International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04 7

Preparation of Edible Grade Dye and Pigments From

Natural Sources Bixa Orellenae Linn
Aminul Islam Chowdhurya, Md. Ashraful Islam Molla a, Mithun Sarkerb , Ashequl Alam Rana a, Swapan Kumer Rayc,
Husna Parvin Nurc and Mohammad Mainul Karima*
a
Department of Applied Chemistry and Chemical Engineering, University of Dhaka, Dhaka-1000, Bangladesh,
b
Department of Applied Chemistry and Chemical Engineering, Noakhali Science and Technology University,
Noakhali, Bangladesh, cFibre & Polymer Research Division, BCSIR labs, Bangladesh Council of Scientific &
Industrial Research (BCSIR), Dhaka-1205, Bangladesh.

Abstract - Annatto is a natural colouring agent obtained from
the outer coats of the seeds of the tropical shrub Bixa orellana.
The major colour principles of annatto are the carotenoids bixin.
Annatto seeds were collected from the local market and it was
ground by mortar and pestle. The ground annatto was taken in a
steam distillation and evaporated. After the evaporation the
sample was dried and ground .The ground annatto seeds are
steam distilled in order to remove the odor. It is possible to
remove upto 6.42 % of the volatile substances from the annatto.
Then different solvents were put on trial and error basis to get
the optimum extract of bixin color. All of the extracting solvents
gave dark red color of bixin. The used solvents are 95 %
ethanol,(cold extraction) 95% ethanol (hot extraction), 10%
glacial acetic acid, 100% glacial acetic acid, 1% alkaline solution
, 0.5% alkaline solution and 0.25 % alkaline solution .Among the
lots of extracting chemical solvents 0.25% alkaline solution gave
the optimum color. It could extract upto 41% of the bixin from
the annatto seeds. The alkaline solution was afterwards
neutralized by acetic acid. Then the collected bixin was taken for
a set of characterizations such as FTIR. S ince pigments are
mostly water insoluble with exceptions of certain pigments of
biological origin. Results of the color extraction from the fresh
and the dried annatto seeds indicated that the extract from the
fresh gave the higher color intensity. From the data it is seen that
the longer the extraction time, the more the concentration of the
gardenia fruit color extracted from either the dried or the fresh
annatto seeds. Owing to the volatile essential oil present in the
bixa seeds studied, the efficiency of the color extraction might be
interfered. Therefore, the investigations on the color extraction
from the annatto seeds with different levels of fat content were
performed. Food colorants may be classified into synthetic,
nature-identical, inorganic, and natural colorants. Colorant
shows activity when applied to a substance. Colorants become the
most sensitive part of any commodity not only for its appeal but
also, it enhances consumer acceptability The color from bright
green spinach, ruby red strawberry, and deep orange pum
making a light – screen support; and to increase acceptability of
food as an appetizing item. Though chemically very similar,
differences in their chemical properties present several
challenges to the analytical chemist with respect to
stereochemistry, solubility, chromatographic behaviour and
stability. This review focuses on the chemistry, stability, and
analysis of annatto pertaining to its use as a permitted.
Index Term-- annatto, bixin, achiote, genotoxicity.
I. INDRODUCTION
Bixin is used both as a spice and a dye since ancient time
[1] [2].Color is the first sensory quality by which foods are
judged, since food quality and flavor are closely associated
with color. In addition, many colorless convenience foods
such as confectionary products, snacks, beverages and gelatin
desserts, the food color was added in order to identify the taste
and increased the palatability. The color generally added to the
food is either natural or synthetic food colors. Since more and
more synthetic food colors were reported unsafe (FAO, 1963;
FAO, 1985; JECFA, 1964) therefore, the search for more
natural food colors is of vital importance. Nowadays, bixin is
also widely applied in food industry and in cosmetics as an
effective natural coloring agent [3] [4]. Food, pharmaceutical,
and cosmetic manufacturers can draw from an array of dyes
and pigments, synthetic or naturally derived, to color their
products. Although synthetic colors have been favored over
the past 100 years, processors have recently turned to naturally
derived colorants as a viable alternative, responding to an
ever-increasing consumer demand for natural products. All
these food dyes and colors are either of the two types. One, the
natural food colors or the natural food dyes. And second, the
artificial or synthetic food colors. The natural food colors are
obviously safer than the artificial food colors. Natural food
colors are extracted from edible food products. They might be
bought from the market or alternately they can also be made in
the household kitchens. In recent years, several beneficial
biological activities of bixin were reported such as free-radical
quenching ability [5] [6], regulation immunoglobulin
production [7] and protective effect on radiation-induced
genotoxicity [8].
Annatto, also called as Roucou, is a derivative of the
achiote trees of tropical regions of the Americas. It is widely
used to produce a red food coloring and also as a flavoring. Its
scent is described as "slightly peppery with a hint of nutmeg"

