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WO2012088276A2 - Phospholipid production and composition manipulation through media manipulation - Google Patents

Phospholipid production and composition manipulation through media manipulation Download PDF

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Publication number
WO2012088276A2
WO2012088276A2 PCT/US2011/066499 US2011066499W WO2012088276A2 WO 2012088276 A2 WO2012088276 A2 WO 2012088276A2 US 2011066499 W US2011066499 W US 2011066499W WO 2012088276 A2 WO2012088276 A2 WO 2012088276A2
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WIPO (PCT)
Prior art keywords
group
fermentation medium
enzyme
lipids
phospholipids
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Application number
PCT/US2011/066499
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French (fr)
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WO2012088276A3 (en
Inventor
Joseph Miller
John R. Palmer
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J3H, Inc
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Publication of WO2012088276A2 publication Critical patent/WO2012088276A2/en
Publication of WO2012088276A3 publication Critical patent/WO2012088276A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6481—Phosphoglycerides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/007—Other edible oils or fats, e.g. shortenings, cooking oils characterised by ingredients other than fatty acid triglycerides
    • A23D9/013—Other fatty acid esters, e.g. phosphatides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • A23D9/02—Other edible oils or fats, e.g. shortenings, cooking oils characterised by the production or working-up
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/006—Refining fats or fatty oils by extraction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

Definitions

  • the invention relates to phospholipid production via culturing microorganisms, and more specifically to phospholipid production and composition manipulation through chemical, biochemical, and enzymatic additions to the fermentation medium.
  • the phospholipids can comprise saturated or unsaturated omega-3, -6, and/or -9 fatty ackJ side chains.
  • Phospholipids are important components to the human diet, since they deliver more bioavailability of general omega-3 fatty acids, such as EPA and DHA, than glycerides and esters.
  • general omega-3 fatty acids such as EPA and DHA
  • the importance of omega-3 fatty acids to the human diet has been well documented.
  • DHA and EPA have been linked to decreased risk of Alzheimer's disease, decreased risk of certain cancers, and decreased risk of cardiovascular disease, as well as promoting general health in these areas. Further, DHA is used as a supplement to infant formula and prenatal vitamins for improved cognitive development and visual acuity.
  • EPA has been linked to decreases in inflammation and as a treatment for schizophrenia.
  • DHA and EPA primarily originate from marine animal origins or microalgae. Lipids derived from certain marine animals, such as krill, Mysis, and marine roe and milt, deliver more bioavailability of general omega-3s (including DHA and EPA) bonded to phospholipids, however, they are delivered in low levels (less than 35% by weight of total lipids) or low purities (less than 45% phospholipids per gram of oil). Further, EPA and DHA supplied in high concentrations (up to 90% total by weight) are in the form of glycerides or esters, which are not as readily absorbed by the digestive system or cellular structure.
  • Nitrogen starvation during fermentation of marine microorganisms is well known to increase the concentrations of omega-3 fatty acids in the neutral lipid fraction.
  • U.S. Patent No. 5,340,594 discusses nitrogen limitation during fermentation to curb cell growth and to increase omega-3 fatty-acid production. The nitrogen limiting conditions are run for about 6 - 24 hours and the microorganisms are harvested at this time to maximize the amount of omega-3 fatty acids in non-polar lipid form.
  • Phospholipids are becoming increasingly valuable for not only their ability to deliver more bioavailable general omega-3 fatty acids but also for their ability to form unique liposome structures. These liposome structures are valuable in the targeted therapy, biologies, gene therapy, and individualized medicine industries.
  • the current art is not focused on phospholipid production, and especially to methods of enhancing phospholipid production during microorganism fermentation.
  • the art is not focused on creating tailored phospholipids with desired fatty acid and polar group components during microbial oil production. Instead, the art is focused on producing higher concentrations of omega-3 fatty acids in neutral lipid form.
  • Phospholipids are usually treated as a byproduct of neutral lipid production and either land-filled or used as animal feed in the aquacuiture industry because of their high functional (binding) or caloric content. (0006] Therefore, it is desirable to create a method of producing a microbial oil comprising high concentrations of phospholipids, and with specific fatty acid chains and polar groups. Such a method would create higher fractions of phospholipids and would eliminate the need to conduct separate esterfication, transesterfication, or acylation steps, after separating the phospholipids from the microbial oil, to create a phospholipid with desired fatty acid chains and polar groups. It is also desirable to use the phospholipids in manufacturing dietary supplements and liposomes. Such products would be beneficial in the nutraceutical and drug deliver industries.
  • the invention disclosed herein provides a method for culturing a microorganisms, such as microalgae, yeast, bacterial, or fungi, to produce a microbial oil comprising both neutral lipids and polar lipids.
  • the polar lipids can comprise phospholipids. Further, when optimized, the polar lipids can be present at a higher weight percent than the neutral lipids.
  • the method comprises adding to the fermentation medium an additional nitrogen source and optionally whole and/or protein fractions while culturing the microalgae.
  • the additional nitrogen source shifts the microbial oii production towards polar lipids, while the whole and/or protein fractions shift the phospholipid product towards a desired polar group, such as serine.
  • the nitrogen source and protein source can be the same or different component.
  • Additional components such as transferase enzymes, esterase enzymes, and phosphodiesterase enzymes may be added to the fermentation medium to assist in the production of phospholipids with tailored fatty acid and polar groups.
  • the method comprises: (a) culturing a microorganism in a fermentation medium; (b) producing a microbial oil comprising neutral lipids and polar lipids; (c) extracting said microbial oil from said fermentation medium; and (d) separating said polar lipids from said neutral lipids; wherein said fermentation medium comprises an additional nitrogen source and optionally whole proteins or protein fractions.
  • the polar lipids can comprise phospholipids.
  • the microorganism can be microalgae, yeast, bacteria, or fungi.
  • the fermentation medium can also comprise enzymes.
  • a phospholipid composition comprising phospholipids derived from fermenting microorganisms in the presence of an additional nitrogen source and optionally whole proteins or protein fractions.
  • a lipid composition comprising the above phospholipids and a structured lipid.
