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Name:
1. Cathryn Choe
2. Clarissha Vallerie Widjaja (Group Leader)
3. Farah Ula Nabilah
4. Fong Shi Hui
5. Helena Wiranata

Student ID:
1. 0338139
2. 0340155
3. 0340810
4. 0338278
5. 0338467

Module Code: FSC60104

Names of partners: -

Email (Individual/Group Leader): Contact No (Individual/Group Leader) :

1. cathrynquerern.choe02@sd.taylors.edu.my 1. +60166822693
2. clarisshavalleriewidjaja@sd.taylors.edu.my 2. +6281805882858
3. farahula.nabilah@sd.taylors.edu.my 3. +601120968557
4. fongshihui@sd.taylors.edu.my 4. +60176200168
5. helenawiranata@sd.taylors.edu.my 5. +601114248131
Title of experiment:
Practical 6 - Determination of Total Anthocyanin & Evaluation of Its Chemical Properties

Module Lecturer: Dr Chan Sook Wah

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Lab Report 6: Determination of Total Anthocyanin & Evaluation of Its Chemical
Properties

Objectives
• To determine the total anthocyanin in food samples by differential pH.
• To evaluate the chemical properties of anthocyanin.

Introduction

Anthocyanins are red, blue, or purple pigments that are water-soluble and can be found in plants.
They are in glycosylated forms and belong to the phenolic group. Anthocyanins present highly in
flowers, tubers, and fruits such as berries, currants, and grapes. Anthocyanin is included as a
flavonoid compound that has a flavylium (2-phenyl chromen plum) ion. The basic molecular
structure of anthocyanin can be seen in figure A. One of the examples of anthocyanins is peonidin
that has magenta color, it is also a type of anthocyanin that can be easily found in plants. (Khoo,
Azlan, Tang and Lim, 2017)

Figure A. Basic anthocyanin structure (Khoo, Azlan, Tang and Lim, 2017)

Flowers and fruits that are reddish, purplish, and bluish in color usually contained anthocyanins.
Some of the flowers can be used as folk medicine, colorant, and food. While fruits that contain
anthocyanins are consumed because of their beneficial effects and some are strong antioxidants.
Moreover, some tubers like black carrot, red cabbage, and purple potato hold a great benefit in
preventing diseases. Many studies have been conducted to determine plant anthocyanins medicinal
value. Studies find that anthocyanins have anticancer, antidiabetic, antimicrobial, anti-obesity
effects, anti-inflammatory, and can help prevent cardiovascular diseases (CDVs). (Khoo, Azlan,
Tang and Lim, 2017)

Factors that affect the stability of anthocyanins vary from the pigment type, copigments,
temperature, light, pH, metal ions, oxygen, enzymes, and also antioxidants. The B-ring in the
anthocyanin structure as well as hydroxyl or methoxyl groups also influence its stability. (Khoo,
Azlan, Tang and Lim, 2017)

The pH condition of the solution can influence the color of anthocyanins due to the ionic nature of
the molecular structure. Anthocyanins appear red in acidic condition, have a purple hue when the
pH is neutral, and change to blue in higher pH condition (basic). The pigments of red color in
anthocyanins are in flavylium cation forms which have higher stability at lower pH level and more
soluble in water. When pH level increases, it forms colorless carbinol pseudobase and chalcone
structure, then followed by anionic quinonoidal species formation. This happens because of the
competition of the kinetic and thermodynamic between the flavonium ion hydration reaction. The
anthocyanin pigments are mostly more stable in low pH conditions compared to basic conditions,
moreover it degrades at higher pH level. (Khoo, Azlan, Tang and Lim, 2017)

Anthocyanins also change their color in a solution by copigmentation and temperature influences.
The reinforcement of anthocyanins by metallic ions or flavonoids is called copigmentation of
anthocyanin aglycon. It helps the stabilization of the color of the plants including leaves, flowers,
and fruits. Metal ion assists the stabilization of carbinol pseudo-base structures in the delphinidin
equilibrium mixture at corresponding PH level. It also helps in providing blue hue. The
copigmentation of anthocyanins with flavonoids changes the flower's color and increases the
intensity. Moreover, glycosylation and acylation help increase the color strength of anthocyanins.
(Khoo, Azlan, Tang and Lim, 2017)

