INTRODUCTION:

Pigments are ascribed to dwell as indispensible compounds in textile, food, pharmaceutical and printing industries^1-3 In comparison with the synthetic chemical based pigments naturally derived pigments are characterized by attractive attributes such as safety, lower toxicity, bright and natural colors, prolific nutrition and pharmacological traits⁴. Bio-pigments are naturally obtained from microbes, plants and animals. Howbeit, the reproductive and growth processes of these flora and fauna can be affected by the origin, season, climate and other criteria, projecting into a scenario of inadequate raw material.

Hence, the extraction of proficient volume of pigments from these sources is an expensive process. However, on the contrary microbes attract the recent attention of industrialists and scientists for their eminent characteristic features viz. cost effectiveness, requiring meagre raw material, higher outturn of products. Thus, channelizing a streamline for industrial scale production⁵. Modernisation in biotechnology has established and contributed a safe, diverse and reliable production of microbial pigments by fermentation technology. Marine actinobacteria are splendid sources of secondary metabolites for wide range of commercial applications^1,6,3,7. However, the exploration of actinomycete pigments from widespread arena and ecological springs is highly mandate for enhancing and extending their multifaceted biotechnological scopes. Amidst the broad channel of pigments, carotenoids have attracted recent attention from the industrial arena.

Carotenoids are natural pigments affiliated to terpenoids subfamily containing fourty atoms of carbon with eight units of isoprene and are comprised of orange, yellow, and red colors with the broadly complexed polyene chain. This structural trait is amenable to their functional activity as a provitamin A- nutrient and antioxidant with their potentiality to defend from UV rays. Carotenoids can also contribute to regulation of genes and programmed cell death or apoptosis in mammals⁸.A metabolic processes indispensable for maintainence of animal health. Due to these biological⁹. The carotenoid global market is dwelled to reach 1.5 billion dollars in the year 2018 and will exceed to a value of 2.0 billion dollar in the year 2022. Inspite of the fact that natural carotenoids demand is rapidly higher, the supply of these carotenoids is inadequate owing to the inefficient method of production¹⁰. Carotenoid pigments are commonly derived from natural means and sources with meagre outturn owing to their complexed and multi-processesing strategy and meagre concentrations in raw materials, which is influenced by undesirable environmental ambience¹¹. Hence the carotenoids production by microbes is an striking alternative, as it saves the time involved in extraction of pigments after their growth and harvest.

Owing to the higher cost of current industrial pigment production processes, there emerges a need to develop a robust and low-cost processes replacing the other synthetic ones. In recent years, appreciable research has been projected on the conversion of agro waste – a cheap and abundantly available substances – into valulable products via an economically feasible system¹². To discern the effect of distinct agro wastes such as Corn cob, ground nut shell, soybean meal and pumpkin kernals were screened out for recruiting the best substrate aiding the pigment production. Actinobacteria are competent to produce diverse colored biochromes as noteworthy antimicrobial and antioxidant compounds. A plenty of these bio-pigments are reported to be intracellular compounds and the reports on the extracellular bio-pigments from marine mangrove ambience is very few. Hence, this study targets to unravel the unknown potency of extracelluar yellow pigmental metabolic activity of S. coelicolor strain SPR7 as promising candidate for dwelling as antibacterial and antioxidant drug compounds by in vitro appraisals.

Bioeconomic strategy is an highlighting vision for natural compound production and bioremediation¹³. Making a natural compound, to be a marketable drug would be completely possible by amalgamation of in vitro conformation. These affinities poses up an interest to explore broadly on pharmacological arena integrating the analysis of drug properties in preliminary stages of drug discovery Interestingly, the yellow pigment of S. coelicolor strain SPR7, was also investigated to be dying agent for cotton fabrics. There are wide range of scientific reports attesting, on microbial pigments produced by fungus, bacteria and plants as robust dye amenable to textile dying prospectives and usage of mordents to impart color into the cloths is quite common in the dying processes^14-16. Howbeit, this study targeted to impart the natural color of the yellow pigment into the cotton materials in the absence of mordents with enhanced color fastness.

The assessment and identification of microbial pigments is necessitous to explore novel sources of bioactive metabolites for all these applications. Recently, several biological analysis have been carried out with multifarious analytic approaches such as UV-Visible spectroscopy and FTIR for the microbial pigmental metabolites¹. In the current study, the techniques such as UV-Visible and FTIR analysis were collaborated to discern the functional groups and nature of the active metabolites present in the yellow bio-pigment sample from S. coelicolor strain SPR7. Hence, with all these strategies it is highly evidential that the isolated yellow bio-pigment would be a compound of interest attracting the attention of pharmaceutical and textile industries in the future, post to executing further scientific investigations.

