growth of  Rhodotorula glutinis yeast extracted from lemon fruit

TABLE OF CONTENTS 1.      Introduction                                                                                                    21.1  Objective                                                                                                   32.      Literature review                                                                                             42.1  Colour Pigment                                                                                         42.1.

1 Natural colour pigment                                                                    42.1.2 Synthetic colour pigment                                                                  52.

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1.3 Microbial pigment                                                                            5            2.2 Lemon fruits                                                                                              62.3 Rhodotorula glutinis                                                                                  62.

4 Pigment stability                                                                                        7                        2.4.1 pH stability                                                                                 72.4.2 Heat stability                                                                              73.      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                                                                   8                              3.3.2 Heatstability                                                                  9            3.4 Analysis                                                                                              9            3.5 Statistical analysis                                                                               9            3.

6 Expected outcome                                                                              94.      References                                                                                                      10 Introduction In biology, theterm “pigment” can be defined as any colored molecule in a cell, regardless ofwhether 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 arewidely used to colour foodstuffs in order to make the processed food moreattractive 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 thatcan produce pigments in the fruit is Rhodotorulaglutinis, part of the Basidiomycota phylum. It produce orangered color, which called torularhodin. Various strains of Rhodotorula presentimportant features such as the production of large amounts of carotenoids,single-cell proteins from ethanol, acetic acid and acetaldehyde(AyerimHernández-Almanza et.

all,2014).              Synthetic colouring in foods hasbeen used in the food coluring industry since long time ago. The issue isartificial colour addictive tends to impact undesirable taste, negative healthissues related to the consumption such as allergenic and intolerancereactions(Malik K.

,2012). In order to overcome this issue, food industrieshave started to search for alternative food colouring originating from naturalsources. One of the way is by using fruit colouring pigments as source ofpigments.            The aim of the study is to identifythe colour pigments in the lemon fruit for using in food industries. Motheranalysis of this study is to investigate the effect of heat and pH stabilitytoward the extraction.

      Objective 1) To obtain thegrowth of  Rhodotorula glutinis yeast extracted from lemon fruit.2) To determinethe colour of pigment produced from cultured R. glutinis.3) To determinethe pH and heat stability on extracted pigment.                   Literature Review 2.1 Colour Pigment             Pigments are applied not assolutions because it is not soluble,but as finely ground solid particles mixedin 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 healthissues. Majority of synthetic pigmnets are brighter and last longer comparednatural pigments.

             2.1.1Natural colour pigment Pigments have many groups. It canclassified in different groups that comprises several compound with specificcharacteristics such as isoprenoid derivatives (carotenoids and iridoids),benzopyran derivatives (oxygenated heterocyclic compounds like anthocyanins andothers flavonoid piments), quinones (benzoquinone, naphthoquinone, andanthraquinone), tetrapyrrole derivatives (chlorophylls and heme colours),N-heterocyclic compounds different from tetrapyrroles (purines, pterins,flavins, phenazines, phenoxazines, and betalains) and melanins. Among naturalpigments from plant sources, the main are either water- or lipid-solublerepresented by carotenoids, chlorophylls, anthocyanins and betalains wichdiffer both in structure and metabolic pathway (Neri-Numa et al, 2017).            In the commercial,carotenoids andbelatains are used in large scale of yellow and orange with natural green andblue colorants are few, thus making room to search many types of pigmentsources such as plant and microorganisms. Furthermore, these compounds havedrawn attention to the food, not only because of their coloring properties, butdue 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 onlycan make food colouring, but also can give more benefits to the consumers.               2.1.2 Synthetic colour pigment Synthetic organic pigments arederived from coal tars and petrochemicals. Many synthetic food colors can causecancer, asthma, hyperactivity and laziness.

For example, Tartrazine which is synthetic lemon yellow azo dye known to cause asthma, allergicreactions because of its nitrous derivatives (Mark, 2012). Moreover manystudies have proved that food dyes can cause harm and adversed effect onchildren. Then there is FD&C Red 40 (Allura Red) that cause hyperactivity in children and immune system tumors inmice. Red 40 contains p-Cresidine, which the U.S. Department of Health and HumanServices says is“reasonably anticipated” to be a human carcinogen (Pletcher, 2015).             2.1.

3 Microbial pigment.             Microbial pigments is microorganismsthat can produce colour pigments, specifically carotenoids,the most widespreadgroup of naturally occurring pigments. More than 750 structurally differentyellow, orange, and red colored molecules are found in both eukaryotes andprokaryotes. Microorganism’sbacteria, algae and fungi produce variety of pigments and therefore, are thepromising source of food colorants(Aberoumand A, 2011).

 Carotenoids protectcells against photooxidative damage and found important applications in foodand nutrition, and as potent antimicrobial agents. These microbial pigments have desirable properties likestability to light heat and pH (Ahmad et al, 2013). Microbial pigments alsopossesses anti-cancer properties and rich of pro-Vitamin A. Futhermore, it alsohas various benefits as they can grow in room temperature, humidity conditionand fast with different colour uses. As conclusion, microbial pigments has manyadvantages over natural and synthetic as they can be produced under controlledcondition in a very less time.   2.2 Lemon fruits             Lemon fruits is one of the citrusfruit which has sour taste and high in acid.

The flesh of the lemon fruits isrich dietery source of carotenoids. It contains orange-yellow colour in lemonfruit. In ripening lemon fruits,carotenoids accumulate to even higher levels in chromoplasts. Researchcharacterized the carotenoid pathway in orange-coloured fruit, with the intentto investigate ways to increase ?-carotene accumulation in the fruit (Guzman etal, 2010).  2.