At higher temperature, solutions of anthocyanins are less stable. For example, when a low pH
level solution is heated up to 40°C, anthocyanin color changes from red to orange. However, when
anthocyanin-rich extract solution undergoes heat treatment, the degradation of anthocyanin
pigments may not happen due to the phenolic compounds that are contained in the extract are
enzymatically degraded by polyphenol oxidase. Also, the enzymatic reaction can be inactivated
with mild heat treatment up to 50°C. Hence, in the food industry, some raw materials will go
through mild heat treatment like blanching to help prevent oxidation of anthocyanins by
polyphenol oxidase. (Khoo, Azlan, Tang and Lim, 2017)

Anthocyanins are commonly used as natural colorants of red, blue, and purple colors in the food
industry to increase consumer acceptability of the products. Besides, served as natural colorant,
anthocyanins also have other properties like antioxidants and also provide many health benefits,
such as an antimicrobial effect and it also helps in preventing chronic diseases. (Khoo, Azlan,
Tang and Lim, 2017)
Apparatus and Instruments

• Analytical balance
• Blender
• Disposable plastic cuvettes
• Dropper pipette
• Filter paper
• Filter funnel
• UV-Vis spectrophotometer
• Pipettes
• Volumetric flasks
• 0.45 µm syringe filter

Reagents and Materials

• pH 1.0 Buffer – Dissolve 1.49 g KCl into 100 mL deionized water. Carefully pour 1.7 mL
concentrated HCl into 100 mL deionized water for 0.2 N. Mix 25 mL of the KCl solution
with 67 mL of the 0.2 N HCl solution. Adjust pH 1.0 ± 0.1 if necessary
• pH 4.5 Buffer – Dissolve 1.64 g sodium acetate in 100 mL deionized water. Adjust to pH
4.5 ± 0.1 with HCl
• Extracting Solvent – 0.01% (v/v) HCl in methanol

Sample

Red cabbage

Sample Preparation

1. Weigh ca. 10 g of fruit or vegetable containing anthocyanin (record exact weight) and place
in blender.
2. Add ca. 50 mL extracting solvent (0.01% HCl in methanol) and blend thoroughly. Transfer
the homogenate into 100 mL volumetric flask with the aid of dropper pipette and funnel.
Make up to volume with the same extracting solvent.
3. Filter through filter paper followed by syringe filter to obtain a clear filtrate.

Procedure

1. Pipette a 1.0 mL aliquot of extract containing anthocyanin into a 25 mL volumetric

flask. Dilute to volume with pH 1.0 buffer and mix.
2. Pipette a second 1.0 mL aliquot of extract containing anthocyanin into a 25 mL
volumetric flask. Dilute to volume with pH 4.5 buffer and mix.
(Note: Solutions are stable at room temperature for 4 hours.)
4. Zero spectrophotometer with buffer.
5. Measure the absorbance of the pH 1.0 and pH 4.5 sample preparations at 510 nm and
700 nm (for turbidity correction).
(Note: Dilute sample further if absorbance is greater than 1.0 AU.)

Calculations:

Calculate the difference in absorbance between the two samples using the following equation:

A = (A510nm pH1.0 - A700nm pH1.0) - (A510nm pH 4.5 - A700nm pH 4.5)

Calculate the %w/w of total anthocyanins in the sample:

A V
Total anthocyanin as Cyanidin - 3 glucoside (% w/w) = x MW x DF x x 100
εL Wt

where,
A = Differences in Absorbance at pH 1.0 and pH 4.5
ε = Cyd-3-glu molar absorbancy (26,900)
MW = anthocyanin molecular weight (449.2)
DF = dilution factor (in this experiment = 25)
V = final volume (mL)
Wt = sample weight (mg)
L = cell path-length (usually 1 cm)

(Note: High levels of fructose, sulphite and ascorbic acid may interfere with the assay. It is
recommended that these factors be tested in model solutions if they are likely to be present in
the samples in significant amounts.)

SECTION 2

Evaluation of Chemical Properties of Anthocyanin

Principle

The colour and stability of an anthocyanin in solution is highly dependent on the pH. They are
most stable and most highly coloured at low pH values and gradually lose colour as the pH is
increased. Anything which interrupts the conjugated double bond system of the anthocyanin
causes a loss of colour. The presence of the positive oxonium ion next to the C-2 position
makes anthocyanins particularly susceptible to nucleophilic attack by compounds such as
sulphur dioxide or hydrogen peroxide. Some metals, such as Fe+3 and Al+3 form deeply
coloured coordination complexes with anthocyanins that have ortho-dihydroxy groups on the
B-ring. Such metalo-anthocyanin complexes have been found to produce discolouration in
some canned fruit products.
Apparatus and Instruments

• Scanning spectrophotometer – capable of scanning from 400 – 600 nm.

