A non-fluorescent pigment produced from the fluorescent Bacillus endophyticus AVP-9 which possess abundant lutein content considered as a xanthophyl-type compound with potentially beneficial pharmacological properties for enhancing human health. In this context, bacterial lutein was extracted by employing different solvents to isolate from Bacillus endophyticus was examined in this study. Chromatographically purified lutein was spectrally characterized using ultraviolet-visible spectrometry and spectral analysis was carried through liquid chromatography-mass spectrometry, functional group identification was done by using fourier transform-infrared spectroscopy, structural determination by nuclear magnetic resonance spectrometry. By using hexane and ethyl acetate (7:3) with the flow rate of 5ml/min, was proven to be an excellent solvent phase for separation of lutein, meaning that up to 90% pure lutein generated with a 60% estimated yield. The purified compound identified as a β, ε-3, 31-diol (lutein) having molecular weight of 568.6g/mol with the molecular formula C40H56O2 which exhibits potential antibacterial activity against E. coli (35mm) followed by S. typhi (33mm), S. flexneri (30mm), P. aureginosa (29mm), M. lutea (28mm), P. vulgaris (24mm), S. aureus (21mm) and lowest antibacterial activity against V. cholera (19mm). Purified pigment shows concentration dependent antioxidant activities with IC50 values of pigment was found to be 4.5 mg/ml. Whereas anti-proliferation activity pigment shows considerable effect MCF-7 and LNCaP cell lines, when treated with 100µg/ml, cell viability was decreased to 36.363% at IC50 value of 21.935 µg/ml and for LNCaP, the cell viability was decreased to 42.449% at IC50 value of 56.937 µg/ml.
Research Article
Extraction, purification, characterization and evaluation of antibacterial and anticancer activity of β,ε-Carotene-3,3′-diol (Lutein) from Bacillus endophyticus- AVP9
https://doi.org/10.21203/rs.3.rs-1398101/v1
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Bacillus
Biological activity
Characterization
Pigment
Lutein
Xanthophyl
Cancer
Natural sources have yielded over 750 different carotenoids with a variety of structures (Wang et al., 2006). Carotenoids with different molecular structures were produced by various sources ranging from bacteria to higher plants. Carotenoids have recently been linked to a slew of positive pharmacological benefits. As a result, determining the therapeutic potentials of various carotenoids attained more
prominence in the medical research. C40 carotenoids with skeletons made up of 40 carbon atoms, such as carotene, - zeaxanthin, lutein, cryptoxanthin, astaxanthin, fucoxanthin, and canthaxanthin, as well as the open chain carotenoid lycopene (Palumpitag et al.,2011; Vechpanich and Shotipruk., 2011; Alisa et al., 2009; Dias et al.,2009; Khachik et al., 1995; Khachik and Beltsville., 2001; Olmedilla et al., 2003). Lutein is a carotenoid that benefits human health by preventing eyesight loss caused by age-related macular degeneration (ARMD), Cardiovascular diseases, and malignancy (Piccaglia et al., 1998). Lutein has sparked increased curiosity as a result of these factors, owing to its potential in neutraceuticals and medicinal uses. Lutein can be found in a variety of forms in nature, with lutein fatty acid esters being one of the most common. Solvent extraction is the most popular method for obtaining lutein fatty acid esters (Kiyotaka et al., 2008). To acquire purified lutein for human health uses, a purification step is usually necessary. When purifying lutein, crystallisation is a common occurrence, although it results in low yield and purity. Re-crystallization can reach high purity, but it needs numerous processes, making the process more difficult and lowering the final yield (Craft and Soares et al.,1992; Molnar et al., 2004; Aman et al.,2005; Khachik et al.,1995).
Apart from the carotenoids produced from higher plants, and other sources, it is becoming difficult to find the natural source for producing unique carotenoids which is easily reproduced with minimum efforts. Bacteria are one such source to produce unique level of carotenoids is an urgent need of hour.
