Method for extraction of Docosahexaenoic Acid (DHA)
Field of the Invention:
The present invention is related to the method of extracting Docosahexaenoic acid (DHA) from
fungal micro floral strain Schizochytrium sp. More specifically the invention is related to the
preparation of enriched DHA oil by single stage SCF-CO2 extraction and further enrichment by
liquid-liquid extraction and deodorization of Docosahexaenoic acid.
Background of the Invention:
Lipids consists of the fatty acids and these fatty acids are essential for growth, survival and
reproduction of all living organisms. The fatty acids are either saturated or unsaturated. The
saturated fatty acids contain chain having carbon-carbon single bond while unsaturated fatty
acids contain double bond between the carbon atoms. Polyunsaturated fatty acids, termed as
PUFAs hereafter, are those in which more than one such double bond is found.
PUFAs are grouped in two series on the basis of the position of the terminal bond being 3C or
6C from the terminal carbon atom of the fatty acid chain. Also they are generally classified into
2 main groups, the omega-6 (66 or n-6) and omega-3 ((o3 or n-3 series). Among the omega-6
fatty acids arachidonic acid is the major precursor of many prostaglandins and eicosanoids and
among the omega-3 fatty acids docosahexaenoic acid have been termed as "Essential Fatty
Acids" as it bears many health supplementing properties. Besides, the Docosahexenoic acid
(DHA) the Eicosapentanoic acid (EPA) is considered extremely essential for the health of
animals and human beings. The molecular structure of both Omega-3 fatty acids (DHA and
EPA) is such that the first double bond follows the third carbon atom from the methyl end of the
fatty acid structure). DHA contains 22 carbon atoms, between which six double bonds are found.
EPA contains 20 carbon atoms, between which five double bonds occur.
The Omega-3 and omega-6 fatty acid are produced commercially from the selected seed plants
and some marine sources. Among the marine sources fish oil contribute major fraction of PUFA.
It has been estimated that in spite of their extraction from natural source the supply will become
2
inadequate to meet the future demands (Gill and Valivety 1997). In the recent time the use of
these polyunsaturated fatty acids (PUFAs) acids has increased markedly due to their health
supporting potential. Interest in them arises from their potential in therapeutic, food and
nutritional applications. For humans DHA and EPA have been shown to be important in brain
development in children, prevention of atherosclerosis, prevention of night blindness,
neurological disorders and even for possible prevention of cancer (Bajpai, P. and P. K. Bajpai.
1993, Barclay, W. R. et al. 1994,; Singh, A. and O. P. Ward. 1997). In addition to the need of
these fatty acids for human health the Omega-3 PUFAs have been shown to enhance growth and
reproduction in crustacean animals, such as prawns, which are very important as aquaculture
animals for human consumption (Harrison, K. E. 1990). The Omega-3 PUFA particularly DHA
is a major structural fat in the brain and retina accounting for up to 93-97% of fat brain and
retina. It is also a key component of the heart. DHA is a functional fatty acid that cannot be
synthesized by human body itself but supplied by consuming foods through essential fatty acids
ALA (a-Linolenic acid: 18:3 co-3) and linoleic acid: 18:2 co-6 which will convert to DHA to an
extent relatively less than 0.4% in the body, so DHA needs to be supplied through food
supplements.
Recent findings have shown a correlation between low levels of DHA and certain behavioral
and neurological conditions associated with aging such as dementia, depression, memory loss
and visual problems demonstrating the physiological importance of DHA for humans. Due to the
proven health supplementing potential of DHA, World Health Organization has recommended
that 1-2 % (2.2g to 4.4g) daily intake of energy out of 2000 calorie diet should be supplemented
from the omega-3 fatty acids. The intake of omega-3 PUFAs via our diet occurs mainly through
the consumption of sea food, which is characteristically rich in omega-3 PUFAs. The average
intake varies among the populations. This strong demand has resulted in the introduction of large
scale marine fish farms and the normal growth and development of several marine fish larvae
depend on the supplementation of the omega-3 polyunsaturated fatty acids in the diet especially
DHA and Eicosapentaenoic acid (Rodriguez et al.l998). At present selected fish oils and micro
algal species are the main industrial sources of DHA. Fish oils with the highest levels of EPA
and DHA include Mackerel, Herring and Salmon. Some fish such as Cod and Haddock store
most of the fat in the liver. The best sources though are cold-water fish such as tuna, mackerel,
3
sardines, herring and lake trout. But to obtain the maximum benefits of DMA from fish oils, one
has to preferably eat raw fish or boiled fish and moreover one should eat the skin behind the gills
around the fins and along the belly as these areas are where most of the oil is stored. Like all
PUFA oils the DHA and EPA are prone to oxidation in presence of light, heat and oxygen. As
the fish oil contains large quantity of polyunsaturated fatty acid they become rancid quickly.
Rancid fish smell fishy and hence are not very appetizing. Though one major source of EPA and
DHA for human consumption is in the form offish oil, fish oil has the disadvantage of an odour,
which is disagreeable to many human consumers. In addition to the above, the fish derived DHA
and EPA is also highly seasonal and variable in their omega-3 PUFA contents. Besides, most of
the fish oil is hydrogenated and the omega-3 PUFAs are destroyed in hydrogenation. The fish oil
often is unusable for human consumption because of contamination with marine pollutants such
as PCB's and high level of heavy metals. Due to these limitations the use of fish oils bearing
PUFA or their inclusion in infant formulas has many disadvantages. As the fish oils generally
contain Eicosapentanoic acid, an undesirable component in infant formulas because it leads to
reduced arachidonic acid levels in infants. This has been correlated with the reduced rates of
infant weight gain.
