FORM 2
THE PATENT ACT 1970
(39 OF 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
TITLE OF THE INVENTION NATURAL BLUE PIGMENT AND PROCESS FOR PRODUCING THE SAME
APPLICANT
NORTH MAHARASHTRA UNIVERSITY, JALGAON

TURAL BLUE PIGMENT AND PROCESS FOR PRODUCING THE SAME
e following specification particularly describes the invention and the manner in ch it is to be performed
FIELD OF THE INVENTION
The invention relates to a natural blue pigment and a process for producing the same. More specifically, the invention relates to a natural blue pigment from cyanobacteria and a process for production of the said pigment, wherein mass cultivation of the cyanobacteria Spirulina is carried out and the natural pigment Phycocyanin is further extracted from the said Spirulina biomass
BACKGROUND OF THE INVENTION
Cyanobacteria are found in a variety of habitats, predominantly in extreme environments such as alkaline lakes, thermal springs, etc. Spirulina is a kind of cyanobacteria propagating in the tropical alkaline lake-water of high salt concentration Nutritional studies show that Spirulina contains high contents of protein having well balanced amino acid composition together with other nutritional components well adapted for human health. It is often called a perfect food since it has the nutrient compositions so well balanced that there is no possibility of health troubles due to the mechanism of unbalanced diet even when only spirulina is taken for a long time. Cyanobacteria are known to produce a variety of pigments that have recently gained importance in different fields.
Phycocyanin (PC) is a blue, light-harvesting pigment in Cyanobacteria and in two eukaryotic algal genera, Rhodophyta and Cryptophyta. This pigment gives many cyanobacteria their bluish color and it is because of these pigments that cyanobacteria are also known as blue-green algae. Phycocyanin is water soluble, strongly fluorescent and has antioxidant properties.

Phycocyanin and related phycobiliproteins are utilized in a number of applications in foods and cosmetics, biotechnology, diagnostics and medicine. Phycocyanins also have a wide range of applications in Therapeutic, Cosmetic, Food & Beverage Industry, FRET assays, Fluorescence microscopy, Fluorescence in Situ Hybridization (FISH), Fluorescence Activated Cell Sorting (FACS), Fluorescence Correlation Spectroscopy (FCS), Labeling of proteins, antibodies and nucleic acids as well as in gel electrophoresis, isoelectric focusing and gel exclusive chromatography. However, there are few companies presently producing and selling phycobiliproteins, phycobiliprotein derivatives or phycobiliprotein applications, because of the difficulties encountered in the extraction of Phycocyanin on an industrial scale.
The functionality of Phycocyanin as a fluorescent dye has been improved by chemical stabilisation of Phycocyanin complexes, while protein engineering has also introduced increased stability and novel biospecific binding sites into Phycocyanin fusion proteins. The structure of Phycocyanin has been found similar to the natural antioxidant, bilirubin, found in humans, thereby suggesting the health benefits of Phycocyanin uptake by humans.
Phycocyanins are called as accessory pigments and are situated in the cell membrane of the organism. Therefore, they are difficult to extract by ordinary procedures and with very low yields. The known methods of destroying cell membranes in order to extract the pigment include (1) mechanical grinding with glass homogenizer, (2) destruction of cell membranes by supersonic wave treatment, and (3) dissolution of cells by successive treatments of various enzymes such as lysozyme and other digestive enzymes. However, the method (1) is inefficient for destroying cell membranes, hence low value for industrial application, the method (2) has a high opportunity of microbial contamination during long supersonic wave treatment time, and the method (3) is not only appropriate for purity of the pigment since the enzyme hydrolysate has many problems in connection with industrialization since the condition of enzyme reactions is optimal for contaminating microbial growth.

A study of the prior art regarding extraction of Phycocyanin revealed many different and lengthy procedures of extraction, with less yield in each case. CN1958598 discloses a technique for extracting phycocyanin from Spirulina, said technique comprising placing water-containing Spirulina at below 0°C to freeze, defreezing at above 0°C, and breaking the cell walls to precipitate phycocyanin. CN1295080 describes an invention to extract Phycocyanin by placing Spirulina in specially compounded salt solution so that phycocyanin in spirulina is dissolved into the solution. Indian patent application 2504/DEL/96 describes an improved process for preparation of blue colorant Phycocyanin from Spirulina. Indian patent application 1029/DEL/96 describes an improved method of effluxing of Phycocyanin from Spirulina sp. Indian patent application 1358/DEL/95 describes an improved process for rapid effluxing of Phycocyanin into water from wet biomass of Spirulina without cell disruption. Indian patent application 1359/DEU95 describes an improved process for preparation of natural blue colorant Phycocyanin from Spirulina. IN184767 describes an improved process for the extraction of Phycocyanin from the alga Spirulina and Indian patent application 548/DEL/2004 describes a process for preparation of food colorant from spirulina.
The present invention therefore discloses a process for cultivation of spirulina on a large scale and extraction of the pigment Phycocyanin from the culture growth.
Blue pigments for food products are, of course, known. However, there are few natural blue pigments which are acid soluble, have appropriate hue and, are approved for use in foods and cosmetics, biotechnology, diagnostics and medicine. It is a further object to provide an effective method for the preparation of the pigment, in a form suitable for use in foods and cosmetics, biotechnology, diagnostics and medicine

