1Govt. Institute of Science, Chhatrapati Sambhajinagar, MS, India-431004.
2Neoliva Lifesciences, Nanded, MS. India -465001.
Phytochemical research is expanding in a search of good alternative for chemical entities used in modern industries. A versatile bioactive compound from algae such as chlorophyll, xanthophyll, phycobiliproteins and carotenoids have been proved to contribute significantly in nutritional, medicinal, therapeutic and cosmetic industry. This group of biomolecules proved to have a prominent, promising future in agriculture, health and biotechnology. The present study characterizes the biophysical and biochemical properties of algae pigment Phycocyanin from Spirulina platensis from the Lonar lake of Maharashtra.
Most of the aquatic inhabitant are fulfilling the need of growing human population from very indigenous oxygen supply to the numerous useful biomolecules. The search towards the underutilized abundant natural alternative resources for food, feed and drug become imperative. Research community has been attracted towards the most diverse aquatic group the algae, to meet this rising demand especially for food and medicine. Naturally algae are found to adopt with numerous extremes habitat such as artic ecosystem, fresh water, sea water, hot springs, saline water and hyper saline lakes etc. with high growth rate. (Arwind et.al 2021). Unlike plants, algae do not have structural features but contributes for the major oxygen production through photosynthesis.
Ancient civilization noted with the numerous evidences of the use of algae as a source of nutrition especially a protein as well as medicine. Spirulina from Texcoco lake in the ‘Chad’ region of central Africa was utilized as the main source of protein supplement for worriers in the form of dried cakes called ‘dihe’. Various sea weeds and algae are important constituents in the soups and salads to enhance flavour and nutritional value in western countries. Thus, algae are emerged as a super food revealing this traditional treasure by modern biotechnology research.
Algal phytopigments such as chlorophyll, xanthophyll, carotenoids, Phycobiliproteins etc. have been studied for their nutritional, medicinal properties. Most of them have marked their industrial significance with prominent antioxidant, antimicrobial, anti-inflammatory as well as radioprotective properties. Natural fluorescence of these pigments is useful in imaging and biosensing.
Phycocyanin a group of phycobiliproteins from blue green algae such as Spirulina from different sources is found to have pharmacological properties. This work is devoted to study the physicochemical and toxicological characterization of the pigment phycocyanin from Spirulina platensis, from the unique site, the Lonar lake from Maharashtra, India. The lake was created approximately fifty thousand years ago because of the impact of the hypervelocity meteorite by creating a crater in basalt rock. Because of its high alkalinity of water, Lonar lake is called as soda lake. It is having diameter of 2 km with the depth 450ft, without any natural outlet. (Adam C. Maloof et al. 2010). The Lonar lake is an exceptional ecosystem because of its salinity and alkalinity which provides unique habitat for flora and fauna to create large diversity. Cyanobacteria found to have remarkable adoptability with extreme environmental conditions like pH, temperature, salinity and alkalinity, these organisms are found to adopt high salinity by producing osmolytes to regulate internal solute concentration. (H. Kauss 1978), (Keller et al. 1999). The ionic constituents of the lake include chlorides, sulphates, sodium. carbonates and bicarbonates. Soluble carbonates provide high buffering capacity to the water. (Foti et al. 2008). The unique equilibrium between HCO3–, CO32–, and CO2 is the reason behind high productivity of this ecosystem. (Grant et.al, 2004) supporting the growth of cyanobacteria, Spirulina platensis, marked as a major primary producer at this ecosystem (Deshmukh and Puranik 2020).
2: MATERIAL AND METHODS
2.1 Collection of Sample and physicochemical analysis
As the hemo- cytometric measurement of water from Lonar lake was reported highest during the month of July -August. (Deshmukh and Puranik 2020), the water sample was collected in the month of August, with high biomass. (Fig-1) Water was analysed preliminary at the collection site and recorded with a temperature 360c and the pH 8.6 (pH strip Fisher Scientific 20105). Further analysis was carried out in the laboratory. Physicochemical parameters such as colour, odour, temperature, pH, total dissolved solutes (TDS), total dissolved oxygen (TDO), concentration of Sulphate, phosphate, magnesium calcium and nitrates etc. of the water samples were recorded.
