Title of Invention

A PROCESS FOR THE PREPARATION OF CAROTENOID RICH SPIRULINA

Abstract

A process for the preparation of carotenoid rich Spirulina The present invention relates to a process for preparation of carotenoid rich Spirulina. The colour reduced, carotenoid rich Spirulina finds application in food products as a nutritional supplement at higher proportions. The novelty of the invention is that treatment of Spirulina biomass with mix of miscible solvent and water facilitated the extraction of blue colour phycobiliproteins and green colour mainly chlorophyll and the resulting Spirulina powder enriched with 0-carotene. The blue colour obtained from Spirulina biomass without homogenization of cells can be concentrated and the process can be scaled up easily by known methods. The green colour (mostly chlorophyll a ) obtained also with out homogenization can be concentrated and stabilized by known methods. The Spirulina biomass after pigments extraction obtained as byproduct with enriched p-carotene (3-4 times) and also rich in protein (42-45%) can be incorporated in food products at higher levels (2-5 times) than that of using the original (untreated) Spirulina powder.

Full Text

present invention relates to a process for preparation of carotenoid rich Spirulina. The colour reduced, carotenoid rich Spirulina finds application in food products as a nutritional supplement at higher proportions. The unprocessed biomass otherwise imparts undesirable dark colour to the food products thereby limiting its application. The pigments extracted during this process can be concentrated and further finds application as food or cosmetic colourants^ Spirulina is recognized as a unique health food throughout the world. Besides its high protein content (55-65%), it has also been reported to be a good source of carotene (precursor of vitamin A), B vitamins, pigments-phycobilins and chlorophyll, minerals and also a good source for gamma linolenic acid - an important nutraceutical. Spirulina has shown to possess cholesterol reducing effect. The Harvard University School of Dental Medicine showed reduced oral cancer cells when Spirulina extracts were used (Babu M et al 1995, Evaluation of chemoprevention of oral cancer with Spirulina. Nutrition and Cancer, 24: 197-202). In Japan, the reports showed that Spirulina reduced kidney nephrotoxicity from mercury and three pharmaceutical drugs in experimental rats (Yamane Y 1988. The effect of Spirulina on nephrotoxicity in rats. Annual symposium of the Pharmaceutical Society of Japan April 15, 1988). Spirulina may also have positive effects against diabetes and reduce high blood pressure (Takai et al 1991. J.Jap. Soc.Nutr. Food Sci 44:273-277). Research in Japan showed that Spirulina increased Lactobacillus in rats 3 times more over a control group (Takai Y et al. 1987, effects of Spirulina on caecum content in rats. Chiba Hygiene College Bulletin, Japan Feb 1987, Vol 5, N0.2). In France, pharmaceutical compounds containing Spirulina accelerated wound healing (Clement G et al. inventors, Institute Francais de petrol, assignee, wound treating medicaments containing algae, Fr.M.5279 lnt.cl.A61 K 11 Sep 1967). A study in Mexico also hinted to the beneficial effect when Spirulina was incorporated in the diet of undernourished children and adults. In Togo, rapid recovery of malnourished infants was reported in a village clinic. In our country large scale studies with preschool children showed carotenes in Spirulina helped to recover from symptoms of vitamin A deficiency (Seshadri, CV 1993. Large scale nutritional supplementation with Spirulina alga. All India project , MCRC, Madras). In Romania Fica V et al 1984 (Observations on the utilization of Spirulina as nutritive factor in treating diseases accompanied by a nutritional deficiency, Clinica 11 Medicala, Spitalui clinic Municipiului Bucuresti, Med Interna 36 (3) 1984) Spirulina tablets were prescribed to patients with nutritional deficiencies and the patients gained weight, and their health improved. In China, Spirulina has been prescribed as a baby nourishing ingredient in baked barley sprouts. 