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Suji (Dracaena angustifolia (Medik.) Roxb.) leaves are famous chlorophyll source used as food colorant in Indonesia and other south-east Asian countries. Its chlorophyll has unique characteristics which can degrade through enzymatic and non-enzymatic reactions. This article summarizes traditional application of Suji leaves, the characteristics of Suji leaf chlorophyll, postharvest stability, and several ways to retain its green color. Potential development of Suji leaf extract as food colorant or food ingredients are also discussed.
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Stability of Chlorophyll as Natural Colorant: A Review for Suji
(Dracaena angustifolia (Medik.) Roxb.) Leaves’ Case
DIAS INDRASTI1,2, NURI ANDARWULAN1,2*,
EKO HARI PURNOMO1,2 and NUR WULANDARI1,2
1Department of Food Science and Technology, Faculty of Agricultural
Engineering and Technology, Bogor Agricultural University, Bogor 16002, Indonesia.
2Southeast Asian Food and Agricultural Science and Technology (SEAFAST) Center,
Bogor Agricultural University, Bogor 16680, Indonesia.
Abstract
Suji (Dracaena angustifolia (Medik.) Roxb.) leaves are famous
chlorophyll source used as food colorant in Indonesia and other
south-east Asian countries. Its chlorophyll has unique characteristics
which can degrade through enzymatic and non-enzymatic reactions.
This article summarizes traditional application of Suji leaves, the
characteristics of Suji leaf chlorophyll, postharvest stability, and several
ways to retain its green color. Potential development of Suji leaf extract
as food colorant or food ingredients are also discussed.
Current Research in Nutrition and Food Science
Journal Website: www.foodandnutritionjournal.org
ISSN: 2347-467X, Vol. 06, No. (3) 2018, Pg. 609-625
Article History
Received: 08 October 2018
Accepted: 13 December 2018
Keywords
Degradation;
Enzymes;
Food;
Green;
Kinetic;
Postharvest;
Storage
CONTACT Nuri Andarwulan andarwulan@apps.ipb.ac.id Department of Food Science and Technology, Faculty of Agricultural
Engineering and Technology, Bogor Agricultural University, Bogor 16002, Indonesia.
© 2018 The Author(s). Published by Enviro Research Publishers.
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Doi: doi.org/10.12944/CRNFSJ.6.3.04
Introduction
Chlorophyll, as a natural pigment, plays an important
role for the green color appearance in plants. The
green color of chlorophyll has been long time used as
a natural colorant.1 Chlorophyll demand continuously
increases inline with increasing awareness for
using natural colorants.2,3 From health aspect,
chlorophyll and chlorophyllin are provide benefits
to human body.4,5 Chlorophyll have antioxidant and
antiinflamatory properties that prevent chronic
diseases such as cancer.6–8 However, changes of
chlorophyll structure into its derivatives made it loses
its activities.9–12
Exploration for chlorophyll content in several plants
has been done worldwide.13–17 One of the green
plants that contain high chlorophyll content is Suji.18,19
Suji (Dracaena angustifolia (Medik.) Roxb.) plant is
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famous for its color and as medicinal plant. Suji own
fresh green color and easily extracted using water.20
Chlorophyll water extract of Suji leaf is added to the
food processing as natural colorant in various food
and non food products.
Chlorophyll in Suji and other plants easily degraded
due to enzymatic reactions and non-enzymatic
reactions influenced by environmental conditions10.
Because of its role as a light capturing unit in
photosynthesis process,21 the presence of light
and heat radiation will excite the transfer of
energy and increase the reactivity of chlorophyll
thus its easily to undergo degradation process.
Chlorophyll degradation progress rapidly as the
chlorophyll structure changes into its derivative
compounds that result in lost of its green color.
Chlorophyll degradation during storage or post-
harvest processing of horticultural produces causes
significant losses.22,23 The discoloration of green
vegetables to dark-green, yellow, or even black
primarily due to the degradation of chlorophyll.
Although potential as natural colorant, due to its
chlorophyll content, Suji plants are only grown as
ornamental in indoor or garden and has not been
utilized optimally. In this review, authors will describe
Suji leaf characteristics; postharvest chlorophyll
stability and its possible degradation pathway and
kinetics; and ways to maintain green color stability.
Futher, the potency of development natural green
colorant from Suji leaf will be explained at the end
of this review.
Botany of Suji
The genus Dracaena consists of about 150
species.24 The Suji (Dracaena angustifolia (Medik.)
Roxb.) plants has been classified in the family of
Agaveceae among the Characteristics of flowers
with six stamens, pediculate inflorescences, and
plants with rosettes of fleshy fibrous leaves.25 Others
recognized it as a distinct family, Dracaenaceae,
along with the genera Sansevieria and Pleomele.26
Suji plant image is presented in Fig. 1.
Suji is an evergreen shrubby plant with a rhizomatous
rootstock. The stems are grayish, smooth, and 1-3
meters tall with no or few branches.27 The plant
has linear-lanceolate leaves, acuminate, length of
15-30 cm, 2-4 cm broad, sessile, margin entire. Its
ribbon-shaped leaves elongated with size 17×2.5
cm and has dark green color. The leaf is smooth
in both surface and sticking alternately in the stem
with intervals of 0.5 cm28 followed by 2-5 yellowish-
white flowers together in terminal wide-spreading
panicles and globose compressed 3-lobed berry.29,30
Dracaenas propagation can be done with seeds,
transplantation, and grafting28 but commercially
they propagated using vegetative method by cutting
relatively large stems.