We have been trying to identify unique or unusual forms of carotenoids from orange pigment- producing bacteria that have been designated as rare or novel species using 16S rRNA analysis. As a result of this screening, lutein was isolated from Bacillus endophyticus AVP-9. The goal of this study was to extract, characterize, and test the antibacterial, anticancer activities of intracellular lutein from Bacillus endophyticus AVP-9.
Chemicals Reagents
All the chemicals and solvents which were used in the extraction by solvents, purification and characterization by chromatographic process is of high purity supplied by Sigma-Aldrich and hi-media.
Experimental Microorganism
Bacillus endophyticus AVP 9, an orange fluorescent phosphate solubilizing rhizobacteria isolated from chilli rhizosphere of Guntur District, Andhra Pradesh which was taken from research supervisor’s culture collection as subject microorganism in present study.
Extraction of intracellular pigment
Pre culture at the concentration of 1% v/v was inoculated 100 ml of sterilized nutrient broth incubated at 37oC for 2 days (Trivedi et al., 2017). The broth was centrifuged for 10min after the incubation in 4oC at 10,000 rpm. The cell-free supernatant was discarded after centrifugation and pellet were sonicated separately with different solvents such as 70% ethanol, 100% ethanol, chloroform, acetone, petroleum ether, 70% methanol, 100% methanol, and distilled water at a speed of 3 cycles per minute with a time span of 30sec (Prasanna Reddy and Rao 2009), and centrifuged for 15 min at 11,000 rpm. The colorless pellet was removed after centrifugation and colorful pigment supernatant (named as AVSR-2) was air dried and re-suspended in 1ml of DMSO and the absorbance was measured at 450 nm (Mitra, 2012).
Phase separation of lutein
An equal amount of hexane was added to acetone extract in a separating funnel and thoroughly mixed. An equal amount of distilled water and 1% w/v sodium chloride solution were added to separating funnel to facilitate organic and aqueous phase separation. The pigment was gradually transported to organic phase (hexane). In aqueous layer, acetone and water-soluble impurities were removed. The organic phase is rinsed four or five times with distilled water for complete removal traces acetone. The aqueous phase yellow pigment was treated with 9:1; v/v 1 N HCl and concentrated in a water bath at 40°C by solvent Meyer and Abdallah, (1978).
Purification
Thin layer chromatography
Thin layer chromatographic (TLC) plates covered with silica gel (TLC silica gel 60, 25 Aluminum sheets 20 x 20 cm, Merck, USA) are used to screen for suitable mobile phase. The extract was spotted onto TLC plates and then placed in the chamber with various mobile phase compositions of hexane and ethyl acetate. The different combinations of mobile phases were examined for better separation in day light (Perez-fons et al., 2011).
Procedure for packing and preparing column
In hexane, a 5g of silica gel was suspended. The suspension was then degassed overnight with the help of a sonicator. For analysis, the slurry was transferred to chromatographic column stabilized by allowing a solvent mixture of (1:2 v/v) ethyl acetate and n-hexane to pass through the column bed in a decreasing eluent flow, followed by 0.5 ml of extract solution in the semi-preparative glass column. The ethyl acetate: hexane (3:7 v/v) mobile phase was then allowed to flow normally. Fractions were collected at an interval of one-minute, air dried to get lutein and stored in a clean glass vial for the structural study (Latha and Jeevaratnam, 2010).
Characterization of pigments
Ultra violet-Visible spectrophotometry
UV-Visible light spectrum of the pigment was analyzed by in 3ml of hexane solution at a concentration of 1mg/ml and measured by using Shimadzu UV-1800 spectrophotometer (Shimadzu, Japan). A Perkin-Elmer LS 50 B luminescence spectrometer was used to investigate the compound's standardized excitation in the range of 200 to 600 nm. Narrow (1.0 mm) excitation slits were employed to ensure a monochromatic light source (Sujak et al., 2000).
High performance liquid chromatography (HPLC)
Pigment extract was analyzed by high performance liquid chromatography, a volume of 20µl at a concentration of 1mg/ml of pigment sample and a combination of pigment and lutein standard having 20µl of 1mg/ml (10µl each) were injected in to the system separately and read the absorbance at a wavelength of 455nm using mobile phase as a gradient solvent mixture of acetonitrile: methanol (9:1v/v) at 1 ml/min flow rate by an Agilent 1100 with a Lichrocart C-18 column (30cm length) (Aman et al., 2005; Kand et al., 2013).
