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Study of Extraction Method and The Stability of Purple Pigment


From Various Natural Sources for Food Colouring

NUR AFIQAH BINTI AB RAZAK

ABSTRACT: Food are important substances needed for the human body to grow and live and nowadays, the food available in the market
are tainted with so many additives that the foods entering human body is deemed as unsafe and have a long adverse effect such as cancer
and asthma attack. Synthetic food colouring is one of the contributor of this problem and consumers are growing aware of this issue.
Consumers demand a safer food colouring thus this study is done regarding natural food colouring available to be extracted. The aim of
this experiment is to study the extraction method of purple pigment from natural sources of dragon fruit, purple cabbage, and purple sweet
potato, to study the effect of different storage parameters on the stability of purple pigment incorporated in food product, and to study the
effect of microencapsulation of purple pigment extracted. The raw materials are extracted to obtain the purple pigment through different
suitable methods, and later encapsulated into powder form. A muffin is cooked and the extracted food dye is fused into the muffin. The
resulted muffin will be tested through several tests such as light presence test, storage time test, temperature test, and taste and colour test.
Results show that the liquid colour extraction of all sample degrades over 4 weeks and PSP is not a suitable candidate for this colour
extraction. Purple cabbage and dragon fruit is successful in the extraction the purple colour in both liquid and solid form. The stability test
for liquid all varies as anthocyanin in each raw material differs according to the nature of raw material and extraction method. The stability
test of coloured muffin temperature test is found that the muffin placed at 4oC surrounding stand a better chance of surviving longer than
the muffin placed at 27oC. As for the presence of light test, the result obtained is that light condition has no effect on the storage life of
muffin. Last but not least, for the taste and colour test for coloured muffin, colour of the muffin is best retained in the refrigerator at 4oC
and the taste test shows that the taste of the muffin is not influenced by the raw material used in the experiment.

KEYWORDS: dragon fruit, purple cabbage, purple sweet potato (PSP), anthocyanin, natural food colouring, stability tests

I. INTRODUCTION unappetizing. Thus, food manufacturers are “obligated” to add back


the lost nutrients, colour, texture, and flavour in order for them to be
Foods are substances that living organisms consume in sold off to consumers, hence, making them loaded with food
order to give additional nutrition to their body and to produce energy additives (Mercola, 2011).
for them to survive. According to Lambert (2016), humans in the
olden days started to search for food by hunting wild animals. This The objectives of this research are aimed to study the
habit then transformed and changed to human growing their own extraction method of purple pigment from natural sources dragon
crops and fast forward later, humans start to process new food such fruit, purple cabbage, and purple sweet potato. To study the effect
as cheese, bread, crackers, etc. (Lambert, 2016). Now, in the modern of different storage parameters (time, temperature, taste, and colour)
days, food has not only become a necessity, but also a luxury to the on the stability of the purple pigment incorporated in food product
human race when their taste buds are tickled with rich flavours, and to study the effect of microencapsulation of purple pigment
different textures, and eye-popping colours. While these enhance extracted.
the experience of tasting food, it also comes with negative setbacks.

This study focuses on how colour affects the food that are
consumed. As mentioned by Rebecca et al., (2008), food colouring II. METHODOLOGY
is a common pre-requisite to compensate for process-related losses
A. Purple Sweet Potato Extraction
to improve overall appearance of food and this factor is important
to meet customers’ expectation. This defines that when foods are PSP extraction method were referred from Xiu-li et al.
being processed, typically in industrial scale, it losses certain (2015). Based on the written research, every 50 gram of PSP were
characteristics like their natural colour, and this result will make the cut and mashed using a blender mixed with 500 milliliter of distilled
food appear dull and unattractive. Food colouring is important water. The sample were then stirred at 37oC for 1 hour and the
because consumers find that food is more appealing with more homogenate were filtered. After filtration, the filtrate was stored at
colour (Gray, 2013). The two main choices of food colorant origins a refrigerator of temperature 4oC in the dark prior to usage.
from natural sources and synthetic made colorant and both came
with their pros and cons.

Mercola (2011) said, texture, valuable nutrients, fibers, natural B. Purple Cabbage Extraction
variation, and flavours are among the things that are lost, when
foods are processed. After processing, the food would most likely For this extraction method, it is based from the research of
appear black, uninteresting and people would find them entirely Ho et al. (2013). For every 40 gram of purple cabbage weighed, 80
millilitre of absolute ethanol were used. Both materials were mixed
2

and heated until it reaches 50oC using a double-container boiler on


a hot plate. After heating of the material, it was stirred rapidly by a
magnetic stirrer for 30 minutes. Lastly, the impurities were filtered
out using a filter paper to separation the extraction product needed
to proceed the experiment.