Dracaena plants easily found in tropical zone from
Africa to the Pacific islands and cultivated intensively
in Southeast Asia, including Indonesia, Malaysia,
and Vietnam. It has different names including Nam
ginseng (Vietnam), Suji (Indonesia, Malaysia), chang
hua long xue shue (Chinese), saiheva and si-ei
Fig. 1: Suji (Dracaena angustifolia (Medik.) Roxb.) plant,
chlorophyll structure and its derivatives20
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(Papua New Guinea).27,29 Common names for this
plant is Dragon’s blood palm according to the deep
red liquid exudes from injured bark.25 Some literature
also named Suji as Pleomele angustifolia Roxb. or
Pleomele angustifolia N.E. Brown.8,20 It is planted
near rivers and water stream, or as decorative plant
in a garden. The preferred habitats are rainforests or
semi-deserts.31 Various species of Dracaena have
an importance in floriculture, medicinal, or social
functions in many society. Despite this importance,
the plant is still underutilized and not cultivated
intensively yet.
Traditional Application of Dracaena Plants
Suji has been use as medicinal plant in Asia region
since ancient times. Diseases are treated using
various parts of this plant. Known locally as Nam
ginseng, Suji roots and rhizomes are used as tonic
and leukemia treatment in Vietnam.32 Its roots is
effective in treating stomachache while its leaves
are used for anti-inflammatory and anti-dysentry.28
The roots are applied to prevent insect bites by
Philippines.27 Sundanese, who inhabit West Java
region in Indonesia, treated cough diseases and
lung disorders using Suji leaves.33 Boiled juice of
squeezed leaves is given to asthma patients who
have shortness of breath. Leaf decoction is drunk
to increase appetite and body weight.29 In the field
of cosmetics, Suji extract is believed to help fertilize
hair growth, render the hair long and pliant.27
Green color from Suji leaves extract has been use
for coloring food and non-food. In Indonesia and
Malaysia, green color of Suji leaves water extract
has been added in food preparations. The extract
give fresh color for various drinks, sweets, puddings,
and desserts. Suji leaves usually mixed with
Pandanus leaves extract as the best combination
to provide fresh green flavored desserts.34 Suji leaf
water extract is used for coloring an Indian pastry
made of glutinous rice. The leaf extract also use in
preparation of tumpeng ponco warno for Javanese
society in Indonesia. Tumpeng ponco warno is rice
featuring identical mountain cones and has five
colors: red, blue, yellow, green, and white.35 Besides
as coloring agent used in porridge and traditional
cakes, Balinese people cooked Suji leaf shoots
and eaten as a side dish with rice.36 For non-food
applications, green color of Suji leaf extract is used
as dye for paper, castor oil, and coconut oil. Suji
extract has been applied for coloring fabrics in home-
made Indonesian batik production.37
Phytochemicals in Suji plants
Suji plants contain many phytochemical components.
The presence of alkaloids, flavonoids, tannins,
terpenoids, saponins, polyphenol, monoterpenoid,
sesquiterpenoid, and glycosides are found in its
root, rhizome, stem, and leaf based on qualitative
screening.28,29,38,39 The ergosterol peroxide, linoleic
acid, and E-phytol content in Suji plant showed its
ability as antituberculosis.27 The methanol extract of
underground parts of Suji plants own eight steroidal
saponin and several recent compounds, including
three spirostanol sapogenenins (namogenins A-C),
four spirostanol saponins (namonins A-D), two
furostanol saponin (namonins E-F), and a pregnan
glycoside (namonin F). Some of them indicated
effective ability as antiproliferative against HT-1080
fibrosarcoma cell cultured in vitro.32,40 Min et al.
(2010)41 isolated six steroidal saponins from fresh
stems of Suji plant, named angudracanosides A-F.
Those compounds had antifungal activity against
Cryptococcus neoformans. The methanol extract of
whole Suji plant has two steroidal saponins, named
drangustosides A-B. These compounds showed anti-
inflammatory activity due to superoxide formation and
elastase discharge by human neutrophils in reaction
to formyl-L-methionyl-L-leucyl-L-phenylalanine,
fMLP/CB.42 Saponins in the form of steroidal
saponins in Dracaena plant suspected to have
anti-inflammatory and analgesic characteristics27.
The ethanol extract of Suji leaves has antibacterial
activity against bacteria S. dysenteriae ATCC 13313
and potential as a supplier of potassium in patients
with hypokalemia dysentry.39
Dracaena angustifolia leaf extracts could activate
cholinesterase activity and could be applied to
build reactivators of cholinesterase prevented by
organic phosphorous compounds.43 Suji leaves
have an in vitro cholesterol-lowering activity. The
compounds estimated to play a role in lowering
cholesterol levels are phenolics, flavonoids, and
vitamin C. Hydroxyl groups in cholesterol react
with ketones in flavonoids to form hemiasetal. The
research used a spectrophotometer to measure
free cholesterol, not flavonoid-bound cholesterol.
