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Microbiology and Biotechnology Letters

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Microbial Biotechnology (MB)  |  Cell Culture and Biomedical Engineering

Microbiol. Biotechnol. Lett. 2022; 50(3): 375-386

https://doi.org/10.48022/mbl.2205.05010

Received: May 23, 2022; Revised: August 14, 2022; Accepted: August 16, 2022

Evaluation of Antioxidant Potential and UV Protective Properties of Four Bacterial Pigments

Rupali Koshti1, Ashish Jagtap1, Domnic Noronha1, Shivali Patkar1, Jennifer Nazareth1, Ruby Paulose1, Avik Chakraborty2, and Pampi Chakraborty1*

1Department of Microbiology, St. Xavier’s College (Autonomous), 5, Mahapalika Marg, Mumbai, Maharashtra, PIN: 400001, India
2Radiation Medicine Centre, Bhabha Atomic Research Centre, J. Wadia Road, Parel, Mumbai, Maharashtra, PIN: 400012, India

Correspondence to :
Pampi Chakraborty,      pampi.chakraborty@xaviers.edu

Abstract

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In the present study, four distinctly colored bacterial isolates that show intense pigmentation upon brief ultraviolet (UV) light exposure are chosen. The strains are identified as Micrococcus luteus (Milky yellow), Cryseobacterium pallidum (Yellow), Cryseobacterium spp. (Golden yellow), and Kocuria turfanensis (Pink) based on their morphological and 16S rDNA analysis. Moderate salinity (1.25%), 25-37℃ temperature, and pH of 7.2 are found to be the most favorable conditions of growth and pigment production for all the selected isolates. The pigments are extracted using methanol: chloroform (1:1) and the purity of the pigments are confirmed by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). Further, Fourier transform infrared (FTIR) and UV-Visible spectroscopy indicate their resemblance with carotenoids and flexirubin family. The antioxidant activities of the pigments are estimated, and, all the pigments have shown significant antioxidant efficacy in 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. The UV protective property of the pigments is determined by cling-film assay, wherein, at least 25% of UV sensitive Escherichia coli survive with bio-pigments even after 90 seconds of UV exposure compared to control. The pigments also hold a good sun protective factor (SPF) value (1.5-4.9) which is calculated with the Mansur equation. Based on these results, it can be predicted that these bacterial pigments can be further developed into a promising antioxidant and UV-protectant for several biomedical applications.

Keywords: Bio-pigment, antioxidant, UV-protective pigments, carotenoids, flexirubin, SPF

Graphical Abstract


Introduction

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Bacteria are known to be a potential source of biopigments. Due to their anti-oxidant, anti-cancer, antimicrobial, and anti-inflammatory properties, bio-pigments have emerged as pharmaceuticals since the last decade [1−3]. Several bacterial pigments are extensively studied; such as carotenoids, melanins, flavins, quinones, violacein, etc. [4−6]. Recent studies have focused on treating many diseases like cancer, leukemia, cardiovascular diseases, diabetes mellitus, neurodegenerative diseases, etc. with bio-pigments [7−11]. Carotenoids are considered to be one of the most abundant types of pigment in bacteria. They get oxidized readily and thus can prevent reactive oxygen species (ROS) formation. Carotenoids obtained from Haematococcus pluvialis and Phaffia rhodozyma are being utilized as food additives for animals and fish, as they act as antioxidants and vitamin supplements [4, 5, 12]. Violacein can be used as an antiviral, antibacterial, antiulcerogenic, antileishmanial, and antitumor agent [13]. Quinones, mostly isolated from marine bacteria exhibit antiviral, antibacterial, and insecticidal activities, and have many commercial applications as natural and artificial dyes [14]. Another important natural pigment is flexirubin with terminal alkyl substitution consisting of ω-phenyl octaenic acid, produced by Cytophaga sp., Sporocytophaga sp., and Chryseobacterium sp. According to recent reports, this pigment acts as an antioxidant and is found with carotenoid in some bacterial species [15].

There are several bacterial pigments and their analogs that have been screened for their antioxidant and radioprotective ability [16]. Pigments like carotenoid, naphthoquinone, and violacein have been shown to possess a potent antioxidant activity [13, 17, 18]. Studies revealed that yellow pigment called staphyloxanthin, from Staphylococcus aureus prevents carbon tetrachloride-induced oxidative stress in Swiss albino mice [19]. Again, violacein showed protection against oxidative damage in gastric ulceration by stimulating mucosal defence mechanisms [20]. So far, only a few microorganisms have been evaluated for the production of bio-pigments with antioxidant and radioprotective properties. Additional microbial taxa or poorly explored habitats should be investigated for the biopigment producers particularly for the above-mentioned purposes.

Ultraviolet (UV) radiation causes adverse biological effects such as photo-aging, skin cancer, generation of ROS, and damages the DNA [21]. Excess production of ROS in the body is associated with aging and chronic cardiovascular and neurodegenerative diseases, diabetes mellitus, and kidney diseases [22]. Carotenoids and melanins offer a selective advantage to the host organisms as they shield them from UV by absorbing in the UV range and by scavenging the free radicals [23, 24]. Many synthetic UV-protective agents present in sunscreens, and also synthetic antioxidants are known to cause side effects and have safety issues [25, 26]. Hence, there is an increased interest to explore natural alternatives that are effective and safe.

