Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF PURE PHYCOCYANIN CONCENTRATE

Abstract

An improved process for the preparation of pure phycocyanin concentrate The present invention relates to an improved process for the preparaion of pure phycocyanin concentrate. The process is an improved process for the concentration and purification of phycocyanin obtained from blue-green algae employing aqueous two-phase extraction. In the present invention, freshly harvested Spirulina biomass is washed two to three times to remove the culture media components. The biomass is homozinised in a homoziniser at the pressure of 100 - 400 kg/m2 for 10-15 min. Then the solution is filtered using cotton bed filter to remove the chlorophyll and cell debris and then centrifuged to remove the traces of these contaminants. The phycocyanin obtained is used for the preparation of aqueous two phase system.

Full Text

The present invention relates to an improved process for the preparaion of pure phycocyanin concentrate. The present invention more particularly relates to an improved process for the concentration and purification of phycocyanin obtained from blue-green algae employing aqueous two-phase extractioi> 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. C-phycocyanin, a blue colored, red fluorescing biliprotein of algae, was first reported by Lemberg in 1928. C-phycocyanin is present in all the species of cyanophyceae family (eg;Spirulina sp). C-Phycocyanin comprises of a protein and chromophore, the protein moiety consists of alpha and beta sub units of molecular weights in the range of 18000 and 20000 each. 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 is in native form. The maximum absorbance for C-phycocyanin is 620nm and that of total protein is at 280nm. The food grade C-Phycocyanin purity (defined as the relationship of absorbance at 620nm to that of 280nm) is of 0.7 and reactive grade purity above. 3.9 is used in cosmetics and molecular genetics industries. The production of C- phycocyanin by Spirulina represents a very interesting case because both the industrial application and commercial value of this product are considerable C-phycocyanin is presently purified by ammonium sulfate precipitation, centrifugation, gel filtration, ion exchange chromatography, and membrane processes. Thus the purification involves many techniques. Methods for the concentration and purification of phycocyanin from the cell culture and broth are known in the art. 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 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, product with better stability, reduce capital and operational costs as compared to other processes. Consequently, process problem associated with the scaling up of the purification can be eliminated to a large extent by using aqueous-twophase systems. Aqueous two phase systems (ATPS) has been recognized as a superior and versatile technique for the downstream processing of biomolecules. The major advantages of ATPS are high capacity, biocompatible environment, low interfacial tension, high yields, lower process time and energy, and high selectivity. Aqueous two phase systems are mainly of two types polymer - polymer and polymer salt. The physical properties of ATPS, such as density, viscosity, and interfacial tension, determine the phase separation and also contribute to the biomolecule partition behavior. Aqueous two-phase systems are well known for their utility in the extraction and purification of biological materials such as enzymes/proteins, nucleic acids, viruses, cell organelles etc. 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 again utilize chromatographic method which are not commercially feasible for the production of food colour, moreover these chromatographic purification methods need one more concentration step and are limited in scale operations by the cost of the resins. Reference may be made to a Japanese patent (1979, 79076867) wherein phycocyanin is stabilized in solutions by the addition of gelatin. The probable drawback here is stabilization in the form of phycocyanin solution is prone to microbial attack and oxidative degradation (which is often indicated by precipitation during storage) since gelatin is neither an antimicrobial or an antioxidant. Reference may be made to an improved process for the preparation of phycocyanin from Spirulina (Manoj et al., 1996, Indian Patent 2504/del/96, Gazette dated 15/1197) wherein, the Spirulina biomass is homogenized and phycobiliproteins are released into homogenate. The homogenate is filtered to separate cell debris from supernatant solution. The separated phycocyanin solution is then subjected to precipitation in order to obtain phycocyanin in solid form. The main drawback of this invention is even after centrifugation, phycocyanin solution is still not free of chlorophyll, cell debris. Also, phycocyanin needs to be made from the precipitation agent, since it has to be used in food/pharmaceutical applications. 