TY - JOUR
AU - Miranda, Claudio D
AB - Abstract The interest in and demand for natural dyes has increased significantly in recent years; however, very few natural blue dyes are commercially available, because blue colored compounds in nature are relatively rare. In this study, a blue pigment-producing bacteria from Lake Chungará (Atacama Desert, Chile) was isolated, and its blue pigment was purified and chemically characterized. The pigment-producing strain was identified as Pseudarthrobacter sp. by 16S rRNA gene sequencing. The pigment was separated from the filtered culture medium by column chromatography/solid-phase extraction using different resins (ionic exchange, C-18, size exclusion). The strain produced up to 2.5 g L−1 of blue pigment, which was very soluble in water, partially soluble in methanol and insoluble in other organic solvents. The pigment was analyzed and characterized by analytical HPLC, UV–Vis, FT-IR, and H-NMR, and purified by semi-preparative HPLC. The pigment was non-toxic to brine shrimp (LD50 > 2.3 g L−1) and was stable at pH 6–10 at temperatures below 60 °C. HPLC analysis shows that the pigment is composed of four major blue fractions. The physicochemical properties and structural analysis demonstrate that this pigment belongs to the indochrome isomers, whose properties have yet to have been characterized. The high solubility in water, good stability in neutral and basic pH, and negligible toxicity of the blue pigment make it a good candidate suitable for several industrial and possibly some food applications. Electronic supplementary material The online version of this article (10.1007/s10295-018-2088-3) contains supplementary material, which is available to authorized users. Introduction Natural pigments and synthetic dyes are used extensively in various fields, such as food production, textiles, paper production, printing inks, cosmetics, and pharmaceuticals [30]. However, consumers have developed aversion towards the use of synthetic colorants in foods due to their possible negative consequences such as carcinogenic effects, skin allergies, neurological disorders in children and the production of toxic waste [1, 22]. As a result, the use of synthetic dyes has declined globally and many have been banned. In response interest in and demand for natural dyes has increased over recent years [2, 23]. Thus, the replacement of artificial colorants by their natural counterparts has become a major challenge for the food, pharmaceutical, and cosmetics industries [22]. A significant number of red, orange and yellow natural colorants have already been approved for use, but only a few blue colorants are commercially available, because blue colored compounds in the nature are relatively rare [3, 20]. Considerable effort has been devoted to the search for natural blue pigments from plant, animal and microbial sources. Pigments from microbial sources are advantageous in terms of production, when compared to similar pigments extracted from vegetables or animals [23]. Specifically, the bacterial pigment offers certain distinctive advantages such as production independent of weather conditions, short life cycles, the potential to produce pigments of different shades and colors, and the easier scaling-up of production [24]. Therefore, screening of new blue pigment-producing strains is of great importance. Nevertheless, the number of studies on blue bacterial pigments is limited, mainly because only a very few bacteria are capable of producing blue pigment [30]. For edible natural pigments, solubility, coloring, stability and safety are key factors in order to be eligible for application in the industry [7, 31]. The solubility determines the type of substrate where the pigment can be applied. In many cases, water solubility of a pigment is preferred when applied to many food, cosmetics and pharmaceuticals [21]. On the other hand, microbial pigments tend to have low stability and variation in shades due to changes in pH, temperature, and light [18]. Thus, the lack of stability of microbial pigments is often a major factor in their failure to reach the market [26]. So far, several blue bacterial pigments of a polar nature