Thus, in the present study, the objective is to isolate and characterize bacterial pigments from coastal soil and water samples and to monitor their antioxidant properties by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays.

Materials and Methods

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Sample collection, isolation, and identification of the bacterial cultures

Sample collection. The pigment-producing bacteria were screened from the water and soil samples. The samples were collected from various locations in Mumbai, India. The co-ordinates of the sample collection locations are- Dadar sea beach (DSB)- 19.020758°, 72.829176° and Napean sea Road (NSR)- 18.958871°, 72.799343°.

The pigment-producing bacteria were screened from the water and soil samples. The samples were collected from various locations in Mumbai, India. The co-ordinates of the sample collection locations are- Dadar sea beach (DSB)- 19.020758°, 72.829176° and Napean sea Road (NSR)- 18.958871°, 72.799343°.

Isolation. For isolation of the bacteria, 1 g each of the soil samples was mixed with 50 ml of saline. From this, 100 μl was added on to the nutrient agar plates (M001, Hi-Media, India), by spread plate technique, and incubated at room temperature (RT) for 48 h. After 48 h, the plates were given a 30 s UV exposure (253.7 nm) by keeping the plate 1 meter away from the UV source in the laminar airflow (ESCO, Singapore) and incubated further for 24 h at room temperature. Only the bacterial colonies that showed intense color after this exposure were selected for further study.

Identification. The cultures were identified using morphological characteristics and 16S rDNA sequencing. The DNA of these cultures was extracted using the phenol-chloroform method and the purified DNA was then subjected to a polymerase chain reaction (PCR) (ESCO Thermal cycler, Singapore) using 27f and 1492r primers [27]. The amplicon was sequenced, and then similar sequences were searched through NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were aligned and a phylogenetic tree was prepared with MEGA software.

Optimization of growth conditions. Optimization of growth parameters like temperature, pH, and salt concentration was carried out simultaneously to assess the optimum growth of the bacteria. The time of UV exposure required for maximum pigment production was also determined. The nutrient broth was inoculated with respective pigment-producing isolates (0.5% v/v, 0.2 OD) and incubated at 4℃, 25℃, 37℃, and 55℃ to check its optimum temperature for growth. Similarly, nutrient broth with differing pH (3, 4, 5, 6, 7, 8, and 9) were inoculated with respective strains and incubated up to 48 h at shaking condition (200 rpm). To investigate the effect of salt concentration on bacterial growth, nutrient broth was mixed with different concentrations of NaCl (0.07%, 0.015%, 0.31%, 0.63%, 1.25%, 2.50%, 5.00%, and 10%) and inoculated with pigment-producing isolates (0.5%v/v, 0.2 OD). After 18 h of incubation at shaking conditions (200 × g), the optimum growth was measured by taking absorbance at 530 nm (BioTek Instruments Inc., USA).

To determine the optimum time of UV exposure required for obtaining higher pigment production, the cell suspension was exposed to UV radiation (253.7 nm) for 0 s, 30 s, 60 s, 90 s, and 120 s and then incubated for 48 h at optimum growth conditions. After growth, the cell pellet was used for pigment extraction. The maximum pigment production was then determined by checking the absorbance at their respective λmax (BioTek Instruments Inc.).

Extraction of the pigments. The solvent extraction method was used for extracting the pigments [13]. The nutrient broth was inoculated with Yellow, Milky yellow, and Pink cultures. After incubation at 37℃ for 48 h, the cell pellet was isolated from bacterial suspension by centrifugation at 4000 × g for 10 min. The harvested cell pellet was solubilized in 30 ml methanol and heated in a water bath for 30 min at 60℃. This solution was centrifuged again and the supernatant was filtered and collected in Petri dishes and kept at RT for the evaporation of methanol. For Golden yellow, the bacterial suspension was centrifuged, as described above and the supernatant was added to a separating funnel, and dissolved in Methanol: Chloroform (1:1). This mixture was stirred strongly for 5 min and kept undisturbed for 1 h to obtain separate layers. Since the pigment got diffused in the solvent layer, it was collected in Petri plates and kept at RT for solvent evaporation. The resulting pigments were stored in sterile centrifuge tubes after measuring the weight of the pigments.

Characterization of the pigments

Thin-layer Chromatography (TLC) and High-performance Liquid Chromatography (HPLC). The pigments obtained after extraction were subjected to TLC and HPLC to assess its components. The pigments were spotted on a TLC plate and methanol was used as a mobile phase. After the run, the TLC plate was dried at RT and placed in an iodine chamber for the development of spots.

