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Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T
Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Original Research Paper
Isolation and characterization of flexirubin type pigment from Chryseobacterium 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, Malaysia Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia c Department of Microbiology, Karpagam University, Coimbatore-641 023, Tamil Nadu, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 February 2014 Received in revised form 14 February 2014 Accepted 21 February 2014
The pigment from Chryseobacterium sp. UTM-3T was isolated and characterized with the aim to develop a 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 and chemical properties revealed that the pigment has similar properties as most of natural pigment. It was insoluble in water and most organic solvents, but soluble only in acetone and alkaline aqueous and DMSO. 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 were then calculated. The color of the pigment was in the range of yellow. This is the first report on the characterization of pigment from Chryseobacterium sp. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Chryseobacterium Flexirubin Characterization Physic-chemical properties Color analysis
1. Introduction The present trend throughout the world is shifting towards the use of eco-friendly and biodegradable natural colorants due to its pharmacological activities. The demand for natural colorants is increasing day by day and are sourced from ores, insects, plants and microbes. Among microbes, bacteria have the immense potential to produce diverse bioproducts and one such bioproduct is pigment. The production and application of bacterial pigment as natural colorants is being investigated by various researchers (Venil et al., 2013). Pigments produced by bacteria are of traditional use in oriental countries and have been a subject of intense research in the present decades because of its potential for applications. Most researchers have focused on the production of yellow, violet and red pigment production from different bacteria. However, the study of pigment (flexirubin) from Chryseobacterium is very limited. Flexirubins are the unique type of bacterial pigments with terminal alkyl substitution consisting of ω-phenyl octaenic acid chromophore esterified with resorcinol and are used in the treatment for chronic skin disease, eczema, gastric ulcers etc (Kim, 2013). Recent studies of the genus Chryseobacterium have
n
Corresponding author. E-mail address: [email protected] (W.A. Ahmad).
documented the significance of its bioactive compounds as biocontrol agent, antioxidants, prebiotics, sulfobacin A and protease producer (Scheuplein et al., 2007; Wang et al., 2007; Chaudhari et al., 2009; Wang et al., 2011; Kim et al., 2012) which substantiate that it is a novel source of bioactive compounds. The flexirubin was first isolated in 1974 from Flexibacter elegans (Reichenbach and Kleinig, 1974) which turned out to be member of a novel class of pigments (Achenbach et al., 1974, 1976). Similar pigments were later identified in a number of other bacteria viz., Cytophaga sp, Sporocytophaga sp and Chryseobacterium sp. Each genera produce specifically modified species of flexirubin which serve as excellent chemosystematic markers (Reichenbach and Kleinig, 1974). According to the survey on the occurrence of the novel compounds, the distribution of flexirubin is rather limited. In the present study, the pigment from Chryseobacterium sp. UTM-3T was characterised and the properties of the pigment were studied to reveal 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 (¼ CECT 8497T ¼KCTC 32509T) isolated from the Orchard in Universiti Teknologi Malaysia, Malaysia was used in this study. The 16S rRNA
http://dx.doi.org/10.1016/j.bcab.2014.02.006 1878-8181/& 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
2
C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
sequence of the bacteria was submitted to GenBank under Accession No. KF751867. The strain was grown in Nutrient broth followed by incubation at 30 1C for 24 h with an agitation speed of 150 rpm. All chemicals used were of analytical grade. The bacterial cell was cultivated in 20 mL NB in 100 mL Erlenmeyer flask at 30 1C and 200 rpm agitation speed until it reached 1.0 optical density at 600 nm (OD 600). 2 mL of the culture was transferred as inoculum into 100 mL of the fresh medium in 500 mL Erlenmeyer flask and incubated at 30 1C at the shaking speed 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 and the supernatant was discarded. The cell pellet was then rinsed with deionized water, followed by centrifugation at 8000 rpm for 5 min to recover the cells by decanting the supernatant. The recovered cells were extracted using 5% acetone according to the method of Williams et al. (1956). The mixture of cells and acetone was treated by ultrasonication until the cells were completely bleached. The pigment was then separated from the cells by centrifugation at 10,000 rpm for 5 min. By this method, it was confirmed that there was no residual pigment in the cell pellets after the extraction and the resulting pigment was concentrated using rotary evaporator (STRIKE 300, Stereo Glass, Italy) at 50 1C, dried for 3 days at 40 1C followed by separation using column chromatography using petroleum ether: benzene: acetone (10:40:5). Fraction no. 4 and 5 contained the yellowish-orange pigment (flexirubin type of pigment) were pooled and characterized (Fig. 1). 2.3. Structural characterization of the pigment The purified yellowish-orange pigment was determined for maximum 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 mg KBr (Scharlau), pressed under 0.414 bar followed by analysis using the FTIR spectrophotometer (Shimadzu, FTIR 8300) between 4000 and 400 cm 1. The powdered pigment was analyzed using the NMR spectroscopic analysis for 1H (400 MHz) and 13C (100 MHz) using Bruker Avance 400 NMR spectrometer. The chemical shifts were reported in parts per million relative to tetramethylsilane in
