FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION
PREPARATION OF EPA AND DHA
2. APPLICANT
(a) NAME: PRAJ INDUSTRIES LIMITED
(b) NATIONALITY: Indian Company
(b) ADDRESS: PRAJ House, Bavdhan
Pune- 411021 .INDIA
-3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION FIELD OF INVENTION
The invention relates to a process for the preparation of a composition of EPA and DHA from a fish oil obtained from blue fishes like sardine, anchovy, etc. It particularly relates to the preparation of said compositions useful in preparations of nutraceutical products.
BACKGROUND
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are members of omega-3 fatty acids. It is established that the omega-3 fatty acids reduce inflammation, helping to prevent inflammatory diseases like heart disease and arthritis. They are also essential for the development of brain, impacting behavior and cognitive function, and are especially necessary during fetal development. EPA is used for high blood pressure in high-risk pregnancies, schizophrenia, personality disorder, cystic fibrosis, Alzheimer's disease, depression, and diabetes. DHA is used for treating type 2 diabetes, coronary artery disease, dementia, and attention deficit-hyperactivity disorder. DHA is also used for improving vision, preventing an eye disease called age-related macular degeneration, preventing and treating depression, and reducing aggressive behavior in people in stressful situations. EPA and DHA present in fish oil preparations are used for a variety of conditions like asthma, cancer, menstrual problems, hot flashes, hay fever, lung diseases, lupus erythematosus, and kidney

disease. EPA and DHA are also used in combination for migraine headache prevention in adolescents, skin infections, Behcet's syndrome, high cholesterol, high blood pressure, psoriasis, Raynaud's syndrome, rheumatoid arthritis, Crohn's disease, and ulcerative colitis. Generally EPA and DHA are required in its ethyl ester form for effective utilization. In general products containing with 60 % EPA/ DHA [38 % EPA to 22 % DHA], 50 % total EPA/ DHA [30 % EPA to 20 % DHA] and 30% EPA/ DHA [18 % EPA to 12 % DHA] are in demand in the market. Common sources of EPA and DHA fatty acids include fish oils, algal oil, egg oil, squid oils, krill oil, etc. Also blue fish oils obtained from sardines, anchovy, etc are rich in EPA and DHA glycerides. Traditionally, the fish oil is converted into its ethyl esters using mainly two methods: 1] sulphuric acid catalyzed esterification and 2] refining of fish oil to remove free fatty acids followed by its transesterification using a basic catalyst. The drawback of first method is, sulphuric acid is highly corrosive to equipment and it requires longer duration to convert the crude fish oil to their ethyl esters. In the case of second method, additional refining step is required to convert crude fish oil to its ethyl ester forms. Hence, there is need to develop an eco- friendly method to convert the crude fish oil fatty acids to their ethyl esters. Herein a process for the preparation of compositions EPA and DHA is disclosed having several advantages over known methods and is industrially scalable, economic and safe, yielding high quality EPA and DHA containing fractions.

DESCRIPTION OF DRAWINGS
The invention discloses a process for preparation of a composition of EPA and DHA from fish oil feedstock. The features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings, wherein:
FIGURE 1 and FIGURE 2 depicts a process flow diagram for the preparation of a composition of EPA and DHA from a fish oil feedstock. Different elements of the process are identified and directional movements of different streams and components formed are shown to describe the features of embodiments of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of the invention, the process comprises providing a fish oil [obtained for 'blue fishes' such as anchovies, sardines, cods, mackerels, herrings and like] having EPA and DHA in triglyceride forms; transesterifying said triglycerides with an alcohol using an acid catalyst at desired temperature and pressure forming alkyl esters of EPA and DHA; subjecting said esters to first molecular distillation at desired conditions forming a first distillate stream and a first residue stream; subjecting said first residue stream to a second molecular distillation at desired conditions forming a second distillate stream and a second residue stream; subjecting said second distillate stream to urea adduction in ethanol forming a first

