Title of Invention	"AN IMPROVED PROCESS FOR THE PREPARATION OF STABLE PHYCOCYANIN"
Abstract	The present invention provides an improved process wherein a high osmotic and protein stabilizing agent in combination with antimicrobials or antioxidants is used for the stabilization and prevention of precipitation of phycocyanin - a natural blue colourant.

Full Text

This invention relates to an improved process for the preparation of stable phycocyanin . The present invention particularly relates to production of stable phycocyanin using a high osmotic and protein stabilizing agent in combination with antimicrobials or antioxidants or a mixture thereof. The phycocyanin thus obtained by this method finds application in food and beverages as colourant. Food colours are of the utmost importance of the food industry because of the powerful influence they exert over consumers' reactions to food products. Though natural colours have become increasingly popular over recent years, they have their limitations in processed food - with stability at the forefront. Natural colours are generally more sensitive to heat, light and pH than their synthetic counterparts. Therefore, they are added at the lowest possible temperatures and as late as possible during processing. Moreover, natural '""colours offer maximum stability at specific pH levels. In many applications water soluble colours are preferred (Rush, S. 1986, Food Ingredients & Processing International p23-27). In essence the natural colours are far more prone to deterioration than synthetics (Food ingredients and Processing International 1988 p.16-20). Despite these shortcomings, the demand for replacing synthetic colours with natural colours has been growing over last decade and will continue to grow. Due to consumer demand and pressures new sources of natural colours with better stability and tinctorial strength are being explored. The instability of the common natural colour has been studied to seek the means of stabilization. The methods employed for improving the stability of natural colours include formulation with, .addition of antioxidant and addition of a combined antioxidant and an emulsifier etc. (Food Marketing & Technology 1994 8(3) 8-10). Phycocyanin is a useful colourant which is recently attracting attention as one of many safe biocolourants for processed foods. & Patents on the use of algal colours mainly phycocyanin from Spirulina platensis and phycoerythrin from Phorphyridium, in foods include colouring of fermented milk products such as Yoghurt (JP 1979,^95770), ices (JP 1987, 06691), chewing gum (JP 1979 138156), soft drinks (JP 1979, 79 -76867) and alcoholic drinks (JP 1980, 077890). Phycocyanin colouration is unique -however it is very unstable in solutions during storage. On standing in solutions the blue colour fades and the original colour can never be recovered from the decolourised solution as the colour change is irreversible. Phycocyanin is the main light harvesting pigment in the blue green algae (Cyanophyta). Phycocyanin is built up of bilins which are open chain tetrapyrroles, covalently linked via one or two thioether links to the cysteine residues in the apoproteins (Killika, S.D. O'Carra P. and Murphy, R.F. 1980, Biochem J. 187, 311-320). The main bilin chromophore is known as phycocyanobilin. The apoproteins are built of equimolar amounts of two dissimilar subunits (alpha and beta), these subunits have molecular masses in the range of 17,000 - 20,000 Da. Each subunit is linked to one or more bilins. Phycocyanin can constitute a major portion of the algal cell protein. This pigment is highly soluble in water but lyophilization reduces the solubility. Powdered phycocyanin is-, stable during storage but it is not easily soluble. Moreover a convenient method for supplying the water soluble colourant for the use of food and cosmetic industries would be as concentrated solutions. The concentrations of this colourant required to colour food or cosmetics is 0.1% w/w (Arad. S.M. & Yaron, A. 1992, Trends in Food Science and Technology (3) 92-97). Procedures currently reported for the preservation of phycocyanin are the following. Phycocyanin when