have been described, including anthraquinone, indochrome, bactobilin, pyocyanin, and actinorhodin [20]. Among these, chromophore indochrome produced by Arthobacter polychromogenes and Streptomyces coelicolor has a higher polarity and occurs in association with the insoluble pigment, indigoidine [8, 27]. Indochrome and amylocyanin are blue pigments composed of various isomers belonging to the structural class of pyridine alkaloids, containing 3,3′-bipyridyl chromophores [29]. Even though many blue pigments of this class isolated from bacteria have been described, information available concerning the stabilities and biological activities of most of the 3,3′-bipyridyl pigments is still scarce [20]. In this work, we isolated a bacterial strain capable of producing blue pigment from a Chilean altiplanic lake in the Atacama Desert. The water-soluble pigment was purified and identified and its production, toxicity, solubility, and stability were characterized. Materials and methods Isolation and identification of pigment-producing bacteria The blue pigment-producing strain used in this study was isolated from a water sample collected from Lake Chungará (Atacama Desert, northern Chile; 18°16′2.15″S 69°9′48.08″W). The isolated strain that showed a blue halo around the colony on an R2A agar plate was subcultured, purified, and named strain 34LCH1. The growth and production of blue pigment by the strain 34LCH1 were observed at 25 °C in different nutritive broths including tryptone yeast extract salts (TYES) broth (0.4% tryptone, 0.05% yeast extract, 0.05% MgSO4·7H2O, 0.02% CaCl·2H2O, pH 7.2), modified M1 Medium (6 ml of 100% glycerol, 5 g of starch, 3 g of yeast extract, 1.5 g of peptone, 0.3 g tryptone, 1 g of arginine, 0.5 g of K2HPO4, 0.1 g of MgSO4, 0.001 g of FeSO4, and 1 L of natural seawater), Marine Broth 2216 (Difco), R2A Broth (Difco), Nutrient Broth (Oxoid), and Tryptic Soy Broth (BD). The pure culture was preserved in TYES broth supplemented with glycerol (15%) and stored at − 80 °C prior to use. The cultures were also preserved by free drying followed by storage at room temperature. Taxonomic identification of strain 34LCH1 was based on the sequencing of the 16S rRNA gene. Crude DNA extracts were obtained using the Wizard genomic purification kit (Promega, Madison, WI, USA). Polymerase chain reaction (PCR) was performed using primers 27F 5′- AGAGTTTGATCMTGGCTCAG-3′, 1492R 5′- TACGGYTACCTTGTTACGACTT-3′, and 907R 5′- CCGTCAATTCMTTTGAGTTT-3′ [15]. PCR product was verified in 1% agarose gels, purified and sequenced by Macrogen (Seoul, Korea). Identification of partial sequences was initially estimated by comparison to 16S rRNA Gene Reference Sequence database (refseq_rna) recorded in the NCBI BLAST (http://www.ncbi.nlm.-nih.gov/). Sequences used in tree construction are the same as those in the BLAST database. Sequences were aligned using CLUSTAL W and phylogenetic tree was constructed according to the Neighbor-Joining method using MEGA X software, with bootstrap values based on 1000 replications [11]. The evolutionary distances were computed using the Maximum Composite Likelihood method. The partial 16S rRNA gene sequences were submitted to the Genbank database and assigned accession number MG754403. Bacterial growth and pigment production kinetics To determine the growth of strain 34LCH1 and pigment production kinetics, 250 ml flasks containing 50 ml of TYES were incubated for 55 h in triplicate (22 °C, 150 rpm). Every 5 h, an aliquot from culture was taken to determine the bacterial count and for pigment content analysis. The bacterial count was made using the micro-droplet technique in plates with TYES medium in triplicate. Pigment was analyzed spectrophotometrically using absorbance analysis, aliquots of 1 ml were filtered (0.22 μm), and a volume of 150 μl was transferred to a microplate and analyzed at 590 nm in a plate reader (Infinite M200 pro, Tecan). For HPLC pigment analysis, the culture liquid was centrifuged (5000 rpm for 20 min), filtered (0.45 and 0.22 μm), and stored at 4 °C. Then, the aliquots for each time period were taken in triplicate and the pigment was retained in ODS (C18, octadecylsilica) solid-phase extraction (SPE) columns (Sep-Pak 6 cc Vac Cartridge, 55-105 μm, Waters). Finally, the pigment was eluted with 95% methanol, dried in a centrifugal evaporator (centrivap, Labconco) and weighed. 