27 out of 30 children aged between 2 and 6 received in a short period from bad apetite, night sweat, diarrhea and constipation (Miao Jian Ren. 1987 Spirulina in Jiangxi China. Academy of Agricultural Science, Jiangxi Province, China. Paper presented at Soc. Appl. Algology, Lille, France Sep 1987). In 1989, the National Cancer Institute announced that chemicals from blue green algae were found to be remarkably active against the AIDs virus and 100 types of cancer (Gustafson et al 1989). The sulfolipid portions of the glycolipids in Spirulina can prevent viruses from either attaching to or penetrating into cells resulting in prevention viral infection (Gustafson et al 1989. AIDs -antiviral sulpholipids from Cyanobacteria (blue- green algae), J.National Cancer Institute, August 16, 1254). The above reports substantiate the nutritional and pharmaceutical benefits of Spirulina owing to its unique constituents and composition. In majority of the above instances Spirulina was consumed mostly in the form of tablets or capsules. It can be mentioned here that consumption of Spirulina as a medical or pharmaceutical method reduces its wide use. On the other hand its consumption can be increased to few folds if it is possible to incorporate in food products, such that it can be consumed in a natural way rather than use in an artificial way in form of tablets or capsules. Spirulina also forms a good source for natural colourants. There is a growing concern in the consumers for the ingredients in their foods and require their foods to be as natural as possible. Food colours plays an important role both in their acceptance and quality. The growing awareness of the beneficial effects of natural colours brought considerable demand for natural colours and their sources. Microalgae such as Spirulina forms one such natural source for colourants such as phycobiliproteins, chlorophylls and carotenoids. Algal colorants are used in various food preparations like fermented milk products, milk shakes, desserts, ice creams, chewing gums, soft drinks and alcoholic drinks etc (F.J.Francis, Lesser known food colourants, Food Technology 1987, 62-67., Arad SM and Yaran A, Natural pigments from red microalgae for the use in foods and cosmetics, Trends in Food Science and Technology 1992, 92-97 and in cosmetics Bioconsult, Agro-lnd Hitech, 1991,2:50-52). Phycobilins are deeply coloured, water soluble fluorescent pigment -protein complexes. They act as accessory light harvesting pigments in algal phytosynthesis. They are found in all bue-green, red and cryptomonad algae. They are classified into three major groups. Phycoerythrins (PEs), Phycocyanins (PCs) and allophycocyanins(APCs). The phycoerythins are red with a bright orange fluorescence, while the phycocyanins and allophycocyanins are blue and fluoresce red. The range of colors found within these groups of pigments is large, depending on the source of the biliprotein and the culture medium in which it is grown and isolated. The individual phycocyanin and phycoerythrin have been classified according to their algal origin and absorption characteristics (Bennet A, Siegelman HW 1979 In the porphyrins. Vol 6, D.Dolphin (Ed). Pp 493-520. Academic Press, NewYork.). Owing to their intense colour and fluorescence properties, phycobiliproteins are widely used as fluorescent markers and find applications in flow cytometry, fluorescence activated cell sorting, histochemistry and in immunodiagnostics etc besides being used as colourants in foods and cosmetics (Glazer, A.N and Stryer, L.Phycofluor probes, TIBS, 423-427, 1984., Glazer, A.N Phycobiliprotein - a family of valuable, widely used fluorphores J.Appl.Phycology 6:105-112, 1994., Oi V.T, Glazer, A.N and Stryer, L.Fluorescent phycobiliprotein conjugates for analyses of cells J.Cell.Biol, 93: 981-986, 1982). Most of the procedures currently reported for the extraction of phycobiliproteins involves disruption of cells. Procedures currently reported for the extraction of phycobiliproteins in microalgae are the following Reference may be made to the method of Yamanaka et al 1981 (Yamanaka & Glazer, Arch. Microbiol, 130,23,1981) wherein the method involves incubation of harvested cells (by centrifugation) with 30mm Sodium Phosphate buffer pH 6.8 containing mannitol, disodium EDTA & egg white lysozyme (5 mg/ml). Cells were sonicated and solubilised using Triton x 100 and the phycocyanin is purified by sucrose gradient centrifugation. The drawback in this method is that expensive chemicals like lysozyme is used for treatment and the triton x 100 and steps like sucrose gradient centrifugation is difficult to do on large scale limits to only laboratory studies of purification of phycocyanin. Reference may be made to the method of Rosinski et al 1981 (Rosinski et al, Ann.Bot, 47,1,1981) wherein algal cells were treated with Triton x100 in place of homogenization by pressure and