MATERIALS AND METHODS:

Sample collection, Isolation and fermentative conditions:

The sediment samples were collected from Jaladi mangroves, Kundapura, Karnataka, India. Sterile polythene bags were used for sample collection and transported under the controlled storage conditions. Actinomycetes isolation was accomplished by using different media viz. Starch casein agar, actinomycetes isolation agar and International Streptomyces Project 2 medium^17-19. Ultimately, starch casein broth (SCB) was recruited for further studies. Molecular characterization was performed by 16SrDNA sequencing and the respective sequence was deposited in GenBank with the accession number (MH712067).

Influence of distinct agricultural wastes on biochrome production:

To enhance the pigment production, broad panel of experiments for optimization were performed using classical strategy, which comprised of changing a single factor at a time and at pre-defined levels remaining factors are retained constant. To discern the effects of distinct agricultural wastes on pigment production, the classical method was followed. The raw agro based wastes such as corn cob and ground nut shells (carbon sources); soybean meals and pumpkin kernals (nitrogen sources) were collected from the local industries. The collected agro wastes were ground into tiny particles (3mm). The response of these wastes were scrutinized for the enhanced bio pigment production. Carbon and nitrogen sources evincing high productive outturn were selected as the productive carbon and nitrogen sources for the fermentation^20-22.

Extraction and purification of biochrome by column chromatography:

The pigment producing actinomycete, VITJM7 was inoculated in 500 ml erlenmeyer flask in 250 ml of the production medium comprising of corn cob and soybean meal as carbon and nitrogen sources replacing starch and casein in SCB. The culture was incubated at 28ºC for 98 h²³and the crude supernatant was used for the extraction of extracellular pigments to perform further analysis. The pigment was extracted by distinct polarity solvent viz. ethyl acetate, chloroform, methanol and n-butanol in 1:1 ratio (W/V). Post to extraction the solvent was removed by rotary evaporation, whereby the resulting residue was dissolved in methanol of analytical grade. The extracted crude pigment was subjected to silica gel G60 chromatography (70-230 mesh) column (40 mm x 400 mm) by hexane: methanol (1:3) solvent elution system. Post to the elution process, 12 fractions were collected. All the fractions were assayed for their antibacterial traits against the strains viz., Escherichia coli, Bacillus subtilis and Staphylococcus aureus. The fractions exhibiting efficient antibacterial activity were pooled and subjected to purity analysis by TLC with solvent systems of chloroform: methanol: distilled water (2:10:1) and ammonia: ethanol: distilled water (1:8:1). The bands were visualized by UV light and the R_f factors were determined²⁴. The purified pigment was used for further characterizations and applications.

Characterization of biochrome:

The UV-Visible Spectroscopic analysis was performed to discern the λmax of the bio-pigment. The purified pigment was subjected to spectral analysis in the wavelength range of 200-800nm by UV-visible spectrometer (Evolution 300UV-VIS spectrophotometer, Thermo Scientific).The FT-IR spectra of the pigment sample was characterized to unravel the presence of functional groups. The sample preparation was performed as described previously. The measurements were recorded in the range of 400 to 4000 cm^-1 with a resolution of 4 cm^-1 by PerkinElmer (spectrum RX I).

Applications of biochrome:

Antioxidant Assay:

The applicability of the bio-pigment to dwell as a potential antioxidant was scrutinized by DPPH (2, 2-diphenyl-1-picrylhydrazylradical) scavenging assay²⁵. The purified pigment sample was prepared in distinct concentrations (20-100 µg/ml) by Milli- Q water and prepared sample of 2 ml was added to 1ml, DPPH solution (0.2mM). The assay mixture was subjected to incubation for 30 min in dark at room temperature. Post to incubation timeline, the absorbance was recorded at 517nm by UV-visible spectrometer (Evolution 300UV-VIS spectrophotometer, Thermo Scientific). Ascorbic acid and methanol were used as the positive control and blank solution. The scavenging percentage was measured as follows:

Ac-At

DPPH activity = --------------- X 100

Antibacterial activity of bio-pigment:

The pigment was also tested for antibacterial activity by well diffusion method. Three strains (Escherichia coli, Staphylococcus aureus, Bacillus subtilis) were used against the bio-pigment to estimate its antibacterial property. The cultures were plated on autoclaved Muller Hinton agar plate by spread plate technique and incubated for 48hrs at 28ºC. After an incubation of 48 hrs, the zone of inhibition was recorded against the organisms to scrutinize the antibacterial activity of the yellow pigment. The well added with the methanol dwelled as the control for reference²⁶.