Carbonyl groups on flavonoids reacted with hydroxyl
groups on cholesterol to form hydrogen bonds. The
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compound that bounded with a sample, or called free
cholesterol, reacted with anhydrous acetic acid and
concentrated sulfuric acid.44
Although comprehensive information on the chemical
composition of the dragon’s blood resin is available,
too few studies of the relationship to its medical
function, specific compounds, and systematics have
been completed.25 Further research needed to study
the mechanism of specific compound from Suji plant
responsible to health benefits.
Suji Leaf Chlorophyll Characteristics
Green color of Suji plants comes from the chlorophyll
compound contained inside. Chlorophyll located
in the intercellular lamella organelle called a
Table 1: Chlorophyll content of several plants
Plant Chlorophyll content (mg/kg fresh weight)
a b Total Ratio a/b
Chicory (cv. Anivip)17 2383.1 897.4 3280.5 2.7
Chicory (cv. Monivip)17 1422.6 581.8 2004.4 2.4
Dandelion leaf17 1805.4 677.1 2482.5 2.7
Garden rocket17 2612.4 983.8 3596.2 2.6
Wild rocket17 2160.1 872.2 3032.3 2.5
Black locust leaf50 12864.6* 3385.4* 16250 3.8
Scots pine50 2908.6* 881.4* 3790 3.3
Sow thistle leaf50 10652.8* 4097.2* 14750 2.6
Green peas109 140.1 90.6 230.7 1.5*
Pistachio15 3.6 1.8 5.4* 2.0*
Spinach60 790.7 292.7 1083.4* 2.7*
Green papric60 57.9 28.2 86.1* 2.0*
Broccoli110 218 90.6 308.6 2.4*
Pink Lady apple (flesh)13 0.4 0.1 0.5* 3.2*
Fuji (I) apple (flesh)13 0.9 0.2 1.1* 3.6*
Reina de Reineta apple (flesh)13 1.2 0.3 1.5* 3.8*
Green Golden Delicious apple (flesh)13 3.5 0.9 4.4* 3.8*
Green Doncella apple (flesh)13 5.2 1.1 6.3* 4.7*
Granny Smith apple (flesh)13 6.3 1.8 8.1* 3.5*
Golden Rosett apple (peel)13 4.9 1.7 6.6* 2.9*
Golden Montana apple (peel)13 4.5 1.2 5.7* 3.9*
Golden Delicious apple (peel)13 18.3 5.3 23.6* 3.5*
Royal Gala apple (peel)13 7.8 2.6 10.4* 2.9*
Fuji (F) apple (peel)13 14.2 3.7 17.9* 3.8*
Ariane apple (peel)13 9 2.2 11.2* 4.0*
Starking Red Chief apple (peel)13 28.7 8 36.7* 3.6*
Pink Lady apple (peel)13 31.8 8.5 40.3* 3.7*
Fuji (I) apple (peel)13 51.5 15.1 66.6* 3.4*
Reina de Reineta apple (peel)13 29.4 8.9 38.3* 3.3*
Green Golden Delicious apple (peel)13 35.4 11 46.4* 3.2*
Green Doncella apple (peel)13 52.8 11.3 64.1* 4.7*
Granny Smith apple (peel)13 237.1 70 307.1* 3.4*
*calculated based on chlorophyll a, chlorophyll b, and total chlorophyll content or ratio of chlorophyll a/b.
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chloroplast.45,46 Its existence is protected by proteins
that form a protein-chlorophyll complex.47–49 The
complex is surrounded by protein-lipid bilayer thus
making chlorophyll stable in it.50
Based on its structure, chlorophyll is a porphyrin
containing a tetrapyrrole base ring that binds to each
other through a methyne bridge (-C=) and binds Mg
ion in the center.10 Close to the third pyrrole ring is
located the fifth isocyclic ring while in the fourth ring
is attached the propionic acid substituent esterified
by a hydrophobic phytol group.3,51,52 Chlorophyll in
plants mostly consists of two basic types namely
chlorophyll a and chlorophyll b.53,55 Chlorophyll a
has a methyl group (CH3) attached to R1 position
(Fig.1) thus its chemical formula is C55H72O5N4Mg
and has blue-green color. Chemical formula of
chlorophyll b is C55H70O6N4Mg which binds to the
formyl group (CHO) on R1 position (Fig. 1), and
has green-yellow color.52,56 Although chlorophyll a
is less polar than chlorophyll b, both are insoluble
in water but very soluble in ethanol and methanol.20
Chemical structure of chlorophyll and its derivatives
are given in Fig. 1.