suspended the cells at 1g wet weight / 15ml in an extraction medium containing 0.75 M potassium phosphate buffer pH 6.8, 1%(w/v) Triton x 100. This was followed by centrifugation to remove cell debris. The drawback in this method is that separation of phycocyanin from buffer and triton x100 and it is limited to lab level only. Reference may be made to the method of Glauzer et al 1992 (Glauzer, M. et al, FEBS, 297, 19-23, 1992) wherein Phycobiliprotein extraction method involved use of protease inhibitors like 1mm phenyl methyl sulphonyl fluoride(PMSF), 1mm EDTA, 1mM sodium azide) in potassium phosphate buffer and phycobiliprotein was extracted by homogenizing the cells. The drawback in this method is that addition of protease inhibitors to large scale process not only adds to the cost but also separation of these inhibitors is crucial and again limits its application to lab level. Reference may be made to the Japanese patent (JP 79164016) wherein the method involves acidification of the blue green algae cells with acid and separation of algal cells followed by washing with organic solvents (acetone and/or methanol). The drawback in this method is that procedures involving acidification of cells followed by solvent extraction will result only in the isolation of the tetrapyrrole prosthetic group. The algal cells treated with acid may not be useful as a by product and the important constituents such as protein, carotenoids may be effected by this treatment. Reference may be made to the French patent (2453199) wherein the method involves crushing of blue green algae cells followed by heat treatment at 100-170° C. The blue pigment is extracted. The drawback in this method is that methods involving heating of cells (100-170°C) can denature the protein part of Phycobiliproteins and only the phycocyanobilin (chromopore) part may be extracted. Heat treatment of cells will effect the other valuable constituents of the algal biomass. Reference may be made to the method of the Rebeller et al 1980 (Rebeller et al 1980 French Patent 2453199) wherein extraction of phycobiliproteins from cyanobacterial cells involves treatment of cells with CaCIa (0.8 - 8.0 g/1) followed by extraction of phycocyanin with a 0.001 - 0.1N alkaline solution containing sodium or potassium carbonate or bicarbonate. The drawback in this method is that methods involving extraction of pigments into alkaline solution containing sodium or potassium carbonate or bicarbonate salts can not be used directly unless concentrated due to salts and instability of pigments under alkaline conditions. Reference may be made to the method of Sarada et al (Indian patent DEL/1029/96) wherein phycobiliproteins {phycocyanin) was extracted without homogenisation of cells by osmotic principle and extraction into aqueous phase. The drawback in this method is that it is a two step process while the present invention is a single step process. Procedures currently reported for the extraction of chlorophyll (green colour ) in microalgae and plant material are the following Reference may be made to the process of AOAC 1999 (Chlorophyll in plants, ln:AOAC Official Method Number 940.03 16th Chapter, 16th Edition, Volume 1, Patricia Cunniff (Editor), AOAC International Suite 500, pp26-28, USA) wherein the plant material is extracted with 85% acetone for chlorophyll estimation. The drawback in this method is that carotenoids are also extracted along with chlorophyll and hence the procedure is not selective for chlorophyll alone. The procedure is limited to quantitative estimation of chlorophyll in the plant materials. Reference may be made to the method of Reinhard Eder 1996 (Pigments, In: Handbook of Food Analysis Volume 1, Leo M.L Nollet (Editor), Marcel Dekker, 937- 1014) wherein the food material is either disintegrated and the chlorophylls are extracted in a blender or ground in a mortar and pestle with the solvent acetone or methanol (80-90%). The drawback in this method is that methanol may cause some allomerization reactions and is limited for only quantitative estimations of chlorophyll in plant and food materials. The drawback here is that it also extracts carotenoids along with the chlorophylls. The another drawback of using methanol in foods lies in its serious toxicity. Reference may be made to the method of Arne Jensen 1978 (Chrophylls and carotenoids In: Handbook of phycological methods Helleburst JA and Craige JS (Editor), Cambridge University Press, pp59-70) wherein chlorophyll extraction from algae consists