• Volumetric flasks, 25 mL
• Pipettes, 2.0 mL, 5.0 mL
• Large test tubes

Reagents and Materials

• Buffer solutions – Prepared buffer solutions of pH 1.0, 3.0, 5.0, 7.0 and 9.0 according to
the standard procedure.
• FeCl3 solution – 0.6% FeCl3 solution in 95% ethanol
• Hydrogen peroxide solution – 3.0% (v/v) H2O2 solution
• Sodium metabisulphite solution – 50, 100, 200, 500, 1000 ppm

Sample

Plants extract containing anthocyanin from previous experiment.

Procedures

A. Effect of pH

1. Label volumetric flask as follows: pH 1, 3, 5, 7 and 9.

2. Transfer 2.0 mL of the extract containing anthocyanin to each 25 mL volumetric flask
(use pipette filler). Make up to volume with the appropriate buffers.
3. Mix and allow to stand at room temperature for 30 minutes.
4. Observe and record the colour for each solution.
5. Scan the solution from 400 – 600 nm and print the absorption spectra.

B. Effect of Fe+3

1. Transfer 2.0 mL of the extract containing anthocyanin into a large test tube.
2. Add in 5.0 mL FeCl3 alcoholic solution. Mix well and allow to stand for a while.
3. Repeat steps 1 – 2 but add in 95% ethanol instead of FeCl3.
4. Observe and record the colour of the solutions.
5. Scan the solution from 400 – 600 nm and print the absorption spectra.

C. Effect of hydrogen peroxide

1. Transfer 2.0 mL of the extract containing anthocyanin into a large test tube.
2. Add in 5.0 mL of 3.0% H2O2 solution. Mix well and allow to stand for a while.
3. Repeat steps 1 – 2 but add in distilled H2O instead of H2O2.
4. Observe and record the colour of the solutions.

D. Effect of sodium metabisulphite

1. Label large test tubes as follows: 0, 50, 100, 200, 500, 1000 ppm. Add 5.0 mL of
appropriate metabisulphite solution to each of the test tube. For 0 ppm, add in distilled
H2O instead.
2. Transfer 2.0 mL of the extract containing anthocyanin to the tubes. Mix well and allow
to stand for a while (approx. 30 min).
3. Observe and record the colour of the solutions.
Results
Section 1 : Determination of Total Anthocyanin by pH Differential Spectrophotometry
Method

Table 1.0: The absorbance value of the pH 1.0 and pH 4.5 sample at 510 nm and 700 nm

Wavelength pH 1.0 pH 4.5

Trial 1 Trial 2 Average Trial 1 Trial 2 Average

510nm 0.874 0.872 0.873 0.034 0.04 0.037

700nm 0.634 0.630 0.632 0.006 0.008 0.007

Calculation

ΔA = (A510nm pH 1.0 – A700nm pH1.0) – (A510nm pH 4.5 – A700nm pH4.5)

ΔA = (0.873–0.632) – (0.037–0.007)

ΔA = 0.241 – 0.03

ΔA = 0.211

Total anthocyanin as Cyanidin - 3 glucoside (% w/w) = 𝛥𝐴x MW x DF x 𝑉 x 100

𝜀𝐿 𝑊𝑡

0.211 25
= x 449.2 x 25 x x 100
(26900)(1) 10000

= 7.84 x 10-6 x 449.2 x 25 x 2.5 x 10-3 x 100

= 0.022 % w/w
Section 2: Evaluation of Chemical Properties of Anthocyanin

Figure 1.0: Absorption spectra from 400-600nm of effect of 95% ethanol on the extract
containing anthocyanin.

Figure 2.0: Absorption spectra from 400-600nm of effect of FeCl3 on the extract containing
anthocyanin.
Figure 3.0: Image of the effect of Water (Left) and Hydrogen Peroxide (Right) on the extract
containing anthocyanin.

Figure 4.0: Effect on different pH on anthocyanin extract solution after 30 minutes.

