Original Research Paper

Isolation and characterization of flexirubin type pigment fromChryseobacterium sp. UTM-3T

Chidambaram Kulandaisamy Venil a, Zainul Akmar Zakaria b, Rajamanickam Usha c,Wan Azlina Ahmad a,n

a Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysiab Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysiac Department of Microbiology, Karpagam University, Coimbatore-641 023, Tamil Nadu, India

a r t i c l e i n f o

Article history:Received 5 February 2014Received in revised form14 February 2014Accepted 21 February 2014Available online 25 March 2014

Keywords:ChryseobacteriumFlexirubinCharacterizationPhysic-chemical propertiesColor analysis

a b s t r a c t

The pigment from Chryseobacterium sp. UTM-3T was isolated and characterized with the aim to developa natural colorant for application in bio-pigment production. The pigment was characterized by UV–Vis,FTIR, NMR and LC–MS which confirms that it belongs to flexirubin type of pigment. The physical andchemical properties revealed that the pigment has similar properties as most of natural pigment. It wasinsoluble in water and most organic solvents, but soluble only in acetone and alkaline aqueous andDMSO. The pigment was stable in UV, sunlight and in dark conditions, stable in the range of 25–100 1C.Ln, an and bn values of the CIELAB color system for the pigments were measured and hue, chroma werethen calculated. The color of the pigment was in the range of yellow. This is the first report on thecharacterization of pigment from Chryseobacterium sp.

& 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The present trend throughout the world is shifting towards theuse of eco-friendly and biodegradable natural colorants due to itspharmacological activities. The demand for natural colorants isincreasing day by day and are sourced from ores, insects, plantsand microbes. Among microbes, bacteria have the immensepotential to produce diverse bioproducts and one such bioproductis pigment. The production and application of bacterial pigment asnatural colorants is being investigated by various researchers(Venil et al., 2013). Pigments produced by bacteria are of tradi-tional use in oriental countries and have been a subject of intenseresearch in the present decades because of its potential forapplications. Most researchers have focused on the production ofyellow, violet and red pigment production from different bacteria.However, the study of pigment (flexirubin) from Chryseobacteriumis very limited.

Flexirubins are the unique type of bacterial pigments withterminal alkyl substitution consisting of ω-phenyl octaenic acidchromophore esterified with resorcinol and are used in thetreatment for chronic skin disease, eczema, gastric ulcers etc(Kim, 2013). Recent studies of the genus Chryseobacterium have

documented the significance of its bioactive compounds as bio-control agent, antioxidants, prebiotics, sulfobacin A and proteaseproducer (Scheuplein et al., 2007; Wang et al., 2007; Chaudhariet al., 2009; Wang et al., 2011; Kim et al., 2012) which substantiatethat it is a novel source of bioactive compounds. The flexirubin wasfirst isolated in 1974 from Flexibacter elegans (Reichenbach andKleinig, 1974) which turned out to be member of a novel class ofpigments (Achenbach et al., 1974, 1976). Similar pigments werelater identified in a number of other bacteria viz., Cytophaga sp,Sporocytophaga sp and Chryseobacterium sp. Each genera producespecifically modified species of flexirubin which serve as excellentchemosystematic markers (Reichenbach and Kleinig, 1974).According to the survey on the occurrence of the novel com-pounds, the distribution of flexirubin is rather limited. In thepresent study, the pigment from Chryseobacterium sp. UTM-3T wascharacterised and the properties of the pigment were studied toreveal its potential as a substitute for synthetic colorants.

2. Materials and methods

2.1. Bacteria, media and culture conditions

The pigmented bacteria, Chryseobacterium sp. UTM-3T (¼CECT8497T¼KCTC 32509T) isolated from the Orchard in UniversitiTeknologi Malaysia, Malaysia was used in this study. The 16S rRNA

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Biocatalysis and Agricultural Biotechnology

http://dx.doi.org/10.1016/j.bcab.2014.02.0061878-8181/& 2014 Elsevier Ltd. All rights reserved.

n Corresponding author.E-mail address: azlina@kimia.fs.utm.my (W.A. Ahmad).

