Background of the invention:

Fatty acids are the molecular level constituents of Fats and Lipids and are found in all living species & used essentially for different & diverse metabolic functions including energy cycles, reproduction and storage.

Fatty acids have been broadly classified as saturated, i.e., with no double bonds and unsaturated i.e., with one or more double bonds, also known in literature as polyunsaturated fatty acids (PUFA). Some of the commonly used fatty acids derive their nomenclature based on the distance from the corresponding carboxylic acid, hence two well known (PUFA's) are also named as omega-3-fatty acids, examples of these are docosahexanoic acid (DHA) and Eicasopentenoic acid (EPA).

1. Literature points to a host of publications, (US patent no 5,484,611,Asian J. of Pharma& chemical research Vol 3, Issue 4, 2010; J. Agric. Food chem. 2008, 56, 3933;Process Biochem. 42 (2007) 1537; Process Biochem. 42 (2007) 1537)which have reviewed the utility of DHA in the growth of infant brain, retinal functions & improvement in certain cardiac impairments. Majority of them indicate Fish oil as the primary sources of DHA. However, the Fish oil as a source has disadvantages like extensive purification, unpleasant tastes and odors, and subject to environmental bioaccumulation of heavy metalcontaminants (US patent no 5,130,242)

The present invention also is directed towards:

(i) Betteringthe strain yields using UV-irradiation

(ii) Lowering the fermentation cycles, using graded oxygenation techniques i.e., shifting
dissolved O2 level methods

(iii) Yielding consistent mixture of fatty acids

(iv) Overcoming the use offish oil derived DHA

Summary of the invention:

The present invention relates to a novel mutant strain of Schizochytriumlimacinum capable of yielding DHA in economically better yields using the "shifting dissolved oxygen" technology.

Another aspect of the invention relates to the novel two stages 02 supply aimed at achieving high concentration and high productivity of DHA. In the first 40 hrs aeration was controlled at 3000 lpm to obtain high cell growth (18-20%) followed by 1000 1pm for DHA accumulation.

Detailed description of the invention:

The micro algae Schizochytriumlimacinum, described in this study was isolated during June 2010 from seawaters of Chorao mangroves, Goa, India and identified morphologically. The Schizochytriumlimacinum was subjected to subculturingand subsequent UV radiation.

Initially 8 mutants were identified based on the invitro assay of enhanced DHA content. From these eight, one mutant strain designated as VIV -1 was chosen for its high DHA productivity (14-15 g/L compared to the wildstrain5g/L) using novel shifting dissolved oxygen level techniques.

One embodiment of the invention is directed to the novel mutant strain of Schizochytriumlimacinumcapable of producing high levels of DHA.

Another embodiment of the invention provides a process for growing the mutant strain VIV -ISchizochytriumlimacinum wherein the said process comprises a culture medium comprising a carbon source, a nitrogen source, a vitamin source, sea water, agar & antibiotic, followed by transferring the cells to a fresh liquid culture medium comprising Nitrogen source &vitamin.

The carbon source is selected from the group comprising dextrose, glucose, starch, glycerol, molasses and corn steep liquor (J. Agric. Food chem. 2008, 56, 3933).

The nitrogen source,which is optionally supplemented with vitamins, is selected from the group comprising ammonium nitrate, corn steep liquor and yeast extract.

In yet another embodiment of the invention the seawater samples are grown in the novel media composition of the modified brothcomprising:

(i) Dextrose - 60 g/L
(ii) Yeast extract - 5 g/L
(iii) Peptone - 5 g/L
(iv) NaCl - 16.0 g/L
(v) MgCl2 - 8.0 g/L
(vi) Na2S04 - 3.0 g/L
(vii) CaCl2 - 2.0 g/L
(viii) KC1 - 1.0 g/L
(ix) Trace elements - 5.0 ml/L
(x) Vitamins - 1.0 ml/L
(xi) Agar - 2.5%

Another embodiment of the invention is directed to the novel fermentation process, which employs shifting oxygen level methodology for enhanced DHA production.

The solvents employed for the extraction of lipids are petroleum ether, toluene, n-hexane, ethylacetate and mixtures thereof.

In another embodiment, the invention is directed to the fatty acid profile of the crude oil obtained from the mutant strainVIV - I. The fatty acid profile comprises of Myristic acid, Pentadecanoic acid, Palmitic acid, stearic acid, oleic acid, alpha linolenic,Eicosatrionicacid& DHA.

Fatty acid patternof the purified DHA estimated by gas chromatographic methodology as the corresponding methyl esters:

In yet another embodiment, the invention is directed to the usage of the Roux bottles during inoculums preparation and in fermentation procedures. The Roux bottle usage increases the multiplication of the cells in a shorter time period thereby reducing the number of microbiological steps for inoculum preparation.

The following examples describe the invention further, not limiting the scope of the invention.

Ex 1: Sample procurement& Shifting dissolved 02 level technique:

The seawater samples obtained from chorea mangroves, Goa, India were grown on modified Vishmac broth. Samples collected were transferred to sterile glass vials & sent to our laboratory within 18 hrs for isolation. The vial contained 2 ml of sterile sea water.