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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04
and flavor as "slightly sweet and peppery". It is commonly
found in Latin America and Caribbean cuisines as both a
coloring agent and for flavoring. Central and South American
natives use the seeds to make a body paint, and lipstick. For
this reason, the achiote is sometimes called the lipstick -tree.
Achiote originated in South America and has spread in
popularity to many parts of Asia. The heart-shaped fruits are
brown or reddish brown at maturity, and are covered with a.
short, stiff hairs. When fully mature, the fruits split open b.
exposing the numerous dark red seeds. While the fruit itself is c.
not edible, the orange-red pulp that covers the seed is used as d.
a commercial food coloring and dyes (similar to turmeric).e.
The achiote dye is prepared by stirring the seeds in water or f.
oil. g.
The use of food additives to enhance the microbial safety
of foods is of great interest to the food industry. Essential oils
of spices such as cloves, garlic, mustard, and cinnamon are
known to contain compounds that possess antimicrobiala.
activities. Understanding how natural antimicrobials work and
affect the growth of microorganisms can lead to new b.
technologies for their use in maintaining the safety and quality c.
of foods [9].The increasing use of natural extracts in foods d.
suggests that, in order to find active compounds, a systematic e.
study of medicinal plants is very important. Bioautography as
a method to localize antimicrobial activity on a chromatogramf.
has found widespread application in the identification of new
antimicrobial agents [10, 11]. Annatto extracts are naturalg.
coloring agents obtained from seeds of the tropical bush Bixa
orellana L. The pericarp of annatto seeds is composed of up h.
to 80% of the carotenoid cisbixin IUPAC: methyl hydrogen i.
9¢-cis-6,6¢-diapocarotene-6,6¢- dioate, also known as 9¢-cis-
bixin or R-bixin [12]; the remaining 20% include trans- and
cis-norbixin (6,6¢-diapocarotene-6,6¢-dioic acid and 9¢-cis-
6,6¢-diapocarotene-6,6¢-dioic acid), several apocarotenoids,j.
volatile compounds [13], and other uncharacterized
substances. When treated with alkali, bixin and all its isomeric k.
forms are transformed to their water-soluble norbixin l.
counterpart. The major coloring component of commercialm.
water-soluble annatto extracts is 9¢-cis-norbixin. Besides its n.
uses in food as a natural colorant and spice, the medicinal u ses
of annatto include anecdotal treatment of diabetes [14], skin
infections, burns, fever [15], measles, gonorrhea [16],
diarrhea, and asthma [17]. Previous reports indicate that
commercial annatto extracts have biological activities against
microorganisms of significance to food fermentation,
preservation, and safety [18]. However, there is no report
describing the nature of the antimicrobial compound(s) in
commercial annatto preparations. The studies presented here
were designed to isolate and identify the main antimicrobial
compound(s) present in water soluble annatto extracts.
Annatto and its extracts are designated collectively as E160b
and are permitted as a food additive in the European Union
and elsewhere, and have widespread use in the food industry
for the colouring of many commodities including flour and
sugar confectionery, dairy and savoury products, soft drinks
and fish.