Biocatalysis and Agricultural Biotechnology 3 (2014) 103–107

sequence of the bacteria was submitted to GenBank under Acces-sion No. KF751867. The strain was grown in Nutrient brothfollowed by incubation at 30 1C for 24 h with an agitation speedof 150 rpm. All chemicals used were of analytical grade.

The bacterial cell was cultivated in 20 mL NB in 100 mLErlenmeyer flask at 30 1C and 200 rpm agitation speed until itreached 1.0 optical density at 600 nm (OD 600). 2 mL of the culturewas transferred as inoculum into 100 mL of the fresh medium in500 mL Erlenmeyer flask and incubated at 30 1C at the shakingspeed of 200 rpm for 24 h and the pigment yield was calculated.

2.2. Isolation and purification of the pigment

The culture broth was centrifuged at 8000 rpm for 10 min andthe supernatant was discarded. The cell pellet was then rinsedwith deionized water, followed by centrifugation at 8000 rpm for5 min to recover the cells by decanting the supernatant. Therecovered cells were extracted using 5% acetone according to themethod of Williams et al. (1956). The mixture of cells and acetonewas treated by ultrasonication until the cells were completelybleached. The pigment was then separated from the cells bycentrifugation at 10,000 rpm for 5 min. By this method, it wasconfirmed that there was no residual pigment in the cell pelletsafter the extraction and the resulting pigment was concentratedusing rotary evaporator (STRIKE 300, Stereo Glass, Italy) at 50 1C,dried for 3 days at 40 1C followed by separation using columnchromatography using petroleum ether: benzene: acetone(10:40:5). Fraction no. 4 and 5 contained the yellowish-orangepigment (flexirubin type of pigment) were pooled and character-ized (Fig. 1).

2.3. Structural characterization of the pigment

The purified yellowish-orange pigment was determined formaximum wavelength, λmax using UV–Vis spectrophotometer(Perkin Elmer Precisely Lambda 25) between 800 and 200 nm.The dried bacterial pellet (1 mg) was finely ground with 200 mgKBr (Scharlau), pressed under 0.414 bar followed by analysis usingthe FTIR spectrophotometer (Shimadzu, FTIR 8300) between 4000and 400 cm�1. The powdered pigment was analyzed using theNMR spectroscopic analysis for 1H (400 MHz) and 13C (100 MHz)using Bruker Avance 400 NMR spectrometer. The chemical shiftswere reported in parts per million relative to tetramethylsilane in

deuterated dimethyl sulfoxide (DMSO) as solvent. The powderedpigment was dissolved in DMSO prior to analysis using the LC–MS(LCQ Deca XP) using electrospray ionization as the ion source.

2.4. Physico-chemical properties of the pigment

2.4.1. Pigment solubility0.05 g of the pigment was added to 10 mL of water, organic

solvents (acetone, benzene, chloroform, ethyl acetate, ethanol,methanol, petroleum ether, hexane and DMSO), alkali (Na2CO3,NaOH) with stirring for 1 h and left for 30 min. Then the solutionwas filtered and absorption were recorded at λmax to attainsolubility of the pigment.

2.5. Stability of the pigment

2.5.1. TemperatureA 0.005 g of pigment solution was treated in a thermostatically

controlled bath at 25, 50, 75 and 100 1C and the samples were heldat each temperature for 0.5, 1, 1.5, 2, 2.5, 3 h, respectively, and thencooled immediately in an ice bath. The absorption spectra of thesolutions was recorded at λmax.

2.5.2. LightA 0.005 g of the pigment solution were held under sunlight,

dark and under ultra-violet light for 5 days and the absorbancewas determined at λmax.

