INTRODUCTION
Hylocereus polyrhizus or more commonly known as the pitaya or
the dragon fruit is a member of the Cactaceae family from the genus Hylocereus.
The flesh of the fruit is red-purple in colour when ripened with minute
black seeds interspersed and has gained a growing interest for cultivation
in Malaysia (Hoa et al., 2006). This fruit has gained much interest
in the society because of its exotic features, attractive colours, nutritional
value and pleasant taste (Le Bellec et al., 2006).
Natural dyes are colorants obtained from biological matter through mechanical
retention, covalent chemical bonds formation or complexes with salts or
metal formations, physical absorption, or by solutions. Natural colorants
have a vast economic significant because the dye trade has a world market
worth £2.5 billion/year.
Betalains were thought to be flavonoids but they have been found to contain
nitrogen and do not change colour reversibly in the same way as anthocyanins
do to pH. Betalains were first extracted from the red beet (Beta vulgaris)
and is used mainly for food colouring. The extract contains red and yellow
pigments, namely the betacyanins and betaxanthins, respectively. Betacyanin
is the major component (95%) of the red pigments in the extract. Never
occurring together in the same source, the betalains are found in place
of the anthocyanin group of pigments in plants of the Caryophyllales Family
(Strack et al., 2003; Kimler et al., 1971; Cai et al.,
2005). However, there is a demand for alternative source other that the
red beet because of the unfavourable earthy flavour caused by geosmin
and pyrazine derivatives, as well as high nitrate concentrations associated
with the formation of carcinogenic nitrosamines (Esquivel et al.,
2007). Dragon fruit is one of the new focuses for the next source
of red dye because it is rich in betalains which are the similar array
of colour pigments found in beetroot and devoid of the mentioned drawbacks.
The objective of this research is to study the colorant of the dragon
fruit peel at various temperatures, pH and length of time to heat exposure
to determine the optimal condition for extracting the dye as natural colorant.
The extraction method employed the use of distilled water, since betacyanin
peel is water soluble. Stability of the colorant towards light was studied,
spectrophotometric and pH analysis was carried out for all samples.
MATERIALS AND METHODS
Plant material: Dragon fruits were obtained from Multi Rich farm
in Nilai, Negeri Sembilan, Malaysia on 21 February 2008. All fruits were
freshly harvested and transported to the postharvest laboratory in University
of Malaya for experiment. Fruits were treated with Benomyl 0.05% and air
dried overnight. Fruit pulp was cut into small cubes, frozen under liquid
nitrogen and stored in -20°C until used.
Sample measurements: Absorbance for all samples were measured
at 538 nm using a spectrophotometer (Pharmacia, Ultrospec II) to determine
total betalain concentration while pH was measured using a Hanna pH meter.
All extracts were filtered into test tubes using mira cloth to remove
the peel and obtain the aliquot. Resulting aliquots in each experiment
was allowed to cool before taking spectrophotometric measurements. All
experiments were carried out in triplicates.
Determination of total betacyanin concent in samples: The absorbance
readings obtained was used to calculate the total betalain concentration
for each sample using the following formula (Herbach et al., 2007):
Where:
| A |
= |
Absorbance |
| DF |
= |
Dilution factor |
| MW |
= |
Molecular weight of betanin |
| |
= |
550 g mol-1 |
| ε |
= |
Molar extinction coefficients |
| |
= |
60,000 L/mol cm in H2O |
| ι |
= |
Pathlength of cuvette = 1 cm |
Determination of optimal temperature: Ten gram of dragon fruit
peel was immersed into 30 mL of distilled water and the dye was extracted
at RT for 5 min. The pH and absorbance of the aliquots were measured.
The experiment was repeated at 50, 80 and 100°C. The best result was
subsequently used in the next experiment.
Determination of optimal length of time to heat exposure: Ten
gram of peel was added into a beaker containing 30 mL of boiling distilled
water. The solution was left for 1 min. The pH and absorbance of the aliquots
were measured. The experiment was repeated at 2, 3, 4, 5 and 10 min at
the same temperature. The best result was subsequently used in the next
experiment.