C. Dragon Fruit Extraction

The extraction from flesh of dragon fruit is based on the (a) (b)
method used by Tang & Norziah (2007). The first step to this
extraction is to thaw the fruit beforehand. This would make the fruit
softer and easier to be processed for the next step. Secondly, the
solvent was prepared with a mixture of 80% acetone, 80% methanol,
and water. After the preparation of solvent, it was then shake well
inside a flat bottom flask along with the raw material with a ratio of
1:2 for sample to solvent.

After shaking, the sample is stirred using a magnetic stirrer


for 1 hour until the material and solvent mixed evenly. Then, it is
allowed to rest for about 15 minutes. Finally, to filtrate the product, (c) (d)
filter paper was used to separate extraction and it was stored at -20oC
Fig. 1: Liquid extraction of purple sweet potato with (a) distilled water, (b)
prior to usage. solvent at initial extraction, and (c) distilled water, (d) solvent at the end of
storage time.

D. Stability Tests On Coloured Muffin

Temperature Test 3

The stability test was based on Tantitunovant (2008) with 2.5


modifications to adapt to the food product. Three muffins were
covered with packaging for protection purpose and placed in three 2
different environment temperatures such as room temperature,
1.5
60oC, and 4oC. The changes were observed and recorded with image
recording every 7 days for 2 weeks. 1

0.5
Presence of Light Test 0
The stability test was based on Tantitunovant (2008) with 0 7 14 21 28
modifications to adapt to the food product. Two muffins were
covered with packaging for protection purpose and placed in a dark A B C D
environment, and an open environment with both at room
temperature. The changes were observed and recorded with image (a)
recording every 7 days for 2 weeks.
3.5

Taste and Colour Test 3


2.5
The stability test was based on Tantitunovant (2008) with
modifications to adapt to the food product. Four muffins were 2
covered with packaging for protection purpose and placed in two 1.5
different places. Two muffins were placed in a dark environment at
room temperature and the other two were placed in an open space. 1
The changes were observed, smelled, tasted, and recorded with
0.5
image recording every 7 days for 2 weeks.
0
0 7 14 21 28
III. RESULTS AND DISCUSSION
A B C D
A. Liquid Colour Extraction

Purple Sweet Potato (b)


Fig. 2: (a) Absorbance value of PSP with distilled water against time, (b)
Absorbance reading for PSP with solvent over time.
3

The PSP extraction was done and the results is shown in


3.5
Fig. 1. Fig. 1 (a) and (b) shows that from the start of the extraction,
the colour of the samples leads more toward brown colour instead 3
of the expected result of purple. This can be seen from during the
extraction that when the PSP is blended, the purple colour degrades 2.5
and loses it colour eventually. 2
Over time, the final result after four weeks is displayed on 1.5
fig. 1 (c) for distilled water sample, and (d) for solvent sample. The
obvious difference in colour change for the two weeks of 1
observation is PSP with distilled water. While the solvent mixture 0.5
also loses colour, but it does not differ as much as the distilled water
mixture. In fig. 1 (c), it can also be seen that the liquid has two 0
separate layers that is the water layer on top, and the pure PSP 0 7 14 21 28
colouring rests at the bottom.
A B C D
This indicates that the sample is not well mixed and
centrifugation should be performed as one of the extraction method
in order to separate pure PSP extraction from the raw material. (a)

For the spectrophotometer reading of both samples, fig. 2 1


(a) show a constant increasing reading for all samples at different
conditions. This indicates that the different storage condition of all
samples does not affect the colouring any different than the other. 0.8
While for fig. 2 (b), the readings decrease over time expect for the
reading at the third week, where the both sample kept in the 0.6
refrigerator spiked to increase. Light exposure was proved to be a
significant factor in determining the instability of anthocyanin when 0.4
the line in fig. 2 (b) at day 21 increase dramatically due to
overexposure of samples before the reading was taken. 0.2
Based on He at al. (2015), presence of hydroxyl group in
anthocyanin molecule is the contributing factor of colour instability 0
to heat. In light of it, ring-opening and degradation were found to be 0 7 14 21 28
the main factors responsible for the colour change at higher
temperature while is agreed by fig. 2 (a) that bottle A and B has A B C D
higher reading when kept at room temperature instead of
refrigerator. (b)

Fig. 4: (a) Absorbance value of purple cabbage with distilled water against
time, (b) Absorbance reading for purple cabbage with solvent over time.
Purple Cabbage
Fig. 3 shows the result for purple cabbage liquid extraction.
Fig. 3 (a) shows that initially, the extracted colour of purple cabbage
with distilled water is absolutely purple. Meanwhile, the colour is
not the same for liquid extraction of purple cabbage with solvent.
Fig. 3 (b) show a lighter shade of purple compare to distilled water
extraction. During extraction process, purple cabbage extracted with
distilled water shows a darker purple colour but it starts to degrade
its colour over time.