2.2: Identification and Cultivation of Spirulina platensis
Preliminary microscopic analysis of water to mark the presence of Spirulina platensis was carried. Spirulina was identified and authenticated from the research laboratory of Neoliva Lifesciences Nanded. Isolated Strain of Spirulina platensis was selectively cultured in Zarrouk’s medium which is a standard medium for Spirulina cultivation. (Zarrouk C. 1966) Spirulina platensis was repeatedly cultured for its purity check in modified Zarrouk’s medium (C. Rajasekaran et.al.2016) slightly modified by replacing KNO3 instead of NaNO3. All the chemicals were purchased from HI media, Mumbai.
2.3: Optimization of Culture Conditions
Classical method of formulation and optimization of culture medium was used to obtain mass production and high protein content, using few cost-effective alternative components to rise the nutrient content of standard Zarrouk’s medium. Final composition was decided by observing the ratio of dry biomass, growth time and amount of phycocyanin obtained. The inoculum about 10% v/v was taken in a conical flask (Set of 3 flasks) containing 150 ml of modified Zarrouk’s medium from the primary culture at a temperature 30 ± 2 °C, pH 8.6 The flasks were kept on orbital shaker for agitation at 80 rpm. The illumination cycle of cool white fluorescent light of 2500 Lux was selected for the photoperiod of 12/12 hours; light and dark cycles were maintained.
Growth density was analysed spectrophotometrically (Chemito UV 2100) at 540nm with a regular interval of 3 days by the method of Dalgaard and Koutsoumanis (2001) and the absorbance was converted to biomass. On the 30th day the estimated biomass was used for further studies.
2.4: Extraction and Purification of the phycocyanin
2.4.1: Isolation of C- Phycocyanin from Spirulina
The powder of Spirulina platensis was mixed in 100 ml of distilled water in proportion of 1:25 and mixed well. The mixture was incubated at 4°C for 24 hours. After incubation sample was centrifuged at 10,000 g for 15 min at 4°C. The pellet containing cells was discarded and the supernatant containing phycocyanin pigment was used for further procedure.
Precipitation with ammonium sulphate was carried out as the sample containing pigment was treated with 60% concentration of ammonium salt of amount 36.1 g for 100 ml. The powder was added gradually with continuous stirring. Complete dissolving of salt yielded precipitate containing phycocyanin. The precipitate was further centrifuged at 12000 g for 20 min at 4°C. The supernatant was discarded and collected the pellet with phycocyanin.
Dialysis was performed by using dialyses membrane-70, MWCO: 12–14 kD procured from Hi-Media . Ammonium Sulphate Extract (ASE) was prepared by using 0.05M phosphate buffer of pH-7. ASE (5 ml) is filled in pre-activated membrane and suspended in same buffer for overnight on magnetic stirrer at 4°C. Separation of colour pigments was achieved by spotting the sample over pre-coated TLC plates of size 2x10cm, 1cm from the bottom end and run using Hexane: acetone (75:25) and 5% Methanol: Toluene (95:9) as mobile phases respectively. Ion exchange chromatography was performed using 30ml DEAE Sepharose column. The column was pre-equilibrated with 20 mM Tris–HCl buffer (pH 8.1). Sample was loaded at a flow rate of 200 cm/h. Then the column was washed with 10 bed volumes of the same buffer. The column was first eluted with 10 bed volumes of 0.15 M NaCl in 20 mM Tris–HCl (pH 8.1), and the C-PC was then eluted with 0.23 M NaCl in 20 mM Tris–HCl (pH 8.1). Finally, the column was eluted with 1 M NaCl at a flow rate of 400 cm/h.
The purified protein solution is firstly frozen at -200C for 24h. Then it is used to lyophilize by process of sublimation using pilot food freeze dryer of Ref Vac: Freeze Drying Systems Pvt. Ltd., Vadodara (Gujrat), India.
2.4.2 : Protein Estimation
Protein estimation was done by the Folin-Lowry method. Spirulina powder in water (1mg/ml) as sample and BSA (Bovine Serum Albumin) of same concentration was used as standard solution. Absorbance at 660nm is recorded to generate the standard graph as concentration against optical density (X and Y axis respectively) and by plotting the reading of unknown in graph, concentration of protein present in test sample was determined.
Estimation and purity were determined by calculating the ratio A620/A280
Where A620 = PC absorber peak (λ max)
A280 = other proteins especially aromatic amino acids.
