OBJECT OF THE INVENTION:

The present invention relates to a novel mutant strain of Schizochytriumlimacinum capable of yielding DHA. The present invention also relates to process for isolating novel mutant strain of Schizochytriumlimacinum capable of yielding DHA.

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 and 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).

Known literature discloses 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 metal contaminants (US patent no 5,130,242)

The present invention also is directed towards:

• Bettering the strain yields using UV-irradiation
• Lowering the fermentation cycles, using graded oxygenation techniques i.e., shifting dissolved O2 level methods
• Yielding consistent mixture of fatty acids

• Overcoming the use of fish oil derived DHA

According to present invention there is provided a novel mutant strain of Schizochytriumlimacinum capable of yielding DHA

Further according to the present invention there is provided a process for isolating novel mutant strain of Schizochytriumlimacinum capable of yielding DHA which comprises

i) procurement & Shifting dissolved O2 level
ii) mutation of SL
iii) fermentation
iv) selection of best colonies by serial Petri dish dilution followed by large scale fermentation
v) fermentation broth working

Another aspect of the invention relates to the novel two stages O2 supply aimed at achieving high concentration and high productivity of DHA. In the first 40 hrs aeration was controlled at 3000 Ipm to obtain high cell growth (18-20%) followed by 1000 Ipm 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 Schizochytriumlimacinumwas subjected to sub culturing and 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 - I 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 isolation by growing mutant strain VIV - I Schizochytriumlimacinum 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 broth comprising:

Dextrose - 60 g/L
Yeast extract - 5 g/L
Peptone - 5 g/L
NaCI - 16.0 g/L
MgCI2 - 8.0 g/L
Na2S04 - 3.0 g/L
CaCI2 - 2.0 g/L
KCI - 1.0 g/L
Trace elements - 5.0 ml/L
Vitamins - 1.0 ml/L
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 strain VIV - I. The fatty acid profile comprises of Myristic acid, Pentadecanoic acid, Palmitic acid, stearic acid, oleic acid, alpha linolenic, Eicosatrionicacid& DHA.

Fatty acid pattern of 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.

We shall now describe the present invention with reference to the accompanying examples, which are given by way of illustrations but does not limit the scope of the invention.

Example 1:
Sample for 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 seawater.

The contents of the vial were aseptically transferred to 2000 ml shake flask containing:
350 ml of sterile pre-culture medium consisting of:

Tropic marine salt - 16.7 g
Glucose - 30 g
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" Petri dish.

The Petri dish was then exposed intermittently to UV light (Three cycles). Eight colonies were selected based on their growth profile and subsequently sub cultured/ 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.

Example 2:
Process for the mutation of SL

The isolated organisms obtained were grown in modified broth having the following ingredients.

Dextrose - 60 g/L
Yeast extract - 5 g/L
Peptone - 5g/L
NaCI - 16.0 g/L
MgCI2 - 8.0 g/L
Na2S04 - 3.0 g/L
CaCI2 - 2.0 g/L
KCI - 1.0 g/L
Trace elements - 5.0 ml/L
Vitamins - 1.0 ml/L
Agar - 2.5%

A dilute suspension of a 60 hrs culture was spread plated on a Petri dish (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 sub culturing & 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 mutants designated as VIV -1 was finally chosen based on its DHA yields for the large-scale fermentation.

Example 3: Fermentation Process
The steam pre sterilized 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 were 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.

0-24 hrs 1.0 WM
25-48 hrs 1.0 WM
49-72 hrs 1.5 WM
73 - Till cycle end 0.4 WM

Dextrose dosing To maintain levels at 10-14% g/L WM: Volume of air/Vol. of media/minute
Example 4:

This example shows the Flow Charts

i) Selection of best colonies by serial Petri dish dilution (Best colony): Selection of best colonies by serial Petri dish dilution (Best colony —► then slant)
Start
Roux bottle X 2
1 L shake flask X 2
20 L fermentor (inoculation)
200 L seed tank (Seed media)
Fermentor2000 L
Dextrose dosing after 48 hrs till end

ii) Large-scale fermentation method
Petri dish isolate (single colony slant)—►
Roux bottle (~ 200 ml) Total vol 1.0 L, Solid media 0.2 L
28° 7 days
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)

iii). Fermentation Broth working:
Fermentation broth -3000 L
Flocullation by addition of Chitosin

Layer separation Aq. Layer —► Wet biomass

Cell disruption under pressure
I
Solvent extraction (~ 2000 L of Toluene)

Layer separation (Removal of residual water)
—►
Drying the organic layer (MgSO4) Vacuum distillation Recovery of Toluene

Crude oil (about 200 Kg 20% - 23% DHA content)

Dil. Acid wash To remove gums Dil. Alkali wash To remove soaps

Hot water wash To neutralize the crude oil Winterification(chilling)

Centrifuging
DHA (about 40-45kgs 32-40%)

WE CLAIM:

1 A novel mutant strain of Schizochytriumlimacinum capable of yielding DHA.

2. A process for isolating novel mutant strain of Schizochytriumlimacinum capable
of yielding DHA which comprises of:

i) procurement & Shifting dissolved O2 level
ii) mutation of SL
iii) fermentation
iv) selection of best colonies by serial Petri dish dilution followed by largescale fermentation
v) fermentation Broth working

3. A process as claimed in claim 2 wherein fermentation process consists of i. the steam pre sterilized 4000 L reactor, having about 2500 L of the growth medium was inoculated with about 200 L of the seed media, ii. 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 fermenter was programmed on the following parameters.

0-24hrs 1.0 WM
25-48hrs 1.0 WM
49-72hrs 1.5 WM
73 - Till cycle end 0.4 WM
dextrose dosing to maintain levels at 10-14% g/L and DHA assay is not less than 32%.

4. A process as claimed in claim 2 wherein 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.

5. A process as claimed in claim 4 wherein the medium is free of bacterial growth that was confirmed microscopically.

6. A process as claimed in claim 5 wherein the medium was sterilized and the wild strain inoculated medium was incubated at RT for about 48-50 hrs

7. A process as claimed in claim 2 wherein the Petri dish containing media was exposed intermittently to UV light (Three cycles), eight colonies were selected based on their growth profile and subsequently sub cultured/ one mutant was identified which exhibited enhanced DHA activity and was selected for fermentation using shifting dissolved O2 level techniques.

8. A process as claimed in claim 7 wherein 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.

9. A process as claimed in claim 8 wherein stepwise aeration strategy was evolved for efficient DHA production using a 3.0 KL reactor, in the present embodiment to the effect of different O2 supply conditions as admeasured by volumetric oxygen mass transfer coefficient (KLa)

10. A process as claimed in any of the claims 1 to 9 wherein the isolated organism is obtained and is grown in modified broth having the following ingredients.

Dextrose - 60 g/L
Yeast extract - 5 g/L
Peptone - 5 g/L
NaCI - 16.0 g/L
MgCI2 - 8.0 g/L
Na2S04 - 3.0 g/L
CaCI2 - 2.0 g/L
KCI - 1.0 g/L
Trace elements - 5.0 ml/L
Vitamins - 1.0 ml/L
Agar - 2.5%