After a month of observation, fig. 3 (c) shows the


(a) (b) appearance of the two separate layers alike the PSP sample and fig.
3 (d), while loses colour, is still in its homogenous state. This can be
mentioned that while distilled water extracted clearer purple colour,
the liquid would lose its homogeneity and destabilize the sample.
Solvent extraction of purple cabbage is believed to be more stable
when kept at storage. In fig. 3 (c), the four samples show different
shades of colour due to its different storage conditions. Bottle A is
observed to be the darkest colour and the condition is normal light
condition and room temperature. The sample that loses most colour
is bottle D. Solvent extraction after four weeks does not resulted in
a huge colour difference compared to initial month.
(c) (d)
Spectrophotometer reading in fig. 4 (a) shows an increasing
Fig. 3: Liquid extraction of purple cabbage with (a) distilled water, (b) reading trend while in fig. 4 (b) shows a total opposite trend. The
solvent at initial extraction, and (c) distilled water, (d) solvent at the end of
increase in absorbance is the effect of polymerization reactions
storage time.
between the byproducts and other composition in the extract (He et
4

al., 2015), while the decrease in absorbance during the observation


period is caused by the degradation of anthocyanins (Janna et al., 4.5
2007). 4
3.5
Thus, the findings indicate that distilled water extraction
3
might trigger the polymerization reaction or could even be the
component that made the reaction with the polymers inside purple 2.5
cabbage. Meanwhile, solvent extraction affects the anthocyanin 2
degradation in purple cabbage which is why the samples in fig. 3 (d) 1.5
loses colour compared to the initial result. Light exposure is what 1
makes the reading of both bottle D in fig. 4 (a) and (b) drops during
0.5
the first week that effectively degrades anthocyanin.
0
0 7 14 21 28
Dragon Fruit A B C D

(b)

Fig. 6: (a) Absorbance value of dragon fruit with distilled water against time,
(b) Absorbance reading for dragon fruit with solvent over time.

Dragon fruit extraction was done and the recorded result is


displayed in fig. 5. Based on fig. 5, the initial colour extraction for
(a) (b) distilled water extraction of dragon fruit is purple and as for solvent
extraction, the colour is dark red. During extraction process, the
colour of the dragon is in deep purple and the colouring can easily
stain the apparatus used. This shows that the dragon fruit is rich with
its anthocyanin pigmentation and it can easily be extracted from the
fruit.

As it can be seen when from fig. 5 (c) and (d), like other
raw material samples, most of the colour of dragon fruit extraction
also degrades. In fig. 5 (c), it shows the colour of distilled water
extraction of dragon food after 4 weeks. Bottle A and B degrades
(c) (d) colour the most compared to the other bottle while bottle C and D
still remains purple, even though the colour changed. The distilled
Fig. 5: Liquid extraction of dragon fruit with (a) distilled water, (b) solvent water extraction also exhibited that the two separate layers appears
at initial extraction, and (c) distilled water, (d) solvent at the end of storage to act like the other raw material samples. Bottle A and B shows
time.
colour toward pink colour while bottle C and D is still considered as
purple.
4
As for fig. 5 (d), the obvious difference is the colour change
3.5
from red to blue for bottle A and B. The two samples that turned
3 yellow after a month shared a same storage condition, which is kept
2.5 at 27oC. For bottle C and D from fig. 5 (d), they are the best sample
to retain the original colour of dragon fruit. As expected, solvent
2 extraction samples are more homogenized compared to distilled
1.5 water extraction sample because there are no existing separate layers
to the sample.
1
0.5 Dragon fruit spectrophotometer reading was also taken
consecutively every week for one month and based on fig. 6 (a), it
0 can be seen that the reading for dragon fruit extracted with distilled
0 7 14 21 water has the most stable reading throughout the weeks. It can be
observed that there is no substantial change in the absorbance
A B C D reading compared to all other absorbance reading and this is because
as explained by Rebecca et al., (2008), betanin has the ability to
(a)
regenerate by recondensation of hydrolysis products. Which meant
that, the colouring may undergo a few structural
adjustment/regeneration to stabilize itself in water

As for fig. 6 (b), the absorbance of reading for both sample at


room temperature is around the same range while the samples kept
in refrigerator decrease in the spectrophotometer reading. The
5

reason the reading of the spectrophotometer decrease is because of Purple Cabbage


the degradation of anthocyanin that are kept in the refrigerator.

