Exploration of the Bioactivity of Pigmented Extracts from Streptomyces Strains Isolated Along the Banks of the Guaviare and Arauca Rivers (Colombia)

Abstract

Pigments are chemical compounds that impart color through mechanisms such as absorption, reflection, and refraction. While traditional natural pigments are derived from plant and insect tissues, microorganisms, including bacteria, yeasts, algae, and filamentous fungi, have emerged as promising sources for pigment production. In this study, we focused on pigment production by 20 Streptomyces isolates from our in-house actinobacteria strain collection, sourced from the Guaviare and Arauca Rivers in Colombia. The isolates were identified via 16S rRNA gene sequencing, and the bioactivities—including antioxidant, antibacterial, and cytotoxic properties—of their extracts obtained across four different culture media were assessed. Promising pigmented hydroalcoholic extracts demonstrating these bioactivities were further analyzed using LC-MS, leading to the annotation of a variety of pigment-related compounds. This study revealed that culture media significantly influenced both pigment production and bioactivity outcomes. Notably, anthraquinones, phenazines, and naphthoquinones were predominant pigment classes associated with cytotoxic and antimicrobial activities, while carotenoids were linked to antioxidant effects. For instance, S. murinus 4C171 produced various compounds exhibiting both cytotoxic and antioxidant activities. These findings highlighted a growth medium-dependent effect, as pigment production, coloration, and bioactivity outcomes were influenced by growth media. These results demonstrate the significant potential of Streptomyces isolates as sources of bioactive pigments for diverse applications.

1. Introduction

Pigments are colored compounds that should not be physically or chemically affected by the medium in which they are incorporated [1], and they have been used since ancient times by civilizations such as China, India, and Egypt [2]. Currently, they are widely employed in various industries, including food, textiles, paints, cosmetics, pharmaceuticals, and plastics [3]. The global market for natural pigments in the food industry is estimated to reach USD 2.5 billion by 2025 [2]. Pigments can be classified, according to their origin, as natural or synthetic. Natural pigments are further categorized based on their specific origin (plant, mineral, microbial, or animal), color formation, chemical constitution, and application method. Examples of natural pigments include carotenoids, melanins, chlorophylls, flavonoids, anthocyanins, betalains, and quinones. Synthetic pigments, on the other hand, are generally classified into cationic, anionic, and nonionic types, and by their applications as direct, acidic, basic, chrome mordants, azo, sulfur, reactive, and dispersed [4].

Despite their origins, pigments can be harmless or toxic to some degree. However, consumers often prefer natural pigments because they are considered safe, non-toxic, non-carcinogenic, and biodegradable [5]. In contrast, synthetic pigments can pose serious human health hazards and environmental risks [3,6] owing to their non-biodegradable nature [7]. They can cause adverse effects [8], such as decreased light penetration in water bodies [9] and increased chemical and biochemical oxygen demand (BOD and COD), and some are bioaccumulative, toxic, mutagenic, or carcinogenic [10], even at very low concentrations.

Natural pigment production is widespread among plants, microalgae, fungi, bacteria, and insects. Among bacterial groups, pigment production is particularly notable in actinobacteria [11], which is highly profitable and biotechnologically valuable [12,13]. Actinobacteria produce metabolites with unique chemical structures and diverse bioactivities, including antibiotics, biopesticides, phytohormones, antitumor compounds, antiviral agents, enzymes, enzyme inhibitors, anti-inflammatory agents, and biosurfactants [14,15,16]. Streptomyces, in particular, is known to produce a large number of specialized metabolites with different properties [13,17,18,19]. In this regard, several genera such as Streptomyces, Nocardia, Micromonospora, Thermomonospora, Actinoplanes, Microbispora, Streptosporangium, Actinomadura, Rhodococcus, and Kitasatospora [20] include microorganisms that produce a wide variety of pigments. The most reported pigment-producing genus is Streptomyces, including species such as S. griseus, S. griseoviridis, S. coelicolor [21,22], S. cyaneus [23], S. vietnamensis [24], S. echinoruber [25], S. shaanxiensis [26], and S. caeruleatus [27].

Microbial pigments are emerging as alternatives to synthetic pigments, offering several advantages such as abundant raw materials independent of season, high yields, higher concentrations with simpler purification processes, regulatory approval, high stability to environmental factors [6,28,29,30,31], and biodegradability [32]. Additionally, some microbial pigments possess antioxidant, anti-inflammatory, anticancer, and antimicrobial properties [30,33], with potential applications in food colorants, nutraceuticals, and cosmetics [32].

The bioactive properties of pigments derived from actinomycetes have attracted significant attention due to their potential applications in medicine and industry. These pigments exhibit a wide range of activities, including antimicrobial, antioxidant, and cytotoxic effects, making them valuable for various uses. For instance, pigments from actinomycetes like Gordonia terrae have demonstrated strong antimicrobial activity against Gram-positive bacteria and fungi, including Bacillus subtilis and Candida albicans [34]. Marine actinomycete strains VES 01 and VES 04 also exhibited notable antibacterial effects against Escherichia coli and B. subtilis, with significant cytotoxicity observed in the Brine Shrimp Lethality Test [35]. Additionally, pigment extracts from G. terrae and marine actinomycetes have shown antioxidant activity, with IC₅₀ values supporting their potential as natural antioxidants [34]. Studies on Streptomyces strains further revealed their ability to produce bioactive pigments with anticancer and antioxidant properties, underscoring their biotechnological significance [36]. Beyond scientific applications, actinomycete pigments have also been explored in non-scientific fields, such as painting, highlighting their versatility [37]. Their potential for eco-friendly applications, including fabric dyeing, has also been demonstrated [38]. While the bioactive properties of actinomycete pigments are promising, further research is required to fully elucidate their mechanisms of action and to optimize their production for commercial use.

Actinobacteria are widely distributed across diverse natural ecosystems, including soil, plants, limestone, freshwater, seawater, sponges, volcanic caves, deserts, air, and insects [16]. In recent years, studies focusing on actinobacteria from unexplored or underexploited habitats as new sources of specialized metabolites have increased [13]. Colombia, recognized for its megadiversity in fauna and flora, also harbors a rich diversity of microorganisms [39]. In the present study, two rivers located in tropical zones were selected as sources of actinomycetes. These rivers have average temperatures ranging from 25 °C to 30 °C and receive substantial solar radiation throughout the year, supporting rich biodiversity and a variety of ecosystems. However, little is known about the biodiversity in these watersheds, particularly their microbiota. Given this context, we hypothesized that the unique environmental conditions of these tropical rivers might induce the production of diverse pigment-related metabolites in Streptomyces strains growing along the riverbanks. We further posited that the pigmented extracts from these strains could exhibit multifaceted properties due to the presence of bioactive specialized metabolites. Therefore, the aim of this study was to explore the biological activity of pigmented extracts from Streptomyces isolated from the Guaviare and Arauca rivers in Colombia, focusing on their antimicrobial, antioxidant, and anticancer properties, in support of the further hypothesis that these strains could lead to the discovery of novel bioactive compounds.