of repeated extraction of algal biomass with 90% acetone or methanol followed by centrifugation or filtration of the extracts. The absorbance of the extract is measured depending on the algal species for chlorophyll a, b, ci and c2. The method is limited for quantitative estimation of chlorophyll but it also extracts carotenoids from algae. The another drawback of using methanol in foods lies in its serious toxicity. Reference may be to the method of Sarada et al 2002 (Indian patent NF/23/02) wherein the method involves chlorophyll extraction from dry biomass of Spirulina with a mix of aqueous solution and missible solvent. The drawback in this method is that it is limited to only dry biomass while the present invention is for fresh biomass and for simultaneous extraction of the pigments. Most of the above mentioned procedures focused on chlorophyll extraction as a quantitative tool and unfortunately ignores simultaneous extraction of carotenoids.In the present study, the main objective is to extract the color components (pigments) of Spirulina without homogenization of cells and without the loss of valuable nutrient like carotenoids (a precursor for vitamin A). If the above cited procedures are followed for chlorophyll extraction in Spirulina, the loss of carotenoids will be too high and the efficacy of Spirulina as a nutritional supplement in food products is lost. Also if the above procedures are followed the valuable blue colour pigment is also lost its original colour. Therefore the present invention is focused on first blue colour extraction without homogenizing the cells followed by green colour extraction by a suitable aqueous solvent mixture resulting in color reduced p-carotene enriched Spirulina powder (without significant loss of carotenoids during pigment extractions). Moreover Spirulina does not contain distinct cell wall unlike plant cells therefore exhaustive treatments were not required for pigments extraction. The carotenoid rich Spirulina obtained after pigments extraction can be supplemented at higher levels to enrich the nutrient proportion in food products in an efficient manner. The main object of the present invention is to provide a process for preparation of carotenoid rich Spirulina which obviates the drawbacks as detailed above. Accordingly a process for the preparation of carotenoid. rich Spirulina which comprises: a) Characterized in that dissolving Spirulina in ethanol water mixture, centrifuging the suspension for the removal of blue and green pigment to obtain Spirulina Biomass, b) dispersing Spirulina biomass in polar solvent and water in 1:5 ratio for a period of 1-8hrs, c) drying the biomass at 40-50°C for 1-3 hours, d) recovering carotenoid rich Spirulina by conventional method In an embodiment for the preparation of carotenoid rich Spirulina wherein polar solvent used may be acetone and ethanol. In an another embodiment a process for the preparation of carotenoid hch Spirulina with protein 40-45% and carotenoid content 0.5-1.4% . The novelty of the invention is that treatment of Spirulina biomass with mix of miscible solvent and water facilitated the extraction of blue colour phycobiliproteins and green colour mainly chlorophyll and the resulting Spirulina powder enriched with p-carotene. The blue colour obtained from Spirulina biomass without homogenization of cells can be concentrated and the process can be scaled up easily by known methods. The green colour (mostly chlorophyll a ) obtained also with out homogenization can be concentrated and stabilized by known methods. The Spirulina biomass after pigments extraction obtained as byproduct with enriched p-carotene (3-4 times) and also rich in protein (42-45%) can be incorporated in food products at higher levels (2-5 times) than that of using the original (untreated) Spirulina powder. The loss of p- carotene due to pigment extractions was minimal (negligible). The colour reduced, p- carotene enriched Spirulina powder obtained as byproduct after pigments extraction finds applications in a wide range of food products as a nutritional supplement to meet the requirements of different people which was otherwise limited to few food preparations besides as tablets and capsules. 