In higher plants, chlorophyll generally composes 0.6
to 1.2%-wt of leaf wight on a dry matter basis.57 The
chlorophyll content of several plants (vegetables,
Fig. 2 The PCA plot of chlorophylls content (mg/kg dry matter) of several plants
(Note. Pr1: Suji leaf 19; X1: Suji leaf111; Zn1: Chicory (cv. Anivip), Zn2: Chicory (cv. Monivip), Zn3:
Dandelion leaf, Zn4: Garden rocket, Zn5: Wild rocket 17; dR1: Broccoli 110; Dp1: Golden Rosett apple
(flesh), Dp2: Golden Montana apple (flesh), Dp3: Golden Delicious apple (flesh), Dp4: Royal Gala
apple (flesh), Dp5: Fuji (F) apple (flesh), Dp6: Ariane apple (flesh), Dp7: Starking Red Chief apple
(flesh), Dp8: Pink Lady apple (flesh), Dp9: Fuji (I) apple (flesh), Dp10: Reina de Reineta apple
(flesh), Dp11: Green Golden Delicious apple (flesh), Dp12: Green Doncella apple (flesh), Dp13:
Granny Smith apple (flesh), Dp14: Golden Rosett apple (peel), Dp15: Golden Montana apple (peel),
Dp16: Golden Delicious apple (peel), Dp17: Royal Gala apple (peel), Dp18: Fuji (F) apple (peel),
Dp19: Ariane apple (peel), Dp20: Starking Red Chief apple (peel), Dp21: Pink Lady apple (peel),
Dp22: Fuji (I) apple (peel), Dp23: Reina de Reineta apple (peel), Dp24: Green Golden Delicious
apple (peel), Dp25: Green Doncella apple (peel), Dp26: Granny Smith apple (peel) 13)
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fruits, and nuts) are varied and the ratio of chlorophyll
a/b in plants ranged from 1.5-4.7 with an average
value of 3.1 (Table 1). The ratio can be varies due
to growth conditions and external factors, especially
high-light intensity and sun exposure.58
Clustered of chlorophyll content and its chlorophyll
a/b ratio of several greeny plants was evaluated using
Principal Component Analysis (PCA) to explore its
relationship. The vertical axis indicates chlorophylls
content (right: higher chlorophyll content, left: lower
chlorophyll content) whereas the horizontal axis
illustrated its chlorophyll a/b ratio (upper: higher ratio,
lower: lower ratio). PCA plot projected by PC1 and
PC2 showed variance occurred for different plants
(Fig. 2). Suji leaf (Pr1 and X1) separated from others
and located in negative quadrant. It revealed that
Suji leaf has higher chlorophyll content than other
plants with high chlorophyll b content that made its
chlorophyll a/b ratio is low.
The difference of chlorophyll content in green leaf is
influenced by growth location, leaf age in one tree,
and leaf position57. Leaf age is determined from the
position of leaf out on the stem from the shoot. The
older age leaves position will be further away from
the shoots. Jokopriyambodo et al. (2014)8 examined
that the chlorophyll content of mature Suji leaf extract
was twice as of immature leaf extract. Ozgen and
Sekerci (2011)59 research on spinach plant found that
the end portion of a leaf far from the stem also has
higher chlorophyll and phytochemical content than
the base of the leaf attached to the stem.
Suji leaves have 73.25% moisture content which
contains 2524.6 ppm chlorophyll a and 1250.3
chlorophyll b.18 Whilst spinach, the common source
of chlorophyll, is contain 1083.4 ppm of total
chlorophyll.60 The high amount or chlorophyll content,
especially chlorophyll a, has implication on the green
appearance of plant and a large ratio of chlorophyll
a/b indicates the convenience of chlorophyll to
dissolve in organic solvents (more hydrophobic).
For example, black locust leaf and Fuji (F) apple
(peel) which have the same chlorophyll a/b ratio of
3.8,13,50 however chlorophyll a and chlorophyll b of
black locust leaf were higher than those in apple
peel thus this leaf has greener color than apple skin.
The Suji leaves have high chlorophyll content with
small difference between chlorophyll a and b (Table
1). The high Chlorophyll content shown by Suji leaves
dark green appearance while its low Chlorophyll a/b
ratio make Suji’s chlorophyll can be easily extracted
using water. These two properties made Suji leaves
has good potency to be utilize as food colorant or
food ingredient.
The lower chlorophyll a/b ratio indicates the
convenience of chlorophyll to dissolve in aqueous
solvent. Chlorophyll a is less polar than chlorophyll
b thus lower ratio of chlorophyll a/b resulted in
higher solubility in water-based solvent. This
property is important in food processing because
water is widely used as a medium to dissolve food
products. Cassava leaf and Suji leaf contained high
chlorophyll content with different chlorophyll a/b ratio.
Cassava leaf has higher chlorophyll a/b ratio than
it in Suji leaf14. The lower ratio made chlorophyll in
Suji leaves more convenient to be extracted using
aqueous solvent. It is characterized by the juice
of Suji leaves that are darker green color than the
water of the cassava leaves or spinach leaves at
the same amount.
Postharvest Stability of Suji Leaf Chlorophyll
Similar to other green leafy plants, degradation of
chlorophyll in Suji leaf occurs during senescence
or postharvest processing.61 Degradation was
directly happened after leaves were harvested
through enzymatic and non-enzymatic reactions.62–64
Enzymatic degradation of chlorophyll occurs due to
the presence of chlorophyll degradation enzymes
that are endogenously present in plant tissues. The
enzymes degrade chlorophyll during senescence
or ripening of green fruits/vegetables.65 While non-
enzymatic degradation affected by environmental
factors and usually develop during post-harvest
processing.66
Enzymatic degradation of chlorophyll
Several types of identified chlorophyll-degradation
enzymes present in plant tissues include
peroxidase,67 Mg-dechelatase,68 pheophorbide a
oxygenase, red chlorophyll-reductase catabolite69,
and chlorophyllase.70 Hörtensteiner and Kräutler
(2011)62 and Ankita and Prasad (2015)71 mentioned
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that in the plant tissue, the initial phase of chlorophyll
degradation begins with activity of chlorophyll b
reductase enzyme and 7-hydroxy methyl chlorophyll-
reductase which transforms chlorophyll b to
chlorophyll a. The conversion encrypted by Non
Yellow Coloring 1 (NYC1) gene62,72 While peroxidase
enzyme starts chlorophyll degradation process by
oxidizing chlorophyll a to 132-hydroxychlorophyll a.73
Chlorophyll a lost its phytol group due to the
activity of the Chlorophyllase enzyme and forms
chlorophyllide a.74,75 chlorophyllase enzyme is
actually a glycoprotein compound in the thylakoid
membrane. Schwartz et al. (2008)57 explains that
chlorophyllase is an esterase enzyme that catalyzes
detachment of phytol groups from chlorophyll
and pheophytin structures. It activity is limited to
porphyrins with carboxyloxy groups at C-10 and
hydrogen at C-7 and C-8 positions. The enzyme
is active in temperature range 60-82.2°C and it is
damaged at higher temperatures (>100°C).10 The
presence of chlorophyllide is desired to maintain the
green color in heated vegetables.
