INTRODUCTION
Fruits of the genus Hylocereus (Berger) Briton and Rose originated
from Latin America and are known as red pitaya belonging to the Cactaceae
family (Stintzing et al., 2002). This vine-like epiphytic cacti
is also cultivated in Vietnam, Malaysia, Taiwan, China, Okinawa, Israel
and Southern China. Producing a deep purple-coloured flesh comparable
to red beet or amaranth, fruits from Hylocereus polyrhizus are
highly appealing in the European and United States market.
Food colouring is a common pre-requisite to compensate for process-related
losses to improve overall appearance of food and this factor is important
to meet customers` expectations. Natural food dye is gaining popularity
especially in the food and beverage sectors due to strong demand for more
natural products by health-conscious consumers (Herbach et al.,
2007). Some examples of natural food colorants which have already been
used in the food industry include anthocyanin, curcumin, beetroot red,
caramel, lycopene, paprika extract and chlorophyll. In addition, natural
colours and pigment from fruits and vegetables may contribute additional
nutritional value to food coloured as observed in cactus fruits (Mohammer
et al., 2005).
Dragon fruit is one of the new focus for the next source of food dye
is because it is rich in betalains which are the similar array of colour
pigments found in beetroot. Beetroot has been the most important betalain
source for natural red colouring and is mainly composed of the red-purple
betanin and the C15-isomer isobetanin. However, there is a demand for
alternative compounds 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). Hence, fruits from the Cactaceae family have been suggested
as a promising betalain source being devoid of the mentioned drawbacks.
Betalains are nitrogenous vacuolar pigments and important chemotaxonomical
markers found in 13 families within the plant kingdom and in some members
of the Basidiomycetes (Stintzing et al., 2004). Betalains have
never been found co-existing with the widely distributed plant pigment
anthocyanon which explains their roles as markers. Some advantages that
betalains possess over anthocyanins include, being more water soluble,
a tinctorial strength up to three times higher than anthocyanins and a
wider pH stability range from pH 3 to 7 making it suitable for application
in a broad palette of low-acid and neutral food (Stintzing and Carle,
2007).
Betalains are divided into the red-purple betacyanins and yellow-orange
betaxanthins which comprise about 55 different structures and promise
a great variation of colour array to the food industry. In dragon fruit
alone, there are at least seven known betalain namely; betanin, isobetanin,
phyllocactin, isophyllocactin, betanidin, isobetanidin and bougainvillein-r-I
(Stintzing et al., 2002) all of which have identical absorption
spectra (λmax) that contribute to the deep-purple colour
observed in the fruit pulp.
This study is aimed at exploring the possibilities of water extraction
method and heat to obtain significant pigment concentration and to test
the stability of samples stored at different temperatures.
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 Biotechnology laboratory
in the 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 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 using Whatman paper No. 1 (9 cm) to remove the
pulp and obtain the aliquot. All experiments were carried out in triplicates.
Efficiency of water volume to extract pigment: Five grams of pulp
were immersed in 5, 10, 15 and 25 mL of Sterilized Distilled Water (SDW)
for 10 min to determine which ratio of weight: volume yields highest pigment
concentration. The pH and absorbance of the aliquots were measured. The
best weight: volume ratio was then used for subsequent experiments.
Efficiency of temperature to extract pigment: Five grams of pulp
were immersed in 5 mL SDW and pigments were extracted at room temperature
(RT), 40, 60, 80 and 100 °C for 10 min to determine which temperature
yields highest pigment concentration. The pH and absorbance of the aliquots
were measured. The best temperature to obtain highest pigment concentration
was then used for subsequent experiments.
Stability of pigments extracted with water
Sample preparation: Five grams of pulp were immersed in 5 mL of SDW
and pigments were extracted at 100 °C using a waterbath for 10 min.
All solutions were filtered to remove the pulp. The pH and absorbance
of the aliquots were measured before used for experiments.
Stability test samples: Five milliliter of aliquots were stored
at RT, 4 and -20 °C in test tubes. The test tubes that contained the
samples for dark storage were wrapped with aluminium foil. All samples
were stored for one week and then taken out for further analysis.
