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Extraction and Purification of Phycocyanin and Their Radical- Scavenging Activity from Multi - Stress Spirulina Isolated from Marine Water

T. Srinivasan*, S. Illanjiam

Department of Microbiology, Hindustan College of Arts and Science, Padur, Kelambakkam, Chennai - 603 103, Tamil Nadu, India

Abstract: The S. platensis were isolated from the marine water were mass mass cultivated and extracted they were partially purified of phycocyanin pigment by SDS -PAGE . In the present study the extracts were partially purified and with some organic impurities and subjected to 7.5 % of SDS-PAGE gel the strain S-I resembles the almost the molecular weight around 110 kda when compared to standard reference strain under multi stress condition. Similarly in the present study, reported that the higher amount of scavenging activity was recorded at 100μg ml-1 (70.54%) in S1-I and the control was recorded at 84.6%.

Keywords: Multi-stress, S. platensis, Scavenging Activity

Received November 02, 2020; Revised December 03, 2020; Accepted December 10, 2020

Citation: T. Srinivasan, S. Illanjiam, Extraction and Purification of Phycocyanin and Their Radical- Scavenging Activity from Multi - Stress Spirulina Isolated from Marine Water. Applied Ecology and Environmental Sciences, 9 (1). 73-75. January 2021. doi:10.12691/aees-9-1-10

1. Introduction

The name “Spirulina” is derived from the Latin word for “helix” or “spiral”, referring to the physical structure of the organism. It is motile multicellular filamentous blue-green algae and reproduced by binary fission. The ability of Spirulina to grow in hot and alkaline environments ensures its hygienic status, as no other organisms can survive to pollute the waters in which this alga thrives.

Spirulina platensis was efficiently cultivated and harvested in Zarrouk's medium. The nutritional Analysis of Spirulina platensis was done initially to establish its significant role in nutraceuticals and its need for commercialization. Inducer studies were done with respect to light inclination, Urobilin and H2O2 to check any possibility of improvement in yield of Spirulina platensis. The corresponding factors for phycocyanin content increase and its associated antioxidant potential were studied. The study focused its concern on propagating Spirulina platensis as a efficient source of nutrition with induced antioxidant property and revealed that additional light and H2O2 promotes the nutrient composition of Spirulina platensis.

Phycocyanin (PC) from Spirulina platensis (CCC540) by using ammonium sulphate precipitation, followed by a single step chromatography by using DEAECellulose-11 and acetate buffer. Precipitation with 65 % ammonium sulphate resulted in 80 % recovery of phycocyanin with purity of 1.5 (A620 /A280). Thro1ugh chromatography an 80 % recovery of phycocyanin with a purity of 4.5 (A620 /A280) was achieved. In SDS_PAGE analysis, the purified PC showed the presence of two subunit a (16 kD) and b (17 kD) [1].

2. Materials and Methods

2.1. Mass Cultivation of Spirulina platensis in Zarrouk’s Medium

The S. platensis cultivation was carried out in 1000L shallow glass tanks during the February- March 2019. of S. platensis was inoculated in medium. The growth medium was mixed to prevent algal accumulation at the surface and to ensure uniform distribution of nutrients. The growth parameters such as biomass various methods described earlier.

2.2. Harvesting and Processing of S. platensis

After the inoculation, when the culture medium turns light green into dark green colour with algal mat, then the algal biomass was ready to harvesting process. The biomass was filtered through the fine nylon filter by gravity filtration. Filtered algal slurry was washed twice with sterile distilled water to remove the impurities.

2.3. Purification

The crude extract was subjected to a single step precipitation using 65 % (NH4)₂ SO4 and kept overnight at 4°C. The pellet was recovered by centrifugation at 27,000 rpm for 15 min at 4°C and dissolved in 10 ml of the same extraction buffer and termed as ammonium sulfate extract (ASE).

2.4. SDS-PAGE

An 7.5 % continuous PAGE under non-denaturing conditions was carried out to reconfirm the purity of phycocyanin. The bands were visualized by Coomassie blue staining. Molecular weight of the purified phycocyanin was determined by running Novex Sharp pre stained protein marker along with the sample.