filtrate stream; subjecting said first distillate stream to a third molecular distillation at desired conditions forming a third distillate stream and a third residue stream; and subjecting said third residue stream to urea adduction in ethanol forming a second filtrate stream.
Present invention facilitates efficient recovery of EPA and DHA from fish oil by a process comprising six steps namely: 1] transesterification, 2] first molecular distillation, 3] second molecular distillation of first residue stream, followed by 4] urea adduction, 5] third molecular distillation of first distillate stream and followed by 6] urea adduction. Each step has one or more elements for performing specific or optional functions as required for achieving selective isolation of EPA and DHA from a fish oil feedstock. A person skilled in the art may appreciate different variations and/ or combinations of these elements that may be used to perform the objects of the invention disclosed herein.
STEP 1: TRANSESTERIFICATION OF FISH OIL
Crude fish oil contains EPA and DHA in triglyceride forms. To get ethyl esters of these omega-3 fatty acids, a transesterification reaction is performed with ethanol in acidic medium at elevated temperature and under nitrogen pressure. Acid catalysts like methanesulfonic acid (MSA) used for effective transesterification. After this, said reaction mass is washed with dilute solution of sodium bicarbonate to remove any traces of

the catalyst and glycerol. This ethyl ester mass is then dried under vacuum and at elevated temperature to obtain a dried mass.
STEP 2: FIRST MOLECULAR DISTILLATION
)n this step, said dried mass obtained in step 1 is subjection to a first molecular distillation in which two fractions are obtained from said dried mass. A first residue stream comprising about 19 % to 25 % EPA by weight and about 9 % to 20 % DHA.by weight is separated from a first distillate stream comprising about 5 % to 12 % EPA by weight and about 0.5 % to 3 % DHA by weight. These two streams are again separately processed to enrich EPA and DHA further to desired purity and quality.
STEP 3: SECOND MOLECULAR DISTILLATION OF FIRST RESIDUE
STREAM
The first residue stream obtained in step 2 is further subjected to a second
molecular distillation step at desired temperature, vacuum and agitation
conditions. Fractionation in this step gives a second distillate stream and a
second residue stream. The second distillate stream comprises about 25 %
to 31 % EPA by weight and about 19 % to 24 % DHA by weight.
STEP 4: FIRST UREA ADDUCTION
Urea adduction removes the saturated and monounsaturated fatty acids
form polysaturated fatty acids. In this step urea adduction is performed on

said second distillate stream obtained after second molecular distillation to get more enrichment in EPA and DHA. It is carried out in ethanolic solution of urea. Then said second distillate stream is thoroughly mixed with said urea-ethanol solution (reaction mass having ethyl ester to urea to ethanol weight composition of between about 1:1:6 to about 1:5:8) and allowed to cool to a lower temperature for a few hours. Upon filtration, it affords a fourth residue stream, and a first filtrate stream rich in EPA and DHA. This first filtrate stream is subjected in a rotary evaporator to remove any residual ethanol and further washed with 10 % by weight sodium chloride solution in hot water to remove any residual urea, followed by drying to remove any moisture. Then forth residue stream so obtained is also washed with 10 % by weight sodium chloride solution in hot water. After this step, said first filtrate stream comprises about 20 % to 45 % EPA by weight and about 14 % to 35 % DHA by weight.
STEP 5: THIRD MOLECULAR DISTILLATION OF FIRST DISTILLATE
STREAM
The first distillate stream obtained in step 2 is further subjected to a third
molecular distillation step at desired temperature, vacuum and agitation
conditions. Fractionation in this step gives a third distillate stream and a
third residue stream. The third residue stream comprises about 20 % to 25
% EPA by weight and about 8 % to 12 % DHA by weight.

