WO2018097931A1

WO2018097931A1 - Extraction of free dha from algal biomass

Info

Publication number: WO2018097931A1
Application number: PCT/US2017/058806
Authority: WO
Inventors: Hiroaki Hara
Original assignee: Fpn Llc
Priority date: 2016-11-28
Filing date: 2017-10-27
Publication date: 2018-05-31

Abstract

A method of free DHA (Docosahexaenoic Acid) fatty acid separation and purification from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipids (triglycerides) and polar lipids (phospholipids) are separated without trans-esterification. A mixture is formed that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water. That mix is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid. The liquid DHA fatty acid may then be separated.

Description

EXTRACTION OF FREE DHA FROM ALGAL BIOMASS

BACKGROUND OF THE INVENTION

1. Field of the Invention.

[0001] The present invention relates to the field of extracting DHA from an algal biomass. More particularly, the present invention relates to separating free fatty acid from the cell structure by the use of lipase hydrolysis process so that all lipids (triglycerides) and polar lipids (phospholipids) are separated without using a trans-esterifi cation method.

2. Description of the Prior Art.

[0002] DHA, an omega-three (Ω-3) fatty acid (FA), has been documented as essential for healthy brain development in children and proven in clinical trials to reduce cardiovascular diseases, lower blood pressure and prevent development of hypertension. It is an essential FA for human health. The accretion of DHA in membranes of the central nervous system is required for optimum development of retinal and brain functions.

[0003] Feeding elevated amounts of DHA oil has reportedly improved memory function in mice. If the mice study is expandable to humans, feeding elevated amounts of free-DHA to infants and children has the potential to improve brain function and IQ levels. Since there is no reported toxicity associated with free-DHA, supplementing a diet with free-DHA for extended periods of childhood is a reasonable mechanism for enhancing child development

[0004] DSM Nutritional Products, a division of Royal DSM, formerly known as Martek Bioscience pioneered the production of DHA (docosahexaenoic acid) containing oil from a dinoflagel- late micro algae, Crypthecodinium cohnii (C. cohnii), over two decades ago and successfully marketed the DHA containing oil worldwide. There are number of species of micro algae many are in the class of dinoflagellates that contains DHA and in some cases DHA and EPA in their lipid content, and culturing of such algae, and the extraction of Omega 3 (Ω3) Fatty acid especially DHA is becoming an important technology to secure the Ω3 in our diet, be it may a direct additive to infant formula, as a supplementary capsule formula, or a source of fish and animal feed as the farmed animal diet. For human consumption, it is vital to extract DHA containing oil rather than dried micro algae biomass, which is of generally unknown toxicity and biological characteristics.

[0005] The extraction technique that is described in the Martek patent (US Patent 5, 130,061) states that supercritical carbon dioxide (scCC ) can be used for extraction. Other commercially used method is the fractional distillation method using hexane or other organic solvent as the intermediary agent. The fractional distillation method is an established method that is less expensive than the scCC , however, the treatment of organic solvent poses the environmental and operational hazard concerns.

[0006] Even as late as 2002, Martek stated (US Patent 6,372,460) that the extraction process leaves significant polar lipids, (PL), mostly trapped in the biomass, which suggests that the combined lipid form of DHA is more difficult to extract than the free-FA form of DHA. This '460 patent provided the animal feed powder that contains residual DHA as a valuable co-product of DHA oil extract.

[0007] In order to extract all the fatty acid in the biomass, there is a standard method called trans-esterification and saponification method. This process for selecting and/or purifying DHA from the other fatty acids through alcohol ester formation (e.g. DHA-Ethyl Ester) includes additional steps and capital equipment, which makes it economically less desirable. The end result includes fatty acids other than DHA, that are of little or no dietary value, and are not PUFA, require further selection/purification steps such as chromatography or fractional distillation.

[0008] For bioavailability in infant formula, free-DHA is the best form of DHA, though it may require anti-oxidants for longer shelf life. The normal pathway for a baby to utilize this FA is to ingest the oil in the form of triacylglyceride (TAG). Babies' lipase enzymes then cleave the bond of each FA from the glycerol arm of the TAG, separating them into free-FAs. The free-FA is then recombined into the baby's own cell membranes in organs where it is needed, such as the brain and eyes. As the free-FA is not water soluble, it is transported by serum albumin in the bloodstream. To maximize assimilation efficiency, free-DHA is the preferred form of nutrient supplement rather than an unrefined powder form of DHA, or even the TAG oil form of DHA. Currently, free-DHA in a purified form is available only in small quantities, and at high cost, from chemical companies such as Sigma Aldrich.