  • the structured lipid can comprise tailored phospholipids.
  • the structured lipid further comprises a first fatty acid carbon chain derived from a first lipid source, a second fatty acid carbon chain derived from a second lipid source, and a third fatty acid carbon chain derived from a third lipid source.
  • the lipid sources can be plant based, marine animal based, or marine plant based.
  • R 1 -acyl / R 2 -acyl Refers to a carbon chain with the terminal carbon part of the carbonyl group that makes up the acyl.
  • C16 Refers to a carbon chain with 16 carbons.
  • CtBj Refers to a carbon chain with 18 carbons.
  • C20 Refers to a carbon chain with 20 carbons.
  • C22 Refers to a carbon chain with 22 carbons
  • Omecia-3 Fattv Acids A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-3 position; that is, the third bond from the methyl end of the fatty acid, including:
  • DHA Docosahexaenoic acid.
  • ALA a-Linolenic acid
  • Omeaa-6 Fattv Acids A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-6 position; that is, sixth bond counting from the end opposite the carboxyl group of the fatty acid, including:
  • GLA Gamma-linolenic acid
  • DGLA Dihomo-gamma-linolenic acid
  • DPA Docosapentaenoic acid
  • Omeqa-9 Fattv Acids A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-9 position; that is, ninth bond counting from the end opposite the carboxyl group of the fatty acid.
  • a method of producing phospholipids comprises: (a) culturing a microorganism in a fermentation medium; (b) producing a microbial oil comprising neutral lipids and polar lipids; (c) extracting said microbial oil from said fermentation medium; and (d) separating said polar lipids from said neutral lipids; wherein said fermentation medium comprises an additional nitrogen source and optionally whole proteins or protein fractions.
  • the polar lipids can comprise phospholipids and can be present at a higher weight percent than said neutral lipids in said microbial oil.
  • lipid fraction of the microbial oil can range from about 40 wt.% to about 70 wt.% neutral lipids, from about 25 wt.% to about 55 wt.% polar lipids, and the balance lipid fractions.
  • Additional weight percent fractions can include: from about 40 wt.% to about 50 wt.% neutral lipids, from about 45 wt.% to about 55 wt.% polar lipids, and balance lipid fractions; from about 50 wt.% to about 60 wt.% neutral lipids, from about 35 wt.% to about 45 wt.% polar lipids, and balance lipid fractions; and from about 60 wt.% to about 70 wt.% neutral lipids, from about 25 wt.% to about 35 wt.% polar lipids, and balance glycolipids.
  • the weight percent ratio of polar lipids to neutral lipids can range from about 0.36:1 to about 1.2:1.
  • the nitrogen sources aid in shifting production towards the polar lipids.
  • the proteins can be whole or fractions.
  • Example proteins include amino acids, such as, proteinogenic amino acids, peptides, polypeptides, peptones, carnitine, GABA, L-DOPA, hydroxyproline, and selenomethionine, ornithine, homoserine, lanthionine, 2-aminoisobutyric acid, and dehydroalanine.
  • the proteinogenic amino acids can include: alanine, arginine, asparagines, aspartic acid, cysteine, glutamic acid, giutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and salts thereof.
  • the peptides can include: substance P, kassinin, neurokinin A, eledosin, neurokinin B, VIP, PACAP, Peptide PHI, GHRH 1-24, glucagon, secretin, NPY, PYY, APP, PPY, POMC, enkephalin, prodynorphin, calcitonin, amylin AGG01 , BNP, and lactotripeptkjes.
  • the proteins can also be hydrolyzed proteins, protein hydrolysates, protein isolates, and protein concentrates.
  • the proteins can be added at the start of the fermentation or during exponential growth (see example below).
  • the nitrogen source can be either the above protein sources, including the above amino acids, or can be a separate source such as: glutamate, monosodium glutamate, glutamic acid, ammonia, ammonium hydroxide, ammonium carbonate, ammonium chloride, ammonium nitrate, nitrate, urea, tryptone, peptone, casein, hydrolysate, creatine, com steep liquor, and combinations thereof.
  • glutamate monosodium glutamate, glutamic acid, ammonia, ammonium hydroxide, ammonium carbonate, ammonium chloride, ammonium nitrate, nitrate, urea, tryptone, peptone, casein, hydrolysate, creatine, com steep liquor, and combinations thereof.
  • glutamate monosodium glutamate, glutamic acid, ammonia, ammonium hydroxide, ammonium carbonate, ammonium chloride, ammonium nitrate, nitrate, ure
  • Schizochytrium is cultured in an artificial seawater based fermentation medium containing 25 g L of NaCI; 5 g/L gS0 4 .7H 2 0; 1 g/L of KCI; 200 mg/L of CaCI 2 ; 5 g/L of glucose; 5 g/L of glutamate (N-source); 1 g/L of KH 2 P0 4 ; 5 ml of Pll metals; 1 ml of A-vitamin solution; and 1 ml of antibiotics.
  • the Schizocbytrium cells began exponential growth, whereby the cells multiply at an exponential rate.
  • the glutamate nitrogen source can remain constant at 5 g L (via replacement addition) or can be increased by adding additional bubbled nitrogen, ammonia, ammonia slats, glutamate, protein sources from above, and/or any of the above described nitrogen sources to the fermentation medium above what is required to maintain the initial 5 g/L concentration of glutamate.
  • the cells can be harvested after 3 days of culturing and placed in fresh medium with constant or increased levels of nitrogen and/or protein source.
  • the concentration of nitrogen (in glutamate form) after 3 days can range from about 5 g L to about 15 g/L, including about 7 g L, 9 g/L, 11 g L, and 13 g L. The concentration of nitrogen will vary depending on the source.
  • the concentration of nitrogen can remain elevated throughout the culturing (e.g. higher than 5 g/L), however, this will require adjustments to the other fermentation medium ingredients to maintain proper pH.
  • microbial oil production is shifted towards the polar lipid fractions and away from the neutral lipid fractions.
  • the fermentation medium can also contain additional components such as carbon sources and microbial growth factors.