Consequently, pigmented ethanolic extracts were obtained and evaluated for their antioxidant capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, antibacterial activity by the disk diffusion assay, and cytotoxic activity by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against human dermal fibroblasts (HDFas), cervical–uterine cancer (HeLa), and MCF-7 (breast cancer) cell lines. Therefore, the aim of this study was to explore and characterize the bioactive properties of pigmented extracts of selected Streptomyces strains isolated from these unique Colombian habitats. By assessing their antioxidant, antibacterial, and cytotoxic activities, this study seeks to identify promising sources of natural pigments with potential applications in various industries, particularly cosmetics and pharmaceuticals. This study contributes to the growing field of natural product chemistry and highlights the significance of Actinobacteria as sources of novel bioactive agents and valuable resources for biotechnological advancements.

2. Materials and Methods

2.1. Reagents and Culture Media

The components and culture media used were as follows: agar (DIBICO, Santa Cruz, Mexico D.F., Mexico), starch (Carlo Erba, Eure, France), glucose, CaCO₃, K₂HPO₄, (NH₄)₂SO₄, CuSO₄.5H₂O, FeSO₄.7H₂O, KNO₃, MgSO₄.7H₂O, MnCl₂.4H₂O, ZnSO₄.7H₂O (PanReac, Barcelona, Spain), yeast extract (OXOID, Hampshire, UK), malt extract (CDH, New Delhi, India), NaCl (Honeywell, Seelze, Germany), KH₂PO₄ (DUKSAN, Kyungkido, Korea), and TSA (Criterion, Santa Maria, CA, USA). DPPH, ABTS, trolox, methanol, and ethanol were purchased from MilliporeSigma (St. Louis, MO, USA). MTT was acquired from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

2.2. Culture Preparation

2.2.1. Streptomyces-Related Strains

The Bioprospecting Research Group at Universidad de La Sabana has a collection of 380 isolates from sediments along the Guaviare riverbank and 780 isolates from the Arauca riverbank, all exhibiting Streptomyces-like morphological characteristics. These isolates have been cultured, isolated, and cryogenically preserved at 1 × 10⁹ spores/mL and –80 °C in ISP2 medium supplemented with 40% glycerol. A subset of 20 pigmented isolates was reactivated on semi-solid isolation medium across four different culture media (see

Table 1. Composition of the culture media used (g/L) [40,41,42].

Composition/Medium	ISP2	ISP4	Modified ISP9	Starch Nitrate
Agar	15	15	15	15
Starch	0	10	0	20
CaCO3	0	2	0	3
CuSO4.5H2O	0	0	0.00064	0
yeast extract	4	0	0	0
malt extract	10	0	0	0
FeSO4.7H2O	0	0.01	0.00011	0.01
Glucose	4	0	10	0
K2HPO4	0	1	5.65	1
KH2PO4	0	0	2.38	0
KNO3	0	0	0	2
MgSO4.7H2O	0	1	1	0.5
MnCl2.4H2O	0	0.001	0.00079	0
NaCl	0	0.5	0	0
(NH4)2SO4	0	2	2.64	0
ZnSO4.7H2O	0	0.001	0.00015	1

). For this, 100 µL of spore suspension (pH 7) was massively seeded onto the media and incubated at 30 °C for 7 days (Friocell, Querétaro, Mexico). Once the strain fully colonized the agar surface, 1 cm² sections were used to initiate liquid cultures.

Media in which the isolates failed to grow or produce pigment were discarded and re-evaluated using liquid culture. Hence, a 1 cm² section was cut from each solid medium and transferred to 3 mL of the corresponding liquid medium in 15 × 1 cm glass test tubes. These cultures were then incubated at 30 °C for 7 days with agitation at 150 rpm (New Brunswick Scientific, CT, USA). This culture (1 mL) was subsequently mixed with fresh medium (9 mL) and incubated under the same conditions (30 °C, 150 rpm) in 50 mL glass Erlenmeyer flasks for 7 days. Finally, the 10 mL culture was transferred to 90 mL of fresh medium in 500 mL Erlenmeyer flasks and incubated for an additional 7 days at 150 rpm and 30 °C.

Pigmentation was assessed by measuring the color coordinates of each culture (solid or liquid) at three random points on the surface of the liquid media or biomass powder placed in a Petri dish. This process followed the previously described method [43,44]. A CR-400 colorimeter (Konica Minolta Sensing Americas Inc., Ramsey, NJ, USA) equipped with a D65 illuminating lamp and a 2° observer was used to measure the L* (lightness), a* (redness/greenness), and b* (yellowness/blueness) values. The culture coloration was then determined using these coordinates and processed through the software available at http://colordesigner.io/convert/hextolab (accessed on 25 September 2024).

2.2.2. Pathogenic Bacterial Strains

On the other hand, the pathogens employed in this study included Klebsiella pneumoniae ATCC 11486, Escherichia coli ATCC BAA 2469, Pseudomonas aeruginosa ATCC 27853, Bacillus subtilis ATCC 55033, Staphylococcus epidermidis ATCC 14990, and Staphylococcus aureus ATCC BAA 44. For each pathogen, 100 µL of cryopreserved strains in TSA medium with 40% glycerol (at 1 × 10⁹ CFU/mL) was thawed and added to 10 mL of TSA medium and then incubated at 30 °C for 24 h. The bacterial concentration was adjusted to an optical density (OD) of 600 nm, with values between 0.8 and 1.0, equivalent to a 0.5 McFarland standard. Subsequently, 100 µL of the bacterial suspension was spread uniformly onto TSA agar plates. After incubation at 4 °C for 10 min, sensi-disks containing different concentrations of the extracts were placed on the semi-solid medium for antibacterial assays (vide infra in 2.4.2 Antibacterial Activity).

2.3. Obtention of Extracts from Pigmented Strains

The final culture volume (100 mL) for each strain was divided into 50 mL Falcon tubes and centrifuged at 6000× g for 10 min (Thermo Scientific, Waltham, MA, USA). Depending on whether the pigment was intracellular or secreted into the medium, the relevant phase (supernatant and/or sediment) containing the pigment was independently lyophilized for 24 h (Labconco, Kansas City, MO, USA). A double extraction was then performed on the lyophilized material from each Falcon tube. First, 75% ethanol (10 mL) was added, vortexed, and agitated at 150 rpm for 24 h. The mixture was then centrifuged at 6000× g for 5 min, and the supernatants were collected in amber vials. The procedure was repeated with an additional 75% ethanol (5 mL) for the second extraction.

2.4. Biological Activity Tests

2.4.1. Antioxidant Capacity

Two methods were performed to determine the antioxidant capacity, i.e., DPPH- and ABTS-related procedures, using the respective stable radicals for scavenging capacity. In both cases, the antioxidant capacity of the resulting ethanolic extracts was evaluated at concentrations of 5000, 500, 50, and 5 ppm in 96-well plates in triplicate, using trolox as a positive control. Given the pigmentation of the extracts, the respective solutions at the final concentration were scanned in the visible spectrum (400–800 nm) to assess potential interferences. None of the pigmented extract solutions, even at the highest concentrations, showed absorption at wavelength above 515 nm. For DPPH, the ethanolic extract (100 µL) and 0.2 mM DPPH (100 µL) were added to a 200 µL well. The plate was incubated for 30 min in the dark, and the OD was measured at 515 nm. For the ABTS method, aqueous solutions of 7 mM ABTS and 2.45 mM ammonium persulfate were separately prepared and subsequently mixed (1:1 ratio) and incubated in the dark for 16 h. The resulting ABTS⁺ radical was adjusted to an OD of 0.7. Finally, the ethanolic extract (10 µL) and ABTS⁺ radical solution (190 µL) were added to a 200 µL well, incubated for 30 min in the dark, and the OD was measured at 734 nm (Bio-Rad, Hercules, CA, USA) [43,44]. The results were expressed as trolox equivalent antioxidant capacity (TEAC), which corresponds to micromoles of trolox equivalents per liter (µmol TE)/L).

