EP2370587A1 - Compositions contenant de l'acide docosahexaènoïque et leur procédé de production - Google Patents

Compositions contenant de l'acide docosahexaènoïque et leur procédé de production

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Publication number
EP2370587A1
EP2370587A1 EP09764852A EP09764852A EP2370587A1 EP 2370587 A1 EP2370587 A1 EP 2370587A1 EP 09764852 A EP09764852 A EP 09764852A EP 09764852 A EP09764852 A EP 09764852A EP 2370587 A1 EP2370587 A1 EP 2370587A1
Authority
EP
European Patent Office
Prior art keywords
nutrient medium
fermentor
microalgae
biomass
medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09764852A
Other languages
German (de)
English (en)
Inventor
Trine Huusfeldt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Marenutrica GmbH
Original Assignee
Marenutrica GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Marenutrica GmbH filed Critical Marenutrica GmbH
Priority to EP09764852A priority Critical patent/EP2370587A1/fr
Publication of EP2370587A1 publication Critical patent/EP2370587A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • 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
    • 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
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6434—Docosahexenoic acids [DHA]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a method of producing an edible composition comprising biomass from at least one heterotrophic microalgae having a content of docosahexaenoic acid (DHA) .
  • the invention further relates to the composition obtained by said method and to the use of the composition as a dietary supplement or a pharmaceutical composition.
  • DHA docosahexaenoic acid
  • DHA docosahexaenoic acid
  • mammals like fish, obtain most of the DHA from dietary sources, such as algae capable of synthesizing DHA.
  • 4,670,285 discloses the use of fish oil from fish such as menhaden and herring as a source of C22 omega-3-fatty acids. Indeed, fish oils are the primary commercial source of omega-3-fatty acids. Often, however, fish oils are unusable for human consumption because of contamination with environmental pollutants such as polychlorinated biphenyls (PCB 1 s) or heavy metals.
  • PCB 1 s polychlorinated biphenyls
  • Microalgae biomass is particularly suitable for the extraction and purification of individual DHA due to its stable and reliable composition and the use DHA, is encouraged by e.g. the WHO and the American Heart Association.
  • the DHA obtained by said method has to be recovered by extraction using organic solvents, such as hexane .
  • organic solvents such as hexane .
  • Such extraction steps are not only expensive and time consuming but also involves potential toxic solvent.
  • n-hexane in 1994 was included in the list of chemicals on the Toxic Release Inventory [N-Hexane Chemical Backgrounder” . National Safety Council . Retrieved on 25 May 2007] .
  • a prolonged culture period is required in order to obtain the described DHA concentrations.
  • PUFA's polyunsaturated fatty acids
  • EPA eicosapentaenoic acid
  • microalgae The concept of culturing microorganisms in open or closed systems and harvesting a product from the microorganisms is not new.
  • algae or phytoplankton are grown and harvested, either as animal food or as sources of chemicals such as carotenes.
  • numerous factors are critical in cultivation of very condition-sensitive marine microorganisms, such a microalgae.
  • One of the largest problems in cultivation of microalgae is the fact that very high density cultivation (greater than about 50 g/L microbial biomass) can lead to a decrease in omega-3 fatty acids productivity.
  • It is a second aspect according to the present invention to provide a method for providing an edible composition comprising biomass from at least one heterotrophic microalgae having a content of, i.e. containing, docosahexaenoic acid (DHA), where the need for recovering the desired composition from the biomass by extraction using one or more organic solvents is eliminated.
  • DHA docosahexaenoic acid
  • It is a fifth aspect of the present invention to provide a fatty acid composition comprising microalgae biomass having a high concentration of DHA which is free of undesirable contaminants, such a heavy metals and/or organic solvents.
  • the new and unique way in which the current invention fulfils one or more of the above-mentioned aspects is to provide a method for manufacturing an edible composition comprising biomass from at least one heterotrophic microalgae having a content of docosahexaenoic acid (DHA) , said method comprises cultivating said microalgae in three different nutrient media in the following steps:
  • the cultivation of the microalgae is preferably carried out in any convenient fermentor.
  • the term fermentor refers in the present application to any device or system that supports a biologically active environment, and includes e.g. bioreactors and microorganism reactors.
  • step a) the heterotrophic microalgae adapt themselves to the growth conditions in the first nutrient medium. It is during this period that the individual algae are maturing but not yet are able to divide.
