1.      Introduction                                                                                                    2

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1.1  Objective                                                                                                   3

2.      Literature review                                                                                             4

2.1  Colour Pigment                                                                                         4

2.1.1 Natural colour pigment                                                                    4

2.1.2 Synthetic colour pigment                                                                  5

2.1.3 Microbial pigment                                                                            5

            2.2 Lemon fruits                                                                                              6

2.3 Rhodotorula glutinis                                                                                  6

2.4 Pigment stability                                                                                        7

                        2.4.1 pH stability                                                                                 7

2.4.2 Heat stability                                                                              7

3.      Material and Method                                                                                      8

            3.1 Material                                                                                               8

            3.2 Sample preparation                                        

                              3.2.1 Preparation of Acidified PDA                                       8

                              3.2.2 Food preparation                                                            8

            3.3 Method

                              3.3.1 pH stability

                              3.3.2 Heat
stability                                                                  9

            3.4 Analysis                                                                                              9

            3.5 Statistical analysis                                                                               9

            3.6 Expected outcome                                                                              9

4.      References                                                                                                      10




In 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).


            Synthetic 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

            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 fruit.

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

3) To determine
the pH and heat stability on extracted pigment.




















Literature Review


2.1 Colour Pigment


            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 colur pigments which can bring health
issues. Majority of synthetic pigmnets are brighter and last longer compared
natural pigments.


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.



            2.1.2 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).


            2.1.3 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. Futhermore, 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.




2.2 Lemon fruits


            Lemon fruits is one of the citrus
fruit which has sour taste and high in acid. The flesh of the lemon fruits is
rich dietery 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).



2.3 Rhodotorula glutinis


 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.

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. 






2.4 Pigment stability

            2.4.1 pH stability

                        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.


Heat stability

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


















3. Materials And Method




Lemon fruit(buy
from Giant Nilai), tartaric acid, 6 plates, peptone solution.


Sample preparation


               3.2.1 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


3.2.2 Food preparation

            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.

3.3 Method

            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

pH stability


                  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.

Heat stability


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




                  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.


Statistical analysis


                  Statistical analysis for all
the data will be perform using the MINITAB Statistical Softwarre fo 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.


Expected outcome


                  The expected outcomes are to
obtain all objectives of the experiment. The yeast need to be cultured and the
colour pigments need to be produced.














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