2.4.2. Antibacterial Activity

The ethanolic pigmented extracts were evaluated against the pathogens K. pneumoniae ATCC11486, E. coli ATCCBAA2469, P. aeruginosa ATCC27853, Bacillus subtilis ATCC 55033, S. epidermidis ATCC14990, and S. aureus ATCCBAA44 using antibiograms by disk diffusion. Solutions of the extract (30 mg/mL) were prepared in 75% ethanol, and pathogen dispersions were prepared using the MacFarland scale 0.5% in 0.85% saline solution. Mass seeding of pathogenic bacteria was performed using a sterile swab in TSA plates. The 1 cm diameter cellulose disks were impregnated with 5, 15, and 30 µL of the pigment solutions to examine the variation in bioactivity depending on the pigmented extract amounts in the range of 150 µg to 900 µg. The tests were performed in triplicate. Finally, the TSA plates were incubated at 37 °C for 24 h (Friocell, Querétaro, Mexico) using the extraction solvent (75% ethanol) as a negative control and the respective antibiotic as a positive control (i.e., ciprofloxacin (5 µg) against K. pneumoniae and E. coli, gentamycin (10 µg) against B. subtilis, and vancomycin (30 µg) against S. aureus and S. epidermidis). The antibiograms were photographed in the colony counter ProtoCOL3 (Microbiology International, Frederick, MD, USA) to measure the inhibition halos using the ImageJ v1.45s program [45].

2.4.3. Cytotoxic Activity

The cytotoxic activity of the ethanolic pigmented extracts was evaluated against cancer lines such as HeLa (cervical–uterine cancer) and MCF-7 (breast cancer), and healthy cell lines like HDFas (human dermal fibroblasts), using the standard MTT assay. Cells were cultured in DMEM medium for HeLa and HDFas, and RPMI for MCF-7, with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, and incubated at 37 °C in a 5% CO₂ incubator. After incubation, cells were treated with different concentrations of the pigmented extracts in triplicate (5–500 ppm (ppm equivalent to mg/L or µg/mL)) and incubated for 24 h. Following the 24 h treatment, MTT solution (100 µL) at 5 mg/mL was added and incubated for 4 h. MTT was removed, and dimethyl sulfoxide (DMSO) (100 µL) was added to each well. The assay was measured at an OD of 570 nm using a plate reader (Bio-Rad, CA, USA), with DMSO as the control [46]. The cell viability percentage (CVP) was calculated by using the mean OD of four wells in the indicated groups using the following formula: CVP = (OD_treatment − DO_blank)/ (DO_control − DO_blank) × 100%.

2.5. Statistical Analysis

One-way analysis of variance (ANOVA) was conducted on the bioactivity data after confirming their normal distribution using the Shapiro–Wilk test (p > 0.5), followed by Tukey’s HSD post hoc test for multiple comparisons to identify significant differences between treatment/dose means (n = 3). Statistical analyses were performed using the packages and protocols included in the InfoStat statistical software v29.09.2020 (National University of Córdoba, Córdoba, Argentina).

2.6. High-Performance Liquid Chromatography Coupled with Mass Spectrometry

The hydroalcoholic extracts were chemically characterized using a Shimadzu Co., LCMS-2020 system (Kyoto, Japan). This system featured a single quadrupole analyzer and a dual ion source (DUIS) that performs electrospray (ESI) and atmospheric pressure chemical (APCI) ionizations. The testing procedure employed a Phenomenex Synergy RP C18 column (150 × 4.4 mm, 2.6 µm). The column was heated to 30 °C and maintained a flow rate of 0.7 mL/min. Two mobile phases, labeled A and B, consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. A gradient elution was conducted over 30 min, starting at 10% B and gradually increasing to 100% B until 25 min, and then returned to 10% B until 30 min. The DUIS was operated in both positive- and negative-ion modes, employing a scan range of 200–1000 m/z, a CDL temperature of 250 °C, a detector voltage of 1.2 kV, a nebulizing gas flow rate of 1.3 L/min, and a drying gas flow rate of 8.0 L/min [47,48]. The characterization was supplemented by HRMS using a Shimadzu HPLC system coupled with a Bruker MicrOToF-Q II spectrometer equipped with a Quadrupole-Time of Flight (QToF) analyzer and ESI (Billerica, MA, USA). Chromatographic parameters identical to those previously mentioned were employed, operating ESI in both positive- and negative-ion modes, scanning between 100 and 1000 m/z, and setting the capillary voltage at 4.5 kV, desolvation line temperature at 400 °C, nebulizer gas (N₂) at 4 Bar, drying gas at 8 L/min, and quadrupole and collision energies at 6 and 12 eV, respectively.

The raw data were pre-processed using MZmine 3 software (v3.3.0) [49], and the detected features were annotated (level 3) from the MS data using the StreptomeDB v3.0 database [50]. The possible metabolites responsible for the pigmentation or tested bioactivities were searched and additionally filtered using StreptomeDB (v2.0) and other databases such as PubChem (v2023) [51], LOTUS (v2021) [52], and the natural products atlas [53,54].

2.7. Molecular Identification

At the end of the seven-day incubation period, a spore solution of each isolate was prepared, and genomic DNA was extracted using a Quick-DNA Fungal/Bacterial Microprep kit (Zymo Research Corporation, Irvine, CA, USA) according to the manufacturer’s instructions. Molecular identification was carried out by sequencing a 1500 bp fragment of the 16S ribosomal RNA gene obtained by PCR amplification using the universal primers 1493R and 27F under the following PCR cycling conditions: initial denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 2 min, with a final extension of 72 °C for 7 min (Bio-Rad, CA, USA). The amplification products were verified by agarose electrophoresis (Bio-Rad, CA, USA). The sequence obtained was compared in the GenBank nucleotide sequence database. A phylogenetic tree was constructed using the maximum likelihood method and Hasegawa–Kishino–Yano model [55] in MEGA X [56].

3. Results and Discussion

3.1. Pigmentation of the Strains in the Different Culture Media

When evaluating pigment production of each strain in different culture media (see Table 2), it was observed that the highest number of pigments were produced in ISP2, which contains malt and yeast extract providing amino acids, peptides, vitamins, and minerals essential for bacterial growth [57,58,59,60], while glucose supplies the necessary energy for specialized metabolite synthesis like pigments [61,62]. This medium has previously been used to produce red pigments with anticancer activity from Streptomyces A 16-1 [63] and Streptomyces sp. PM4 [64], brown pigments with antibacterial and anti-HIV activities from Streptomyces sp. S45 [65], melanoid pigments like eumelanin with antiproliferative and antioxidant capacities from S. parvus BSB49 [66], and pyomelanin from Streptomyces sp. MPPS2 [67].

