Title of Invention

A PROCESS FOR THE PREPARATION OF PURIFIED PHYCOCYANIN

Abstract

This invention relates to an improved process for the preparation of purified phycocyanin which comprises homogenizing freshly harvested blue green algal biomass using water then centrifuging to get extract, adding to the said aqueous alkali metal sulfate solution and a solvent (capable of partitioning phycocyanin from other impurities) selected from ethylene glycol, polyethylene glycol under stirring, separating the top solvent phase rich in phycocyanin by conventional methods such as decantation or pipeting, allowing to settle the phycocyanin by gravitation and then separating purified phycocyanin by conventional centrifugation method.

Full Text

This invention relates to an improved process for the preparation of purified phycocyanin. More particularly this invention relates to the purification of C-Phycocyanin a natural blue food colorant from blue green algae. The phycocyanin obtained by this purification method finds application as food colourant. Presently, there is a general tendency to move towards the use of natural rather than synthetic dyes in food and cosmetics. A number of synthetic pigments are being excluded from food and cosmetics use on the grounds that they are toxic, carcinogenic or otherwise unsafe. Microalgae grown in open ponds is a potential source of food colorant. The colors produced from microalgae are used in various food preparations such as fermented milk products, sweets, cake decoration, milk shakes (Anon 1987 Chemical Engineering p19 -22: Anon 1993 Food Ingredients and Analysis International p33; Paul Colins and Jane 1995, Food Tech.Europe p64-70; Guadin.C, 1988, Algal Biotechnology, Stadler T (Eds) Elsievier Applied Science p33-40) Phycobiliproteins are the principal photoreceptors for photosynthesis in blue green, red and some other types of algae. These proteins with covalently linked bile pigment chromophores may comprise up to 24% of the dry weight of the cells and well over half of the total soluble (Bennett. A and BogoradL, 1973 J. Cell Biol.,58, p419-422 ). Phycobilisomes are bound to the external face of the thylakoid membranes, where they serve as light absorbing antennae to fiinnel excitation energy into the reaction centers of photo system II. They show maximum absorption in the 470-650 nm region, in the valley between the blue and far red absorption peaks of chlorophyll a. These 600 nm dia hemidiscoidal or hemispherical peripheral membrane complexes contain hundreds of tetra pyrrole chromophores and have a mass comparable to that of ribosomes ( AR Grossman et al,1993 Microbiological Reviews, p725-749). Phycocyanin is used as a natural food colourtant especially for products such as ice cream, yogurt and there is potentially large market for the phycobilins (Borowitzka MA; 1994, Algal Biotechnology in Asia Pacific Region, Phang et al (eds) p 5 - 15). 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 ( Gnatt et al 1979, 1979, Plant Physiology, 63, p615-620, Gnatt and Lipschltz 1974, Biochemistry, 13, 2960-2966). Subjecting Phycocyanin to additional purification steps, like sucrose gradient centrifugation, gel filtration and column chromatography can further reduce the presence of these contaminants which occur in small amounts. But scale up of these methods is very difficult as they are more analytical ( Bennett and Bogorad 1971, Biochemistry, p3625 -3634, Yamanaka et al 1978, J Biol Chem. 253, 8303) than preparative methods) and uneconomical to produce Phycocyanin on large scale as food colorant Recent developments 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 down stream processing of biomolecules ( Albertson, 1986, Partition of cell particles and macromolecules, 3rd ed.New York, Raghavarao KSMS et al 1995, Advances in Applied Microbiology 41,97-171) 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. In the method of Bennetand Bogord (1971, Biochemistry,p3625-3634) portions of the algal slurry and the supernatant solution was saturated 70% ammonium sulfate, allowed to stand for lhour, centri&ged and the phycocyanin rich pellet was resuspended in 0.1 M acetate and dialyzed against 21it of the same buffer and after dialyses 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 1hour, centrifuged and the pellets were resuspended in 0.005M phosphate buffer. Further purification has been carried out using 2.5x25cm brushite column. Method of Gnatt and Lipschltz (1974, Biochemistry, 13, p2960-2966) utilizes sephadex chromatography and sucrose gradient centrifugation. In another method of Gnatt and Lipschuhz(1972,J.Cell.Biol. 54,p313-317), the phycocyanin is purified using only sucrose gradient centrifugation and stored in 0.75M phosphate buffer. Method of Yamanaka et