[0009] In contrast to the production of nutritional supplements from micro-algae, bio-diesel technology uses vegetable or micro-algal oil to generate fuel containing high proportions of fatty acid methyl esters (FAMEs) that are not of nutritional value as they produce toxic methanol when metabolized. Similarly processed fatty acid ethyl esters (FAEEs) using ethanol instead of methanol are not as toxic, but still result in the formation of undesirable ethanol when metabolized by human.

[0010] DSM has been cultivating C. cohnii to produce DHA containing TAG oil called Life's DHA^®. The process involves spray drying the biomass and then separating the TAG from biomass via a high-temperature two-phase solvent extraction process. Chinese competitors use the same spray drying and solvent extraction method, while some do not even extract the oil and sell the oil-containing dried biomass directly. The putative DHA-synthase that is contained in the algal biomass products, most likely, has been denatured through high temperature processing and the application of organic solvents. The so-called DHA rich feed derived from the extracted residual biomass may no longer contain active DHA synthase. As a result, the biomass products sold by DSM and its Chinese competitors are not marketed as synthase-enriched biomass.

SUMMARY OF THE INVENTION

[0011] The present invention is a method of free DHA fatty acid separation and purification from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipids (triglycerides) and polar lipids (phospholipids) are separated without trans-esterifi cation. A mixture is formed that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water. That mix is frozen to at least lower than 0 degrees C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid. The liquid DHA fatty acid may then be separated.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is directed to an enzymatic process to maximize the amount of free-DHA extracted from biomass oil. The invention processes algal biomass from C. cohnii and other algal species (e.g., Skizochytrium sp., Pavlova sp., T. Isochrysis, etc.) that are known to be rich in DHA using the growth system described herein. The process provides the separation of free fatty acid from the cell structure by the use of lipase hydrolysis process so that all oils (triglycerides) and polar lipids (phospholipids) are separated without using saponification nor trans- esterification method. After the lipase hydrolysis is completed or almost completed, the entire mix of lysed biomass that consists protein, polysaccharide, lipase protein, freed fatty acids, and water is then frozen to at least lower than 0°C but not as cold as DHA's freezing temperature of - 44°C so that every material other than free fatty acid DHA become frozen solid, and thereby separates the remaining liquid DHA fatty acid.

[0013] Growth conditions suitable for C cohnii is shown as an example:

• Growth Temperature: 25^°C

• Growth Mode: semi -continuous nutrient addition/carbon only addition/harvest cycles to generate sufficient biomass for testing.

• Organic Nutrient Media: Glucose and organic N source (5: 1 w:w ratio), lx addition to start, and once growing exponentially adding another lx to increase biomass.

• Bubbling of Growth System to ensure adequate oxygen diffusion to the cells, which is hampered by both the organic-rich media and the production of extracellular polysaccharides at high cell densities (de Swaaf et al. 2003).

• C. cohnii is scaled up in volume in a 20L carboy used to inoculate the larger bioreactor.

Growth is monitored in each bioreactor. As the culture reaches exponential growth, an additional lx nutrient media may be added to yield a higher biomass/volume, and once growth ceases, an additional addition of glucose is only made to ensure nitrogen limitation and 'fattening' of the cells to maximum lipid content. Once reaching stationary phase induced by nitrogen limitation, cells are harvested.

[0014] In a first step of the process of the present invention, C. cohnii is harvested from a bioreactor by continuous centrifugation. Roughly three-quarters of each growth volume is harvested so that the remaining fraction serves as the inoculum for the next growth/harvest cycle upon refilling with filtered and pasteurized seawater.

[0015] The concentrated biomass is then further centrifuged to increase solids content to >10%. At this point, some material is used as 'wet biomass' for testing with no further manipulation. Additional material is lyophilized to produce intact dried powder for testing, presumably containing active DHA synthase. The lyophilization process maintains the sample at near freezing temperature and maintains the integrity of enzymes in the biomass.

[0016] The total lipids of C. cohnii are extracted from biomass using the approach of Bligh and Dyer 1959, modified for the microalga Isochrysis galbana (Devos et al. 2006). The biomass is ground and then sonicated in a chloroform: methanol: water mix (1 :2:0.2 v/v) from which a chloroform phase containing the lipids is separated. The chloroform-lipid extract is passed through a silica gel cartridge from which the three main lipid classes are separated using the following solvents: chloroform for neutral lipids (primarily TAGs), a chloroform: methanol mix (5: 1 v/v) for glycolipids and methanol for phospholipids.