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 5 gm of fresh Spirulina was dispersed in 50 ml of 40% mix of ethanol and water solution at room temperature for a period of 4hr. The biomass was separated from the blue pigment by centrifugation at SOOOrpm for 15min followed suspension in aqueous mix of ethanol and water at 70% for period of 3hrs. The biomass was separated from the green pigment by centrifugation at SOOOrpm for 10min followed by drying at 50°C for 1 hour. The blue pigment content is estimated by the method of Tandeau De Marsac.N & Houmard.J, 1988, (Methods in Enzymology 167:318-328). The green pigment is estimated by the method of A. Jensen 1978 (In: Handbook of Phycological methods Eds: J.A.Hellebust and J.S.Craige, pp 60-64). The blue and the green pigments content thus obtained is given in table 1. The carotenoid content of the biomass was found to be 0.6 % with a protein content of 38%. The colour of the treated Spirulina biomass was determined employing a colour meter by using Hunter system (Lab) of colour measuring system. The colour of the samples were reported in terms of (Table Removed) Spirulina powder after 30.86 1.26 9.24 0.62 pigments extraction The undesirable green colour, as represented by the negative 'a' values (Table 1) reduced significantly due to treatment as mentioned in the example. Example 2 5 gm of fresh Spirulina was dispersed in 50 ml of 30% mix of ethanol and water solution at room temperature for a period of 5hr. The biomass was separated from the blue pigment by centrifugation at 6000rpm for 15min followed suspension in aqueous mix of ethanol and water at 50% for period of 4hrs. The biomass was separated from the green pigment by centrifugation at SOOOrpm for 10min followed by drying at 45°C for 2 hour. The blue pigment content is estimated by the method of Tandeau De Marsac.N & Houmard.J, 1988, (Methods in Enzymology 167:318-328). The green pigment is estimated by the method of A. Jensen 1978 (In :Handbook of Phycological methods Eds: J.A.Hellebust and J.S.Craige, pp 60-64). The carotenoid content of the biomass was found to 0.8 % with a protein content of 43%. The blue and the green pigments content thus obtained is given in table 2. The colour of the treated Spirulina biomass was determined employing a colour meter by using Hunter system of colour measuring system. The colour of the samples were reported in terms of L, a, b. Table 2.Pigment extraction and colour parameters of control and treated Spirulina Samples Sample L a b p -carotene % pigment mg/100mg Extracted Blue colour pigment -- - - - 58 Green colour pigment -- - - -- 75 Spirulina powder control 28.67 -6.80 6.97 0.18 Spirulina powder after 27.45 0.67 10.04 0.85 The undesirable green colour, as represented by the negative 'a' values (Table 2) reduced significantly due to treatment as mentioned in the example. Example 3 5 gm of fresh Spirulina was dispersed in 50 ml of 28% mix of ethanol and water solution at room temperature for a period of 5hr. The biomass was separated from the blue pigment by centrifugation at 6000rpm for 15min followed suspension in aqueous mix of acetone and water at 50% for period of 3hrs. The biomass was separated from the green pigment by centrifugation at 5000rpm for 10min followed by drying at 50°C for 1 hour. The blue pigment content is estimated by the method of Tandeau De Marsac.N & Houmard.J, 1988, (Methods in Enzymology 167:318- 328). The green pigment is estimated by the method of A. Jensen 1978 (In :Handbook of Phycological methods Eds: J.A.Hellebust and J.S.Craige, pp 60-64). The carotenoid content of the biomass was found to 0.9% with a protein content of 40%. The blue and the green pigments content thus obtained is given in table 3. The colour of the treated Spirulina biomass was determined employing a colour meter by using Hunter system (Lab) of colour measuring system. The colour of the samples were reported in terms of L, a, b (Table Removed) The undesirable green colour, as represented by the negative 'a' values (Table 3) reduced significantly due to treatment as mentioned in the example. Example 4 5 gm of fresh Spirulina was dispersed in 50 ml of 15% mix of ethanol and water solution at room temperature for a period of 5hr. The biomass was separated from the pigment by centrifugation at GOOOrpm for 15min followed suspension in aqueous mix of ethanol and water at 60% for period of 3hrs. The treated biomass was separated from the pigment by centrifugation at SOOOrpm for 10min followed by drying at 50°C for 1 hour. The blue pigment content is estimated by the method of Tandeau De Marsac.N & Houmard.J, 1988, (Methods in Enzymology 167:318-328). The green pigment is estimated by the method of A. Jensen 1978 (In :Handbook of Phycological methods Eds: J.A.Hellebust and J.S.Craige, pp 60-64). The