OBJECTS OF THE INVENTION
Accordingly, the main object of the present invention is to provide a natural blue pigment from cyanobacteria with a process for production of the said pigment, wherein mass cultivation of the cyanobacterium Spirulina is carried out and the natural blue pigment Phycocyanin is obtained from the said Spirulina biomass directly without using any extraction process.
SUMMARY OF THE INVENTION
To meet the above objects and to overcome the problems existing in the prior art, the present invention describes said process comprising:
1. Growing the cyanobacteria in specific culture media under sterile conditions
2. Harvesting the cell biomass aseptically
3. Standing the biomass under oxygen deprived condition for specific time interval
4. Allowing the pigment to leach out into the medium
5. Collecting the pigment
6. Purifying the pigment by conventional method as per requirement
DETAILED DESCRIPTION OF THE INVENTION
In one form of the invention, the present invention describes a natural blue pigment from cyanobacteria with a process for production of the said pigment, wherein mass cultivation of the cyanobacteria Spirulina is carried out and the natural blue pigment Phycocyanin is obtained from the said Spirulina biomass directly without using any extraction process
In one more form of the invention, the Spirulina is cultivated in minimal medium

In yet another form of the present invention, the Spirulina may be cultivated in tap water
In one more form of the present invention, the Spirulina may be cultivated in BG-11
In one more form of the present invention, the Spirulina may be cultivated in Zarouck's medium
In one more form of the present invention, the Spirulina may be cultivated at alkaline PH
In one more form of the invention, the time for batch cultivation may be between 0-30 days but more preferably between 5-15 days
In yet another form of the invention, the culture may be cultivated in continuous mode
In yet another form of the invention, the biomass may be harvested by filtering the medium
In one more form of the invention, the filtration may be carried out by filter paper, cheese cloth, or sieve
In another form of the invention, the filtered biomass may be allowed to stand in oxygen deprived condition
In yet another form of the invention, the filtered biomass may be allowed to stand in oxygen deprived condition for 3-15 days preferably between 5-10 days
In one more form of the invention, the cells of the biomass may be allowed to rupture in oxygen deprived condition to release the blue colored pigment phycocyanin
DETAILED DESCRIPTION WITH RESPECT TO EXAMPLES
The following examples are for the purpose of illustration of the present invention and should not be construed to limit the scope of the present invention.

EXAMPLE 1
Growth of culture
Spirulina spp. was isolated. from the water sample of alkaline Lake, Lonar. The identification of the blue-green algae was done' as per the monograph, 'Cyanophyta' (Desikachary 1959). The cyanobacterium was maintained on BG-11 media (Kaushik 1987). Each liter of BG-11 medium contains (g L"1) of NaN03, 1.5; K2HP04, 0.04; MgS04.7H20, 0.075; CaCI2.2H20, 0.036; citric acid, 0.006; ferric ammonium citrate, 0.006; EDTA, 0.001; Na2C03, 0.02 and 1 mL of trace solution A5 containing (g L"1) of H3BO3, 2.86; MnCI2, 1.81; ZnS04.7H20, 0.222; Na2Mo04.2H20, 0.39; CuS04.5H20; 0.079 and Co(N03)2.6H20, 0.0494. The pH of the media was adjusted at 10.36 (lake water pH).The flask was exposed to a light intensity of 1200-1500 lux with a light/dark cycie of 16/8 h. After incubation of 12 days the ceiis were harvested by centrifugation.
Example 2
Extraction of phycobiliprotein by conventional method as described in literature
The pellet of the culture grown by the method described above was washed with distilled water and resuspended in 5 mL of 0.05 M phosphate buffer. The content was sonicated (Sonics and Materials Inc., USA) with 30 Hz frequency at a pulse of 10/5 min. Freezing thawing of the sonicated content was done. The extract was centrifuged at 5000 rpm (Sorvall, USA, Model RC2). The supernatant was collected and the concentration of phycobiliproteins was measured spectrophotometrically at required pH with 0.05 M phosphate buffer as blank. The maximum amount of phycocyanin produced from optimized media composition was 23.8 mg/L at 0.058 g/L of CaCI2.2H20 and 0.115 g/L of Na2C03, respectively
Example 3
Extraction of phycobiliprotein by the method described in the present invention
The growth of the culture as described in example 1 was collected. It was kept in oxygen less condition for a specific period. Phycocyanin was released into the