Fabric dying:

The colored pigment extracted from the isolate, SPR7 was recruited for dying absorbent cotton cloth. The dying process was performed according to the protocol proposed by Hernandez et al. without any mordanting pre-treatment Post to dying, the samples were washed by boiling in lissapol for 5min and rinsed with cold water¹⁵.

RESULTS AND DISCUSSION:

Isolation and Identification of potent isolate:

The sediment samples were collected from the Jaladi mangroves, (Karnataka, India) and out of 20 different actinobacterial isolates only the strain VITJM7 produced extracellular yellow pigment with whitish cellular biomass in the culture. Out of all three media used starch casein, enhanced the number of actinomycete isolates. BLAST analysis bioinfomatics tool displayed highest similarity (98%) with Streptomyces coelicolor. The molecular phylogenetic tree was generated, aiding the identification of the potential isolate as S. Coelicolor strain SPR7. As a typical strategy, there area other species of Streptomyces taxas such as Streptomyces fradiae, Streptomyces torulosus and Streptomyces bellus^27-29.

Effect of distinct agricultural wastes on biochrome production:

The effect of tropical agro-industrial residues such as corn cob, ground nut shell, soybean meal, pumpkin seeds was assessed for selecting the luxuriant substrate aiding the pigment production. In the midst of carbon sources, Corn cob dwelled as the most apposite agro-industrial based residue accounting to the highest significant production of pigments (1.8 OD units/gds) by SPR7 strain, in compared to the other tested carbon source, ground nut powder (Fig 1a-d). A typical strategy has been well enlightened²¹, who recounted a significant influence of corn cob in the production of Monascus red and orange pigments by a red yeast, Monascus purpureus. In the context of nitrogen sources, soybean meal evinced a higher outturn scale of pigment (2 OD units/gds) at the dual wavelengths of 410nm and 495nm in contrast with pumpkin kernals (Fig 1e-h). Interestingly, a yellow bio pigment generated by Thermomyces sp. displayed the similar productive yield by the incorporation of soybean meal and soy peptone in the fermentation media³⁰. These results clearly emphasizes the indispensible contribution of agro based wastes for the proficient production of secondary metabolites like pigments and showcasing the bio-based synergy between the agro wastes and microbial productivity.

Characterization of purified biochrome:

The λmax of the purified yellow pigment was observed at 410nm and 495nm with absorbance 2.05 and 1.46 respectively (Fig. 2). Several other taxas of marine bacteria have showed a similar results of the UV-visible spectrum with maximum absorbance at 430, 451and 480 nm signifying the presence of distinct types of pigment in the purified sample³¹.

Fig. 1a-d 3D surface plots of carotenoid pigment production by agro-based carbon sources, a) and b) Corn cob; c) and d) Ground nut shell powder.

Fig. 1e-h 3D surface plots of carotenoid pigment production by agro-based nitrogen sources, e) and f) soybean meal; g) andh) pumpkin kernal.

The FT-IR spectrum was scrutinized correspondingly for all the values of standard (Fig. 3). A broad stretching peak at 3389.67 cm^-1 for Symmetrical Hydroxyl groups stretching (-OH), 2928.35 for methyl groups (-CH) stretching vibrations, 2636.72 and 2210.63 cm⁻¹ for asymmetrical C-H stretching. Peaks for CO and CN groups were observed at 1636.75 cm^-1 and 1738.61cm^-1. Peak at 1999.84 and 1555.84 cm^-1attributed for characteristic C=C and N=N stretching.1316.28 cm^-1for C-OH stretching, 1038.25 and 1021.34 cm^-1 for C-H stretching. These bands were consistent with spectral vibrations obtained for microbial pigments recorded earlier³².

Fig. 2: UV- VIS spectral profiles of carotenoid production from Streptomyces coelicolor SPR7.

Fig. 3: FT-IR spectra of carotenoid production by Streptomyces coelicolor strain SPR7

APPLICATIONS OF YELLOW BIOCHROME:

Anticancer and Antimicrobial analysis of pigment:

Oxidative stress can be induced majorly by the environmental related factors viz. smoke, ozone and UV-radiation. Metabolic processes is amenable to cause oxidative stress in every cell. Antioxidant potentiality of an antioxidant compound can be ascribed to its ability to scavenge the reactive oxygen species (ROS) or free radicals present in the surroundings. A light sensitive colored chemical compound, DPPH (2,2-diphenyl-1-picryl-hydrazyl) is composed of free radicals with higher stability. The scavenging potential of any compound is defined by its competency to liberate these ROS or free radicals into hydrogen ions³³.

