The 10th SCIENCE and TECHNOLOGY SYMPOSIUM FOR OISP STUDENTS

Ho Chi Minh City University of Technology (HCMUT), VNU-HCM

Study on Betacyanin extraction methods and its content in red dragon fruit peel and
beetroot.
(Khảo sát điều kiện chiết xuất và xác định hàm lượng betacyanin trong vỏ thanh long
ruột đỏ và củ dền)
Phan Thuy Han1,2,3, Nguyen Thi Minh Thu1,2,3, Tran Hien1,2,3, Nguyen Dong Minh1,2,3, Ta Thi Minh Ngoc1,3*
1
Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), 268 Ly Thuong
Kiet Street, District 10, Ho Chi Minh City, Vietnam
2
Office for International Study Programs, Ho Chi Minh City University of Technology (HCMUT), 268 Ly
Thuong Kiet Street, District 10, Ho Chi Minh City, Vietnam
3
Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam
*Corresponding author: [email protected]

Abstract
Betacyanin is used as a food colorant to increase the sensory properties of much different food such as
meat, sausages, beverages, candies, etc. Besides its coloring effect, betacyanin is currently of particular
interest because they also bring many valuable biological activities for health. Beetroot as well as red
dragon fruit skin is known as a rich source of betacyanin which could become potential raw materials for
the production of this food coloring additive. This study focuses on investigating the conditions affecting
the extraction process, comparing the betacyanin content in beetroot and red dragon fruit skin. Samples
from beetroot and red dragon fruit skin were pretreated with two methods: frozen or blanched before
extracted with McIlvaine citric phosphate pH 5.0 as a buffer solution. The betacyanin content was then
evaluated for 1, 2, 3, 4, and 5 hours. All experiments were repeated three times, the data was analyzed
with a significance level of 5%. The betacyanins extraction process reached the highest yield at 31.69 ±
1.32mg/100g from blanched beetroot with solid to liquid ratio R S/L 1:4 after 4 hours. It was approximately
8 times higher than that of fruit peels (4.08 ± 0.18 mg/100g) RS/L 1:4 after 3 hours.
Keywords: Betacyanin, beetroot, red dragon fruit skin, content, extraction.
1. Introduction and manufacturers are seeking methods to utilize the by-
Betacyanin is well-known as one of the most copious natural products, which is the peel of red dragon fruit (Hylocereus
red pigments that exist in many types of vegetables such as polyrhizus) in this case. It also contains a significant amount
carrot, beetroot, red Amaranthus, dragon fruit, and some of betacyanins (18.67 mg/100g) [12].
tubers. They are a water-soluble nitrogen-containing The objective of this study was to evaluate the effects of three
substance that provides the red-violet color, compared to the variables – time, solvent-to-materials ratio (w/v), and initial
yellow-orange color from betaxanthins – another betalain treatment (blanching, freezing, none processing) – on
derivative [1]. Not only do they cause non-toxin or allergic betacyanin extraction from beetroots and red dragon fruit
reactions but they also demonstrate many biological activities peels, to obtain the extracts, which can be further processed
for the human body, preventing chronic degenerative diseases to use as natural food additives.
– hypertension, dyslipidemia, cancer, and cardiovascular
disease [2].
2. Methods and Implementation
In contrast with synthetic colorants which associate with the Materials
terms ‘artificial’ and ‘unhealthy’, plant-derived pigments are Red dragon fruit and beetroot were purchased from a local
more preferred by consumers for their organic, healthful, and supermarket in Ho Chi Minh city. The McIlvaine citric –
benign characteristics. Therefore, the industrial application of phosphate buffer solution was obtained with 0.1M citric acid
betacyanins for natural food coloring is widely adopted and 0.2M sodium phosphate dibasic (Na2HPO4) from China.
despite their high cost and poor stability. According to
Stintzing & Carle [3], the betacyanin can be stable in a wide Sample preparation
range of pH 3 to 7, which makes it apt for application in a The procedure was followed by Lopez et al. 2009 [13] with
broad palette of low-acid and neutral food, such as meat, ice some modifications. The raw beetroot and red dragon fruit
cream, banana spread [4], ready-to-serve beverage [5], cow were washed under running water to remove dust. Beetroot
milk [6], jelly gummies [7], strawberry and peach yogurt [8], was manually peeled and then cut into small rectangular
pork loin [9] or even as the indicator for intelligent packaging pieces 1 x 0.5 x 3 (cm). The pulp and the green part of the
for fish freshness [10]. The most significant betalains source dragon fruit were eliminated to obtain the peel with outer and
is red beet (Beta vulgaris L. ssp. Vulgaris) [11],thereby it has inner layers then they were cut into pieces the same size as
been the dominant subject of betacyanins–related literature. the beetroot. Each sample was weighed 10g. Five peel
However, to address the global food waste crisis, scientists samples and five beetroot samples were blanched in water
The 10th SCIENCE and TECHNOLOGY SYMPOSIUM FOR OISP STUDENTS
Ho Chi Minh City University of Technology (HCMUT), VNU-HCM