  • step a) During this initial step of the algae growth cycle, synthesis of RNA, enzymes and other molecules occurs.
  • the medium used in step a) is designed specifically in order to ensure that the optimal conditions are providing for the algae. It must be understood that this phase is very important in building new healthy cells that will be able to complete the fermentation. If the conditions in step a) is not optimal each individual cell will not be healthy at the end of step a) and the two following steps will as a result be less efficient and optimal, eventually resulting in reduced concentration of the composition according to the invention.
  • step b) the heterotrophic microalgae undergoes a period characterized by cell doubling and rapid growth.
  • the actual rate of this growth depends upon the growth conditions, which affect the frequency of cell division events and the probability of both daughter cells surviving.
  • the medium used in step b) is designed specifically in order to ensure that optimal conditions are provided for the algae during this growth cycle, which preferably is exponential.
  • step c) the production of DHA of the microalgae induced by the imposition of a stationary phase of heterotrophic microalgae.
  • This is according to one embodiment of the invention obtained by using a nutrient medium, which is depleted of a nitrogen source.
  • this could in addition or alternatively also be achieved by lowering or raising the pH-value and/or lowering the temperature of cultivation step c) .
  • each nutrient medium provides optimal conditions for the heterotrophic microalgae in the respective step, it is possible to obtain an improved biomass production, (greater than about 100 g/L microbial wet-biomass) , compared with the conventional methods, without observing a decrease in the content of the DHA contained in the microalgae.
  • the different steps of the heterotrophic microalgae lifecycle, corresponding to the different steps in the method according to the invention is completed faster and therefore more economical than in the prior art.
  • step a) provides healthy heterotrophic microalgae that will be able to complete fermentation in step b) and c) .
  • this phase has always been performed in the same medium as the following exponential phase, step b) .
  • the inventors have now surprisingly found that when two different nutrient mediums are provides, one for each step, a healthier cell are obtained in step a) which not only has an increased growth rate but said cells are also capable of surviving longer and under harsher conditions; e.g. in respect of increased sear forces in the medium.
  • the method according to the invention can optionally comprise the additional steps of harvesting the biomass obtained after step a) and/or step b) .
  • the harvested biomass obtained can be transferred/added to the second and third nutrient medium, respectively.
  • samples can advantageously be obtained from the nutrient medium containing the heterotrophic microalgae in order to determine when the heterotrophic microalgae is ready to be transferred to or begin step b) .
  • conventional methods of determination cell motility and/or the size of the algae can preferably be used.
  • step a) it is desirable to obtain cells having a size of about 2-10 ⁇ m in step a) , since this indicates that the algae have adapted to the first nutrient medium.
  • the first microalgae suspension or biomass from the first microalgae suspension is either transported/added to the second nutrient medium or the second nutrient medium is transferred to e.g. the first microalgae suspension.
  • the mixing of the first microalgae suspension with the second nutrient medium can be an instant mixing, i.e. they are simply placed in the same container, or as an alternative can step b) be modified in that the biomass from the first microalgae suspension can be retained in the fermentor, and the first nutrient medium can continuously be removed from the fermentor while the second nutrient medium is transferred to the fermentor. Said removal/transfer is continued until the nutrient medium in the fermentor substantially consists of the second nutrient medium.
  • This modified step b) ensures that the microalgae is transferred to the second nutrient medium without increasing the working volume.
  • the microalgae will consistently have access to nutrient medium and the transfer of the microalgae from one medium to the next therefore eliminate the risk of a new lag phase, while the algae is adjusting to the new medium.
  • the second nutrient medium is designed for optimally stimulating the exponential phase. Thereby is ensured that a desired density of the biomass is obtained faster and more economically .
  • samples can also be obtained during step b) in order to establish when the desired biomass of between 10 and
  • Crypthecodinium cohnii will preferably have a size of 10-20 ⁇ m.
  • the second microalgae suspension or biomass from the second microalgae suspension is mixed with the third nutrient medium, in a similar way as the first microalgae suspension was mixed with the second nutrient medium.
  • step c) be modified in a similar way as step b) described above.
  • the biomass from the second microalgae suspension is retained in the fermentor and the second nutrient medium can continuously be removed from the fermentor while the third nutrient medium is transferred to the fermentor. Said removal/transfer continues till the second nutrient medium in the fermentor substantially consists of the third nutrient medium.
  • step c) the second microalgae suspension has been introduced to the third nutrient medium, inducing production of DHA and thereby the composition according to the invention.
