both microbial and enzymatic activities. However,
et al., 2014), coconut water is believed to be useful thermal processing could also initiate various types of
in preventing and relieving many health problems, chemical reactions, with some causing deterioration
including dehydration, constipation, digestive on the quality and sensorial attributes (Aguiar et al.,
problems, fatigue, heatstroke, diarrhea, kidney stones 2012; Igual et al., 2014). Non-enzymic browning,
and urinary tract infections (Campbell-Falck et al., which could be caused by phenols oxidation, ascorbic
2000). Presence of natural cytokinin nucleotides acid (AA) degradation and Maillard reaction (Li et al.,
(kinetin, kinetin riboside and trans-zeatin riboside-5′- 2008; Lima et al., 2009), and rancidity (Fan, 2002),
monophosphate) in coconut water boosts the benefits during thermal processing are two main reasons for
of drinking coconut water for its anti-ageing effect rejections by consumers. Phenolic compounds in
(Ge et al., 2006). Mandal et al. (2009) had isolated coconut water could be oxidised in the similar way
three antimicrobial peptides from coconut water, as tea catechins during pasteurisation, which leads to
hence indicating the potential of coconut water as a brown discolouration (Kim et al., 2007). Apart from
natural source for antibiotics. Recently, Chang and that, brown discolouration during thermal treatment
Wu (2011) have detected the presence of (+)-catechin could also be due to the occurrence of Maillard
and (−)-epicatechin, which have anti-microbial, reaction e.g. during pasteurisation, since protein
antioxidant and anti-cancer activities, in coconut and reducing sugars are available in coconut water
water. Study done by Ismail et al. (2007) has proven (Jayalekshmy and Mathew, 1990; Tan et al., 2014).
that coconut water has the potential to be a natural Hence, antioxidant and anti-browning agents could
alternative to carbohydrate-electrolyte sports drinks be added to control the occurrence of non-enzymic
for its rehydration ability. Knowing the greatness of browning (Damasceno et al., 2008) and formation of

*Corresponding author.
Email: [email protected]
Tel: +604-6534302; Fax: +604-6573678
632 Tan et al./IFRJ 22(2): 631-637