Figure 5.0: Effect of addition of sodium metabisulphite solution on anthocyanin, from left to
right, flasks with sodium metabisulphite concentration 0, 50, 100, 200, 500 and 1000ppm.
Discussion
Section 1

Red cabbage is an excellent source of food colorant and rich in a number of bioactive substances,
including anthocyanins (Ahmadiani 2015). Cyanidin-3 glucoside can determine the total
anthocyanin in red cabbage. According to Francis and Markakis (1989), Cyanidin-3 glucoside is
the most common anthocyanin in nature.

The pH differential method has been widely accepted by natural product chemists for years. It is
a method to measure the monomeric anthocyanin pigments and the results may not seem to be
correlated with the color intensity of the juice or wine samples as they are judged visually. The
use of the method is in research and for quality control of anthocyanin-containing fruit juices,
wines, natural colorants and other beverages. (Lee et al., 2005; Wrolstad, 1993)

Based on the result in Section 1, the total anthocyanin as Cyanidin-3 glucoside is 0.022% w/w.
The anthocyanin undergoes a structural transformation with changes in pH. Anthocyanin
pigments can be described as being indicators. At pH 1.0, anthocyanin exists in high coloured
oxinium or flavilium form which will indicate a larger absorbance at 510nm and 700nm.
However, at pH 4.5 nearly all monomeric anthocyanins are in the hemiketal form, a small
proportion are in the quinoidal form or the flavylium form, which will make a small contribution
to the absorbance (Lee et al., 2005). The difference in absorbance at the wavelength of maximum
absorption will be proportional to anthocyanin content. The wavelength of maximum absorption
for anthocyanins is 510 nm. (Wrolstad, 1993)

Section 2

Chemical properties of anthocyanin are the key factor driving the stability and color of
anthocyanin. Alternation in acid concentration (pH) will significantly affect the anthocyanin
pigment either synergistically or antagonistically by influencing the conjugated double bond
system. Moreover, in this experiment the stability and color anthocyanin may also be modified in
line with the addition of metal ion (Fe3+), hydrogen peroxide, as well as sodium metabisulphite.
Effect on pH

Observation made by adding anthocyanin into several solutions with distinct ranges of pH:
1,3,5,7, and 9. Difference in color can be explained into certain classification of pH as shown in
Figure 4. Reddish color may appear in pH 1 and 3, yet the bleaching effect can be noted as the
solution in pH 3 has lighter red color compared to red in pH 1. On the other hand, purple color
range can be noticed in pH 5, 7, and 9. Discoloration occurred slightly from pH 5 to pH 7
resulted in lighter violet color at higher pH. However, thicker purple color posed on pH 9 as the
basic solution.

Reason behind this phenomenon was due to the molecular structure of anthocyanins having an
ionic nature (TURTURICĂ, OANCEA, RÂPEANU and BAHRIM, 2015). According to Khoo,
Azlan, Tang and Lim (2017), four anthocyanin structures exist in equilibrium in aqueous media
such as flavylium cation, carbinol pseudobase, quinonoidal base, and chalcone. At the pH 1 the
predominant species is the red-colored flavylium cation. The stability of anthocyanin is higher at
acidic condition. When flavylium cation is formed, it enables the anthocyanin to be highly
soluble in water. The decrease in water concentration increases the rate of deprotonation of the
flavylium cation, thus reducing color stability. Based on these statements, it can be concluded
that the discoloration from pH 1 to 3 may happen due to this reason.

Further, the purple color on higher pH value resulted from the formation of a blue quinonoidal
base. This blue quinoidal species is unstable only at lower pH (pH 4-5). However, in neutral pH
(pH 7), anionic quinonoidal and chalcone species are formed from further deprotonation of
quinonoidal that will give purple coloration (Khoo, Azlan, Tang and Lim, 2017). Besides, red
cabbage anthocyanin extract contains various types of anthocyanin pigment that are capable of
forming several ranges of blue and red shades (Ahmadiani, M.Sc., 2015). Therefore, when the
red shades of flavylium cation vanished significantly, the blue shades will appear more and
combination of pigments resulted in purple coloration.