Fourier transform Infrared spectroscopy
Infrared spectrum of pigment was collected using Potassium Bromide (KBr) pellets at 32 average scan rate on a Nicolet Magna-IR 380 spectrophotometer (Department of Physics, Acharya Nagarjuna University) (Andrew et al., (2002).
Liquid Chromatography-Mass Spectrometry (LC-MS)
Pigment were examined using an APCI temperature of 400°C with detection voltage of 1.6 kV and curved desolvation line of 250°C, and a block temperature of 200°C the positive ion measuring mode in Apex mass spectrum. The nebulizer had a gas flow rate of 2.5 ml/min. Scanning masses between 200 and 800 m/z yielded full scan spectra (Boonnoun, Opaskonkun and Prasitchoke, 2012).
Nuclear Magnetic Resonance Spectrometry
The structure of the pigment was characterized by using Varian INOVA model for both and H and C13-NMR analysis. Using a CP/MAS solid probe and a Nano probe, all spectra were obtained in CDC13 at 25°C (Boonnoun et al., 2012).
Biological-Activities Antibacterial activity
The purified pigment was dissolved in 3ml of DMSO at a concentration of 1mg/ml and tested for antibacterial activity using the agar cup technique against the human pathogenic bacteria such as Proteus vulgaris (ATCC 13315), Staphylococcus aureus (ATCC 15748) E. coli (ATCC 8739), Shigella flexneri (ATCC 12022), Salmonella typhi (ATCC 65154) and Micrococcus lutea (ATCC 10240). A molten agar was poured on to the sterilized petri plates previously seed with 0.2ml of 24hr young culture of test organisms and allowed to solidification. Wells were punctured by using 6mm of sterilized stainless steel borer and 50µ1 of pigment sample was loaded on to each well. 50µl of DMSO serves as a control sample and the plates were incubated for 24-hours at 37oC. Antibacterial activity was observed in terms of zone of inhibition around the loaded well (Banerjee and Chatterjee, 2011).
Antioxidant properties
The DPPH radical scavenging assay was used to determine the antioxidant properties of the isolated lutein from Bacillus endophyticus AVP 9. Using methanol, distinct pigment concentrations (0.25 – 10µg/ml) were achieved. 3 ml of freshly made DPPH methanol solution (40µg/ml) was mixed with 1 ml of each dilution and incubated in the unlighted area for 30 min at room temperature. The blank was pigment in 3ml of methanol without DPPH, and the standard was BHT (10µg/ml). The control was a methanol solution containing DPPH (without pigment), and the blank was pigment in 3ml of methanol without DPPH. The absorbance was measured using UV/VIS spectrophotometer at 517 nm (ELICO SL-210, India). Using the formula below, the percent inhibition of DPPH radicals was computed (Mohana et al, 2013).
Absorbance Blank
Anticancer activity Maintenance of cell lines
MCF-7 and LNCaP cell lines were bought from NCCS in Pune, India, and grown in DMEM medium
supplemented with 10% Fetal Bovine Serum and antibiotic streptomycin (0.5 ml-1) with 5 percent CO2 at 37°C temperature.
MTT Assay
The MTT test, which consisted of three separate evaluations with six centralizations of mixes in triplicates, was used to determine cell feasibility. Trypsinization of MCF-7 and LNCaP cells was performed, and tryphan blue readings were collected to determine the number of viable cell count. Using a hemocytometer, cells were counted and seeded at a thickness of 5.0 X 103 cells/well in 100µl media in 96 well plate culture medium, medium-term brooded at 37oC. Following incubation, the old media was replaced with new media containing 100µl of the test compound in reagent wells of 96 plates, with different test compound convergences. After 48 hours, discard the pharmacological arrangement and replace it with new media. MTT solution (0.5mg/ml) was poured into each well, and the plates were incubated at 37°C for 3 hours. The production of chromophore formazan crystals is aided by the conversion of MTT salt to chromophore formazan crystals by cells with metabolically robust mitochondria at the end of the incubation time. Using a microplate, the optical thickness of solubilized crystals in DMSO was determined at 570 nm. The portion reaction bends for each cell line were used to construct the centralization of test tranquillize estimated to repress cell development by half quality, and the following equation was used to determine rate development restraint (Venkanna et al., 2014; Linnewiel-Hermoni et al., 2015).
