ABSTRACT: reactions (Malik K., 2012). In order to overcome



biology, the term “pigment” can be defined as any colored molecule in a cell,
regardless of whether or not it is soluble. Pigments are colored by selective
absorption, structural color results from selective reflection. There are two
pigments, which are natural and synthetic pigments. Natural and synthetic
pigments are widely used to colour foodstuffs in order to make the processed
food more attractive to consumers (Carocho, Morales et. all, 2015). Natural pigments can be obtained from plants,
microorganisms and insects or animals tissues (Boo et al., 2011).
In this research, the source of the pigment is microorganisms. One of yeast
that can produce pigments in the fruit is Rhodotorula
glutinis, part
of the Basidiomycota phylum. It produce orange red color, which called
torularhodin. Various strains of Rhodotorula present
important features such as the production of large amounts of carotenoids,
single-cell proteins from ethanol, acetic acid and acetaldehyde(Ayerim Hernández-Almanza
et. all,2014).

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colouring in foods has been used in the food coluring industry since long time
ago. The issue is artificial colour addictive tends to impact undesirable
taste, negative health issues related to the consumption such as allergenic and
intolerance reactions (Malik K., 2012). In order to overcome this issue, food
industries have started to search for alternative food colouring originating
from natural sources. One of the way is by using fruit colouring pigments as
source of pigments.




          The aim of the study is to identify
the colour pigments in the lemon fruit for using in food industries. Mother
analysis of this study is to investigate the effect of heat and pH stability
toward the extraction.




1) To obtain the growth of Rhodotorula glutinis yeast extracted from lemon

2) To determine the colour of pigment
produced from cultured R. glutinis.

To determine the pH and heat stability on extracted pigment.




5.1) Colour


          Pigments are applied not as solutions
because it is not soluble, but as finely ground solid particles mixed in a
liquid. Colour pigments may be synthetic and natural in the food products, but
most of food products using synthetic color pigments which can bring health
issues. Majority of synthetic pigments are brighter and last longer compared
natural pigments.




5.1.1) Natural Colour Pigment


          Pigments have many groups. It can
classified in different groups that comprises several compound with specific
characteristics such as isoprenoid derivatives (carotenoids and iridoids),
benzopyran derivatives (oxygenated heterocyclic compounds like anthocyanins and
others flavonoid piments), quinones (benzoquinone, naphthoquinone, and
anthraquinone), tetrapyrrole derivatives (chlorophylls and heme colours),
N-heterocyclic compounds different from tetrapyrroles (purines, pterins,
flavins, phenazines, phenoxazines, and betalains) and melanins. Among natural
pigments from plant sources, the main are either water- or lipid-soluble
represented by carotenoids, chlorophylls, anthocyanins and betalains wich
differ both in structure and metabolic pathway (Neri-Numa et al, 2017).

          In the commercial,carotenoids and
belatains are used in large scale of yellow and orange with natural green and
blue colorants are few, thus making room to search many types of pigment
sources such as plant and microorganisms. Furthermore, these compounds have
drawn attention to the food, not only because of their coloring properties, but
due to their biological activities such as antioxidant, anticancer,
anti-inflammatory, antiobesity, anti-angiogenic and neuroprotective activities
(Neri-Numa et al, 2017). In the food field, natural colour pigments not only
can make food colouring, but also can give more benefits to the consumers.


5.1.2) Synthetic Colour Pigment


          Synthetic organic pigments are
derived from coal tars and petrochemicals. Many synthetic food colors can cause
cancer, asthma, hyperactivity and laziness. For example, Tartrazine which is
synthetic lemon yellow azo dye known to cause asthma, allergic reactions
because of its nitrous derivatives (Mark, 2012). Moreover many studies have
proved that food dyes can cause harm and adversed effect on children. Then
there is FD&C Red 40 (Allura Red) that cause hyperactivity in children and
immune system tumors in mice. Red 40 contains p-Cresidine, which the
U.S. Department of Health and Human Services says is “reasonably
anticipated” to be a human carcinogen (Pletcher, 2015).


5.1.3) Microbial Pigment


          Microbial pigments is microorganisms
that can produce colour pigments, specifically carotenoids, the most widespread
group of naturally occurring pigments. More than 750 structurally different
yellow, orange, and red colored molecules are found in both eukaryotes and
prokaryotes. Microorganism’s bacteria, algae and fungi produce variety of
pigments and therefore, are the promising source of food colorants (Aberoumand
A, 2011). Carotenoids protect cells against photooxidative damage and
found important applications in food and nutrition, and as potent antimicrobial
agents. These microbial pigments have desirable properties like stability to
light heat and pH (Ahmad et al, 2013). Microbial pigments also possesses
anti-cancer properties and rich of pro-Vitamin A. Furthermore, it also has
various benefits as they can grow in room temperature, humidity condition and
fast with different colour uses. As conclusion, microbial pigments has many
advantages over natural and synthetic as they can be produced under controlled
condition in a very less time.