The chemical purity of the pigments was assessed using HPLC. The extracted pigments were dissolved in 50% dimethyl sulfoxide (DMSO) and then analyzed by HPLC using Eurosphere C-18 reversed-phase cartridge {Dimension: 300 mm (length) × 4 mm (diameter), particle size: 5 μm} in KNAUER, Germany system. The mobile phase contained Solvent A: 0.1% formic acid in HPLC water and Solvent B: 0.1% formic acid in methanol. At a flow rate of 0.5 ml/min, the elution gradient was as follows: for zero time and 2 min, 95% solvent A and 5% solvent B; for 1 min and 20 min gradient changes to 5% solvent A and 95% solvent B. The UV detector, connected in series, was set to absorbance at 255 nm. The ChromGate software was used for the analysis of the data.

UV-visible and Fourier Transform Infrared (FTIR) analysis. The dried pigments were dissolved in 1ml of 5% methanol solution (1 mg/ml) and its UV-visible absorption range was determined by performing their spectral analysis in a UV-Visible spectrophotometer (Jasco V-530). To predict their structural conformation and functional group, the extracted pigments (1 mg/ml of pigment dissolved in methanol) were analyzed in the range of 4000 cm-1 to 400 cm-1 by FTIR spectroscopy (Horiba FT720). The samples were mixed homogenously with dried crystals of KBr to make pellets and immediately used for the FTIR analysis to avoid any water absorption.

Estimation of polyphenols in pigments by Folin-Denis method. The polyphenol estimation of the respective pigments was performed in a 96-well plate by the Folin-Denis method [17]. All four pigments (1000 μg) were dissolved in 1 ml of 5% methanol for this assay. A standard curve of tannic acid (20−100 μg/ml) was plotted, and the reaction set up contained- 100 μl pigment + 50 μl Folin-Denis reagent + 50 μl saturated Na2CO3. The absorbance was taken at 760 nm (BioTek Instruments Inc.) and the concentration of polyphenols present in the pigment was calculated from the standard graph and was represented in μg tannic acid equivalent (TAE).

Evaluation of the antioxidant potential. The antioxidant potential of the pigments was determined using FRAP, ABTS, and DPPH assays.

FRAP assay. FRAP was evaluated using the method described before [28]. The reaction set up contained- 180 μl FRAP reagent + 18 μl D/W + 6 μl pigments. 250 μg/ml, 500 μg/ml, and 1000 μg/ml of the pigments (dissolved in 5% methanol) were used for this assay and ascorbic acid (200 μg/ml) was used as a positive control. FeSO4·7H2O (100−1000 μmoles/l) was used to prepare the standard curve and the readings were taken at 595 nm (BioTek Instruments Inc.). The equation of the standard graph was used to determine the FRAP activity and it was represented as μmoles Fe2+ equivalent.

ABTS assay. The potential of the pigments to neutralize ABTS+ radicals was evaluated by ABTS assay [29]. 250 μg/ml, 500 μg/ml, and 1000 μg/ml of the pigments (dissolved in 5% methanol) were used for this assay. Ascorbic acid (200 μg/ml) was used as a positive control for this assay. 50 μl of pigment was added to 150 μl of ABTS solution and kept for 15 minutes in dark. Then the absorbance was taken at 734 nm (BioTek Instruments Inc.). The following formula was used to determine the percent antioxidant activity:

ABTS scavenging activity (%) =Absorbance of control -Absorbance of test or standard samplesAbsorbance of control×100

DPPH assay. The free radical scavenging ability of the pigment extracts was measured by DPPH assay [29]. For the assay, DPPH (0.1 mM in methanol) was used as the stock reagent. 250 μg/ml, 500 μg/ml, and 1000 μg/ml of the pigments dissolved in 5% methanol were used for this assay. 50 μl of pigment was added to 150 μl of DPPH solution and kept for 30 min in dark at room temperature. The readings were then taken at 517 nm (BioTek Instruments Inc., USA). The antioxidant activity was calculated as follows-

DPPH scavenging activity (%) =Absorbance of control-Absorbance of test or standard sampleAbsorbance of control×100

Ultraviolet protection assay with cling film. The ultraviolet protective ability of the pigments was evaluated by using the method described previously [26]. Initially, 500 μl of UV susceptible E. coli (OD 0.1) was taken in a sterile watch glass and covered with a cling film such that a concave depression was formed at the center of the film. 600 μl of the extracted pigment solutions (60 mg/ml) were placed in the depression on the film and this set up was exposed to UV light at different time intervals i.e. 90 s and 120 s. UV susceptible E. coli in a watch glass, with sterile distilled water in the depression on the cling film and without the cling film were used as controls. After exposure for the respective time interval, 100 μl of culture sample from each watch glass was added (neat or diluted) on the nutrient agar by pour plate method, incubated at 37℃ overnight, and % survival was determined using the following formula-

% viability=Number of cfu in UV treated sampleNumber of cfu in UV untreated sample×100

Evaluation of Sun Protection Factor (SPF). The SPF of the pigments was determined using the Mansur equation [30]. The absorption spectrum between 290−320 nm, at 5 nm interval was taken and methanol was used as blank. The SPF factor for each pigment was calculated as follows:

SPF (spectrophotometric) = CF×∑ 290 320EE(λ)×I(λ)×Abs(λ)

EE × I values are constants [30]. CF is correction factor (=10), I is the solar intensity spectrum, Abs is the absorbance of the sunscreen product, and EE is the erythemal effect spectrum.