Determination of optimal pH value: Distilled water at different
pH was prepared by adding 1 M of citric acid into distilled water until
the desired pH is obtained. Ten gram of peel was added into 30 mL of pH
2 distilled water and heated to 100°C. The extraction was carried
out for 5 min while, maintaining the temperature through out. The pH and
absorbance of the aliquots were measured. The experiment was repeated
using distilled water with different pH: 3, 4, 5 and 6. The best result
was subsequently used in the next experiment.
Determination of colour stability: Ten gram of peel was added
into 30 mL of pH 5 distilled water and heated to 100°C. The extraction
was carried out for 5 minutes while maintaining the temperature through
out. The pH and absorbance of the aliquots were measured. A control was
prepared with 10 g of peel immersed in 30 mL of distilled water for 5
min. Both aliquots were exposed to light for 12 h each day.
Determination of resuspended colour stability: Extracts were prepared
from heating 10 g of peel in 30 mL of pH 5 distilled water heated to 100°C
for 5 min. The aliquots were evaporated in the oven overnight to dry.
Every 1 g of dried aliquots collected was resuspended in 50 mL water.
The pH and absorbance for the samples were taken before and after drying.
The control for this experiment was dried extract from 10 g of peel in
30 mL of distilled water at RT for 5 min.
RESULTS
Determination of optimal temperature: Figure 1
shows that the total betacyanin content obtained from 10 g of peel in
30 mL of distilled water at different temperatures were significantly
different. The total betacyanin content obtained at RT, 50, 80 and 100°C
were 5.34, 10.47, 12.69 and 24.03 mg L-1, respectively. The
highest yield of betacyanin content was obtained from sample heated at
100°C.
|
| Fig. 1: |
Total betacyanin content from 10 g peel in 30 mL distilled
water at different temperatures |
|
| Fig. 2: |
Total betacyanin content from 10 g peel in 30 mL distilled
water at 100°C for different length of time |
|
| Fig. 3: |
Total betacyanin content from 10 g peel in 30 mL of
different pH distilled water at 100°C for 5 min |
Determination of optimal length of time to heat exposure: Figure
2 shows that the total betacyanin content obtained from heating 10
g peel in 30 mL of distilled water at 100°C at 1, 2, 3, 4, 5 and 10
min were significantly different. The total betacyanin content obtained
at 1, 2, 3, 4, 5 and 10 min were 10.54, 13.95, 18.37, 19.03, 27.98 and
26.25 mg L-1, respectively. The highest yield of betacyanin
content was obtained from samples heated at 100°C for 5 min.
Determination of optimal pH value: Figure 3
shows that the total betacyanin content obtained from heating 10 g peel
in 30 mL of distilled water at 100°C for 5 min in different pH were
significantly different. The total betacyanin content extracted in 2,
3, 4, 5 and pH 6 distilled water were 12.45, 23.14, 16.79, 25.74 and 22.70
mg L-1, respectively. The highest yield of betacyanin content
was obtained from samples heated at 100°C for 5 min in pH 5 of distilled
water.
Determination of stability to light: Figure 4
shows the betacyanin content of sample and the control monitored over
a period of 14 days. The graph shows that the betacyanin content decreased
over time. On day 1, the total betalain content of samples was 22.12 mg
L-1 while, the control yielded 3.97 mg L-1. At day
14, the betacyanin content of samples decreased to only 1.13 mg L-1.
The control was terminated at day 6 when absorbance readings were zero
indicating that betacyanin was no longer present.
|
| Fig. 4: |
Total betacyanin content of samples kept in 16 h of
light exposure daily for 14 days |
|
| Fig. 5: |
Total betacyanin content of samples resuspended in
distilled water, exposed to light for 7 days |
Determination of resuspended colour stability: Figure
5 shows the betacyanin content of sample and the control monitored
over a period of 7 days. The graph shows that the betacyanin content decreased
over time. On day 1, the total betalain content in samples was 39.97 mg
L-1 while, the control had 10.12 mg L-1. At day
7, the betacyanin content in samples decreased to only 3.12 mg L-1
while, control decreased to 3.03 mg L-1. The sharp decrease
observed can be explained by the fact that the dried extract was exposed
far longer to both heat and oxygen during the drying process.