2.4.3: Spectroscopic studies and molecular weight determination
Maximum absorbance was determined by taking the spectrum in the range of 550nm-650nm. Peak reading was used to determine the λmax of C-PC (Eppendorf Bio Spectrometer, Germany).
Purified cyanobacterial protein was qualitatively evaluated using SDS-PAGE for their molecular weight to confirm the purity of C-PC, 12 % SDS-PAGE was performed. The bands were visualized by Coomassie blue staining. The molecular weight of the purified C-PC was determined by running a pre-stained protein marker (PUREGENE) along with the sample.
NMR Spectral characterization was carried out at Molecular Structural Laboratory, National Chemical Laboratory (NCL) Pune, to confirm the structural properties of the isolated phycocyanin and compared with the standard available.
3: RESULTS
Figure 3.1: Lonar Lake (District. Buldhana, Maharashtra, India.)
Figure 3.2: Water Collection Site (Lonar Lake)
The water from Lonar Lake is highly alkaline, with a pH of 8.6 , exhibits light green colour shows high turbidity, (Figure 3.2) indicates the presence of blue-green algae (Yannawar & Bhosle 2013). The algal blooms and the activity of other organisms give it a slightly foul smell. The Total Dissolved Solids (TDS) in the water sample are significantly higher than the normal portable range of 500mg/L. Additionally, the Total Dissolved Oxygen (DO) level is very low. While high concentrations of inorganic substances such as Phosphate, Sulphate, Magnesium, Calcium, and nitrates, indicating a heavy inorganic load in the water as shown in table 3-a .
Table 3-a : Physicochemical parameters of Lonar Lake Water
|
Parameter |
value |
|
Colour |
Light Green |
|
Temperature |
360c |
|
pH |
8.6 |
|
Odour |
Slightly Foul |
|
TDS |
13989 mg/l |
|
TDO |
0.89 mg/l |
|
Sulphate |
23mg/l |
|
Phosphate |
0.42 mg/l |
|
Magnesium |
148.5 mg/l |
|
calcium |
118 mg/l |
|
Nitrate |
4.1 mg/l |
3.2: Identification of Spirulina platensis
Initial serial dilutions of water sample cultured in Zarrouk’s medium, a standard medium for Spirulina cultivation (Zarrouk C,1966) in laboratory, to obtain the initial growth of cyanobacteria. Microscopic observations confirmed the presence of Spirulina in the medium. which was authenticated from the PG and Research Laboratory, Department of Botany, Government Institute of Science, Aurangabad.
Figure 3.3: Preliminary Microscopic observation of Spirulina
Isolated Strain of Spirulina platensis was selectively cultured in Zarrouk’s medium Spirulina platensis was repeatedly cultured for its purity check in modified Zarrouk’s medium (C. Rajasekaran et.al.2016) [11] It further slightly modified by replacing KNO3 instead of NaNO3. All the chemicals were purchased from HI media, Mumbai.
A newly formulated medium, consisting of specific components such as single super phosphate (2.5 g/litre), sodium nitrate (2.0 g/litre), muriate of potash (1.0 g/litre), sodium chloride (0.5 g/litre), magnesium sulphate (0.1 g/litre), calcium chloride (0.05 g/litre), and sodium bicarbonate (5 g/litre). This innovative medium yielded a dense green mass within a remarkably short period of 5 to 8 days, under optimal incubation conditions (figure -3.4) This medium exhibited significantly enhanced growth, as well as increased protein concentration, when compared to the traditionally used media.
Figure 3.4: Spirulina cultivation with different constituent ratio
After 30 days incubation period, the cellular morphology of the obtained Spirulina was analysed using a light microscope (figure 3.5). After 30 days of controlled cultivation under all standard growth conditions the expected biomass was measured. Algal biomass is measured directly by measuring total Suspended solids (TSS) and total volatile suspended solids (VSS) The values were 0.25g/L(TSS) and 0.09 g/L(VSS) which was quite sensible biomass index for algae production previously given by (Rameshprabu Ramraj et al 2015). Spirulina fine powder was obtained from dry algal mass (Figure 3.6). This breakthrough in medium formulation and cultivation technique holds promise for enhanced Spirulina production, with potential applications in various industries.
Figure3.5: Microscopic confirmation of Spirulina Figure: 3.6: Spirulina Biomass andPowder
3.3: Extraction of the Phycocyanin from Spirulina platensis
The coloured pigments in the sample were primarily identified by preliminary TLC test. According to TLC Method described by S. Sathya in 2016 plates were then allowed to run, leading to the separation of the pigments for identification purposes.
