WO2010066737A1

WO2010066737A1 - Compositions containing docosahexaenoic acid and method for its production

Info

Publication number: WO2010066737A1
Application number: PCT/EP2009/066637
Authority: WO
Inventors: Trine Huusfeldt
Original assignee: Marenutrica Gmbh
Priority date: 2008-12-08
Filing date: 2009-12-08
Publication date: 2010-06-17
Also published as: EP2370587A1; EP2194138A1

Abstract

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. The method according to the invention utilises three different nutrient media in three different steps, where each nutrient medium is designed for providing optimal conditions for the heterotrophic microalgae in the respective step. Thereby is achieved an improved biomass production compared with the conventional methods, without observing a decrease in the content of the desirable products.

Description

COMPOSITIONS CONTAINING DOCOSAHEXAENOIC ACID AND METHOD

FOR ITS PRODUCTION

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.

Beneficial effects of increased dietary intake of long chain omega-3 fatty acids in humans, such as docosahexaenoic acid (DHA), are well documented. The effects include the reduction of cardiovascular and inflammatory diseases (i.e. arthritis and atherosclerosis), reduction of depression, increasing length of gestation in the third trimester, and inhibiting tumor growth.

Although a metabolic pathway exists in mammals for the biosynthesis of DHA from dietary linolenic acid, this pathway is bioenergetically unfavorable [Crawford, P. AOCS. Short

Course in Polyunsaturated Fatty Acids and Eicosanoids, pp. 270-

295 (1987)] . It is therefore believed that mammals, like fish, obtain most of the DHA from dietary sources, such as algae capable of synthesizing DHA.

In this respect several heterotrophic marine microorganisms have been found to produce high levels of the important essential fatty acids, including that of genus Crypthecodinium [Jiang and Chen, Process Biochemistry 35 (2000) 1205-1209; Jiang and Chen, Journal of Industrial Microbiology & Biotechnology, (1999) Vol. 23, 508-513; Vazhappilly and Chen, Journal of the American Oil Chemists Society, (1998) Vol. 75, No. 3 p 393-397] . The DHA becomes increasingly concentrated in organisms as it moves up the food chain. Conventionally humans obtained most of the beneficial omega-3 fatty acids from fish. U.S. Pat. No. 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 (PCB¹ s) or heavy metals.

There are furthermore many problems associated with the recovery of fish oils containing DHA for food uses. However, the use of fish oil as a food additive is limited due to problems associated with its typical fishy smell, unpleasant taste, and poor oxidative stability which makes them unsatisfactory for use in edible products.

Therefore, alternative sources are of interest. 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.

There have been several attempts in the art to use marine microorganisms for the production of significant quantities of DHA. One such an attempt is disclosed in EP 0 515 460, which describes that it is possible to produce oils containing a high proportion of DHA during cultivation of microorganisms in a fermentor .

However, the DHA obtained by said method has to be recovered by extraction using organic solvents, such as hexane . Such extraction steps are not only expensive and time consuming but also involves potential toxic solvent. As an example can be mentioned that 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] . It should furthermore be noted, that in order to obtain the described DHA concentrations, a prolonged culture period is required .

Another problem with the prior art is that most microorganisms produce two or more polyunsaturated fatty acids (PUFA's) in their lipids. This is often undesirable, as the complexity of the organism's lipid profile will limit the use of the oils in some food and pharmaceutical applications. This is e.g. due to the presence of other undesirable PUFAs in the oil or due to ratios of the different PUFAs falling out of the desirable range for the specific application.

As one example can be mentioned, that eicosapentaenoic acid (EPA) a component of fish oil is an undesirable component in e.g. infant formulas because of its prolonged anticoagulant effects and its depression of arachidonic levels in infants. This has been correlated with reduced rates of infant weight gain (Carlson et al . INFORM 1:306.)