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, sephadex G200, DEAE, Sephadex G25, SephadexG200 and freeze drying, however, the drawback of this invention is that it involves many steps and also it is difficult to scale up the process. Also the presence of buffer salts used during the purification of phycocyanin makes the product undesirable for food/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 processes: the first process 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 process 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 process 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 process that the aqueous solution obtained in the third process 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 Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima. J Chem Technol biotechnol 76: 1273-1280. This work deals about the partitioning of phycocyanin from the cell debirs and achieved a purity of phycocyanin of 3.8 after ultrafiltration. The main object of the present invention is to provide an improved process for the separation, extraction, concentration and purification of phycocyanin. Another object of the present invention is to separate and extract phycocyanin free from chlorophyll, cell debris and other contaminants to one of the phases employing aqueous two-phase extraction 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 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 concentrate the phycocyanin solution using a novel athermal process such as osmotic membrane distillation to remove excess water load during subsequent processing steps such as freeze drying etc. Yet another object of the present invention is to facilitate the easy processing of phycocyanin by integrating aqueous two-phase extraction with membrane based separation process such as ultrafiltration and osmotic membrane distillation. Accordingly, an improved process for the preparaion of pure phycocyanin concentrate which comprises : a) harvesting the Spirulina biomass by known method, b) washing the said biomass with plain water by known method for 10-15 minutes, c) adding water at a ratio of 1:1 to the said biomass d) homogenizing the biomass mechanically for a period of 5-10 minutes at a pressure in the range of 100-400 kg/cm2 e) adjusting the pH of the above obtained homogenized biomass to 4-5 by known methods, f) filtering the above obtained biomass and further passing through activated carbon at a flow rate of 50/100 ml/min, g) centrifuging the above obtained biomass to obtain phycocyanin solution , h) adding phase forming components selected from polyethylene glycol ( PEG) 1550, PEG 4000, PEG 6000 and PEG 20,000 at a level of 6-12% (w/w), and salt selected from Potassium phosphate, Ammonium sulfate, Magnesium sulfate, Sodium sulfate, Sodium citrate potassium phosphate at a level of 10-15% (w/w) i) mixing above obtained mixture for a period of 1-2hr at 400-600 rpm in a magnetic stirrer followed by equilibrating using separating funnel for at least 24 h and separating the top phase containing pure phycocyanin having purity in the range of 3.8 to 4.3 using spectrophotometric method at 620/280 nm, j) filtering the pure phycocyanin solution by ultrafiltration followed by osmotic membrane distillation to obtain pure phycocyanin concentrate at a range of .004% to .014% , In an embodiment of the process wherein the harvesting of the biomass may be carried out by using vibro filter. In an another embodiment of the process wherein phase forming agent used may be selected from PEG 4000 and salt selected may be Potasium phosphate. In the present invention, freshly harvested Spirulina biomass is washed two to three times to remove the culture media components. The biomass is homozinised in a homoziniser at the pressure of 100 - 400 kg/m2 for 10-15 min. Then the solution is filtered using cotton bed filter to remove the chlorophyll and cell debris and then centrifuged to remove the traces of these