In order to meet the expected rise in demand and to circumvent the drawbacks of fish oils,
alternative production processes for the PUFAs are currently being developed. In this perspective
research on the extraction of PUFA oil from micro organism is being considered.
The Micro-organisms containing EPA and DHA can be cultivated on a large scale and the oil
derived from them is suitable for use in human nutrition and animal feeds (Bajpai, P. and P. K.
Bajpai. 1993). Among the microorganism several single-celled plants, the algae and fungi
contain high levels of EPA and DHA and have been considered for the said purposes (Singh, A.
and O.P. Ward, 1997). Microorganisms can be easily cultivated on a large scale using cheap
nutrients. Several groups of microorganisms contain high amounts of EPA and DHA. Such
organisms can be used directly as feed, or the said PUFAs can be extracted from them for further
use. Search for microorganisms containing high amounts of DHA and EPA has shown that
thraustochytrid protists contain the highest amounts of DHA and EPA. Their cells are used in
animal feeds or for extraction of PUFAs for commercial use (Lewis, T.E. et al., 1999, US Patent
No. 6,451,567). Among the microorganisms, the marine algae are thought to be the primary
4
producers of the omega-3 polyunsaturated fatty acids in the marine food chain. These marine
microorganisms represent the greatest percentage of the undescribed marine species (Colwell
1997) of which the marine microalgae form a large potential source of Docosahexaenoic acid.
The potential source of omega-3 oils is the group of micro-heterotrophs called Thraustochytrids.
The Thraustochytrids are a group of non-photosynthetic, heterotrophic organisms presently
classified under the Stramenophila kingdom, together with oomycetes and Labyrinthulids and
have been studied as potential omega-3 producers for industrial use due to their high lipid
content and high levels of DHA. These species are sometimes also classified as marine fungi.
Thraustochytrids are the common name of the Microheterotrophs that feed as saprobes or
occasionally as parasites. Thraustochytrids have a wide geographical distribution with strains
isolated from Antarctica, North Sea, India, Japan and Australia (Lewis et al., 1999). Ihey are
rarely found on living plants and appear to be inhibited by plant anti-microbial agents. Members
of these groups are often abound on dead autochthonous as well as allochthonous plant material
such as macro algae and submerged Mangrove leaves. They are common in the neritic and the
oceanic water column and the sediments including the deep sea.
The micro flora particularly Shizochytrium could provide a stable supply of fatty acids on a
mass scale and the oil produced from it have less fishy smell besides having higher content of
DHA as compared to the fish oil. Besides the Shizochytrium high levels of DHA are also found
in dinoflagellates, such as Crypthecodinium cohnii and the Amphidinium species. Data available
in the scientific literature demonstrate the large variation in biomass, lipid and maximum DHA
yields obtained for different Thraustochytrid strains. For example, Schizochytrium aggregatum
produced a biomass of 0.9g per litre after 10 days (Vazhappilly and Chen, 1998), while a
biomass of 48g per litre after 4 days was achieved using Schizochytrium sp. SR21 (Yaguchi et
al., 1997). It is very clear from the various studies conducted so far that the development of a
microbial PUFA production process requires the selection of the proper microorganism and the
optimized culture techniques.
The application of a strain of thraustochytrid for in the food industry such as food-additives,
nutritional supplements as additives for infant milk formula, feedstuffs and drug additives has
been described in The Japanese Patent No. 9633263. The strain contains at least 2% of dry wt as
DHA. The production of DHA by fermentation is also described in Japanese patent No. 980
5
3671. This also describes the Docosapentaenoic acid (DPA) another PUFA, from lipids of
thraustochytrid protists. Cells of thraustochytrid protists may be directly used as feed in
aquaculture (US Patent 5,908,622). Alternatively, DHA and EPA may be extracted from
thraustochytrid cells, using appropriate technologies (Japanese Patents JP 103105555 and JP
10310556). US Patent No. 5,340,594 describes a process for production of whole-celled or
extracted microbial products using thraustochytrid protists with a high concentration of the
omega-3 PUFAs. There is a potential for the use of thraustochytrid protists as human
Nutraceutical (Application A428 of Australia New Zealand Food Authority (ANZFA). The
production of DHA and EPA from thraustochytrid protists requires that they are grown in
suitable conditions in fermentors, to yield commercially useful amounts of the two PUFAs. The
issue of providing suitable conditions for growth and production of DHA and EPA in
thraustochytrids has been addressed in several research papers and patents (US Patent No.
5,340,742). US Patent No. 6,461,839 provides a method of producing PUFAs in Labyrinthula
sp., using a culture medium containing oil or fatty acid as a carbon source. Yokochi et al., (1999)
describe salinity, temperature, carbon source, oil and nitrogen sources for production of high
amounts of DHA in the thraustochytrid Schizochytrium limacinum. Optimal pH and medium
ingredients have also been described for Thraustochytrium aureum (lida T. et al.l996).
Several investigations that are carried lead to the isolation of many strains of Thraustochytrid
micro floral organisms of mainly genus Thraustochytrium and Schizochytrium as alternative
source for the industrial production of PUFAs containing primarily DHA as key constituent
which is used for human and animal in the form of feed and health supplements. The process
developed so far for extraction and isolation of PUFAs are much complex and have used toxic
organic chemicals and solvents including chlorinated solvents in huge quantity to get desired
strength of DHA in PUFAs as well as characteristic micro floral or marine fishy odour free
PUFAs.
Object of the Invention:
Aim of the preset invention is to extract and prepare docosahexaenoic acid oil using green
solvent supercritical fluid carbon dioxide (SCE-C02) extraction method;
6
Further object of the invention is to reduce the use of toxic organic chemicals and solvents
including chlorinated solvents;
Another object of the invention is to prepare odour free DHA oil having 40-45% content of DHA
and liquid-liquid extract containing 55-80% DHA.