Hence supported by Remini et al. (2018), the structure of anthocyanin is strongly dependent on
the pH solution and as consequence so is its color stability, which is highly related to the
deprotonation and hydration equilibrium reaction constant values as presented in Figure 6.
 Figure 6. Chemical reactivity of Anthocyanins depending on pH (Remini et al., 2018)

Effect on the addition of Fe3+

The analysis of color in the addition of Fe 3+ will be observed through the comparison of Figure 1
and Figure 2. In Figure 1, the lowest absorbance value was noted from 440nm – 460nm and at
600nm. The low peak at 440nm – 460nm means low absorbance level of purplish-blue color.
This would mean the higher the percentage of blue color to appear as the solution color which
can be catched by eyes. While at 600nm, low absorbance value of yellow color will also impart
yellow color solution. Marginal difference can be observed in Figure 2 that the lowest peak that
fell on 400nm and 480nm represents the lowest absorbance level in purple and blue color.
Meaning the color appears will be in the wider range of purple to blue color (light wavelength
refers to Figure 7).
 Figure 7. Visible wavelength light on color interpretation

The formation of metal complexes, mainly with Fe 3+, to the anthocyanin complex as depicted at
Figure 8 impart special traits in the absorbance value graph, such as hyperchromic and
bathochromic shifts, which are presented in Figure 2. As in Figure 1, the highest peak of
absorbance level fell on approximately 530nm (green wavelength) at 0.20 value. Whilst, UV
spectrophotometer result on the addition of Fe 3+ showed great hyperchromic state that the
absorbance level soared to 1.4 value, as well as bathochromic shift which the highest absorbance
value fell on 570nm-580nm.

Thus, the addition of Fe3+ in this experiment will preserve the blue color of anthocyanin. Even
though, decline intensity of particular visible color wavelength was noted due to the increase of
absorbance level. This outcome is supported by Ratanapoompinyo, Nguyen, Devkota and
Shrestha (2017) that the chelation of metal ions by anthocyanins was reported to stabilize the
quinonoidal base and protect the anthocyanin molecule from nucleophilic attack.

Figure 8. Mechanism of formation of the complex anthocyanin-metal

Effect on Hydrogen Peroxide

The pigment from anthocyanin still remains boldly strong in the test tube where water was added
and has discoloured to a translucent pale pink when reacted with hydrogen peroxide as seen in
Figure 3. The colour is degraded due to oxidation caused by the reaction with hydrogen peroxide
that breaks chemical bonds in anthocyanin(Dangles and Fenger, 2018).

By a Baeyer-Villiger oxidation process, the hydrogen peroxide will attack the carbon-2 and the
peroxyl group in anthocyanin will begin a realign to form a seven carbon ring with carbon-2 as a
carbocation (Blaine Stebbins, 2017).

Water is a common isolation medium for anthocyanin, therefore it can retain its vibrant pinkish
red colour because it retains its stability when in water (Khoo, Azlan, Tang and Lim, 2017).

Effect on Sodium Metabisulphite

In Figure 5, it is observed there is a gradual change from the left where the colour is more stable
and darker purple shade and gradually fading to a light pink tone towards the right.

Sodium metabisulphite is an inorganic compound and is known as a reducing type bleaching

agent (Barberá, Metzger and Wolf, 2000). The pigment from purple cabbage is produced by the
chromophore. All chromophores will consist of an unsaturated bond. Reducing bleaching agents
such as sodium metabisulphite work by releasing hydrogen atoms that can change unsaturated
bonds to become single bonds. This process causes the colour loss (Sodium Metabisulfite Food
Grade, 2020). Therefore as the ppm of sodium metabisulphite increases in concentration, the
decolourization due to bleaching becomes more prominent.
Conclusion
In brief, total anthocyanin in red cabbage due to differential pH is 0.022% w/w. Differential pH
will lead to structural tranformation of anthocyanin pigment. Chemical properties of anthocyanin
can be determined through the alternation of pH. With the highest stability on acidic condition,
anthocyanin at pH 1 showed intense red. Whilst, changes of red color due to flavylium cation to
purplish blue color at pH 5 and above because of the formation of quinonoidal base. Moreover, at
pH 9, intense purple was observed.
Beside pH alternation, the addition of particular substance can either lead to discolouration or
enhancement of certain colour range. The addition of Fe3+ is noted to bring hyperchromic and
bathochromic shifts that eventually preserve blue colour. In the other hand, the mixture of
anthocyanin either with hydrogen peroxide or sodium metabisulphite resulted in discoloration to
transparent solution. Color degradation in the incorporation of hydrogen peroxide was influenced
by the oxidation which break the anthocyanin structure. While sodium metabisulphite released
hydrogen atom that threaten unsaturated bond in anthocyanin structure. Thus, the relation of
decolorization and increased amount of sodium metabisulphite were in parallel line.

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