  • Carbon sources can be glucose, corn starch, ground com, potato starch, wheat starch, molasses, grain, and combinations thereof.
  • Microbial growth factors can be yeast, vitamins, com steep liquor, and combinations thereof, though it should ne noted here that these are added for general nutritive purposes and are not added to influence the composition of the resulting polar lipids within the total lipids.
  • the fermentation medium can also comprise specific enzymes to assist in creating a tailored phospholipid with specific fatty acid-acyl side chains (R 1 -acyl and R 2 - acyl terminal positions on the phospholipid) and polar groups.
  • the enzymes can comprise transferase enzymes that promote acylation of the fatty acids or esterase enzymes that promote esterfication of the fatty acids.
  • the transferase enzymes will assist in replacing the R 1 and/or R 2 -acyi side chains on the phospholipids with a desired R and/or R -acyl side chain.
  • the transferase enzyme in combination with C20 and higher fatty acids (in free, ester or glyceride form), will inhibit C18-acyl fatty acid carbon chains aggregation, thereby promoting C20-acyl or higher fatty acid carbon chain aggregation.
  • the esterase enzyme acts in a similar fashion, however, the rearrangement is done via esterfication and not acylation of the R 1 -acyl and R 2 -acyl terminal positions.
  • Example transferase enzymes can include: acyltransferases, glyceronephosphate O-acyltransferase, and lecithin-chloesterol acyltransferase.
  • Example esterase enzymes can include: lipases and phospholipases, including phospholipase A1 , phospholipase A2, and phospholipase B. The enzymes can be added at the beginning of the culturing or during the exponential growth phase. For example, the enzymes can be combined with the nitrogen source and protein source, and added at the outset.
  • the fermentation medium can comprise phosphodiesterase enzymes, such as phospholipase C and phospholipase D, that promote esterfication of the polar group on the phospholipids.
  • phosphodiesterase enzymes such as phospholipase C and phospholipase D
  • the phosphodiesterase enzyme in combination with a serine moiety such as serine salt or phosphatidylserine
  • the specific chemistry associated with phospholipase C and phospholipase D is known to those skilled in the art.
  • the enzymes can be added at the beginning of the culturing or during the exponential growth phase.
  • the enzymes can be combined with the nitrogen source and protein source, and added at the outset.
  • Phaeodactylum microalgae is cultured in an artificial seawater based fermentation medium containing 25 g/L of NaCI; 5 g/L MgS0 .7H 2 0; 1 g/L of KCI; 200 mg/L of CaCi 2 ; 5 g/L of glucose; 5 g/L of glutamate (N-source); 1 g/L of KH 2 P0 4 ; 5 ml of Pll metals; 1 ml of A-vitamin solution; 1 ml of antibiotics; transferase enzymes; and Krill oil comprising C22 carbon chains (the C22 carbon chains can be present in free fatty acid, esters, or glyceride form).
  • Phaeodactylum is known to produce high concentrations of EPA relative to DHA. After 3 days of culturing at 27°C and a pH of about 7, the Phaeodactylum cells began exponential growth, whereby the cells multiply at an exponential rate. Also, the transferase enzymes in combination with the Krill oil, promote acylation at the R 1 and R 2 -acyt side chains (C20-acyls) via aggregation with C22-acyl chains on the phospholipids being produced. At this point, serine amino-acids in conjunction with a phosphodiesterase enzyme are introduced to the fermentation medium.
  • the serine amino-acids act as both the additional nitrogen source and serine moiety to promote the production of phosphatidylserine.
  • the serine amino acids can be added as the protein source and serine moiety with additional glutamate or similar nitrogen containing salt, as the additional nitrogen source.
  • glutamate, similar nitrogen containing salt, or amino-acid can be added as the additional nitrogen source and marine animal lipids (e.g. Atlantic mackerel) as the serine moiety.
  • multiple combinations of added nitrogen, proteins, and serine moieties can be used.
  • the cells can also be harvested after 3 days of culturing and placed in fresh medium, prior to the addition of the nitrogen source, serine moiety, and phosphodiesterase enzyme.
  • the microorganism can be microalgae, yeast, bacteria, or fungi.
  • the microalgae can include species from the genera Thraustochytrium, Schizochytrium, and Crypthecodinium, including Crypthecodinium cohnii (C. cohnii).
  • members of the class Dinophyceae, Bacillariophyceae, Chlorophyceae, Prymnesiophyceae, and Euglenophyceae can produce suitable phospholipids with high concentrations of DMA.
  • the microalgae can include species from the genera Thraustochytrium Schizochytrium, Phaeodactylum, Nannochioropsis, Porphydrium, and Monodus, including Phaeodactylum tricomulum, Porphyridium cruentum, and Monodus subterranous (described in Chemicals from Microalgae. Edited by Zvi Cohen, Taylor & Francis Ltd., 1999, hereby incorporated by reference in its entirety).
  • Additional microalgae that produce DHA and EPA can include Odentella aurita (described in Braud JP, "Simultaneous culture in pilot tanks of the microalgage Chondrus crispus and the microalgage Odentella aurita producing EPA", 1998), Pavolova lutheri (described in Guiheneuf et al., "Effect of UV stress on the fatty acid and lipid class composition in two marine microalgae: Pavlova lutheri and Odentella aurita", Springer Science and Business, 2010), Isochysis galbana (described in Chemicals from Microalaaqe. edited by Zvi Cohen, Talory and Francis Ltd., 1999), Nannochloropsis (described in Chemicals from Microalaaqe). and Porphyridiu cruentum (described in Chemicals from Microalgae).
  • the microalgae can be Chaetoceros calcitrans, Chaaotoceros gracilis, Nitzichia cloesterium, Skeletonema costatum, Thalassiosira pseudonana, Dunafiella tertiolecta, Nannochloris atomus, Chroomonas salina, Nannochloropsis oculata, Tetraselmis chui, Tetraselmis suecica, Pavlova salina; all described at www.fao.org docrep/003/w3732e w3732e07.htm.
  • the above references are hereby incorporated by reference in their entirety.
