Title of Invention

A PROCESS FOR THE PREPARATION OF FOOD COLOURANT FROM SPIRULINA.

Abstract

The present invention relates to a process for the preparation of food colourant from Spirulina. The present invention more particularly relates to an integrated process for the preparation of phycocyanin using aqueous two-phase extraction (ATPE) and osmotic membrane distillation (OMD). The major advantages of ATPE are high capacity, biocompatible environment, low interfacial tension, high yields, lower process time and energy, and high selectivity. 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.

Full Text

The present invention relates to a process for tlie preparation of food colourant from Spirullna. The present invention more particularly relates to an integrated process for the preparation of phycocyanin using aqueous two-phase extraction (ATPE) and osmotic membrane distillation (OMD). The practice of coloring food dates back to ancient times and today color has become a vital constituent of food. Color is one of the first characteristics perceived by the sense and is indispensable for rapid identification and ultimate acceptance of a product. 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. Microalgae grown in open ponds is a potential source of food colorant. The micoalgae contains phycobiliproteins that have applications in food preparations like fermented milk products, sweets, milk shakes and cake decorations. Cyanobacteria and algae possess a wide range of coloured components including carotenoids, chlorophyll and phycobiliproteins. The principal phycobiliproteins are Phycocyanin, allo-phycocyanin and phycoerythrin. C-phycocyanin is a blue colored, red fluorescing biliprotein, present in all the species of cyanophyceae family (eg;Spirulina sp). C-Phycocyanin comprises of a protein and cht'omophore, 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 Spirullna 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. Phycocyanin is used as a colorant in food (chewing gums, dairy products gellies etc) and cosmetic such as lipstick and eye liners in Japan, Thailand and China. It was also shown to have therapeutic value (Immunomodulating and anti cancer activity). Owing to its fluorescence properties it is used in phycofluor probes for immuno-diagnostics. The purification of C-phycocyanin is important because of its high commercial importance. There are several methods such as freezing and thawing, homogenization in buffer solutions etc for the extraction of Phycocyanin and almost all these methods suffer from the problem of chlorophyll contamination. The other contaminants are small fragments of cell wall and membranes, glycogen storage particles. The purification of Phycocyanin using sucrose gradient centrifugation, gel filtration and column chromatography can further reduce the presence of these contaminants but scale up of these methods is very difficult as they are more analaytical than preparative methods. 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 two-phase systems. Recent development in biotechnology utilizes the potential of Liquid-liquid extraction using aqueous two-phase systems and has recognized this technique as a superior, economical and versatile technique for the downstream processing of biomolecules. The major advantages of ATPE are high capacity, biocompatible environment, low interfacial tension, high yields, lower process time and energy, and high selectivity. 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. Procedure currently used for the purification of Phycocyanin mainly makes use of chromatographic separation, SDS gel electrophoresis or sucrose gradient centrifugation or ammonium sulfate precipitation or the combination of these methods. Reference may be made to Bennet and Bogord 1971, Biochemistry P-3625-3634 wherein portions of algal slurry and the supernatant solution was saturated by 70% ammonium sulfate, allowed to stand for Ihour, centrifuged and the Phycocyanin rich pellet was re-suspended in 0.1 M acetate and dialyzed against 2lit of the same buffer and after dialysis Phycocyanin is passed through a 2.5 X 40cm G-100 column. The Phycocyanin bands were pooled and again brought to a saturation of 70% ammonium sulfate allowed to stand for 1 hour, centrifuged and the pellets were re-suspended in 0.005M phosphate buffer. Further purification has been earned out using brushite column. Reference may be made to Gnatt and Lipschultz, 1972 J. Cell.Bio.54,P-313-317, wherein the Phycocyanin is purified using only sucrose gradient centrifugation and stored in 0.75 M phosphate buffer. In another reference of Gnatt and Lipschultz 1974, Biochemistry, 13,P2960-2966, Phycocyanin is purified by sephadex chromatography and sucrose gradient centrifugation. Reference may be made to Yamanaka et al 1978, J. Bio. Chem, 253 P-8303-8311 wherein the Phycocyanin purification is carried out by sucrose step gradient and was recovered from the 0.75M sucrose layer, free of detectable chlorophyll which was stable for a period of about 2 weeks at 4°C as a concentrated solution. 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 utilization of chromatographic method is 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 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 free from the precipitating agent, since it has to be used in food/pharmaceutical applications. Reference may be made to Glauser et al 1992, FEBS letters 297, PI9-23, wherein ion exchange chromatography using Diethyl amioethyl cellulose (DEAE)-650 was used for the purification of Phycocyanin. 