Stability of pigments from juice concentrate
Sample preparation: Approximately 500 g of fruit pulp were sieved
to obtain concentrated juice. Five milliliter of juice was poured into
test tubes and extracted at 100 °C for 10 min while another 5 mL of
juice was poured into test tube at room temperature as control. The pH
and absorbance at 538 nm of each solution were determined before used
for experiments.
Stability test samples: Five milliliter of juice were stored at
RT, 4 and -20 °C in test tubes. The test tubes that contained the
samples for dark storage were wrapped with aluminium foil. All samples
were stored for one week and then taken out for further analysis.
Determination of total betalain concentration in samples: Absorbance
values of samples were measured using a spectrophotometer (Pharmacia,
Ultrospec II) at 538 nm against a blank of SDW. The absorbance obtained
was then used to calculate the total betalain concentration using the
following formula (Herbach et al., 2007).
| A |
= |
Absorbance |
| DF |
= |
Dilution factor |
| MW |
= |
Molecular weight of betanin 550 g mol-1 |
| ∈ |
= |
Molar extinction coefficients 60,000 L mol-1 cm in H2O |
| l |
= |
Path length of cuvette = 1 cm |
RESULTS
Efficiency of water volume to extract pigment: Figure
1 shows that the pH for 5 g of pulp extracted in different volumes
of SDW did not show any significant differences while Fig.
2 shows the total betalain concentration for the same solution. The
total betalain concentration obtained from 5 g of pulp in different volumes
of SDW was 25.5, 25.3, 24.9 and 24.2 mg L-1, respectively and
the highest yield would be samples which had 5 g of pulp extracted in
5 mL of SDW.
|
| Fig. 1: |
pH of 5 g pulp in extracted with different volumes of
SDW. The pH for samples in different volumes of SDW did not show any
significant changes |
|
| Fig. 2: |
Total betalain concentration of 5 g pulp in different
volumes of SDW. The highest yield of pigments was obtained by extracting
5 g of pulp in 5 mL of SDW |
Efficiency of temperature to extract pigment: Figure
3 shows the pH of 10 g pulp extracted in 10 mL of SDW at different
temperatures and the results showed that pH did not vary much. Figure
4 shows that the total betalain concentration obtained after the heat
treatment at RT, 40, 60, 80 and 100 °C was 26.2, 25.6, 26.1, 25.7
and 26.2 mg L-1, respectively. The highest yield was samples
that were extracted at RT and 100 °C.
Stability of pigments extracted with water
Samples extracted at room temperature: Figure 5
shows the pH of samples at day 0 and 7 after being stored in different
temperatures and conditions. The pH did not exhibit any significant change
after 1 week of storage. Figure 6 shows the total betalain
concentration of the same samples and there was a significant increase
after one week of storage indicating that pigments under went structural
changes or degradation. The highest increase was observed in samples that
were stored at 4 °C in the dark, from 26.20 mg L-1 at day
0 to 94.78 mg L-1 at day 7 while the lowest increase, thus
the most stable, was observed in samples that were stored at -20 °C
exposed to light, from 26.32 mg L-1 at day 0 to 63.12 mg L-1
at day 7.
|
| Fig. 3: |
pH of 10 g pulp in 10 mL of SDW at different temperatures.
There was no significant changes in pH value after samples were subjected
to different temperatures for pigment extraction |
|
| Fig. 4: |
Total betalain concentration of 10 g pulp in 10 mL of
SDW at different temperature. There was no significant difference
in all samples extracted at different temperatures but the highest
pigment yield was obtained in samples extracted at room temperature
and 100 °C |
|
| Fig. 5: |
pH changes in samples extracted with SDW at room temperature
stored in different conditions and temperatures. The pH value of samples
did not show any significant change after one week of storage |
|
| Fig. 6: |
Total betalain concentration in samples extracted at
room temperature and stored in different conditions and temperatures.
