Title of Invention

A PROCESS FOR PREPARATION OF NATURAL BLUE COLOURANT FROM SPIRULINA SPECIES

Abstract

The present invention relates to a process for the preparation of phycocyanin a natural blue colorant from spirulina species, which involves washing of the biomass, homogenization of the cells followed by acidification. Sedimenting and centrifuging the homogenized solution in order to remove the contaminants such as cell debris, chlorophyll, and subjecting the obtained supernatant to adsorption in a packed bed column of suitable adsorbents before lyophilizing the phycocyanin solution to powder form. The purity of phycocyanin (ratio of the absorption of 620 nm to 280 nm)is=4.0.

Full Text

The present invention relates to a process for the preparation of natural blue colorant from spirulina species. The present invention more particularly relates to a process for the preparation of phycocyanin, a natural blue colorant, from spirulina species. The practice of coloring food dates back to ancient times and today color has become a vital constituent of food. In recent years, interest in the use of natural colorants has increased considerably mainly because of the apparent lack of toxicity and environmentally more safe. Natural coloring compounds used in food, cosmetics and molecular genetics industries are products of great commercial significance. Phycocyanin, a blue colored, red fluorescing biliprotein of algae, was first reported by Lemberg in 1928. Phycocyanin is present in all the species of cyanophyceae family (eg;Spirulina sp). Phycocyanin comprises of a protein and chromophore. Phycocyanin content varies in the range of 10 to 15% of Spirulina bio-mass, based on the culture conditions. This colorant is highly stable at pH 5 to 8. Furthermore, it exhibits a strong red fluorescence when the protein is highly concentrated and in native form. The maximum absorbance for phycocyanin is 620 nm and that of total protein is at 280 nm. The purity of food grade Phycocyanin (defined as the ratio of absorbance at 620 nm to that of 280 nm) is 0.7 and reactive grade phycocyanin of purity > 3.9 is used in cosmetics and molecular genetics industries. Methods for the concentration and purification of phycocyanin from the cell culture and broth are known in the art. Phycocyanin is presently purified by ammonium sulfate precipitation, centrifugation, gel filtration, ion exchange chromatography, and membrane processes. Such purification involves many techniques. Almost all the methods suffer from the problem of cell debris and chlorophyll contamination during the processing of phycocyanin. Subjecting the separated phycocyanin to additional processing steps such as precipitation, centrifugation, gel filtration, column chromatography leads to considerable reduction in contaminants. However, scale-up of these methods is extremely difficult and uneconomical to produce phycocyanin on large scale. Hence there is a need for an improved process for the concentration and purification of phycocyanin which has potential for easy scale-up, easy handling of the product, for obtaining product with better stability and to reduce capital as well as operational costs as compared to other processes. Reference may be made to Anon 1987 Chemical Eng. P-19-22 wherein one of the commercialized technologies for the production of phycocyanin utilizes gel filtration as the final purification step to remove the impurities. The drawback is to utilize chromatographic methods which are not commercially feasible for the production of food colour, moreover these chromatographic purification methods need one more concentration step. Reference may be to Wenhui et al., 2001 CN1291616 wherein, separating and purifying high-purity high-activity phycocyanin includes such steps as preparing mashed spirulina, extracting, salting out, dialysis, concentrating and purifying by chromatographic techniques like sephadex G200, DEAE, Sephadex G25, and freeze drying. However, the drawback of this invention is that it involves many steps, which are difficult to scale up. Also the presence of buffer salts used during the purification of phycocyanin makes the product undesirable for food and pharmaceutical applications. Reference may be made to Mikiyoshi, 2001 JP190244, A method for producing a blue coloring matter which is characterized by comprising the following process steps: the first step that the dried powder of spirulina is suspended in water to mix with an ion exchange resin, and a soluble protein containing phycocyanin to be the blue coloring matter is bound to the ion exchange resin; the second step that the ion exchange resin bound with the soluble protein is washed with water using a filter with a mesh enough to hold the above ion exchange resin; the third step that an aqueous solution showing blue color is collected through separating the soluble protein from the above ion exchange resin by using an eluent, the fourth step that the aqueous solution obtained in the third step is evaporated to dryness to obtain the objective blue coloring matter as blue powder. This process is difficult to handle large amount of phycocyanin. Also evaporation of phycocyanin solution at high temperature is not desirable since phycocyanin is unstable at high temperature. Reference may be made to 'Affinity precipitation using chitosan' as a ligand carrier, Biotechnology and bioengineering, Vol 33, 1989 (216-220) by Sensatad C and Mattiason B, wherein chitosan has been used effectively for the purification of the enzyme trypsin and found that their results were similar to that of the gel filtration. Reference can be made to 'Chitin-the undisputed biomolecule of great potential' ( Rudrapatnam et al. Critical reviews in Food Science and Nutrition, 43, (1) 21-87 2G03) wherein chitosan has been used in pharmaceutical industry for possible biomedical applications. Its polymeric cationic character along with its possession of a potentially reactive functional group have given it unique possibilities for utilization in controlled drug release technologies. The United States Food and Drug Administration have approved chitosan as a feed additive. Chitosan is also used as a food quality enhancer in certain countries like Japan, Italy and Norway. Reference may be made to 'Practical application of aqueous two-phase systems for the development of a prototype process for phycocyanin recovery from Spirulina maxima'. J Chem Technol Biotechnol 76: 1273- 1280. This work deals about the partitioning of phycocyanin from the cell debris and a purity of phycocyanin of 3.8 after ultrafiltration was achieved. This work contains three step