The predominant colors produced were black and brown. These dark pigments, possibly melanin or melanoid, can be useful criteria for taxonomical studies [68]. However, varied colors such as yellow, pink, and purple were also observed in other culture media. The common nutrient for these particular pigments is starch as a carbon source, which can induce the expression of genes involved in specialized metabolite biosynthesis and ensure a sustained release of glucose for growth and metabolite production [69,70,71,72]. This carbon source has been used in starch casein nitrate medium to produce red pigment with antimicrobial and anticancer activities from Streptomyces sp. JAR6 [73], yellow pigments with antimicrobial activity from S. coeruleorubidus [74] and S. parvulus C5-5Y [75], and with anticancer activity from S. griseoaurantiacus JUACT 01 [46] and S. aurantiacus AAA5 [76].

Table 2. Pigmentation produced by the strains in the different culture media ((B) = biomass, (L) = liquid media, or (–) = no pigmentation).

Strain	ISP2		ISP4		Modified ISP9		Starch Nitrate
	Color	Lab *	Color	Lab *	Color	Lab *	Color	Lab *
2	(L)	9.99, 6.44, 7.49	–		–		–
94	(L)	12.45, 4.60, 4.79	–		(L)	20.82, 10.16, 13.96	–
144	(B)	4.29, 1.43, 0.50	(L)	18.68, 7.69, 10.92	–		(L)	19.51, 14.60, 23.45
197	–		–		(B)	1.39, 0.29, −1.61	–
220	(L)	46.45, 12.34, 38.52	–		–		–
246	–		–		–		(L)	53.76, 11.77, 59.26
247	(B)	11.76, 11.34, 0.97	–		–		–
263	(B)	8.30, 16.56, 3.98	–		–		–
290	–		(L)	47.14, 9.75, 52.21	–		–
308	(B)	3.99, 0.00, 1.01	–		–		–
356	(B)	22.63, 16.05, 15.99	–		–		(B)	53.97, 19.95, 8.64
381	(B)	20.96, 26.86, 16.83	–		–		–
443	(L)	56.26, 9.31, 54.02	–		–		–
626	(L)	36.62, 25.26, 40.00	–		–		–
864	(L)	28.83, 9.35, 18.34	–		–		–
1B18	(B)	9.74, 1.80, 3.00	–		(B)	14.14, 6.74, 12.82	–
1B247	(L)	29.17, 5.97, 11.69	–		–		–
3C110	(B)	7.28, 0.00, 1.45	–		–		–
4C168	(L)	38.26, 8.17, 34.19	–		–		–
4C171	–		(L)	56.97, 2.36, 53.98	–		–

* Lab-derived color coordinates were measured at three random points on the surface of the liquid media or biomass powder in a Petri dish, following the described procedure [77,78].

Table 2. Pigmentation produced by the strains in the different culture media ((B) = biomass, (L) = liquid media, or (–) = no pigmentation).

Strain	ISP2		ISP4		Modified ISP9		Starch Nitrate
	Color	Lab *	Color	Lab *	Color	Lab *	Color	Lab *
2	(L)	9.99, 6.44, 7.49	–		–		–
94	(L)	12.45, 4.60, 4.79	–		(L)	20.82, 10.16, 13.96	–
144	(B)	4.29, 1.43, 0.50	(L)	18.68, 7.69, 10.92	–		(L)	19.51, 14.60, 23.45
197	–		–		(B)	1.39, 0.29, −1.61	–
220	(L)	46.45, 12.34, 38.52	–		–		–
246	–		–		–		(L)	53.76, 11.77, 59.26
247	(B)	11.76, 11.34, 0.97	–		–		–
263	(B)	8.30, 16.56, 3.98	–		–		–
290	–		(L)	47.14, 9.75, 52.21	–		–
308	(B)	3.99, 0.00, 1.01	–		–		–
356	(B)	22.63, 16.05, 15.99	–		–		(B)	53.97, 19.95, 8.64
381	(B)	20.96, 26.86, 16.83	–		–		–
443	(L)	56.26, 9.31, 54.02	–		–		–
626	(L)	36.62, 25.26, 40.00	–		–		–
864	(L)	28.83, 9.35, 18.34	–		–		–
1B18	(B)	9.74, 1.80, 3.00	–		(B)	14.14, 6.74, 12.82	–
1B247	(L)	29.17, 5.97, 11.69	–		–		–
3C110	(B)	7.28, 0.00, 1.45	–		–		–
4C168	(L)	38.26, 8.17, 34.19	–		–		–
4C171	–		(L)	56.97, 2.36, 53.98	–		–

* Lab-derived color coordinates were measured at three random points on the surface of the liquid media or biomass powder in a Petri dish, following the described procedure [77,78].

It should be noted that pigment production in the starch nitrate culture medium is slow, but it has the advantage that pigment production is greater than the amount of biomass produced. This may be due to the fact that the biosynthesis of specialized metabolites likely competes with biomass formation, slowing growth [79]. Additionally, some strains that produced pigment in agar lost their production in liquid medium, and when culture volumes were increased, a decrease in pigment production was observed, or the coloration became less visible compared to the culture medium color.

Evaluating different culture media is crucial as the production of specialized metabolites, including pigments, is influenced by changes in the primary carbon and nitrogen sources. These changes can either increase the supply or limit access to key precursors [80,81,82], triggering the activation of biosynthetic enzymes or simultaneously affecting both processes [79,83]. Bacteria often prefer certain single carbon sources over others, preventing the synthesis of enzymes necessary for the catabolism of non-selected carbon sources through carbon catabolite repression (CCR) [84]. Similarly, nitrogen source preference (nitrogen repression) affects the expression of primary nitrogen metabolism and subsequently the biosynthesis of specialized metabolites, which require nitrogen compound precursors [79,85].

3.2. Molecular Identification

The characterization of Streptomyces strains using 16S rRNA gene sequencing is a fundamental approach in microbial taxonomy and phylogenetics. This method provides a high level of accuracy in identifying and classifying bacterial species based on their genetic sequences [80,81]. In this study, a BLAST search of the GenBank database using the 16S rRNA gene sequences for each pigmented strain confirmed that all identified isolates belong to the genus Streptomyces. The 16S rRNA gene sequence similarities between the strains and type strains of the genus Streptomyces ranged from 98% to 100%. This high similarity level indicated a close genetic relationship among the strains, aligning with previous findings in the literature where 16S rRNA gene sequencing has been effectively used to delineate species within the Streptomyces genus (

Table 3. Molecular identification of strains (n = 18) with pigmentation.