extracted from cyanophycean-- alga consists of a protein plus the chromophore linked by a peptide linkage. In many applications the protein becomes denatured and precipitates which also degrades the colour. To avoid this kind of degradation the phycocyanin has to be stabilised. Reference may be made to Japanese patent (JP 1980, 077890; JP 1978, 53101 575) wherein the phycocyanin is stabilised at low pH and high alcohol concentrations by pronase hydrolysis of the proteins in the phycobiliproteins. The drawback is pronase hydrolysis of phycocyanin to increase the stability of phycocyanin will remove the protein part and only the prosthetic group, Nthe tetrapyrrole group1 can be extracted into alcohol. Reference may be made to Japanese patent (JP 1979, 79 076 867) wherein to prevent precipitation of phycocyanin, gelatin was added which can stabilize the phycocyanin solution. The drawback is that addition of gelatin to phycocyanin can prevent the precipitation of phycocyanin but it may not be effective against the oxidative degradation of the protein part of the pigment and also to the microbial growth which often develops in aqueous solution of phycocyanin. Reference may be made to Japanese patent (JP 1979 138156) wherein addition of antioxidants such as Ascorbate and erythroborate are described for stabilization of phycocyanin. The drawback is that in solutions phycocyanin will get precipitated during storage which will affect the shelf life and therefore the addition of antiocidants alone is not helpful to prevent the deterioration. The main object is to provide an improved process for the preparation of stable phycocyanin. Another object of the present invention is to provide a process wherein a high osmotic and protein stabilizing agent in combination with antimicrobials or antioxidants is used for the stabilization and prevention of precipitation of phycocyanin - a natural blue colourant which obviates the drawbacks as detailed above. The phycocyanin used in present invention has been obtained from cyanophycean algae collected from water bodies of Mysore. Accordingly the present invention provides an improved process for the preparation of a stable phycocyanin characterized in that blending the said phycocyanin with synergistic composition comprising a high osmotic and protein stabilizing agent , an antimicrobial agent and an antioxidant, said process comprises of blending phycocyanin in the range 2.5% -15% with 25 - 75% high osmotic and protein stabilizing agent glycerol and 0.02 - 0.2% of antimicrobials selected from sodium benzoate, potassium bisulphate and 0.02 - 0.2% of antioxidants selected from L - cysteine, mannitol, at a temperature ranging 27° to 45°C , at least for 3 minutes to get stable phyconyanin. glycerol and 0.02 - 0.2% of antimicrobials selected from sodium benzoate, potassium bisulphite or Q.02 - 0.2% of antioxidants selected from L-cysteine, mannitol below the temperature of 45 ^C at least for 3 minutes to get a stable phycocyanin The high osmotic and protein stabilising agent used in the present invention has prevented the precipitation of phycocyanin protein in the suspension and the high osmotic character prevents microbial spoilage. /=» The addition of antimicrobial and antioxidants enhanced the stability and keeping quality of phycocyanin through the synergistic action. The degradation of the protein in the phycocyanin is inhibited by the high osmotic and protein stabilizing property of glycerol and oxidative degration is inhibited by the presence of antioxidants or the presence of antimicrobials prevents the microbial degradation. The main type of degradation involved in the storage of phycocaynin is oxidative the combination of high osmotic and protein stabilizing agent and antioxidant will contribute more stabilizing affect. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention Example-1 Stock solution of 2.5% (w/v) phycocyanin was prepared in 25% glycerol(v/v). The phycocyanin solution in 25% glyceroi was divided into two groups and stored at ambient (27+2°C) and refrigerated (8il°C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UV160A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.3S (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, ry Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 25% glycerol on the stability of phycocyanin Phycocyanin Preservation rate (%) Ambient Temp. Refrigerated temp. Control Degraded 35% 25% glycerol Degraded 70% Colour stability of Phycocyanin in 25% glycerol (-b* value, Bfueness of the sample) (Example Removed) Example-2 Stock solution of 5.0% (w/v) phycocyanin was prepared in 50% glycerol(v/v). The phycocyanin solution in 50% glycerol was divided into two groups and stored at ambient (27+.2°C) and refrigerated (8il°C) temperature. Stability studies of the pigment were carried out as described here.r.An aliquote of the • ' .v -••„. sample from the stock phycocyanin solution was' suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UV160A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 50% glycerol on the stability of phycocyanin Preservation rate (%) Ambient Temp. Refrigerated temp. Control Degraded 35 50% glycerol 75 72.5 Colour, stability of Phycocyanin in 50% glycerol (-b* value, Bmeness of the sample) Weeks Ambient temp. Refrigerated temp. Control 50% glycerol Control 50% glycerol 0 -2.029 -1.122 -1.145 -0.952 1 -1.095 -1.304 -1.875 -0.732 2 -0.577 -1.066 -1.875 -0.91 3 Degraded -1.645 -2.981 -0.654 4 -1.709 -2.752 -0.622 5 -1.075 -3.248 -0.718 6 -1.082 -2.822 -0.7365 Example-3 Stock solution of 7.0% (w/v) phycocyanin was prepared in 25% glycerol(v/v) containing sodium benzoate at 2000 ppm level. The phycocyanin solution in 25% glycerol with sodium benzoate was divided into two groups and stored at ambient (27±2°C) and refrigerated (8+:l0C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UV160A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 25 % glycerol/sodium benzoate on the stability of phycocyanin Phycocyanin preservation rate (%) Ambient Temp. Refrigerated temp. to Control Degraded 35% 25% glycerol + 91% 93% sodium benzoate Colour stability of the pigment (-b* value, Blueness of the sample) (Example Removed) Example-4 Stock solution of 10.0% (w/v) phycocyanin was prepared in 50% glycerol(v/v) containing sodium benzoate at 2000 ppm. The phycocyanin solution in 50% glycerol with sodium benzoate was divided into two groups and stored at ambient (27 + 2°C) and refrigerated (8+_l°C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (nig/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UVI60A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 50% Glycerol/Sodium Benzoate on the stability of Phycocyanin Phycocyanin preservation rate (%) Ambient Temp. Refrigerated temp. '0 Control Degraded 35% 50% glycerol + 87% ^ 77% sodium benzoate Colour stability of the pigment (-b* value, Blueness of the sample) Weeks Ambient temp. Refrigerated temp. Control 50% glycerol Control 50% glycerol +sodium benzoate +sodium benzoate 0 -2.029 -0.508 -1.145 -1.235 \ rt;U095 -0-904 -1-875 -1.185 2 -0.577 -0.904 -1.875 -1.723 3 Degraded -0.88 -2.981 -1.052 4 -0.763 -2.752 -1.171 5 -0.673 -3.248 -1.653 6 -0.753 -2.822 -1.059 Example-5 Stock solution of 6.0% (w/v) phycocyanin was prepared in 25% glycerol(v/v) containing potassium bisulphite at a concentration of 2000 ppm. The phycocyanin solution in 25% glycerol with potassium bisulphite was divided into two groups and stored at ambient (27 + 2°C) and refrigerated (8il°C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UV160A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45* days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 25% glycerol/Pot. bisulphite on the stability of phycocyanin Phycocyanin preservation rate (%) Ambient Temp. Refrigerated temp. Control Degraded 35 25% glycerol 76 94.5 + Potassium bisulphite Colour stability of Phycocyanin in 25% glycerol/Pot.bisulphite (-b* value, Blueness of the sample) Weeks Ambient temp. Refrigerated temp. Control 25% glycerol Control 25% glycerol +Pot."bisulphite +Pot.bisulphite 0 -2.029 -1.695 -1.145 -1.24 1 -1.095 -1.273 -1.875 -1.491 2 -0.577 -1.481 -1.875 -1.306 3 Degraded -1.503 -2.981 -1.009 4 1.678 -2.752 -1.54 5 -1.511 -3.248 -1.742 6 -2.008 -2.822 -1.114 Example-6 Stock solution of 12.0% (w/v) phycocyanin was