Statistical analysis. The statistical analysis was performed with Microsoft Excel and SigmaStat3.5 software. Experimental data is depicted as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by a post hoc test (Student-Newman-Keuls) was applied to the ‘UV protective assay’ and ‘UV exposure optimization data’. The differences were deemed significant when p < 0.05.

Results and Discussion

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Identification of the pigment producing bacteria and optimization of the growth conditions

Many bacterial colonies were obtained on the agar plate after isolation from different samples. The plates were exposed to UV light for 30 s and incubated overnight. Only four colonies showed intense pigmentation after UV exposure. They were named - Milky yellow, Pink, Golden yellow, and Yellow according to their color (Figs. 1A−D). UV-C exposure can lead to a stress response for bacteria which can cause cell death and mutation depending on the exposure time. Some bacteria have been shown to increase pigment production after UV exposure too [31]. Therefore, the UV exposure time for the cultures was determined, to get intense pigmentation without causing any potential detrimental effect on the cell number. It was observed that C. pallidum (Yellow) showed a better amount of pigment after 60 s of UV exposure followed by 24 h of incubation. M. luteus (Milky yellow) needed 100 s, K. turfanensis (Pink), and Chryseobacterium spp. (Golden yellow) required 120 s of UV exposure to enhance the pigment production compared to unexposed cultures (Fig. 1S). It was evident from the UV exposure study, that the chosen isolates were UV resistant to a great extent. M. luteus and Kocuria spp. are known for their UV resistance [32, 33], but this study confirms that Chryseobacterium spp. isolated from the coastal regions can also tolerate UV exposure.

Figure 1.(A-D) Pigment producing bacterial isolates on nutrient agar plates. Effect of (E) pH and (F) salt concentration on cell growth for the selected isolates after 24 h and 18 h of incubation respectively.

The colony characteristics and Gram’s nature of the isolates are depicted in Table 1. After 16S rDNA sequencing, the phylogenetic tree was constructed and the strains were identified to be M. luteus (Milky yellow), K. turfanensis (Pink), Cryseobacterium spp. (Golden yellow), and C. pallidum (Yellow) (Fig. 2).

Table 1 . Colony characteristics of the isolates.

Name of the OrganismsGram’s natureMorpho-logyColorOpacityConsistencyElevationBorderSize
Cryseobacterium pallidum-Short rodsYellowTranslucentButyrousFlatIrregular2 mm
Cryseobacterium spp.-CoccobacilliGolden yellowTranslucentButyrousFlatIrregular2 mm
Micrococcus luteus+Cocci in diploid and clusterMilky yellowOpaqueMucoidSlightly raisedIrregular3 mm
Kocuria turfanensis+Cocci in clusterPinkOpaqueMucoidSlightly raisedIrregular2 mm


Figure 2.Phylogenetic tree indicating close evolutionary relationship of (A) Milky yellow pigmented isolate to M. luteus, (B) Pink pigmented isolate to K. turfanensis, (C) Yellow pigmented isolate to C. pallidum, and (D) Golden Yellow pigmented isolate to Chrysobacterium spp.

To obtain the pigments in high concentration, certain growth parameters of the bacterial cultures such as the temperature of incubation, UV exposure, salt concentration, and pH of the medium were optimized [31]. Yellow pigment producer showed higher growth when incubated at room temperature, whereas, for Golden yellow, Milky yellow, and Pink, the optimum temperature for growth was 37℃. It was observed that all the cultures showed higher growth between pH 7 and pH 8, indicating that neutral or slightly alkaline pH was the optimum pH for the growth of these organisms, but Pink pigment-producing bacteria had shown growth between pH 3 to pH 9 in considerably higher rate. It was observed that all the cultures showed higher growth between pH 7 and pH 8, indicating that neutral or slightly alkaline pH was the optimum pH for the growth of these organisms, but pink pigment-producing bacteria had shown growth between pH 3 to pH 9 in considerably higher rate (Fig. 1E). After pH and temperature, the salt concentration was optimized for the isolates. All the organisms were able to survive in a wide range of salt concentrations and could tolerate up to 5% NaCl in the medium. The result also confirmed that M. luteus and K. turfanensis could tolerate even 10% salt concentration (Fig. 1F). It was observed that changes in pH, temperature, and salt concentrations didn’t alter the pigment production rate in the chosen organisms (data not shown). Overall, the selected strains showed tolerance to a broad range of salt concentrations, temperature, and pH which could be attributed to their habitat. The pigment yield was also investigated at the optimum growth conditions for all four strains. K. turfanensis and M. luteus strains produced 350 mg and 330 mg of pigments per liter of the inoculated broth respectively. Again, C. pallidum and Cryseobacterium spp. gave relatively less yield i.e. 280 mg and 292 mg per liter respectively after 48 h of incubation.