Strain	Identification	Similarity	Strain	Identification	Similarity
2	Streptomyces griseochromogenes	99.93%	381	Streptomyces albospinus	99.18%
94	Streptomyces tendae	99.86%	443	Streptomyces murinus	98.96%
144	Streptomyces humi	99.55%	626	Streptomyces albospinus	99.85%
197	Streptomyces misionensis	99.72%	864	Streptomyces lacticiproducens	99.17%
220	Streptomyces argillaceus	100.00%	1B18	Streptomyces lactacystinicus	99.35%
246	Streptomyces murinus	99.93%	1B247	Streptomyces mediolani	99.64%
290	Streptomyces noursei	99.24%	3C110	Streptomyces fodineus	99.86%
308	Streptomyces humi	99.64%	4C168	Streptomyces murinus	99.93%
356	Streptomyces hygroscopicus	99.93%	4C171	Streptomyces murinus	99.86%

). However, strains 247 and 263 were not able to successfully amplify the 16S rRNA gene, and these strains were not accurately identified.

A phylogenetic tree (Figure 1) was constructed based on 16S rRNA gene sequences of Streptomyces species using the maximum likelihood method and Hasegawa–Kishino–Yano model [55] in MEGA X [56].

The bootstrap consensus tree, inferred from 1000 replicates, is presented in Figure 1, with the percentage of replicate trees supporting the clustered taxa shown next to the branches [86]. In the phylogenetic tree, some strains cluster closely together, such as strains S. lacticiproducens 864, S. albospinus 381, and S. humi 144, and strains S. murinus 4C168, S. humi 308, and S. mediolani 1B247. However, there is no clear pattern in strain relationships, with the most promising yellow pigment-producing strains (S. noursei 290, S. murinus 246, and 4C171) being relatively distant from each other.

Some strains with identical BLAST identifications are found in different branches of the phylogenetic tree and exhibit varying bioactivities. This suggests that more extensive morphological, biochemical, or sequencing analyses might be necessary for accurate identification. Additionally, the novel sources of isolation could indicate that some strains represent new identifications. The use of 16S rRNA gene sequencing is well documented in the literature for its robustness in identifying bacterial strains [87]. This gene serves as a reliable molecular marker due to its presence in all bacteria and the slow rate of evolutionary change in its sequence, making it ideal for phylogenetic studies. Similarly, the utility of 16S rRNA gene sequencing in the classification of Streptomyces species has been previously demonstrated, highlighting its effectiveness in distinguishing between closely related strains [88]. However, despite its strengths, 16S rRNA gene sequencing has limitations [87]. The resolution of this method may not be sufficient to differentiate between very closely related species or strains with highly similar sequences. Consequently, additional genetic markers or whole-genome sequencing might be required for more precise identification and classification. Hence, while 16S rRNA gene sequencing is a powerful tool for the identification and characterization of Streptomyces strains, combining this method with other genetic and phenotypic analyses enhances the accuracy and depth of microbial taxonomy studies [89]. This integrative approach is crucial for uncovering the full potential of Streptomyces species in producing bioactive compounds.

3.3. Biological Activity Assessment

3.3.1. Antioxidant Capacity

In most pigmented extracts, a higher test concentration exhibits a higher antioxidant capacity, indicating a dose-dependent response [90]. Differences in antioxidant capacities determined by the two assays (Figure 2) can be greater in highly pigmented sources [91]. According to the DPPH assay, extracts with the highest antioxidant potential (TEAC > 20 µM, p < 0.05) are from strains S. humi 144 in ISP4, S. misionensis 197 in modified ISP9 (mISP9), S. hygroscopicus 356 in ISP2, and S. lactacystinicus 1B18 in mISP9. These extracts have dark brown or black colors, possibly indicating melanin pigments. In the case of the ABTS assay, almost all extracts at maximum concentration exhibit good antioxidant capacities, except for strains S. murinus 246 and S. humi 144 in starch nitrate medium, and strains S. noursei 290 and S. murinus 4C171 in ISP4, which resulted in TEAC < 20 µM (p < 0.05). Notably, three of these are yellow pigments with low antioxidant capacity in both methods (see Tables S1 and S2).

The strain S. tendae 94-derived extract shows slight changes in antioxidant capacity between ISP2 and mISP9 media in both methods. The strain S. humi 144-derived extract evaluated in three media shows significant differences (p < 0.05): in ISP2 and ISP4, the DPPH-scavenging variation is slight (6.86 µM) and larger in ABTS (16.95 µM). However, in starch nitrate medium, the change was considered drastic with null DPPH and low ABTS-scavenging ability. Similarly, strain S. hygroscopicus 356-derived extracts show high DPPH capacity when cultured in ISP 2 and almost null in starch nitrate, while the ABTS assay shows antioxidant capacity with a difference of 9.59 µM.

Antioxidants are crucial for preventing oxidative stress, which can cause damage to lipids, proteins, and DNA, leading to diseases such as cancer, neurodegeneration, and cardiovascular conditions [92,93]. Various in vitro and in vivo methods are utilized to evaluate antioxidant capacity, playing a significant role in understanding how antioxidants function [94]. Two commonly used methods for evaluating antioxidant capacity are the DPPH and ABTS assays (see Figure 2). Differences between the results from these methods may arise because the ABTS assay is applicable to both hydrophilic and lipophilic antioxidant systems, while the DPPH assay is applicable only to hydrophobic systems [91,95,96]. Additionally, it is suggested that the ABTS assay better reflects antioxidant contents than the DPPH assay [97], being highly sensitive, practical, fast, very stable [98], and useful for high-moisture or highly pigmented nutrients [99].

The choice of culture media significantly influences the specialized metabolite profiles produced by microorganisms and the respective antioxidant capacities [80,81,82]. This variation can explain differences in antioxidant activity observed between methods, as hydrophilic antioxidants may not be detected using the DPPH method, while the ABTS method is more effective at measuring their scavenging capacity [91]. In this context, the production of a carotenoid-like red pigment from Streptomyces sp. LS1 was notably higher when using a modified Luria–Bertani medium (LBM) for 5 days compared to three other media. However, after optimization through a one-variable-at-a-time (OVAT) approach, the best production conditions were achieved with specific parameters: yeast extract (3 g/L), fructose (8 g/L), NaCl (3 g/L), pH 7, shaking at 200 rpm, temperature at 30 °C, and an incubation time of 72 h [100]. In general, the growth of Streptomyces strains and their ability to produce diffusible pigments are influenced by several factors, e.g., NaCl concentration tolerance, pH, incubation time, temperature, and carbohydrate assimilation [101].

The antioxidant activity of pigmented extracts and pigments from Streptomyces strains has attracted significant attention due to their potential health benefits and industrial applications [32]. These species, a well-known genus of actinomycetes, produce a wide range of pigments, including melanin, carotenoids, anthraquinones, and prodiginines [36]. These pigments are often linked to the organism’s defense mechanisms, such as protection against UV radiation and oxidative stress. Prodigiosin, a red-pigmented tripyrrole compound, is one of the most well-known pigments produced by Streptomyces [102]. Its antioxidant activity is primarily attributed to its ability to donate hydrogen atoms or electrons, neutralizing reactive oxygen species (ROS) and preventing cellular damage from oxidative stress [103]. For instance, prodigiosin from Streptomyces sp. WMA-LM31 exhibited 62% free radical scavenging activity against DPPH [104]. On the other hand, melanin-like pigments produced by Streptomyces also display significant antioxidant properties [105]. The phenolic structure of melanin likely contributes to its ability to quench free radicals and act as a potent antioxidant, as demonstrated by eumelanin from Streptomyces lasalocidi NTB 42, which showed DPPH and ABTS radical scavenging activities with IC₅₀ values of 70.6 μg/mL and 176.8 μg/mL, respectively [106]. Previously, Streptomyces strains have reported melanin with antioxidant capacity, such as Streptomyces girseorubiginosus D5 isolated from desert soil in India [107], Streptomyces glaucescens NEAE-H isolated from Gulbarga region in India [41], and Streptomyces parvus BSB49 from Bayburt province (Turkey) [66]. Overall, pigments from Streptomyces have shown antioxidant properties, likely due to their molecular structures that enable them to neutralize free radicals and reduce oxidative stress. These antioxidant capacities, linked to various naturally occurring bioactive moieties, span IC₅₀ values ranging from 1.3 to 4330 μM [108].

