Analysis of Carotenoids and Identification of Mangrove Sediment Bacteria of Segara Anakan, Cilacap

The excessive consumption of artificial dyes can lead to negative effects on human body. Thus, the invention of natural dyes, such as carotenoids, is needed in order to reduce the negative impacts. Carotenoids are yellow, orange, and reddish yellow pigments produced by plants, animals, algae, and microorganisms. This study was aimed to analyze the carotenoid pigments of mangrove sediment bacteria in Segara Anakan, Cilacap, and to identify species of bacteria that can produce carotenoids. Carotenoids were analysed by using Thin Layer Chromatography (TLC) and UV-Vis spectrophotometer. Meanwhile molecular identification of bacteria was carried by 16S rDNA PCR and DNA sequence was analysed through a program called Basic Local Alignment Search Tool (BLAST). The kinship of bacteria was shown in Phylogenetic tree by using Bioedit and MEGA 5 software. Qualitative analysis by using TLC produced several pigments like: β-carotene, β-isorenieraten, lycopene, flavonoids, chlorophyll a, chlorophyll b and a feofitin with Rf value of 0.36-0.95. Quantitative analysis showed that bacteria KH (greenish yellow), KT (dark yellow) and KM (light yellow) produced carotenoids at 95.30 g.g-1, 110.34 g.g-1 and 25.349 g.g-1. KH, KT and KM were suspected to be bacteria known as Streptomyces chartreusis, Bacillus megaterium and Streptomyces chromofuscus with similarity of 99%. Mangrove sediment bacteria had the potential of producing carotenoids as an alternative of eco-friendly natural dyes.


Introduction
In the market, dyes are very often added to food. The use of dyes that is excessive, inappropriate and harmful needs to be avoided because it can have negative impacts on health. Natural dyes can be used as an alternative solution in dealing with the harmful effects of the use of artificial dyes. Natural dyes can be obtained from plants, animals, micro-algae (cyanobacteria), heterotrophic and phototrophic bacteria, and fungi (Britton, 1998;Kurniawan et al., 2010;Gupta et al., 2011). Natural dyes that have been mass produced are β-caroten from Dunaliella, astaxanthin from Haematococcus, and lutein from Chlorophycea (Campo et al., 2007). Bacteria that are known to be the producers of carotenoid are Streptomyces sp., Brevibacterium maris, Paracoccus alcaliphilus, Rhodotorula rubra, Bradyrhizobium sp. (Hannibal et al., 2000;Simova et al., 2004;Baskar et al., 2010;Arlita et al., 2013).
Bacteria are potential organisms utilized in the health and industry sector. Natural color pigment or substance that can be produced by bacteria has positive impacts on human health. Palanichamy et al. (2011) said Streptomyces sp. isolated from sediments on the coast of Chennai, India, is potential as a pigment producer. The new acyclic carotenoids and monocyclic C40 carotenoids were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens and novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes respectively (Shindo and Misawa, 2014).
The utilization of bacteria as the producers of carotenoid pigment can be an alternative to gain dyes that are eco-friendly and safe to be consumed by human. This research is aimed to analyse carotenoid pigment produced by mangrove sediment bacteria of Segara Anakan, Cilacap and to identify bacteria that can produce carotenoid pigment.

Material and Methods
The content of carotenoid was measured by spectrophotometry method (Dharmaraj et al., 2009). Carotenoid was extracted in accordance with Dharmaraj et al. (2009) with modifications. Bacterial isolates were inoculated in a starch casein medium with pH 7 of 100 ml, and incubated in a shaker with a rotation speed of 180 rpm and temperature of 28C for 120 hours. Furthermore, the solution was centrifuged at 6000 rpm for 15 minutes. Bacterial cells were taken and extracted by using maceration method with acetone and methanol (7:3). Then, the extract is centrifuged and dried by using nitrogen gas.