[0017] Each of the fractions is then dried down under a stream of nitrogen prior to methylation of the fatty acids. An internal standard (C19:0) is added to each sample prior to further processing. For each lipid fraction, fatty acid saponification is carried out in methanolic NaOH (0.5 M) at 80°C for 15 min. Methylation of the free fatty acids is achieved using 12% methanolic boron trifluoride at 80°C for 20 mins. The fatty acid methyl esters (FAME) are then extracted twice with isooctane, washed with water and dried using sodium sulfate.

[0018] The fatty acid profile of each lipid fraction is then analyzed as FAMEs by gas chromatography linked to a mass spectrometer (Shimadzu GC/MS QP2010 Ultra), equipped with auto sampler, and a BPX70 column (SGE Analytical Science) using an injector at 250 °C, column oven initially at 90 °C and 1.17 ml min^"1 helium carrier flow rate. Quantification is by reference to the response of the internal standard and calibrations using the Supelco 37-component FAME mix. Fatty acid profile resulted in this process shows 40-45% of total FA from the sampled C Cohnii is of DHA.

[0019] Microbial sources of lipase are generally the most applicable to technological applications of this type because of the great variety of catalytic activities available, the high yields possible, regular supply due to absence of seasonal fluctuations and rapid growth of microorganisms on inexpensive media. Microbial enzymes are also more stable than their corresponding plant and animal enzymes and their production is more convenient and safe. These are commercially available from a variety of suppliers and may exist in free form or bound to a substrate (immobilized) that allows their recovery following the reaction process.

[0020] Lipases exhibit positional-, substrate- and stereo-specificity toward their substrates (reviewed in Kapoor and Gupta 2012). Lipases are either 'non-specific' and target all hydroxyl groups producing mainly free fatty acids; or are ' 1,3-specific' targeting specific bonds, generally positions 1 and 3 (sn-1,3) in TGs, producing free fatty acids and mono- and diacylglycerides. In addition, lipases may be 'fatty acid selective' . Several lipase formulations discriminate against Q3-PUFAs, releasing saturated and monounsaturated fatty acids to a greater degree than Ω3- PUFAs (Ustun et al. 1997). Others distinguish between Q3-PUFAs, for instance Thermomyces lanuginosus lipase hydrolyzes EPA more effectively than DHA from TGs in anchovy oil (Akanbi et al. 2013). However, the selectivity of specific lipase enzymes may be dependent, to a large extent, on the particular reaction conditions and testing can be useful for specific characterizations.

[0021] Candidate lipases tested for their capability to hydrolyze TGs and PLs from C. cohnii are chosen on the basis of previous evidence of relevant reaction capabilities and potentially include the following commercially available products:

Product Origin Manufacturer Significance

1. Lipase F Rhizopus niveus Amano Enzyme High DHA enrichment from Isochrysis PL¹

2. Lipase M Mucor javanicus Amano Enzyme High DHA enrichment from Isochrysis PL¹

3. Lipozyme TL-IM Thermomyces lanuginose Novozymes Effective for DHA from anchovy oil²

4. Lipase PS C-l^* Burkholderia cepacia Amano Enzyme Most effective for DHA from sardine oil³

5. Lipase CR^* Candida rugosa Fluka Switzerland Most effective for DHA from salmon oil⁴ References 1. Devos et al 2006; 2. Akanbi et al. 2013; Gamez-Meza et al. 2003; 4. Kahveci et al. 2010.

^* denotes immobilized enzyme.

[0022] Specific quantities (units of enzyme (U), generally the amount that liberates 1 μπιοΐ min^"1 of fatty acid at 37°C) of each lipase product are initially prepared in phosphate buffer of optimum pH and concentrations, as specified by the manufacturers. Enzyme reactions are carried out in glass vessels at set temperature; again, initially as advised by the product manufacturers. The reactions are started by the addition of substrate and the reaction rates determined by subsampling from the reaction mixture.

[0023] Reaction mixture subsamples collected over the course of the reactions (e.g. at 2 hour intervals over a 12-hour total reaction time) are used to determine the production rate of DHA from each of the substrates, for each of the lipase products.

[0024] The quantities of fatty acids (including DHA) released by the enzymes are determined using the approach of Williams et al. (1995). The technique uses the fact that methanolic-NaOH methylates only fatty acids that are esterified to the glycerol backbone and converts free fatty acid to aqueously soluble sodium salts; in contrast, methanolic-HCl methylates both esterified and free fatty acids. After lipase action the difference in fatty acid composition of the two meth- ylation reactions is a quantitative measure of the fatty acid released by the enzyme(s). Complex media are the norm naturally in either the digestive systems of higher animals or when lipases are used for extracellular digestion of natural substrates by, for instance, fungi, from whom many commercially available lipid products are produced. Lipases are most effective in biphasic media in which interfacial activation of the enzyme molecules is enabled.