Chlorophyllide which lost its Mg2+ called
pheophorbide.75 The Mg-dechelatase enzyme
or Mg-dechelatase substance will release
Mg2+ in chlorophyll a and form pheophorbide a
structure.73 Mg-dechelatase is active on artificial
chlorophyll substrate (chlorophyllin) but not on
natural compounds of chorophyllide.69 While Mg-
dechelatase substance reacts to chlorophyllin and
chorophyllide. It indicates that on in vivo the loss
of Mg atoms from the chorophyllide structure is
more due to the activity of low molecular weight
compounds.62 Pheophorbide a decomposes into a
fluorescent chorophyll catabolite, which is generally
colorless, through the formation of red chlorophyll
catabolite by pheophorbide a oxygenase enzyme
and red chlorophyll reductase.76
Schelbert et al. (2009)77 reported the role of
pheophytinase enzymes (pheophytin pheophorbide
hydrolase, PPH) in converting pheophytin to
pheophorbide. The presence of lipoxygenase,
peroxidase, and polyphenol oxidase (PPO) enzymes
in plant tissues could degrade the quality of
vegetables/fruits and responsible in chlorophyll
degradation.62,67,78–80 The lipoxygenase plays an
important role in the establishment of hydroperoxides
and free radicals that induce yellow color or
colorless appearance.10 Peroxidase enzyme is an
oxidoreductase enzyme found in many organelles
including chloroplasts.81 Although the peroxidase
enzyme has a significant role in fruit ripening, it
is also responsible for chlorophyll degradation
through oxidation of phenolic compounds by
hydrogen peroxide.67,82,83 Further, the phenoxy radical
oxidize chlorophyll into colorless compounds10.
The chlorophyll-oxydase enzyme is located in the
thylakoid membrane of chloroplasts.83 This enzyme
oxydize chlorophyll into chlorophyll a-1 compounds
that are suspected to be intermediates in the
chlorophyll degradation process.73
Non-enzymatic degradation of chlorophyll
Environmental factors or processing of horticultural
products (green fruits and vegetables) makes
chlorophyll unstable. The presence of heat, acid or
low pH, light, or microbial agents made chlorophyll
degraded through several processes.66,84,85 The
replacement of Mg2+ with two H+ ion in chlorophyll
structure known as pheophytinization.86 The change
of chlorophyll into pheophytin is influenced by pH
or acid condition. Eckardt (2009)87 asserted that
formation of pheophytin becomes the first stage
of chlorophyll degradation during senescence,
followed by the release of phytol groups. The original
blue-green chlorophyll a turns into a gray-colored
pheophytin a and green-yellow chlorophyll b turns
into a brown-colored pheophytin b. The conformation
of pheophytin from chlorophyll a occurs 2.5-10 times
faster than that of chlorophyll b.57
The green color of chlorophyll in the vegetables/fruits
was turning to olive-green during food processing.
The organic acids from the food product were
released during processing and decresed pH while
chlorophyll is stable at alkaline condition.84 During
fermentation, chlorophylls change to become
pheophytins and pyropheophytins. Decreasing
pH and green color changing accelerated with
the presence of acid-producing microbes.88 The
presence of heat during fermentation is thought
to affect pyropheophytin formation. Pheophytins
and pheophorbides as derivatives products from
chlorophyll degradation were found in the final
product.57
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Thermal process makes protein in the chlorophyll-
protein complex denatured thus chlorophyllase
enzymes meet their substrate. Its reaction de-
esterificate phytol groups in chlorophyll and produces
chlorophyllide structure.74 The chromophore
character of chlorophyll in de-esterification reaction
is unchanged and the color remains green.3 The
phytol group (which makes chlorophyll fat soluble)
will be detached from the chlorophyll structure thus
increasing the polarity of chlorophyll. Minguez-
Mosquera et al. (2008)89 mentioned that pheophytin
can also be a substrate to de-esterification reaction
and form pheophorbide structure. Ramírez et al.