3. Results & Discussion

3.1. Isolation and Identification of Spirulina from Different Marine Water Bodies

The microalgal genera were identified based on their morphological characters like such as colour of scum, shape, filamentous, type of floating and presence of vacuoles under the low and high power of light microscope and pH tolerance of the algae. Among the five samples Spirulina species were isolated from three locations viz., Ennore,(S1) Kovalam (S2), Kalpakkam (S3).

3.2. Mass Cultivation of Spirulina Platensis S-I in Zarrouk’s Medium

The mass cultivation of S.platensis S-1in seawater medium were conducted period of February - March. The growth was increased with increase in growth period upto 30 days. In February March 2019 average day time temperature normally above 39°C. The S.platensis S-I showed highest level of biomass (0.432 g ml-¹).

3.2.1. Growth of Spirulina Platensis S-I Strain in Outdoor Condition

Table 1.

3.2.2. Estimation of Antioxidant Activity against DPPH Radical-Scavenging Activity of Spirulina S1 in Zarrouk’s Medium (In-Vitro)

The antioxidant activity of mutant strains of Spirulina S1 against DPPH free radical were determined and results are present in Table 2.

The antioxidant activity of Spirulina S-I strains were studied at different concentrations (viz., 10, 25, 50, 75 and 100 μg ml⁻¹) of Spirulina organic extract and compared with positive control. The Spirulina strain S-I was recorded the higher level of free radical scavenging activity against DPPH. The S-I strains, the higher amount of scavenging activity was recorded at 100μg ml-1 (70.54%).

Table 2.

3.2.3. Extraction and Purification of Phycocyanin Pigment

Extraction and purification of phycocyanin, was completed by major steps: crude extract preparation ammonium sulfate precipitation. Purity was also reconfirmed by the presence of single bands of a - subunit (112kDa) during SDS-gel electrophoresis

Figure 1.

In the later 1980s, an important experiment on large-scale (3000 mE) cultivation of Spirulina in seawater medium with a fully mechanized process of agitation, harvesting and spray- drying was initiated in Huilai, Guangdong Province, by the South China Sea Institute of Oceanology, CAS and sponsored by the National Key Program [2].

[3] have reported that, the procedure for the cultivation of Spirulina in untreated, fertilised seawater was developed. Use of this cultivation system has resulted in production of 7.3 g/m²/day in seawater supplemented with urea, and 5.2 g/m²/day in seawater supplemented with nitrate. The advantages of cultivating Spirulina in seawater medium (compared to freshwater medium) are: (1) lower fertilizer cost; (2) the medium after harvest can be repeatly used; (3) the pH of the seawater medium constantly remains at 9 - 10 and need not be adjusted; (4) seawater culture is not easily polluted by heavy metals and contaminants. Hence the yield and quality are both improved [2]. They have studied that, Spirulina cells are grown in outdoor ponds for mass cultivation, they are exposed to various stress conditions, including high temperature stress. During daylight hours in tropical countries, the cells are exposed to high temperatures of around 40°C. The temperature fluctuation in outdoor mass cultivation has a serious effect on biomass yield and the biochemical content of the cells. Temperature increase from 35°C to 40°C, the level of the polyunsaturated fatty acid γ-linolenic acid (GLA) in Spirulina platensis decreases approximately 30%, compared to the level found in cells grown at an optimal temperature (35°C).

Similarly, in the present study reported that, the mass cultivation of Spirulina S1 in Zarrouk’s medium was conducted in glass tank with surface area 4m² (1000 L), during summer season at two different period of (February - March). In season, February - March, 2019 average day time temperature normally above 39°C. The highest biomass and proximate composition of Spirulina S1 was recorded.

The multi stress tolerant Spirulina strain S1 was found to be suitable for mass cultivation in Zarrouk’s medium during this season. The study proved this season in India is suitable for mass production of Spirulina in Zarrouk’s medium. [4] have reported that, the Spirulina was cultivated under salt stress conditions (0.02 M as control), 0.04 and 0.08 M NaCl led to a remarkable alteration of algal.