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II. M ATERIALS AND M ETHODS
Materials
Fresh Bixa seeds were purchased from the local market
and 10.295 g of bixa was weighed and ground in the mortar
and pestle. The following analytical grade chemicals were
used:
Absolute Alcohol.
Zinc sulphate (ZnSO4 .7H2 O)
Ammonium carbonate ((NH4 )2 CO3 )
Commercial Zinc oxide (ZnO, Fluka , Switzerland)
Hydrochloric acid (Merk, Germany)
Sodium hydroxide (NaOH, Merk, Germany)
KBr (Merk, Germany)
The following instruments were used to prepare and
characterize the photocatalysts and to monitor the adsorption
and the photo catalytic degradation of the dyes:
Thermostated Mechanical Shaker (SWB-20, HAAKE, Fisons
Ltd, Germany)
Digital Balance (Model: FR-200, JAPAN)
Muffle Furnace (ISUZU Seisakusho)
Microwave Oven (NDO-450ND, EYELA)
Magnetic Stirrer (EYLA: Heating Stirrer, RCH-10
Cooperation Bangladesh-Japan-1990)
UV- Visible Recording Spectrophotometer (UV-1650 PC,
Shimadzu, Japan).
UV-Light Source wavelength 254 nm (Applied Photophysics,
England)
pH Meter ( HM-26S, TOA Electronics Ltd, Japan)
Laboratory Centrifuge Machine (Y J 03-043-4000 China and
Max. Energy-200J, 50Hz, 220-240V.240W Manufactured to
BS 440L-D NICKEL ELECTRONIC Ltd, Weston-S- Mare,
Avon, England)
Laser Induced Breakdown Spectrophotometer (LIBS, 200 mJ
Nd: YAG Laser)
FT-IR Spectrometer (IR-Prestige, Shimadzu, Japan)
Scanning Electron Microscopy, SEM (Philips XL30, Holland)
Deionizer, (Puric, model-s, Takashima Keiki Co Ltd. Japan)
Magnetic Stirrer (EYLA: Heating Stirrer, RCH-10
Cooperation Bangladesh-Japan-1990).
Steam distillation
Steam distillation is a special type of distillation (a
separation process) for temperature sensitive materials like
natural aromatic compounds. Many organic compounds tend
to decompose at high sustained temperatures. Separation by
normal distillation would then not be an option, so water or
steam is introduced into the distillation apparatus. By adding
water or steam, the boiling points of the compounds are
depressed, allowing them to evaporate at lower temperatures,
preferably below the temperatures at which the deterioration
of the material becomes appreciable. After distillation the
vapors are condensed as usual, usually yielding a two -phase
system of water and the organic compounds, allowing for
simple separation. When a mixture of two practically
immiscible liquids is heated while being agitated to expose the
surfaces of both the liquids to the vapor phase, each
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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04
constituent independently exerts its own vapor pressure as a
function of temperature as if the other constituent were not
present. Consequently, the vapor pressure of the whole system
increases. Boiling begins when the sum of the partial pressures
of the two immiscible liquids just exceeds the atmospheric
pressure.
9
The extracted bixa and photo degradated product yields
at different times of photolysis were analyzed by FT-IR
spectrometer for their characterization. The FT-IR spectra of
the samples were recorded in the region of 4000 cm-1 – 400
cm-1 with 2 cm-1 resolution. Attenuated total reflection (ATR)
was used to record the spectra of the. In this mode, a small
amount of solid sample was put on the ZnSe crystal, which
allowed total reflection when the IR radiation was allowed to
pass at a predefined incident angle.