Meaurement of carotenoid's quantity
Meaurement of carotenoid's quantity (Sahara et al., 2013), was done by using UV-Vis Scan Spectrophotometer with a wavelength of 400-600 nm. Total carotenoid was counted by using sample comparison with the standard of β-carotene curve (Musfiroh et al., 2008).

Molecular identification of mangrove sediment bacteria that produce carotenoids
DNA was extracted in accordance with Margavey et al. (2004) with modification. A total of 1 ml of liquid culture of bacteria was put into a 1.5-mL tube, and then it was centrifuged for 5 min. Next, pellet was homogenized with 400 μlTE and, again, centrifuged for 5 min. 400 μl of SET solution and 50 μl of Lysozim solution were added and incubated at a temperature of 37 0 C for 1 h. 50 μl of SDS solution were added and then incubated at a temperature of 65 o C for 1 h. Furthermore, 167 μl of NaCl solution were added and incubated at the the temperature of 65 o C for 1 h. 400 μl of cool chloroform solution were added and then incubated at room temperature for 30 min. After 30 min, the mixture was centrifuged for 10 minutes and supernatant obtained was moved to a new tube and then a solution of isopropanol in the ratio of 1:1 was added and incubated at -20 0 C for 24h. The samples then were centrifuged for 5 min. The pellet was homogenized with cool ethanol 70%. The ethanol was evaporated and the pellet formed was dried by wind. The pellet then was resuspended with 50 μl of TE solution.
The results of DNA sequences in bacteria were compared to DNA sequences in the DNA data center. The search was performed with the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information, National Institute for Health, USA (www.ncbi.nlm.nih.gov) (Atschul et al., 1997). Then, a phylogenetic tree was made from DNA sequent by employing Bioedit and MEGA 5 software (Zhang et al., 2008).

Analysis of carotenoid pigments
Observations based on the color samples of the bacterium used three test samples. Bacteria that contained carotenoid pigments had several color (Figure 1.).
The value of Rf (Table 2.) shows that the extract with KH code allegedly contained β-carotene, flavonoids, feofitin a, chlorophyll a, and chlorophyll b pigment. The one with KT code was suspected to contain β-carotene, flavonoids, and chlorophyll a pigment. While extract with KM code was suspected to contain β-Isorenieraten pigment, chlorophyll a, chlorophyll b and lycopene pigment. research would only discuss about carotenoid pigment groups that were β-carotene, β-Isorenieraten, and lycopene. The movement of nonpolar carotene was faster, marked with the higher Rf than that of other pigments (Rizkina et al., 2013) so that easily identified trough visual observation. According to Gross (1991), yellow-orange dye is a sign of carotene pigment group, green-blue is chlorophyl a, while light yellow is xanthophyll pigment group. Carotenoids are divided into two main groups namely polar carotenoid (xanthophill) and non-polar carotenoid non polar (carotene).
The result of TLC was confirmed by employing UV-Vis spectrophotometer. Based on the analisys of the rough extract of KH, KT dan KM pigment, a spectrum pattern with 3 peaks in the wavelength of 400-600 nm (Figure 2.) could be obtained. They were indicated as a carotenoid pigment (Sahara et al., 2013).
The result of measurement shows that KH extract was indicated for containing carotenoid pigment with three peaks, which were 468.5, 439.5 and 416.0 nm. KT extract shows that maximum wavelengths were 653.5, 474.5 and 364.5. In addition, apparently KM extract contained carotenoid pigment that had wavelengths of 467.5, 439.0 and 416.0. Those three extracts samples are included in carotenoid wavelength category, which is 400-600 nm (Sahara et al., 2013). Besides the comparation between the wavelength of the samples and that of the references, the fathoming of carotenid content was also held by comparing the curve of spectrophotometer with the standart carotenoid curve of Laughlin et al. (2002). Laughlin et al. (2002) states that pure β-carotene, when it is measured with the wavelength of 210-700 nm, shows a peak in the wavelength of 452 nm.
It is supported by Barredo (2012) who explain that maximum wavelengths of β-carotene pigment's are 430, 453 and 447 nm; those of Lycopene are 445, 457 and 504 nm; those of Zeaxanthin are 427, 453 and 447 nm; and those of Lutein are 427, 448 and 472 nm. While Cantaxanthin and Astaxanthin only has one maximum wavelenght, that is 470 and 477 nm, repectively. The result of β-carotene degree determining by using ray spectroskopy method appeared on the pigment of sediment bacteria as shown on Figure 3 and Table 2.
The regression equation for the curve above in the concentration range of 5-50 ppm was y= 0.0662x -0.0068 with R 2 = 0.99. the result shows that the level of beta carotene of KT was higher than that of KH and KM. β-carotene concentration in KH and KT were exceed the "limit of quantification" while the concentration of KM could be counted accurately. The problem related to the limited supply of rough extract of bacteria that produced KH and KT pigment curbed us in validating the consentration of β-carotene of KH dan KT extract. KT extract's level of β-carotene is higher than that of other extracts seen from the consentration of the measurement result by using regression equation. Besides, the dark yellow dye owned by KT extract indicated that its carotene content was high. It is supported by Britton et al. (1995) that carotene pigment has orange hue. The production of pigment by microorganisms is affected by several factors such as pH, temperature, source of carbon and nitrogen, and time of incubation (Chintapenta et al., 2012).
Carotenoid content produced by KH, KT and KM bacteria was less than that produced by Micrococcus sp. Isolated from the sediment, Canada. A research conducted by Ibrahim (2008) shows that Micrococcus sp. produces 430 g.g -1 of carotenoid. It means sediment bacteria can produce carotenoid more than carrot and purple carrot, that is between 200 and 300 μg.g -1 (Lee et al., 2011). Ibrahim (2008) says that there are some factors affect the production of carotenoid besides light, that are pH, rate of centrifuge and the temperature of incubation.