  • the microbial oil must be isolated and purified from the above marine biomasses. Impurities, such as bacteria, particulates, and extraction chemicals, are almost always present when the microbial oils are extracted. Extraction of the microbial oil, neutral lipids, and polar lipids from the microalgae can be done using known methods, including polar and non-poiar solvent extraction, spray drying, super critical extraction, centrifuge, enzymatic extraction, mechanical press, extrusion, sonication, decanter extraction, and combinations thereof.
  • One method of extracting polar lipids is to spray dry the marine biomass, which will lyse the cells, and then use a non-polar solvent, such as hexane, to remove the fatty acid polar lipid portion, including the phospholipids.
  • a non-polar solvent such as hexane
  • Another method is to pretreat the biomass to deactivate any potential phospholipase, which would otherwise degrade the phospholipids.
  • the neutral and polar lipids are extracted from the biomass using known techniques, including polar and non-polar solvent extraction, spray drying, super critical extraction, centrifuge, enzymatic extraction, mechanical press, extrusion, and decanter extraction.
  • the polar lipids, including phospholipids are then isolated and purified from the total lipid fraction with water wash, acetone, or other solvents that cause separation of the neutral from polar and glycolipids.
  • the phospholipids are then dried using known methods including wiped-film evaporation.
  • Any bacteria present in the mircobiomass or phospholipid can be inactivated using an anti-bacterial agent or by UHT treatment prior to processing.
  • Particulates can be filtered out using various filtration methods, such as centrifuge, filter press, cyclone filtration, gravity decanter, or filter media. Extraction of the solvents can be removed using flash distillation, evaporation, and gravity decanting.
  • the phospholipids can be combined with additional compounds, such as fat- soluble vitamins (e.g. A, D, E, K, T), CoQ10, and Resveratrol by solubilizing with TPGS.
  • additional compounds such as fat- soluble vitamins (e.g. A, D, E, K, T), CoQ10, and Resveratrol by solubilizing with TPGS.
  • the phospholipids can be combined with additional structured lipids to make a lipid composition.
  • the structured lipid can comprise tailored phospholipids and further comprises a first fatty acid carbon chain derived from a first lipid source, a second fatty acid carbon chain derived from a second lipid source, and a third fatty acid carbon chain derived from a third lipid source. Structured lipids and methods for making the same are disclosed U.S. Provisional Application No. 61/333,173, which is herein incorporated by reference in its entirety.
  • liposomes comprising the above disclosed phospholipids and lipid compositions. Such liposomes are useful in drug delivery and targeted therapeutics. Liposomes and methods for making the same are disclosed in U.S. Provisional Application No. 61/420,962.

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Abstract

Disclosed is a method for producing phospholipids by culturing microorganisms in a fermentation medium that has been manipulated through chemical, biochemical, and enzymatic additions. Chemical additions can include nitrogen addition to aid in shifting microbial oil production towards phospholipids. Biochemical additions include specific amino acids and enzyme additions include specific transferase enzymes esterase enzymes, and phosphodiesterase enzymes. The microorganisms can be microalgae, yeast, bacteria, or fungi.

Description

PHOSPHOLIPID PRODUCTION AND COMPOSITION MANIPULATION THROUGH
MEDIA MANIPULATION
[0001] The invention relates to phospholipid production via culturing microorganisms, and more specifically to phospholipid production and composition manipulation through chemical, biochemical, and enzymatic additions to the fermentation medium. The phospholipids can comprise saturated or unsaturated omega-3, -6, and/or -9 fatty ackJ side chains.
BACKGROUND OF THE TECHNOLOGY
[0002] Phospholipids are important components to the human diet, since they deliver more bioavailability of general omega-3 fatty acids, such as EPA and DHA, than glycerides and esters. The importance of omega-3 fatty acids to the human diet has been well documented. DHA and EPA have been linked to decreased risk of Alzheimer's disease, decreased risk of certain cancers, and decreased risk of cardiovascular disease, as well as promoting general health in these areas. Further, DHA is used as a supplement to infant formula and prenatal vitamins for improved cognitive development and visual acuity. EPA has been linked to decreases in inflammation and as a treatment for schizophrenia.
[0003] Currently, DHA and EPA primarily originate from marine animal origins or microalgae. Lipids derived from certain marine animals, such as krill, Mysis, and marine roe and milt, deliver more bioavailability of general omega-3s (including DHA and EPA) bonded to phospholipids, however, they are delivered in low levels (less than 35% by weight of total lipids) or low purities (less than 45% phospholipids per gram of oil). Further, EPA and DHA supplied in high concentrations (up to 90% total by weight) are in the form of glycerides or esters, which are not as readily absorbed by the digestive system or cellular structure.
10004] Nitrogen starvation during fermentation of marine microorganisms is well known to increase the concentrations of omega-3 fatty acids in the neutral lipid fraction. For example, U.S. Patent No. 5,340,594 discusses nitrogen limitation during fermentation to curb cell growth and to increase omega-3 fatty-acid production. The nitrogen limiting conditions are run for about 6 - 24 hours and the microorganisms are harvested at this time to maximize the amount of omega-3 fatty acids in non-polar lipid form.
SUMMARY OF THE INVENTION
[0005] Phospholipids are becoming increasingly valuable for not only their ability to deliver more bioavailable general omega-3 fatty acids but also for their ability to form unique liposome structures. These liposome structures are valuable in the targeted therapy, biologies, gene therapy, and individualized medicine industries. Unfortunately, the current art is not focused on phospholipid production, and especially to methods of enhancing phospholipid production during microorganism fermentation. Moreover, the art is not focused on creating tailored phospholipids with desired fatty acid and polar group components during microbial oil production. Instead, the art is focused on producing higher concentrations of omega-3 fatty acids in neutral lipid form. Phospholipids are usually treated as a byproduct of neutral lipid production and either land-filled or used as animal feed in the aquacuiture industry because of their high functional (binding) or caloric content. (0006] Therefore, it is desirable to create a method of producing a microbial oil comprising high concentrations of phospholipids, and with specific fatty acid chains and polar groups. Such a method would create higher fractions of phospholipids and would eliminate the need to conduct separate esterfication, transesterfication, or acylation steps, after separating the phospholipids from the microbial oil, to create a phospholipid with desired fatty acid chains and polar groups. It is also desirable to use the phospholipids in manufacturing dietary supplements and liposomes. Such products would be beneficial in the nutraceutical and drug deliver industries.