Brief description of drawings:
Figure 1: Schematic Diagram of SCF-C02 Extraction of Shizochytrium;
Figure 2: Flow diagram of DHA extraction and oil preparation;
Figure 3: Bar diagram of recovery of DHA and its assay content from Schizochytrium biomass
by single Stage Process of SCF-CO2 System;
Figure 4: Fig. 4: Bar Diagram of recovery of DHA after winterization of SCF-CO2 Extract in
Acetone and Ethanol
Summary of the Invention:
The present invention is a method for extraction of docosahexaenoic acid (DHA) from single cell
fungal micro floral strain oiSchizochytrium SP. the said method comprising the steps of;
a) Single stage primary SCF-CO2 extraction to achieve up to 36-45% docosahexaenoic acid
extract (DHAE), with or without the use of co-solvent or convention downstream
process, using liquefied extraction gases;
b) Secondary two stage or liquid-liquid extraction process carried at optimized pressure
150-225 bar at temperature up to 60°C to enrich the contents of docosahexaenoic acid up
to 55-80% from docosahexaenoic acid taken from the above single stage SCF-CO2
extraction process;
c) Deodorization of docosahexaenoic acid extract by treating with co-solvent;
7
d) Solvent Winterization of docosahexaenoic acid for preparing docosahexaenoic acid oil.
The present invention discloses a novel process for extraction of docosahexaenoic acid extract
(DHAE), constituting polyunsaturated fatty acids (PUFAs) with docosahexaenoic acid as major
constituent, from a new patented fungal Micro flora strain of Schizochytrium sp. Tne algal
biomass Schizochytrium sp. is extracted for the first time by a process SCF-CO2 at pressures
from 175-625 bar at temperatures 60-70 °C to get the initial concentration of DHAE with
docosahexaenoic acid up to 36-45% in the extract formed by initial single stage extraction and
multi-separator separation of SCF-CO2 extractor system. The single stage extract is also
enriched to 55-80% DHA extract by a second stage called two stage extraction technique
wherein the initial extract obtained is processed again by liquid-liquid SCF-CO2 extraction at
pressures ranging from 80-375 bar at temperature up to 60°C. The enriched DHA containing
extract prepared by second stage liquid-liquid extraction is converted to DHA oil by co-solvent
winterization.
The present invention is a first ever novel method for preparing DHAE extract from
microalgae species Shizochytrium by high pressure supercritical carbon dioxide with or
without the use of CO solvent and further enrich the DHA extract by second stage liquid-liquid
extraction and solvent winterization. The DHA oil is prepared using multi separator system
thus getting the enriched as well as the deodorized oil. The solvent winterization is carried out
to remove the lower saturated fats and waxes for increasing the concentration of PUFA in the
DHA extract using multi separator SCF-C02 process is reported for the first time. The DHA
extract prepared by SCF-C02 extraction is isolated and fractionated in the single step having
different content of DHA. The same is converted to DHA oil by using co-solvent ethanol and or
acetone for winterization.
In single stage extraction, the highest concentration of DHA achieved is up to 36-45% with a
maximum recovery of 84%. The DHA extraction from Micro floral biomass (Shizochytrium) at
different extraction pressure is almost constant without much variations. The optimum pressure
for the DHA extraction recovery is found to be 425 bar where DHA recovery content is about
37.7%. In fact the recovery at 400 to 425 bar varies to a small extent. The enrichment of DHA
content done by second stage liquid-liquid extraction yield 55-80% DHA extract containing
8
Docosahexaenoic acid. The solvent were used in the winterization step only to remove lower
molecular weight saturated fatty acids, phosphatides and waxes. The DHA oil prepared by this
method bear DHA content from 40-45% for the oil prepared from the single stage SCF-CO2
extract. Antioxidant like tocopherol, rosemary extract and ascorbic acid or its esters are added to
prolong the life of DHA oil. The DHA oil is stored at a temperature 0-5°C in fully filled
containers which are blanketed with nitrogen. The DHA oil so obtained by the present method of
single stage SCF-CO2 and liquid-liquid extraction and solvent winterization is very useful for
direct formulation to make variant dosage forms like softgel capsules, powders and granules for
their application of food, beverage or pharmaceutical industry.
Detailed Description of Invention:
The polyunsaturated fatty acids and their components like EPA and DHA are good lipid soluble
and their extractions are carried using various organic solvents like hexane, dimethyl ether,
diethyl ether, ethyl acetate, chloroform, dichloromethane, methanol, ethanol and mixtures
thereof Though the extraction of PUFA oil through solvent extraction is well known art and
being used in the industry for preparation of DHA oil but due to the adverse effect of the
solvent residue retained in the extracted oil a number of attempts have been made to search
alternatives for the extraction of PUFA oils. This has been further required due to the
restrictions in use of organic toxic solvents due to health consciousness. For achieving this inert
liquefied gases like CO2, CH2CH2, CH3CH3, N2O, N2 or mixtures thereof in supercritical
condition have gained popularity in applications like extraction of non-polar and moderately
polar lipids like polyunsaturated fatty acids. Since liquid CO2 and N2 are readily available as
inexpensive source for extraction/isolation of botanicals like PUFA. Owing to these
characteristic of Liquid, supercritical CO2, N2 and mixture thereof were preferred for conducting
the present extraction of PUFA oil., more precisely the extraction of docosahexaenoic acid
extract, referred as DHAE, from a patented single-celled fungal micro floral strain of
Schizochytrium WZU4771 using advanced technology of multiple-three separator supercritical
fluid carbon dioxide (SCF-CO2) system of very high extraction pressure of up to 450 bar.