free fatty acids (Das Purkayastha et al., 2012) during this study were of analytical grade, except for AA and
thermal processing. SMB, which are of food grade.
Various approaches to control the development
of browning and rancidity have been carried out, Coconut water
e.g. thermal inactivation of enzymes and addition Green coconuts (aged 6 months old from coconut
of chemical additives. Nowadays, antioxidants are variety of Malayan Tall) were purchased from Anba
one of the common methods to preserve foodstuffs Coconuts located at Abu Siti Lane, Penang. Surface
by retarding deterioration, rancidity and/or of the coconut husks was cleaned with distilled water
discolouration (Arogundade and Mu 2012). Among and 1% bleach (Clorox) before a stainless steel knife
the wide variety of additives, sulphites and AA are was used to perforate the fruit mesocarp. GCW was
the popular additives used in food industry. Browning manually extracted from the coconut fruit and filtered
inhibition by sulphites is caused by the reaction through muslin cloth. The filtered GCW from several
between sulphite ions and quinines (Danilewicz et al., fruits (3 to 5 coconuts with similar maturity age) was
2008). Despite its effectiveness, usage of sulphites as pooled in a 2 L Schott bottle and kept temporary in
antioxidants is governed by restrictions set by World an icebox with ice packs. All GCW was prepared
Health Organisation (WHO) due to health-related on the same day of purchase and extraction. Sample
issues incurred by sulphite-sensitive individuals preparation was triplicated.
(Li et al., 2008). Increased suspicious of toxic and
carcinogenic effects of synthetic antioxidants resulted Thermal treatment of GCW
in demand for healthier and natural alternatives, such The effect of AA and SMB on brown discolouration
as AA. The effectiveness of AA as anti-browning of GCW was investigated by adding AA (0, 50 and
agent is reflected by its ability to reduce phenoxyl 100 ppm) and SMB (0, 15 and 30 ppm) to the GCW
radicals and quinone forms of phenolics back to samples. GCW samples (80 mL) with added AA and/
colourless diphenols in a coupled oxido-reduction or SMB were transferred into individual 100 mL
reaction (Louarme and Billaud, 2012). Despite the Schott bottle. The GCW samples were subjected to
efficiency of AA, its use is limited by the organoleptic elevated thermal processing (121°C for 5 min at 15
consideration. Campos et al. (1996) reported that the psi) using a SX-700 high-pressure steam steriliser
flavour quality of coconut water was altered when (Tomy, Japan). After the thermal treatment, all GCW
AA exceeded 200 ppm. samples were cooled in ice water prior to browning
To our knowledge, very little studies have been index and malonaldehyde measurements.
performed on coconut water processing, with majority
of studies on coconut water were related to enzyme Browning index
inactivation, physicochemical properties of coconut Brown discolouration of GCW samples was
water and health benefits of coconut water. The aim determined using browning index measurement and
of this paper was to provide information on the effect was performed using a CM-3500d spectrophotometer
of AA and sodium metabisulfite (SMB) in brown (Konica Minolta, Japan) at wavelength of 420 nm
discolouration and rancidity of green coconut water according to method as described by Tan et al.
(GCW) during elevated thermal processing (121°C (2012). GCW was transferred into a 10 mm optical
for 5 min at 15 psi). In addition, the effectiveness quartz cell (Konica Minolta, Japan) to measure
between these additives in reducing the development absorbance. The absorbance at wavelength 550 nm
of brown discolouration and rancidity of GCW during was also recorded to correct for any turbidity in the
elevated thermal processing will also be compared. GCW (Tan et al., 2012). Browning index was defined
based on the following equation:
Materials and Methods
Browning index = Abs420 – Abs550 (Eq. 1)
Materials
Ascorbic acid (AA), trichloroacetic acid where, Abs420 is the absorbance at 420 nm and
(TCA), butylated hydroxytoluene (BHT) and Abs550 is the absorbance at 550 nm.
1,1,3,3-tetraethoxypropane (TEP) used in this
study were purchased from Sigma-Aldrich Co., Measurement of malonaldehyde using TBA-based
St. Louis, USA. Sodium metabisulfite (SMB) and assay
2-thiobarbituric acid (TBA) were purchased from Measurement of malonaldehyde in GCW was
Merck KGaA, Darmstadt, Germany and Alfa Aesar, based on the method as described by and Fan (2002)
Lancashire, UK, respectively. All chemicals used in with slight modification. GCW (5 g) was added to
 Tan et al./IFRJ 22(2): 631-637 633

a test tube containing 5 mL of either Solution A comparison to AA using the following equation:
(containing 20% TCA and 0.01% BHT) or Solution
B (containing 20% TCA, 0.01% BHT and 0.65% Relative effectiveness = CX2 / CX1 (Eq. 5)
TBA). Samples were heated at 95°C for 35 min in
a WB22 water bath (Memmert, Germany). After where, CX2 is the coefficient of X2, which
heating, samples were cooled in ice water for 15 represents SMB, and CX1 is the coefficient of X1,
min and absorbance at 440, 532 and 600 nm were which represents AA.
recorded using UV-1650 PC spectrophotometer
(Shimadzu, Japan). Results and Discussion
Standard solutions of TEP were prepared
according to the method as described by Pegg Effect of AA and SMB on brown discolouration
(2001) to give final concentrations of 0.001, 0.004, All GCW samples were subjected to elevated
0.006, 0.008 and 0.01 mM in distilled water. All the thermal processing (121°C for 5 min at 15 psi) using
standard solutions were added with 5 mL of 0.65% an autoclave. All processed GCW showed brown
TBA. Standards were heated at 95°C for 35 min in discolouration with different extent of browning
a water bath. After heating, standards were cooled intensity. The amount of AA and SMB added had an
in ice water for 15 min and absorbance 532 was influence on the magnitude of brown discolouration
recorded. Standard curve with absorbance value of GCW, with higher AA and SMB concentrations
against standard concentration (in M) was plotted. yielded GCW with lower browning index (Table 1).
TBA value, expressed as mg malonaldehyde Table 1. Browning index and TBA value obtained from
equivalents/kg sample, was calculated according to GCW samples added with AA and SMB prior to elevated
the following equations: thermal processing (121°C for 5 min at 15 psi)

A = (Abs532sol.B – Abs600Sol.B) – (Abs532Sol.A –

Abs600Sol.A) (Eq. 2)
B = (Abs440Sol.B – Abs600Sol.B) × 0.0571 (Eq. 3)
TBA value = [S-1 × MW × 106 × (A – B)] / m (Eq. 4)

where, Abs440 is the absorbance at wavelength

440 nm, Abs532 is the absorbance at wavelength 532
nm, Abs600 is the absorbance at wavelength 600
nm, S-1 is the gradient of the standard curve, MW is
the molecular weight of malonaldehyde (72.03), 106
converts the units so that results can be expressed as
mg malonaldehyde equivalents/kg sample and m is
the sample mass (in g).