A pH meter is an electronic instrument used to measure the

pH (acidity or alkalinity) of a liquid (though special probes are
sometimes used to measure the pH of semi-solid substances).
A typical pH meter consists of a special measuring probe (a
glass electrode) connected to an electronic meter that
measures and displays the pH reading.

III. RESULT AND DISCUSSION

Steam distillation is employed to remove of essential oils, for
instance, perfumes from Bixa powder. The percentage of the
essential oil content was calculated by the following formula:
% of essential oil =
weight of bixa taken  weight of residue
 100
weight of bixa taken
Fig. 1. Steam distillation method

Characterization of the bixin product by FT-IR

T ABLE I
REMOVAL OF THE ESSENTIAL OIL FROM BIXA P OWDER BY STEAM DISTILLATION

Sl. Dis tillatio n A n n at t o W t. of Steam v o latile

No . time (h r) Us ed (g ) Res id u e (g ) Su b s tan ces (%)

01. 2.5 10.0295 9.4672 5.61

02. 3 10.0150 9.4194 5.95

03. 3 10.0251 9.3815 6.42

Table I show that it is possible to remove 4-6% essential oil from Bixin to get odorless residue by steam distillation method.

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T ABLE II
EXTRACTION OF BIXIN BY USING DIFFERENT EXTRACTING SOLVENTS
Sl. Extracting Extracting Amount Extraction Extraction Amount of Extract
Solvent Solvent of Bixa method time (hr) extract (g) (% )
No. Used (ml) used (g)

01 95% ethanol 250 2.0023 Cold 48 0.3636 18.15

Extraction

02 95% ethanol 250 2.0008 Ho t 48 0.2774 17.32

extract io n

03 10% glacial 50 2.0347 Cold 48 0.2714 13.34

acetic acid+ 90% Extraction
absolute alcohol

04 Glacial acetic 50 2.0072 Cold 48 0.3699 18.43

acid Extraction

05 Hot water 100 4.8603 Ho t 24 1.5303 31.48

extract io n

06 Alcoholic sodium 200 12.0315 Cold 24 4.6953 39.02

hydroxide Extraction

Table II show the extraction of bixin by using 95 % ethanol
(cold extraction) 95% ethanol (hot extraction), 10% glacial
acetic acid, 100% glacial acetic acid, 1% alkaline solution,
0.5% alkaline solution and 0.25 % alkaline solution as
solvents. It is seen that the extraction rate is minimum in 10%
glacial acetic acid+ 90% Absolute alcohol and upto 39.02%
bixin extractable using the cold extraction method by
employing alcoholic sodium hydroxide as extracting solvent
which is the maximu m extraction rate.

Thin Layer Chromatography (TLC)

Fig. 2. Standard Curve of Bixin

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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04 10
Chemicals: Bixin was obtained from BCSIR food division lab optimal chromatographic conditions adopted for the study
and solvents were obtained from Merck, Darmstadt, Germany. were achieved by adjusting the instrumental parameters as
Extraction and Scanning Conditions: such;
Standard Solution: 1% stock solution of standard bixin was C Distance from the base edge = 20 mm
prepared. Different application volumes of standard bixin C Band length = 8 mm
were applied on the TLC stationary layer starting with 100 nL C Track distance = 20 mm
to 500 nL to illustrate the standard curve (Figure 2). C ¹ of applications = 8
Bixa Samples: Five grams aliquots of seeds powder of 2 2 3 C After development in CHCl – MeOH (95:5), the plates
Bixa orellana were extracted with EtOH, CH Cl and 2 2 was measured at 460 nm with a TLC Scanner III (Camag,
EtOH-CH Cl (1:1). The extracts were evaporated in vacuo at Switzerland).
400 C. 1% solution of each extract was prepared. 10 μL bixin C Figure 4 represents densitometric TLC chromatograms of
samples were applied bandwise onto the plates using the Ethanol sample (a), Chloroform sample (b) and Chloroform:
automated TLC sampler III (Camag, Switzerland). The Ethanol, 1:1.

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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04 12

Fig. 3. Densitometric T LC chromatograms of Ethanol sample (a), Chloroform sample (b) and Chloroform: Et hanol, 1:1(c)
T he identification and quantitative determination of bixin, indicaxanthin, betanin and isobetanin upon extraction of these sp ecific constituents was studied. T he
standard bixin has been subjected to T LC densitometric evaluation under the suit able conditions proved that this technique is suitable for the detection of these
molecules under the selected conditions. Different application volumes of standard bixin were applied on the TLC stationary layer to illustrate the standard curve
(Figure 2). T he samples under investigation were analyzed under the same conditions as the standard (Figure 3). Concentrations of bixin in the different prepared
samples were calculated.

T he results indicated that the highest percent of bixin was found in sample extr acted with 100 % EtOH (1.37 %) followed by the sample extracted by 3
EtOH/CHCl (1:1) (1.23 %) and finally the sample 3 extracted with 100 % CHCl (1.16 %). These results are in agreement with tha t obtained previously by HPLC
[12].