(sample: water ratio 1:10) at 90 ± 1 oC for 1 min, without coefficient [17]. In the regard to its stability, the temperature
stirring. Five other peel samples and beetroot samples were is the most critical effect on the structure of betalains. Too
frozen at -10oC in the freezer for 8h hours then were naturally high temperature leads to the modification in structure and
defrosted to the room temperature (30 ± 1oC). Another 5 chromatic aspects through isomerization, deglycosylation,
samples of fruit peels and beetroot were kept raw as reference hydrolysis, and dehydrogenation processes, which decrease
samples. the stability of the red pigment, and decarboxylation which
astonishingly benefits its stability [3].
Extraction process
After preparation, all samples were immersed into the Oxygen is another major factor accelerating betacyanin
McIlvaine buffer solution pH 5.0 with ratios of 1:1, 1:2, 1:3, degradation. The rapid destruction of betalains in the
1:4, and 1:5 (w/v) for each treatment. The process occurred at presence of reducing or oxidizing agents, including molecular
50 ± 1oC in a closed thermostatic tank for 5 hours. oxygen, suggests that either active oxygen species or an
oxidation-reduction mechanism is involved in the
Analysis of juice colorant by spectrophotometry degradation [18]. To minimize chromatic shifts of
betacyanin-containing solutions, betacyanin degradation may
For the determination of the concentration of betacyanin, be prevented by minimum thermal exposure and the
1000 μL. of extracted beet juice or 2000 μL .of extracted exclusion of light and oxygen [3].
fruit juice was added to 0.1M McIlvaine buffer solution at pH
5.0 and the volume made up to 2 mL. Then the absorbance of 3. 1 Effect of time and ratio solid/liquid (R S/L) on
the diluted juice was measured after 1, 2, 3, 4, and 5 hours at the extraction of betacyanins
538 nm wavelength against the McIlvaine buffer as blank by For the raw beetroot, the highest yield was 5.68 ±
a spectrophotometer (Jinghua 752, China). The betacyanin 0.48mg/100g with RS/L of 1:4 after 5 hours. The R S/L 1:1
content (BC) was expressed as mg/100g fresh weight and illustrated a steady downward after the peak at 4 hours which
calculated by using the following the equation [14]–[16]: can be attributed to the thermal degradation of these pigments
A × MW ×V × DF ×100 at more extended treatment as proposed by [19]. While the
BC (mg/100 g)= extraction curve of betacyanins of other ratios showed quite
ε × L ×W
an alike pattern with a linear increase, similar to the result of
Where: untreated samples with pH 5.0 from the experiment [20].
A is the absorbance at 538 nm
MW is the molecular weight of Betacyanins (550 g.mol-1)
V is solution volume (ml)
DF is a dilution factor
ε is the molar extinction coefficient of Beta-cyanins (60,000
L.mol-1.cm-1)
L is the path length of the cuvette
W is sample weight (g)

Statistical analysis
All the experiments were performed in triplicate and the
results were expressed as the mean ± SD of three replicates.
Data were analyzed using the two-way analysis of variance
(ANOVA) in Stargraphics .The multiple range test LSD with
a significance level at p-value < 0.05, was applied to the
results to test the significant difference.

3. Results and Discussion

According to Das et al. 2019 [14], the highest extracted
betacyanin content could be achieved by acidifying the
medium to pH 5.0 - 6.0 – the optimal pH range of betalains
[13]. Furthermore, using the acidifying agent such as citric
acid as the chelating agents and partially neutralizing the
electrophilic center of betalains, improve its stability [16].
The extraction temperature also has a remarkable effect on
the extraction efficiency. The low temperature of 10 oC is not
enough for extraction, the range from 20oC to under 55oC is
recommended. The relatively high temperature is beneficial Fig 1. Extracted betacyanin content from raw beetroot (left) and fruit
peel (right) with various treatment methods, Results are presented as
for the extraction performance as it softens the plant tissues, means ± standard deviations (n = 3). Superscript lowercase letters
and increases the permeability, solubility, and diffusion indicate a significant difference (p≤0,05) among different periods within
The 10th SCIENCE and TECHNOLOGY SYMPOSIUM FOR OISP STUDENTS
Ho Chi Minh City University of Technology (HCMUT), VNU-HCM