contaminants. The phycocyanin obtained is used for the preparation of aqueous two phase system. The novelty of the present invention is that higher purity of phycocyanin is achieved in a single stage and also the phycocyanin gets concentrated in one of the phases. The contaminants like chlorophyll and cell debris gets settled at the interface. The following examples are given by way of illustration of the present invention and should not be constructed to the limit the scope of the present invention. Example 1 Around 6kg of Spirulina biomass was harvested using conveyor filter and washed with tap water to remove the culture media components. To the biomass an equal amount of water was added and then homosinised at the pressure of 100- 400 kg/cm2 for about 10 min. The solution obtained was filtered and centrifuged at the rate of 50 ml/min using cotton bed filter to remove the chlorophyll and other impurities and disc type centrifuge at around 12,000 g to remove remaining traces of these impurities. Phycocyanin obtained was added to 6% w/w PEG and 15% w/w potassium phosphate to which 79% w/w of phycocyanin was added to have a total of 100%. The mixture was mixed using magnetic stirrer at 500 rpm at about one hour at room temperature. The mixture was allowed to equilibrate in a separating funnel for about 24 hrs the top and bottom phase were separated and the amount and purity of phycocyanin (620/280nm) present in each of the phases are estimated spectrophotometrically. The top PEG rich phase has a high amount of phycocyanin and bottom salt rich phase has other protein with residual impurities settling at interface. The partition coefficient (the ratio of phycocyanin concentration in top phase to that of the bottom phase) of the phycocyanin was estimated. The top phase containing phycocyanin was dialyzed against distilled water and lyophilized. PEG/Salt/ Phycocyanin 6/15/79 Crude Phase Top Phase Bot Phase V (ml) 24.4 70 Volume Ratio 0.3486 Total phycocyanin Mg/ml 1.4195 0.0061 0.7 Purity (620/280) 4.317 1.74 K 232.8 K- Partition coefficient Example 2 Around 6kg of Spirulina biomass was harvested using conveyor filter and washed with tap water to remove the culture media components. To the biomass an equal amount of water was added and then homosinised at the pressure of 100- 400 kg/cm2 for about 10 min. the solution obtained was filtered and centrifuged at the rate of 50 ml/min using cotton bed filter to remove chlorophyll and other impurities and disc type centrifuge at around 12,000 g to remove remaining traces of these impurities. Phycocyanin obtained was added to 7% w/w PEG and 8.03% w/w potassium phosphate to which 84.97% w/w of phycocyanin was added to have a total of 100%. The mixture was mixed using magnetic stirrer at 500 rpm at about one hour at room temperature. The mixture was allowed to equilibrate in a separating funnel for about 24 hrs the top and bottom phase were separated and the amount and purity of phycocyanin (620/280nm) present in each of the phases are estimated spectrophotometrically. The top PEG rich phase has a high amount of phycocyanin and bottom salt rich phase has other protein with residual impurities settling at the interface. The partition coefficient (the ratio of phycocyanin concentration in top phase to that of the bottom phase) of the phycocyanin was estimated. The top phase containing phycocyanin was subjected to ultra filtration and concentrated using osmotic membrane distillation. The concentrated sample was lyophilized. PEG/Salt/ Phycocyanin 7/8.03/84.97 Crude Phase Top Phase Bot Phase V (ml) 26 64 Volume Ratio 0.41 Total phycocyanin mg/ml 1.1065 0.0072 0.4097 Purity (620/280) 3.808 0.581 1.0 K 153.96 K = Partition coefficient Example 3 Around 6kg of Spirulina biomass was harvested using conveyor filter and washed with tap water to remove the culture media components. To the biomass an equal amount of water was added and then homogenized at the pressure of 100- 400 kg/cm2 for about 10 min. the solution obtained was filtered and centrifuged at the rate of 50 ml/min using cotton bed filter and disc type centrifuge at around 12,000 g to remove remaining traces of impurities. Phycocyanin obtained was added to 6% w/w PEG and 13% w/w potassium phosphate to which 81% w/w of phycocyanin was added to have a total of 100%. The mixture was mixed using magnetic stirrer at 500 rpm at about one hour at room temperature. The mixture was allowed to equilibrate in a separating funnel for about 24 hrs