3.3.2. Antibacterial Activity

Among the six pathogenic microorganisms tested, only three—B. subtilis ATCC 55033, S. epidermidis ATCC 14990, and S. aureus ATCC BAA44—demonstrated sensitivity to the pigmented extracts (see

Table 4. Antibacterial activity of the pigmented extracts.

Pigmented Extracts	Tested Bacterial Strains						Pigmented Extracts	Tested Bacterial Strains
	1	2	3	4	5	6		1	2	3	4	5	6
2-A	–	–	–	–	–	–	356-A	–	–	–	–	++	–
94-A	–	–	–	–	–	–	356-D	–	–	–	–	–	–
94-C	–	–	–	–	–	–	381-A	–	–	–	–	–	–
144-A	–	–	–	–	–	–	443-A	–	–	–	–	–	–
144-B	–	–	–	–	–	–	626-A	–	–	–	–	–	–
144-D	–	–	–	–	–	–	864-A	–	–	–	–	–	–
197-C	–	–	–	–	–	–	1B18-A	–	–	–	–	–	–
220-A	–	–	–	–	–	–	1B18-C	–	–	–	–	–	–
246-D	–	–	–	+	++	++	1B247-A	–	–	–	–	++	+++
246-A	–	–	–	–	+++	++	3C110-A	–	–	–	–	++	–
263-A	–	–	–	–	–	–	4C168-A	–	–	–	–	–	–
290-B	–	–	–	+++	++	++	4C171-B	–	–	–	++	+++	+++
308-A	–	–	–	–	–	–

Pathogenic microorganisms: (1) Klebsiella pneumoniae ATCC11486, (2) Escherichia coli ATCCBAA2469, (3) Pseudomonas aeruginosa ATCC27853, (4) Bacillus subtilis ATCC 55,033, (5) Staphylococcus epidermidis ATCC14990, (6) Staphylococcus aureus ATCCBAA44. +++ (inhibition diameter > 15 mm), ++ (10–15 mm), + (1–10 mm), – (inhibition diameter < 1 mm). Extract amount for all antibiograms = 900 µg. Pigmented extracts listed according to a code comprising the codified strain (Table 2) hyphenated to the culture medium type, i.e., ISP2 (A), ISP4 (B), modified ISP9 (C), starch nitrate (D). For more information, all the antibiograms are presented in the Supplementary Materials.

).

Pigmented extracts from strains S. murinus 246 grown in starch nitrate medium, S. noursei 290, and S. murinus 4C171 in ISP4 medium exhibited antimicrobial activity against these three bacteria and show promising potential as antibiotics from the lowest concentration tested (150 µg). To a lesser extent, extracts from strains S. murinus 246 and S. mediolani 1B247 grown in ISP2 showed activity against S. epidermidis ATCC 14,990 and S. aureus ATCC BAA44 from an intermediate concentration of 450 µg of extract. Extracts from strains S. fodineus 3C110 and S. hygroscopicus 356 in ISP2 exhibited activity only against S. epidermidis ATCC 14,990 at the highest concentration tested (900 µg). In most cases, changing the culture medium did not affect antibacterial activity. However, strain S. hygroscopicus 356 inhibited the growth of S. epidermidis ATCC 14,990 in ISP2 medium but not in starch nitrate medium, suggesting that the same strain may produce different specialized metabolites with varying bioactivities depending on the medium.

The discovery and development of new antibiotics are crucial due to the increasing threat of antimicrobial resistance (AMR). This problem is exacerbated by the overuse and misuse of antibiotics, inappropriate waste management, and environmental transmission [109,110]. It is estimated that at least 700,000 people worldwide die each year as a result of drug-resistant infections [109]. Additionally, the high numbers of secondary infections during COVID-19 pandemic [111,112] and the massive use of antibiotics as (co)treatment [113] worsened the situation, adding to the ongoing emergence of AMR [114], with likely unfavorable outcomes in clinical, economic, and societal aspects [109,115,116].

The three susceptible bacterial strains are characterized by their yellow pigmentation and the use of starch as a carbon source. S. epidermidis and S. aureus are of clinical interest. In this regard, S. epidermidis is part of the human skin microbiome but can cause opportunistic infections, such as nosocomial infections, particularly in immunocompromised patients [117,118]. It may outcompete other bacteria in skin wounds and can acquire antibiotic resistance through horizontal gene transfer from other Staphylococcus species [119,120]. On the other hand, S. aureus ATCC BAA44 is an Iberian MRSA clone isolated from a hospital. It can lead to a range of infections, including those affecting the skin, subcutaneous tissues, and potentially invasive infections such as osteomyelitis, meningitis, pneumonia, lung abscess, empyema, and endocarditis [121,122,123].

The antibacterial activity of pigmented extracts and pigments from Streptomyces strains has been widely documented, making these microbes a rich source of bioactive compounds with potential for pharmaceutical development [105]. Streptomyces species are known to produce a variety of pigmented secondary metabolites, many of which exhibit strong antibacterial properties. Notably, previously reported antibiotic yellow pigments include D-actinomycin from S. parvulus C5-5Y, isolated from the leather industry [75], a fraction from S. hygroscopicus subsp. ossamyceticus (D10), isolated from desert soil, which was active against vancomycin-resistant S. aureus [124], a crude pigment from Streptomyces sp. D25 with activity against S. aureus MTCC96 [125], and grixazone A and B from S. griseus IFO13350, obtained from the Institute of Fermentation, Osaka, Japan (IFO), which was active against B. subtilis ATCC6633 and S. aureus 209P JC-1 [126].

3.3.3. Cytotoxic Activity

When evaluating the pigmented extracts against three cell lines—human dermal fibroblasts (HDFas), cervical cancer cell line (HeLa), and breast cancer cell line (MCF-7) (see Figure 3)—the MCF-7 cell line was the most resistant to the extracts. Extracts from strains S. murinus 246 in starch nitrate and S. murinus 4C171 in ISP4 medium showed promising anticancer activity against HeLa and MCF-7 without being toxic to the HDFa cell line. Extract Streptomyces noursei 290 in ISP4 medium demonstrated good anticancer activity against HeLa at all concentrations evaluated and against MCF-7 at maximum concentration, although it was toxic to HDFas. Strain S. murinus 443 in ISP 2 also showed promise at all concentrations without being toxic to HDFas.