Characterization of the pigments

The pigments were isolated by solvent extraction which is a crude method of isolation. Therefore, the components of the extracted pigments were determined using TLC and HPLC. For TLC, only one spot was obtained for Golden yellow, yellow, milky yellow and pink pigment samples after exposure to iodine vapor and their obtained Rf values were 0.79, 0.51, 0.75, and 0.622, respectively (Fig. S2). This indicated that the pigments contain a single constituent and are pure. Our HPLC data also supported this fact. All four pigments showed retention time (Rt) in the range of 3.3−3.5 min respectively, in the HPLC analysis. This analysis ascertains a satisfactory level of pigment purity through the solvent extraction method. The gradient mode and mobile phase (H2O: Methanol: Formic acid) were found to be the most appropriate for the separation of pigment extracts. Due to the moderate hydrophilicity of both the carotenoid and flexirubin pigments, they elute early with the solvent phase of intermediate polarity (Fig. 3).

Figure 3.HPLC chromatograms of the pigments; (A) Milky yellow pigment isolated from M. luteus, (B) Pink pigment isolated from K. turfanensis, (C) Golden yellow pigment isolated from Chryseobacterium spp., and (D) Yellow pigment isolated from C. pallidum.

Characterization of the pigments was done using FTIR and UV-visible spectroscopy. Through UV-vis spectroscopy (Fig. 4), it was observed that pink pigment isolated from K. turfanensis absorbed in the UV range (259 nm) as well as in the visible range (471 nm). Milky yellow and golden yellow pigments showed absorption maxima at 260 nm and 256 nm respectively. Yellow pigment isolated from C. pallidum showed the highest absorption at 256 nm and 400 nm respectively (Fig. 4). These results are characteristic of photo-protective pigments such as carotenoids and flexirubin [15, 34].

Figure 4.UV Visible spectra of the bio-pigments: Milky yellow pigment isolated from M. luteus, pink pigment isolated from K. turfanensis, golden yellow pigment isolated from Chryseobacterium spp., and yellow pigment isolated from C. pallidum are dissolved in 5% methanol and their spectral analysis were done.

FTIR of all four pigments was carried out to understand their chemical characteristic (Fig. 5). FTIR results for the pink pigment showed the C-O stretching frequency at 1649 cm-1 which represents the carboxyl group, indicating the presence of chelating groups or ester group or long-chained aliphatic group. The broad hump at 3788 cm-1 represented the presence of the -OH/ NH functional group. Milky yellow pigment showed a similar spectrum, only with the reduced intensity at 1075 cm-1 peak, which may be due to the smaller contribution of the aliphatic chain. The broad hump was also shifted to 3509−3336 cm-1 which is mostly for -OH linkage and small water adsorption. These peaks indicated that milky yellow and pink possibly have the carotenoid structure [35]. Golden yellow showed a strong peak at 1634 cm-1 which represents the aliphatic carbonyl group, most probably in amide linkage or ester linkage with a long aliphatic chain. The peak at 1412 cm-1 also represented the C-H stretching frequency of -CH3 situated in the fatty acid/long-chain group which confirms the ester formation. The asymmetric C-H stretching frequency at 1075 cm-1 was also elevated from the aliphatic hydrocarbon chain. Therefore, the golden yellow pigment from Chryseobacterium spp. also appears to be of the carotenoid class of pigment. For Yellow, the peak at 3533 cm-1 attributed to the hydroxyl stretching of absorbed water and the absorption band at 3288 cm-1 was due to the presence of -OH groups. The peaks at 1641 cm-1 and 1229 cm-1 corresponded to the C-O stretching frequencies. There was a little shift observed in the position of carbonyl and the hydroxyl absorption spectra of Yellow which can be due to the strong chelating structure of the pigments. The absorption bands in the range of 1109 cm-1 to 672 cm-1 were due to asymmetric C-H stretching in alkyl hydrocarbons. Therefore, from these results, it can be concluded that yellow pigment from C. pallidum has structural similarities with the flexirubin class of pigments [15].

Figure 5.FTIR Analysis of pigments; Milky yellow pigment isolated from M. luteus (Red), Pink pigment isolated from K. turfanensis (Blue), Golden yellow pigment isolated from Chryseobacterium spp. (Black), and Yellow pigment isolated from C. pallidum (Green).

Estimation of the polyphenol content and antioxidant activity of the pigments

Natural pigments are known to contain polyphenols and studies prove that these polyphenols contribute to antioxidant activity [36]. Therefore, in this study, the total amount of polyphenols present in the pigments was determined by the Folin-Denis assay. Pink showed the highest amount of polyphenol content (263 μg TAE), followed by Yellow (217 μg TAE), Golden yellow (101 μg TAE), and Milky yellow which had the least polyphenol content (71 μg TAE) (Fig. 6A). Since all the pigments were found to contain a high amount of polyphenol, this result indicated that these pigments could be potent antioxidants.

Figure 6.Estimation of the polyphenol content in pigments through (A) Folin-Dennis assay and evaluation of its antioxidant potential by (B) FRAP assay, (C) ABTS assay, and (D) DPPH assay.