a ratio and treatment. Superscript uppercase letters illustrate significant betacyanins may result in low yields, and require longer
differences (p ≤ 0,05) among different ratios within the same
extraction times [26]. Therefore, the degradation of these
temperature and treatment.
polysaccharides is a fundamental step to improving the
In figure D of raw fruit peel, all samples of different ratios release of betacyanins, few methods had been applied for this
reached their maximum release of the red pigment at 4 hours purpose such as heat treatment, freezing, and enzyme
(which the highest yield was 2.74 ± 0.18 of RS/L 1:4) before utilization. Preheating the plant tissue for a short time before
declining with the same possible cause. the extraction process is helpful to avoid enzymatic
In figures B and E, the ratio of 1:5 led to the highest amount dilapidation of pigments by inactivating enzymes, also
of betacyanins from freezed beetroot (9.12 ± 0.92) and fruit reduces microbial load, increases the tissue permeability, and
peel (6.23 ± 0.17). The extracted pigment amount of five facilitates the betacyanin extraction [27], [28] although it may
different ratios accelerated in the first two hours (figure 2c) destroy some pigments [24]. According to Zaidel et al. 2017
and four hours (figure 2d) and then dramatically fell. Because [29], the highest pectin amount in red dragon fruit peels was
as the time increased, the diffusivity of the pigments in the 20.34%. The effect of near-freezing temperature storage in
liquid phase decreased due to the concentration of these apricot studied by Fan et al. 2019 [30] indicated that it
solutes in the solid phase is lower at this stage of the delayed the degradation of pectin fraction and protected cell
extraction and transport phenomenon was based on the wall structure from loss.
internal diffusion of the solute into the solvent [21].
However, the lower concentration of solvent RS/L requires
greater time and temperature. It means more samples, longer
extraction time, and/or higher extraction temperature [4].
It is possible to observe in figures C and F that as the
sample/solvent ratio increased, higher was the betalain
content obtained. The highest content of betacyanin from
blanched beetroot (31.69 ± 1.32) and peel (4.08 ± 0.18) was
obtained after 4 hours and 3 hours using a R S/L 1:4 and from
this time the extraction reached equilibrium concentration.
The increase in the volume of solvent allows for greater
hydration and swelling of the solid matrix, which results in
improved extraction yield [22]. Another factor that can
contribute to the increase in the removal of pigments is the
reduction of the viscosity of the medium with the increase of
the solvent/sample ratio, which improves the intensity of
cavitation [23].
The result of two-way ANOVA showed the p-value <0.05,
indicating that there was no interaction between the two
factors time and solid-liquid ratio.

3.2 Effect of pre-treatment methods on the

extraction of betacyanins
The treatment before the extraction process improved the
efficiency of red pigment release compared to the untreated
samples (5.68 ± 0.48 for beetroot and 2.74 ± 0.18 for fruit
peel). It can be obvious to recognize that the preheated
samples result in the highest yield for beetroot (31.69 ± 1.32
mg/100g). Whereas, freezing was proved to be the optimal
treatment for red dragon fruit peel samples at 6.23 ± 0.17
mg/100g. This was in accordance with to result of 6.7 ± 0.2
mg/100g of [24]. It is also noteworthy that in almost all cases,
the untreated samples were significantly smaller in the
number of released betacyanins than the treated ones. This
suggested that the pretreatment was significantly effective in
improving the extraction yield. Fig 2. Extracted betacyanin content from beetroot (left) and
fruit peel (right) over time with different ratios and methods.
The difference in the suitable extraction methods between
Results are presented as means ± standard deviations (n = 3).
beetroot and red pitaya peel could due to the distinct of Superscript lowercase letters indicate a significant difference (p
pectins amount. Pectins are a group of polysaccharides that ≤ 0,05) among different treatments within the same ratio and
are rich in galacturonic acids and found in the middle lamella time.
of the plant cell wall or intercellular lamellar region [25]. It
can function as a barrier to the diffusion of intracellular
compounds during extraction. For this reason, extraction of
4. Conclusions
The 10th SCIENCE and TECHNOLOGY SYMPOSIUM FOR OISP STUDENTS
Ho Chi Minh City University of Technology (HCMUT), VNU-HCM

The betacyanins extraction process reached the highest yield betalains can be used as antioxidant colorants protecting
at 31.69 ± 1.32mg/100g from blanched beetroot with solid to food constituents from oxidative damage,” Acta Hortic.,
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times higher than that of fruit peels (4.08 ± 0.18 mg/100g) 10.17660/ActaHortic.2015.1067.44.
RS/L 1:4 after 3 hours. This study may provide convenient
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