The contents of the vial were aseptically transferred to 2000 ml shake flask containing:

1. 350 ml of sterile preculture medium consisting of:
(i) Tropic marine salt - 16.7 g
(ii) Glucose - 30 g
(iii) Yeast extract - 10 g/1000 ml to pH 6.0

The flask was incubated at RT & examined for growth after 72 hrs.

The organism was isolated in axenic cultures by subculturing again as above but with 0.04% admix of penicillin to prevent the growth of bacteria. Absence of bacterial growth was confirmed microscopically.

The medium was sterilized and the wild strain inoculated medium was incubated at RT for about 48-50 hrs

A 1:100 suspension (in distilled water) was inoculated by "Spread technique" on MV agar placed in a 5" petridish.

The petridish was then exposed intermittently to UV light (Three cycles). Eight colonies were selected based on their growth profile and subsequently subcultured/ one mutant was identified which exhibited enhanced DHA activity and was selected for fermentation using shifting dissolved O2 level techniques.

Based on the taxonomical understanding of the micro algal growth which occurs in broadly two stage wise phase, oxygenation was planned to match the same to achieve high concentration of the cell mass and consequently high productivity of DHA.

For the first 37-45 hrs oxygenation was controlled at 30001pm to obtain high cell growth, subsequently the oxygenation was dipped to 10001pm for yielding higher accumulated DHA.

The final maximum lipid concentration and DHA content reached to 44.6 g L"1,15 g L"1.

In the present embodiment the stepwise aeration strategy was evolved for efficient DHA production using a 3.0 KL reactor. In the present embodiment the effect of different O2 supply conditions as admeasured by volumetric oxygen mass transfer coefficient (KLa) has been presented.

Ex 2: Process for the mutation of SL:The isolated organism obtained were grown in modified broth having the following ingredients.
(i) Dextrose - 60 g/L
(ii) Yeast extract - 5 g/L
(iii) Peptone - 5 g/L
(iv) NaCl - 16.0 g/L
(v) MgCl2 - 8.0 g/L
(vi) Na2S04 - 3.0 g/L
(vii) CaCl2 - 2.0 g/L
(viii) KC1 - 1.0 g/L
(ix) Trace elements - 5.0 ml/L
(x) Vitamins - 1.0 ml/L
(xi) Agar - 2.5%

A dilute suspension of a 60 hrs culture was spread plated on a petridish (having the same constituents as above) and then intermittently exposed to UV light. Eight mature colonies based on their size and morphology were selected for further subculturing& growth using a seed media essentially equivalent to A.

This process was repeated and the seed media cells were analyzed for DHA content using extraction techniques & GC. One of the mutant designated as VIV -1 was finally chosen based on its DHA yields for the large scale fermentation.

Ex 3: Fermentation Procedure:

The steam presterilised 4000 L reactor, having about 2500 L of the growth medium was inoculated with about 200 L of the seed media.

The contents were stirred at 120 rpm and a temperature of 28°C, the aeration of the contents was simultaneously undertaken and the pH of the contents maintained at 5.5 - 6.5 with liquor ammonia dosing. The fermentor was programmed on the following parameters.

(i) 0-24 hrs 1.0 VVM
(ii) 25-48 hrs 1.0 VVM
(iii) 49-72 hrs 1.5 VVM
(iv) 73 - Till cycle end 0.4 VVM
(v) Dextrose dosing To maintain levels at 10-14% g/L VVM: Volume of air/Vol. of media/minute

Ex 4: Flow Charts:

i) Selection of best colonies by serial petridish dilution (Best colony):

Selection of best colonies by serial petridish dilution (Best colony then slant)

Start

y Roux bottle X 2

1 L shake flask X 2

y 20 L fermentor (inoculation)

200 L seed tank (Seed media)

Fermentor2000 L

-►Dextrose dosing after 48 hrs till end

ii) Large-scale fermentation methodology:

Petridish isolate (single colony —► slant)

y Roux bottle (~ 200 ml) Total vol 1.0 L, Solid media 0.2 L
28° 7 days
1.0 L (Shaker)
28° 48-72 hrs

30.0 L (Mini fermentor)
28° 48 hrs 300 L (Seed fermentor)
28° 3.0 KL (Main fermentor - 5 days)
iiO.Fermentation Broth workup:
Fermentation broth -3000 L

Flocullation ► by addition of Chitosin
I
Layer separation—Aq. Layer
I
Wet biomass
I
Cell disruption under pressure
I
Solvent extraction —► (~ 2000 L of Toluene)
I
Layer separation —► (Removal of residual water)
I
Drying the organic layer —►(MgSCv)
I
Vacuum distillation —►Recovery of Toluene
I

Crude oil —► (about 200 Kg 20% - 23% DHA content) Dil. Acid wash —►To remove gums
I
Dil. Alkali wash-*- To remove soaps
I
Hot water wash -►To neutralize the crude oil
I
Winterification(chilling)
I
Centrifuging
I
DHA ► (about 40-45kgs 32-40%)

Claims:

1. A novel mutant strain of Schizochytriumlimanicumis claimed.

2. A process of fermentation, which comprises the use of shifting dissolved oxygen
methodology.

3. The process of fermentation of claim 2 wherein the fermentation is carried out based on the isolated mutant STRAIN.

4. The process of fermentation of claim 3 wherein the DHA assay is not less than 32%.