20 μl of filtered aqueous pigment solution was analyzed by reversed phase in a Waters HPLC equipment comprising an AF in-line degasser, 600 pump and controller and 2998 diode array detector (DAD) with analytical flow cell. Chromatographic running conditions were 25 °C, a flow rate of 1 ml/min, mobile phase acetate buffer 100 mM pH 5.0, 1–100% methanol and an analytical Xterra MS ODS column (5 µm, 4.6 × 250 mm). Pigment characterization Pigment composition was studied using analytical HPLC as previously described in Section “Kinetics of bacterial growth and pigment production”. Solubility was assessed in a series of organic solvents including methanol, ethanol, isopropanol, acetonitrile, acetone and ethyl acetate, by observing the color of the solvent after a period of time (30 min). UV–Vis spectra of crude 1 pigment (see “Pigment isolation and purification” section) in a buffer composed of 50 mM sodium citrate, 50 mM sodium dihydrogenphosphate and 50 mM boric acid previously adjusted with concentrated NaOH or HCl to pH 4–12 were recorded using a plate reader (Tecan Infinite M200 PRO). Pigment stability was evaluated by decay of the absorbance (600 nm) during a timed incubation in the dark as function of pH and temperature. The crude 1 pigment was evaluated at an initial concentration of 1.1 mg ml−1 in 50 mM citrate/borate/phosphate buffer and a pH between 4 and 12. At 23.1 ± 0.1 °C, absorbance was measured using the plate reader. Stability at 60 ± 1 and 80 ± 1 °C was assessed in crude 2 pigment (see “Pigment isolation and purification” section) at pH 7.0, 50 mM phosphate buffer using a water bath to maintain constant temperature. Half-life (t1/2) was calculated as the time at which the initial absorbance was half its initial value. Solid stability was assessed visually at ambient temperature. Brine shrimp acute toxicity bioassay Brine shrimp toxicity study was assessed as described by [28] with some modifications. Between 10 and 30 Artemia sp. nauplii (48 h grown) in 0.2 µm filtered seawater (3.5% salinity, pH 7.8) were placed in each well of a 12-well plate together with crude 1 pigment, F2 or sodium dodecyl sulfate (SDS) solution in seawater. Plates were incubated for 24 h at 25.0 ± 0.1 °C, after which the number of live and dead nauplii in each well was counted and the percentage of dead brine shrimp was plotted against the pigment concentration to obtain the concentration at which 50% of nauplii die (LC50), by fitting to a dose–response curve with Origin 8.0 (OriginLab). Purification and structural identification of blue pigment Pigment isolation and purification Pigment was obtained from a 30-h culture of strain 34LCH1 in TYES broth (25 °C, 150 rpm), which was tangentially filtered (Reexeed-21SX, Asashi Kasei, 30 kDa) to obtain a cell-free blue permeate. In order to achieve pigment purification, the permeate was sequentially passed through solid-phase extraction or chromatographic columns. In the first stage, the blue liquid was passed through anion exchange resin (Amberlite IRA-400, Cl− form, Aldrich) and then a cationic resin (Amberlyst 15, Na+ form, Merck). The pigment was extracted in an ODS (40–63 μm, LiChoprep, Merck), washed with water to remove salts and eluted with a mixture of water and methanol: 70/30 (in volume). The eluate was retained in an anion exchange resin (40 µm, Accell Plus QMA, Cl− form, Waters), washed with methanol, water and then eluted with 5% NaCl. The NaCl was removed by washing with water in an ODS column and the pigment eluted with water and methanol: 20/80, rota-evaporated and precipitated with acetone, centrifuged, decanted and dried in a centrifugal evaporator, obtaining crude 1 pigment. The solid crude 1 was then dissolved in water and purified, as a single blue fraction, by means of size exclusion chromatography (SEC) (Toyopearl HW-40F, Tosoh) with water as the eluent. The pigment was further concentrated, precipitated with acetone, recrystallized twice in ethanol/water and vacuum dried, obtaining