One single assay is usually not reliable enough to conclusively determine the antioxidant potential of the compounds [37]. Therefore, we have determined the antioxidant potential through three different assays. The reducing power of the compounds was evaluated using the FRAP assay, which in turn indicates its antioxidant potential [38]. Studies indicate that not all antioxidant compounds react at the same rate with FRAP and its sensitivity depends upon the type and amount of polyphenols present [39]. Pink showed the highest ferric reducing ability, followed by Golden yellow, Milky yellow, and Yellow (Fig. 6B).

The free radical scavenging potential of any antioxidant compound is evaluated using DPPH and ABTS assays. Antioxidants donate hydrogen to free radicals and thus scavenge them [29]. Studies have reported that higher molecular weight phenolics react more readily with ABTS+, whereas, the steric accessibility of DPPH allows small compounds to react rapidly with it [38, 39]. It was observed that Yellow showed the highest ABTS scavenging ability (92 ± 0.75%), followed by Golden yellow (76 ± 6%), Milky yellow (75 ± 4%), and Pink (61 ± 3%) (Fig. 6C). The positive control, ascorbic acid (200 μg/ ml) showed 72 ± 3% free radical scavenging activity in this assay. The results of DPPH assay indicated that Milky yellow had the highest (29.6 ± 0.32%) antioxidant activity, followed by Yellow, Pink, and Golden yellow (Fig. 6D). The ascorbic acid also showed 87 ± 4% of free radical scavenging activity in this assay. It was also observed that the DPPH and ABTS scavenging efficacy of the pigments increased with the increasing concentration.

Ultraviolet protection assay and SPF values of the pigments

These pigments showed absorbance in the UV range, its UV-protective property was tested against UV susceptible E. coli by performing a cling film assay. Previously, Devihalli C. Mohana et al. has reported that carotenoids from Micrococcus spp. protect UV-sensitive S. faecalis from cell death, when exposed to UV light [26]. Similar results were observed in our study, where, at least 30−45% of UV-susceptible E. coli were viable after 90 s of UV exposure in presence of pigments compared to controls (Fig. 7A). With the increase of UV exposure up to 120 s, comparatively lesser protection was seen, i.e. a minimum of 3% E. coli cell viability was observed over the controls. However, the Milky yellow pigment isolated from M. luteus showed the highest protection over control even after 120 s of UV exposure (Fig. 7B). The SPF values for the pigments were also determined and they were found to be following with the UV-protective assay data. Milky yellow (4.9) had the highest SPF, followed by Yellow (2.45), Pink (2.26), and Golden yellow (1.48) (Fig. 7C). SPF of the bacterial pigments could be determined directly or by adding the pigments with sunscreens having known SPF by Mansur equation. In the present study, the SPF values were determined without incorporating any sunscreen in it. The obtained SPF values are comparable with the previously published data [40, 41].

Figure 7.Analysis of the pigments’ UV protective efficacy by cling film assay. (A) 90 s UV exposure, (B) 120 s UV exposure (*p < 0.05), (C) SPF values of the pigments evaluated using the Mansur equation.

In the present study, we have investigated the antioxidant potential of bacterial pigments. For that purpose, four pigments were extracted from bacteria such as M. luteus (Milky yellow), C. pallidum (Yellow), Cryseobacterium spp. (Golden yellow), and K. turfanensis (Pink). Our study demonstrated the optimization of the pigment production process based on UV-exposure, pH, temperature, and salt concentration. It also described a separation process for each of the pigments including the less-studied pigment from K. turfanensis. The characterization of the pigments was done by UV-Vis spectroscopy, HPLC, and FTIR. These pigments belonged to the carotenoids and flexirubin family. Pigments from K. turfanensis (pink) and C. pallidum (Yellow) showed significant levels (p < 0.05) of polyphenols and antioxidant potential compared to the other two pigments. Again, M. luteus (Milky yellow) and C. pallidum (Yellow) showed a higher amount of UV-protective activity in UV-cling film assay. A few studies have reported that incorporating UV-protective pigments in commercially available sunscreens increases the sunscreen’s SPF by 10−60% [42, 43]. Since these pigments were UV-protective and had good SPF, after appropriate toxicity studies, these pigments could be incorporated into sunscreens. Moreover, the pigments can be used as safe and natural antioxidants for several therapies.

Acknowledgments

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The authors would like to thank Dr. Ankan Dutta Chowdhury, Sizouka University for his help in FTIR analysis. The authors also express their sincere gratitude to the Department of Microbiology, St. Xavier’s College (Autonomous), Mumbai and Radiation Medicine Centre, BARC, Mumbai for providing laboratory facilities, and their valuable expertise for this work.