the top and bottom phase were separated and the amount and purity of phycocyanin (620/280nm) present in each of the phases are estimated spectrophotometrically. The top PEG rich phase has a high amount of phycocyanin and bottom salt rich phase has other proteins. The partition coefficient (the ratio of phycocyanin concentration in top phase to that of the bottom phase) of the phycocyanin was estimated. The top phase containing phycocyanin was subjected to ultra filtration and concentrated using osmotic membrane distillation. To the concentrated sample glycerol was added in which even the residual amount of PEG gets settled down. In the presence of glycerol the phycocyanin is stable and this can be kept at room temperature. PEG/Salt/ Phycocyanin 6/13/81 Crude Phase Top Phase Bot Phase V (ml) 20 66 Volume Ratio 0.3 Total phycocyanin mg/ml 1.3911 0.0114 0.4097 Purity (620/280) 3.896 0.622 1.0 K 121.93 K = Partition coefficient The main advantages of present invention are: 1. The formation of ATPS considerably reduces the volume of the phycocyanin that is to be processed for product recovery. 2. Higher concentration of phycocyanin can be achieved since most of it gets partitioned to the top phase. 3. The trace residual impurities such as chlorophyll, cell debris present get settled at the interface and hence no contamination of these compounds occurs. 4. The other proteins get partitioned to the bottom phase. 5. Additional steps for the purification of phycocyanin can be eliminated and also the process is less tedious. 6. All the operations are carried out at room temperature and hence is more economical. 7. The process is simple and easy to scale-up 8. Simple and inexpensive steps for the removal of contaminants such as chlorophyll and cell debris. 9. Substantial reduction in volume of the product, which results in easy handling of the product for further processing methods like ultrafiltration, osmotic membrane distillation and dialysis. 10. The final product retains its spectral properties thereby rendering this product to be used both as the food and reactive grade. We claim: 1. An improved process for the preparaion of pure phycocyanin concentrate which comprises ; a. harvesting the Spirulina biomass by known method, b. washing the said biomass with plain water by known method for 10-15 minutes, c. adding water at a ratio of 1:1 to the said biomass, d. homogenizing the biomass mechanically for a period of 5-10 minutes at a pressure in the range of 100-400 kg/cm2 e. adjusting the pH of the above obtained homogenized biomass to 4-5 by known methods, f. filtering the above obtained biomass and further passing through activated carbon at a flow rate of 50/100 ml/min, g. centrifuging the above obtained biomass to obtain phycocyanin solution , h. adding phase forming components selected from Polyethylene glycol (PEG) 1550, PEG 4000, PEG 6000 and PEG 20,000 at a level of 6-12% (w/w), and salt selected from Potassium phosphate, Ammonium sulfate, Magnesium sulfate, Sodium sulfate, Sodium citrate potassium phosphate at a level of 10-15% (w/w) i. mixing above obtained mixture for a period of 1-2hr at 400-600 rpm in a magnetic stirrer followed by equilibrating using separating funnel for at least 24 h and separating the top phase containing pure phycocyanin having purity in the range of 3.8 to 4.3 using spectrophotometric method at 620/280 nm, j. filtering the pure phycocyanin solution by ultrafiltration followed by osmotic membrane distillation to obtain pure phycocyanin concentrate at a range of .004% to .014%., 2. A process as claimed in claim 1 wherein the harvesting of the biomass is carried out by using vibro filter. 3. A process as claimed in claim 1 wherein phase forming agent used is selected from PEG 4000 and salt selected is Potasium phosphate. 4. An improved process for the preparation of pure phycocyanin concentrate substantially as here in described with reference to the examples 1 to 3 accompanying this specification.

Documents:

390-DEL-2003-Abstract-(10-12-2008).pdf

390-del-2003-abstract.pdf

390-DEL-2003-Claims-(10-12-2008).pdf

390-del-2003-claims.pdf

390-DEL-2003-Correspondence-Others-(10-12-2008).pdf

390-del-2003-correspondence-others.pdf

390-del-2003-correspondence-po.pdf

390-DEL-2003-Description (Complete)-(10-12-2008).pdf

390-del-2003-description (complete).pdf

390-del-2003-form-1.pdf

390-del-2003-form-18.pdf

390-del-2003-form-2.pdf

390-DEL-2003-Form-3-(10-12-2008).pdf

390-del-2003-form-3.pdf