DISCUSSION
The optimal temperature, time and pH to obtain highest yield of betacyanin
content from dragon fruit peel was heating samples at 100°C, for 5
min with pH 5 distilled water. Heating of water during betacyanin extraction
from peel resulted in betacyanins being drawn out from the matrix constituents
more effectively. Samples extracted at 100°C gave highest yield despite
exposure to extreme heat and this can be explained by the fact that betanin
has the ability to regenerate by recondensation of hydrolysis products
associated with a colour regain (Stintzing and Carle, 2007). As betacyanins
undergo thermal treatment, it is known that the pigments will experience
degradation and fluctuating chromatic stability (Herbach et al.,
2006b). As shown in Fig. 1, samples extracted at 100°C
gave a comparable high yield of betacyanin content to samples heated at
other temperatures. It is possible that the occurring pigment after 100°C
thermal treatment is isobetanin, the isomer of betanin (Herbach et
al., 2006b). The structural alterations that occur after heat treatment
will give a different pigment configuration but all betacyanins still
gives a same wavelength colour which is detected at 538 nm using the spectrophotometry
method.
The best length of time for heat exposure to obtain high yield of betacyanin
content was for 5 min. When samples were heated for 10 min, it yielded
a lower betacyanin content as indicated in Fig. 3. This
shows that prolonged heating has detrimental effects betacyanin retention
and stability. Although, regeneration of the pigment is possible, it becomes
less efficient as pigments are exposed to heat in an extreme manner (Herbach
et al., 2004). The pH determination in this experiment showed that
pH 5 is the best pH condition to extract and obtain highest yield of betacyanin
content. According to previous studies, betalain pigments favor a pH range
of 4 to pH 6 in the presence of oxygen and also under anaerobic conditions
(Herbach et al., 2006a).
According to Herbach et al. (2006b), non-treated pitaya juice
is able to regenerate 3% of its pigment while heat-treated juice recorded
a 10% regeneration in pigment regeneration. This phenomenon can be associated
to the ability of betacyanins to regenerate as long as the basic building
blocks are present: cyclo-DOPA ring and betalamic acid, which forms the
betacyanin chromophores. When pitaya juice is subjected to heat, the betacyanins
will undergo multiple structural adjustments like isomerization, deglycosylation,
decarboxylation and hydrolysis (Herbach et al., 2006a) to stabilize
and regenerate itself as oppose to non-treated juice without any alteration
of physical condition. This further reinforces the possibility of heat
treated dragon fruit juice for use as potential dye and heat treatment
can improve pigment yield.
The stability test in this study showed that betacyanin content decreased
upon prolonged light exposure. Earlier studies also showed that betacyanins
are light-sensitive pigments and tend to degrade due to light absorption
in the visible light and ultra-violet range the betalain molecules (Cai
et al., 2005; Herbach et al., 2006b). This phenomenon leads
to an excitation of electrons of the betalain chromophore, causing it
to transcend to higher energy states. In this condition the reactivity
of the molecules are higher, in other words, the activation energy of
the molecules are lowered causing the ready degradation of the pigments.
The dried extract that was resuspended in distilled water showed an intense
purple-red aliquot and significantly high content of betacyanin. These
results indicate that pigment extracted from dragon fruit peel are highly
promising to be sourced as natural dye although the stability pales compared
to maintaining the pigments in the form of juice. This can be explained
by the fact that the extracts were exposed to further heat in the drying
oven overnight. Temperature is regarded as the most crucial factor governing
betalain stability and even though being heat liable pigments, pigments
lose stability at elevated temperatures (Herbach et al., 2006c).
It is therefore the resuspended dried extracts would be far less stable
even though pigment retention after drying is significant.
CONCLUSION
The flesh and peel from dragon fruit is an avenue where there are wide
array of betacyanins to be exploited as natural food dye or colourant.
From this study, samples yield highest betacyanin content at 100°C
in pH 5 distilled water and heated for 5 min. The pH and pigment retention
capacity observed in this study revealed that betacyanin have high tolerance
towards factors such as temperature and light which is most important
in food colouring stability. Stability tests carried out showed gradual
degradation but further studies with strictly controlled environment and
conditions will ascertain the stability of the pigments. When pigments
were dried and resuspended in this study, the resulting aliquot showed
comparable pigment retention which is promising to developing a natural
dye in powder form which is preferred by consumers. Further studies and
experiment are needed to ascertain and confirm these initial findings.
Thus, the potentials and promising findings so far on the peel of dragon
fruit makes the crop a new valuable source of water-soluble and natural
dye for health conscious consumers along with the food additive industry.
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