crude 2 pigment. Semi-preparative reversed-phase purification was performed using a Waters HPLC as described previously for analytical HPLC (Section “Pigment isolation and purification”) with some modifications: semi-preparative flow cell and a Waters Atlantis dC18 (ODS, 10 µm, 19 × 250 mm) column in gradient mode, using as mobile phase 100 mM pH 6.0 citrate buffer with 0–5% methanol, 5 ml/min at 25 °C. To collect each one of the main fractions (F1, F2, F3 and F4), the solvent and buffer were removed by rotary evaporation and reverse phase SPE on ODS, respectively. Remaining moisture in the coloured solution was removed using a Centrifugal Vacuum Concentrators (CentriVap. Labconco. Mod. 7310031). Spectroscopic characterization IR spectra of F1 and F2 were determined using an FT-IR spectrophotometer (Vector 22, Bruker) as KBr disks. 1H-NMR experiments were performed using a Bruker AVANCE III HD-400 (400 MHz) instrument in D2O solution of F1, F2 and F3. Mass spectra of F1, F2 and F3 were obtained using an AB Sciex Triple Quad 4500 model by direct infusion in positive electrospray ionization mode (ESI +). UV–Vis spectra of fractions (F1–F4) were obtained using a Waters 2998 DAD detector during chromatographic purification at pH 6.0 (see Section “Pigment isolation and purification”). Results and discussion Isolation and identification of pigment-producing bacteria A preliminary comparison against the 16S rRNA Gene Reference Sequence GenBank database indicated that the strain is closely related to Pseudarthrobacter sp (formerly Arthrobacter). Indeed, 16S rRNA gene sequence of the strain 34LCH1 showed a 99.7, 98.8, 98.9, and 99.1% similarity with Pseudarthrobacter phenanthrenivorans, Pseudarthrobacter polychromogenes, Pseudarthrobacter oxydans, and Pseudarthrobacter siccitolerans, respectively. These results were confirmed in the phylogenetic analysis, in which the strain 34LCH1 was clustered with other species of Pseudarthrobacter (Fig. 1). Fig. 1 Open in new tabDownload slide Phylogenetic tree based on 16S rRNA gene sequence comparison showing the position of strain 34LCH1 and closely related species of the genus Pseudarthrobacter and Arthrobacter. The scale bar represents a 2% nucleotide sequence divergence. NCBI accession numbers are presented in parentheses The best growth and blue coloration of strain 34LCH1 were observed in TYES medium, which was used for subsequent experiments. The cells of strain 34LCH1 form round colonies with a slightly concave center after of 2 days of growth on TYES agar medium (Fig. 2a). After 24 h, the blue pigment begins to appear around the colonies and from it the pigment diffuses until that the agar of the petri dish is fully colored after 72 h, indicating the polar nature of the pigment. In TYES broth, the blue coloration is observed after 20 h of culture (Fig. 2b). Fig. 2 Open in new tabDownload slide Pseudarthrobacter sp. 34LCH1 colonies on a TYES media and b liquid culture Bacterial growth and pigment production kinetics The bacterial growth curve (Fig. 3) shows an exponential phase after 15 h of incubation, a stationary phase that becomes more noticeable after 30 h and, finally, a death phase after 40 h of culture. Maximum blue pigment production was reached after 35 h of culture, up to 0.18 absorbance units at 600 nm. Concordantly, the quantitative analysis of crude 1 pigment production allowed to estimate a production of 2.5 g per liter of culture after 35 h. Similar values were maintained until 55 h of culture. Other authors have described a high production of bacterial pigments, including prodigiosin (2.95 g L−1), violacein (3.5 g L−1), and indigoidine (3.9 g L−1) [6, 17, 32]. Thus, the level of pigment production by strain 34LCH1 is considered relatively high, even taking into account that it has not been made using an optimized production process. Optimization could increase production by several times the values obtained in the experiments presented here. Fig. 3 Open in new tabDownload slide Microbial growth kinetics and production of blue pigment of the strain Pseudarthrobacter sp. 34LCH1 in TYES medium at 22 °C. Means and standard deviations are based on three replicates Analytical