Still another problem with the known techniques is that they often use photosynthetic alga such as Emiliania spp . When these species are utilized for the production of DHA, a high yield of DHA may be accomplished but since the algae requires a number of very specific conditions for growth, e.g. light, such processes is complicated, expensive and time consuming, and therefore not suitable for industrial production.

The concept of culturing microorganisms in open or closed systems and harvesting a product from the microorganisms is not new. There are numerous systems used throughout the world in which algae or phytoplankton are grown and harvested, either as animal food or as sources of chemicals such as carotenes. However, 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.

This may be due in part to several factors including the difficulty of maintaining high dissolved oxygen levels due to the high oxygen demand developed by the high concentration of microorganisms in the media. Conventional methods to maintain higher dissolved oxygen level include increasing the aeration rate and/or using pure oxygen instead of air for aeration and/or increasing the agitation rate in the fermentor/bioreactor .

These solutions generally increase the cost of compound production and capital cost of fermentation equipment but can also cause additional problems. For example, increased aeration can easily lead to severe foaming problems in the fermentor, since high cell densities and increased mixing can lead to microbial cell breakage due to increased shear forces in the media. This causes the lipids, e.g. DHA to be released in media where they can become oxidized and/or degraded by enzymes. Microbial cell breakage is an increased problem in cells that have undergone nitrogen limitation or depletion to induce lipid formation, resulting in weaker cell walls.

Consequently, a system in which precise control of these operating conditions is accomplished will permit not only large scale culturing of algae which cannot now be grown optimally by existing commercial technology but also the mass production of compositions comprising omega-3 fatty acids which are not currently available on an economic basis in the marketplace. It is therefore a first aspect of the present invention to provide a method for cultivating a heterotrophic microalgae where the optimal conditions are provided throughout the growth of the algae and during production of the desired composition.

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.

It is a third aspect according to the present invention to provide a method for providing an improved heterotrophic microalgae biomass production.

It is a fourth aspect of the present invention to provide a composition comprising microalgae biomass having a high concentration of DHA and wherein the composition lacks the undesirable fish odor/taste and which has a reasonable shelf life.

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.

It is a sixth aspect according to the present invention to manufacture said composition over a relatively short period of time and at the same time obtain a high yield, utilizing an inexpensive medium and simple steps for production.

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:

a) cultivating the microalgae in a first nutrient medium in order to obtain a first microalgae suspension, said first nutrient medium is arranged for preparing the microalgae for growth,

b) cultivating the first microalgae suspension obtained in a) in a second nutrient medium in order to obtain a second microalgae suspension, said second nutrient medium is arranged for stimulating the growth of the microalgae, and

c) cultivating the second microalgae suspension obtained in b) in a third nutrient medium in order to obtain a third microalgae suspension, wherein the content of nitrogen sources in said third medium is limited in order to induce said microalgae to produce docosahexaenoic acid at a concentration of at least 20 grams per liter of nutrient solution.

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.

In step a) according to the present invention 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.

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.

In 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.

In 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. However, 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) .

Adding a different nutrient media in each of the three different steps, where 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. At the same time 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. In this respect it is important to notice that the content of the relevant medium will depend on the type of microalgae however the person skilled in the art will based on the information in the present application be able to modify the media disclosed in this application and arrive at an optimal medium for any specific microalgae.

As stated earlier it is important that step a) provides healthy heterotrophic microalgae that will be able to complete fermentation in step b) and c) . Conventionally 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. This will have the advantage that any undesirable water-soluble compounds which e.g. can have a negative effect on the growth of the heterotrophic microalgae or the production of the composition according to the invention easily can be removed and thereby reduce the undesirable effect thereof.

During step a) 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) . In this respect conventional methods of determination cell motility and/or the size of the algae can preferably be used.

Using a preferred specie Crypthecodinium cohnii, it is desirable to obtain cells having a size of about 2-10 μm in step a) , since this indicates that the algae have adapted to the first nutrient medium.