prepared in 50% glycerol(v/v) containing potassium bisulphite at a concentration of 2000 ppm. The phycocyanin solution in 50% glycerol with potassium bisulphite was divided ^into two groups and stored at ambient (27+2°C) and refrigerated (8±1°C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UV160A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 50% Glycerol/potassium bisulphite on the stability of phycocyanin Phycocyanin preservation rate (%) Ambient Temp. Refrigerated temp. Control Degraded 35 50% glycerol 90 86 + Pot bisulphite ° Colour stability of Phycocyanin in 50% glycerol/Pot.bisulphite (-b* value, Blueness of the sample) (Example Removed)Example-7 Stock solution of 14.0% (w/v) phycocyanin was prepared in 50% glycerol(v/v) containing L-cysteine at a concentration of 2000 ppm. The phycocyanin solution in 50% glycerol with L-cysteine was divided into two groups and stored at ambient (27+ 2° C) and refrigerated (8±.1°C) temperature. Stability studies of the pigment were carried out as described here. An aliquote of the sample from the stock phycocyanin solution was suitably diluted, the phycocyanin content (mg/ml) was determined by measuring the optical density at 620 and 650nm using a glass cuvette with a 1cm light path in a Shimadzu Spectrophotometer model UVI60A. The phycocyanin content was calculated using standard formula OD620 - 0.7 X OD650 / 7.38 (Tandaeu DMN and Joumard, J. 1988, Methods in enzymology, Volume 167, p.318-328, Lester Packer & Alexander Glazer (Eds.) Academic Press). Phycocyanin dissolved in aqueous solution served as control. The colour stability of the pigment was followed by measuring the tristimulus values every week. The preservation rate was calculated from the percentage of the amount of pigment retained in the sample at the end of the experimental period. The samples were stored for 45 days and the pigment content and the tristimulus values were measured weekly in the sample. Effect of 50% Glycerol/L.cysteine on the stability of phycocyanin Phvcocyanin preservation rate (%) Amoient Temp. Refrigerated temp. Control Degraded 35 50% glycerol 86 98.55 + Cysteine,,, Colour stability of Phycocyanin in 50% glycerol/Cysteine (-b* value, Bfueness of the sample) Weeks Ambient temp. Refrigerated temp. Control 50% glycerol Control 50% glycerol +Cysteine +Cysteme 0 -2.029 -2.646 -1.145 -2.699 1 -1.095 -2.286 -1.875 -2.153 2 -0.577 -3.041 -1.875 -2.13 3 Degraded -2.173 -2.981 -2.52 4 2.346 -2.752 -2.302 5 -2.449 -3.248 -2.309 6 -2.212 -2.822 -2.553 The main advantages of the present ' invention are (1) No need to remove the protein part of the pigment for the preservation. (2) Enhances the shelf life of the pigment. (3) Phycocyanin is easily misible. (4) All the ingredients used in this system are non-toxic to the digestive system and does not irritate skin or sensitive tissues and all are generally recognised as safe food substance. (5) The stable phycocyanin thus obtained has no hazardous health effect. It proves to be ecofriendly and environmentally safe constitutent. We Claim: 1. An improved process for the preparation of a stable phycocyanin characterized in that blending the said phycocyanin with synergistic composition comprising a high osmotic and protein stabilizing agent , an antimicrobial agent and an antioxidant , said process comprises of blending phycocyanin in the range 2.5% - 15% with 25 - 75% high osmotic and protein stabilizing agent glycerol and 0.02 - 0.2% of antimicrobials selected from sodium benzoate, potassium bisulphate and 0.02 - 0.2% of antioxidants selected from L - cysteine, mannitol, at a temperature ranging 27° to 45°C , at least for 3 minutes to get stable phyconyanin. 2. An improved process for the preparation of stable phycocyanin as substantiated herewith with reference to examples.

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367-del-2000-abstract.pdf

367-del-2000-claims.pdf

367-del-2000-complete specification(granted).pdf

367-del-2000-Correspondence-Others.pdf

367-del-2000-correspondence-po.pdf

367-del-2000-description (complete).pdf

367-del-2000-form-1.pdf

367-del-2000-form-19.pdf

367-del-2000-form-2.pdf

367-del-2000-form-3.pdf