process like aqueous two phase extraction, followed by ultrafiltration and precipitation in order to obtain phycocyanin of purity 3.8. The yield of the product obtained in this case is just 29%. The main object of the present invention is to prepare the natural blue colorant phycocyanin. Another object of the present invention is to separate and extract phycocyanin free from chlorophyll, cell debris and other contaminants Still another object of the present invention is to extract phycocyanin without the use of precipitating agents. Yet another object of the present invention is to obtain a high purity of phycocyanin in a single step and hence decreasing the other processing steps involved. Still another object of the present invention is to facilitate the processing of phycocyanin without very harsh processing conditions that might degrade the protein. Yet another object of the present invention is to carry out the purification in large scale without product deterioration. Accordingly, the present invention relates to a process for the preparation of natural blue colorant phycocyanin from spirulina species, which comprises the steps of; a) washing of the biomass for 10-15 minutes to remove the adhering culture media components, b) adding water to the biomass at a ratio of 1:1, and then homogenizing the biomass for 5-10 minutes in the pressure range of 100-400 kg/cm2to obtain cell homogenate, c) adjusting the pH of the homogenate solution to 4-5, d) sedimenting the cell debris by adding 4-5 milliliter of 1 % Chitosan solution to the above100 milliliter of the homogenized solution, e) centrifuging the above solution at 2000-3000 revolution per minute for 5 minutes, f) adsorbing the contaminants such as other proteins, chlorophyll and cell debris by passing the supernatant solution through activated charcoal in packed bed mode one or two times, g) lyophilizing the obtained phycocyanin solution with purity 3.98 (ratio of absorbance at 620 nm / absorbance at 280 nm ) to get phycocyanin powder. In an embodiment of the present invention, the Spirulina species used is blue green algae such as Spirulina platensis, Spirulina maxima . In the present invention, freshly harvested Spirulina biomass is washed two to three times to remove the adhering culture media components. The biomass is homogenised at the pressure of 100 - 400 kg/cm2 for 10-15 minutes, then the pH of the homogenate is adjusted to 4.5 in order to release the phycocyanin from the cells, 4-5 milliliter of 1% chitosan is added wherein all the cell debris settles down, which is further centrifuged at 2000 -3000 revolution per minute for 4-5 minutes. The obtained supernatant is passed through activated charcoal bed once or twice, which results in phycocyanin having a purity of 3.98 Novelty The novelty of the present invention lies in achieving high purity of phycocyanin above 3.98 (ratio of absorbance at 620 nm /ratio of absorbance at 280 nm) by using simple process steps such as sedimentation and adsorption. The following examples are given by way of illustration of the present invention and should not be constructed to limit the scope of the present invention. Example 1 10 kg of Spirulina biomass was washed with water to remove the culture media components. Water was added to the biomass at a ratio of 1:1 and homogenised at the pressure of 300 kg/cm2 for 10 minutes. 100 milliliter of 1% Chitosan was added to the homogenized solution for the settling of cell debris and the homogenate was centrifuged at 3000 revolution per minute for 5 minutes. The supernatant obtained was passed through activated charcoal bed twice. The yield of phycocyanin obtained was 70%. The purity of phycocyanin solution was estimated and a purity (Ratio of absorbance at 620 nm/280nm) of 3.98 (as shown in the table below) was observed. (Table Removed) Example 2 6 kilograms of Spirulina biomass was washed with water to remove the culture media components. Water was added to the biomass at a ratio of 1:1 and homogenised at the pressure of 300 kg/cm2 for 10 minutes. 60 milliliter of 1% Chitosan was added to the homogenized solution for the settling of cell debris and the homogenate was centrifuged at 2000 revolution per minute for 5 minutes. The supernatant obtained was passed through activated charcoal bed twice. The yield of the phycocyanin obtained was 74%. The purity of phycocyanin solution was estimated and a purity (Ratio of absorbance at 620 nm/280nm) of 3.96 (as shown in the figure below) was observed. (Figure Removed) The main advantages of present invention are 1. High purification of phycocyanin is achieved 2. Simple process steps such as sedimentation and adsorption are used 3. Additional steps such as precipitations, gel filtration, ion exchange chromatography for the purification of phycocyanin are eliminated and hence the process is less tedious. 4. All the operations are carried out at room temperature 25 ± 1 degree C. 5. The process is simple, less time consuming and easy to scale-up 6. The final product retains its spectral properties thereby rendering this product to be used both as reactive grade, pharmaceutical grade and also in food. We claim: 1. A process for the preparation of natural blue colorant phycocyanin from wet biomass of any species containing phycocynin, which comprises the steps of; a) washing of the biomass for 10-15 minutes to remove the adhering culture media components, b) adding water to the biomass at a ratio of 1:1, and then homogenizing the biomass for 5- 10 minutes in the pressure range of 100-400 kg/cm2to obtain cell homogenate, c) adjusting the pH of the homogenate solution to 4-5, d) sedimenting the cell debris by adding 4-5 milliliter of 1 % Chitosan solution to the above 100 milliliter of the homogenized solution, e) centrifuging the above solution at 2000-3000 revolution per minute for 5 minutes, f) adsorbing the contaminants such as other proteins, chlorophyll and cell debris by passing the supernatant solution through activated charcoal in packed bed mode one or two times, g) lyophilizing the obtained phycocyanin solution with purity 3.98 to get phycocyanin powder. 2. A process as claimed in claim 1 wherein the biomass obtained from the different species of blue green algae selected from Spirulina platensis, Spirulina maxima

Documents:

746-del-2005-abstract.pdf

746-DEL-2005-Claims-(06-07-2011).pdf

746-del-2005-claims.pdf

746-del-2005-Correspondence Others-(06-07-2011).pdf

746-del-2005-correspondence-others.pdf

746-del-2005-description (complete).pdf

746-del-2005-Form-1-(06-07-2011).pdf

746-del-2005-form-1.pdf

746-del-2005-form-18.pdf

746-del-2005-form-2.pdf