deuterated dimethyl sulfoxide (DMSO) as solvent. The powdered pigment 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 solubility 0.05 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 solution was filtered and absorption were recorded at λmax to attain solubility of the pigment. 2.5. Stability of the pigment 2.5.1. Temperature A 0.005 g of pigment solution was treated in a thermostatically controlled bath at 25, 50, 75 and 100 1C and the samples were held at each temperature for 0.5, 1, 1.5, 2, 2.5, 3 h, respectively, and then cooled immediately in an ice bath. The absorption spectra of the solutions was recorded at λmax. 2.5.2. Light A 0.005 g of the pigment solution were held under sunlight, dark and under ultra-violet light for 5 days and the absorbance was determined at λmax. 2.6. Determination of color value of the pigment The absorbance values of pigment solutions at 450 nm were adjusted 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 CIELAB color system (Hunter Associates Laboratory, Inc., Virginia, USA). These values were then used to calculate chroma (Cn) and hue angle (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 represent yellow and blue, respectively. Chroma values denote the saturation or purity of the color. Values close to the center at the same Ln value indicate dull or gray colors, whereas values near the circumference represent vivid or bright colors. Hue angle values represent 0 for redness, 90 for yellowness, 180 for greenness, and 270 for blueness. 2.7. Statistical analysis All experimental results were centered at using three parallel measurements of mean 7standard deviation. Analysis of variance and probability tests were conducted to determine the degree of differences between the means obtained with P o0.05 regarded as significant while P o0.01 was considered very significant. 3. Results and discussion 3.1. Structural determination of the pigments
Fig. 1. Procedure for extraction and purification of pigment.
The orange pigment exhibited peaks at 450 nm when extracted in acetone (Fig. 2). The FTIR spectrum for the purified pigment is shown in Fig. 3. In the spectra of datiscetin, the peak at 3753 cm 1 is attributed to the hydroxyl stretching of absorbed water and the absorption band at 3454 cm 1 is due to the OH groups. The peaks between 1737 cm 1 and 1217 cm 1 corresponds to C–O stretchings. The shifts in carbonyl and the hydroxyl absorption spectra of the pigment can be related to strong chelation (Nagy et al., 1998;
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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Fig. 2. UV–Vis spectrum of the pigment.
Fig. 3. FTIR spectrum of the pigment.
Deveoglu et al., 2012). Also the absorption bands at 893 to 526 cm 1 are due to asymmetric C–H stretching in alkyl hydrocarbons which corresponds to the flexirubin class of pigment (Bej, 2011). The 1H NMR and 13C NMR spectrums of purified orange pigment are given in Fig. 4, in DMSO and its resonances have been completely assigned. Some flexirubin type pigment contains chlorine at ring A. The pigment showed certain structural peculiarities not found so far in other organisms. The chlorinated series was found in addition at B ring to the normal flexirubin. The mass spectrum obtained from the LC–MS analysis exhibited a single major peak of molecular ion at m/z 159 (m þ1) 145 (Fig. 4), which is characteristic for the flexirubin class of pigment (12). The pigment exhibited an immediate color shift from characteristic orange to brown when flooded with 20% KOH and reverted to its initial color when flooded by an acidic solution once excess KOH was removed (Fautz and Reichenbach, 1980). The addition of 20% KOH changed the color of the pigment to dark
orange and also broadened the peak, thus confirming that it is a flexirubin type of pigment (Weeks, 1981). Flexirubin type of pigment contains ω-phenyloctaenic acid chromophore esterified with resorcinol carrying two hydrocarbon chains. Their chemical structures are characterized by non-isoprenoid ω-phenyl-substituted polyene carboxylic acids (Reichenbach and Kleinig, 1974). This basic chemical structure may be modified by variation of the length and branching of the hydrocarbon chains on the resorcinol and by the introduction of additional substituents on the ω-phenyl ring, specifically methyl and chlorine, chlorinated counterparts are found in every flexirubin producing organism. In this way, a large variety of different flexirubin type pigments arise (Stafsnes and Bruheim, 2013). Flexirubin has been proven to be located in the outer membrane of the bacteria and the concentration was found to be about 10 times as high as the phospholipid content in the outer membrane of ordinary Gram negative bacteria. Thus flexirubin functions as photoprotective compound or is involved in the
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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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 time at 3.5 min.