[0025] The major focus of research on commercial micro-algal lipid production to date has been on supplying the biodiesel industry with TGs, that through transesterification reactions produce the monoalkyl esters that comprise biodiesel (reviewed in Chisti 2007). Several studies have investigated the potential to use enzyme-based processes to carry out the transesterification of micro-algal-derived lipids to biodiesel. For instance, an immobilized lipase originating from Burkholderia sp. C20, was used successfully for the transesterification of oil from the microalga Chlorella vulgaris (Tran et al. 2012). In close to commercial scale application, immobilized lipase from Candidia sp. 99-125 was used to catalyze the transesterification of oil extracted from heterotrophic Chlorella protothecoides (Li et al. 2007). Pertinent to the present invention is the finding that transesterification by the Burkholderia sp. C20 lipase was more efficient in disrupted micro-algal biomass containing 71 % water, than in extracted micro-algal oil (Tran et al. 2013). The present invention provides for free fatty acids production using lipase hydrolysis that is equally effective in unprocessed wet micro-algal biomass; a biotechnological advance that has not been reported previously.

[0026] The present invention uses a lipase-based approach similar to that which has been applied in biodiesel-research to the rapidly expanding demand for PUFAs as nutritional supplements. However, in contrast to the transesterification reactions required for biodiesel production, few studies have reported the use of lipases to hydrolyze micro-algal lipids to their free-fatty acid form, especially to free-DHA (Devos et al. 2006). Although, a similar approach has been investigated for extracted fish and other oils (e.g. Gamez-Meza et al. 2003, Akanbi et al. 2013). Several studies have made use of the selectivity of certain lipases against the ester bonds between glycerol DHA and EPA, in order to enrich the Ω-3 PUFA content of extracted TAGs from fish oil (e.g. Shimada et al. 1997).

[0027] Lipase, water and the biomass mix can be homogenized through a high pressure homoge- nizer such as GEA Niro-Soavi homogenizer. The output of such homogenization is lysed cells and lipase as part of water mix slurry. Once lipase enzyme cleaves TAG bonds and PL bonds, and frees up the fatty acids inside the cells, the target DHA can be extracted from the mix. The biomass mix slurry is comprised of water, protein, polysaccharides, and remnants of PL and TAG such as phosphine, and glycerin. Components of the mix can be separated in a cold extraction process.

[0028] Phase separation is a common separation method, whereby a target substance is of a certain phase such as liquid or gas, while the rest of the material in the mix is of a different phase such as solid or liquid, respectively. Solvent can be used to separate the target substance from mix components that are not soluble in the solvent. In the case of lipase freed DHA, the target free DHA substance has a significantly lower melting temperature— -44°C— compared to the melting temperatures of the other substances in the above-mentioned biomass mix slurry. In the case of C. Cohnii, most of the remainder FA component is solid saturated FA at room temperature except for a very small portion that is EPA (Eicosapentaenoic acid), another Ω3 FA, which is less than 1% of the total FA component, and oleic acid, which is a monounsaturated fatty acid that has a melting temperature of 13°C. Saturated FAs have relatively high temperature melting point and, at the water freezing temperature of 0°C, all of the otherwise liquid material in the biomass mix slurry becomes solid except the free DHA and small amount of EPA in the case of C. cohnii. Further with respect to this invention, the melting temperature of EPA is -54°C, which is well below the water freezing temperature.

[0029] The liquid DHA may be separated from solid mush by gravitational force to let the liquid settle through a filter material. Frozen crystals in the biomass mush under freezing temperature will act as a tortuous path against the liquid DHA inside and, depending on the viscosity of liquid DHA at or below freezing temperature, it may take a long time to achieve separation by that method. Therefore, to accelerate the liquid/solid separation, if desired, centrifugal force may be employed in a manner well known to those skilled in the art, such as by rapid rotation with a centrifuge of a container including the combination. Mechanically compressing the frozen mush to squeeze the liquid out through filter means is another option.