[0007] The invention disclosed herein provides a method for culturing a microorganisms, such as microalgae, yeast, bacterial, or fungi, to produce a microbial oil comprising both neutral lipids and polar lipids. The polar lipids can comprise phospholipids. Further, when optimized, the polar lipids can be present at a higher weight percent than the neutral lipids. The method comprises adding to the fermentation medium an additional nitrogen source and optionally whole and/or protein fractions while culturing the microalgae. Surprisingly, it has been found that the additional nitrogen source, whether with or without a protein source, shifts the microbial oii production towards polar lipids, while the whole and/or protein fractions shift the phospholipid product towards a desired polar group, such as serine. Note that the nitrogen source and protein source can be the same or different component. Additional components, such as transferase enzymes, esterase enzymes, and phosphodiesterase enzymes may be added to the fermentation medium to assist in the production of phospholipids with tailored fatty acid and polar groups. [0008] In one aspect, a method of producing phospholipids is disclosed. The method comprises: (a) culturing a microorganism in a fermentation medium; (b) producing a microbial oil comprising neutral lipids and polar lipids; (c) extracting said microbial oil from said fermentation medium; and (d) separating said polar lipids from said neutral lipids; wherein said fermentation medium comprises an additional nitrogen source and optionally whole proteins or protein fractions. The polar lipids can comprise phospholipids. The microorganism can be microalgae, yeast, bacteria, or fungi. The fermentation medium can also comprise enzymes.
[0009) In another aspect, a phospholipid composition is disclosed comprising phospholipids derived from fermenting microorganisms in the presence of an additional nitrogen source and optionally whole proteins or protein fractions.
[0010] In a further aspect, a lipid composition is disclosed comprising the above phospholipids and a structured lipid. The structured lipid can comprise tailored phospholipids. The structured lipid further comprises a first fatty acid carbon chain derived from a first lipid source, a second fatty acid carbon chain derived from a second lipid source, and a third fatty acid carbon chain derived from a third lipid source. The lipid sources can be plant based, marine animal based, or marine plant based.
DEFINITIONS
[0011J While mostly familiar to those versed in the art, the following definitions are provided in the interest of clarity.
[0012] R1-acyl / R2-acyl: Refers to a carbon chain with the terminal carbon part of the carbonyl group that makes up the acyl. [0013] C16: Refers to a carbon chain with 16 carbons.
[0014] CtBj Refers to a carbon chain with 18 carbons.
[0015] C20: Refers to a carbon chain with 20 carbons.
[0016] C22: Refers to a carbon chain with 22 carbons
[0017] Omecia-3 Fattv Acids: A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-3 position; that is, the third bond from the methyl end of the fatty acid, including:
[0018] DHA: Docosahexaenoic acid.
[0019] EPA: Eicosapentaenoic acid.
(0020] SPA: Stearidonic acid
[0021] ALA: a-Linolenic acid
[0022] ETA: Eicosatetraenoic acid
(0023] DPA: Docosapentaenoic acid
[0024] Omeaa-6 Fattv Acids: A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-6 position; that is, sixth bond counting from the end opposite the carboxyl group of the fatty acid, including:
[0025] GLA: Gamma-linolenic acid
[0026] DGLA: Dihomo-gamma-linolenic acid
[0027] CLA: Calendic acid
[0028] DPA: Docosapentaenoic acid
10029] Omeqa-9 Fattv Acids: A family of unsaturated fatty acids that have in common a final carbon-carbon double bond in the n-9 position; that is, ninth bond counting from the end opposite the carboxyl group of the fatty acid. DETAILED DESCRIPTION OF THE INVENTION
[0030] In one aspect, a method of producing phospholipids is disclosed. The method comprises: (a) culturing a microorganism in a fermentation medium; (b) producing a microbial oil comprising neutral lipids and polar lipids; (c) extracting said microbial oil from said fermentation medium; and (d) separating said polar lipids from said neutral lipids; wherein said fermentation medium comprises an additional nitrogen source and optionally whole proteins or protein fractions. The polar lipids can comprise phospholipids and can be present at a higher weight percent than said neutral lipids in said microbial oil.
[0031] Known microorganism fermentation methods result in a microbial oil with approximately 80 wt. % or higher neutral lipids, about 15 wt. % or less polar lipids, and remainder glycolipids and other lipid fractions. However, the disclosed methods can result in a production shift towards polar lipids and away from neutral lipids. For example, the lipid fraction of the microbial oil can range from about 40 wt.% to about 70 wt.% neutral lipids, from about 25 wt.% to about 55 wt.% polar lipids, and the balance lipid fractions. Additional weight percent fractions can include: from about 40 wt.% to about 50 wt.% neutral lipids, from about 45 wt.% to about 55 wt.% polar lipids, and balance lipid fractions; from about 50 wt.% to about 60 wt.% neutral lipids, from about 35 wt.% to about 45 wt.% polar lipids, and balance lipid fractions; and from about 60 wt.% to about 70 wt.% neutral lipids, from about 25 wt.% to about 35 wt.% polar lipids, and balance glycolipids. The weight percent ratio of polar lipids to neutral lipids can range from about 0.36:1 to about 1.2:1. The nitrogen sources aid in shifting production towards the polar lipids. [0032| The proteins can be whole or fractions. Example proteins include amino acids, such as, proteinogenic amino acids, peptides, polypeptides, peptones, carnitine, GABA, L-DOPA, hydroxyproline, and selenomethionine, ornithine, homoserine, lanthionine, 2-aminoisobutyric acid, and dehydroalanine. The proteinogenic amino acids can include: alanine, arginine, asparagines, aspartic acid, cysteine, glutamic acid, giutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and salts thereof. The peptides can include: substance P, kassinin, neurokinin A, eledosin, neurokinin B, VIP, PACAP, Peptide PHI, GHRH 1-24, glucagon, secretin, NPY, PYY, APP, PPY, POMC, enkephalin, prodynorphin, calcitonin, amylin AGG01 , BNP, and lactotripeptkjes.