  • the culture is grown for a number of hours depending on the used heterotrophic microalgae.
  • heterotrophic microalgae are cultivated for a time sufficient to produce the composition according to the invention, usually from about 120 to about 240 hours, although this time is subject to variation.
  • the culture is grown until the desired concentration of DHA contained in the microalgae is at least 5- weight% of the nutrient medium, preferably at least 25-weight% of the nutrient medium.
  • the pH-value in at least the first and second nutrient medium is maintained at a substantially constant value.
  • microalgae is very susceptible and sensitive to any mechanical force, especially when the cell density is high, such mechanical forces can lead to cell breakage causing the lipids, such as the DHA, to be released in media. It is therefore not beneficial simply to add e.g. a pH regulator directly to the fermentor, as it is not possible to obtain a homogenous nutrient medium without causing significant cell damage.
  • the inventor of the present invention has surprisingly found that if a specific nutrient medium recycling system is used where the microalgae is retained in the fermentor and a separate container is placed in fluid communication with the fermentor, it is possible to continuously remove spent culture medium from the fermentor, replenish the spent medium in the separate container e.g. by adjusting the pH-value, and recycle the replenished medium to the fermentor.
  • This advantageously embodiment will works as a kind of perfusion culture where spent media and/or toxic byproducts constantly are removed from the fermentor containing the microalgae and replenished medium is continuously added to said fermentor at the same flow rate.
  • replenished any adjustment to the nutrient medium which will renew the medium either completely or in part and/or adjust a specific parameter in the medium e.g. the pH-value.
  • the term therefore comprises the addition of nutrients, adjusting the pH-value in the medium and/or removal of any undesirable metabolites in the medium.
  • the present embodiment further has the advantage that residual nutrients can be used when the medium is recycled to the fermentor and that depleted nutrients or additional nutrient can be added to the medium in the separate container.
  • Nutrients in a media is never consumed completely which is undesirable from a cost-effectiveness point of view, because the residual nutrients are wasted with the effluent media and the cost of nutrients is the major contributor to the cost of the nutrient media. This problem is conveniently meet by the present embodiment according to the invention.
  • the flow rate of removal of the spent medium and addition of replenished medium is substantially identical in order to maintain the volume in the fermentor at a steady state.
  • the spend medium is preferably removed from an upper part of the fermentor and the replenished medium is preferably added to a lower part of the fermentor, as this will ensure a flow though the fermentor.
  • the microorganisms are preferably retained in the fermentor using filtration, e.g. by adding a filter in the fluid communication where the nutrient medium is removed from the fermentor and transferred to the separate container, since the key to successful recycling is the retention of at least the majority of the cells in the fermentor, allowing operation at relatively high flow rates without the danger of washout or cell breakage.
  • the microalgae is very sensitive to the application of physical stirring, as the algae cell breakage can cause lipids, e.g. DHA, to be released in the nutrient media where they can become oxidized and/or degraded by enzymes, resulting in e.g. a fishy smell and/or taste.
  • the inventor of the present invention has surprisingly discovered that when a nutrient medium recycling system as described above is used, it is not necessary to have any physical means e.g. a stirrer or mixer in the fermentor, in order for the mixing or stirring of the nutrient medium with the biomass to be efficient. The recycling of the nutrient medium is sufficient.
  • DO dissolved oxygen
  • DO is preferably not constant throughout the cultivation of the microalgae.
  • DO is preferably provided in a first period of time, preferably about 0.5 h, interrupted by a second period of time, preferably about 3 h, where no DO is added to the fermentor.
  • the heterotrophic microalgae used for production is preferably a Dinophyceae. From said class heterotrophic microalgae within the genus of Crypthecodinium and Schizochytrium are preferred, as these have proven capable of producing relatively large amounts of DHA. However, the specie Crypthecodinium cohnii is according to one embodiment of the present invention the most desirable organism to utilize for the production of DHA.
  • the heterotrophic microalgae In the marine environment, the heterotrophic microalgae is usually found in full salinity seawater and, as such, is adapted to growth in an environment with a high chloride concentration .
  • seawater has a salinity of between 3.1% and 3.8%. This means that every 1 kg of seawater has approximately 35 grams of dissolved salts, which consist mostly, but not entirely, of the ions of sodium chloride: Na + , Cl .
  • the three nutrient mediums according to the present invention comprises salt at a concentration corresponding to the salinity of seawater, i.e. preferably between about 2% and about 4%, preferably between about 2.1% and about 3.0%, especially about 2.6%.