The FTIR report and characterization:

b) C=O stretching vibration : in the region of 1680-1710

cm—1

c) For C-O : C-O stretching and OH bending vibration

is in the region of 1210-1320cm1

d) C-O-H bending: in the region of 1395- 1440 cm—1

2) For Ester group – COOR:

–1
a) C=O stretching vibration: 1735-1750 cm

b) C-O stretching: 1000-1300 cm—1

1) For -COOH group :

a) –OH stretching vibration : absorption in the region of

2500-3300 cm-1

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Fig. 4. Chemical structure of bixin

Fig. 5. FT IR peak of BIXIN at normal T emperature

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3) For unsaturated C=C: The IR region is 1640- 1660 cm—1

Fig. 6. IR peak of heat treated bixin

Figure 5 show the FTIR peak of bixin at normal Temperature. IV. CONCLUSION
The Results of FTIR clearly characterize the presence of pure Importance of natural dye is more relevant world wide
bixin in the extracted sample. in the context of increasing environment consciousness. The

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 International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:10 No:04 15
non-toxic, biodegradable and eco friendly properties make [12] Corradi, C. G. Micheli, and G. Sprocati, Study of saffron used in
compound foods through identification of its coloring, bittering and
them exceedingly popular amongst nature loving and health
odorous principles. Chem. Abstr. 1981, 95:218933.
awarded people. In spite of all, the present international [13] Deven, T . K. and A. I. Scott, Handbook of Naturally Occuring
consumption of natural dye is about 10000 t, which is about Compounds, Vol. II:T erpenes, 1972.
1% of the synthetic dye consumption. It is all due to the [14] Springer Verlag, New York, Duguenois, P. Saffron in the modern
pharmacy: Identification and purity analysis, 1972, 429.
existing limitations and technical drawbacks of these dyes, [15] Chem. Abstr. 79: 45885 FAO, 1963. Specification for Identify and
which restrict their uses in textile dyes. In view of above, Purity of Food Additives, Vol. II. FAO, Rome, 67 FAO, 1985,
adequate R&D efforts have been made to tackle some of the FAO/WHO Food Additives Data System, Food and Nutrition paper,
impediments of natural dyes. Standardization of extraction and 30/rev.FAO, Rome, 287.
[16] O. Gisvold and E. H. Roger,T he Chemistry 528 Kasetsart J. (Nat. Sci.)
dyeing profiles of some of the prominent dyes has been 32 (4) of Plant Constituents, Applied Science Publisher Ltd., London,
developed under different conditions to achieve maximum 1983, 471.
yield and reproducible results. The paper deals with the [17] FAO, Specification for Identity and Purity and Toxicological Evaluation
standardization of extraction and dyeing profiles for of Food Colors, 1964.
[18] Joint FAO/WHO Expert committee on food additive, FAO, Nutrition
traditionally known dye from the seeds of annatto (Bixa
Meeting report series No.38, 246.
Orellenae Linn.). Annatto is mainly valued for its principle [19] K. Nobuo, K. Suzuki and S. Asao, Crocin extraction from Gardenia
colourings component bixin, which imparts the red colour on jasminoides fruit. Chem. Abstr. 1976, 85.
food items. Standard profiles have been developed for the
extraction of red color dye from annatto using aqueous solvent
methodologies. The steam distillation can eliminate 4-6% of
essential oil which is removed from the turmeric. Then the
solvent extraction is done and maximum yield is obtained by
using sodium hydroxide solution. But it is not satisfactory
because of cost management. So the selected solvent is 0.25%
alkaline solution. By using this solvent it is possible to extract
39-41% of the bixin. The extracting bixin is then purified. The
best purifying solvent was acetone it removed about 27%
impurities from the extracting s olvent. The characterization is
done by FT-IR and UV spectrophotometer and theses showed
that the purified sample contained the bixin. The pH of this
product was 6.8. This product is heat sensitive i.e. heat
unstable and light sensitive.

ACKNOWLEDGM ENT
This investigation was supported partly by Special Allocation
Project (BS-117) of year 2008-09 from the Ministry of
Science and Information & Communication Technology,
Bangladesh and by Laboratory Facilities from the Department
of Applied Chemistry and Chemical Engineering, University
of Dhaka, Dhaka-1000, Bangladesh.

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