(00331 The proteins can also be hydrolyzed proteins, protein hydrolysates, protein isolates, and protein concentrates. The proteins can be added at the start of the fermentation or during exponential growth (see example below).
[0034] The nitrogen source can be either the above protein sources, including the above amino acids, or can be a separate source such as: glutamate, monosodium glutamate, glutamic acid, ammonia, ammonium hydroxide, ammonium carbonate, ammonium chloride, ammonium nitrate, nitrate, urea, tryptone, peptone, casein, hydrolysate, creatine, com steep liquor, and combinations thereof. During fermentation, the nitrogen source is kept constant or increased, which is opposite known techniques of nitrogen starvation to increase neutral lipid production.
[0035] For example, Schizochytrium is cultured in an artificial seawater based fermentation medium containing 25 g L of NaCI; 5 g/L gS04.7H20; 1 g/L of KCI; 200 mg/L of CaCI2; 5 g/L of glucose; 5 g/L of glutamate (N-source); 1 g/L of KH2P04; 5 ml of Pll metals; 1 ml of A-vitamin solution; and 1 ml of antibiotics. After 3 days of culturing at 27°C and a pH of about 7, the Schizocbytrium cells began exponential growth, whereby the cells multiply at an exponential rate. At this point, the glutamate nitrogen source can remain constant at 5 g L (via replacement addition) or can be increased by adding additional bubbled nitrogen, ammonia, ammonia slats, glutamate, protein sources from above, and/or any of the above described nitrogen sources to the fermentation medium above what is required to maintain the initial 5 g/L concentration of glutamate. Alternatively, the cells can be harvested after 3 days of culturing and placed in fresh medium with constant or increased levels of nitrogen and/or protein source. The concentration of nitrogen (in glutamate form) after 3 days can range from about 5 g L to about 15 g/L, including about 7 g L, 9 g/L, 11 g L, and 13 g L. The concentration of nitrogen will vary depending on the source. Note that the concentration of nitrogen can remain elevated throughout the culturing (e.g. higher than 5 g/L), however, this will require adjustments to the other fermentation medium ingredients to maintain proper pH. By maintaining or increasing the nitrogen concentration in the fermentation medium through nitrogen addition, microbial oil production is shifted towards the polar lipid fractions and away from the neutral lipid fractions.
[0036] The fermentation medium can also contain additional components such as carbon sources and microbial growth factors. Carbon sources can be glucose, corn starch, ground com, potato starch, wheat starch, molasses, grain, and combinations thereof. Microbial growth factors can be yeast, vitamins, com steep liquor, and combinations thereof, though it should ne noted here that these are added for general nutritive purposes and are not added to influence the composition of the resulting polar lipids within the total lipids.
[0037] The fermentation medium can also comprise specific enzymes to assist in creating a tailored phospholipid with specific fatty acid-acyl side chains (R1-acyl and R2- acyl terminal positions on the phospholipid) and polar groups. When a specific fatty acid side chain is desired, the enzymes can comprise transferase enzymes that promote acylation of the fatty acids or esterase enzymes that promote esterfication of the fatty acids. The transferase enzymes will assist in replacing the R1 and/or R2-acyi side chains on the phospholipids with a desired R and/or R -acyl side chain. For example, if a C20 or higher side chain phospholipid is desired and C18 or lower fatty acids are currently present on the R1 or R2-acyl terminal chains, the transferase enzyme in combination with C20 and higher fatty acids (in free, ester or glyceride form), will inhibit C18-acyl fatty acid carbon chains aggregation, thereby promoting C20-acyl or higher fatty acid carbon chain aggregation. The esterase enzyme acts in a similar fashion, however, the rearrangement is done via esterfication and not acylation of the R1-acyl and R2-acyl terminal positions. [Example transferase enzymes can include: acyltransferases, glyceronephosphate O-acyltransferase, and lecithin-chloesterol acyltransferase. Example esterase enzymes can include: lipases and phospholipases, including phospholipase A1 , phospholipase A2, and phospholipase B. The enzymes can be added at the beginning of the culturing or during the exponential growth phase. For example, the enzymes can be combined with the nitrogen source and protein source, and added at the outset. [0038] Further, the fermentation medium can comprise phosphodiesterase enzymes, such as phospholipase C and phospholipase D, that promote esterfication of the polar group on the phospholipids. For example, if a serine polar group is desired and a choline polar group is current present on the phospholipids, the phosphodiesterase enzyme in combination with a serine moiety (such as serine salt or phosphatidylserine), will replace the choline polar group with the serine polar group on the phospholipid. The specific chemistry associated with phospholipase C and phospholipase D is known to those skilled in the art. The enzymes can be added at the beginning of the culturing or during the exponential growth phase. For example, the enzymes can be combined with the nitrogen source and protein source, and added at the outset.
[0039] An example of enzyme addition is as follows: Phaeodactylum microalgae is cultured in an artificial seawater based fermentation medium containing 25 g/L of NaCI; 5 g/L MgS0 .7H20; 1 g/L of KCI; 200 mg/L of CaCi2; 5 g/L of glucose; 5 g/L of glutamate (N-source); 1 g/L of KH2P04; 5 ml of Pll metals; 1 ml of A-vitamin solution; 1 ml of antibiotics; transferase enzymes; and Krill oil comprising C22 carbon chains (the C22 carbon chains can be present in free fatty acid, esters, or glyceride form). Phaeodactylum is known to produce high concentrations of EPA relative to DHA. After 3 days of culturing at 27°C and a pH of about 7, the Phaeodactylum cells began exponential growth, whereby the cells multiply at an exponential rate. Also, the transferase enzymes in combination with the Krill oil, promote acylation at the R1 and R2-acyt side chains (C20-acyls) via aggregation with C22-acyl chains on the phospholipids being produced. At this point, serine amino-acids in conjunction with a phosphodiesterase enzyme are introduced to the fermentation medium. The serine amino-acids act as both the additional nitrogen source and serine moiety to promote the production of phosphatidylserine. Further, the serine amino acids can be added as the protein source and serine moiety with additional glutamate or similar nitrogen containing salt, as the additional nitrogen source. Alternatively, glutamate, similar nitrogen containing salt, or amino-acid can be added as the additional nitrogen source and marine animal lipids (e.g. Atlantic mackerel) as the serine moiety. Thus, multiple combinations of added nitrogen, proteins, and serine moieties can be used. The cells can also be harvested after 3 days of culturing and placed in fresh medium, prior to the addition of the nitrogen source, serine moiety, and phosphodiesterase enzyme.