STEP 6: SECOND UREA ADDUCTION
In this step urea adduction is performed on said third residue stream obtained after third molecular distillation to get more enrichment in EPA and DHA. It is carried out in as in step 3. Upon filtration, it affords a fifth residue stream, and a second filtrate stream rich in EPA and DHA. This second filtrate stream is subjected in a rotary evaporator to remove any residual ethanol and further washed with 10 % by weight sodium chloride solution in hot water to remove any residual urea, followed by drying to remove any moisture. Then fifth residue stream so obtained is also washed with 10 % by weight sodium chloride solution in hot water. After this step, said second filtrate stream comprises about 18 % to 45 % EPA by weight and about 5 % to 16 % DHA by weight.
ADVANTAGES OF THE DISCLOSED PROCESS
• MSA as a catalyst used in transesterification step is eco-friendly.
• The transesterification conditions used are unique for efficient conversion of triglycerides present in the fish oil to corresponding fatty acid ethyl esters.
• The molecular distillation conditions used for isolation of EPA/ DHA fractions are unique and efficient to get desired amounts of EPA/ DHA in said fractions [purified products].
• , The urea adduction conditions used are effective in creating highly
enrich EPA/ DHA fractions.

• Besides the process provides efficient recovery of EPA/ DHA in final products.
EXAMPLES
Examples provided below give wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various experimental results is given in the examples and tables, which demonstrate the advantageous and novel aspects of the process for the preparation of a composition of EPA and DHA from fish oil.
EXAMPLE 1
About 100 kg of crude sardine fish oil was reacted with 30 kg of absolute ethanol in the presence of 3 kg of methanesulfonic acid [MSA] at temperature of about 100 °C and under nitrogen pressure of about 3 kg/cm2 for about 3 hours. Next, on the completion of reaction, ethanol was distilled using a rotary evaporator at 90 °C under vacuum at about 10 mbar, forming a crude ethyl ester mixture of EPA and DHA in residue. This mixture was washed with about 26 kg of sodium bicarbonate [10 % by weight] in hot water solution. At this glycerol and neutralized catalyst settled at the bottom of mixture were removed. The mixture was further washed with hot water to remove any traces of catalyst and glycerol. The ethyl ester mixture was then dried under vacuum at about 10 mbar and at temperature

of about 90 °C. This process afforded 98 kg of ethyl esters with glyceride content less than 1 % by weight. This dried mixture was then subjected to a first molecular distillation step (short path distillation) at temperature of about 150 °C under vacuum between 30 - 50 mTorr, at a flow rate about 27 kg/hour of said dried mixture and at agitation of about 280 rpm. This step afforded about 23 % of first residue stream containing about 21 % EPA and about 21 % DHA by weight; and first distillate stream containing about 12 % EPA and about 3 % DHA by weight. Next, said first residue stream was further subjected to a second molecular distillation step at temperature of about 185 °C under vacuum between 30 - 50 mTorr, at a flow rate about 15 kg/hour of said residue and at agitation of about 280 rpm. This step afforded about 18 % of second distillate stream containing about 27 % EPA and about 24 % DHA by weight. In the next step, said second distillate stream was further processed to enrich EPA and DHA content using a urea adduction step. Herein about 35 kg of urea was completely dissolved in about 140 kg of ethanol [95 % ethanol pure by volume] at temperature of about 70 °C. Then said second distillate stream was thoroughly mixed with said urea-ethano! solution and allowed to cool to about 5 °C and slowly stirred at this temperature for about 3 hours. Upon filtration with step afforded a first filtrate stream rich in EPA and DHA. This stream was subjected in a rotary evaporator to remove any residual ethanol and further washed with 15 kg of sodium chloride solution [10 % by weight] in hot water to remove any residual urea, followed by drying to remove any