Percentage Inhibition = 100(Control-Treatment)/Control
Experimental organism
B.endophyticus AVP 9 (KF527823) is an orange fluorescent rhizobacillus isolated from chill rhizosphere by Nokku Pradeep Kumar and Amrutha .V. Audipudi., (2014) was selected as experimental organism in present investigation. B.endophyticus AVP 9 was cultured by quadrant streak method and colonies of pure culture are observed to appear in orange colour and emit bright orange fluorescence under U.V light at 350nm (Figure 1).
Extraction and phase separation of lutein
Pigment was extracted from broth using different solvent systems such as 70% ethanol, 100% ethanol, 100% chloroform, 100%, hexane, petroleum ether, methanol and distilled water (Figure 2). Initial standardization and percentage (%) recovery of pigment were analyzed at 450nm in terms of optical density (O.D). As O.D of pigment extraction was reported high in hexane (0.95) compared to other solvents (Figure 3). Hexane was considered as suitable solvent to yield maximum recovery of pigment. Hexane extract was subjected to dual phase separation using hexane and ethyl acetate. Pigment was resolved as hexane and ethyl acetate epiphase and soluble impurities as acetone and water hypophase. Hypophase with acetone and repeated wash with water help to nullify/remove soluble impurities and traces of acetone was removed. Dual resolution of yellow colored pigment was observed in epiphase. Pigment was resolved as yellow colour pigment in ethyl acetate and yellow colour pigment in hexane were collected and concentrated by rota evaporation at 40oC.
Thin layer and column chromatography
Different hexane: ethyl acetate ratios were examined for their effects. The mobile phase included a greater volume ratio of ethyl acetate, yellow spot of the sample was pushed upwards from the base line, as seen in (Figure 4). The best mobile phase ratio (hexane: ethyl acetate v/v) was discovered to be 70:30, resulting in the clearest separation. The ability to purify the lutein extract solution using a 70:30 v/v mixture of hexane and ethyl acetate was demonstrated in the preceding experiment. Two primary colour bands were seen on the 8 mm x 240 mm open column filled with silica and a 70:30 v/v combination of hexane and ethyl acetate. The yellow fractions eluted from the sample were mostly free lutein and its deteriorated form, as described above, and they accounted for a sizable portion of the total. Therefore the eluted fraction collected from this band air dried and characterized further.
UV-Visible Spectroscopy
U.V. Visible spectra of hexane dissolved AVSR 2 showed two major peaks at 360nm and 522 nm (0.8AU). Absorption spectrum of AVSR 2 did not show any excitation indicating that AVSR2 does not have any fluorescence property (Figure 5).
High performance liquid chromatography
HPLC chromatogram of purified pigment AVSR 2 resolved into 10 peaks at 455nm (Figure 6). At 455nm, AVSR 2 showed major peak with retention time of 3.528 and % peak area of 56.41, whereas a major peak of 3.611 was obtained when the purified pigment extract spiked with the lutein standard, suggesting that the purified pigment was similar to the lutein standard.
Fourier transform infrared spectroscopy
Fourier Transform Infrared spectrophotometer (FTIR) analysis of lutein shows a major peak is clearly visible at 1888.31 and –OH stretching band at 1463.336 is observed, and confirms the compound as lutein (Figure 7).
Liquid Chromatography
Mass spectral analysis of AVSR 2 showed a single major peak with mol.wt of 568.6g/mol at 7.324 min. Mass spectrometry represents a molecular weight of 568.6g/mol (M+H) +], with a molecular formula of C40H56O2 when compared to LC-MS result of luetin standard from NIST library (Figure 8).