9
The extraction of these PUFA oil from Shizochytrium biomass has been conducted in high
pressure supercritical CO2 extractor. The High pressure extractors as used in the present
invention are designed independently as parallel systems connected each to independent high
pressure CO2 pumps having CO2 flow rate up to 0.8-1.6/min with a design pressure of both
pumps and extractors 690 bar while the respective High Pressure, Medium Pressure and Low
Pressure separators are set to pressures of 450, 240 and 83 bar. The extraction of micro floral
biomass in powder form of 1-2 mm particle size is conducted by placing the algal meal in a
basket which is further loaded into an extractor vessel. In the process of extraction, the extractors
are confined to various extraction pressures between pressures of operation 175-450 bar while
temperatures are controlled using heat exchangers in-between 50-70*^0. Though, the extractions
are also attempted at a temperature higher than 70'^C, for the purpose of invention, higher
temperatures SO'^C are not preferable as the facts that the PUFA and its constituents DHA is
highly susceptible for temperatures and oxygenated-air. The pressure parameters of each
extractor and separators are regulated to maintain the set pressure conditions by high pressure
auto control valves in concordance of temperatures regulation through heat exchangers.
Principally, the extraction of active ingredient DHA in DHAE from fungal biomass is carried by
diffusion of dense fluid or solvent supercritical fluid carbon dioxide through biomass particle and
a dissolved DHAE in supercritical fluid carbon dioxide is pushed to the H.P separator first. For
the present invention, the well optimized extraction conditions of extraction vessel is found to be
between 175-425 (bar) at temperatures 50-60°C while high, medium and low pressure separators
respectively are maintained at around optimum pressure (bar) and temperature (°C) 90-350 and
60, 50-130 bar and 50°C, 40-45 bar and 12°C. An invention is further coincides wherein the
multiple separators are very much crucial in enhancing the DHAE with DHA up to 36-45% at
first stage itself and same is achieved by varying the pressures (bar) and temperatures (°C) of
respective H.P, M.P and L.P. separators individually between 90-175 and 50-60, 50-110 bar and
40°C, 43±2 bar and 12°C. The multi (three) separator SCF-CO2 system allows to change the
pressures and temperatures to a broad margin so that allowing for, simultaneously at single
stretch, the removal and push of unwanted lower chain fatty oils/waxes present in DHAE
collected in the H.P in to M.P and L.P separators thus enrich the content of DHA in DHAE to
36-45% in H.P separator.
10
The extraction is carried out using supercritical fluid extraction from the fungal based biomass
Schizochytrium sp. to achieve, in single stage as one-go primary extraction, a DHAE constituting
a high concentration of 36-45% DHA which cannot be possible such high concentrations by
normal conventional multistage organic-solvent extraction techniques. The primary extract
produced has such a nature in the form of soft and creamy viscous liquid but not in an oil form.
Hence that a primary extract produced is called as docosahexaenoic acid extract (DHAE).
Further enrichment of DHA in DHAE extract is carried by a process, a second stage liquid-liquid
extraction which is a repeated extraction to get DHA by reloading the DHAE of primary
extraction process in the extractor vessel at optimized conditions of low pressures between 80-
250 bar at temperature 40 to 55°C. The pressure and temperature of extraction vessel are set
between 90-150 bar up to the temperature 60°C while the conditions of individual separators
High Pressure, Medium Pressure and Low Pressure are set at respective pressures (bar) and
temperatures (°C) between 70-110 and 45-60; 60-90 and 50; and 45 and 8-12. One aspect such
embodiment is that to make and collect the product enriched with DHA from the extractor vessel
itself where the product is loaded. A secondary process is done to get enriched concentrations of
DHA in DHAE up to 55-80%. In the present invention, desired industrial concentrations of
DHAE with DHA of 36-45% is easily produced by single stage extraction and adjusting the
pressure and temperature of extractor and separators to accomplish the 36-45% DHA in the
extract. However, to get rid of free fatty acids and the undesired odour it needs a further
refining so that DHAE extracts which have more acceptable odour and have higher content
DHA is prepared for a better market and as consumer acceptable product. So it is needed to
produce further an improved flowable DHAE product as in oil form that is free from
characteristic obnoxious odor of microflora due to assimilated phospholipids and rancid free
fatty acid and volatile secondary oxidation products. The saturated fats and lower chain fatty
acids keep the DHAE extract in soft frozen creamy form instead of it in oil form at temperatures
less than 35°C.The SCF-CO2 plant design and process Flow diagram of DHA extraction from
Shizochytrium biomass and its conversion to oil is presented in Fig 1 and Fig 2 respectively. To
prepare the DHA oil as available in the market having better odour, improvement experiments
11
are also conducted in furtherance using DHAE of primary stage to carry dephospiiolipidization
and deodorization in simpler fashion using two stage supercritical liquid-liquid extraction
process of DHAE by adding 0.2% alkali particularly sodium hydroxide and solvent
winterization. The solvent ethanol and or acetone were used for this purpose of winterization of
oil. The lower molecular weight fatty acid and phosphatides get precipitate during the solvent
winterization at 0-5 °C. The precipitated material is discarded and the DHA oil is isolated. This
is further put for deodorization with nitrogen or liquid carbon dioxide for removal of odour
causing chemicals. In the investigation using three separator system SCF-C02 extraction the
second stage liquid-liquid extraction is carried out at a pressure ranging from 80-250 Bar to get
DHA concentration from 55-80%. This is further converted to oil by solvent winterization by
removing the precipitated impurities. The second stage liquid-liquid extraction is not only carried
to enrich the DHA content but also results in the deodorization. Incorporation of liquid -liquid
extraction and or solvent winterization makes product suitable for market acceptability.