Identification of sediment bacteria producing carotenoid pigment
Bacteria used in the process of identification were labelled KM, KH and KT. KT bacteria (Figure 4.) were assumed as a part of Bacillus megaterium and the level of similarity was 99%. The result of the research shows that Bacillus megaterium could produce dark yellow pigment. Based on the research held by Mitchell et al. (1986) in Khaneja et al. (2009) it is known that Bacillus megaterium can produce red pigments. According to Khaneja et al. (2010) there are some Bacillus spesies that can produce carotenoid, such as B. marisflavi, B. indicus, B. firmus, B. altitudinis and B. safensis with yellow, orange and pink detected on the wave length of 455, 467 and 492 nm. It is supported by the research of Duc et al. (2006) that discovers Bacillus indicus produces yellow carotenoid. Nugraheni et al. (2010) in their research about the characteristic of carotenoid of bacterial symbionts in seagrass, Thalassia hemprichii, find that B. licheniformis produces orange carotenoid. Bacillus Genus produce carotenoid pigment with optimum pH, that is 7 (Khaneja et al., 2010).
KM and KH bacteria were Streptomyces chromofuscus and S.chartreusis that were 99% similar. Some types of Streptomyces that have been examined previously are also reported for containing certain kinds of carotenoid for instance the one identified by Dharmaraj et al. (2009), Streptomyces sp., isolated from Callyspongia diffusa sponge produces lycopene pigment with the maximum wave length is 445 nm and it's Rf value is 0.40. Next, Dharmaraj et al. (2009)    S.coelicolor and S. violaceoruber bacteria from the sediment of sea and soil in India as the producers of actinorhodin pigment that has red-blue dye. Bose et al. (2013) in their research state that Streptomyces sp. T1027, lighted by a lamp during the culture, can produce β-carotene pigmen. Baskar et al. (2010) also discovered that Streptomyces sp. isolated from some spesies of fish such as Tendania anhelan, Epinephelus diacanthus, Cyprinus carpio and Osepherenemous gourami can produce several tyoes of carotenoid like phytoene, phytofluene, αcarotene, β-carotene and β-isorenieratene.