The present invention relates to process for obtaining natural mixed carotenoids rich Spirulina biomass. Background of the Invention
The blue green algal form Arthrospira or so-called Spirulina has attracted considerable interest for its commercial production for functional or designer food additives and also as a tool for Research and Development (R&D) and its usage thereafter for curbing and curtailing various ailments in living creatures like the human, animal, fishes and birds etc mainly as chemo-preventive and therapeutic ingredient. The presence of many commercial units all over the Globe along with its market flooded with plenty of Spirulina based health and dietary food supplements, cosmetic and feed products are the witness of the fact (www.spirulinasource.com).
Numerous Arthrospira (Spirulina) species like Arthrospira platensis, Arthrospira fusiformis, Arthrospira maxima and Arthrospira pacifica has been collected, isolated, cultured and produced under a variety of culture conditions (light, temperature and pH) and environmental regimes around the World, both at lab and outdoor levels in all sort of optimum growing conditions and units with altogether different array and a varieties of culturing medium's like that of Zarrouk's, Ripley Fox, CFTRI, MCRC, Earthrise etc (www.spirulinasource.com). The usage and focus on culturing and taming various GRAS (Generally Regarded

As Safe) species of Arthrospira (Spirulina) alga worldwide in all sort of culture mediums, culturing units like glass or plastic tubes to flasks or from cement, mortar to brick based circular, rectangular to raceway type ponds, poly propylene based close indoor and outdoor units to photo bioreactors (PBRs) and various culture conditions like light, temperature, pressure and pH regimes has been done primarily to achieve higher biomass rich in protein, phycocyanin, chlorophyll, amino acids, vitamins and other cellular ingredients. It is very well depicted in booklets or literatures and even on labels being provided along with various formulation as launched and prevailing world wide mainly in the form of over scores of food, feed and cosmetic products with various national and international specifications and guidelines (www.spirulinasource.com).
Various mixed carotenoids have been scientifically proven and are used in food, feed, cosmetics and various medicinal sectors quite effectively as chemo-preventive and therapeutic, colouring ingredients. Studies have time and again proven the importance, impact and incorporation of all vitamins and also the natural mixed carotenoids predominantly of beta-carotene, lutein, lycopene and zeaxanthin in all sort of natural biomasses and also in man made variety of health additives, antioxidants, anti aging, food, feed and also as cosmetic colouring agents. The usage of natural mixed carotenoids zeaxanthin and lutein mainly for eye care (as anti macula degradation agent), trans and cis beta carotenes as potent

natural antioxidants or free radical scavenger or cancer prevention, Lycopene in prostrate cancer treatment and astaxanthin in cancer prevention, anti aging and feed additive for aqua culture fishes and prawns has been immense and also goes well with a successful revenue generation portfolio of huge and an ever increasing consumer market demands for human and animal health care sectors. Spirulina vitamins like B-complex and folic acid add value to various products in term of their therapeutic and chemo preventive agents (www.spirulinasource.com).
Many efforts have been done to produce spirulina containing one or two ingredients in higher amount. In CN1092104 germanium and selenium containing spirulina has been produce by cultivating spirulina in germanium dioxide and sodium selenite.
Manufacturing method of Iron-rich spirulina has been disclosed in CN1144844, in which ferrous methionine has been used in the medium for cultivating the alga.
CN 1584011 disclosed spirulina containing arginine, ornithine or citrulline by adding L- arginine, L-omithine and L-citrulline into culturing liquid.
In JP61158777 Algae (preferably Spirulina, or Chlorella) is cultivated in a medium containing preferably 10-100 ppm water-soluble chromium (III) compound not extremely inhibiting growth of algae, so that it contains chromium and that gives algae having improved glucose tolerance factor.

RU2198215 disclosed method of vanadium-enriched Spirulina in which vanadium salt taken among the group: sodium vanadate, vanadium nitrate, vanadyl sulfate is taken in the concentration 100-200 mg/1 during cultivation of alga.
In all above patents content of micronutrient in the alga can increase by cultivating the alga in to desired micronutrient.
AU2004322412 disclosed method for mass production of spirulina using seawater-based medium composition of pH ranging between 6.5 and 8.0 comprising sodium bicarbonate of concentration ranging between 1.2 to 3.0 % w/v, nitrogen of concentration ranging between 0.1 to 0.3 % w/v, phosphorus of concentration ranging between 0.1 to 0.3 % w/v, potassium of concentration ranging between 0.1 to 0.3 % w/v seawater and composition thereof.
MD3101F, MD3128F, MD3129F, MD 3171F and MD2386 disclosed process for obtaining spirulina biomass with the use of nutrient medium containing various salts e.g. NaHC03, K2HPO4, NaN03? NaCl, K2S04, CaC1.6H20, MgS04.7H20, H3BO3, MnCl2.4H20, ZnS04.7H20, CuS04.5H20, M0O3, KN03, FeS04.7H20, Fe-EDTA.
All the above stated patents disclosed method for obtaining higher biomass of spirulina. None of the patent disclosed medium or process that increases natural mixed carotenoids, vitamins and carbohydrates content in the said alga.