References

Go to
  1. Sen T, Barrow CJ, Deshmukh SK. 2019. Microbial pigments in the food industry - Challenges and the way forward. Front. Nutr. 6: 7.
    Pubmed KoreaMed CrossRef
  2. Ramesh C, Vinithkumar NV, Kirubagaran R, Venil CK, Dufossé L. 2019. Multifaceted applications of microbial pigments: Current knowledge, challenges and future directions for public health implications. Microorganisms 7: 186.
    Pubmed KoreaMed CrossRef
  3. Dufossé L. 2006. Microbial production of food grade pigments. Food Technol. Biotechnol. 44: 313-321.
  4. Soni SK. 2007. Microbes: A source of energy for 21st century. New India publishing agency.
  5. Rao MPN, Xiao M, Li W-J. 2017. Fungal and bacterial pigments: Secondary metabolites with wide applications. Front. Microbiol. 8: 1113.
    Pubmed KoreaMed CrossRef
  6. Tuli HS, Chaudhary P, Beniwal V, Sharma AK. 2015. Microbial pigments as natural color sources: current trends and future perspectives. J. Food Sci. Technol. 52: 4669-4678.
    Pubmed KoreaMed CrossRef
  7. Díaz-Ruiz C, Montaner B, Pérez-Tomás R. 2001. Prodigiosin induces cell death and morphological changes indicative of apoptosis in gastric cancer cell line HGT-1. Histol. Histopathol. 16: 415-421.
  8. Pandey R, Chander R, Sainis KB. 2007. Prodigiosins: a novel family of immunosuppressants with anti-cancer activity. Indian J. Biochem. Biophys. 44: 295-302.
  9. Kavitha R, Aiswariya S, Ratnavali CM. 2010. Anticancer activity of red pigment from Serratia marcescens in human cervix carcinoma. Int. J. PharmTech. Res. 2: 784-787.
  10. Rodrigues AL, Trachtmann N, Becker J, Lohanatha AF, Blotenberg J, Bolten CJ, et al. 2013. Systems metabolic engineering of Escherichia coli for production of the antitumor drugs violacein and deoxyviolacein. Metab. Eng. 20: 29-41.
    Pubmed CrossRef
  11. Tanaka T, Shnimizu M, Moriwaki H. 2012. Cancer chemoprevention by carotenoids. Mol. Basel Switz. 17: 3202-3242.
    Pubmed KoreaMed CrossRef
  12. Lampila LE, Wallen SE, Bullerman LB. 1985. A review of factors affecting biosynthesis of carotenoids by the order Mucorales. Mycopathologia 90: 65-80.
    Pubmed CrossRef
  13. Numan M, Bashir S, Mumtaz R, Tayyab S, Rehman NU, Khan AL, et al. 2018. Therapeutic applications of bacterial pigments: a review of current status and future opportunities. 3 Biotech. 8: 207.
    Pubmed KoreaMed CrossRef
  14. Soliev AB, Hosokawa K, Enomoto K. 2011. Bioactive pigments from marine bacteria: Applications and physiological roles. Evid. Based Complement. Alternat. Med. 2011: 670349.
    Pubmed KoreaMed CrossRef
  15. Venil CK, Zakaria ZA, Usha R, Ahmad WA. 2014. Isolation and characterization of flexirubin type pigment from Chryseobacterium sp. UTM-3T. Biocatal. Agric. Biotechnol. 3: 103-107.
    CrossRef
  16. Rajagopal L, Sundari CS, Balasubramanian D, Sonti RV. 1997. The bacterial pigment xanthomonadin offers protection against photodamage. FEBS Lett. 415: 125-128.
    CrossRef
  17. Giuliani C, Tani C, Maleci Bini L, Fico G, Colombo R, Martinelli T. 2018. Localization of phenolic compounds in the fruits of Silybum marianum characterized by different silymarin chemotype and altered colour. Fitoterapia 130: 210-218.
    Pubmed CrossRef
  18. Konzen M, De Marco D, Cordova CAS, Vieira TO, Antônio RV, Creczynski-Pasa TB. 2006. Antioxidant properties of violacein: Possible relation on its biological function. Bioorg. Med. Chem. 14: 8307-8313.
    Pubmed CrossRef
  19. Kurjogi MM, Sanakal RD, Kaliwal BB. 2010. Antibiotic susceptibility and antioxidant activity of Staphylococcus aureus pigment staphyloxanthin on carbon tetrachloride (ccl4) induced stress in swiss albino mice. Int. J. Biotechnol. Appl. 2: 33-40.
    CrossRef
  20. Antonisamy P, Kannan P, Aravinthan A, Duraipandiyan V, Valan Arasu M, Ignacimuthu S, et al. 2014. Gastroprotective activity of violacein isolated from Chromobacterium violaceum on Indomethacin-induced gastric lesions in rats: Investigation of potential mechanisms of action. ScientificWorldJournal 2014: 616432.
    Pubmed KoreaMed CrossRef
  21. Afaq F, Zaid MA, Khan N, Dreher M, Mukhtar H. 2009. Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin. Exp. Dermatol. 18: 553-561.
    Pubmed KoreaMed CrossRef
  22. Liguori I, Russo G, Curcio F, Bulli G, Aran L, Della-Morte D, et al. 2018. Oxidative stress, aging, and diseases. Clin. Interv. Aging 13: 757-772.