  • the mediums according to the invention can also comprise other salts naturally occurring in seawater, either alone or in combination with sodium chloride.
  • the first and second nutrient mediums will contain a nitrogen source, preferably in the form of a yeast extract, as this is an inexpensive nitrogen source.
  • the yeast extract is preferably a water-soluble extract of autolyzed yeast cells suitable for use in culture media and could e.g. be a commercially available yeast extract from DIFCO, or MARCOR.
  • yeast extract is an organic nitrogen source it will also contain a number of other micronutrients . However, a person skilled in the art could easily determine other organic nitrogen sources.
  • the third nutrient medium further comprises a omega-3 or omega- 6 plant oil, such as rapeseed oil and groundnut oil, since the inventors surprisingly have found that the severe foaming problems in the fermentor using the conventional mediums are reduced and in some cases completely eliminated.
  • a omega-3 or omega- 6 plant oil such as rapeseed oil and groundnut oil
  • the first nutrient medium comprises a plant oil e.g. rapeseed oil
  • microbial cell breakage a problem in cells that have undergone nitrogen limitation or depletion in order to induce lipid formation, when increased shear forces are applied in the media. This is due to the fact that nitrogen limitation or depletion results in weaker cell walls.
  • a plant oil such as rapeseed oil
  • the present inventors have found that the addition of a plant oil, such as rapeseed oil, to the third nutrient medium decrease the microbial cell breakage even when increased shear forces are applied, thereby reducing the amount of lipids in the medium which can become oxidized and/or degraded by enzymes .
  • composition according to the invention which have fewer undesirable oxidation and/or decomposition products than the extracted oils obtained in the prior art, thereby eliminating the fishy odor and the unpleasant taste normally associated with fish oils.
  • carbon sources for use in the second and third nutrient medium can preferably be provided in the form of glucose.
  • the carbon sources in the first nutrient medium can e.g. be hop and/or malt.
  • the first nutrient medium can in a preferred embodiment comprise 2-4% NaCl, 0.1-2.0% yeast extract, 0.1-2.0% plant oil (rapeseed oil), 10-50% malt, 0.5-3.0% hop, and the remaining being distilled water, as the inventors surprisingly have found that such a nutrient medium ensures that the heterotrophic microalgae are healthier, has a longer lifetime than previously known and is capable of meeting harsher conditions such as increased shear forces in the medium.
  • the second nutrient medium comprises in a preferred embodiment 2-4% NaCl, 0.5-30% glucose and 0.1-2.0% yeast extract, and the remaining being distilled water.
  • This nutrient medium has the benefits compared to conventional mediums, that the heterotrophic microalgae growths under optimal nutrient conditions, thereby ensuring a faster and more economical production of the desired algae-biomass .
  • the third nutrient medium which is designed to initiate a stationary phase of the heterotrophic microalgae, comprises 2- 4% NaCl, 0.5-50% glucose, 0.1-2.0% plant oil, such as rapeseed oil, and the remaining being distilled water. As is evident the third nutrient medium is depleted of a nitrogen source. However, this could in addition or alternatively also be achieved by lowering or raising the pH-value and/or lowering the temperature of the cultivation process in step c) .
  • An alternative way of providing nitrogen deficiencies in the third nutrient medium can be obtained by having a ratio of the carbon source to the nitrogen source, which promotes the efficient production of the composition according to the invention.
  • a preferred ratio of carbon source to nitrogen source is about 10-15 parts glucose to 1 part yeast extract.
  • the cultivation in step a) and b) can be carried out at any life-sustaining temperature.
  • C. cohnii will grow at temperatures ranging from about 15°C to 34°C.
  • the temperature is maintained at about 25 °C to 30 °C, and most preferably at about 27°C in step a) and b) .
  • Heterotrophic microalgae which grow at 27°C are preferred, because they will have a faster doubling time, thereby reducing the fermentation time.
  • Appropriate temperature ranges for other microorganisms are readily determined by those of skill in the art.
  • step c) The production of the composition according to the invention in step c) is preferably achieved at a temperature ranging from about 13 °C to about 18 °C, with a preferred temperature at about 15 °C, as the inventors have found that this temperature promotes a faster production of said composition.
  • the cultivation can in all three steps be carried out over a broad pH range, typically from about pH 5.0 to 9.0. Preferably, a pH of about 7.5.