Objects of the Invention:
The main object of the present invention is to produce higher concentrations of natural mixed carotenoids predominately zeaxanthin followed by trans and cis beta-carotene along with appreciable amounts of lycopene, astaxanthin and their isomers.
An object of the present invention is to generation of the final Spirulina biomass more or less is "Golden Yellow" in colour and with fewer smell of typical sea weed type than the traditional high seaweed type smelling bluish green colour biomass.
Another object of the invention is the decrease quantity and ratio of nitrogen related cellular ingredients such as proteins and pigments like phycocyanin and Chlorophyll-a.
A further object of the current invention is to cultivate higher natural mixed carotenoids containing spirulina either solely in plain water with prescribed medium or with an artificial osmotically balancing water prepared with addition of crude sea salt, or with naturally occurring sea water or with any inland saline or brackish water from any source like well, pond or even from a lake as a prime base at various salinity gradients as high as upto 5% level or even higher.
An additional object of the invention is to disclose productive and more cost effective method for production of natural mixed carotenoids rich spirulina.

Yet another object of the invention is to obtain natural mixed carotenoids rich spirulina at suitable artificial or natural light (as required) and temperature regimes at naturally prevailing atmospheric pressure regimes both at laboratory and at outdoor levels.
Furthermore object of the invention is to obtain higher yields of vitamins like B3, B6 and folic acid along with increased carbohydrate contents. Summary Of The Invention:
According the invention, a new culturing media has been developed using salt solution complex comprising (a) carbon source like sodium bicarbonate, sodium acetate as a key constituent in addition to (b) sodium chloride, di potassium hydrogen phosphate, sodium nitrate, potassium sulphate, magnesium sulphate, calcium chloride, ferric EDTA, trace elements, urea or ammonium chloride and potassium chloride.
The proposed invention highlights and depicts that with Arthrbspira (Spirulina) species and its growth-associated biomass generation can be accumulated with enriched quantities of natural mixed carotenoids and xanthophylls like zeaxanthin, astaxanthin, lycopene, beta-carotene and its isomers if grown in a osmotically balanced and nutritionally active newly invented Spirulina Medium with or without a osmotically balanced and active saline gradients. Traces of other carotenoids and xanthophylls have also been procured.

This novel discovery thus enables production of the above-mentioned natural mixed carotenoids and xanthophylls enriched "golden yellow" coloured biomass with fewer seaweed type odour possessing quite less proteins, phycocyanin, Chl-a with much higher quantities of carbohydrates and vitamins -B3, B6 and Folic acid namely from four species of Arthrospira (Spirulina) namely Arthrospira platensis, Arthrospira fusiformis, Arthrospira maxima and Arthrospira pacifica. Detailed Description:
The nature of the invention and the manner in which it is to be performed is clearly described in the specification. The invention has various components and they are clearly described in the following pages of complete specification.
The mother culture of the Arthrospira (Spirulina) species as depicted in current invention are first cultured and maintained in Zarrouk's medium, but only upto lab level. Zarrouk's medium is one of the main historical designed and opted mediums for growing and maintaining Arthrospira (Spirulina) species cultures at the laboratory level studies with minimum care and without much damage or deviations in the overall nature and biochemical structure of Arthrospira (Spirulina) alga.
They are than subsequently cultured and maintained in invented spirulina media so as to achieve its desired growth-associated biomass generation

accumulated with enriched quantities of natural mixed carotenoids like zeaxanthin, astaxanthin, lycopene, beta-carotene and its isomers.
The algal species of Arthrospira used in the present invention are Arthrospira platensis, Arthrospira fusiformis, Arthrospira maxima and Arthrospira pacifica.
In the present invention carbon source is selected from group consisting bicarbonate, carbonate, acetate, glucose and sucrose or combination of thereof.
In the present invention sodium bicarbonate, sodium acetate, or combination of thereof are primarily used as a carbon source.
The salts or chemicals used in the present invention are sodium chloride, di potassium hydrogen phosphate, sodium nitrate, potassium sulphate, magnesium sulphate, calcium chloride, ferric EDTA, trace elements, urea or ammonium chloride and potassium chloride.
Trace elements include zinc (Zn), copper (Cu), cobalt (Co), molybdenum (Mo) and magnesium (Mn).
Different concentrations of prescribed salts or chemicals are taken into consideration as inorganic and organic nutrients as ratio's in invented spirulina medium (Table-I).