NMR Spectroscopy
The 1H NMR spectra of lutein (CDCL3) exhibited the following characteristics: 1S/C40H56O2/c1 -29(17- 13-19-31) 1S/C40H56O2/c1-29 InChI (17-13-19-31) 1S/C40 = InChI (3) 21-23-37-33(5) 25-35(41) 30.15-11-12-16 27-39(37,7) 8) 30.15-11-12-16 (2) 18-14-20-32(4) 22-24-38-34(6) 26-36(42) 28-40(38,9)10/h11-25,35-37,41-42H,2628H2,1-10H3/b12-11+,17-13+,18-14+,23-21+,24-22+,29-15+,30-16+,31-19+,32-20+/t35,36+,37?/m0/s1. 10/h11-25,35-37,41-42H,26 28H2,1-10H3/b12-11+,17-13+,18-14+,23-21+10/h11-25,35-37,41-42H,2628H2,1-10H3/b12-11+,17-13+,18-14+,23-21+,10/h11-25,35-37,41-42H,2628H2,1-10H3/b12-11+,17-13+,18-14+,23-21+,10/h11-25 AVSR 2 was identified as Lutein (,-carotene-3,3'-diol) with the chemical formula (C40H56O2) based on the spectral features of 1H and C13NMR spectrum and the NIST database (Figure 9a and 9b).
Biological properties Antibacterial activity
Lutein shows highest inhibition against E. coli (35mm) followed by S. typhi (33mm), S. flexneri (30mm), P. aureginosa (29mm), M. lutea (28mm), P. vulgaris (24mm), S. aureus (21mm) and lowest antibacterial activity against V. cholera (19mm) (Figure 10).
Antioxidant and anticancer activity
Antioxidant activity of yellow pigment was concentration dependant. The inhibitory percentage of yellow pigment were determined to be 82% and the standard BHT was 95% in an antioxidant activity assay, and the results were compared to BHT> Yellow pigment (Figure 11). Yellow pigment with anti-proliferation action has a significant effect on MCF-7 and LNCaP cell lines. Yellow pigment was used to treat MCF-7 cells at a concentration of 100µg/ml, and cell viability was reduced to 36.363% at an IC50 value of 21.935µg/ml, and for LNCaP, cell viability was reduced to 42.449% when cells were treated with yellow pigment at a concentration of 100µg/ml, with an IC50 value of 56.937µg/ml (Figure 12).
Carotenoids are the most common pigment group produced by a wide range of organisms, including non- phototrophic prokaryotes and higher plants (Kim et al., (2007). Carotenoids are pigments that appear yellow, orange, or red in colour and are essential for the survival of non photosynthetic bacteria by protecting them from photo oxidative damage and allowing them to absorb visible light (Stafsnes et al., 2010; Luckner, 1990; Britton et al., 1995). Carotenoids are commercially employed as food colourants, animal feed supplements, nutraceutical, cosmetic, and pharmaceutical applications due to their biological qualities (i.e. antioxidant). Carotenoids are primarily extracted from plant tissues, microorganisms, or chemical synthesis in commercial manufacture. Because of the rising global market for these chemicals, microbial production of carotenoid pigments and diversity of carotenoid structures holds a lot of promise in terms of both production efficiency and diversity of carotenoid structures (de Haan et al.,1991; Vandammen, 1992; Buzzini and Martini, 1999; Buzzini, 2001; Frengova et al.,2003).
A culture collection of coloured bacteria can be a significant resource for isolating novel carotenoids with interesting features, particularly if the collection exhibits a wide range of carotenoid structures. Bacterial culture collections have the benefit of being simple to screen employing high throughput technologies, such as growing in well plate formats with robotic carotenoids extraction and quick resolution LC-MS analysis.
Bacillus endophyticus AVP 9 Kf527823, potential phosphate solubilizing rhizobacteria isolated from chilli rhizosphere is a gram positive, motile and rod shaped bacterium exhibited orange fluorescence under long wavelength UV light (Nokku Pradeep Kumar and Amrutha .V. Audipudi., 2014). B endophyticus. In present investigation, B. endophyticus AVP 9 was selected as experimental microorganism to characterize fluorescence pigment and its efficacy in pharmaceutical field.
