Bioactive Pigments from Isolated Bacteria and Its Antibacterial, Antioxidant and Sun Protective Application Useful for Cosmetic Products

Abstract

Bacterial pigments are the unique and sustainable source of bioactive colour compounds used in cosmetics, food, textiles, printing and pharmaceutical products. Here, we report the pigment-producing isolates and their biological activities that could be benefited for different industries including cosmeceuticals. In this study, a total of 19 pigment-producing bacteria were isolated and purified from collected soil and water samples. The colour production ability of purified bacteria was observed up to 5 transfers. Of the 19 isolates, two isolates lost colour production ability in subsequent transfers. Crude pigments extracted from the remaining 17 isolates showed sunscreen activity in the range of 0.4–8.34. However, only 6 of them showed significant antibacterial and antioxidant activities. In the media optimization experiment, these 6 bacteria showed optimum growth in neutral to alkaline pH, while optimum temperatures for growth were different for different bacteria. One isolate produces the promising pigment, out of all six potential pigments. It is stable up to 5 transfers, having antioxidant and antibacterial activity with Sun protective activity; the strain was identified using 16srRNA gene sequencing and obtained accession number as MK770403 (probable strain is Staphylococcus xylosus) from National Center for Biotechnology Information (NCBI) database. The results of this study suggested that these bioactive pigments can further be developed and used as antibacterial, antioxidant and sun-protective ingredients in cosmeceuticals.

Electronic supplementary material

The online version of this article (10.1007/s12088-020-00870-x) contains supplementary material, which is available to authorized users.

Exposure to ultraviolet (UV) rays of the Sun can cause many health issues like pigmentation [1], erythema [2], immunosuppression [3], skin cancer [4], photoaging [5], etc. Generation of reactive oxygen species (ROS), apoptosis, alteration at the cellular level, DNA damage, etc. are responsible for these acute and chronic effects [6]. Sunscreens are being widely used for protection against harmful UV rays. Owing to adverse side effects such as acne, itching, burning, redness of the skin, etc. of synthetic photo-protective products, people are turning towards natural sunscreen products. Due to the increasing awareness and demands of natural sunscreens, researchers also focus on the discovery of new and cost-effective natural sunscreens agents [7]. Many microorganisms are an inhabitant of high-UV rays sites, produce pigments that can protect them against UV damage [8]. Bacterial pigments namely prodigiosin, violacein, melanin, etc. are reported for their UV protection properties [9–11]. Pigments of microalgae namely phycocyanin, lutein, ß-carotene, Zeaxanthin, etc. are reported for various biological activities and used in pharmaceutical, nutraceutical and cosmeceutical industries [12, 13]. Besides, unique characteristics of bio pigments such as antimicrobials, antioxidants, anticancer, anti-obesity and anti-inflammation make them essential in many areas of use, such as food, textiles, printing, pharmaceutical, cosmetics, etc. [14, 15]. Some of the UV protecting bacterial pigments have antibacterial and antioxidant activities also. This study aimed to explore the bioactive bacterial pigments with antibacterial, antioxidant activities along with photoprotection.

Pigment producing bacteria were isolated from soil samples collected from sites that are naturally disturbed or disturbed by anthropogenic activities. Earlier reports suggested that bacteria produce pigments to protect themselves from the harsh conditions; hence, the chances of getting pigmented bacteria were more from these sites. Total of 15 soil samples and 1 water sample was collected from different location of Gujarat (Table S1). A total of 19 pigment-producing bacteria were isolated and purified from the collected samples (Figure S1). The isolates were given experimental codes (Table S2). The effect of standard culture conditions on pigment production properties of isolated bacteria was observed on solid (Nutrient agar plate) as well as liquid (Nutrient broth flask) media up to five transfers as it was observed that location induced secondary metabolite production (in this case pigments) stops in standard culture conditions. Results of the experiment showed that JVP coded isolate stops producing colour in the first transfer in both the media. TLY also lost its colour production in the first transfer but only in liquid media. Hence, these two isolates were eliminated from further experiments. UWGY and UUY coded isolates lost its colour production after second and third transfers respectively in liquid media only. Also, a change in colour production was noticed in JFO after 3rd transfer i.e. orange to yellow in liquid media (Table S3). Therefore, UWGY, UUY, and JFO are useful only in batch culture or on solid medium.