5.2) Lemon


          Lemon fruits is one of the citrus
fruit which has sour taste and high in acid. The flesh of the lemon fruits is
rich dietary source of carotenoids. It contains orange-yellow colour in lemon
fruit. In ripening lemon fruits, carotenoids accumulate to even higher levels
in chromoplasts. Research characterized the carotenoid pathway in
orange-coloured fruit, with the intent to investigate ways to increase
?-carotene accumulation in the fruit (Guzman et al, 2010).


5.3) Rhodotorula


          Rhodotorula glutinis is a pigmented yeast, part of the Basidiomycota phylum, easily
identifiable by orange/red colonies in the media (Arendrup, 2014). It can live
at various place and condition including in soil, seawater, plants, dairy
product and household environment (Vishniac, 2010). Futhermore, it is possible
for laboratory specimens to become contaminated with this yeast. In
humans, Rhodotorula species can be recover from cultures of skin,
nails, and respiratory, gastrointestinal, and urinary tracts and are generally
thought to be commensals. It reproduce by budding, ovoid to ellipsoidal or
elongate. Rhodotorula strains to to cycloheximide and some strains able to grow
in high NaCl and high glucose.

          R. glutinis secretes the
enzyme alpha-L arabinofuranosidase. It prefer to grow at pH of 5.2, a
temperature of 28C and is an aerobic yeast. It will produce orange/red colonies
so it is easily detected.


5.4) Pigment


5.4.1) pH


          Some yeast can only growth in certain
pH. For Rhodotorula glutinis, it can grow at environment pH like in
soil, sea water and human body. Latha (2005) found that the R. glutinis
which was able to grow and form pigments under a wide range of initial pH
conditions from 2.5 to 9.5. The cell dry weight increased gradually with an
increase in the pH of the modified Czapek dox broth, which the most optimum pH
of growth is at pH 5.5.



5.4.2) Heat


          ?-Carotene synthesis by R. glutinis is
increased at lower temperature. It produced optimum orange-coloured colonies at
30 C. Maximum temperature for R. glutinis to grow is 45 C and the lowest
is 5 C.




6.1) Materials:


In this study lemon fruits (buy from Giant, Nilai) and tartaric acid
will be used. Aside from that, 6 plates are needed along with peptone solution.
The materials should be transported to the Food Biotechnology Laboratory of the
Department of Food Science and Technology, Islamic Science University of
Malaysia in baskets for further processing.


6.2) Sample


6.2.1) Preparation
of Acidified PDA


          Add tartaric acid to the molten PDA
at 48 OC to reach a pH of 3.5. It has been determined that 1.85 ml of a 10%
sterile solution of tartaric acid will decrease the pH of 100ml PDA medium to
3.5. ix gently to avoid introduction of air bubbles. Divide bottle contents
among six petri plates. Store plates at room temperature on a bench counter for
24-48 hours to allow the free moisture on the agar surface to dry.

6.2.2) Food


          Transfer packages of lemon fruit to
the refregirator. Refrigerate the product 24-48 hours to allow thawing. After 2
days, cut the fresh lemon fruit into halves. Extract juice using a manual juice
extracter. Receive juice in clean container to minimize cross contamination
between samples. Refrigerate the juice in a closed, properly labelled container
until analysed in the subsequent laboratory period.


6.3) Methodology:


          Prepare 10-1 and 10-2
dilutions of the lemon juice sample using the 9-ml peptone blanks. After that,
dispense 0.1 ml of the 100 dilution (undiluted) onto two plates
containing acidified PDA. Repeat for the 10-1 and 10-2
dilutions. This is total six plates. Next, spread the inocula evenly on the
plates using a sterile bent glass spreader. Finally incubate plates, right-side
up at 25 C for 5 days. The plate should remain undisturbed until counted.


Stability to Various Physical and Chemical Conditions:

6.3.1) pH


          From six plates of agar, add 3 of
them different volume of tartaric acid until the pH turn to 4.0, 3.5, and 3.0.
The optimum grow of R. glutinis will
be counted as the best growth.




6.3.2) Heat


          For determining heat stability of the
yellow pigment solution, different agar are incubate at temperatures of 35, 37
and 39 OC. The optimum grow of R. glutinis will be counted as
the best growth.


6.4) Analysis


          The colonies of fungi will be count
using aerobic plate count. The count of fungi will be categories based on
effect of pH and heat stability in the acidified PDA agar.


6.5) Statistical


          Statistical analysis for all the data
will be perform using the MINITAB Statistical Software for windows. There are
two categories which are difference of pH and heat stability. One-way analysis
of variance (ANOVA) will be perform and the significance was given in terms of
p-values, with differences at the 95% confidence level being considered statistically
significant for the result of all analysis.









            At the end of
experiment, all of the objective which are expected to be successfully
achieved, and the null hypothesis is expected to be accepted.

            If there is a
possibility that the experiment failed, it might happen so because there might
be one or two or even more miscalculation occurring at the methods section due
to any mistakes during the experiment. The experiment will be conducted again
if the objective is not achievable, where some steps will be changed to suit
the experiment requirement. 

































































































































































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4.   Hernández-Almanza,
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