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 with separation of Phycocyanin from cell homogenate by ATPE and Ultra filtration wherein differential partitioning of Phycocyanin and cell debris was achieved in ATPE with the help of centrifugation and removal of Phycocyanin from phase components was achieved by ultra filtration. Since this work involves expensive operations such as centrifugation and ultra filtration , hence this process is not economically viable and cannot be implemented for large scale separation of Phycocyanin. Reference may be made to Nagaraj et al. 2003(435/DELy03) for separation of Phycocyanin from homogente wherein leaching was carried out by acidified water and solid separation was achieved by deep-bed filtration and centrifugation and purification was achieved by adsorbing impurities on charcoal while concentration was achieved by osmotic membrane distillation. This process is not suitable for achieving high purity of Phycocyanin. Furthermore this process involves expensive operation such as centrifugation and deep bed filtration which are difficult to scale up and hence cannot be used on large scale for extraction, purification and concentration of Phycocyanin. Reference may be made to Chethana et al. 2003 (390/DEL/03) for separation of Phycocyanin from homogenate wherein leaching was carried out by acidified water and solid separation was achieved by centrifugation and purification was achieved by ATPE. This process is suitable for achieving high purity of Phycocyanin however results have shown extensive material loss during processing. Furthermore, process involves expensive operation for solid separation, hence may not be suitable for purification of Phycocyanin on large scale. The main object of the present invention is to provide a process for the preparation of food colourant from Splrulina. Another object of the present invention is to separate and extract Phycocyanin free from soluble and insoluble contaminants. Still another object of the present invention is to recover Phycocyanin without the use of precipitating agents. Another object of the present invention is to obtain complete leaching of Phycocyanin from the cells, solid removal and concentration. Yet another object of the present invention is to obtain a high purity of Phycocyanin. Still another object of the present invention is to recycle the PEG obtained after complete purification and concentration of Phycocyanin. Yet another object of the present invention is to concentrate the Phycocyanin Still another object of the present invention is to facilitate the easy processing of Phycocyanin by integrating vahous steps such as leaching, solid separation, concentration, purification and product recovery Accordingly, the present invention provides a process for the preparation of food colourant from Spirullna, comprising; a) harvesting the biomass of spirulina, b) washing the biomass with water to remove the culture media components for about 10-15 minutes c) adding water to the biomass at a ratio of 1:1 and tlien homogenizing the biomass for about 5-10 minutes, at a pressure range of 100-400 kg/cm2, d) adjusting the pH of the above homogenized solution to 4-5, e) adding of the phase forming components such as Polyethylene Glycol at a level of 6-12%(w/w) optimally/ potassium or sodium phosphate at a level of 10-15%(w/w), f) mixing the components obtained from the above step for a period of 50-60 min. at a speed of at 400-600 rpm, g) equilibrating the mixture in the separating funnel for 4-6h, h) purifying the top phase by adding the water and salts selected from a group of K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 or combination of these salts to the top phase (obtained from the first extraction) wherein it again forms a two phase and the soluble impurities (allophycocyanin, nucleic acids, proteins and carbohydrates) present in the top phase gets partitioned to the bottom phase, i) adding salts selected from a group comprising K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 and combination of these salts to the top phase (obtained from second extraction) and subjecting it to Osmotic membrane distillation will result in the formation of dispersion wherein Phycocyanin precipitates at the interface, j) centrifuging the dispersion to obtain the colourant. In an embodiment of the process the micro-alga source of food colourant is from different species of blue green algae such as Spirulina platensis and Spirulina maxima. In an another embodiment of the process the phase forming polymer is polyethylene glycol 1500 and 4000 or combination thereof. In an another embodiment of the process the food colourant is phycocyanin. In the present invention, freshly harvested Spirulina biomass is washed two to three times to remove the culture media components. The biomass is homogenized in a homogenizer at the pressure of 100 - 400 kg/m2 for 10-15 min. The cell homogenate obtained is used for the preparation of aqueous two-phase system. The novelty of the present invention is that fact that ATPE is able to do the job of different processes such as filtration, centrlfugation extraction adsorption in a single stage. Another novelty lies in increasing the leaching efficiency and purification by ATPE simultaneously. Novelty lies in the formation of a new phase systems by the addition of salt in such a way that differential partitioning of Phycocyanin, the product to one phase and the impuhties like allophycocyanin, nucleic acids, proteins, and carbohydrates to the other phase occurs simultaneously. Novelty also lies in the fact that OMD was effectively employed to achieve concentration of Phycocyanin and isolation of Phycocyanin from phase