Table 1 Solubility of the pigment. Tests
Response
Solubility in water Solubility in organic solvents Acetone Benzene, chloroform, ethyl acetate, ethanol, methanol, petroleum ether, hexane Solubility in 1 mol NaOH at 100 1C Solubility in 1 mol Na2CO3 DMSO
þþ þþ þþ þ
þ þ Strong positive response. þ Positive response. Negative response.
respiratory chain. Their presence and high concentration in the outer membrane suggests that they may have a structural function (Irschik and Reichenbach, 1978). 3.2. Physico-chemical properties of the pigment
Fig. 5. Effect of light on the stability of the pigment. Error bar represents standard deviation of the means (n¼ 3).
only 0.04 was lost from 25 to 50 1C. The color of the pigment was slightly changed to pale yellow and the results indicated that the pigment was slightly degraded at 100 1C.
3.2.1. Solubility of the pigment The 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) and aqueous acid but soluble in alkaline solutions (Na2CO3 and NaOH) and slightly soluble in DMSO Table 1.
3.4. Effect of light on the stability of the pigment
3.3. Effect of temperature on the stability of the pigment
3.5. Color characteristic of the pigment
The effect of temperature on the pigment stability was determined to ascertain the applicability of bacterial pigment. The pigment solutions were thermostatically treated in a controlled water bath at 25, 50, 75, 100 1C by incubating at specified time intervals. After being treated for 3 h from 25 to 100 1C, the absorbance of the pigment decreased with a loss of 0.027, while
Several models have been developed for the analysis of color, but the CIELab system is the one that nowadays presents a high acceptance since the color perception is uniform which means that the Euclidean distance between two colors corresponds approximately to the color difference perceived by the human eye (Hunt, 1991). The CIELab is an international standard for color
The absorbance of the pigment slightly declined when exposed under sunlight and UV for 5 days, and remains same when exposed in dark. The color of the pigment has no obvious change and this indicated that light had less effect on the pigment (Fig. 5).
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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particularly assessment of its potential in therapeutic uses and its applicability in food, textile industry etc.
Table 2 The color characteristics of the pigment. CIELAB color values
Pigment
Ln
an
bn
Chromaa
Hue angleb
Acknowledgement
3.58
1.21
5.41
5.543
77.39
The authors are thankful to the Ministry of Agriculture, Malaysia for the Techno fund grant (TF0310F080; RJ130000. 7926.4H002), Research University Grants (Q.J130000.2526.02H84 and Q.J130000.2526.03H83) and Universiti Teknologi Malayisa for the Post Doctoral Fellowship to Dr. C.K. Venil. References
Fig. 6. Two dimensional plot for the pigment.