(2015)16 found that all cholorophyll in processed
olives contain no Mg ion (pheophytin form). Pumilia
et al. (2014)15 mentioned that 85% decrease
of pheophytin a and b levels during 60 min
roasting of pistachio nuts as well as an increase in
pyropheophytin a and b levels 10-12 times higher
than in raw pistachio. The addition of blansir time
resulted in decreased levels of chlorophyll a and b
in spinach and increased levels of chlorophyll a and
b and pheophytin a and b due to pheophytinization
reactions.90 In refrigerating and freezing process,
which generally maintains post-harvest quality
of horticultural products, chlorophyll damage still
occurs. Spinach stored at freezing temperatures
continues to undergo changes in its green color.91
High-pressure treatments do not cause chlorophyll
degradation in some vegetables, while combination
of high-pressure and high-temperature treatment
rapidly degrade chlorophyll.60 Although chlorophyll
b is more resilient than chlorophyll a at 70°C but
both are degraded at higher temperatures. The
alternative thermal processing to extend fruit shelf-
life is microwave heating. Nevertheless, processing
of kiwi fruit with microwave still eliminates most of its
chlorophyll content and pheophytin a becoming the
major component in the processed kiwi.11
Chlorophyll degradation reactions occur in the isocyclic
ring (cyclopentanone/ring V) are differentiated
into epimerization, decarbomethoxylation, and
allomerization.89 Epimerization is an isomerization
reaction between H and CO2CH3 molecules on an
isocyclic ring of chlorophyll. This reaction does not
cause discoloration. Decarbometoxylation is an
oxidative reaction that replaces the carbomethoxy
group in C-132 (COOCH3) with H ion without altering
the basic porphyrin structure. This reaction produces
pyro-derivative compounds that have same color
and spectroscopic character as their precursors
(chlorophyll, pheophytin, chlorophyllide, and
pheophorbide). While allomerization reactions occur
when the isocyclic ring is oxidized by an oxygen
triplet molecule (3O2) or release of carbomethoxy
group at C-13.2,57
Chemical degradation of chlorophyll in Suji leaf
and other green plants can go through several
processes, but in general the hypothetical pathway of
chlorophyll degradation in Suji leaf suggested takes
place through two paths. The first pathway is through
the formation of pheophytin due to the activity of
enzymes or acid conditions and the second pathway
is through the formation of chlorophyllide as a result
of specific reactions by chlorophyllase enzymes (Fig.
Fig. 3: General pathway of chlorophyll degradation in plant or processed food
617
ANDARWULAN et al., Curr. Res. Nutr Food Sci Jour., Vol. 6(3), 609-625 (2018)
3). Pheophytin and chlorophyllide then degraded
into pheophorbide which loss it green color property.
Pheophorbide can be further degraded to colorless
products at a final step.
Chlorophyll Degradation Kinetic
Degradation kinetics of chlorophyll and its derivative
products during processing can be used as a model
for predicting shelf life.92 Kinetic model are able to
estimate the shelf life of a product based on different
variables that can affect food degradation.93 Kinetics
of chemical reaction described as the measurement
of reaction velocity and analysis of experimental data
to determine factors affecting the reaction, such as
reaction rate, reaction order, equations that give
information about the speed dependence on product
concentration that will affect the reaction, and the
effect of temperature on reaction rate.94
Chlorophyll a damages 2.5 times faster than
chlorophyll b with activation energy ranged from
4.80±0.91 to 14.0±0.71 kcal/mol for chlorophyll a
and 6.84±0.29 to 11.0±1.06 kcal/mol for chlorophyll
b at various pH values.84 Chlorophyll’s green color
loss, due to heat and oxidation, mostly follows the
Arrhenius first order reaction. A slight change in
temperature will leads to increasing chlorophyll
kinetic.93
Various factors that affect the rate and kinetics of
chlorophyll degradation were thermal and acidic
condition, microbial growth, and light intensity.88,95–98
The green color of blanched green peas was rapidly
disappearing with decreasing pH.84 Gunawan and
Barringer (2000)88 found that acid compounds
containing benzene rings induced color changes
faster than acid compounds with simple carbon
chains because of their hydrophobicity. Their study
showed that microbial growth accelerates the color
change by produce acid and cause occurence of
holes in the broccoli surface. Ghidouche et al. (2013)4
predicted the shelf life of multiple colorants by
increasing the intensity of light. The result concluded
that the light treatment has higher damage effect to
the chlorophyll structure than temperature treatment.
The increase of light intensity would increase the
chlorophyll degradation rate. Photodegradation
rate of chlorophyll was easily to changes of light
intensity and can be described using the first order
reaction model.94 The presence of lipid protected
chlorophyll from photo-oxidation. Triolein acid gave
better protection and retard the kinetic rate of
photodegradation than oleic acid in a paraffin oil
system, possibly due to rivalry between lipids and
chlorophyll for singlet oxygen.99
A first-order kinetic mechanism was suitable for
illustrating the discoloration processes of virgin olive
oils under non-oxygen thermal auto-oxidation.93 The
kinetic constants for chlorophylls degradation was
3.6 times lower than the respective constants for
carotenoids. Chlorophyll has higher activation energy
(16.03 ± 0.26 kcal.mol-1) than carotenoids (15.45
± 0.17 kcal.mol-1). It indicates that small changes
in temperature can increase the kinetic constant
of chlorophyll. Chlorophyll structure is also more
resistant to color changes due to heat.93 Decreased
retention of microencapsulated Zn-chlorophyll
pigments followed first-order kinetics with velocity
constants (k) 1.5×10-3 day-1 and half-life predictive
for more than 15 months.