carotenoid content of the biomass was found to 1.2 % with a protein content of 35%. The blue and the green pigments content thus obtained is given in table 4. The colour of the treated Spirulina biomass was determined employing a colour meter by using Hunter system (Lab) of colour measuring system. The colour of the samples were reported in terms of L, a, b (Table Removed) The undesirable green colour, as represented by the negative 'a1 values (Table 4) reduced significantly due to treatment as mentioned in the example. At optimal conditions 95% of blue pigment and 80% of green pigment were extracted which can be concentrated by the known methods of Marco Rito-Palomares et al 2001,J.Chem.Technol Biotech 76:1273-1280 and also by the conventional ammonium sulphate precipitation (Tandeau De Marsac.N & Houmard.J, 1988, (Methods in Enzymology 167:318-328). The colour parameters of the samples (Spirulina and treated Spirulina) are shown in Table 1, which reports the colour in terms of Hunter L, a,b values. The L value of the sample representing the lightness or brightness of the samples that increases to the maximum of about 30% (Example 1) when the treatments are given. The negative values for 'a' indicates the greenness of the samples. From a very green sample with a = -6.8 unit (example 2) the treatment with water and acetone or ethanol and combination there of drastically reduced the greenness of the samples to a 'a' value of 0.26 to 1.93 indicating significant reduction in green colour. In both cases, the untreated samples possess a highly undesirable green colour with a tinge yellow colour. The treatments, as cited in Examples 1 to 4, shows significant reduction in green colour. Hence, the treatments shifts the colour from bluish green to yellowish brown. The Spirulina biomass thus obtained after the pigments extractions was enriched with carotenoid content retaining 75-85% of its protein. It can be clearly concluded that the process for preparation of carotenoid rich Spirulina suggested in the present patent markedly increases the application of the p -carotene enriched Spirulina sample along with two natural colourants extracted from it. The two natural colourants extracted can be purified and stabilized by known methods and finds application as food or cosmetic colourants The main advantages of the present invention are 1. Treatment of Spirulina powder with the mix of aqueous solvent system separated phycocyanin very efficiently without homogenizing the cells. 2. The loss of the other pigments such as chlorophyll and p-carotene by this treatment is negligible. 3. Second step of treatment could effectively separate the green colour without effecting the other nutrients. 4. The Spirulina biomass obtained after the pigments separation is enriched with pcarotene (3-4 fold) with a retention of 75-85% of protein. 5. The Spirulina biomass thus obtained after pigments can be used in higher proportions in various food products including biscuit as a nutritional supplement which was not possible with the untreated Spirulina sample due to the dark (greenish brown) colour it imparts to the food items 6. The colorants separated by this process can be easily purified by known methods 7. The process is easy to scale up for both colorants separation as well as for pcarotene enriched biomass. 8. The solvents used for colorants separation can be recovered and reused. 9. The carotenoid enriched Spirulina powder can be used in food, pharmaceutical and other related products at a high level for the nutritional and health benefits and also for improved consumer acceptance . WE CLAIM: 1. A process for the preparation of carotenoid rich Spirulina which comprises: a) Characterized in that dissolving Spirulina in ethanol water mixture, centrifuging the suspension for the removal of blue and green pigment to obtain Spirulina Biomass, b) dispersing Spirulina biomass in polar solvent and water in 1:5 ratio for a period of 1-8hrs, c) drying the biomass at 40-50°C for 1-3 hours, d) recovering carotenoid rich Spirulina by conventional method 2. A process for the preparation of carotenoid rich Spirulina as claimed in claim 1 wherein polar solvent used is acetone, ethanol and a combination thereof 3. A process for the preparation of carotenoid rich Spirulina contains protein 40-45% and carotenoid content is 0.5-1.4% . 4. A process for the preparation of carotenoid rich Spirulina as substantially herewith references to examples.

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