measurement since 1976. Ln is the luminance component (from 0 to 100), and an (from green to red) and bn (from blue to yellow) are the two chromatic components (Papadakis et al., 2000). The CIELab parameters of the pigment are presented in Table 2. Ln value was 3.58 and the pigment exhibited hue angle of 77.39 and chroma of 5.543 which represents yellow. As shown in Fig. 6 the pigment falls on the yellow region in two dimensional plot. 4. Conclusions In this study, the pigment was isolated from Chryseobacterium UTM-3T. The pigment was purified and identified as flexirubin type of pigment. Further the physico-chemical properties were characterized and found the pigment was stable towards temperature and light. From the results of this study it is concluded that an interesting yellow pigment could be isolated and purified from Chryseobacterium sp. by submerged fermentation. However, large scale fermentation should be carried out for industrial applications. Further studies are required for this pigment
Achenbach, H., Kohl, W., Reichenbach, H., 1976. Flexirubin, einneuartiges Pigment aus Flexibacterelegans. Ckem. Ber. 109, 2490–2502. Achenbach, H., Kohl, W., Reichenbach, H., Kleinig, H., 1974. ZurStruktur des Flexirubins. Tetrahedron Lett., 2555–2556 Bej, A.K., 2011. Anticancer and Antimicrobial Compounds from Antarctic Extremophilic Microorganisms. Patent No. 20110301216. Chaudhari, P.N., Wani, K.S., Chaudhari, B.L., Chincholkar, S.B., 2009. Characteristics of sulfobacin A from a soil isolate Chryseobacterium gleum. Appl. Biochem. Biotechnol. 158, 231–241. Deveoglu, O., Cakmakci, E., Taskopru, T., Torgan, E., Karadag, R., 2012. Identification by RP-HPLC-DAD, FTIR, TGA and FESEM-EDAX of natural pigments prepared from Datisca cannabina L. Dyes Pigm. 94 (3), 437–442. Fautz, E., Reichenbach, H., 1980. A simple test for flexirubin-type pigments. FEMS Microbiol. Lett. 8, 87–91. Hunt, R.W.G., 1991. “Measuring Colour”, in Multiple Access, second ed. Ellis Horwood Ltd., Chichester. Irschik, H., Reichenbach, H., 1978. Intracellular location of flexirubins in Flexibacter elegans (cytophagales). Biochem. Biophy. Acta Biomembr 510, 1–10. Kim, H.S., Sang, M.K., Jung, H.W., Jeun, Y.C., Myung, I.S., Kim, K.D., 2012. Identification and characterization of Chryseobacterium wanjuense strain KJ9C8 as a biocontrol agent of Phytophthora blight of pepper. Crop Protect 32, 129–137. Kim, S.K., 2013. Marine Biomaterials: Characterization, Isolation and Applications. CRC Press, Taylor & Francis. Nagy, L., Mehner, H., Christy, A.A., Sletten, E., Edelmann, F.T., Andersen, Q.M., 1998. Preparation and structural studies on organotin (IV) complexes with flavonoids. J. Radioanal Nucl. Chem. 227, 89–99. Papadakis, S.E., Malek, S.A., Kamdem, R.E., Yam, K.L., 2000. A versatile and inexpensive technique for measuring color of foods. Food Technol. 54, 48–51. Reichenbach, H., Kleinig, H., 1974. The pigments of Flexibacter elegans: novel and chemo systematically useful compounds. Arch. Microbiol. 101, 134–144. Scheuplein, R.J., Mizutani, A., Yamaguchi, S., 2007. Studies on the nonpathogenecity of Chryseobacterium proteolyticum and on the safety of the enzyme: protein-glutaminase. Regul. Toxicol. Pharmacol. 49, 79–89. Stafsnes, M.H., Bruheim, P., 2013. Marine biomaterials: characterization, isolation and applications. Book Chapter: Pigmented Marine Heterotrophic Bacteria: Occurrence, Diversity, and Characterization of Pigmentation. 117–148. Venil, C.K., Zakaria, Z.A., Ahmad, W.A., 2013. Bacterial pigments and their applications. Process Biochem. 48 (7), 1065–1079. Wang, S.L., Liang, Y.C., Liang, T.W., 2011. Purification and characterization of a novel alkali-stable α-amylase from Chryseobacterium taeanense TKU001, and application in antioxidant and prebiotic. Process Biochem. 46 (3), 745–750. Wang, S.L., Yanga, C.H., Lianga, T.W., Yen, Y.H., 2007. Optimization of conditions for protease production by Chryseobacterium taeanense TKU001. Bioresour. Technol 99, 3700–3707. Weeks, O.B., 1981. Preliminary studies of the pigments of Flavobacterium breve NCTC 11099 and Flavobacterium odoratum NCTC 11036. In: Reichenbach, H., Weeks, O.B. (Eds.), The Flavobacterium-Cytophaga Group. Weinheim: Gesellschaftfür Biotechnologische Forschung, pp. 109–114. Williams, R.P., Green, J.A., Rappoport, D.A., 1956. Studies on pigmentation of Serratia marcescens. I. Spectral and paper chromatographic properties of prodigiosin. J. Bacteriol. 71, 115.