Higher natural mixed carotenoids containing spirulina using modified spirulina medium is cultivated either solely in plain water or in an artificial osmotically balancing water based medium achieved by addition of crude sea salt, or in naturally occurring sea water or in any inland saline or brackish water from any source like well, pond or even from a lake as a prime base at various salinity gradients as high as upto 5% level or even higher.
The process for cultivating higher carotenoids containing spirulina comprises following steps:
(1) Spirulina is cultivated in above stated Medium at 28±4°C temperature,
45±15 Klux Light intensity and 9.5±0.5 alkaline pH in suitable and
appreciable volume holding glass, plastic or cemented containers or ponds.
(2) The media is added as per alga need and stirred at regular intervals either
manually or mechanically.

(3) The alga is finally harvested and subjected to drying as and when highest level of natural mixed carotenoids got accumulated in the algal cells furnishing it a "Golden Yellow" colour.
Spirulina cultures harvested at commercial scale in culture conditions as above from suitably stirred culturing ponds under artificial / natural conditions to procure biomass with higher levels of Natural Mixed Carotenoids levels along with other key cellular ingredients under optimum drying conditions.
Dried coloured Spirulina biomass rich in Higher Natural Mixed Carotenoids and other key cellular ingredients subjected to QA/QC approval and for optimised vacuum packaging.
Determination of high value of mixed carotenoids both quantitatively and qualitatively is being estimated and done with the help of Jasco HPLC. It has been carried out with freshly harvested cell pellets via centrifugation at 4°C. Prior to their analysis and quantifications by Jasco HPLC, the pellets are subjected to sonication in appropriate organic solvent and are filtrated through 0.2-micron membranes. All extracted mixed carotenoids along with xanthophylls are finally pooled to a known volume. The same procedure is also later followed with sun, oven and spray dried final algal biomass. The depicted picks of all mixed carotenoids and xanthophylls and HPLC chromatograms are analyzed, quantified and correlated with prescribed reference standards.


The prominent identity revealing cellular constituents of such Blue Green Algae's (BGA's) like phycocyanin's, Chlorophyll-a and high protein levels dip to their lowest levels especially by various nutrient depletions at regular intervals.

This also revealed their sacrificing patterns and behaviours as tamed by pathways of autotrophy, mixotrophy and heterotrophy independently or in combinations of any of the two or even the three in continuum with invented spirulina media quite successfully at pilot scales. The newly designed innovative taming process or methodology with or without the base of a suitable salinity oriented osmotically balanced and active nutrient pool supplemented with requisitioned organic and inorganic nutrients as depicted in invented spirulina media yielded much higher levels of specific growth rates of biomass enriched with higher quantities of almost all natural mixed carotenoids along with xanthophylls than the traditional culturing and growth regimes as currently or being followed in past. This rare feat has been pioneered both at laboratory and pilot plant scale batch and continuous culturing and biomass production levels both in open outdoor and indoors suitable culturing units or even in so called closed bioreactors and even in photo bioreactors with the prescribed Arthrospira (Spirulina) sought after natural and artificial light, temperature, even pressure regimes and pH zones.
The present breakthrough further reflects that appropriate carbon/nitrogen (C/N) ratio along with fitting phosphate (P) dosings of the organically or inorganically independently or in combinations of the two with appropriate dosing of other remaining enriched growth medium to the algal growth phase in a suitable salinity and osmotically active liquid or solid base. It adds a significant thrust on

the biosynthesis of mixed carotenoids and associated xanthophylls of the mentioned blue green algae. The higher C/N ratio along with appropriate dosing and depletion of other much-needed nutrients generated higher biomass laden with enhanced quantities of natural mixed carotenoids dominated by astaxanthin and zeaxanthin as against traditional trans beta-carotene and its isomers. The light dependent and independent mixed carotenoids and xanthophylls producing ability of such and other blue greens can be successfully tapped from large commercial productions units with higher revenue generation and profits.
The invention is illustrated more in detail in the following examples. The examples describe and demonstrate embodiments within the scope of the present invention. These examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope.
Example 1: Spirulina culture media containing sodium bicarbonate as carbon source

Pigment profile of Arthrospira species with traditional Spirulina media vs. invented Spirulina Media Sodium Bicarbonate and Acetate as Carbon source:

The main advantages of present invention are,
1. Total natural mixed carotenoids content of spirulina increases upto 50 %,
but individual contents of mixed carotenoids natural like Zeaxanthin,
Astaxanthin increased to as high as 80 - 320%. Lycopene was obtained at
appreciable levels for the first time in above mentioned spirulina species,
without addition of any extra /intra cellular stimulant like nicotine.
2. In the present invention the final biomass is more or less "Golden Yellow"
in colour and with fewer smell of typical seaweed type than the traditional
bluish green colour with strong seaweed type smell.
3. The vitamin contents increased upto 750% especially that of folic acid.