[0040] The microorganism can be microalgae, yeast, bacteria, or fungi. The microalgae can include species from the genera Thraustochytrium, Schizochytrium, and Crypthecodinium, including Crypthecodinium cohnii (C. cohnii). Also, members of the class Dinophyceae, Bacillariophyceae, Chlorophyceae, Prymnesiophyceae, and Euglenophyceae can produce suitable phospholipids with high concentrations of DMA. For phospholipids containing EPA, the microalgae can include species from the genera Thraustochytrium Schizochytrium, Phaeodactylum, Nannochioropsis, Porphydrium, and Monodus, including Phaeodactylum tricomulum, Porphyridium cruentum, and Monodus subterranous (described in Chemicals from Microalgae. Edited by Zvi Cohen, Taylor & Francis Ltd., 1999, hereby incorporated by reference in its entirety).
[0041] Additional microalgae that produce DHA and EPA can include Odentella aurita (described in Braud JP, "Simultaneous culture in pilot tanks of the microalgage Chondrus crispus and the microalgage Odentella aurita producing EPA", 1998), Pavolova lutheri (described in Guiheneuf et al., "Effect of UV stress on the fatty acid and lipid class composition in two marine microalgae: Pavlova lutheri and Odentella aurita", Springer Science and Business, 2010), Isochysis galbana (described in Chemicals from Microalaaqe. edited by Zvi Cohen, Talory and Francis Ltd., 1999), Nannochloropsis (described in Chemicals from Microalaaqe). and Porphyridiu cruentum (described in Chemicals from Microalgae).
[0042] Further, the microalgae can be Chaetoceros calcitrans, Chaaotoceros gracilis, Nitzichia cloesterium, Skeletonema costatum, Thalassiosira pseudonana, Dunafiella tertiolecta, Nannochloris atomus, Chroomonas salina, Nannochloropsis oculata, Tetraselmis chui, Tetraselmis suecica, Pavlova salina; all described at www.fao.org docrep/003/w3732e w3732e07.htm. The above references are hereby incorporated by reference in their entirety.
[0043| Cultivation techniques of the above microalgae are well known. For example, Thraustochytrium and Schizochytrium have been well documented in U.S. Patent No. 5,340,594, hereby incorporated by reference in its entirety. Cultivation of C. cohnii has been well documented in U.S. Patent Nos. 5,397,591 and 5,492,538 and Japanese Patent Publication (to Kokai) No. 1-199588 (1989), all hereby incorporated by reference in their entirety.
[0044] The microbial oil must be isolated and purified from the above marine biomasses. Impurities, such as bacteria, particulates, and extraction chemicals, are almost always present when the microbial oils are extracted. Extraction of the microbial oil, neutral lipids, and polar lipids from the microalgae can be done using known methods, including polar and non-poiar solvent extraction, spray drying, super critical extraction, centrifuge, enzymatic extraction, mechanical press, extrusion, sonication, decanter extraction, and combinations thereof.
[0045] One method of extracting polar lipids is to spray dry the marine biomass, which will lyse the cells, and then use a non-polar solvent, such as hexane, to remove the fatty acid polar lipid portion, including the phospholipids. Such process, and other suitable processes, is described in detail in U.S. Patent No. 6,372,460, herein incorporated by reference in its entirety. Another method is to pretreat the biomass to deactivate any potential phospholipase, which would otherwise degrade the phospholipids. The neutral and polar lipids are extracted from the biomass using known techniques, including polar and non-polar solvent extraction, spray drying, super critical extraction, centrifuge, enzymatic extraction, mechanical press, extrusion, and decanter extraction. The polar lipids, including phospholipids, are then isolated and purified from the total lipid fraction with water wash, acetone, or other solvents that cause separation of the neutral from polar and glycolipids. The phospholipids are then dried using known methods including wiped-film evaporation.
[0046) Any bacteria present in the mircobiomass or phospholipid can be inactivated using an anti-bacterial agent or by UHT treatment prior to processing. Particulates can be filtered out using various filtration methods, such as centrifuge, filter press, cyclone filtration, gravity decanter, or filter media. Extraction of the solvents can be removed using flash distillation, evaporation, and gravity decanting.
[0047] The phospholipids can be combined with additional compounds, such as fat- soluble vitamins (e.g. A, D, E, K, T), CoQ10, and Resveratrol by solubilizing with TPGS. [0048] Further, the phospholipids can be combined with additional structured lipids to make a lipid composition. The structured lipid can comprise tailored phospholipids and further comprises a first fatty acid carbon chain derived from a first lipid source, a second fatty acid carbon chain derived from a second lipid source, and a third fatty acid carbon chain derived from a third lipid source. Structured lipids and methods for making the same are disclosed U.S. Provisional Application No. 61/333,173, which is herein incorporated by reference in its entirety.
[0049] Also disclosed are liposomes comprising the above disclosed phospholipids and lipid compositions. Such liposomes are useful in drug delivery and targeted therapeutics. Liposomes and methods for making the same are disclosed in U.S. Provisional Application No. 61/420,962.

Claims

What is claimed is.
1. A method of producing phospholipids comprising:
(a) culturing a microorganisms a fermentation medium;
(b) producing a microbial oil comprising neutral lipids and polar lipids;
(c) extracting said microbial oil from said fermentation medium; and
(d) separating said polar lipids from said neutral lipids; wherein said fermentation medium comprises an additional nitrogen source and optionally whole proteins or protein fractions.