Although specific kinetic study on chlorophyll in
Suji leaf is not available yet, its predictive thermal
degradation might followed the first-order kinetic of
Arrhenius reaction. Discoloration process due to
photodegradation of chlorophyll in Suji leaf will also
follows the first-order reaction. The shelf-life model
for aqueous-based photosensitive food, incuding
chlorophyll from Suji leaves, could be predicted
using formula by Manzocco et al. (2008)100 as follows:
where SLL,T is the product shelf-life at certain light
intensity (L) and temperature value (T), a is the final
color value, T is the absolute temperature (K), Tref is
the reference temperature, and R is the molar gas
constant (8.31 Jmol-1K-1).
The detail mechanism of chlorophyll degradation
of Suji leaf has not been studied. Information on
chlorophyll thermal- and photodegradation of Suji
leaf will give better understanding on degradation
mechanism thus leads to more effective ways to
maintain chlorophyll stability.
618
ANDARWULAN et al., Curr. Res. Nutr Food Sci Jour., Vol. 6(3), 609-625 (2018)
Retaining Green Color Stability of Suji Leaf
Chlorophyll
Utilization of liquid chlorophyll extract from Suji leaf
as colorant has been done since long time ago in
Indonesia. But it is less practical and the color has
lower stability than synthetic colorants.1 Chlorophyll
extraction on a household scale usually done by
grinding green leaves then extracted using water.
The liquid extract then filtered and added to the
food and beverage processing process. Liquid
extraction method has a disadvantage that liquid
extract should be directly used. If the liquid extract
is not immediately used then the green color will
turn brown.
Previous researches on preserving green color
of chlorophyll extract were mainly focused on
maintaining chlorophyll structure, formation of
chlorophyll-derived compounds that were still
green and more resistant to heat (chlorophyllide),
or making metallo-chlorophyll complexes.101
These efforts include: heat treatment to inactivate
chlorophyllase enzyme and minimize chlorophyll
conversion to pheophytin, formation of bright green
metallo-chlorophyll complex by adding Cu2+ or Zn2+
salt, control to the process conditions (temperature,
pH, and ionic strength of food products), and
addition of surface active agents.66 However previous
studies on Suji leaves extract are mainly focused
on producing metallo-chlorophyll complexes with
addition of Zn to substitute Mg in the center of
chlorophyll structure.18,20,102
Addition of CaCO3 and MgCO3 could retained
green color of chlorophyll from Suji extract.103
Those salts completely neutralized the acid in
the plant tissue and avoid acidification thus inhibit
the arrangement of pheophytins in chlorophyll
extract.
104,105
Jokopriyambodo et .al. (2014)
8
produced
powdered colorant from water-insoluble Suji leaf
extract using dried oven. Since powdered Suji
extract has low solubility, the encapsulated powder
by addition various coating materials was expected
to increase its solubility and protect chlorophyll from
environment damage. Therefore, development of
natural colorant through chlorophyll drying process
to become powder made it more convenient for
storage and application. Combination of extraction
using ZnCl2 and selected coating agent by spray
drying method resulted best characteristics in green
color, total chlorophyll, and antioxidant activity. A 30%
n-octenyl succinic anhydride (OSA) modified starch
as filler was able to retain green color of chlorophyll
extract.106 Other extraction technique was maceration
with aqueous solvent and NaHCO3 as a stabilizer and
thus powdered using maltodextrin by freeze dryer.18
Despite green color of chlorophyll powder was faded
due to addition of coating agents, encapsulation
method made it more easily to dissolve in food.
Research on Suji leaf extraction method found that
optimum extraction process of natural pigment from
Suji leaf was done by stabilized aqueous extraction
method with the addition of 700 ppm ZnCl2 combined
with experiment parameters (pH 7 and temperature
of 85°C). These method yields total chlorophyll
content of 47.2975 mg/100 g fresh weight.20
Chlorophyll degradation into pheophytin can also be
affected by the presence of surface active agents for
example detergent. Detergent absorbed on surface
of thylakoid cell membrane, where the chlorophyll
is located, affects the H+ permeability through the
membrane. The presence of cationic detergents
will block the H+ ions on surface membrane diffusing
into the cells to prevent chlorophyll degradation.
In contrast, anionic detergents will attract H+ ions,
increase their concentration on surface membrane,
and increase their diffusing rate. Thus the presence
of anionic detergents can accelerate chlorophyll
degradation. While non-ionic detergents on surface
membrane will reduce its negative charge so that H+
ions will be attracted to the surface and consequently
chlorophyll degradation will decrease.57 Addition of
Tween 80 increased the total chlorophyll content
of extract and retained its green color. As non-
ionic detergent, Tween 80 suppressed pheophytin
formation, help the lipophilic chlorophyll emulsified
in water, and facilitate contact with chlorophyllase
enzymes.19
Potential Development of Natural Green Colorant
from Suji Leaf
Efforts to develop natural colorant from Suji leaves
have not been able to maintain its stability. Further
research needs to be done so that the green color
of colorant is similar to fresh leaf. A comprehensive
quantitative study also needs to estimate shelf-life
and its application in food and non-food products.
Industrial scale-up also needs to be intensively
619
ANDARWULAN et al., Curr. Res. Nutr Food Sci Jour., Vol. 6(3), 609-625 (2018)
undertaken so that the production of natural
colorant can be massively produced and of has high
economic value.