2.6. Determination of color value of the pigment

The absorbance values of pigment solutions at 450 nm wereadjusted to the range of 1.0–2.0 for color analysis. The values of Ln,an, and bn were measured by a ColorFlex EZ meter with the CIELABcolor system (Hunter Associates Laboratory, Inc., Virginia, USA).These values were then used to calculate chroma (Cn) and hueangle (hab) values. Ln indicates lightness from 0 (black) to 100(white). Positives and negatives in an represent red and green,respectively, whereas positives and negatives in bn representyellow and blue, respectively. Chroma values denote the saturationor purity of the color. Values close to the center at the same Ln

value indicate dull or gray colors, whereas values near thecircumference represent vivid or bright colors. Hue angle valuesrepresent 0 for redness, 90 for yellowness, 180 for greenness, and270 for blueness.

2.7. Statistical analysis

All experimental results were centered at using three parallelmeasurements of mean7standard deviation. Analysis of varianceand probability tests were conducted to determine the degree ofdifferences between the means obtained with Po0.05 regarded assignificant while Po0.01 was considered very significant.

3. Results and discussion

3.1. Structural determination of the pigments

The orange pigment exhibited peaks at 450 nmwhen extractedin acetone (Fig. 2). The FTIR spectrum for the purified pigment isshown in Fig. 3. In the spectra of datiscetin, the peak at 3753 cm�1

is attributed to the hydroxyl stretching of absorbed water and theabsorption band at 3454 cm�1 is due to the OH groups. The peaksbetween 1737 cm�1 and 1217 cm�1 corresponds to C–O stretch-ings. The shifts in carbonyl and the hydroxyl absorption spectra ofthe pigment can be related to strong chelation (Nagy et al., 1998;Fig. 1. Procedure for extraction and purification of pigment.

C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 103–107104

Deveoglu et al., 2012). Also the absorption bands at 893 to526 cm�1 are due to asymmetric C–H stretching in alkyl hydro-carbons which corresponds to the flexirubin class of pigment (Bej,2011).

The 1H NMR and 13C NMR spectrums of purified orangepigment are given in Fig. 4, in DMSO and its resonances havebeen completely assigned. Some flexirubin type pigment containschlorine at ring A. The pigment showed certain structural peculia-rities not found so far in other organisms. The chlorinated serieswas found in addition at B ring to the normal flexirubin.

The mass spectrum obtained from the LC–MS analysis exhib-ited a single major peak of molecular ion at m/z 159 (mþ1) 145(Fig. 4), which is characteristic for the flexirubin class of pigment(12). The pigment exhibited an immediate color shift fromcharacteristic orange to brown when flooded with 20% KOH andreverted to its initial color when flooded by an acidic solution onceexcess KOH was removed (Fautz and Reichenbach, 1980). Theaddition of 20% KOH changed the color of the pigment to dark

orange and also broadened the peak, thus confirming that it is aflexirubin type of pigment (Weeks, 1981).

Flexirubin type of pigment contains ω-phenyloctaenic acid chro-mophore esterified with resorcinol carrying two hydrocarbon chains.Their chemical structures are characterized by non-isoprenoidω-phenyl-substituted polyene carboxylic acids (Reichenbach andKleinig, 1974). This basic chemical structure may be modified byvariation of the length and branching of the hydrocarbon chains onthe resorcinol and by the introduction of additional substituents onthe ω-phenyl ring, specifically methyl and chlorine, chlorinatedcounterparts are found in every flexirubin producing organism. Inthis way, a large variety of different flexirubin type pigments arise(Stafsnes and Bruheim, 2013).

Flexirubin has been proven to be located in the outer mem-brane of the bacteria and the concentration was found to be about10 times as high as the phospholipid content in the outermembrane of ordinary Gram negative bacteria. Thus flexirubinfunctions as photoprotective compound or is involved in the

Fig. 2. UV–Vis spectrum of the pigment.

Fig. 3. FTIR spectrum of the pigment.