HPLC chromatogram of the blue pigment at 590 nm indicated that pigment produced by Pseudarthrobacter sp. 34LCH1 was composed of four main blue peaks, called fractions 1–4 (F1, F2, F3 and F4; Fig. S2), in order of decreasing polarity. This pigment contained the same number of fractions previously described for the indochrome pigment A (β-D-pyranose, β-d-pyranose), BI (β-d-furanose, β-d-furanose), BII (α-pyranose-β-pyranose), and BIII (contains α-furanose residue) produced by Pseudarthrobacter polychromogenes (formerly Arthrobacter polychromogenes) [13, 14]. Additionally, indochrome has only been reported to be produced by indigoidine-producing bacteria, and in contrast to indochrome, indigoidine is water insoluble by what it is retained inside the bacterial cells. Thus, and considering that the colonies of the strain 34LCH1 are whitish, the production of indigoidine in this strain can be ruled out. The four main components showed similar UV–Vis spectra (data not shown), which would indicate that they share a common chromophore. Although the pigment appeared to be relatively unstable at pH 5.0, there was no degradation in analytical HPLC experimental time period. At higher pH values, lower retention times were observed, but with a poor resolution of the chromatographic peaks, whereas under more acidic conditions a faster degradation was found, affecting with it the chromatographic analysis. We observed that the relative amounts of soluble pigment involved in fractions 1–4 tend to be maintained without significant predominance of any of them over the culture period, according to the specific chromatographic-peak areas at each measured point during the kinetic analysis. Pigment characterization Crude pigment and its fractions were hygroscopic blue solids, soluble in water and organic solvent/water mixtures, slightly soluble in methanol and insoluble in other less polar solvents, in agreement with that reported for indochrome [14]. Spectrophotometric characterization of crude 1 pigment showed a λmax centered around 600 nm (Fig. S3). At pH less than 6 and greater than 10, a hypsochromic shift was observed (Fig. 4), pH at which the pigment was less stable. The stability of crude 1 pigment was studied because in this purification stage it is more useful for possible applications such as an industrial colorant. Pigment was stable in solid state at ambient temperature for at least 3 months. At neutral-basic pH and 23 °C, the pigment was relatively stable. At pH 7–9, the pigment solution was stable, at least, within 2 weeks at ambient temperature. At pH values greater than 10 and less than 5, the absorbance in aqueous solution at 23 °C decreases rapidly over time, indicating pigment instability at these pH values (Fig. 5b). Additionally, in the short-term the pigment was also stable at pH values between 5.5 and 10.5 (Fig. 5a). Fig. 4 Open in new tabDownload slide Crude 1 pigment maximum absorbance–wavelength as a function of pH for 1.1 mg crude/ml in 50 mM phosphate/citrate/borate buffer Fig. 5 Open in new tabDownload slide Crude 1 pigment absorbance as function of pH. a Degradation of blue pigment at different pH values over 6.5 h (the absorbance values at pH 6 were not included, because these remain stable as well as values obtained at pH 7). b Crude pigment absorbance at 600 nm as function of pH at different times. Experimental conditions: 1.1 mg/ml crude 1 pigment, 50 mM phosphate/citrate/borate buffer, 23.1 °C. Means and standard deviations are based on three replicates Measured half-life at different pH values and 23 °C was 3.1, 6.2, 3.6 and 0.92 h for pH 4.0, 4.5, 11.5 and 12, respectively. It has been reported that indochrome in acid aqueous solution is very labile, and when indochrome degrades in a strong acidic medium it rapidly generates yellow products [14]. Also, nicotine blue II, a blue water-soluble pigment from Pseudarthrobacter oxidans (formerly Arthrobacter oxidans) is unstable at acidic pH [12]. For other pH values, t1/2 could not be obtained during the experimental time period. Half-life measured at 60 and 80 °C and pH 7.0 were 19.5 and 5.0 h, so the pigment was less stable at higher temperatures and very high or very