The Lower extraction pressure in liquid-liquid extraction retains the higher molecular weight
DHA in the extractor itself while the lower molecular weight saturated fatty acids are removed.
The extraction under supercritical condition is carried with or without co-solvent like ethanol,
methanol, and ethanol: water 50:50 or methanol: water 50:50 and mixtures thereof up to volume
of 40% on DHAE quantity loaded in the extractor. During the liquid-liquid extraction
experiments for DHAE either in CO2 fluid medium as such or CO2 fluid with co-solvents in
which again without treatment or pretreatment with either mild acid, alkali or alkaline salts of
0.2% for each set of experiments are also carried to control the levels of peroxide value and free
fatty acid value which are quality parameters for finding the oxidative-degradation status of
DHA oils whose quality can be comparable to readily produced DHA oils of commerce.
At the end of liquid-liquid extraction process, the enriched DHA oils that are produced are
subjected for supercritical nitrogen or CO2 extraction up to 100 bar pressures at temperature 40-
50''C in order to further remove odoriferous volatile substances which could impair the oil
quality. The DHA oil prepared by the present invention contain more green process as the
extraction by SCF-C02 followed by co-solvent winterization uses very less amount of solvent
12
as compared to the conventional methods of preparation of DHA oil which uses large quantity
of solvent for extraction and or refining. The DHA oil prepared by the method of present
invention is a green alternative for existing processes of DHA oil manufacture. The DHA oil so
obtained is added with natural antioxidants for prolonging its life and could be safely used for
food, beverages and pharmaceutical industries.
The DHA oil produced by the method of present invention is environmentally sustainable
method as it uses carbon dioxide in its supercritical state for extraction which is available in
plenty. The use of supercritical carbon dioxide which is recycled will also be helpful in
maintaining green house gas as this will be used as extraction solvent. The Shizochytrium
biomass used for the extraction of DHA oil is replenished as it is a renewable resource. The
process of extraction used for DHA oil will also reduce the burden on petroleum resources as
extraction solvent are replaced with the super critical carbon dioxide. The process of
investigation also uses second stage extraction for further enriching the DHA content. The DHA
oil prepared by present method will certainly use very low amount of solvent which is used for
solvent winterization.
The DHA oil prepared by present investigation could further be converted to food, beverages and
pharmaceutical application by using food grade excipients in different quantity.
Example:
I. Primary Extraction of Schizochytrium Biomass to get DHAE:
The multiple-three separator supercritical CO2 extraction system (Fig.I) with the capacity of
IX12 L extraction vessel is used for the extraction. The basket is filled with Schizochytrium sp.
WZU4771 biomass (l-2mm particle size) up to 1.5-3.0 Kg in basket or till the basket is full and
0.2% alkali of biomass, particularly sodium hydroxide is added. The top basket cover plate is
assembled properly with basket seal. Basket bottom and top plate are assembled with filter paper
Whattman No.l along with frit to avoid any carry over of fine material to the separator vessels.
In the experiments, very much broad parameter changes are made in the extraction vessel at
pressures between 175-450 bar and temperatures up to 70°C preferably 50-60°C while in
13
respective multiple H.P, M.P and L.P. separators are individually controlled between pressures
and temperatures respectively at 110-350 and 60, 50-150 and 60, 40 and 12. The optimized
extraction parameters are set on programmed logic control (PLC) system for extractors (bar/°C)
175-450 bar at temperatures 50-60°C and for high pressure separator 175/60°C, medium pressure
separator 110/40°C and low pressure separator 40/12°C and also carried the experiments at
various conditions tabulated in Table-1 with process flow Fig.2.
The flow rates of CO2 pump are set for 0.8-1.6 kg/min. The total CO2 flow for the extractor is
50±10% kg/kg microflora biomass loaded. Start the CO2 pump with flow rate of 0.8 kg/min and
the flow rate is gradually increased up to a stable flow of CO2 1.6 kg/min by observing pump run
is in healthy condition. Collect the DHAE from the H.P and other lower fatty acids,
phospholipids and or waxes from the M.P and trace volatiles from the L.P separators. Also water
obtained is collected, separately, from medium pressure and low pressure separators by phase
separation. The extraction is also attempted with 10-30% co-solvent (ethanol) for recovering
the maximum DHA from the Shizochytrium. The recovery of active for the different
extraction trial conducted lies between 69-84%.
The extraction parameter set for 12*1 L Supercritical Fluid C02 extractor are also carried in
similar fashion in 3001x3 SCF-CO2 system by loading the biomass in multiple volume of the 12
L extractor. In the commercial extraction using 300L*3 number extractors which are connected
parallel with three independently operated CO2 pumps for each extraction vessel. The separators
for collecting the extract are connected in series to the extraction vessels. The extraction
condition for the 300L extractor are kept same as used for the 12 L extractor except the flow
rates of CO2 which is varied between 10-45Kgs/min.
Table 1: Recovery of DHA and its Concentration from Schizochytrium Biomass by Single Stage
Process of SCF-C02 System.