The initial activated culture of the remaining 17 pigment-producing isolates was inoculated in 300 ml nutrient broth and incubated for 48 h at 37 °C in shaking condition. After an incubation period, cultures were centrifuged and crude pigment (approx. 5000 µg) was extracted from pellet using methanol, centrifuged, filtered and allowed it to dry through evaporation [16] (Figure S2). All these crude extracts of pigment were analyzed to assess Sun Protection Factor (SPF) by a slightly modified protocol of Mansur et al. [17]. All 17 extract showed SPF in a range of 0.4–8.34. Among all, crude extract of BUY1, JUFO, JFY, JUFY1, UUY, JFO, and JVY gave good SPF values. BUY1 and JFY possessed the highest SPF values 8.34 and 7.72 respectively. These two pigments showed the potential to be developed as an ingredient in SPF products. Earlier reported SPF values of orange, red and pink pigments are 1.785, 1.629 and 2.72 respectively [18]. However, crude extracts of orange, red and pink pigments of our study gave more than the above-mentioned values (Table 1).

Table 1.

SPF values of isolated pigments

Sr. no	Sample	SPF20	Difference	SPF 24	Difference
1	BUY1	25.763	5.106	32.387	8.34
2	BUY2	23.348	2.691	27.557	3.519
3	BUP	23.279	2.622	24.935	0.897
4	UUY	23.96	3.303	30.731	6.693
5	UUP	22.313	1.656	27.971	3.933
6	UWGY	26.384	5.727	27.695	3.657
7	JUFO	28.247	7.59	31.697	7.659
8	JUFY1	25.556	4.899	30.455	6.417
9	JUFY2	23.969	3.312	25.901	1.863
10	JUFY3	23.483	2.826	29.006	4.968
11	K1Y	22.658	1.999	29.144	5.106
12	K2O	22.52	1.863	26.867	2.106
13	K2Y	21.071	0.414	27.143	3.105
14	JFO	23.279	2.622	27.55	3.512
15	JFY	28.385	7.728	29.834	5.796
16	JVY	22.106	1.449	29.282	5.244
17	SR	21.692	1.035	26.039	2.001

Bold numbers show higher range of SPF

Concurrently, these extracted crude pigments were evaluated for their antibacterial activity. Gram-positive bacteria—Bacillus subtilis (MTCC 736) and Staphylococcus aureus (NCIM 2079) and Gram-negative bacteria- Escherichia coli (NCIM 2065), Pseudomonas aeruginosa (NCIM 2200) and Salmonella abony (NCIM 2257) were used to assess antibacterial activity using agar well diffusion method [19]. Results revealed that the average zone of inhibition was between 13.00 ± 0.57 and 34.33 ± 0.33 mm (Table 2). The extract of JFO showed activity against all five test bacteria. Hence, JFO could be developed as an antibacterial coloured ingredient in cosmetic products. K2O, SR, and JVY showed activity against 3 test bacteria—E. coli, B. Subtilis, and S. aureus. UUY showed a zone of inhibition against B. subtilis and S. aureus. BUY2 showed a zone of inhibition only against B. Subtilis (Figure S3 and S4). Further, Minimum Inhibitory Concentration (MIC) of crude pigment extracts showing antibacterial activity was analyzed using the broth microdilution method [20]. The highest MIC was > 5000 µg/ml and the lowest MIC was 625 µg/ml. Crude JFO pigment was effective against both Gram-positive as well as Gram-negative test bacteria with different range of MIC (Table S4). Therefore, this pigment could be developed and used as a broad-spectrum antibacterial coloured component in cosmetic products.

Table 2.