low pH values. When the pigment was decomposed due to high temperature at neutral pH or at basic pH at ambient temperature it became colorless, but when it does so at acidic pH the solution turns yellow, as seen for indochrome [14], indicating a different decomposition mechanism. As stability studies were performed on crude pigments, it is not known which compounds of the mixture are those that degrade, nor if other blue compounds are produced due to, for example, isomerization. When solutions that have lost their blue color at high or low pH are returned to neutral pH, they do not recover their original color, confirming that the absorbance change observed is due to pigment degradation. Pigment instability at low pH values would preclude its use as colorant in many food products because of its acidic nature. The same situation has been observed for other natural blue pigments, such as indigo carmine [3]. However, the instability at high or low pH values could be useful in developing a food irreversible pH sensor, i.e., a colorant that indicates, by changing its color irreversibly, when a food such as milk has been acidified by decomposition. On the other hand, we have found that the bacterial strain does not lose the ability to produce the pigment even after making more than 25 successive transfers in TYES agar solid medium. Furthermore, its ability to produce pigment was not lost when revived in solid medium, after having been stored at − 80° C (or freeze-dried) for at least 2 years, indicating that the strain capacity of production is stable over time. Brine shrimp acute toxicity bioassay Crude 1 or F2 at concentrations studied did not cause higher mortality to Artemia nauplii than negative controls. The LC50 value for the positive control (SDS) was 18.9 mg L−1 (R2 = 0.98), in agreement with the reported values [16]. Due to the low pigment toxicity on brine shrimp, only estimated toxicity data are given. Crude 1 showed an LC50 > 2300 mg L−1 and F2 LC50 > 320 mg L−1. As a general guideline, a crude pigment LC50 > 1 g L−1 can be considered as non-toxic for brine shrimp [19]. Although for F2 the LC50 had not been reached, the estimated value is sufficiently large to be considered, in the worst case, as having a low toxicity (caffeine has LC50 = 0.31 g L−1 on Artemia) [19]. It has been seen that the toxicity for Artemia is related to bioactivity [19], so the pigment would be expected to have a low bioactivity. Also, brine shrimp LC50 values correlate with LD50 in mice [25] and is a predictive tool for the toxic potential of plant extracts in humans [10]. So, it is expected that the pigment would not be toxic to other organisms, unless it requires an enzymatic process that Artemia does not have for the toxic to be activated [28]. Although Bycroft [5] describes indochrome A as a nucleoside-type antibiotic, to our knowledge, there are no studies that support this bioactivity or other biological activity of diazaindophenol derivatives. Purification and structural identification of blue pigment Pigment isolation and purification The blue filtered culture medium decomposes turning yellow at room temperature in less than 2 days, so the process must be carried out relatively quickly and keeping the samples frozen. The first column of anionic SPE retained most of the colored substances that are not blue, the blue pigment passing through because of the salts in the culture medium. In the ODS column, the pigment was retained, and after the inorganic salts were removed from the pigment, most of the pigment was thereafter retained in the anion exchange resin, which indicated that the chromophore of the blue substances that form the pigment was anionic. Due to the low solubility of the pigment in methanol, the use of methanol with water was required to efficiently elute from ODS. No attempts were made to perform a chromatographic separation on the anionic resin, so pigment was eluted as a single blue band with 5% NaCl. Crude 1 pigment was obtained by SPE with materials that are reusable (anionic resin and ODS) in a simple process, which would allow this pigment to be isolated at a relatively low cost. When the pigment was precipitated with