B. Powder Colour Extraction

Purple Sweet Potato

Fig. 9: Powder product of purple cabbage with (left) distilled water


extraction and (right) solvent extraction.

L a b

100
Fig. 7: Powder product of PSP with (left) distilled water extraction and (right)
solvent extraction. 80
60
L a b
40
100 20
80 0
0 7 14 21 28
60 -20
40
(a)
20
0 L a b
0 7 14 21 28
60
(a) 50
40
L a b 30
100 20
10
80
0
60 0 7 14 21 28
-10
40 -20

20 (b)

0 Fig. 10: Chromameter reading for purple cabbage powder extraction for (a)
0 7 14 21 28 distilled water extraction, (b) solvent extraction

(b)

Fig. 8: Chromameter reading for PSP powder extraction for (a) distilled water
extraction, (b) solvent extraction
6

Dragon Fruit hue to the spray dried product. The texture of the distilled water
extraction powder is soft while the solvent extraction powder is rough
and when taken out of the spray drier, it clumps in big chunks.

Dragon fruit powders were at two different colour tones.


As seen in fig. 11, the colours are soft purple-pink colour for the
distilled water extraction, and a striking purple for the solvent
extraction. In term of powder texture, the texture of the distilled water
extraction is softer compared to solvent extraction powder. The
solvent extracted powder is seen to be clumping and more hardened
compared to the rest of the other powders.

Powder readings were taken every week for four


Fig. 11: Powder product of dragon fruit with (left) distilled water extraction
consecutive weeks and the reading was recorded and compared to the
and (right) solvent extraction. chromameter chart. Based on Manolopaulou & Varzakas (2016),
parameter L*, a*, and b*, help in determining the colour properties
of the sample. When comparing to the chromameter chart, the hues
L a b of purple range between 0 to 40 for a* and -15 to -60 for b*. When
the reading falls in between both ranges, the colour obtained is in the
100 right purple colour group. L* shows for brightness from 0 (black) to
100 (white).
80
The figures 8, 10, and 12 shows the reading obtained and
60 for PSP, both extraction does not fall in between the range of the
chromameter chart. Thus, it is proven that the colour extracted from
40 PSP powder did not achieve the purple colour. For purple cabbage,
both extraction method of purple cabbage gives reading that fall in
20 between the range of the purple colour and it can be clearly seen from
the colour of the powder and initial liquid extraction. Lastly for
0 dragon fruit, the distilled water extraction falls in all the range of the
0 7 14 21 28
chromameter chart, but the solvent extraction differs. The a* reading
-20
of solvent extraction of dragon fruit goes until 60 and this makes the
colour lean more toward the red hues. Nevertheless, the b* reading
(a) from fig. 12 indicates that it still belongs to the purple shade but
having a stronger red colour to its powder.
L a b

80 C. Muffin Test

60

40

20

0
0 7 14 21 28
-20
Fig. 13: Initial product of muffin after incorporated with extracted colouring
(b)

Fig. 12: Chromameter reading for dragon fruit powder extraction for (a)
distilled water extraction, (b) solvent extraction Fig. 13 shows the original colour of the muffin straight after
it is baked. The colour of the muffin differs greatly from the colour of
the powder. Significantly, the colour for purple cabbage has the most
The colour of the powder after each of the samples are change. The colour is turned grey and this is because the original
spray dried varied differently from each other. Fig. 7 that shows the colour for the muffin batter is slightly yellow, and mixing it with the
powder product of PSP does not represent any colour from the colouring changes it original colour. All colourings are affected by
anthocyanin group which consists of red – purple – blue. From the the original colour of the muffin batter, but the dragon fruit samples
liquid extraction result, it can already be seen that PSP loses its colour are still noticeably in the anthocyanin colour range.
even before it is spray dried, thus spray drying properties does not
necessarily effect the colouring of PSP.