The purified pigment of S. coelicolor strain SPR7 was subjected to antioxidant activity test by virtue of DPPH assay. The scavenging proficiency of purified pigment with DPPH radical was estimated, where a dose influenced increase in the DPPH scavenging profile was observed. The competency of the pigment to scavenge hydroxyl radicals is assigned to the presence of characteristic -O- and –OH- as functional groups in their structures. In comparison with the ascorbic acid (standard), proficient activity was evinced by the pigment sample (Fig. 4). Melanin pigment isolated from Thar desert actinobacteria, Streptomyces girseorubiginosus attributed to a similar valuable antioxidant activity³⁴.

The purified pigment exhibited proficient activity against Staphylococcus aureus. The activity against gram negative bacteria, E. coli was the least; while the zone of inhibition observed against the taxa, Bacillus subtilis was higher in comparison to E. coli (Table 1). The pigment- like antibiotics produced by distinct actinomycetes from Rabat neighbourhood soil, revealed a similar consistent results to that of S. coelicolor strain SPR7²⁷. A typical antibacterial investigation has also been established for actinobacterial genera such as Strepto sporangium from Algerian sediments³⁵.

Fig. 4: Scatter plot depicting anticancer activity of carotenoid pigment and ascorbic acid (Standard).

Table 1: Elucidation of antibacterial activity of purified pigment from Streptomyces coelicolor strain SPR7 against various organisms, expressed as zone of inhibition.

Test bacteria
Zone of inhibition [mean (mm) ± SE]	Escherichia coli	Staphyllococcus aureus	Bacillus subtilis
Zone of inhibition [mean (mm) ± SE]	9.0 ± 0.05	20.1 ±0.17	15 ± 0.09

Fabric dying:

Textile industries project a search for exploration of alternative source for synthetic pigments and hence, the natural colorant compounds produced by microbial sources are safe and cost effective with natural and bright coloring features⁴. The dying traits of the extracted pigment, was tested with distinct types of the textile materials. The textile stuffs were subjected to normal water wash and yellow color of the pigment imparted were retentive by distinct types of textile cloths. Cotton cloths were tested to scrutinize the coloring capacity of the yellow pigment (Fig. 5). Interestingly, the dying process did not require any mordant or fixative as required in conventional dying process. Thereby, it is unerring to propose that the extracted yellow pigment from S. Coelicolor strain SPR7 has prolific fastness and color retentive property inspite of the continuous water wash (thrice). The interactions between textile cloths and natural microbial pigments has been well established in previous reports^6,15. Hence, microbes are highly potentialistic to produce pigments³⁶.

Fig. 5: Fabric dying of cotton cloth by carotenoid pigment a) cotton cloth without carotenoid b) yellow colored cotton cloth post to carotenoid treatment.

CONCLUSIONS:

Pigments are indispensible compounds for multifaceted applications in textile, cosmetics, food and pharmaceutical industries. In comparison to the synthetic pigments, naturally produced pigmental or biochrome colorants are of higher scope due to their non-toxicological and pharmacological attributes, bright coloring, natural fastness and safe to usage. In the current study, an interesting and noteworthy pigmental secondary metabolite from an actinobacterial isolate S. coelicolor strain SPR7 evinced as a yellow pigment was successfully extracted, purified and used to achieve antioxidant, antibacterial and. Moreover, it is a remarkable attribute that beta carotene was non carcinogenic or non tumorigenic. Interestingly, the competency of the biochromes to dwell as a outstanding dying agent is a supplementary for encouraging the global market of textile industries in the upcoming period of demand. The yellow pigment, imparted the cotton fabrics a pleasant color, supporting its requirement in the industrial fields and prospectives. Advanced research to further explore and obtain the commercial grade colorants from S. coelicolor strain SPR7 with chemical and biomolecular characterization would channelize a new avenue in the commercial sectors to support the minimization strategies of noxious effects of synthetic dyes and colorants; thereby retaining a sustainable ecological environment which is away from these chemical dyes and pigment.

CONFLICT OF INTEREST:

There is no conflict of interests among the authors.

ACKNOWLEDGEMENTS:

The authors are thankful the management of Vellore Institute of Technology, Vellore for providing the necessary facility to carry out this work.

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Received on 22.02.2022 Modified on 30.12.2022

Research J. Pharm. and Tech 2023; 16(12):5958-5963.

DOI: 10.52711/0974-360X.2023.00967