Thereafter, 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. As the transfer to the second nutrient medium is a continuous process, 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 . As for step a) samples can also be obtained during step b) in order to establish when the desired biomass of between 10 and

50 g/L nutrient medium is obtained. At this stage Crypthecodinium cohnii will preferably have a size of 10-20 μm.

When the desired biomass concentration is obtained 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.

Alternatively can 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.

During 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.

In general, the 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.

In any case 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.

It is a well known problem with the conventional technology that when cell densities reaches a relatively high level, the accumulation of potentially toxic metabolites or shifting of the optimal pH-value can become significant with the consequence of limiting further growth of the cells. This growth limitation makes it impossible and uneconomical to grow the microalgae on an industrial large scale.

Thus, it is preferably that the pH-value in at least the first and second nutrient medium is maintained at a substantially constant value. However, since 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. By replenished is meant 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 fact that the culture medium is replenished in the separate container, and not directly in the fermentor as in e.g. conventional batch systems, provides a number of benefits.

As a first benefit can be mentioned that only the culture media is removed from the fermentor and then transferred to a separate container. As no cells are present in the separate container the stirring/mixing in the container can take place at high speed, using relatively high forces, making it very easy and convenient to replenish the medium with additional nutrients and/or adjust different parameters such as the pH- value, thereby providing a simple and inexpensive way of controlling the media without physically affecting the algae.

Thus, the use of a separate container for replenishing the nutrient mediums gives an obvious cost benefit by both reducing valuable time for e.g. adjusting pH or adding nutrients, and a the same time obtaining a high cell density, i.e. a relatively high amount of desired composition.

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.

As mentioned previously 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.

However, a specific beneficial effect is obtained when dissolved oxygen (DO) is added to a lower part of the fermentor, irrespectively of the fermentor is using a nutrient recycling system, is a feed-batch fermentor or the like. The DO will due to laws of nature ascends to the top of the fermentor and provide a very gentle mixing with the microalgae.

The addition of 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.

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 .

The vast majority of 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 .

Thus, in order to mimic the natural conditions for the heterotrophic microalgae as much as possible, 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.

Since the 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.

When the first nutrient medium comprises a plant oil e.g. rapeseed oil, this has proven especially beneficial for the exponential growth phase of the heterotrophic microalgae in step b) as said algae can obtain a specific growth rate of at least 0.15 h¹ at 27⁰C and a generation time of at least 3 d¹.

As described earlier is 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. However, 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 .

Thereby is obtained a 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.

It has further been discover by the present inventors that the brown stains which conventionally is present on the agitator and the upper half of a fermentor if the nutrient medium contains a yeast extract are completely eliminated when the nutrient medium comprises a plant oil, such as rapeseed oil.

Conventionally such brown stains are removed using mechanical means, rendering the cleaning both laboriously and expensive. However, using the media according to the present invention eliminates/reduces the need for such mechanical cleaning, whereby e.g. a faster cleaning of the fermentation equipment is obtained.

Those of skill in the art know acceptable carbon sources for use in the second and third nutrient medium. For example, carbon 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) .

In general terms it is desirable to stress the heterotrophic microalgae to such an extend that they start producing the desired composition according to the invention.

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. Using glucose and yeast extract as examples, 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. Generally C. cohnii will grow at temperatures ranging from about 15°C to 34°C. Preferably 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.

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. During the later stages of the fermentation, the culture medium tends to be lowered, if desired, 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. Mostly 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. As examples of 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 inventors have proven that 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) . As used herein, 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.

As one example can be mentioned 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) .

As described above 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. However 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 .

In the embodiments described above it is contemplated that 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%. At this stage the heterotrophic microalgae has a cell size of 20 - 36 μm. Thereby is obtained the advantage over the prior art that additional steps of extracting the desirable compounds are eliminated. Furthermore, since the extraction often involves potential toxic solvents, the final product according to the invention if fee of residues from the organic solvent. As the DHA and other PUFA's are located within the algae, the oils are not subjected to oxidation or degradation, whereby the side- compounds normally associated with the fishy smell and/or taste are not produced. In addition, the composition according to the present invention is cholesterol free, contaminant free [e.g. heavy metals, polychlorobiphenyls (PCBs)], and taste good.