For powder of purple cabbage, fig. 9 displays that the


purple colour is very much visible with the distilled water extraction
in a soft purple colour, and the solvent extraction gives a dark purple
7

Temperature Test results as samples in fig. 15 (c) and in addition, is consistent


throughout the two whole weeks of observation.

The results for this test proved that the presence of light
does not play a part on the shelf-life of the muffin. Samples are
clearly only affected when placed in different surrounding
temperature. Light presence does not necessarily contribute to the
growth of these moulds on the muffin.

(a) (b)
Taste and Colour Test
Fig. 14: Result after two weeks of observation for condition (a) room
temperature and normal light, (b) refrigerator and normal light Colour test is done throughout the two weeks of muffin
observation. For fig. 15 (a) and (b), the colour of the muffins cannot
be observed due to the magnifying growth of moulds on the muffin.
For the temperature test of colouring on muffin, the Thus, the result for colour observation on room temperature after
comparison is between muffin placed at room temperature, 27oC and two weeks is not obtainable.
in the refrigerator at 4oC. Fig. 14 (a) displayed a large growth of
For fig. 15 (c) and (d), the colour of muffin at the end of
microbes on the surface of the muffin. All samples are affected by the
two weeks and the initial of the observation does not show any
mould growth as early as the first week of observation. For fig. 14
significant different. The colours retain itself after two weeks in the
(b), no growth of moulds or microbe can be seen even after two weeks
refrigerator, making it a better option for storage condition.
of observation. All samples in the refrigerator shows consistency in
its colour and texture of the muffin. Taste test for the muffin is only done on the muffins stored
at 4oC because at 27oC after two weeks, the muffin is not safe to be
The surrounding temperature of the muffins are clearly
consumed for the test. Comparison is made for the three different raw
affecting the growth of microbes and moulds. Refrigerate temperature
material by distilled water and solvent.
slows down the effect of the growths significantly compared to the
samples placed at room temperature.
Table 1: Result of Taste Test
Distilled Water Solvent
PSP  Sweeter  Tastes bland
Presence of Light Test
Purple Cabbage  Tastes bland  Tastes normal
Dragon Fruit  Tastes normal  Sweeter

Taste test displays simple differences between the distilled


water group and solvent group. For PSP, distilled water mixture have
a sweeter taste, while the solvent mixture tastes bland. The bland taste
is the same for purple cabbage with distilled water. The solvent
mixture for purple cabbage does not have any distinct taste, and this
(a) (b) result includes the sample for dragon fruit with distilled water.
Meanwhile, the solvent mixture with dragon fruit has a sweeter taste
compared to its distilled water mixture.

The colour test for the samples are only valid for samples
kept in the refrigerator. Thus, it is recommended that the samples at
room temperature are kept with an oxygen diffuser packaging that
would help prevent the growth of microbes and moulds. This addition
will aid obtaining the colour result for samples in room temperature.
As for samples in the refrigerator, the colour test is successfully done
(c) (d) and results showed that no significant colour difference is made
during the span of two weeks of storage.
Fig. 15: Result after two weeks of observation for condition (a) room
temperature and normal light, (b) room temperature and dark, (c) refrigerator As for the taste test, no abnormal taste can be tasted in any
and normal light, (d) refrigerator and dark.
of the muffin. Aside from the small taste difference between distilled
water sample and solvent sample between the raw material, it does
not have much of an effect. This makes up the point that the taste of
For test of presence of light, sample in fig. 15 (a) and (b) is the raw material does not affect the taste of the muffin once the
completely covered underneath the moulds that have grown on the colouring is incorporated. Hence, the natural colourings are suitable
muffin. While fig. 15 (b) is wrapped with aluminium foil, it does not to be used in cooking as it does not disturb the original taste of the
make any difference with the sample placed at normal light foods.
conditions.

Oppositely, samples in fig. 15 (c) and (d) does not at all


host the growth of microbes and moulds. Samples in fig. 15 (d) is
also completely wrapped with aluminium foil, but it shows the same
8

IV. CONCLUSION soccata Rondani. Indian Journal of Plant Protection, 31(1), 73-
77.
Results from this study shows that the extraction of purple
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extraction. This is because the anthocyanin present in PSP is (Hylocereus spp.) components with valorization potential. PhD
unstable and can easily degrade. Even though initially PSP in raw thesis, Ghent University, Belgium, 217 p.
form is purple colour, it loses its colour during the extraction
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to further advance the study of extraction for purple colour for PSP. Colour Changes of Green Vegetables. Curr Res Nutr Food Sci
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material purple cabbage and dragon fruit is successful. Both raw October.02
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