C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 103–107 105

respiratory chain. Their presence and high concentration in theouter membrane suggests that they may have a structural function(Irschik and Reichenbach, 1978).

3.2. Physico-chemical properties of the pigment

3.2.1. Solubility of the pigmentThe results indicated that the pigment was insoluble in water

and most of the common organic solvents (benzene, chloroform,ethyl acetate, ethanol, methanol, petroleum ether, hexane) andaqueous acid but soluble in alkaline solutions (Na2CO3 and NaOH)and slightly soluble in DMSO Table 1.

3.3. Effect of temperature on the stability of the pigment

The effect of temperature on the pigment stability was deter-mined to ascertain the applicability of bacterial pigment. Thepigment solutions were thermostatically treated in a controlledwater bath at 25, 50, 75, 100 1C by incubating at specified timeintervals. After being treated for 3 h from 25 to 100 1C, theabsorbance of the pigment decreased with a loss of 0.027, while

only 0.04 was lost from 25 to 50 1C. The color of the pigment wasslightly changed to pale yellow and the results indicated that thepigment was slightly degraded at 100 1C.

3.4. Effect of light on the stability of the pigment

The absorbance of the pigment slightly declined when exposedunder sunlight and UV for 5 days, and remains same whenexposed in dark. The color of the pigment has no obvious changeand this indicated that light had less effect on the pigment (Fig. 5).

3.5. Color characteristic of the pigment

Several models have been developed for the analysis of color,but the CIELab system is the one that nowadays presents a highacceptance since the color perception is uniform which meansthat the Euclidean distance between two colors correspondsapproximately to the color difference perceived by the humaneye (Hunt, 1991). The CIELab is an international standard for color

Fig. 4. NMR and LCMS of orange pigment. LC–MS chromatogram showing the peak (ion selected at m/z 159) of the purified orange pigment with the retention timeat 3.5 min.

Table 1Solubility of the pigment.

Tests Response

Solubility in water �Solubility in organic solventsAcetone þþBenzene, chloroform, ethyl acetate, ethanol, methanol,petroleum ether, hexane

�

Solubility in 1 mol NaOH at 100 1C þþSolubility in 1 mol Na2CO3 þþDMSO þ

þþ Strong positive response.þ Positive response.� Negative response.

Fig. 5. Effect of light on the stability of the pigment. Error bar represents standarddeviation of the means (n¼3).

C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 103–107106

measurement since 1976. Ln is the luminance component (from0 to 100), and an (from green to red) and bn (from blue to yellow)are the two chromatic components (Papadakis et al., 2000). TheCIELab parameters of the pigment are presented in Table 2. Ln

value was 3.58 and the pigment exhibited hue angle of 77.39and chroma of 5.543 which represents yellow. As shown in Fig. 6the pigment falls on the yellow region in two dimensional plot.

4. Conclusions

In this study, the pigment was isolated from ChryseobacteriumUTM-3T. The pigment was purified and identified as flexirubintype of pigment. Further the physico-chemical properties werecharacterized and found the pigment was stable towards tem-perature and light. From the results of this study it is concludedthat an interesting yellow pigment could be isolated and purifiedfrom Chryseobacterium sp. by submerged fermentation. However,large scale fermentation should be carried out for industrialapplications. Further studies are required for this pigment

particularly assessment of its potential in therapeutic uses andits applicability in food, textile industry etc.

Acknowledgement

The authors are thankful to the Ministry of Agriculture,Malaysia for the Techno fund grant (TF0310F080; RJ130000.7926.4H002), Research University Grants (Q.J130000.2526.02H84and Q.J130000.2526.03H83) and Universiti Teknologi Malayisa forthe Post Doctoral Fellowship to Dr. C.K. Venil.

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Table 2The color characteristics of the pigment.

CIELAB color values

Ln an bn Chromaa Hue angleb

Pigment 3.58 �1.21 5.41 5.543 77.39

Fig. 6. Two dimensional plot for the pigment.

C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology 3 (2014) 103–107 107