    Pubmed KoreaMed CrossRef
  23. Shivprasad S, Page WJ. 1989. Catechol formation and melanization by Na-dependent Azotobacter chroococcum: a protective mechanism for aeroadaptation? Appl. Environ. Microbiol. 55: 1811-1817.
    Pubmed KoreaMed CrossRef
  24. To KY, Lai EM, Lee LY, Lin TP, Hung CH, Chen CL, et al. 1994. Analysis of the gene cluster encoding carotenoid biosynthesis in Erwinia herbicola Eho13. Microbiology 140: 331-339.
    Pubmed CrossRef
  25. Krishnaiah D, Sarbatly R, Bono A. 2007. Phytochemical antioxidants for health and medicine - A move towards nature. Biotechnol. Mol. Biol. Rev. 1: 97-104.
  26. Mohana D, Thippeswamy S, Abhishek R. 2013. Antioxidant, antibacterial, and ultraviolet-protective properties of carotenoids isolated from Micrococcus spp. Radiat. Prot. Environ. 36: 168.
    CrossRef
  27. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. 2008. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ. Microbiol. 74: 2461-2470.
    Pubmed KoreaMed CrossRef
  28. Benzie IFF, Strain JJ. 1996. The ferric reducing ability of plasma (FRAP) as a measure of "Antioxidant Power": The FRAP assay. Anal. Biochem. 239: 70-76.
    Pubmed CrossRef
  29. Shalaby E, Shanab S. 2013. Comparison of DPPH and ABTS assays for determining antioxidant potential of water and methanol extracts of Spirulina platensis. Indian J. Mar. Sci. 42: 556-564.
  30. Dutra EA, Oliveira DAG da C, Kedor-Hackmann ERM, Santoro MIRM. 2004. Determination of sun protection factor (SPF) of sunscreens by ultraviolet spectrophotometry. Rev. Bras. Ciênc. Farm. 40: 381-385.
    CrossRef
  31. Palacio-Barrera AM, Areiza D, Zapata P, Atehortúa L, Correa C, Peñuela-Vásquez M. 2019. Induction of pigment production through media composition, abiotic and biotic factors in two filamentous fungi. Biotechnol. Rep. 21: e00308.
    Pubmed KoreaMed CrossRef
  32. Melin A-M, Perromat A, Lorin C, Déléris G. 2002. γ Irradiation and cellular damage in Kocuria rosea: investigation by one- and two-dimensional infrared spectroscopy. Arch. Biochem. Biophys. 408: 211-219.
    CrossRef
  33. Goldberg S, Freundt E. 2016. Protective qualities of UV-resistant bacteria. Acta Spartae 2: 9-13.
  34. Mathews-Roth MM. 1987. Photoprotection by carotenoids. Fed. Proc. 46: 1890-1893.
    Pubmed CrossRef
  35. Trivedi N, Tandon S, Dubey A. 2017. Fourier transform infrared spectroscopy (FTIR) profiling of red pigment produced by Bacillus subtilis PD5. Afr. J. Biotechnol. 16: 1507-1512.
    CrossRef
  36. Boo H-O, Hwang S-J, Bae C-S, Park S-H, Heo B-G, Gorinstein S. 2012. Extraction and characterization of some natural plant pigments. Ind. Crops Prod. 40: 129-135.
    CrossRef
  37. Barros RGC, Andrade JKS, Denadai M, Nunes ML, Narain N. 2017. Evaluation of bioactive compounds potential and antioxidant activity in some Brazilian exotic fruit residues. Food Res. Int. 102: 84-92.
    Pubmed CrossRef
  38. Sowndhararajan K, Kang SC. 2013. Free radical scavenging activity from different extracts of leaves of Bauhinia vahlii Wight & Arn. Saudi J. Biol. Sci. 20: 319-325.
    Pubmed KoreaMed CrossRef
  39. Magalhães LM, Segundo MA, Reis S, Lima JLFC. 2008. Methodological aspects about in vitro evaluation of antioxidant properties. Anal. Chim. Acta 613: 1-19.
    Pubmed CrossRef
  40. Bana M, Heshmatipour Z. 2014. Isolation of pigment-producing bacteria from surface water and study of sun protection factor (SPF) of the purified pigments. Iran. J. Public Health. 43: 143-143.
  41. Choksi J, Vora J, Shrivastava N. 2020. Bioactive pigments from isolated bacteria and its antibacterial, antioxidant and sun protective application useful for cosmetic products. Indian J. Microbiol. 60: 379-382.
    Pubmed KoreaMed CrossRef
  42. Koli SH, Suryawanshi RK, Mohite BV, Patil SV. 2019. Prospective of Monascus pigments as an additive to commercial sunscreens. Nat. Prod. Commun. 14: 1-7.
    CrossRef
  43. Suryawanshi RK, Patil CD, Borase HP, Narkhede CP, Stevenson A, Hallsworth JE, et al. 2015. Towards an understanding of bacterial metabolites prodigiosin and violacein and their potential for use in commercial sunscreens. Int. J. Cosmet. Sci. 37: 98-107.
    Pubmed CrossRef

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