2. The method of claim 2, wherein said microorganisms are microalgae.
3. The method of claim 1 or 2, wherein said polar lipids comprise phospholipids.
4. The method of claim 1 or 2, wherein said polar lipids are present at a higher weight percent than said neutral lipids in said microbial oil.
5. The method of claim 1 or 2, wherein said whole proteins or protein fractions are selected from the group consisting of: amino acids, proteinogenic amino acids, peptides, polypeptides, peptones, carnitine, GABA, L-DOPA, hydroxyproline, and selenomethionine, ornithine, homoserine, lanthionine, 2-aminoisobutyric acid, and dehydroalanine.
6. The method of claim 5, wherein said proteinogenic amino acids are selected from the group consisting of: alanine, arginine, asparagines, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and salts thereof,
7. The method of claim 6, wherein said amino acids are selected from the group consisting of serine, glycine, and salts thereof.
8. The method of claim 7, wherein said peptides are selected from the group consisting of: substance P, kassinin, neurokinin A, eledosin, neurokinin B, VIP, PACAP, Peptide PHI, GHRH 1-24, glucagon, secretin, NPY, PYY, APP, PPY, POMC,
enkephalin, prodynorphin, calcitonin, amylin AGG01 , BNP, and lactotripeptides.
9. The method of claim 1 or 2, wherein the concentration of said nitrogen is increased during said culturing.
10. The method of claim 9, wherein said proteins are selected from the group consisting of: whole proteins, hydrolyzed proteins, protein hydrotysates, protein isolates, and protein concentrates.
1 1. The method of claim 4, wherein the weight percent ratio of polar lipids to neutral lipids is from about 1.2:1 to about 2:1. 2. The method of claim 1 or 2, wherein said nitrogen source is selected from the group consisting of: glutamate, monosodium glutamate, glutamic acid, ammonia, ammonium hydroxide, ammonium carbonate, ammonium chloride, ammonium nitrate, nitrate, urea, tryptone, peptone, casein, creatine, corn steep liquor, and combinations thereof.
13. The method of claim 1 or 2, wherein said fermentation medium further comprises a carbon source.
14. The method of claim 13, wherein said carbon source is selected from the group consisting of glucose, com starch, ground com, potato starch, wheat starch, molasses, grain, and combinations thereof.
15. The method of claim 1 or 2, wherein said fermentation medium further comprises a microbial growth factor.
16. The method of claim 15, wherein said microbial growth factor is selected from the group consisting of: yeast, vitamins, com steep liquor, and combinations thereof.
17. The method of claim 1 or 2, wherein said nitrogen source in said fermentation medium is present in an amount high enough to favor production of polar lipids over neutral lipids. 8. The method of claim 1 or 2, wherein said culturing or said producing are done under non-nitrogen limiting conditions.
19. The method of claim 1 or 2, wherein said fermentation medium further comprises a transferase enzyme that promotes acylation of fatty acids on said polar lipids.
20. The method of claim 19, wherein said transferase enzyme is selected from the group consisting of: acyltransferases, glyceronephosphate O-acyltransferase, and lecithin-chloesterol acyltransferase.
21. The method of claim 1 or 2, wherein said fermentation medium further comprises an esterase enzyme that promotes esterfication of fatty acids on said polar lipids.
22. The method of claim 21 , wherein said esterase enzyme is selected from the group consisting of: lipases and phospholipases.
23. The method of claim 22, wherein said phospholipase is selected from the group consisting of phospholipase A1 , phospholipase A2, and phospholipase B.
24. The method of one of claims 19-20, wherein said transferase enzyme is selected to target C18 or lower fatty acids.
25. The method of one of claims 21-23, wherein said esterase enzyme is selected to target C18 or lower fatty acids.
26. The method of claim 1 or 2, wherein said fermentation medium contains a phosphodiesterases enzyme that promotes esterfication of the polar group on said polar lipids.
27. The method of claim 26, wherein said phosphodiesterase enzyme is selected from the group consisting of: phospholipase C and phospholipase D.
28. The method of claim 1 or 2, wherein said amino acid is selected from the group consisting of serine, glycine, and salts thereof, wherein said fermentation medium comprises a first enzyme comprising phosphodiesterase enzyme and a second enzyme selected from the group consisting of transferase enzyme and esterase enzyme.
29. A phospholipid composition comprising phospholipids derived from fermenting microalgae in the presence of an additional nitrogen source and optionally whole proteins or protein fractions.
30. The method of any one of the preceding claims, wherein said microorganism is selected from the group of microalgae consisting of: Thraustochytrium, Schizochytrium, Crypthecodinium, Dinophyceae, Baciliariophyceae, Chlorophyceae, Prymnesiophyceae, Phaeodactylum, Nannochloropsis, Porphydrium, Monodus and Euglenophyceae,
31. The phospholipid from claim 29, wherein said microalgae is selected from the group consisting of: Thraustochytrium, Schizochytrium, Crypthecodinium, Dinophyceae, Baciliariophyceae, Chlorophyceae, Prymnesiophyceae, Phaeodactylum,
Nannochloropsis, Porphydrium, Monodus and Euglenophyceae.
32. A lipid composition comprising:
(a) a phospholipid derived from fermenting microorganisms in the presence of an additional nitrogen source and optionally whole proteins or protein fractions; and
(b) a structured lipid, wherein the structured lipid comprises a first fatty acid carbon chain derived from a first lipid source, a second fatty acid carbon chain derived from a second lipid source, and a third fatty acid carbon chain derived from a third lipid source.
33. The lipid composition of claim 32, wherein said microalgae is selected from the group consisting of: Thraustochytrium, Schizochytrium, Crypthecodinium, Dinophyceae, Baciliariophyceae, Chlorophyceae, Prymnesiophyceae, Phaeodactylum,
Nannochloropsis, Porphydrium, Monodus and Euglenophyceae,
34. A liposome comprising the phospholipids from claim 29.
35. A liposome comprising the structured lipids from claim 32.
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