Statistical analysis
A 3×3 factorial design was used to study the
effects of two factors; AA concentration (X1) and
SMB concentration (X2), on browning index and TBA
value of GCW samples. Three AA concentrations (0,
1
Control represents fresh GCW without any thermal processing.
2
AA: ascorbic acid; SMB: sodium metabisulfite.
50 and 100 ppm) and three SMB concentrations (0, 3
TBA (thiobarbituric acid) value expressed as mg malonaldehyde
15 and 30 ppm) were tested. Three replicates were equivalents/kg sample.
carried out for a total of 27 runs. Analysis of variance Comparisons within the same column (a-e) are shown in the
(ANOVA), Tukey’s test for multiple comparisons, table with the data written as mean ± standard deviation of 3
replicates. Means followed by different letter are significantly
interaction plots and regression equations were used
different at P<0.05 level of significance according to Tukey's
for analysing the data. SPSS version 20 (SPSS, USA) Multiple-Range Test.
was used to complete these statistical analyses. The brown discolouration of GCW could be a
Relative effectiveness of SMB as anti-browning consequence of enzymatic browning reactions and
agent (measured by the ability to reduce browning non-enzymatic browning reaction, e.g. Maillard
index) or as antioxidant (measured by the ability reaction and phenol oxidation (Li et al., 2008).
to reduce TBA value) was calculated using the However, since heat treatment at 90°C for 550 s was
coefficients from the regression equations generated. sufficient to inactivate both polyphenol oxidase and
Relative effectiveness of SMB was calculated in
634 Tan et al./IFRJ 22(2): 631-637

Figure 1. Interaction plots for (a) mean browning index and (b) mean TBA value as a function of AA and SMB. Means
with different letter are significantly different at P<0.05 level of significance according to Tukey's multiple-range

peroxidase present in coconut water (Campos et al., and Siriphanich, 2012).

1996) the contribution of enzymatic browning was To further understand the effect of AA and SMB
small. Owing to the elevated thermal processing concentrations on the brown discolouration of GCW,
condition used in this study, brown discolouration interaction plots were constructed and comparisons
of coconut water could be caused mainly by non- between the tested levels of concentration for the same
enzymatic reaction, such as the Maillard reaction, additive were determined using Tukey’s Multiple-
degradation of AA and/or oxidation of phenolic Range Test. When AA and SMB were absence in
compounds (Li et al., 2008; Lima et al., 2009), GCW, the browning index was high and increased by
since the thermal treatment applied was sufficient approximately 8 times, from 0.0085 (fresh untreated
to deactivate the enzymes responsible for brown CGW) to 0.0689. With the addition of AA from 0 to
discolouration (Campos et al., 1996; Tan et al., 50 ppm, browning index was significantly (P<0.05)
2014). (+)-Catechin and (−)-epicatechin found in reduced. However, no significant difference (P>0.05)
coconut water (Chang et al., 2011) could be oxidised was observed when AA concentration was increased
in a similar way as tea catechins during heating or from 50 and 100 ppm (Figure 1A). On the contrary,
pasteurisation, which causes green tea liquor to browning index decreased significantly (P<0.05)
became darker and deeper yellow in colour (Kim et with an increase in SMB concentration from 0 to 30
al., 2007). ppm (Figure 1A).
Addition of AA and SMB into GCW had Based on the data obtained, a regression equation
significant (P<0.05) effect on brown discolouration (Eq. 6) was generated to estimate the degree of
in all samples (Table 2). The effectiveness of AA in effectiveness of AA and SMB in reducing brown
controlling browning was previously demonstrated discolouration of GCW samples as a result of the
on slices of carambola (Weller et al., 1997), white elevated thermal processing. Based on the coefficients
wines (Peng et al., 1998), fresh-cut cantoloupe melon of X1 and X2 shown in Eq. 6, relative effectiveness
(Lamikanra and Watson, 2001) and banana smoothies of SMB in comparison to AA was calculated using
(Wang et al., 2013), while the effectiveness of Eq. 5. SMB was found to be relatively more effective
sulphites in controlling browning had also been (relative effectiveness of ~7×) in reducing the
demonstrated on slices of ivy gourd (Kulkarni and browning index when compared to AA.
Vijayanand, 2012) and coconut husk (Mohpraman
 Tan et al./IFRJ 22(2): 631-637 635