14
%'AjaA0D3J [^ o i ^ m c r i K ^ ^
VHQ liejaAO
'S % P|9!A i" o o o u s o m p ^ m
- t ; r J O T - i r H o i f O ^ t od
.2 S pBj}X3VHa
™ "s
§ S (%'AB55B) ;;;; 37-
UOIPEJJXa rq j o H ^ ^ ^ r j g < ^ ^ 3 ^ ^ g ^ g
* • • ^ ^ ^ ^ ^ ^ ^ ^ — . ^ ^ ^ . v n M ^ MBMPBBaWH— ^ ^ ^ — ^ — ^ — ^ — ' — ^ ^ — ^ « — — « — ^ — — ^ — ^ - ^ - — ^ ^ ^ ^ — ^ ^ ^ ^
< S ! ^ S 'o S S 5 q ") «?
i S ^ 5 5 § ^ S 5 S S
5 ^-joiejadBS rn d r n d t - H d o d
§ -£
5 -r .nin.^^-^.^ *N r^ oi ^ o Q * t » - i oo
J^ X-JOJeJ3oas t (N m m ui 9 • t en
E-jo)ej3d3S z Z 2 Z Z Z Z Z
^ ra 3-J0}EJaa»S 1^ o d o d o d o d o d o d c o
< «
I lu
Q C
— o q t n f H o q i / ) i / } a 5 r ^ r^
T-J0iej3dac "^ i^ "^ ^ u S o o i a ui r^
(uJFpeJjxi) ^^——. ^
0 0 0 00 lo IN g 00 01 •=
^ • -•• '^x^'^'i^'' O
5 E-J0}BJ8daS H K I - I - I - I - H l - .U
u •—* __^^^^_^^^^^^__^_ .^^.^^^^^^^_ ^__^^^^^_ ^^^^..^^^^ ^^^———^^^_ ^^^—^_^^^ ^^^.^^.^^^ ^_^_^___« ^^_-—_—_ tlO
g ^
tf ^ —
" 3-jo;Bjad8S w & ^ r - i i H o o o ^
X ra
Q ^ E
r^ oi m i n o i n m m o c
T i n i p i a r l a r 1^ *^ ^ O O U S O T O O cn m (0
o 2
(V^^''> ? f o 5 j o ^ o ^ o ^ o g o I j o ? j o — .y
S f ^ m-N SQ Su mn CQ oi mn iL nn o^ uO^ - iliII.
o m o o o Q i / i u - ] ^oi/i'
IT) ( N i f t L n o Q r N J r J dsi>0 JOPBJJXJ m m m m * 9 ^ •t infJ
^ — — ^ ^ — — — ^^____ _ _ ^ _ _ +1 £
00 o o Q O o o m o o o o o o >,-
.!» UUUaiUODVHQ r M < N N r M * r M < N r M S£
> — .9^ -E.
™ r M r > i ( N r M ( S ( N f N i i N J;;tl
5 %3JniS!0i^ ^ ^ ^ ^ - ^ ^ ^ ^ >^
i Ei g
m r o m r n m m m m 4-> ^-
%vna 3 - S 3 S 3 3 3 3 2^
Q. -5
(3)1) Wa q q q q q q q q gfc
jo Alt) papeoi I I I I I \ I o3 5
apo3SDjnoswa ZOOZSSi -= ! S5
, , 1 , , , 1 to oc
01 11
1^ o o g j O r H r s i L n c j i ,i"«ir
•ON u o j p e j j x g g ^ g ^ ! ^ ^ ! ^ ; ^ ^^
15
Yields:
The extraction yield from dry microflora biomass with respect to DHA content of biomass >30%
is given below. The DHA content in the DHA extract will depend on the content of DHA in the
initial raw biomass.
I Extraction Yield I DHA content in DHAE
(On loaded biomass basis)
(Overall)
High pressure separator 28-32% Assay 36-45%
Medium pressure separator 5-7.0%) Assay 2-11%
Low pressure separator < 1.0% Assay 1 -6%
2. Second stage Liquid-Liquid Extraction and a Simultaneous Deodorization Process of
DHAE Using Liquid Carbondioxide Media to Get Higher Content-Deodorized DHA Oil:
About 1.0-2.0 kg or up to the half of the extractor volume is loaded in 121x1 extraction vessel
with the DHAE obtained from the primary extraction process. The pressure and temperature of
extraction vessel are set between 90-150 bar up to the temperatures 60°C while the conditions of
individual separators HP, MP and LP are set at respective pressures (bar) and temperatures (°C)
between 70-110 and 45-60; 60-90 and 50; and 45 and 8-12. The SCF-CO2 system CO2 pump is
started to pump the liquid CO2 in to the extraction vessel up to a flow rate starting initially at
0.7Kg/minutes then increased gradually up to a stable flow of CO2 1.2Kg/min after setting the
extraction parameters. Pumps are continued to run to carry liquid-liquid extraction of DHAE up
to a total CO2 flow of 60-80Kg/kg of DHAE extract loaded in the extraction vessel. The
deodorized DHA extract/ oil is separated and collected from the HP separator while lighter oil,
wax and phosphatides are collected in MP separator along with traces of lighter oils collection in
LP separator. The results of liquid-liquid extraction are given in Table 2.
16
Table 2:Liquid-Liquid Extraction Data of DHA
^ B 2 > > i ll
€ < " % s & s s o o ^ c/^
^92 ioOO 429.6 447 167.4 ^16 29.19 ND 420 ms 46.43 45?76 No Co-
(42.96) (44.7) (11.6) (47.5) solvent
1^93 1000 429.6 397 1610 250 34^0 ND 342 2011 46^81 4^6 NoCo^
(42.96) (39.7) (25.0) (58.8) Solvent
394 1000 429.6 ~562 205.0 "183 2610 700 330 1842 42.87 53.80 Ethanol used
(42.96) (56.2) (18.3) (70%) (55.8) as co-solvent
~395 1000 420.0 580 200.0 190 2l0 800 300 204.0 48^6 5000 Ethanol used
(42.00) (58) (19.0) (80%) (68.0) as co-solvent
# Pressure <& temperature varies with ± 5% of set parameters on PLC (Programmable logic
controller), RM= Raw Material Schizochytrium biomass, T= Traces, NA = Not applicable
The yield of extract during second stage liquid-liquid extraction ranges between 47-59 % while
the recovery of active ranges from 45-57 %. The DHA content in the extract ranges from 47-
68%. The active recovery data of DHA extracted from Shizochytrium by SCF-C02 single stage
extraction is presented in Fig. 3.