It was observed that there was a significant increase of total betalain
content after one week of storage in all samples |
|
| Fig. 7: |
pH of samples extracted at 100 °C and stored in
different conditions and temperatures. It was observed that all samples
exhibited a slight increase in pH value after one week of storage |
Samples extracted at 100 °C: The pH for all samples exhibited
a slight increase at day 7. The highest increase of pH was observed in
samples stored at room temperature exposed to light, from pH 5.25 at day
0 to pH 6.02 at day 7. The lowest increase, thus most stable, was observed
in samples that were stored at -20 °C in the dark, from pH 5.34 at
day 0 to pH 5.4 at day 7 (Fig. 7). Figure
8 shows the total betalain concentration of the same samples and there
was a significant increase after 1 week of storage indicating that pigments
under went structural changes or degradation happened. The highest increase
of total betalain concentration was observed in samples that were stored
at room temperature exposed to light, from 25.60 mg L-1 at
day 0 to 129 mg L-1 at day 7. The lowest increase, thus most
stable, was observed in samples that were stored at room temperature in
the dark from 25.80 mg L-1 at day 0 to 67.76 mg L-1
at day 7.
|
| Fig. 8: |
Total betalain concentration of samples extracted at
100 °C and stored in different conditions and temperature. It
was observed that there was a significant increase of total betalain
content after 1 week of storage in all samples |
|
| Fig. 9: |
pH changes of juice concentrate samples stored at different
temperatures and conditions. All samples showed a slight increase
after one week of storage except for samples kept at room temperature
in the dark that showed a slight decrease |
Stability of pigments from juice concentrate
Juice concentrate extracted at room temperature: Figure
9 shows the pH of juice concentrate samples stored in different temperatures
and conditions. All samples except samples kept at room temperature in
the dark, showed a slight increase in their pH value after one week of
storage. Figure 10 shows the total betalain concentration
in the same samples in which all samples showed a decrease in total betalain
concentration after 1 week storage except for samples kept at -20 °C
which showed an increase in total betalain concentration. The highest
decrease was observed in samples stored at room temperature exposed to
light, from 246 mg L-1 at day 0 to 67.76 mg L-1
at day 7.
|
| Fig. 10: |
Total betalain concentration of juice concentrate samples
in different conditions and temperatures. The total betalain concentration
of samples stored in -20 °C (light and dark) showed an increase
in total betalain concentration while all other samples showed a decrease |
|
| Fig. 11: |
pH of juice concentrate samples in different conditions
and temperatures. All samples showed an increase in pH value after
one week of storage |
|
| Fig. 12: |
Total betalain concentration of juice concentrate samples
in different conditions and temperatures. All samples showed a significant
decrease after one week of storage |
Juice concentrate extracted at 100 °C: All samples showed
an increase in pH value. The highest pH increase was observed in samples
that were stored at 4 °C exposed to light, from pH 4.63 at day 0 to
pH 5.07 at day 7 while the lowest increase was observed in samples that
were stored at room temperature exposed to light, from pH 4.66 at day
0 to pH 4.78 at day 7 (Fig. 11). The results showed
that there was a general decrease in total betalain concentration in samples
after one week of storage. The highest decrease was observed in samples
stored at -20 °C in the dark, from 247.75 mg L-1 at day
0 to 163.45 mg L-1 at day 7 while the lowest decrease was observed
in samples that were stored at 4 °C exposed to light, from 246.64
mg L-1 at day 0 to 215.58 mg L-1 at day 7 (Fig.
12).
DISCUSSION
Efficiency of water volume to extract pigment: The best water
volume to be used in extracting pigment from 5 g of pulp is 5 mL of SDW.
It is highly possible that any extraction procedure to obtain best yield
of pigments from dragon fruit pulp is by using the ratio of 1:1 (weight:
volume).
Efficiency of temperature to extract pigment: As betacyanins undergo
thermal treatment, it is known that the betalains (major pigment class
in dragon fruit) will experience degradation and fluctuating chromatic
stability (Herbach et al., 2006a). The betalains will be subjected
to processes like isomerization, deglycosylation, decarboxylation, hydrolysis
and other processes. The specific main pigment known in dragon fruit which
is betanin gives the red-purple colour. As indicated in the result, samples
extracted at 100 °C also gives a comparable high yield of betalain
content at 538 nm to samples subjected to room temperature. It is possible
that the occurring pigment which is scarlet red after 100 °C thermal
treatment is isobetanin, the isomer of betanin.
Stability of pigments extracted with water: All samples extracted
at RT and 100 °C showed a significant increase in total betalain concentration
after one week of storage in the different temperatures. The increase
in samples extracted at 100 °C 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). It is also
possible that when betacyanins are extracted with water, it is drawn out
from its protective matrics where the condition in the pulp is the stable
environment for betacyanins.