Figure 4.8 a) Shows the separation of Phytopigments from Spirulina platensis by using two different solvent methods. Solvent system 1 Hexane: acetone (75:25) gives 4 different spots for coloured pigments while anther solvent system of 5% Methanol: Toluene (95:9), gives only one spot of phytopigment
Figure 3.7: a) TLC of isolated Phycocyanin from Spirulina platensis b) TLC of extract and Standard Phycocyanin
The pigments in the sample were separated based on their affinity for the different mobile phases, resulting in distinct spots on the TLC plate. By comparing the position and appearance of these spots to known standards confirmed the presence of phycocyanin pigments in the sample(figure3.7-a). Comparative TLC pattern that indicating coinciding spot with standard phycocyanin from Spirulina Entrepreneur Patha Peta, Kurnool, Andhra Pradesh. (Figure 3.7 b)
Ammonium sulphate was used for the precipitation. The sample containing pigment was treated with 60% concentration ammonium salt of amount 36.1 g for 100 ml. The powder was added gradually with continuous stirring. The complete dissolving of salt yielded precipitate containing phycocyanin. The precipitate was further centrifuged at 12000 g for 20 min at 4°C. The su A dialysis membrane with a Molecular Weight Cut Off (MWCO) of 12-14 kD, obtained from Hi-Media, was utilized in the experiment. To create the Ammonium Sulphate Extract (ASE), 5 ml of ASE was prepared using a 0.05M phosphate buffer with a pH of 7. The pre-activated membrane was then filled with the ASE and placed in the same buffer. The setup was left overnight on a magnetic stirrer at 4°C, similar method was used by Veronica et.al in 2016. (Verónica Cruz de Jesús, 2016)
Supernatant was discarded and collected the pellet with phycocyanin. (Elin Julianti 2019) Precipitation was observed at 60% and after its centrifuge, yellowish supernatant was obtained along with precipitate which is discarded and pellet is proceed for further purification A deep blue, fine and free flowing powder was obtained after lyophilization.
Figure 3.8: a) Crude extract of Phycocyanin b) Crude extract with ammonium salt
Figure 3.9: a) Dialysis of C-Phycocyanin b) precipitation of C-phycocyanin
Ion exchange chromatography was utilized with a 30 ml DEAE Sepharose column. Prior to loading the sample, the column was pre-equilibrated with 20 mM Tris–HCl buffer at a pH of 8.1. The sample was then introduced to the column at a flow rate of 200 cm/h. Subsequently, the column was washed with 10 bed volumes of the same buffer. This method was described by C.C Moraes in 2009. The elution process involved several steps. Firstly, the column was eluted with 10 bed volumes of 0.15 M NaCl in 20 mM Tris–HCl (pH 8.1). This step allowed the elution of some components. Next, the C-PC, a target compound was selectively eluted from the column using 0.23 M NaCl in 20 mM Tris–HCl (pH 8.1).In the last step of elution, a higher salt concentration was used. The column was eluted with 1 M NaCl at a faster flow rate of 400 cm/h to ensure the complete elution of any remaining bound substances. Finally, the target compound C-PC was successfully separated and purified from the sample. (C.C. Moraes, 2009 ) The purified protein solution was subjected to two-step preservation process. Initially, it was frozen at an extremely low temperature of -20°C for 24 hours. Subsequently lyophilized, in the pilot food freeze dryer Ref Vac: Freeze Drying Systems Pvt. Ltd., Vadodara, Gujarat, India.
Figure 3.10: Lyophilized Phycocyanin Figure 4.13 Estimation of Protein by Folin-Lowry method
3.4: Protein Estimation (Folin-Lowry Method)
The concentration and purity of the isolates were determined with the formula by Boussiba et.al in 1979 and by Bennett et. al. in 1973. The concentration of phycocyanin estimated by Folin Lowry method for all four methods of extraction. At each step of isolation i.e. Crude extract, ammonium sulphate precipitation, Dialysis and column chromatography, Phycocyanin is generally graded according to its purity ratio (A620/A280).