al (1978, J.Biol.Chem. 253,p8303-8311) utilizes sucrose step gradient for the phycocyanin purification and the phycocyanin 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 Gulglieloni and Cohen (1984, Protistologica, 20v,p393-399) used discontinuous sucrose density gradient for the sucrose gradient are dialyzed at ambient temperature against 0.75M potassium phosphate buffer pH7, containing 5x10-1 M phenyl methylsulphonyl fluoride and ImM EDTA. For long term storage, the phycobilisomes are precipitated by ammonium sulfate, pelleted by centrifiigation and stored at -20° C Ion exchange chromatography using Diethyl aminoethyl cellulose (DEAE)-650 was used in the method of Glauser et al (1992, FEBS letters 297,pl9-23). In another method (Glazier, AN and Hixson,CS 1975,J.Biol.Chem 5487 - 95) the phycocyanin purification was carried out by repeated chromatography on hydroxyapatite and by gel Alteration on sephadex G200 at neutral pH. Bryant et al (1976, Arch.Microbiol 110,p61-66) used ammonium sulphate precipitation followed by Diethylaminoethyl cellulose Chromatography. DEAE cellulose chromatography followed by hydroxyapatite column was used for the purification of phycocyanin in the method of Kobayashi et al (1992 Arch.Biochem.Biophys 152, p187-194) Gardner et al (1980, Biochem.Biophys.Acta 624, pl87-192) used DEAE-cellulose column preequilibrated with 20mm Tris-Sulphate, pH8. The Phycocyanin, obtained by the ammonium sulphate precipitation of the elutant was stored at 4°C. Rosiniki et al (1981 Ann.Bot 47, pl-12) purified Phycocyanin by chromatography on a sepharose column. Drawbacks connected with the hitherto processes are as follows: Most of the methods known for the purification of phycocyanin utilises, Ammonium sulphate precipitation followed by DEAE column chromatography or gel Alteration on sephadex G200 or sepharose CL4B column chromatography which are not feasible for the large scale production of Phycocyanin. Chromatographic methods will result in the dilution of the pigment and therefore need further concentration of the pigment, which is not cost effective. Chromatographic procedures are limited in the scale up operation by the cost of resins. For ammonium sulphate precipitation of large quantity of ammonium salt has to be used and will result in the requirement of one more purification step like dialysis. Sucrose step gradient is difficult to scale up and also uneconomical for the large scale purification purposes. Main objective of the invention is to provide an improved process for the preparation of the purified phycocyanin- The natural blue colourant. Another object is to provide simple economic commercially viable environmental friendly system still another object is to prepare purified phycocyanin using aqueous two phase system. One phase of the system consists of alkali metal sulphate and other phase comprises of phycocyanin solution obtained from homogenisation of algal biomass. The process comprises of the following steps: 1. The homogenisation and centrifugation of the harvested blue green algal biomass. 2. Preparation of the phase system containing polyethylene glycol (in the range of 6-20%w/w) and sulphate salts of alkali metals (in the range of 5 -20%) water (in the range of 5 - 20%w/w) and homogenised blue green algal biomass (in the range 30 - 60 % w/w). 3. Permitting the phase system to separate in a separating fiinnel where Phycocyanin partitions to PEG rich top phase and the chlorophyll and other contaminants to the salt rich bottom phase. 4. Separating the PEG rich (Top) phase containing Phycocyanin and allowing the phycocyanin to precipitate which upon centrifugation yield the product (Phycocyanin) free from chlorophyll and other contaminants. Accordingly the present invention provides an improved process for the preparation of purified phycocyanin which comprises homogenizing freshly harvested blue green algal biomass using water then centrifuging to get extract, adding to the said aqueous alkali metal sulfate solution and a solvent (capable of partitioning phycocyanin from other impurities) selected from ethylene glycol, polyethylene glycol under stirring, separating the top solvent phase rich in phycocyanin by conventional methods such as decantation or pipeting , allowing to settle the phycocyanin by gravitation and then separating purified phycocyanin by conventional centrifugation method.. Alkali metal sulphate used is selected from Sodium sulphate, Potassium sulphate and the quantity used is in the range from 5 to 12 %. The invention is further illustrated in the following examples which should not however be construed to limit the scope of the invention. Example -1 50 ml of suspension containing phycocyanin obtained be the homogenisation and centrifugation of freshly