4. The protein, phycocyanin's and Chl-a contents decreased substantially.
5. Carbohydrates profile witnessed a considerable increased in its trend.

We claim:
1. A process of obtaining enhanced natural mixed carotenoids containing
"Golden Yellow" coloured spirulina species with least seaweed type
"smell" accompanied with high "vitamin and carbohydrate" contents by
(a) growing spirulina in an aqueous medium comprising carbon sources sodium chloride, Di-potassium hydrogen phosphate, sodium nitrate, potassium sulphate, magnesium sulphate, calcium chloride, ferric EDTA, trace elements, urea or ammonium chloride and potassium chloride;
(b) providing optimum temperature, light and pH and other cultivation regimes;
(c) stirring the cultures and invented media addition at required intervals;
(d) collecting and drying the golden yellow spirulina species biomass.

2. The process as claimed in claim 1 wherein spirulina species selected from the group consisting Arthrospira platensis, Arthrospira fusiformis, Arthrospira maxima and Arthrospira pacifica.
3. The process as claimed in claim 2 wherein spirulina species Arthrospira platensis and Arthrospim fiisiformis are used more ocassionally.

4. The process as claimed in claim 1 wherein carbon source is selected from group consisting bicarbonate, carbonate, acetate, glucose and sucrose or combination of thereof.
5. The process as claimed in claim 4 wherein 0.05 - 15 gm/litre carbon source is used.
6. The process as claimed in claim 4 wherein sodium bicarbonate, sodium acetate or combination of thereof used as carbon source solely or in combination thereof.
7. The process as claimed in claim 6 wherein 0.50- 15.0-grams/litre sodium bicarbonate is used.
8. The process as claimed in claim 6 wherein 0.05 - 2.50 grams/litre sodium acetate is used.
9. The process as claimed in claim 1 wherein 05.0 - 45.0 grams/litre sodium chloride, 0.02 - 1.50 grams/litre di potassium hydrogen phosphate, 0.03 -1.25 grams/litre sodium nitrate, 0.15 - 0.50 grams/litre potassium sulphate, 0.02 - 1.00 grams/litre magnesium sulphate, 0.01 - 0.04 grams/litre calcium chloride, .0.001 - 0.015 grams/litre ferric EDTA, 0.10 - 1.5 ml trace elements, 0.01 - 0.100 grams/litre urea or ammonium chloride, 0.015 - 2.00 grams/litre potassium chloride are used.

10. The process as claimed in claim 1 wherein zinc (Zn), copper (Cu), cobalt (Co), molybdenum (Mo) and magnesium (Mn) are used as trace elements.
11. The process as claimed in claim 1 wherein aqueous medium is selected from the group consisting plain water, an artificial osmotically balancing water based medium with addition of crude sea salt, naturally occurring sea water, any inland saline water, brackish water from any source like well, pond or even from a lake as a prime base at various salinity gradients as high as upto 5% level or even higher.
12. The process as claimed in claim 11 wherein naturally occurring seawater is used as aqueous medium.
13. The process as claimed in claim 1 wherein the said aqueous media exhibits a pH within the range of 9.5 + 0.5.
14. The process as claimed in claim 1 wherein the temperature during the culturing step is within a range from about 28 ± 4 ° C.
15. The process as claimed in claim 1 wherein the light intensity during the culturing step is within a range from 45 + 15 Klux.
16. The process as claimed in claim 1 wherein natural mixed carotenoids concentration increases up to 50 %.

17. The process as claimed in claim 16 wherein natural mixed carotenoids
include zeaxanthin, trans- beta- carotene, cis- beta- carotene, lycopene,
astaxanthin, and their isomers.
18. The process as claimed in claim 1 wherein quantity of nitrogen related cellular ingredients like proteins, phycocyanin and chlorophyll-a decrease up to lower levels.
19. The process as claimed in claim 1 wherein the final biomass is more or less "Golden Yellow" in colour and with fewer smell of typical seaweed type.
20. The process as claimed in claim 1 wherein vitamins like B3, B6 and Folic
acid along with carbohydrates increase quite substantially.
21. A process for preparation of high mixed carotenoids containing spirulina
species as substantially herein describes with foregoing description and
examples.