components in single step. The following examples are given by way of illustration of the present invention and should not be construed to the limit of 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 homogenized at the pressure of 100-400 kg/cm^ for about 10 min. Cell homogenate obtained was added to 6% w/w PEG and 15% w/w potassium phosphate to which 79% w/w of Cell homogenate 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 6 hrs, the top and bottom phase were separated and the amount and purity of Phycocyanin (620/280nm) present in each of the phases were 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. To the top phase, K2HPO4, and KH2PO4, and water were added to achieve phase composition of 6% PEG and 13% salt and allowed for equilibration. The other contaminants present in the top phase get partitioned to the bottom phase resulting in the increased purity of Phycocyanin. To the top phase containing phycocyanin 10-15% potassium phosphate salts were added and subjected to OMD wherein excess water in the top phase was removed. The partition coefficient (the ratio of phycocyanin concentration in top phase to that of the bottom phase) of the phycocyanin was estimated. The recovery of Phycocyanin in top phase was calculated as percent yield (YT% =(CTVT) X 100 (CiVi) (Table Removed) 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 homogenized at the pressure of 100-400 kg/cm^ for about 10 min. Cell homogenate obtained was added to 12% w/w PEG and 15% w/w potassium phosphate to which 73% w/w of cell homogenate was added to have a total of 100%w/w. The mixture was mixed using magnetic stin-er at 500 rpm at about one hour at room temperature. The mixture was allowed to equilibrate in a separating funnel for about 6 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 spectrophotometncally. 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. To the top phase, K2HPO4, and KH2PO4, and water were added to achieve phase composition of 9.41% PEG and 13% salts and allowed for equilibration. The other contaminants present in the top phase get partitioned to the bottom phase resulting in the increased purity of Phycocyanin. To the top phase containing Phycocyanin 10-15% potassium phosphate salts were added and subjected to OMD wherein excess water in the top phase was removed. The partition coefficient (the ratio of Phycocyanin concentration in top phase to that of the bottom phase) of the Phycocyanin was estimated. The recovery of Phycocyanin in top phase was calculated as percent yield (YT% =(CTVT) X 100 (CiVi). (Table Removed) 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 impurities such as chlorophyll, cell debris present get settled at the interface and hence no contamination of these compounds occurs. 4. The other phycobiliproteins like allo-phycocyanin 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 earned out at room temperature and hence are more economical. 7. The process is simple and easy to scale-up 8. This process provides simple and inexpensive step for the removal of insoluble 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 10. The final product retains its spectral properties thereby rendering this product to be used both as the food and reactive grade. 11.The phase forming polymer, PEG is recovered and recycled. It was found to have the physical properties similar to that of pure (fresh) PEG. 12. Osmotic membrane distillation removes the water at ambient conditions thereby used to alter phase composition to facilitate recovery of Phycocyanin from phase components. We claim: 1. A process for the preparation of food colourant from Spirulina, comprising; a) harvesting the biomass of spirulina, b) washing the biomass with water to remove the culture media components for a period of 10-15 minutes, c) adding water to the biomass in a ratio of 1:1 and then homogenizing the biomass for a period of 5-10 minutes, at a pressure ranging 100-400 kg/cm2, d) adjusting the pH of the above homogenized solution to 4-5, e) adding of the phase forming components such as Polyethylene Glycol in the range of 6-12%(w/w) optimally/ potassium or sodium phosphate in the range of 10-15%(w/w), f) mixing the components obtained from the above step for a period of 50-60 min. at a speed of at 400-600 rpm, g) equilibrating the mixture in the separating funnel for 4-6h, h) purifying the top phase by adding the water and salts which are selected from a group of K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 either or combination of these salts to the top phase to obtain the dispersion, i) centrifuging the dispersion to obtain the colourant. 2. A process as claimed in claim 1, wherein micro-algae source of food colourant is selected from a group of blue green algae comprising Spirulina platensis and Spirulina maxima. 3. A process for the preparation of food colourant from Spirulina substantially as here in described with reference to the examples accompanying this specification.

Documents:

548-DEL-2004-Abstract (26-10-2009).pdf

548-del-2004-abstract.pdf

548-DEL-2004-Claim- (26-10-2009).pdf

548-del-2004-claims.pdf

548-DEL-2004-Correspondence-Others (26-10-2009).pdf

548-del-2004-correspondence-others.pdf

548-del-2004-correspondence-po.pdf

548-del-2004-description (complete).pdf

548-DEL-2004-Description(Complete)- (26-10-2009).pdf

548-DEL-2004-Drawings (26-10-2009).pdf

548-del-2004-drawings.pdf

548-DEL-2004-Form-1 (26-10-2009).pdf

548-del-2004-form-1.pdf

548-del-2004-form-18.pdf

548-DEL-2004-Form-2 (26-10-2009).pdf

548-del-2004-form-2.pdf

548-DEL-2004-Form-3 (26-10-2009).pdf

548-del-2004-form-3.pdf

548-del-2004-form-5.pdf