VINA C.

YU

BSFT-3

EXPERIMENT 3. EXTRACTION OF NATURAL PIGMENTS

Objectives:

1. To study the solubilities of natural pigments.

Review of Related Literature:

1. A REVIEW: SEPARATION AND CHEMICAL PROPERTIES OF ANTHOCYANINS USED

FOR THEIR QUALITATIVE AND QUANTITATIVE ANALYSIS

Summary: Methods of analysis for naturally occurring anthocyanins are described. The
large number of chemical groups which may bind to the flavylium molecule has contributed
to a large variation in structure, making the qualitative analysis of anthocyanins difficult.
Qualitative analysis has generally involved preliminary solvent extraction followed by
chromatographic separation and purification of pigments. Individual anthocyanins are then
characterized by their chromatographic mobility, absorption spectra, and by means of
controlled hydrolysis and oxidation tests. Quantitative analysis of anthocyanins may be
carried out using either differential or subtractive spectral methods. The validity of results
obtained by either of these methods is dependent on the presence or absence of interfering
substances within the samples. Where the quantification of individual anthocyanins is
desired, their separation from a mixture, normally by means of column chromatography, is
first necessary. High resolution of microgram quantities of anthocyanin without the need for
extensive sample purification prior to analysis has led to an increase in the use of HPLC
techniques for quantitative work.

2. DECOMPOSITION OF TURMERIC CURCUMINOIDS AS AFFECTED BY LIGHT,

SOLVENT AND OXYGEN

Summary: Turmeric curcuminoids are important natural pigments but their instability limits
their use in many foods. This study was conducted to examine light-induced degradation of
turmeric curcumin as affected by solvent system and oxygen. The rate constants and half-
lives were determined for purified curcumin, demethoxycurcumin and bis-
demethoxycurcumin exposed to 1450 lux using reversed-phase HPLC. Rates of degradation
of each pigment followed first-order kinetics. Differences in rate constants and half-lives
between the individual pigments were minor in an acetic acid-NaCl solution; however, in
methanol, large differences in stability to light of the individual pigment were observed.
Stability of all the pigments to light was several fold greater in methanol than in acid brine. In
methanol sparged with air, stability to photooxidation was curcumin > demethoxycurcumin >
bis-demethoxycurcumin. In methanol, curcumin was more stable than the other
curcuminoids when sparged with air than when sparged with nitrogen. In methanol sparged
with nitrogen, demethoxycurcumin was the most stable pigment. All of the pigments were
stable in the dark.

3. PRODUCTION OF MONASCUS PURPUREUS PIGMENTS; INFLUENCED BY

AMIDASE AND ACID PROTEASE ACTIVITY

Summary: Monascus purpureus grown on rice secretes several polyketide pigments used
for coloring foods. The amidase enzymes, L-asparaginase and L-glutaminase activities were
identified by rapid plate assay. The pink zone formed around colonies of M.
purpureus (Microbial Type Culture Collection [MTCC] 410), hyper pigment (Central Food
Research [CFR] 410-11) and albino (CFR 410-22) were 2.0, 2.4 and 1.2 cm, respectively.
The L-asparaginase (0.103 IU) and L-glutaminase (0.139 IU) activity was comparatively
more in CFR 410-11 than MTCC 410 and CFR 410-22. Higher acid protease activity (5,170
Units) was observed in CFR 410-22. The mutant CFR 410-11 has secreted more red
pigment cultured on rice (2.248 optical density [OD] units) and in broth (0.841 OD units).
Significant difference in amidase and acid protease activities of MTCC 410 and its mutant
CFR 410-11 and CFR 410-22 have revealed the importance of these enzymes in pigment
production.

Procedure: Weigh five 2g portions of achuete seeds and place in 100ml beakers. To one
beaker, add 10ml water; to the second, 10ml + 0.045g baking soda; to the third beaker, 10
ml water + 0.4g citric acid; to the fourth, 10ml hot oil; and 10 ml of 1% KOH to the last
beaker. Stir occasionally and let stand for 30 minutes. Remove seeds, blot dry and weigh.
Determine the weight loss. Observe the color and appearance of the extracts.

Results and Discussion:

Table 3.1 Effect of different extractants on the pigments of achuete.

EXTRACTING SOLUTIONS COLOR AMOUNT EXTRACTED

Water Dark orange-red 0.1143 g
Water + Baking Soda Dark orange-red 0.0039 g
Water + Citric Acid Dark orange-red 0.0311 g
Oil Dark orange- red 0.0167 g
KOH, 1% Dark red-black 0.1077 g

Pigments are natural substances in cells and tissues of plants and animals that
impart color. According to extraction methods, the natural pigment can be divided into four
maincategories: namely, plant and animal body formed by the juice or liquid solventextractio
n or solid color; body of animal and plant dry powder obtained by grinding pigment; by
microbial fermentation, metabolites separated into liquid or powder for further processing of
the pigment; using natural products as raw material, obtained by the enzyme and the
pigment.
 Annatto, also called Achuete, is a derivative of the achiote trees of tropical regions of
the Americas, used to produce a red food coloring and also as a flavoring. Its scent is
described as "slightly peppery with a hint of nutmeg" and flavor as "slightly sweet and
peppery". Annatto is produced from the reddish pulp which surrounds the seed of the achiote
(Bixa orellana L.). It is used in many cheeses, margarine, butter, rice, smoked fish, and
custard powder. Annatto 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.

As a food additive, annatto has the E number E160b. The fat soluble part of the
crude extract is called bixin, the water soluble part is called norbixin, and both share the
same E number as annatto. Annatto seed contains 4.5-5.5% pigments, which consists of 70-
80% bixin. In the United States, annatto extract is listed as a color additive exempt from
certification and is commonly considered to be a natural color. The yellowish orange color is
produced by the chemical compounds bixin and norbixin, which are classified as
xanthophylls, a type of carotenoid. However, unlike beta-carotene, another well-known
carotenoid, they do not have the correct chemical structures to be vitamin A precursors. The
more norbixin in an annatto color, the more yellow it is; a higher level of bixin gives it a more
reddish shade. Unless an acid-proof version is used, it takes on a pink shade at low pH.

References:

JACKMAN, R. L., YADA, R. Y. and TUNG, M. A. (1987), A REVIEW: SEPARATION AND

CHEMICAL PROPERTIES OF ANTHOCYANINS USED FOR THEIR QUALITATIVE AND
QUANTITATIVE ANALYSIS. Journal of Food Biochemistry, 11: 279308. doi:10.1111/j.1745-
4514.1987.tb00128.x

PRICE, L. C. and BUESCHER, R.W. (1996), DECOMPOSITION OF TURMERIC

CURCUMINOIDS AS AFFECTED BY LIGHT, SOLVENT AND OXYGEN 1. Journal of Food
Biochemistry, 20: 125133. doi:10.1111/j.1745-4514.1996.tb00577.x

DHALE, M. A., PUTTANANJAIAH, M.-K. H., SUKUMARAN, U.-K. and

GOVINDASWAMY, V. (2011), PRODUCTION OF MONASCUS
PURPUREUS PIGMENTS; INFLUENCED BY AMIDASE AND ACID PROTEASE
ACTIVITY. Journal of Food Biochemistry, 35: 12311241. doi:10.1111/j.1745-
4514.2010.00447.x

CHAITANYA, L. G. (2014), Food Coloring: The Natural Way. Research Journal of Chemical
Sciences.