medium due to cell lysis due to which the semisolid cell containing medium became a blue colored liquid due to accumulation of phycobiliproteins. Estimation was carried out by the method of Silvana et al, 2008. The amount of phycocyanin produced by the present invention was 20 mg/L
Example 4
Purification of Phycocyanin by conventional method
Partial purification was performed as per Pate! et al. (2005). The purification steps were performed in the dark at 10-15°C using 10 mM phosphate buffer (pH 6.5) containing 0.01% sodium azide. Appropriate amount of finely powdered ammonium sulphate was added gradually to the cell extract with a continuous stirring for 1 h. The crude cell extract was fractionated by precipitation with ammonium sulphate first at 25% and then at 50% saturation. The precipitate from 25% saturation was discarded and the supernatant was further brought to 50% saturation of ammonium sulphate and allowed to stand for 4 h at 4°C. The precipitated proteins were collected by centrifugation at 10,000 x g for 30 min at 4°C and resuspended in phosphate buffer and dialyzed overnight at 4°C against the same buffer. The purification of CPC was tracked by determining optical density of fractions at 280, 620 and 652 nm using UV visible spectrophotometer (Shimadzu, Japan). The maximum purity ratio obtained by the conventional method was 0.65
Example 5
Checking the purity of Phycocyanin produced by the present invention
The crude Phycocyanin produced by the method as described in the present invention was checked for purity without any steps of purification. The crude cell extract was tracked by determining optical density of fractions at 280, 620 and 652 nm using UV-visible spectrophotometer (Shimadzu, Japan). The purity ratio obtained by the present method was 0.6 after 10 days and 0.77 after 12 days.

Example 6
Testing the food grade quality of phycocyanin
The use of phycocyanin in food and pharmaceutical products is only possible after establishing the process conditions to obtain specific grades of purity. A purity ratio (A615/A280) of 0.7 is considered as food grade, 3.9 as reactive grade, and greater than 4.0 as analytical grade (Silvana et al, 2008). The blue colored liquid after release of Phycocyanin into the medium due to lysis of cells was analysed for its food grade quality. The results showed that even the crude extract without taking any extra effort on purification, was of food grade, with a purity ration of 0.77.
References
Desikachary T V. (1959) Cyanophyta. New Delhi: Indian Council of Agricultural Research.
Kaushik B D (1987). Laboratory methods for blue-green algae. New Delhi associated Publication Company.
Silvana Terra Silveira, Luci Kelin de Menezes Quines, Carlos Andre' Veiga Burkert, Susana Juliano Kalil, (2008) Separation of phycocyanin from Spirulina platensis using ion exchange chromatography Bioprocess Biosyst Eng 31:477-482
Takao Furuki, Shuichi Maeda, Satoshi Imajo, Tetsuya Hiroi, Tsutomu Amaya (2003), Rapid and selective extraction of phycocyanin from Spirulina platensis with ultrasonic cell disruption Journal of Applied Phycology 15: 319-324

We claim,
1. A natural blue pigment from Spirulina
2. A natural blue pigment from Spirulina as in claim 1, wherein, the spirulina may be cultivated aseptically or septically
3. A natural blue pigment from Spirulina as in claims 1 and 2 wherein, the Spirulina may be cultivated in conventional media
4. A natural blue pigment from Spirulina as in claims 1 and 2 wherein, the Spirulina may be cultivated in tap water
5. A natural blue pigment from Spirulina as in claims 1 and 2 wherein, the Spirulina may be collected from the growth medium by filtration
6. A natural blue pigment from Spirulina as in claims 1, 2 and 5, wherein, the Spirulina may be stored in oxygen less environment for a specific period
7. A natural blue pigment from Spirulina as in claims 1 and 6, wherein; the Spirulina may be stored in oxygen less environment for a period between 3-12 days but more preferably between 5 to 10 days.
8. A process for production of natural blue pigment from Spirulina, said process comprising:
a. Growing the cyanobacteria in specific culture media under sterile
conditions
b. Harvesting the cell biomass aseptically

c. Standing the biomass under oxygen deprived condition for specific time
interval
d. Allowing the pigment to leach out into the medium
e. Collecting the pigment
f. Purifying the pigment by conventional method as per requirement
9. A natural blue pigment from Spirulina and a process for producing the same substantially as described herein and illustrated with help of examples