Earlier studies reported that, yellow pigmented Bacillus strains isolated from human (HU16,Hu19), soil fields (SF147, SF188) and rice condiments (RKS469) and orange pigmented Bacillus sp isolated from human (HU 33,HU 36,HU 13, B. indicus sd/3 and B. cibi JG-30 shows high carotenoid extraction in organic solvents (Khaneja et al., (2010). Pink colored Bacillus strains SF 214 (FJ9777603) and Bacillus sp. (SF 237 GQ141979) show poor pigment extraction in organic solvents for both cellular suspensions and homogenization, however water soluble pigments were identified when extracted with French peas, according to the study Khaneja et al., (2010). In contrast, solvent extraction analyses of our results revealed that intracellular orange pigment of B. endophyticus AVP 9 was water insoluble and best extracted in acetone and hence acetone was selected as an ideal solvent.
Perez-fons et al.,(2011) reported that the separation of yellow and orange pigments from aerobic spore formers isolated from soil sample Rf values were of 0.95 and 0.91 were obtained using a TLC method, showing that the orange pigment was more polar. In present research, AVSR 2 pigment of B. endophyticus AVP 9 was separated by phase separation of solvent followed by TLC to yellow pigments with Rf value 0.49 using hexane: ethyl acetate (7:3) as solvent systems respectively (Fig. 12). Similar results were reported by Vora et al, (2014) reported that yellow pigments of S. marcescens separated into yellow bands with Rf value 0.47 in TLC profile. These findings support that yellow pigments varied in polarity ranging from polar to non polar. Sujak et al, (2000) also reported that absorption maxima of The monomeric and aggregated forms of zeaxanthin and lutein have absorption peaks at 450nm and 445nm, respectively. The ultra violet and visible spectra of B. endophyticus AVP 9 revealed a strong absorption peak at 522 nm. Because of its fine structure between 400 and 500nm, its UV/Vis spectra suggest that AVSR 2 is lutein and zeaxanthin. Between 400 and 500nm, the absorption maxima of (all-E)-lutein, (9-Z)-lutein, and (90-Z)-lutein showed hypsochromic shifts of 5nm and 4nm, respectively, and the absorption maxima of (13-Z)-lutein and (130-Z)-lutein showed shifts of 8 and 6nm, respectively (Aman et al., (2005). Two -carotenes with retention periods of 2.99 min-1 were identified as trans- -carotenes and retention times of 3.24 min-1 were identified as the cis- isomer of - carotene in isolated bacterial symbionts from soft corals (Ariska et al., (2017). A cis-subsidiary isomer's peak in the near ultraviolet (349nm) is a distinguishing feature. With retention durations of 2.99 min-1 and 3.24 min-1, bacteria produce essentially identical types of carotenoids, indicating that the pigment belongs to the carotenoid family.
Khachik’s et al.,(1997) Within 8.0 minutes, marigold flower lutein was divided into (13-Z)-lutein, (130-Z)-lutein, (all-E)-lutein, (9-Z)-lutein, and (90-Z)-lutein. Human retina extracts have also been found to have identical isomers. Aman et al., (2005) used methanol and water to separate the
xanthophyll stereoisomers (13-Z)-zeaxanthin and (all-E)-lutein. Similarly, one prominent peak at 3.5 minutes in HPLC analysis of AVSR 2 of B. endophyticus AVP 9 corroborated the presence of lutein in the current study. In LCMS analysis, AVSR 2 of B. endophyticus AVP 9 revealed the presence of a chemical with a mass of 568.6g/mol. Boonnoun et al., (2012) reported two compounds free lutein and anhydrolute in respective to the mass of 568 and 551 g/mol respectively. Analysis of non fluorescent pigment by Gupta et al(2015) reported nine peaks of which the most prevailing compound in terms area was β, ε-3, 31-diol (lutein).