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
Contents lists available at ScienceDirect
Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Original Research Paper
Isolation and characterization of flexirubin type pigment from Chryseobacterium 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, Malaysia Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia c Department of Microbiology, Karpagam University, Coimbatore-641 023, Tamil Nadu, India b
art ic l e i nf o
a b s t r a c t
Article history: Received 5 February 2014 Received in revised form 14 February 2014 Accepted 21 February 2014
The pigment from Chryseobacterium sp. UTM-3T was isolated and characterized with the aim to develop a 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 and chemical properties revealed that the pigment has similar properties as most of natural pigment. It was insoluble in water and most organic solvents, but soluble only in acetone and alkaline aqueous and DMSO. 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 were then calculated. The color of the pigment was in the range of yellow. This is the first report on the characterization of pigment from Chryseobacterium sp. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Chryseobacterium Flexirubin Characterization Physic-chemical properties Color analysis
1. Introduction The present trend throughout the world is shifting towards the use of eco-friendly and biodegradable natural colorants due to its pharmacological activities. The demand for natural colorants is increasing day by day and are sourced from ores, insects, plants and microbes. Among microbes, bacteria have the immense potential to produce diverse bioproducts and one such bioproduct is pigment. The production and application of bacterial pigment as natural colorants is being investigated by various researchers (Venil et al., 2013). Pigments produced by bacteria are of traditional use in oriental countries and have been a subject of intense research in the present decades because of its potential for applications. Most researchers have focused on the production of yellow, violet and red pigment production from different bacteria. However, the study of pigment (flexirubin) from Chryseobacterium is very limited. Flexirubins are the unique type of bacterial pigments with terminal alkyl substitution consisting of ω-phenyl octaenic acid chromophore esterified with resorcinol and are used in the treatment for chronic skin disease, eczema, gastric ulcers etc (Kim, 2013). Recent studies of the genus Chryseobacterium have
n
Corresponding author. E-mail address: [email protected] (W.A. Ahmad).
documented the significance of its bioactive compounds as biocontrol agent, antioxidants, prebiotics, sulfobacin A and protease producer (Scheuplein et al., 2007; Wang et al., 2007; Chaudhari et al., 2009; Wang et al., 2011; Kim et al., 2012) which substantiate that it is a novel source of bioactive compounds. The flexirubin was first isolated in 1974 from Flexibacter elegans (Reichenbach and Kleinig, 1974) which turned out to be member of a novel class of pigments (Achenbach et al., 1974, 1976). Similar pigments were later identified in a number of other bacteria viz., Cytophaga sp, Sporocytophaga sp and Chryseobacterium sp. Each genera produce specifically modified species of flexirubin which serve as excellent chemosystematic markers (Reichenbach and Kleinig, 1974). According to the survey on the occurrence of the novel compounds, the distribution of flexirubin is rather limited. In the present study, the pigment from Chryseobacterium sp. UTM-3T was characterised and the properties of the pigment were studied to reveal 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 (¼ CECT 8497T ¼KCTC 32509T) isolated from the Orchard in Universiti Teknologi Malaysia, Malaysia was used in this study. The 16S rRNA
http://dx.doi.org/10.1016/j.bcab.2014.02.006 1878-8181/& 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
2
C.K. Venil et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
sequence of the bacteria was submitted to GenBank under Accession No. KF751867. The strain was grown in Nutrient broth followed by incubation at 30 1C for 24 h with an agitation speed of 150 rpm. All chemicals used were of analytical grade. The bacterial cell was cultivated in 20 mL NB in 100 mL Erlenmeyer flask at 30 1C and 200 rpm agitation speed until it reached 1.0 optical density at 600 nm (OD 600). 2 mL of the culture was transferred as inoculum into 100 mL of the fresh medium in 500 mL Erlenmeyer flask and incubated at 30 1C at the shaking speed 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 and the supernatant was discarded. The cell pellet was then rinsed with deionized water, followed by centrifugation at 8000 rpm for 5 min to recover the cells by decanting the supernatant. The recovered cells were extracted using 5% acetone according to the method of Williams et al. (1956). The mixture of cells and acetone was treated by ultrasonication until the cells were completely bleached. The pigment was then separated from the cells by centrifugation at 10,000 rpm for 5 min. By this method, it was confirmed that there was no residual pigment in the cell pellets after the extraction and the resulting pigment was concentrated using rotary evaporator (STRIKE 300, Stereo Glass, Italy) at 50 1C, dried for 3 days at 40 1C followed by separation using column chromatography using petroleum ether: benzene: acetone (10:40:5). Fraction no. 4 and 5 contained the yellowish-orange pigment (flexirubin type of pigment) were pooled and characterized (Fig. 1). 2.3. Structural characterization of the pigment The purified yellowish-orange pigment was determined for maximum 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 mg KBr (Scharlau), pressed under 0.414 bar followed by analysis using the FTIR spectrophotometer (Shimadzu, FTIR 8300) between 4000 and 400 cm 1. The powdered pigment was analyzed using the NMR spectroscopic analysis for 1H (400 MHz) and 13C (100 MHz) using Bruker Avance 400 NMR spectrometer. The chemical shifts were reported in parts per million relative to tetramethylsilane in