In general, tested extracts exhibited a dose-dependent response (Figure 3). However, some extracts exhibited an unusual trend. For instance, strain S. tendae 94 in mISP9 showed higher cytotoxicity against MCF-7 at intermediate concentrations than at the highest concentration, while strain S. misionensis 197 in mISP 9 and strain S. tendae 94 in ISP2 showed higher activity at lower concentrations. Half-maximal inhibitory concentrations (IC₅₀) were calculated for those extracts showing a dose-dependent response (see Figures S16–S19). Strain S. murinus 4C171 in starch nitrate had IC₅₀ values of 146.4 ppm against HeLa and 242.6 ppm against MCF-7, which were relatively higher than those reported in the literature, ranging from 0.04 to 48.07 ppm [36].

Strain S. murinus 246 in starch nitrate had an IC₅₀ of 119.3 ppm against HeLa, which is lower than undecylprodigiosin from Streptomyces sp. JAR6 [73] but higher than a yellow pigment from Streptomyces griseoaurantiacus JUACT 01 [46]. For strains showing cytotoxicity, IC₅₀ values were 152.6 ppm for S. mediolani 1B247 in ISP2 and 104.8 ppm for S. noursei 290 in ISP4. These values are higher than those reported for melanin from Streptomyces glaucescens NEAE-H, which were 37.05 ppm and 48.07 ppm against noncancer cell lines [37]. This suggests that higher concentrations are required for cytotoxicity, though these extracts are not entirely safe.

Identifying anticancer compounds is crucial due to the limitations of current chemotherapies, such as multidrug resistance and secondary effects. Natural products, including those from microorganisms, have shown potential in inhibiting cell proliferation and promoting apoptosis [127]. Bacteria are highlighted as sources of specialized metabolites with anticancer properties, potentially leading to the development of more effective cancer therapies [128,129]. Streptomyces species, known for producing clinically significant drugs like doxorubicin and bleomycin [130,131], also produce pigmented compounds with anticancer activity. Notable examples include red pigmented crude extracts from Streptomyces sp. PM4 [64] and Streptomyces sp. A 16-1 [63], pure compounds such as undecylprodigiosin from Streptomyces sp. JAR6 [73], and prodigiosin from Streptomyces sp. WMA-LM31 [104]. Other pigments include a yellow pigment from Streptomyces griseoaurantiacus JUACT 01 [46] with activity against HeLa and liver cancer (HepG2), melanin from Streptomyces glaucescens NEAE-H [41], and eumelanin from Streptomyces parvus BSB49 [66] with activity against skin cancer (HFB4) and HeLa, respectively.

3.4. LC-MS-Based Annotation (Level 3) of Metabolites Possibly Responsible for the Observed Activities by the Different Streptomyces-Derived Extracts

The most promising pigmented extracts (i.e., those that presented one or more bioactivities with high levels among the extracts evaluated) were analyzed by liquid chromatography coupled with mass spectrometry (LC-MS) to chemically characterize such extracts and annotate those detected compounds. The annotated metabolites were searched in the literature and the StreptomeDB database to determine a plausible role in pigmentation and bioactivity, as well as to find previous sources related to Streptomyces species that produce them. In this context, strain S. humi 144, cultured on ISP2 and ISP4 media, exhibited antioxidant capacity, as evidenced by DPPH and ABTS assays. LC-MS-based characterization suggested that this activity may be associated with chlorogenic acid [52], a phenylpropanoid previously identified in Streptomyces sp. PM9 [132], known for its antioxidant efficacy comparable to ʟ-ascorbic acid [133]. This effectiveness is often attributed to the hydroxyl groups in the aromatic ring structure [134]. Additionally, strain S. humi 144 in ISP2 medium demonstrated moderate cytotoxic activity against HeLa cells, possibly due to the presence of certain compounds, detected and annotated in this extract (Table S6), which were previously reported to have cytotoxic activity. In contrast, strain S. noursei 290 in ISP4 showed antibacterial activity against B. subtilis ATCC 55033, S. epidermidis ATCC14990, and S. aureus ATCCBAA44, which may be attributed to the compounds listed in Table S7. This extract also had good anticancer activity against HeLa and MCF-7 cells, potentially due to the detected, annotated compounds listed in Table S8. Furthermore, LC-MS analysis suggested that the pigments responsible for coloration in this strain may include phenazine-1-carboxylate (also known as tubermycin B) with a greenish-yellow hue [135], or tetracenomycin D1, related to tetracenomycin C, a pale yellow antibiotic [136]. Other compounds with potential antimicrobial and cytotoxic activity included pentostatin, phenazine-1-carboxylate, yautomycetin, and tetracenomycin D1.

Strain S. murinus 246 in starch nitrate exhibited antibacterial activity against B. subtilis ATCC 55033, S. epidermidis ATCC14990, and S. aureus ATCCBAA44, possibly due to the action of some of the annotated compounds (Table S9), which are related to antibiotics such as cephamicyn C, lankamycin, and sisomicin. It also showed promising cytotoxic activity against HeLa and MCF-7 cells without toxicity to the normal cell line (HDFas), potentially due to the presence of fumiformamide, maculosin, and salinomycin as annotated compounds with reported cytotoxic activity (Table S10). The pigments responsible might include a xanthone-related compound, such as xantholipin B. Xanthones, derived from the greek word “xanthos”, meaning yellow, are polyketide-derived compounds, firstly isolated from Streptomyces but also found in plants, with a C6-C1-C6 carbon skeleton [137]. Xanthones have various properties, including antimicrobial, antioxidant, anti-inflammatory, and antitumor activities [121,122,123]. Recently, a new polycyclic xanthone, namely sattahipmycin, was identified from marine Streptomyces sp. GKU 257-1 and exhibited antimicrobial and cytotoxic activity [138]. Remarkably, the other S. murinus strain (i.e., 4C171) in the ISP4 medium showed antibacterial activity against B. subtilis ATCC 55033, S. epidermidis ATCC14990, and S. aureus ATCCBAA44, potentially due to a diverse metabolite class listed in Table S11. The extract of this strain exhibited low antioxidant capacity, possibly related to the low action or low concentration of annotated compounds in this extract (Table S12). However, it demonstrated promising anticancer activity against HeLa and MCF-7 cells without toxicity to the healthy cell line (HDFas), likely due to the presence of active metabolites (Table S13). Potential pigments include carotenoids like beta-carotene, γ-carotene, neurosporene, 1,2,1′,2′-tetrahydrolycopene, and lycopene, which have been reported in Streptomyces species without photoinduction [139]. Other possible pigments include resistomycin, a natural antibiotic with cytotoxic activity [36,76,140]; flavonoids such as taxifolin or dihydroquercetin [36], phenazine-1-carboxylate, which has a greenish-yellow coloration [135], tetracenomycin C, which is a pale yellow antibiotic [136], riboflavin, identified in species such as Streptomyces sp. TS-2-2 [141], and the red pigment roseoflavin from species like Streptomyces davaonensis [131,132].