Thus, the main pigment may undergo multiple structural adjustment/regeneration
to stabilize itself in the water as oppose to pigments used directly from
the juice concentrate without any alteration of physical condition. This
regeneration is encouraged when pH value is close to pH 6 with the presence
of the basic building blocks of the betacyanins cyclo-DOPA ring and betalamic
acid, which forms the betacyanin chromophores.
The results show that betacyanins have great pigment retention ability
even after being heated and stored in adverse conditions and exposed to
illumination. Samples which exhibited minimal pH change after one week
of storage were samples extracted at room temperature and kept in 4 °C,
exposed to light while samples which exhibited minimal total betalain
change were samples extracted at room temperature, kept at -20 °C
and exposed to light.
Studies on the stability and the regeneration of betacyanins in dragon
fruit are still at an early stage because this is a relatively new crop
being focused on as a natural food dye source. A noteworthy behaviour
of betalain (the major pigment in Hylocereus polyrhizus) that should
be taken into consideration in future experiments is that, it undergoes
multiple processes like decarboxylation, deglycosylation, hydrolysis,
isomerization, dehydrogenation and others when it is subjected to these
factors : water activity, pH change, antioxidants present, chelating agents,
temperature (heat), illumination and oxygen/nitrogen atmosphere (Herbach
et al., 2006b). The structural alterations that occur will give
a different pigment configuration but all these betacyanins still gives
a purple-red colour which is detected at 538 nm using the spectrophotometry
method.
From the results obtained, it was observed that samples extracted at
room temperature showed minimal pH and total betalain content changes
as compared to samples extracted at 100 °C. This shows that pigments
subjected to heat during extraction exhibit lower pigment stability and
support the findings that heat is an important stability factor as previously
mentioned.
Stability of pigments from juice concentrate: The pH value for
samples extracted at RT and 100 °C showed a general increase after
one week of storage and as the pH increases, there is a decrease in total
betalain content. There is a difference in results between pigments extracted
with water and pigments used straight from juice concentrate where total
betalain concentration showed increase and decrease respectively. This
may be contributed by the extraction method carried out where pigments
were extracted with water first in the previous section. One of the factors
that affect betalain stability is water activity where lower water activity
(aw) improves betalain stability. According to Herbach et
al. (2006b), it is possible that betacyanins in their natural matrices
have superior stability compared to purified solutions. Plant constituents
like sugars, acids and pectic substance will lower the aw value,
thereby stabilizing betalainic pigments from the start of the experiment.
The total betalain content is also much higher after one week of storage
compared to results obtained in previous section. This suggests that for
high total betalain content, juice concentrate is a preferred choice.
It was observed that samples extracted at room temperature showed minimal
pH and total betalain content changes as compared to samples extracted
at 100 °C. This shows that pigments subjected to heat during extraction
exhibit lower pigment stability and support the findings that heat is
an important stability factor as previously mentioned. Samples stored
at -20 °C in both extraction methods showed minimal change compared
to other samples stored at room temperature and 4 °C which suggests
that lower temperature stabilizes the pigments more effectively.
CONCLUSION
The wide array of betacyanins present in Hylocereus polyrhizus
provides an avenue to obtain a new natural food colourant. The results
in this study showed that the best weight: volume ratio to extract pigments
with water is 1:1 and the best temperature to use for high pigment yield
is 100 °C. 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.
Other than that, the results indicated that the betacyanins have the ability
to regenerate under suitable conditions which supports earlier findings
mentioned earlier. The most stable condition for pigment storage observed
in this study where there was least change in pH and pigment concentration,
was samples stored in -20 °C. Visibile colour changes that was observed
when samples were subjected to heat suggests that there is a possibility
that structural conformation occurred but pigment concentration did not.
Overall, this study can conclude that water extraction and heat are viable
methods to obtain high concentration of betacyanins and these pigments
have a great tolerance towards factors that are important when it comes
to food colouring, water extraction could be more economical and heat
may provide an alternative colour for a natural dye. Further studies and
experiment are needed to ascertain and confirm these initial findings.
Thus, the potentials and promising findings so far on Hylocereus polyrhizus
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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