acetone, co-precipitate colorless substances were largely eliminated in recrystallization with ethanol/water mixture, thus obtaining a deeper blue solid. Indochrome crude pigment was also precipitated from ethanol/water mixtures as described by Knackmuss [14]. The fact that pigment components eluted as a single band in size exclusion chromatography confirms that they have similar molecular weights. In indochrome and amylocyanin, the components are isomers [9, 14], so they have the same molecular weight. Due to the high polarity of pigment compounds, they exhibited low retention on ODS, so semi-preparative chromatography was performed with a low organic solvent concentration. Therefore, HPLC purification on ODS column was a slow process (Fig. 6), which could be improved using hydrophilic interaction chromatography (HILIC), more suitable for high polarity compounds [4]. Nevertheless, when semi-preparative HPLC was performed, a good resolution was achieved (Fig. 6), obtaining a separation of the fractions. This reversed-phase HPLC purification is orthogonal to that described in literature, where ion chromatography was employed. Fig. 6 Open in new tabDownload slide HPLC semi-preparative chromatogram of crude 2 pigments at 590 nm. F1, F2, F3, and F4 on the graph indicate the fractions 1, 2, 3, and 4, respectively. Experimental conditions: Gradient mode, mobile phase 0–5% methanol in 100 mM pH 6.0 citrate buffer. 10 μm, 19 × 250 mm ODS column, 5 ml/min and 25 °C Spectroscopic characterization FT-IR spectroscopy and mass spectrometry F1 and F2 FT-IR analysis showed signals at 1654 and 1655 cm−1. Those observed carbonyl stretching absorption wavelengths are in line with those reported (1655 cm−1) for diazaindophenol sodium salts [9, 14]. Mass spectra of compounds showed m/z 542 peak corresponding to sodium salts adducts, [M + Na]+ (542.1 Da, C20H22N3Na2O12), m/z 520 to [M + H]+ (520.1 Da, C20H23N3NaO12) and m/z 462 corresponding to [M-H2O2-Na]+ (462.1 Da, C20H20N3NaO10). Some observed peaks were for F1 (m/z): 542.0, 520.4, 461.9, F2 (m/z): 542.1, 462.1, F3 (m/z): 542.1, 520.5, 462.2. UV–vis spectroscopy Main pigment fractions at pH 6.0 in water/methanol (2%) showed maximum absorbance in the visible spectra at 600.7 nm (F1 and F3) and 593.4 nm (F2 and F4) (Fig. S4) and, thus, F1 and F3 should share a structural feature. According to Knackmuss (14), for indochrome in water/pyridine mixture, symmetric β isomers indochrome A and indochrome BII maximum wavelengths appear at higher values (583 nm and 588 nm) than those of asymmetric indochrome isomers BI and BIII (578 nm and 576 nm) [14]. In the case of indochrome BII, the largest bathochromic shift is due to strong intramolecular hydrogen bonding between the chromophore and the β-d-ribofuranosyl moiety which stabilize the anion [12–14]. In our case, similar to that described by Knackmuss, we find two groups of spectra; however, we cannot find differences between F1 and F3, probably due to the spectra being obtained in a different solvent mixture and at more acidic pH than in the indochrome studies of Knackmuss [14]. H-NMR spectroscopy F1 1H-NMR spectra in D2O showed a singlet proton at 7.72 ppm assigned as vinyl proton of indochrome diazaindophenol aglycone and only one anomeric doublet at 4.59 ppm (Figs. 7, 8, and S1). Habermehl [9] and Knackmuss [14] showed that symmetric β isomers indochrome A and indochrome BII have a one vinyl singlet and one anomeric doublet; however, the symmetric α isomers show two vinyl and one anomeric proton signal (Table S1); thus, F1 may either correspond to indochrome A or indochrome BII. Because F1 and F3 share a common λmax in the visible spectra, F3 must be the other symmetric β isomer. Unlike that reported in the literature for indochrome A and indochrome BII, F1 showed a multiplet at 3.65–4.27 ppm due to d-ribose glycone, while indochrome A presents a multiplet between 3.10 and 3.98 and indochrome BII between 3.44 and 4.05 ppm for the same structure. This difference may be due to the spectra made in different solvents, although other indochrome isomers show H-NMR multiplet to 4.30 ppm (Table S1). Fig. 7 Open in new tabDownload slide H-NMR spectra of