Aman et al., 2005 purified free luetin sample evaluated by 1H-NMR after chromatographic purification showed a peak pattern comparable to that of Aman's and Khachik's study. The purified free luetin sample comprised of just free luetin, free lutein's stereoisomer's, and anhydrolutein, according to the results.
AVSR 2 of B. endophyticus AVP 9 shows the absorption from 786.96 cm_1 to 3630.03 cm_1 and the major peak is clearly visible at 1888.31 cm_1 and –OH stretching band at 1446.61 cm_1. It clearly confirmed that AVSR 2 compound is a lutein. Similarly Bhattacharyya and Dhar, (2016) reported the absorption spectra of marigold derived lutein shows peaks at 721.913 cm_1, 1877.095 cm_1, 1453.336 cm_1 and 2854.000 cm_1. Presence of ester bond at 1742.043 cm_1 and absence of –OH stretching band at 1463.336 cm_1 indicates the presence of lutein ester and lutein respectively. B. endophyticus AVP 9 showed a significant antibacterial activity against pathogenic bacteria. AVSR 2 showed maximum inhibition against AVSR 2 against E. coli (35mm). Zhao et al., (2016) reported that antibacterial activity of orange pigment with the zone of inhibition 5.0mm to 23.0mm against the both gram negative and gram positive bacterial pathogens. Banerjee and Chatterjee, (2011) reported that only fraction C3 of B. cereus M116 (MTCC 5521) showed potential antibacterial activity against B.subtilis, B. cereus, K. pneumoniae,
M. lutea and S. aureus and where as no inhibitory effect was shown on E. coli, S. typhi, P. aeruginosa, A. niger, and S. cerevisiae.
Keceli et al., (2013) The antioxidant activity of carotenoid extracts obtained from 20 different R. glutinis strains was investigated, and it was discovered that none of the carotenoid extracts obtained from M 26, M 31, M 37, and LM 40 strains of R. glutinis were active as antioxidants, inhibiting AAPH-induced oxidation and exerting an antioxidant character when compared to the control. The antioxidant activity of
B. endophyticus AVP 9 AVSR 2 is significant, with IC50 values of 3.5 mg/ml and 4.5 mg/ml, respectively. The IC50 values of red and yellow carotenoids extracted from micrococcus sp isolated from soil were reported to be 3.5 mg/ml and 4.5 mg/ml, respectively, according to Mohana, 2013.
Rezaeeyan et al., 2017 reported the anticancer activity of carotenoids isolated from Kokuria marine QWT 12 with five gradient concentration levels of carotenoid pigment against breast cancer (MDA-MB-468, MDAMB-231, and MCF-7), prostate cancer (DU145, PC3, LNCaP), and lung cancer (A549), shows significant IC50s of 1, 4, and 8 mg/ml of extracted pigments against MCF-7, A-549, MDA-MB-468, and MDA AVSR 2 of Bacillus endophyticus AVP 9 has an IC50 value of 21.935µg/ml against MCF-7 cell lines and 56.927µg/ml against LNCap cell lines, indicating that, yellow pigments have significant anti- proliferative effect against all cell lines examined.
Based on the findings of this study, it can be concluded that the bacterium B. endophyticus AVP 9 has a lot of potential for producing carotenoid-like pigments in its native environment. The findings on B. endophyticus AVP 9 suggest that this pigment could be developed as a therapeutic agent. Bacillus pigments are produced by numerous species of Bacillus and are widely known for their medicinal applications. Further research into the pigment's protein binding capacity may provide more information on the pigment's property. This is the first report to our knowledge on the extraction and characterization of yellow orange carotenoids such as lutein from B.endophyticus AVP9. This research also underlines the potential of this pigment as an antibacterial and anticancer agent in the pharmaceutical industry.
Conflict of interest
Authors declare no conflict of interest
Acknowledgements
The third author is thankful for Department of Science and technology, Ministry of Science and Technology, Government of India, New Delhi, under Inspire Fellowship JRF (No. DST/INSPIRE Fellowship/2015/IF150034) for financial assistance.
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