Average zone of inhibition of pigments against test bacteria

Pigment code	Average zone of inhibition (in mm) ± SEM
	E. coli	S. aureus	B. subtilis	P. aeruginosa	S. abony
BUY1	–	–	–	–	–
BUY2	–	–	15.66 ± 0.33	–	–
BUP	–	–	–	–	–
UUY	–	13.00 ± 0.57	18.33 ± 0.66	–	–
UUP	–	–	–	–	–
UWGY	–	–	–	–	–
JUFO	–	–	–	–	–
JUFY1	–	–	–	–	–
JUFY2	–	–	–	–	–
JUFY3	–	–	–	–	–
K1Y	–	–	–	–	–
K2O	16.00 ± 0.57	20.33 ± 0.33	23.33 ± 0.33	–	–
K2Y	–	–	–	–	–
JFO	25.33 ± 0.88	25.33 ± 0.88	30.66 ± 0.88	21.00 ± 0.57	23.33 ± 0.33
JFY	–	–	–	–	–
JVY	13.33 ± 0.33	13.33 ± 0.33	18.33 ± 0.33	–	–
SR	21.00 ± 0.57	16.66 ± 0.33	19.33 ± 0.33	–	–
Positive control (Streptomycin)	28.66 ± 0.88	27.00 ± 1.15	34.33 ± 0.33	27.66 ± 0.33	28.33 ± 0.57

Antioxidant plays an important role in different health care sectors including cosmeceuticals. Many skin disorders such as skin cancer, acne, etc. are associated with the generation of reactive oxygen species (ROS) [21]. The radical scavenging activities of the aforementioned potential pigments at different concentrations ranging from 312.5 to 5000 μg/ml were carried out using 2-2 diphenyl-1-picrylhydrazyl (DPPH) method in 96 well plates [22]. The results of this experiment indicated that crude extract of pigment coded BUY2, UUY, and SR exhibited moderate antioxidant activity with an EC₅₀ value of 1376.04, 1367.46 and 1793.2 μg/ml, respectively (Fig. 1). Hence, these three pigments could be developed and used as antioxidant ingredients in cosmetic products.

Fig. 1.

Antioxidant activities of potential pigments at different concentrations

In further experimentation, we assessed the effect of temperature and pH on pigment yield. All aforementioned six bacteria were individually streaked on nutrient agar plates by direct streaking method and incubated at a different temperature—17 °C, 25 °C, 37 °C and 45 °C for 48 h. Growth and total yield of pigment (in mg/ml) were assessed. No growth was detected at 45 °C, except bacteria coded JFO and K₂O. All bacteria had different optimum growth temperature (Figure S5). Next, all six bacteria were subjected to 3 different pH: 5 (acidic), 7 (neutral) and 9 (basic) and incubated on their respective optimum temperature. At acidic pH, bacteria coded BUY2, UUY, and JVY did not show any growth while K2O, JFO, and SR showed little growth with pigment production. All the bacteria showed almost similar results at pH 7 and pH 9 (Figure S6).

Based on the results of bioactivity and growth parameters, we found that among all 6 potential bioactive pigment-producing bacteria- K2O is a promising one and was identified using 16 s rRNA gene sequencing (accession number MK770403). BLAST analysis in NCBI revealed the sequence was more likely matched with Staphylococcus xylosus strain KL162 (Figure S7). However, the reported study suggested that this strain does not produce any pigment. So, this isolated pigment-producing bacterium belongs to the same genus and species, but the subspecies or strain might differ. It might be the same strain but there is a possibility of a mutation in gene due to anthropogenic activities, so it started produced pigment.

In summary, the results of the current study concluded that diverse pigment-producing bacteria are mostly present at stress conditions or it can be started producing pigment in stressful environmental conditions. From the results of the SPF experiment, we can conclude that BUY1 and JFY exhibited the highest SPF values; therefore, these two pigments could be developed further for sunscreens products. According to antibacterial observation, JFO gave the broad-spectrum antibacterial activities against all five test bacteria. Further, this pigment could be analyzed against other bacteria and fungi which are responsible for skin diseases and developed as a potential and novel antibacterial candidate. The results of an antioxidant assay depicted that UUY and BUY2 pigments could be utilized as an antioxidant in cosmetic products. K2O produces the promising pigment, out of all six potential pigments. It is stable up to 5 transfers, also possessed antioxidant as well as antibacterial activity against S. aureus, B. subtilis, and E. coli bacteria and average SPF value was 2. Hence, it could be further developed and used as antibacterial, antioxidant and sun-protective ingredients in cosmetic products. These pigments may also possess other pharmaceutical properties, for that extension of this research is required.

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