Even though it is not needed to extract the relevant compositions according to the invention, such an extraction step would also be within the scope of the present application.

In these cases the composition according to the invention can be extracted from the harvested material using an effective amount of solvent. Those of skill in the art can determine suitable solvents.

Absorption of the composition according to the invention is best achieved in the small intestine of the human or animal body. Thus, the 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.

As used herein the term "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.

In the convention fluidized bed 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 μm) , uniformly to complex shaped products. It is possible to build a film thickness of 100 mils (2500 μm) using higher preheat temperatures and multiple dips.

The person skilled in the art would understand that other conventional coating techniques also can be used according to the invention.

The oil drops are preferably encapsulated with a polymerisable material of natural origin such as alginat . Using 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.

The invention will be explained in greater detail below where further advantageous properties and example embodiments are described with reference to the examples.

EXAMBLES:

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:

Each of the following ingredients listed in table 1, except the rapeseed oil, are added one at a time to 500 ml of distilled water with a temperature of 40°C, while it is stirred. One ingredient is completely dissolved before the next ingredient is added.

When all the ingredients are dissolved 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.

Thereafter 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. First nutrient medium Second nutrient medium Third nutrient medium

2.6% NaCI 2.6% NaCI 2.6% NaCI 1% yeast extract 5.0 % glucose 20% glucose 1% rapeseed oil 1 % yeast extract 1 % rapeseed oil 3.5% ethanol 30% malt 2.5% hop Tabel 1

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 * 10⁶ 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 μm.

Step b)

Into a 1000-liter working volume STF was loaded 200 liter of the second nutrient medium obtained in example 1. 300 liter of the first algae suspension obtained in step a) was added to said second nutrient 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 (second nutrient medium) was continuously added to the STF, until a complete volume of 1000 liter of the second algae suspension was obtained.

Step c)

Into a 25,000-liter working volume STF was loaded 1000 liter of the third nutrient medium obtained example 1. The 1000 liter of the second algae suspension obtained in step b) was added to the medium. Agitation was set at 275 rpm, dissolved oxygen (DO) to 4.5 mg/1, the temperature was set to 15°C. The pH-value was continually adjusted to a pH of about 7.5.

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.

Harvest

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 .

Example 3: Monitoring the effect of pH-control in step b)

Into 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.

Dissolved oxygen (DO), 4.5 mg/1, was added in periods of 30 minutes to the bottom of the fermentor, interrupted with 3 h without addition of DO - this cycle was repeated throughout the experiment. The temperature was set to 23.5 °C . 10 percent per volume inoculums from a seed fermentor containing about 14.2 * 10⁴ 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 .

After the 66 h the pH-value was adjusted to about 7.8 by adding 0.5 M NaOH to the separate container, and the pH was continuously adjusted until the present experiment was halted after 258 h. Results

The results is depicted in fig. 1 and clearly shows that during the first 66 h, where the pH-value was not adjusted, the pH- value continued to decrease and the cell density of the microalgae substantially stagnated.

Furthermore, as the pH-value was adjusted to about 7.8 the microalgae immediately started to grow, having a minor lag- phase followed by an exponential phase.

These results clearly shows that it is not possible to grow heterotrophic microalgae on a large-scale basis, as the pH- value will affect the growth to such an extend that it neither beneficial nor economical to manufacture the composition according to the invention on a large-scale basis.

Furthermore, if the pH-value is maintained at the optimal level about 7.8 using the recycling system according to one embodiment according to the present invention, 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

Step c)

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.