3. Two Stage Liquid-Liquid Extraction and a Simultaneous Deodorization Process of
DHAE Using Liquid-Liquid Carbondioxide in Presence of Co-solvents Ethanol and or Ethanol:
Water, Methanol: Water, Acetone to Get Higher Content-Deodorized DHA Oil. The results of
winterization/ deodorization experiment carried using ethanol and acetone are presented in
Table 3 and 4 respectively.
17
About 1.0-2.0 kg or up to the half of the extractor volume is loaded in 121x1 extraction vessel
with the DHAE obtained from the primary extraction process. The pressure and temperature of
extraction vessel are set between 90-150 bar up to the temperatures 60'^C while the conditions of
individual separators HP, MP and LP are set at respective pressures (bar) and temperatures ("C)
between 70-110 and 45-60; 60-90 and 50; and 45 and 8-12. Then the SCF-CO2 system CO2
pump is started to pump the liquid CO2 in to the extraction vessel up to a flow rate starting
initially at 0.7Kg/minutes then increased gradually up to a stable flow of CO2 1.2Kg/min. Then
the co-solvent pump is started to inject the co-solvent ethanol into liquid carbon dioxide line at
similar pressure conditions to that of liquid-liquid carbon dioxide pump at flow rate of 20-
30ml/min up to 90-180 minutes so that ratio of co-solvent to the loaded DHAE primary extract in
the extraction vessel is at the ratio of 1:1, 1:2 to 1:3. The co-solvent pump is stopped after
passing the desired quantity of carbon dioxide. However, the Liquid carbon dioxide pumps are
continued to run to carry liquid-liquid extraction of DHAE up to a total CO2 flow of 60-80Kg/kg
of DHAE extract loaded in the extraction vessel. The deodorized DHA oil is separated and
collected from the HP separator while lighter oil, wax and phosphatides are collected in MP
separator along with traces of lighter oils collection in LP separator. The obtained DHA oil in
ethanol medium from the H.P separator is filtered on hyflow-supercel bed or celite. The filtrate
of ethanol constituting DHA oil is subjected for stripping of ethanol on suitable evaporator like
rotaevaporator under vacuum at temperatures 45-50°C to get deodorized DHA oil. Similar
experimental conditions are maintained to carry above experiment independently using other
solvents like ethanol: water, methanol: water and to process DHA oil.
The DHA extract obtained by the SCF-C02 extraction was converted to DHA oil by solvent
winterization. For this ethanol and acetone was used at 2 to 10 volume of the DHA extract and
mixed. The results of these experiments are presented in Table 3 and table 4 respectively. The
winterization conducted with ethanol resulted in 74-86% recovery of DHA during DHA oil
preparation. However, this step resulted in high peroxide value. To overcome this experiment
were conducted with acetone. The acetone winterization experiment resulted in 95-98% recovery
of active DHA. The comparison of solvent winterization results presented in table 3 and table 4
18
shows that the winterization process with ethanol resulted in peroxide value higher than the
required product quality. While, the winterization step using acetone resulted in the peroxide
value within the specified limit. The recovery data and assay of the DHA oil prepared after
winterization conducted with ethanol and acetone is presented in Fig. 4.
Table 3: Solvent Winterization of DHA Extract with Ethanol
Exp. I Details of DHA SCF- I Details of DHA Oil I Remark
No. CO2 Extract
Qty. Assay Activ Qty. Assay Active PV Yiel Recover
(g) (%) e(g) (g) (%) (g) meq/kg d y(%)
(%)
"01 800 37!0 296 621 4E0 255 106 77^6 861 Overall recovery of active 64.6%'
~02 imOO 38^0 380 660 43^0 284 23J0 66^0 74J Overall recovery of active 56.0%
l i 3 800 36J0 288 610 400 244 5^0 763 847 Overall recovery of active
63.5%
H i 800 37IO 296 590 43^0 254 15^6 73l 85l Overall recovery of active
64.4%
Table 4: Solvent Winterization of DHA Oil with Acetone
Exp I Details of DHA SCF- I Details of DHA Oil I Remark
CO2 Extract
No. Qty. Assay Active Qty. Assa Activ PV Yiel Recover
(g) (%) (g) (g) y(%) e{g) meq/kg d y(%)
(%)
~0\ 50 35.3 17.65 39?7 42.63 16.92 3^9 79^4 95^9 Overall recovery of active"68^%"
1)2 200 36 72 169.3 4L8 708 22 84/7 983 Overall recovery of active 70.6%
~03 250 36 90 201.0 43l 86^6 ND 804 962 Overall recovery of active
69.0%
1)4 400 38 152 321.0 44.0 141.2 4^0 803 97^8 Overall recovery'ofactive
70.3%
19
Industrial application of the invention:
The present invention is a first ever developed novel industrial process using a supercritical fluid
extraction system of 3 x 300 Liters extractors facilitated with advanced multi-separator system.
The process has been developed as eco-friendly, green recyclable carbon dioxide fluid process
and is single stage, straight forward process which full out the drawbacks of previous processes
which are normally developed utilizing combustive processes with complex process of
investigation carried to produce DHA extract in low yield. The DHA extract produced is used
for preparation of DHA oil as carried out in the present investigation by the process of solvent
winterization. The single stage extract obtained is also used for second stage liquid-liquid
extraction to enrich the DHA content in the DHA extract up to 55-80% preferably between 40-
60%. The enriched extract is deodorized to remove the waxes and phosphatides to prolong its life
and added antioxidants particularly tocopherol and ascorbic acid and or its ester. The high
strength and purity DHA oil developed is much cost-effective product derived by organic-green
process using eco-friendly SCF-C02 extraction and solvent winterization which uses very less
amount of solvent as compared to the prevalent process which extract the micro floral biomass
with solvent. The product derived by this process is much useful as alternate direct industrial
ingredient for food, beverage and Nutraceutical applications either directly or by adding food
grade excipients for food and pharmaceutical applications.