Suji leaves have better characteristics than other
chlorophyll sources. The Suji leaves have no
gel-forming components which could prevent the
extraction of chlorophyll from leaf tissue. In addition,
Suji leaves also have non-toxic components such as
cyanide on cassava leaves.107 Its leaf is not bitter but
rather has specific scent.28 Compared to basil, Suji
leaves have a distinctive lighter leaf scent that is
favored and widely applied to various food products
without disturbing the original aroma of the product.
Utilization of Suji plants was limited recently, as an
ornamental plant and food coloring in the household
scale. Nonetheless Suji plants are promising to be
cultivated intensively because its propagation is easy
by stem cuttings or grafting.
Green pigments content in Suji plants is potential
to be used as food coloring materials as well
as functional food ingredients. Marquez and
Sinnecker (2008)108 states that the requirement
for an economical material to be used as a source
of chlorophyll are its high pigment content, its
availability, the ease of harvesting and drying,
efficient extraction mechanisms, and the low activity
of chlorophyllase enzymes. Suji leaf have fullfill
most of the requirement as described above thus its
support the potential of Suji leaves to be developed
as a source of chlorophyll that can be used as food
coloring as well as functional food raw materials.
Conclusion
Chlorophyll from Suji leaves extract has unique
characteristics due to its content and ratio of
chlorophyll a/b. This ratio makes Suji’s chlorophyll
easier to dissolve in aqueous solvent than other
sources. Chlorophyll might be turns into its
derivatives through enzymatic and non-enzymatic
degradation during processing followed first-order
of Arrhenius kinetics reaction. The degradation was
prevent by heat treatment modification, formation of
metallo-chlorophyll complex, control of processing
condition, and addition of antioxidant compounds.
Chlorophyll extract was developed from liquid form
to encapsulated powder using several methods. The
chlorophyll pigments contain in Suji plants is potential
to be used as colorant or food ingredients due to
no gel-forming property, non-toxic components,
and easy to cultivate. Due to limited research on
chlorophyll from Suji leaf, further research needed
to optimize the utilization of its potency and stability.
Acknowledgement
The authors thank the Ministry of Research,
Technology and Higher Education of the Republic
of Indonesia for research funding and Southeast
Asian Food and Agricultural Science and Technology
(SEAFAST) Center, Bogor Agricultural University for
supporting the publication.
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... Several factors (e.g., heat, low pH, light, or enzymes) may degrade Chl during the extraction process. The replacement of Mg 2+ with two H + ions (pheophytinization) triggers the conversion of blue-green Chl a into gray pheophytin a, whereas green-yellow Chl b turns into a brown pheophytin b (Indrasti et al., 2018). The degradation of Chl a occurs 2.5-10 times faster than that of Chl b (Eckardt, 2009). ...
... Reported values are mean ± 95% confidence interval of triplicate measurements. those reported in the literature (Indrasti et al., 2018), and remained constant as the enzyme dose changed. ...
... These results appeared relevant given that Chl a is the least stable form of pigment, as described in literature. Indrasti et al. (2018) reported that Chl a degrades 2.5 times faster than Chl b due to heat, pH, and oxidation phenomena. ...
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... Among antioxidant compounds chlorophylls and carotenoids are of particular interest, as pigments for the pharmaceutical, cosmetic, and food industries (respectively E140 and E160). We found that CL has a total quantity of chlorophylls comparable to spinach, followed by PP and SJ with values major of green papric and VA with values comparable to that reported for apple peel [45]. The quantity of chlorophyll a is slightly below the content of chlorophyll b, probably due to the organic solvent used, which favors the extraction of the most lipophilic chlorophyll b. ...
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... It was proven that particle sizes 40 mesh produced more chlorophyll compared to 40 mesh. However, in smaller sizes, particles tended to cluster and block the solvent so extraction did not take place well [14]. ...
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... Furthermore, the resulting waru leaf extract is solid brown with a characteristic waru odor. The brown color of waru leaf extract is caused by the drying process so that the green color of chlorophyll in the leaves is oxidized [9]. The concentration of waru leaf extract can be reduced by centrifugation so that dissolved particles in the extract can be removed. ...
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Chlorophyll concentrations of biological soil crust (biocrust) samples are commonly determined to quantify the relevance of photosynthetically active organisms within these surface soil communities. Whereas chlorophyll extraction methods for freshwater algae and leaf tissues of vascular plants are well established, there is still some uncertainty regarding the optimal extraction method for biocrusts, where organism composition is highly variable and samples comprise major amounts of soil. In this study we analyzed the efficiency of two different chlorophyll extraction solvents, the effect of grinding the soil samples prior to the extraction procedure and the impact of shaking as an intermediate step during extraction. The analyses were conducted on four different types of biocrusts. Our results show, that for all biocrust types chlorophyll contents obtained with ethanol were significantly lower than those obtained with dimethyl sulfoxide (DMSO) as solvent. Grinding of biocrust samples prior to analysis caused a highly significant decrease in chlorophyll content for green algal lichen- and cyanolichen-dominated biocrusts, and a tendency towards lower values for moss- and algae-dominated biocrusts. Shaking of the samples after each extraction step had a significant positive effect on the chlorophyll content of green algal lichen- and cyanolichen-dominated biocrusts. Based on our results we confirm a DMSO-based chlorophyll extraction method without grinding pretreatment and suggest to insert an intermediate shaking step for complete chlorophyll extraction (see supplement S6 for detailed manual). Determination of a universal chlorophyll extraction method for biocrusts is essential for the inter-comparability of studies conducted across all continents.
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