  • a base such as KOH or NaOH, can be used to adjust the medias pH-value prior to inoculation.
  • inorganic pH controls can be used to correct the pH-values during the different steps. If a nutrient medium recycling system is used, the pH-value can conveniently be adjusted in the separate container as described earlier.
  • One, more or all of the cultivation steps a) , b) and c) can in one embodiment be "fed-batch" processes, i.e. it is based on feeding a growth limiting nutrient substrate to the different cultivation steps.
  • the fed-batch strategy is advantageously in one embodiment, as this will provide a high cell density in the fermentor.
  • the feed solution is highly concentrated to avoid dilution of the bioreactor.
  • the fed-batch process has the advantage that is gives the operator an opportunity of controlling the reaction rate in order to avoid e.g. technological limitations connected to the cooling of the reactor and oxygen transfer.
  • the fed- batch process further allows the metabolic control, to avoid osmotic effects, catabolite repression and production of undesirable side products, such as undesirable PUAF ' s .
  • Different strategies can be used to control the growth in a fed-batch process, e.g. nutrient availability, sedimentation rate, temperature, pH, gas exchange rate and cell integrity.
  • the biomass from the third microalgae suspension obtained in step c) are preferably harvested by conventional means.
  • suitable harvesting techniques can be mentioned centrifugation, flocculation or filtration, however other techniques are well known for the person skilled in the art.
  • the harvested biomass can then be cleaned by washing and/or dried, again using conventional techniques and method.
  • the harvested, washed/cleaned and dried biomass has a water content of about 10-weight% to about 50-weight%.
  • the dried microalgae- biomass can in a first embodiment be used directly as a dietary composition according to the invention.
  • the resultant biomass i.e. the composition according to the invention, comprises at least 25- weight% DHA.
  • the composition according to the invention will normally contain additional PUFAs in addition to the DHA.
  • These PUFAs can e.g. be one or more of the following: PUFAs: octadecanoic acid (18:0), octadecanoic acid (18:1), eicosanoic acid (20:0), and Docosanoic acid (20:0) .
  • the denotation (18:1) octadecanoic acid means that octadecanoic acid is a carboxylic acid with an 18-carbon chain and one double binding. These denotations are well known in the art and the person skilled in the art would easily understand them.
  • the content of DHA is advantageously as high as possible in relation to the other PUFAs, and preferably above 25%, more preferably above 45%, especially above 75% and even more especially above 90% of the weight of total PUFAs.
  • the following fatty acid composition in the composition according to the invention 40% docosahexaenoic acid (22:6), 14% octadecanoic acid (18:0), 22% octadecanoic acid (18:1), 18% eicosanoic acid (20:0), and 6% Docosanoic acid (22:0) .
  • the edible composition comprises biomass from at least one heterotrophic microalgae wherein the microalgae produces the docosahexaenoic acid (DHA) , thus the composition will be comprise a reliably high protein content from the heterotrophic microalgae.
  • the composition can also comprise an alternative protein source or a protein supplement.
  • the proteins preferably have a size between about 10 kDa and about 250 kDa, as these proteins have proven especially beneficial and comparable to conventional vegetable proteins .
  • the entire biomass from step c) is used as the edible composition according to the invention, as the microalgae in this step will have the desired content of DHA, of at least 25-weight%.
  • the heterotrophic microalgae has a cell size of 20 - 36 ⁇ m.
  • composition according to the invention can be extracted from the harvested material using an effective amount of solvent.
  • solvents Those of skill in the art can determine suitable solvents.
  • composition according to the invention can in a preferred embodiment be encapsulated with a coating capable of withstanding the effects of the human/animal stomach acid and provide a controlled release in the small intestine. Delivering the composition directly to the small intestine also eliminate any undesirable taste the product may have or is associated with.
  • Said micro- or nanoencapsulating can according to the present invention be archived by an encapsulation method comprising the following steps: - preparing core particles of the composition according to the invention coating the core particles with a biodegradable release-controlling polymer.
  • encapsulation refers to a range of techniques used to enclose compositions or products in a relatively stable shell known as a capsule, allowing them to, for example, be taken orally or be part of e.g. a gel or cream.
  • the two main types of capsules are hard-shelled capsules, which are normally used for dry, powdered ingredients, and soft- shelled capsules, primarily used for oils and for active ingredients that are dissolved or suspended in oil.
  • One possible coating technique is the fluidized bed coating technique, which is a simple dipping process.