deuterated dimethyl sulfoxide (DMSO) as solvent. The powdered pigment 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 solubility 0.05 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 solution was filtered and absorption were recorded at λmax to attain solubility of the pigment. 2.5. Stability of the pigment 2.5.1. Temperature A 0.005 g of pigment solution was treated in a thermostatically controlled bath at 25, 50, 75 and 100 1C and the samples were held at each temperature for 0.5, 1, 1.5, 2, 2.5, 3 h, respectively, and then cooled immediately in an ice bath. The absorption spectra of the solutions was recorded at λmax. 2.5.2. Light A 0.005 g of the pigment solution were held under sunlight, dark and under ultra-violet light for 5 days and the absorbance was determined at λmax. 2.6. Determination of color value of the pigment The absorbance values of pigment solutions at 450 nm were adjusted 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 CIELAB color system (Hunter Associates Laboratory, Inc., Virginia, USA). These values were then used to calculate chroma (Cn) and hue angle (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 represent yellow and blue, respectively. Chroma values denote the saturation or purity of the color. Values close to the center at the same Ln value indicate dull or gray colors, whereas values near the circumference represent vivid or bright colors. Hue angle values represent 0 for redness, 90 for yellowness, 180 for greenness, and 270 for blueness. 2.7. Statistical analysis All experimental results were centered at using three parallel measurements of mean 7standard deviation. Analysis of variance and probability tests were conducted to determine the degree of differences between the means obtained with P o0.05 regarded as significant while P o0.01 was considered very significant. 3. Results and discussion 3.1. Structural determination of the pigments
Fig. 1. Procedure for extraction and purification of pigment.
The orange pigment exhibited peaks at 450 nm when extracted in acetone (Fig. 2). The FTIR spectrum for the purified pigment is shown in Fig. 3. In the spectra of datiscetin, the peak at 3753 cm 1 is attributed to the hydroxyl stretching of absorbed water and the absorption band at 3454 cm 1 is due to the OH groups. The peaks between 1737 cm 1 and 1217 cm 1 corresponds to C–O stretchings. The shifts in carbonyl and the hydroxyl absorption spectra of the pigment can be related to strong chelation (Nagy et al., 1998;
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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Fig. 2. UV–Vis spectrum of the pigment.
Fig. 3. FTIR spectrum of the pigment.
Deveoglu et al., 2012). Also the absorption bands at 893 to 526 cm 1 are due to asymmetric C–H stretching in alkyl hydrocarbons which corresponds to the flexirubin class of pigment (Bej, 2011). The 1H NMR and 13C NMR spectrums of purified orange pigment are given in Fig. 4, in DMSO and its resonances have been completely assigned. Some flexirubin type pigment contains chlorine at ring A. The pigment showed certain structural peculiarities not found so far in other organisms. The chlorinated series was found in addition at B ring to the normal flexirubin. The mass spectrum obtained from the LC–MS analysis exhibited a single major peak of molecular ion at m/z 159 (m þ1) 145 (Fig. 4), which is characteristic for the flexirubin class of pigment (12). The pigment exhibited an immediate color shift from characteristic orange to brown when flooded with 20% KOH and reverted to its initial color when flooded by an acidic solution once excess KOH was removed (Fautz and Reichenbach, 1980). The addition of 20% KOH changed the color of the pigment to dark
orange and also broadened the peak, thus confirming that it is a flexirubin type of pigment (Weeks, 1981). Flexirubin type of pigment contains ω-phenyloctaenic acid chromophore esterified with resorcinol carrying two hydrocarbon chains. Their chemical structures are characterized by non-isoprenoid ω-phenyl-substituted polyene carboxylic acids (Reichenbach and Kleinig, 1974). This basic chemical structure may be modified by variation of the length and branching of the hydrocarbon chains on the resorcinol and by the introduction of additional substituents on the ω-phenyl ring, specifically methyl and chlorine, chlorinated counterparts are found in every flexirubin producing organism. In this way, a large variety of different flexirubin type pigments arise (Stafsnes and Bruheim, 2013). Flexirubin has been proven to be located in the outer membrane of the bacteria and the concentration was found to be about 10 times as high as the phospholipid content in the outer membrane of ordinary Gram negative bacteria. Thus flexirubin functions as photoprotective compound or is involved in the
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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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 time at 3.5 min.