blue pigment. a F1 and b F2. Spectra in D2O at 400 MHz. Strong signal at 4.8 ppm corresponds to water (HOD) Fig. 8 Open in new tabDownload slide Proposed structures for Pseudarthrobacter sp. 34LCH1 pigment main components. a indochrome A, b indochrome BII, c amylocyanin BIV and d amylocyanin B2A. Amylocyanin BIV consists of two compounds, the one presented here corresponds to the α-d-ribofuranosyl-β-d-ribofuranosyl derivative. F1 may either be indochrome A or BII and F2 may be amylocyanin BIV or B2A F2 showed two vinyl proton signals at 7.68 and 7.77 ppm, assigned as vinyl protons and two doublets at 4.89 and at 4.54 ppm assigned as anomeric protons. Between indochrome isomers, only asymmetric ones show two anomeric doublets; thus, F2 may correspond to an asymmetric d-ribosyl-diazaindophenol derivative. When comparing F2 with indochrome and amylocyanin components spectra, it can be deduced that in F2 one residue is an α-d-ribofuranosyl glycone (anomeric proton at 4.89 ppm) and the other is a β-d-pyranose or β-d-furanose. Thus, we suggest that the two F2 possible structures are amylocyanin B2A (α-d-furanose, β-d-pyranose) and amylocyanin BIV (α-D-furanose, β-d-furanose). However, more experiments are needed to complete structure assignment. No N–H or O–H protons were observed for F1 or F2 because rapid proton interchange with D2O suppressed signals of those acidic protons. F3 showed spectra like F1 and F2, but a low signal precludes further interpretation although, as described, it probably corresponds to a symmetric β-d-isomer. No NMR spectroscopy studies were performed for F4. Spectroscopic characterization, chemical properties and bacterial genus strongly support a glycosyl diazaindophenol structure (Fig. 8) of the Pseudarthrobacter sp. 34LCH1 blue pigment components, very probably being F1 indochrome A or BII and F2 amylocyanin B2A or BIV sodium salts, but 2D NMR and 13C NMR spectra would reliably confirm the proposed structures. Conclusions In this study, a blue pigment producing bacterial strain isolated from Chilean altiplanic lake was identified as Pseudarthrobacter sp. and its pigment was chemically characterized. The water-soluble blue pigment produced by this strain was composed of four main fractions which appear to correspond to sodium salts of ribopyranose and ribofuranose diazaindophenol glycoside isomers. The structural analysis of some of these fractions allows us to identify same compounds present in indochrome pigment, which has been previously reported. The blue pigment reached a yield as high as 2.5 g L−1 after 35 h of bacterial culture. The pigment exhibited a high water solubility and a very low toxicity within preliminary tests. Also, it was stable at temperatures below 60 °C and between pH 6 and 10. These properties suggest its suitability for use in some food, textile, paper, cosmetics, and pharmaceuticals. In addition, the interaction between pH and temperature which can result in a loss of the stability of blue pigment could prove useful in the design of a cold chain temperature monitoring devices for food, drugs, or vaccines during storage and transportation. Further studies are required to determine the optimal culture conditions that would increase the pigment production levels as well as its applicability as industrial dye. Considering that blue colored compounds in the nature are relatively rare and that only a few natural blue colored pigments are commercially available, the blue pigment produced by the strain 34LCH1 is a promising candidate for future industrial applications in the pastry, paper, cosmetic, or textile industries. Acknowledgements The authors thank Dr. Matthew Lee for his support for the review of this manuscript. 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TI - Purification and characterization of indochrome type blue pigment produced by Pseudarthrobacter sp. 34LCH1 isolated from Atacama desert
JF - Journal of Industrial Microbiology and Biotechnology
DO - 10.1007/s10295-018-2088-3
DA - 2019-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/purification-and-characterization-of-indochrome-type-blue-pigment-rFNSiwYC0L
SP - 101
EP - 111
VL - 46
IS - 1
DP - DeepDyve
ER -