  • the fluidized bed is a tank with a porous bottom plate and the polymer is in the form of a powder.
  • the plenum below the porous plate supplies low- pressure air uniformly across the plate.
  • the rising air surrounds and suspends the divided core particles, so the polymer dispersed in the air resembles a boiling liquid.
  • Products that are preheated above the melt temperatures of the powder are dipped in the fluidized bed, where the powder melts and fuses into a continuous coating.
  • a high transfer efficiency results from little drag out and no dripping.
  • the fluidized bed powder coating method is used to apply heavy coats in one dip, 3 - 10 mils (75 - 250 ⁇ m) , uniformly to complex shaped products. It is possible to build a film thickness of 100 mils (2500 ⁇ m) using higher preheat temperatures and multiple dips.
  • the oil drops are preferably encapsulated with a polymerisable material of natural origin such as alginat .
  • a polymerisable material of natural origin such as alginat .
  • alginat as the encapsulating polymer it has been found that the composition according to the invention is protected against the conditions of the stomach and upper intestine, thereby allowing the composition according to the invention to be introduced into the colon where it may offer it's health benefits.
  • the microcapsules thereby obtained can either be used as a dietary supplement or as a pharmaceutical composition for reduction of cardiovascular and inflammatory diseases or reduction of depression or increasing length of gestation in the third trimester or inhibiting tumor growth.
  • Example 1 Preparation of the first, second and third nutrient medium A preparation of the first, second and third nutrient mediums is prepared as follows:
  • the pH of the medium is adjusted to 7.6, using 1 N NaOH.
  • the volume is then brought to 1000 ml by the addition of distilled water.
  • the different nutrient media is sterilized by autoclave treatment at 121° C, at 103 kPa above atmospheric pressure, for 15 minutes.
  • the rapeseed oil is then filter sterilized and separately added.
  • the media are then cooled at stored for later use.
  • Example 2 Manufacture of the composition according to the invention using a fed-batch system
  • Step a) Into a 300-liter working volume STF was loaded 100 liter of the first nutrient medium obtained example 1. 10 percent per volume inoculums from a seed fermentor containing about 4 * 10 6 cells/ml were added to the medium. Agitation was set at 275 rpm, dissolved oxygen (DO) to 4.5 mg/1, the temperature was set to 27°C. The pH-value was continually adjusted to a pH of about 7.5.
  • Fresh medium (first nutrient medium) was continuously added to the STF, until a complete volume of 300 liter of the first algae suspension was obtained and the algae had a size of 5-10 ⁇ m.
  • Fresh medium (third nutrient medium) was continuously added to the STF, until a complete volume of 25,000 liter of the third algae suspension was obtained.
  • the culture was then permitted to grow for an additional time sufficient to ensure that the concentration of DHA was 20 grams per liter of nutrient solution and the concentration of microbial biomass was 100 g/L nutrient medium.
  • the culture was then harvested by centrifugation with the cell pellet retained.
  • the harvested pellet of cells was frozen and dried (lyophilized) to about 20% moisture content.
  • the dried pellet could be used directly as the edible composition according to the present invention or be subjected to a micro- or nano-encsapsulation .
  • a nutrient medium recycling system comprising a fermentor in fluid communication with a separate container for replenishing the nutrient medium, was loaded 35 liter of the relevant nutrient medium obtained in example 1. The working volume of the fermentor was 11-litre.
  • Spent culture medium was continuously removed from the top of the fermentor and transferred to the separate container, where the pH-value was adjusted/controlled.
  • the replenished nutrient medium was thereafter continuously added to the bottom of the fermentor.
  • the fermentor has no agitator, i.e. no means for physically stirring the culture.
  • the temperature was set to 23.5 °C . 10 percent per volume inoculums from a seed fermentor containing about 14.2 * 10 4 cells/ml of Crypthecodinium cohnii were added to the medium.
  • Step a) The microalgae was grown in the first nutrient medium until the cells reached the exponential phase - the pH was continuously adjusted to a pH-value of about 7.8 in the separate container.
  • Step b) When the exponential phase was reached the nutrient medium in the separate container was changed to the second nutrient medium obtained in example 1. During the first 66 h after the pH-value of the medium was not adjusted in the separate container .
  • the microalgae continues to grow at an exponentionel rate, ensuring that a very high biomass and thereby composition according to the invention can be obtained.