On the other hand, strain S. fodineus 3C110 growth in ISP2 medium exhibited antibacterial activity against S. epidermidis ATCC14990, potentially due to those metabolites filtered as bioactives (Table S14). It also showed moderate antioxidant capacity, possibly related to salinomycin, the only antioxidant compound detected in this extract by LC-MS. This 3C110-derived extract was found to be active against HeLa cells at maximum dose, involving the presence of three cytotoxic compounds (Table S15). Additionally, strain S. hygroscopicus 356 in ISP2 medium showed antibacterial activity against S. epidermidis ATCC14990, moderate antioxidant capacity, and reasonable cytotoxic activity against HeLa cells at maximum concentration, whose bioactivity was rationalized by the reported properties of those annotated compounds for this extract (Tables S16–S18). Remarkably, this strain S. hygroscopicus 356 grown in starch nitrate showed no antibacterial activity against the pathogens tested and lower antioxidant capacity compared to ISP2 medium, exhibiting a different profile, and most compounds were not produced and accumulated in such a medium (i.e., starch nitrate), except 2-acetylpyrrole, previously produced by Streptomyces sp. MUM273b [142], which was additionally detected. This extract also showed cytotoxic activity against HeLa cells at maximum concentration but to a lesser degree than in ISP2, involving cytotoxic compounds such as chartreusin, jadomycin A, and pentalenolactone (Table S19).

The LC-MS-based characterization of S. hygroscopicus 356-derived extracts obtained from two culture media (ISP2 and starch nitrate) revealed the plausible production of annotated aromatic polyketide diphenyl ethers, i.e., Violaceol I and II, purple pigments known from co-cultures of Streptomyces rapamycinicus and Aspergillus nidulans [143], or endophytic fungi Aspergillus austroafricanus and Streptomyces lividans [144]. These pigments or related compounds might account for the pinkish-purple color of strain S. hygroscopicus 356 in starch nitrate and its darker shade in ISP2 medium. However, the coloration of this strain 356 in ISP2 differs slightly from purple, possibly due to the presence of other pigments like β-isorenieratene, luteothin, and xanthone-like metabolites.

Particularly, strain S. misionensis 197 in mISP9 medium shows no antibacterial activity against the evaluated pathogens or cytotoxicity against cell lines but exhibits certain toxicity against HDFas. It also showed moderate antioxidant activity, potentially due to antioxidant compounds (Table S20). Similarly, strain S. murinus 443 in ISP2 medium showed no antibacterial activity against the evaluated pathogens and low antioxidant capacity, but it was active against HeLa and MCF-7 cells, possibly related to the presence of previously reported Streptomyces-derived compounds (Tables S21 and S22). Detected pigments in this extract included carotenoids such as β-carotene, γ-carotene, β-isorenieratene, phytoene, 1,2,1′,2′-tetrahydrolycopene, and lycopene [139]. Finally, strain S. lacticiproducens 864 in ISP2 medium exhibits no antibacterial activity against evaluated pathogens, moderate antioxidant capacity, and cytotoxic activity against HeLa and MCF-7 cells at the highest tested concentrations, whose properties could be explained by the detection of bioactive metabolites listed in Tables S23 and S24. In the case of strain S. humi 308 in ISP2 medium, this exhibits only antioxidant capacity, possibly due to the production of phenolic-like antioxidant metabolites [145,146,147].

The LC-MS data of annotated compounds were thoroughly analyzed to determine which pigments could be linked to these bioactivities. The compiled data (Table S25) revealed a wide array of pigment-related compounds spanning various chemical classes, including naphthoquinones (NQs) like jadomycin A and griseorhodin A, benzoquinones (BQs) such as geldanamycin and herbimycin, phenazines (PZs) like phenazine-1-carboxylate and pyocyanine, xanthones (XTs) such as xantholipin B, phenylpropanoids (PPs) like MPBDF, benzopteridines (BPs) such as roseoflavin, carotenoids (CTs) like lycopene, and anthraquinones (AQs) such as rabelomycin, idarubicin, and nogalamycin.

This diversity of the annotated pigment-related compounds suggests that Streptomyces strains are prolific producers of structurally diverse secondary metabolites, many of which are known for their bioactivity, as noted in the StreptomeDB [50]. Among these, anthraquinones and phenazines were particularly prominent, indicating their frequent production across different strains and culture media. The annotated compounds exhibited various bioactivities, including cytotoxic, antimicrobial, and antioxidant properties. Notable strains such as S. murinus 4C171 and S. hygroscopicus 356 produced multiple pigment-related compounds associated with these bioactivities. For example, S. murinus 4C171, cultured in ISP4 medium, produced 13 different bioactive pigments, including rabelomycin (AQ), granaticin (NQ), and lycopene (CT), which exhibited antimicrobial, antioxidant, and cytotoxic activities. Similarly, S. hygroscopicus 356, cultured in ISP2 medium, yielded antimicrobial and cytotoxic anthraquinones such as elloramycin A and urdamycin A, highlighting the strain’s therapeutic potential. In addition, the choice of culture medium significantly influenced the production of specific pigment-related compounds. For instance, S. murinus 4C171 and S. hygroscopicus 356 produced a variety of anthraquinones in ISP4 and ISP2 media, respectively, while starch nitrate medium stimulated the production of unique compounds like jadomycin A (NQ) in S. hygroscopicus 356. ISP2 medium was particularly effective in inducing the production of cytotoxic compounds, as observed in S. lacticiproducens 864 and S. murinus 443, which produced doxorubicinol (AQ) and aclacinomycin A (AQ), respectively. In general, this study demonstrates that the examined Streptomyces strains are a relevant source of bioactive pigments with chemical diversity, contributing to cytotoxic, antimicrobial, and antioxidant activities. Anthraquinones and phenazines dominate the list of bioactive pigments, with several compounds showing strong antibacterial and anticancer potential. The choice of culture medium plays a critical role in determining the type and yield of bioactive compounds, underscoring the importance of optimizing cultivation conditions to fully exploit the pharmaceutical potential of Streptomyces-derived pigments.

4. Conclusions

This study highlights the potential of Streptomyces-derived pigments as a valuable source of bioactive compounds with antibacterial, anticancer, and antioxidant properties. Among the six pathogenic microorganisms tested, several showed sensitivities to pigmented extracts from various Streptomyces strains. Additionally, multiple extracts demonstrated potent anticancer activity against HeLa and MCF-7 cell lines, as well as antioxidant capacity via DPPH and ABTS assays. Notably, strains S. noursei 290, S. murinus 246, and 4C171, which produce yellow pigments and utilize starch as a carbon source, were particularly promising. Extracts from these strains exhibited strong antibacterial and anticancer activities, with selectivity toward cancer cells. Chemical analysis of the extracts identified several bioactive compounds responsible for these effects, including anthraquinone and phenazine-related compounds. The composition and bioactivity of the extracts were also influenced by the culture medium. For instance, strain S. hygroscopicus 356 displayed different antibacterial and antioxidant activities depending on the medium used, with starch proving to be a particularly favorable carbon source for producing yellow-pigmented extracts with pronounced bioactivity. Overall, this study underscores the diverse bioactivities of Streptomyces-derived compounds and their potential applications in pharmaceutical and therapeutic fields. Further research is needed to fully explore the therapeutic potential of these compounds.