3 Rhodotorula glutinis   Rhodotorula glutinis is a pigmented yeast,part of the Basidiomycota phylum, easily identifiable by orange/red colonies inthe media (Arendrup, 2014). Itcan live at various place and condition including in soil, seawater, plants,dairy product and household environment(Vishniac, 2010). Futhermore, it ispossible for laboratory specimens to become contaminated with this yeast. Inhumans, Rhodotorula speciescan be recover from cultures of skin, nails, and respiratory, gastrointestinal,and urinary tracts and are generally thought to be commensals.

It reproduce bybudding, ovoid to ellipsoidal or elongate. Rhodotorula strains to tocycloheximide and some strains able to grow in high NaCl and high glucose.            R.

glutinis secretes the enzyme alpha-L arabinofuranosidase. It prefer togrow at pH of 5.2, a temperature of 28C and is an aerobic yeast. It willproduce orange/red colonies so it is easily detected.      2.4 Pigment stability            2.

4.1 pH stability                        Some yeast can onlygrowth in certain pH. For Rhodotorula glutinis, it can grow at environment pHlike in soil, sea water and human body. Latha (2005) found that the  R. glutinis which was able to grow and formpigments under a wide range of initial pH conditions from 2.5 to 9.5. The celldry weight increased gradually with an increase in the pH of the modifiedCzapek dox broth, which the most optimum pH of growth is at pH 5.

5.             2.4.2Heat stability                        ?-Carotene synthesis by R. glutinis is increased atlower 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 3.

1Material Lemon fruit(buyfrom Giant Nilai), tartaric acid, 6 plates, peptone solution. 3.2Sample preparation                3.2.

1 Preparation ofAcidified PDA                 Add tartaric acid tothe molten PDA at 48 OC to reach a pH of 3.5. It has been determinedthat 1.85 ml of a 10% sterile solution of tartaric acid will decrease the pH of100ml PDA medium to 3.5.

ix gently to avoid introduction of air bubbles. Dividebottle contents among six petri plates. Store plates at room temperature on abench counter for 24-48 hours to allow the free moisture on the agar surface todry.

 3.2.2 Food preparation            Transfer packages oflemon fruit to the refregirator. Refrigerate the product 24-48 hours to allowthawing.

After 2 days, cut the fresh lemon fruit into halves. Extract juiceusing a manual juice extracter. Receive juice in clean container to minimizecross 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-mlpeptone blanks. After that, dispense 0.1 ml of the 100 dilution(undiluted) onto two plates containing acidified PDA. Repeat for the 10-1and 10-2 dilutions.This is total six plates.

Next, spread theinocula evenly on the plates using a sterile bent glass spreader. Finallyincubate plates, right-side up at 25 C for 5 days. The plate should remainundisturbed until counted.Pigmentstability to various physical and chemical conditions3.

3.1pH stability                    From six plates of agar, add 3of them different volume of tartaric acid until the pH turn to 4.0, 3.5, and3.0.

The optimum grow of R. glutiniswill be counted as the best growth.3.3.

2Heat stability                    For determining heat stabilityof the yellow pigment solution, different agar are incubate at temperatures of35, 37 and 39 OC. The optimum grow od R. glutinis will be counted asthe best growth. 3.4Analysis                   The colonies of fungi will becount using aerobic plate count.

The count of fungi will be categories based oneffect of pH and heat stability in the acidified PDA agar. 3.5Statistical analysis                   Statistical analysis for allthe data will be perform using the MINITAB Statistical Softwarre fo windows.There are two categories which are difference of pH and heat stability.

One-wayanalysis of variance(ANOVA) will be perform and the significance was given interms of p-values, with differences at the 95% confidence level beingconsidered statistically significant for the result of all analysis. 3.6Expected outcome                                    The expected outcomes are toobtain all objectives of the experiment. The yeast need to be cultured and thecolour pigments need to be produced.

          References  1.      MárcioCarocho, PatriciaMorales,Isabel C.F.R.Ferreira : Natural food addictives.2015;2.2.

      Boo HO, Hwang SJ, Bae CS, Park SH, Song WS. Antioxidant activityaccording to each kind of natural plant pigments. Kor J PlantRes. 2011;24:134–141.

3.      Ayerim Hernández-Almanza,Julio Cesar Montanez, Miguel A. Aguilar-González, CristianMartínez-Ávila, CristóbalN. Aguilar : Rhodotorulaglutinis as source of pigments and metabolites for food industry;2013:4-5.4.      Malik K.: Ocimum sanctum seeds, natural superdisintregrant, formulation and evaluationof fast melts tablets of nimesulide;42(1):49-59.

5.      AberoumandA : A Review article on Edible Pigments properties and sources as naturalbiocolorants in foodstuff and food industry. World Journal of Dairy and FoodSciences. 2011; 6:71-78.

6.       Ahmad WA, Ahmad WYW, Zakaria ZA, Yosof NZ:Applications of Bacterial Pigments as Colorant. The Malaysian Perspective. NewYork: Springer Briefs in Molecular Science.

2012; 57-74.7.      Guzman I, Hamby S, Romero J, Bosland PW, O’ConnellMA: Plant Sci. 2010 Jul; 179(1-2):49-59.8.      Arendrup MC, Boekhout T, Akova M, Meis JF, Cornely OA,Lortholary O.

  ESCMID and ECMM joint clinical guidelines for thediagnosis and management of rare invasive yeast infections. Clin MicrobiolInfect. 2014;20:76-98.

9.      Vishniac HS, Takashima M. Rhodotorula arctica sp.

nov., a basidiomycetous yeast from Arctic soil. Int J Syst Evol Microbiol.2010;60(Pt 5):1215-8       


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