  • Example 4 Manufacture of the composition according to the invention using a nutrient medium recycling system
  • the microalgae suspension obtained in example 3, was used for the production of the composition according to the invention.
  • the second nutrient medium in the separate container was changed to the third nutrient medium obtained in experiment 1. Recycling of the medium was allowed for about 1 h in order to ensure that the entire nutrient medium in the fermentor was the third nutrient medium; hereafter the temperature was decreased to 15°C.
  • the culture was then permitted to grow for an additional period sufficient to ensure that the concentration of DHA was 20 grams per liter of nutrient solution and the concentration of wet microbial biomass was 100 g/L nutrient medium.
  • the culture was then harvested by centrifugation with the cell pellet retained.
  • the harvested pellet of cells was frozen and dried (lyophilized) to about 20% moisture content.
  • the dried pellet could be used directly as the edible composition according to the present invention or be subjected to a micro- or nano-encsapsulation .

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Abstract

La présente invention concerne un procédé de production d'une composition comestible comprenant de la biomasse provenant d'au moins une microalgue hétérotrophe ayant une certaine teneur en acide docosahexaènoïque (DHA). L'invention concerne également la composition obtenue par ce procédé, et l'utilisation de cette composition comme complément alimentaire ou composition pharmaceutique. Le procédé ci-décrit utilise trois milieux nutritifs différents dans trois étapes différentes, chaque milieu nutritif étant conçu pour apporter des conditions optimales aux microalgues hétérotrophes lors de l'étape correspondante. La production de biomasse est ainsi améliorée par rapport aux procédés classiques, sans observer de réduction de la teneur en produits souhaitables.
EP09764852A 2008-12-08 2009-12-08 Compositions contenant de l'acide docosahexaènoïque et leur procédé de production Withdrawn EP2370587A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP09764852A EP2370587A1 (fr) 2008-12-08 2009-12-08 Compositions contenant de l'acide docosahexaènoïque et leur procédé de production

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP08170974A EP2194138A1 (fr) 2008-12-08 2008-12-08 Composés contenant de l'acide docosahexaénoïque et son procédé de production
EP09764852A EP2370587A1 (fr) 2008-12-08 2009-12-08 Compositions contenant de l'acide docosahexaènoïque et leur procédé de production
PCT/EP2009/066637 WO2010066737A1 (fr) 2008-12-08 2009-12-08 Compositions contenant de l'acide docosahexaènoïque et leur procédé de production

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EP2370587A1 true EP2370587A1 (fr) 2011-10-05

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EP09764852A Withdrawn EP2370587A1 (fr) 2008-12-08 2009-12-08 Compositions contenant de l'acide docosahexaènoïque et leur procédé de production

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WO2013121365A1 (fr) * 2012-02-14 2013-08-22 Seambio Fuel Limited Procédés et systèmes destinés à la culture de microalgues
FR2997959B1 (fr) * 2012-11-09 2016-04-01 Invivo Nsa Utilisation de sucre dans une culture de microalgues pour diminuer leur auto-floculation
CN104046567A (zh) * 2013-03-17 2014-09-17 中国石油化工股份有限公司 养殖微藻的方法和生产油脂的方法
AU2015263044B2 (en) * 2014-05-22 2021-07-01 MARA Renewables Corporation Methods of oil production in microorganisms
FR3025215A1 (fr) 2014-08-27 2016-03-04 Fermentalg Nouveau procede de culture de microalgues
CN114686534A (zh) * 2020-12-30 2022-07-01 嘉必优生物技术（武汉）股份有限公司 一种磷脂型dha的制备方法

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US4670285A (en) 1982-08-06 1987-06-02 The University Of Toronto Innovations Foundation Infant formula
US5407957A (en) * 1990-02-13 1995-04-18 Martek Corporation Production of docosahexaenoic acid by dinoflagellates
WO1999006585A1 (fr) * 1997-08-01 1999-02-11 Martek Biosciences Corporation Compositions nutritives contenant de l'acide docasahexanoique et leurs procedes de production
EP1359224A1 (fr) * 2002-05-01 2003-11-05 Ato B.V. Procédé de production d'acides gras polyinsaturés par des microorganismes marins
JP4733043B2 (ja) * 2003-10-02 2011-07-27 マーテック バイオサイエンシーズ コーポレーション 改変された量の塩化物およびカリウムを使用した微細藻類における高レベルのｄｈａの産生法

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EP2194138A1 (fr) 2010-06-09

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