Table 1 Solubility of the pigment. Tests
Response
Solubility in water Solubility in organic solvents Acetone Benzene, chloroform, ethyl acetate, ethanol, methanol, petroleum ether, hexane Solubility in 1 mol NaOH at 100 1C Solubility in 1 mol Na2CO3 DMSO
þþ þþ þþ þ
þ þ Strong positive response. þ Positive response. Negative response.
respiratory chain. Their presence and high concentration in the outer membrane suggests that they may have a structural function (Irschik and Reichenbach, 1978). 3.2. Physico-chemical properties of the pigment
Fig. 5. Effect of light on the stability of the pigment. Error bar represents standard deviation of the means (n¼ 3).
only 0.04 was lost from 25 to 50 1C. The color of the pigment was slightly changed to pale yellow and the results indicated that the pigment was slightly degraded at 100 1C.
3.2.1. Solubility of the pigment The 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) and aqueous acid but soluble in alkaline solutions (Na2CO3 and NaOH) and slightly soluble in DMSO Table 1.
3.4. Effect of light on the stability of the pigment
3.3. Effect of temperature on the stability of the pigment
3.5. Color characteristic of the pigment
The effect of temperature on the pigment stability was determined to ascertain the applicability of bacterial pigment. The pigment solutions were thermostatically treated in a controlled water bath at 25, 50, 75, 100 1C by incubating at specified time intervals. After being treated for 3 h from 25 to 100 1C, the absorbance of the pigment decreased with a loss of 0.027, while
Several models have been developed for the analysis of color, but the CIELab system is the one that nowadays presents a high acceptance since the color perception is uniform which means that the Euclidean distance between two colors corresponds approximately to the color difference perceived by the human eye (Hunt, 1991). The CIELab is an international standard for color
The absorbance of the pigment slightly declined when exposed under sunlight and UV for 5 days, and remains same when exposed in dark. The color of the pigment has no obvious change and this indicated that light had less effect on the pigment (Fig. 5).
Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i
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particularly assessment of its potential in therapeutic uses and its applicability in food, textile industry etc.
Table 2 The color characteristics of the pigment. CIELAB color values
Pigment
Ln
an
bn
Chromaa
Hue angleb
Acknowledgement
3.58
1.21
5.41
5.543
77.39
The authors are thankful to the Ministry of Agriculture, Malaysia for the Techno fund grant (TF0310F080; RJ130000. 7926.4H002), Research University Grants (Q.J130000.2526.02H84 and Q.J130000.2526.03H83) and Universiti Teknologi Malayisa for the Post Doctoral Fellowship to Dr. C.K. Venil. References
Fig. 6. Two dimensional plot for the pigment.
measurement since 1976. Ln is the luminance component (from 0 to 100), and an (from green to red) and bn (from blue to yellow) are the two chromatic components (Papadakis et al., 2000). The CIELab parameters of the pigment are presented in Table 2. Ln value was 3.58 and the pigment exhibited hue angle of 77.39 and chroma of 5.543 which represents yellow. As shown in Fig. 6 the pigment falls on the yellow region in two dimensional plot. 4. Conclusions In this study, the pigment was isolated from Chryseobacterium UTM-3T. The pigment was purified and identified as flexirubin type of pigment. Further the physico-chemical properties were characterized and found the pigment was stable towards temperature and light. From the results of this study it is concluded that an interesting yellow pigment could be isolated and purified from Chryseobacterium sp. by submerged fermentation. However, large scale fermentation should be carried out for industrial applications. Further studies are required for this pigment
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Please cite this article as: Venil, C.K., et al., Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. (2014), http://dx.doi.org/10.1016/j.bcab.2014.02.006i