Table 2. The results of the factorial experiment for used in this study (Table 1). The amount of AA and
browning index and TBA value obtained from GCW SMB added into GCW had an influence on the level of
samples added with AA and SMB prior to elevated thermal rancidity of GCW samples; with higher AA and SMB
processing (121°C for 5 min at 15 psi) concentrations yielded GCW with lower TBA values.
The interaction plot for AA shows no significant
changes on the TBA value when AA was added at
concentration below 50 ppm (Figure 1B). However,
significant (P<0.05) reduction in TBA value was
observed at AA concentration above 50 ppm. On the
other hand, an increase in SMB concentration from 0
to 30 ppm significantly (P<0.05) decreased the TBA
values (Figure 1B).
Based on the data obtained, a regression equation
(Eq. 7) was generated to estimate the degree of
effectiveness of AA and SMB concentrations in
reducing the TBA value of GCW samples as a result
of elevated thermal processing applied in this study.
Relative effectiveness of SMB was found to be more
effective (relative effectiveness of ~35×) in reducing
the TBA value when compared to AA.

Y = 48.77 – 1.78E-2X1 – 6.24E-1X2 + 3.10E-4X1X2

1
X1, ascorbic acid concentration (0, 50 and 100 ppm); X2, (Eq. 7)
sodium metabisulfite concentration (0, 15 and 30 ppm).
2
TBA: thiobarbituric acid. where, Y is the TBA value (in mg malonaldehyde
Y = 0.0646 – 1.34E-4X1 – 9.29E-4X2 + 4.77E-6X1X2 equivalents/kg sample), X1 is the concentration of
(Eq. 6) AA (in ppm) and X2 is the concentration of SMB (in
ppm).
where, Y is the browning index, X1 is the concentration
of AA (in ppm) and X2 is the concentration of SMB Conclusion
(in ppm).
Significant (P<0.05) decrease in both browning
Effect of AA and SMB on rancidity index and TBA value was observed in GCW samples
GCW have been reported to contain a trace with AA and/or SMB subjected to elevated thermal
amount of oil, and the level of coconut oil has been processing (121°C for 5 min at 15 psi). SMB appeared
shown to increase with maturity of the coconut to be approximately 7 and 35 times more effective
fruit (Santoso et al., 1996; Tanqueco et al., 2007; in suppressing brown discolouration and rancidity,
Tan et al., 2014). Presence of coconut oil could respectively, during elevated thermal processing
cause rancidity to form free fatty acids, which will when compared to AA. Hence, SMB is considered
lead to the deterioration of organoleptic quality of relatively more effective, when compared to AA,
the coconut water (Das Purkayastha et al., 2012). as an anti-browning agent as well as antioxidant for
Study done by Fan (2002) showed that a thermally GCW subjected to elevated thermal processing.
processed apple juice had a higher malonaldehyde
concentration compared to fresh apple juice. To Acknowledgements
overcome this problem, one could either remove the
oil from coconut water during processing or suppress The financial support of Post-Doctoral Fellowship
the occurrence of rancidity during processing with from Universiti Sains Malaysia for Dr. Tan Thuan
the aid of antioxidants, with the latter one being of Chew was gratefully acknowledged. We gratefully
interest in this study. acknowledge and are indebted to the anonymous
Both additives showed significant (P<0.05) effect referees for comments and constructive suggestions
in reducing TBA value in GCW (Table 2). In the provided for improving the manuscript.
absence of AA and SMB, occurrence of rancidity (as
denoted by the increased in TBA value) was detected
as a result of elevated thermal processing conditions
636 Tan et al./IFRJ 22(2): 631-637

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