moisture. Then forth residue stream so obtained was also washed with about 35 kg of sodium chloride solution [10 % by weight] in hot water. After this step, said first filtrate stream [about 9 % recovery] contained about 43 % EPA and about 35 % DHA by weight. The remaining forth residue stream [about 8 % recovery] contained about 16 % EPA and about 15 % DHA by weight. Further said first distillate stream obtained after first molecular distillation was subjected to a third molecular distillation step at temperature of about 150 °C under vacuum between 30 - 50 mTorr, at a flow rate about 69 kg/hour of said distillate and at agitation of about 280 rpm. This step afforded about 23 % of third residue stream containing about 25 % EPA and about 9 % DHA by weight and 45 % of third distillate stream containing about 5 % EPA and about 1 % DHA by weight. In further step, said third residue stream was further processed to enrich EPA and DHA content using a urea adduction step. Herein about 45 kg of urea was completely dissolved in about 181 kg of ethanol [95 % ethanol pure by volume] at temperature of about 70 °C. Then said third residue stream was thoroughly mixed in said urea-ethanol solution and allowed to cool to about 5 °C and slowly stirred at this temperature for about 3 hours. Upon filtration this step afforded a second filtrate stream rich in EPA and DHA. This stream was subjected in a rotary evaporator to remove any residua! ethanol and further washed with 22 kg of sodium chloride solution [10 % by weight] in hot water to remove any residual urea, followed by drying to remove any moisture. Then fifth residue stream so obtained was also washed with

about 45 kg of sodium chloride solution [10 % by weight] in hot water. After this step, said second filtrate stream [about 12 % recovery] contained about 44 % EPA and about 14 % DHA by weight. The remaining fifth residue stream [about 5 % recovery] contained about 5 % EPA and about 1 % DHA by weight.

EXAMPLE 2
Table 2-1 provides conditions used in said first molecular distillation step for different experimental batches [B] and its differential out come. TABLE 2-1:

Stream B1 B2 B3 B4 B5 B6 B7 B8 B9
First
distillate
[recovery
% w/w] 74 62 60 53 48 25 28 64 68
EPA in
first distillate
[% w/wl 13 9 9 8 6 4 5 11 12
DHAin
first distillate
[% w/wl 3 2 2 2 1 0.5 1 3 3
First
residue
[recovery
% w/wl 16 28 30 37 42 65 61 27 23
EPA in
first
residue [%
w/wl 22 25 26 25 25 19 20 25 21
DHAin
first
residue [%
w/wl 19 16 15 13 12 8 9 21 21
Temp. [oC] 150
130 150
Vacuum [mTorr] 30-40 20-50 10-50 20-50 30-50 20-40 20-70 30-50
Flow rate [kg/h] 22 36 32 54 27 54 104 34 27
Agitation [RPM] 280

EXAMPLE 3
Table 3-1 provides conditions used in said urea adduction on said second distillation stream of EXAMPLE 1 for different experimental batches [B] and its differential out come. TABLE 3-1:

Stream B1 B2 B3 B4 B5 B6 B7 B8 B9
Filtrate
[recovery
% w/wl 12 6 5 1 12 9 7 . 8 8
EPA in
filtrate [%
w/w| 36 40 39 21 32 39 45 42 42
DHAin
filtrate [%
w/w] 30 31 31 16 27 33 35 33 34
Residue [recovery
% w/w] 4 11 12 17 5 8 8 7 8
EPA in residue f% w/w] 14 19 23 31 16 14 18 18 17
DHA in residue [% w/w] 12 19 22 26 15 14 17 17 15
Time [h] 12 6 12 6
Temp. [°C1 _ 5 0 5
Urea [%w/wl 1:1 1:2 1:3.5 1:5 1:1 1:2
Ethanol
(95%) [%w/wl 1:8
Agitation [RPM] 50

EXAMPLE 4
Table 4-1 provides conditions used in said urea adduction on said third residue stream of EXAMPLE 1 for different experimental batches [B] and its differential out come. TABLE 4-1:

Stream B1 B2 B3 B4 B5 B6 B7 B8 B9
Filtrate
[recovery
% w/w] 17 11 7 2 15 12 9 10 12
EPA in
filtrate [%
w/w] 29 38 48 18 29 29 44 46 41
DHA in
filtrate [%
w/w] 10 12 16 5 10 13 13 16 13
Residue [recovery
% w/w] 5 11 14 21 8 11 12 11 9
EPA in residue [% w/w] 11 10 12 24 13 6 13 6 8
DHAin residue [% w/w] 4 4 5 8 5 3 5 2 3
Time [h] 12 6 12 6
Temp.
[oC] - 5 0 5
Urea
[%w/w] 1:1 1:2 1:3.5 1:5 1:1 1 2
Ethanol
(95%) [%w/w] 1:8
Agitation [RPM] 50
While the invention has been particularly shown and described with reference to embodiments listed in examples, it will be appreciated that

several of the above disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen and unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims. Although the invention has been described with reference to specific preferred embodiments, it is not intended to be limited thereto, rather those having ordinary skill in the art will recognize that variations and modifications may be made therein which are within the spirit of the invention and within the scope of the claims.

5. CLAIMS
WE CLAIM:
1. A process for preparation of a composition of EPA and DHA comprising:
(a) providing a fish oil having EPA and DHA in triglyceride forms;
(b) transesterifying said triglycerides with an alcohol using an acid catalyst at desired temperature and pressure forming alkyl esters of EPA and DHA;
(c) subjecting said esters to first molecular distillation at desired conditions forming a first distillate stream and a first residue stream;
(d) subjecting said first residue stream to a second molecular distillation at desired conditions forming a second distillate stream and a second residue stream;
(e) subjecting said second distillate stream to urea adduction in ethanol forming a first filtrate stream;
(f) subjecting said first distillate stream to a third molecular distillation at desired conditions forming a third distillate stream and a third residue stream; and

(g) subjecting said third residue stream to urea adduction in ethanol forming a second filtrate stream.
2. The process of claim 1, wherein said fish oil is obtained from sardine, anchovy and other blue fishes.
3. The process of claim 1, wherein said alcohol is ethanol.
4. The process of claim 1, wherein said acid catalyst is methanesulfonic acid.
5. The process of claim 1, wherein said trans-esterification is performed at temperature of about 100 °C under pressure of about 5 kg/cm2 for about 3 hours.
6. The process of claim 1, wherein said first molecular distillation is performed at temperature of about 130 °C to about 150 °C in vacuum of about 10 mTorr to 50 mTorr at a feed flow rate of about 25 kg/hour to about 35 kg/hour and agitation rate of about 280 RPM.
7. The process of claim 1, wherein said second molecular distillation is performed at temperature of about 170 °C to about 185 °C in vacuum of about 10 mTorr to 50 mTorr at a feed flow rate of about 15 kg/hour to about 20 kg/hour and agitation rate of about 280 RPM.

8. The process of claim 1, wherein said third molecular distillation is performed at temperature of about 150 °C in vacuum of about 10 mTorr to about 50 mTorr at a feed flow rate of about 50 kg/hour to about 70 kg/hour and agitation rate of about 280 RPM.
9. The process of claim 1, wherein said urea adduction is performed in ethanol at temperature of about -5 °C to about 5 °C and reaction mass having ethyl ester to urea to ethanol weight composition of between 1:1:6 to 1:5:8.
10. The process of claim 1, wherein said first residue stream comprises about 19 % to about 25 % EPA by weight and about 9 % to about 20 % DHA by weight.
11. The process of claim 1, wherein said first distillate stream comprises about 5 % to about 12 % EPA by weight and about 0.5 % to about 3 % DHA by weight.
12. The process of claim 1, wherein said second distillate stream comprises about 25 % to about 31 % EPA by weight and about 19 % to about 24 % DHA by weight.

13. The process of claim 1, wherein said third residue stream comprises about 20 % to about 25 % EPA by weight and about 8 % to about 12 % DHA by weight.
14. The process of claim 1, wherein said first filtrate stream comprises about 20 % to about 45 % EPA by weight and about 14 % to about 35 % DHA by weight.
15. The process of claim 1, wherein said second filtrate stream comprises about 18 % to about 45 % EPA by weight and about 5 % to about 16 % DHA by weight.

16. A composition of EPA and DHA according to the process of claim 1.
17. A product comprising a composition of EPA and DHA according to the process of claim 1.