We Claim,
1. The method for extracting Docosahexaenoic Acid (DHA) from single cell fungal micro
floral strain of schizochytrium sp. comprising the steps of:
a) Single stage SCF-C02 extraction from Schizochytrium sp. biomass to achieve
upto 36-45% docosahexaenoic acid extract (DHAE), with or without the use of
co-solvent or convention downstream process, using liquefied extraction gases;
b) Liquid-liquid extraction process carried at optimized pressure 80-250 bar at
temperature upto 60°C to enrich the contents of docosahexaenoic acid (DHA)
upto 55-80% from Docosahexaenoic Acid (DHAE) extract taken from the above
single stage SCF-CO2 extraction process;
c) Deodorization of docosahexaenoic acid by treating with co-solvent;
d) Solvent Winterization of docosahexaenoic acid for preparing docosahexaenoic
acid oil.
2. The method of extracting DHA as claimed in claim 1 wherein the Schizochytrium sp.
biomass is filled in the extraction vessel which is set at the pressure between 175-450 bar
and temperature up to 70°C preferably upto 50-60°C.
3. The method of extracting DHA as claimed in claim 2 wherein the initial flow rate of CO2
is 0.8 kg/min which is increased up to stable flow rate of 1.6 kg/min.
4. The method of extracting DHA as claimed in claim 2 wherein the CO2 flow for the
extractor is 50 ± 10% kg/kg of biomass loaded.
5. The method for extracting DHA as claimed in claim 2 wherein the extractor is set at a
pressure 450 bar and temperature 70°C, coupled with advanced three stage separator
system.
21
6. The method for extracting DHA as claimed in cl,aim 5 wherein the three stage separator
comprises of high pressure separator ((H.P.). medium pressure separator (M.P.) and low
pressure separator (L.P.) respectively connected in series with extractor and are
maintained at around optimum pressure bar and temperature 90-125 and 50-60°C, 55-100
and 50°C, 40-45 and 12°C respectively.
7. The method for extracting DHA as claimed in claim 5 wherein the temperature and
pressure of the three stage separators are kept varying of respective H.P., M.P. and L.P.
separators between 90-110 bar and 50-55 °C, 55 bar and 50 °C, 43±2 bar and 12 ''C.
8. The method for extracting DHA as claimed in claim 7 wherein the DHA assay obtained
from High Pressure, Medium Pressure and Low Pressure separator is 36-45%, 2-11% and
1 -6% respectively.
9. The method for extracting DHA as claimed in claim 1 wherein the co-solvents used in the
single stage extraction process are selected from the group of methanol, ethanol, acetone
and a mixture of ethanol and methanol or mixture of solvent.
10. The method for extracting DHA as claimed in claim 1 wherein the second stage liquidliquid
extraction comprises of a repeated extraction of primary extract DHAE using one
liquefied extraction gases carbon dioxide, nitrogen, methane, ethane, nitrous oxide as
such without any co-solvent or with co-solvent.
11. The method of extracting DHA as claimed in claim 10 wherein the extract of first stage
SCF-CO2 is loaded in the extraction vessel having the pressure between 90-150 bar and
temperature up to 60° C coupled with three separators in series i.e. high pressure
separator ((H.P.), medium pressure separator (M.P.) and low pressure separator (L.P.)
which are set at respective pressure and temperature between 70-110 bar and 45-60 °C,
60-90 bar and 50 V , 45 bar and 8-12 °C respectively.
22
12. The method of extracting DHA as claimed in claim 10 wherein the liquid CO2 is pumped
in to the extraction vessel starting initially at rate of 0.7 kg/min and increased up to 1.2
kg/ min.
13. The method of extracting DHA as claimed in claim 10 wherein the total CO2 flow in
extraction vessel is 60-80 kg/kg of DHAE extract loaded.
14. The method of extracting DHA as claimed in claim 10 wherein the deodorization of DHA
is carried out in second stage by flowing of co-solvent into the extraction vessel at flow
rate of 23-30 ml/min up to 90-180 minutes.
15. The method for extracting DHA as claimed in claim 14 wherein the co-solvents are
ethane! or ethanol and water or methanol and water of ratio 50:50 up to volume of 40%
for loaded DHAE.
16. The method of extracting DHA as claimed in claim 9 wherein the liquid-liquid extraction
comprises repeated extraction process of primary extract DHAE in which to enrich
docosahexaenoic acid upto 55-80% by removal of undesired waxes.
17. The method for extracting DHA as claimed in claim 1 wherein the preparation of DHA
oil from the primary extract or liquid-liquid extracted DHA extract is carried out by
solvent winterization by adding 2-10 volume ethanol, methanol or acetone, hexane or a
mixture thereof, preferably acetone or ethanol and separating the precipitated mass after
12-24 hours at low pressure and temperature.
18. The method of extracting DHA as claimed in claim 17 wherein the solvent used for
winterization is acetone which results in 95-98% recovery of active DHA.
23
19. The DHA powder and granules are formulated from the DHA oil extracted from any of
the above claims is prepared using the exciepients selected from starch ester,
microcrystalline cellulose, hydroxy propyl methyl cellulose, light magnesium carbonate,
tribasic calcium phosphate, magnesium stearate and mixtures thereof with or without
rosemary extract, vitamin C palmitate, natural tocopherol and mixtures thereof at
respective concentrations of 0.2, 0.5 and 2% w/w.
Dated this 09"' day of January 2012.