Metabolism as well as an enhancement or induction of biologically active compounds. The salt stress conditions caused growth with decrease in dry weight, chlorophyll a content as well as certain xanthophylls (neoxanthin and violaxanthin) while β-carotene production was stimulated especially at higher salt concentrations. The ethanolic algal extract (100 μg/ ml at 0.08 M NaCl) exhibited the highest antioxidant activity compared with those of the synthetic antioxidant butylated hydroxy anisol as standard (85.0, 89.9 and 86.0, 91.8% respectively). Similarly in the present study, reported that the higher amount of scavenging activity was recorded at 100μg ml-1 (70.54%) in S1-I and the control was recorded at 84.6%.

3.3. Extraction and Partial Purification of Phycocyanin Pigment by SDS- PAGE

An efficient means of separation and purification of C-PC from three different cyanobacterial species, i.e., Spirulina, Phormidium, and Lyngbya spp. is achieved through easy and single step chromatographic method. The results of the purification process corroborate the efficiency of the methods employed. The purity ratio of C-PC at the end of the process was nearly 4.4, which is more than the reported value for pure C-PC [5]. The molecular weight of native C-PC determined by gel filtration chromatography is almost similar for Spirulina (112 kDa) and Phormidium sp. (131 kDa) but lower for Lyngbya sp. (81 kDa). The absence of inorganic impurities was confirmed by IR spectra, which also indicates -helix as the major element of its secondary structure and lack of b -sheets. SDS-PAGE yielded two bands corresponding to the a and b subunits. In the present the extracts were partially purified and with some organic impurities and subjected to 7.5 % of SDS-PAGE gel the strain S1 resembles the almost the molecular weight around 110 kda when compared to standard reference strain.

4. Summary & Conclusion

Spirulina strains, based on their biomass, content of the efficient strains S1 were selected for further studies on Zarrouk’s medium. The strain Spirulina- S1 exhibited the best growth, biochemical constituents and antioxidant activities with multi-stress tolerant ability and selected for mass production process. The mass cultivation was carried out in glass tank (1000ml) at two different periods, February- March 2019. The growth parameters were increased with increase in growth period up to 30th day. The Spirulina S1 showed highest level of biomass (0.432 g ml⁻¹), were recorded in 30 days. The Spirulina S1 were extracted and partially purified for the antioxidant scavenging activity and purification of phycocyanin pigment by SDS -PAGE and light expression studies were done. The multi stress tolerant Spirulina S1 was found to be suitable for mass cultivation in Zarrouk’smedium and the Phycocyanin produced in high amount under the multi stress condition when compared to other strains

References

[1]   | Soletto, D., Binaghi, L., Lodi, A., Carvalho, J.C.M. and Converti, A. Batch and fed batch cultivations of Spirulina platensis using ammonium sulphate and urea as nitrogen sources. Aquaculture, 2005; 243: 217-224.
[2]   | Wu, B., Tseng, CK and Xiang,W. Large- scale cultivation of Spirulina in seawater based culture medium. Botanica Marina, 1993; 36(2): 99-102.
[3]   | Tomaselli L.. Morphology, ultrastructure and taxonomy of Arthrospira (Spirulina) maxima and Arthrospira (Spirulina) platensis.InVonshak, A., ed. Spirulina platensis (Arthrospira): Physiology, cell biology, and biotechnology, 1997; pp: 1-16. London, Taylor and Francis.
[4]   | Shalaby, EA., Shanab, SMM. and Vikramjit Singh. Salt stress enhancement of antioxidant and antiviral efficiency of Spirulina platensis. Journal of Medicinal Plants Res, 2010; 4(24): 2622-2632.
[5]   | Boussiba S., Richmond A. Isolation and purification of phycocyanins from the blue- green alga Spirulina platensis. Arch. Microbiol, 1979; 120: 155-159.