harvested biomass of Spirulina platensis, was added to the other components to yield 100 gm of phase system of total composition of Polyethylene Glycol (PEG,7.5%), Sodium sulphate salt (6.5%). This phase system kept at room temperature was rigorously agitated using a stirrer and allowed to settle for 30 minutes. Phycocyanin partitioned to the PEG rich (top) phase and impurities like chlorophyll partitioned to the salt rich (bottom) phase. The PEG rich (top) phase was collected and kept for one hour for gravity precipitation of Phycocyanin and then centrifuged at 5000 RPM, to get Phycocyanin. The Phycocyanin thus obtained was found to retained its spectral properties. The absorbion spectrum of the product was identitical to the reported pattern in literature (Gnatt etal 1979 Plant physiol 63,615-620.) Example - 2 40 ml of suspension containing phycocyanin obtained be the homogenisation and centrifugation of freshly harvested biomass of Spirulina platensis, was added to the other components to yield 100 gm of phase system of total composition of Polyethylene Glycol (PEG, 10%), Sodium sulphate salt (8.0%). This phase system kept at room temperature was rigorously agitated using a stirrer and allowed to settle for 30 minutes. Phycocyanin partitioned to the PEG rich (top) phase and impurities like chlorophyll partitioned to the salt rich (bottom) phase. The PEG rich (top) phase was collected and kept for one hour for gravity precipitation of Phycocyanin and then centrifuged at 5000 RPM, to get Phycocyanin. The Phycocyanin thus obtained was found to retained its spectral properties. The absorbion spectrum of the product was identitical to the reported pattern in literature (Gnatt et al 1979 Plant physiol 63.615-620.) Advantages of the present purification process can be summarized as 1. The use of Aqueous two phase system is simple, can be carried out without the use of sophisticated equipment and easy to scale up 2. Elimination of the use of sucrose gradient centrifugation or ammonium sulfate precipitation for purification which are cumbersome and prohibitively expensive purification methods to scale up. 3. This purification method helps not only in retaining the biochemical activity but also in enhancing the stability of the product 4. Complete removal of chlorophyll and other impurities (if any) in a single preparation cum purification step. The Phycocyanin solution volume is reduced to about l/4th to l/5th so that very less volume has to be processed in the final concentration step (centrifugation). 5. The absence of the solid supports or adsorptive surfaces as exist in chromatography, makes the present method of using aqueous two phase systems more efficient (due to reduced mass transfer resistance's) for the separation and purification of biomolecules like phycocynian. 6. The problems of toxicity that often characterize the organic solvent extraction process are eliminated as aqueous two phase systems have high water content(85-90 %) 7. The phase system can be reused to certain extent, which favorably adds to the economics of the process. We claim: 1. An improved process for the preparation of purified phycocyanin which comprises homogenizing freshly harvested blue green algal biomass using water then centrifuging to get extract, adding to the said aqueous alkali metal sulfate solution and a solvent (capable of partitioning phycocyanin from other impurities) selected from ethylene glycol, polyethylene glycol under stirring, separating the top solvent phase rich in phycocyanin by conventional methods such as decantation or pipeting , allowing to settle the phycocyanin by gravitation and then separating purified phycocyanin by conventional centrifugation method. 2. An improved process as claimed in claim 1 wherein the alkali metal sulfate used is selected from Potassium sulfate and Sodium sulfate and the amount ranges from 6 to 10 % w/v. 3. An improved process as claimed in claims 1 & 2 wherein the top phase used is selected from Polyethylene glycol and ethylene glycol. 4. An improved process as claimed as in claim 3 wherein the concentration of the top phase compound ranges from 5 % to 15%. 5. An improved process as claimed in claims 1 to 4 wherein separation of pure phycocyanin is effected by the concentration of both the alkali metal sulfate and polyethylene glycol. 6. An improved process for the preparation of purified phycocyanin substantially as herein described with reference to the examples.

Documents:

1119-del-1998-abstract.pdf

1119-del-1998-claims.pdf

1119-del-1998-complete specification [granted].pdf

1119-del-1998-correspondence-others.pdf

1119-del-1998-correspondence-po.pdf

1119-del-1998-description (complete).pdf

1119-del-1998-form-1.pdf

1119-del-1998-form-2.pdf

1119-del-1998-form-4.pdf