[0030] The organic solvent such as ethyl acetate (EA) may be used to facilitate the cold extraction. EA's melting point of -83.6 °C is also much lower than the water freezing point. EA is slightly soluble in water, and DHA is soluble in EA. When the water changes its phase, the solute in EA and the solvent DHA are the only remaining liquid, and easily separated by mechanical ways such as filtration, centrifugation, or just simple gravitational separation. [0031] The present invention combines the steps described above of lipase hydrolysis process and freeze separation process. The invention further provides for the option of carrying out these two primary steps in a single bioreactor made of durable but flexible material such as plastic, rubber. The bioreactor material must withstand freezing temperatures required to turn the components of the mix other than the free DHA. The bioreactor holds the biomass, lipase and water mix. The flexibility of the bioreactor container allows for soft agitation of the mix to promote the lipase hydrolysis reaction. Upon complete cleaving of fatty acid that is determined by the above stated experiments, the flexible bioreactor is placed in a chilling apparatus sufficient to lower and control the environment of the bioreactor, such as a commercial freezer. The bioreactor can be opened at a top to load it with material, and with a filter end including a filter material at a bottom of the bioreactor. A receptacle container for securing the desired target material can be attached to the filtered end. The filtered end receptacle is placed at the bottom of the bioreactor. In the event centrifugal force is used to separate mixture components, the filtered end receptacle container is hung and supported in the centrifuge separator, which may include a centrifuge spinner. The filter material at the bioreactor bottom is a microporous membrane type filter but not limited thereto, and it may be further supported by rigid larger pore grids. The centrifuge separation process is done under the water freezing temperature.

[0032] The filled receptacle container is used to collect free DHA. In the case of C.Cohnii there may be about 1% of EPA in the mix, but no saturated or monounsaturated fatty acid will be present. The remaining frozen mush can be reheated to about 10°C, and the centrifugal separator may be used to separate liquid effluent and solid. The aforementioned bioreactor may be used to just drain the water from the bottom of the bioreactor through the filter. Liquid in this case will be water, or water and dissolved lipase mix. All the remaining fatty acid will be still in the solid phase. The solid may further separated into FAs and other solid such as protein and polysaccharides.

[0033] Various methods can be used to separate the fatty acids. Solvent exchange methods, or fractionating distillation methods are well known. Supercritical C0₂ is an excellent solvent for free fatty acids, though the presence of water prevents this method. Surfactants may dissolve the solid FA into micelles and wash the FA away from the protein and polysaccharides to facilitate the separation. Targeted separation and purification of DHA synthase enzyme may be possible from the residual biomass.

Claims

What is claimed is:

1. A free DHA (Docosahexaenoic Acid) fatty acid separation and purification method from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method.

2. A free DHA fatty acid separation and purification method from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid.

3. A free DHA fatty acid separation and purification method from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid freezes solid, and be separated from the liquid DHA fatty acid by mechanical means.

4. A free DHA fatty acid separation and purification method from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid by mechanical means.

5. A method for extraction and purification of free DHA from Chrypthecodinium cohnii biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that in- cludesbiomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not colder than DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid by mechanical means.

6 A free DHA (Docosahexaenoic Acid) fatty acid separation and purification method from micro-algae biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method.

7. A free DHA fatty acid separation and purification method from micro-algae biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid.

8. A free DHA fatty acid separation and purification method from micro-algae biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid freezes solid, and be separated from the liquid DHA fatty acid by mechanical means.

9. A free DHA fatty acid separation and purification method from micro-algae biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not as cold as DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid by mechanical means.

10. A method for extraction and purification of free DHA from micro-algae biomass that are rich in such fatty acid, whereby free fatty acid is separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not colder than DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid by mechanical means.

11. A method for extraction and purification of free DHA and EPA from micro-algae biomass that are rich in such fatty acid, whereby free omega 3 fatty acids are separated from the cell structure by the use of lipase hydrolysis process so that all lipid (triglyceride) and polar lipid (phospholipid) are separated without using trans-esterification method, then, the entire mix that includes biomass protein, polysaccharide, lipase protein, freed fatty acids, and water is frozen to at least lower than 0 degree C but not colder than DHA's freezing temperature of -44 degree C. so that every material other than DHA free fatty acid is frozen solid, and separate the liquid DHA fatty acid by mechanical means. 12 A method for separation and purification of free DHA and free EPA from micro-algae biomass that are rich in such fatty acids, whereby free fatty acids are separated from all lipid including triglyceride and polar lipid using lipase hydrolysis reaction, then entire mix that includes biomass protein, polysaccharides, lipase protein, freed fatty acids and water and an organic solvent such as Ethyl Acetate is frozen to at least lower than zero degree C. but not colder than DHA and EPA's freezing temperature, so that every material other than DHA and EPA and the solvent is frozen solid and separate the liquid DHA and EPA fatty acids dissolved in the solvent by gravity force or mechanical means, and then raise the temperature of the solvent mix to a boiling temperature of solvent thereby the pure DHA and EPA remain as liquid.