Table 3-b : Concentration and Comparative Purity Analysis
|
Sr. No. |
Method |
C-PC (µg/ml) |
Purity ratio 620/280 |
|
1 |
Crude extract |
76.2 |
0.74 |
|
2 |
Ammonium sulphate precipitation |
84.1 |
0.81 |
|
3 |
Dialysis |
123.8 |
1.16 |
|
4 |
Column Chromatography |
413 |
1.78 |
According to Patil, G et.al., 2006, purity levels of phycocyanin are classified depending on the ratio A620/A280, a purity of 0.7 is considered to be of food grade, 3.9 is considered to be of reactive grade, and 4.0 or greater is considered to be of analytical grade. Out of the four methods, column chromatography estimated the highest purity of phycocyanin. The Purity ratio obtained was 1.78. Hence Food-grade phycocyanin with a purity ratio (A620/A280) of approximately 1.78 was obtained in this study due to the more effective removal of contaminating proteins in the ammonium sulphate precipitation and further column chromatography process. Maximum absorbance was determined by taking the spectrum in the range of 550nm-650nm. λmax of C-PC is 618 nm (figure 3.11). (Eppendorf Bio Spectrometer, Germany).
Figure 3.11: Absorption spectra of isolated Phycocyanin
3.4: Molecular weight Determination
Purified cyanobacterial protein was qualitatively evaluated using SDS-PAGE for their molecular weight to confirm the purity of C-PC, 12 % SDS-PAGE was performed under non-denaturing conditions. The bands were visualized by Coomassie blue staining. The molecular weight of the purified C-PC was determined by running a pre-stained protein marker (PUREGENE) along with the sample. Molecular weight determination of the isolated phycocyanin by SDS -PAGE showed the molecular weight of the standard as well as isolated phycocyanin (figure3.12) Two bands corresponding to two polypeptides 19kDa and 11kDa were observed to coincide with the standard phycocyanin which confirm the purity of isolated phycocyanin.
Figure4.15: Molecular Weight determination of Isolated Phycocyanin
3.5: Structural Analysis
NMR Spectral characterization was carried out at Molecular Structural Laboratory, NCL Pune, to confirm the structural properties of the isolated phycocyanin. NMR spectra of phycocyanin has given the valuable insights into its atomic-level structure, conformational changes. The chemical shifts observed in the spectra indicate the individual nuclei within molecule. The number, position, and intensity of different peaks will provide information about the amino acid sequence and the overall secondary and tertiary structure of the protein.
Figure 4.16: NMR Spectra of Isolated phycocyanin
4. CONCLUSION:
Spirulina platensis was isolated and successfully cultured by using modified Zarrouk’s medium. Microscopic observations shown in figure 3.5 confirmed the morphological identification of Spirulina . The maximum biomass achieved with modified medium and converted to fine spirulina powder(figure 3.6) Preliminary TLC method (figure 3.7 a) and b)) confirmed to mark the presence of phytopigments along with phycocyanin in spirulina whole extract . Further precipitation of phycocyanin (Figure 3.8 a,b,c) is achieved through ammonium sulphate precipitation , dialysis and fine pigment achieved after lyophilization(3.10). Among the four methods used for protein purification viz, Crude extract Ammonium sulphate precipitation Dialysis Column Chromatography, the maximum yield 413 (µg/ml) was achieved by column chromatography. Maximum absorption of isolated phycocyanin was noted at 618 nm. Further molecular weight of phycocyanin was completely matched with pure pigment with two polypeptides of 19 KDa and 11KDa. NMR spectra shown to predict the same structural formula that of pure phycocyanin. It has been concluded that the food grade phycocyanin was isolated from Spirulina platensis from lonar soda lake.
DISCUSSION:
Various pharmacological properties of phycocyanin from algae from various natural resources have been revealed by many researchers . phycocyanin explored with its potent antioxidant ,anti-inflammatory neuroprotective properties make it as a promising ingredient for pharmaceutical as well as nutraceutical formulations. Along with its fluorescent blue colour it proved as a natural colourant in food industry as clean labelled natural ingredient. Its application in many bioassays , diagnostic imagine, biosensor development is based on fluorescence and photo visibility of phycocyanin.
REFERENCES
V. N. Wadekar*, A. L. Shirfule, Biophysical and Biochemical Characterization of phycocyanin from Spirulina platensis from Lonar lake of Maharashtra, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 12, 925-938 https://doi.org/10.5281/zenodo.17830463
10.5281/zenodo.17830463