Harvest

Claims

CLAIM

1. A method of producing an edible composition comprising biomass from at least one heterotrophic microalgae having a content of docosahexaenoic acid (DHA) , said method comprises cultivating, in at least one fermentor, said microalgae in three different nutrient media in the following steps: a) cultivating the microalgae in a first nutrient medium in order to obtain a first microalgae suspension, said first nutrient medium is arranged for preparing the microalgae for growth, b) cultivating the first microalgae suspension obtained in a) in a second nutrient medium in order to obtain a second microalgae suspension, said second nutrient medium is arranged for stimulating the growth of the microalgae, and c) cultivating the second microalgae suspension obtained in b) in a third nutrient medium in order to obtain a third microalgae suspension, wherein the content of nitrogen sources in said third medium is limited in order to induce said microalgae to produce docosahexaenoic acid at a concentration of at least 20 grams per liter of nutrient solution.

2. A method of producing an edible composition comprising biomass from at least one heterotrophic microalgae having a content of docosahexaenoic acid (DHA) , wherein step b) in the method according to claim 1 is modified in that the biomass from the first microalgae suspension is retained in the fermentor, and the first nutrient medium continuously is 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.

3. The method according to claim 2, wherein step c) in the method according to claim 1 is modified in that the biomass from the second microalgae suspension is retained in the fermentor, and the second nutrient medium continually is removed from the fermentor while the third nutrient medium is transferred to the fermentor, said removal/transfer is continued until the nutrient medium in the fermentor substantially consists of the third nutrient medium.

4. The method according to claim 1, 2 or 3, wherein the biomass from the microalgae is retained in the fermentor and the fermentor is placed in a recycling fluid communication with a separate container, wherein the method comprises the following step

- continuously removing nutrient medium from the fermentor during the cultivation in at least one of step a) , b) and c) and transferring the nutrient medium to the separate container, - replenishing and/or monitoring the nutrient medium in the separate container, and

- continuously recycling the replenished nutrient medium to the fermentor.

5. The method according to claim 4, wherein the at least one of the first, second or third nutrient medium is replenished by adjusting the pH-value to a substantially constant value in said separate container.

6. The method according to any of the claims 4 or 5, wherein the at least one of the first, second or third nutrient medium is replenished by adding nutrients to the first and/or second nutrient medium in said separate container.

7. The method according to any of the claims 1 - 6, wherein dissolved oxygen (DO) is added to a lower part of the fermentor in order to provide a stirring effect in the fermentor .

8. The method according to claim 7, wherein DO is added to the fermentor 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.

9. The method according to any of the claims 1 - 8, wherein the method further comprises harvesting the biomass from the third microalgae suspension.

10. The method according to claim 9, wherein the method further comprises cleaning and/or purifying and/or drying the harvested biomass.

11. The method according to any of the preceding claims, wherein the heterotrophic microalgae is of the class Dinophyceae, preferably of the genus Crypthecodinium or Schizochytrium, preferably the species Crypthecodinium cohnii .

12. The method according to any of the preceding claims, wherein the temperature in the first cultivation step a) and/or second cultivation step b) are about 25°C to about 30°C, preferably about 27°C.

13. The method according to any of the preceding claims, wherein the temperature in the third cultivation step c) are about 13°C to about 18°C, preferably about 15°C.

14. The method according to any of the preceding claims, wherein the first nutrient medium comprises 2-4% NaCl 0.1- 2.0% yeast extract, 0.1-2.0% rapeseed oil, 10-50% malt, 0.5-3.0% hop, the remaining being distilled water.

15. The method according to any of the preceding claims, wherein the second nutrient medium comprises 2-4% salt, 0.5-30% glucose, less than 0.1% rapeseed oil, and 0.1-2.0% yeast extract, the remaining being distilled water.

16. The method according to any of the preceding claims, wherein the third nutrient medium comprises 2-4% salt, 0.5-50% glucose and 0.1-2.0% rapeseed oil, the remaining being distilled water.

17. The method according to any of the preceding claims, wherein the first, second and third culture medium has a pH value between 5.0 and 9.0, preferably, a pH value about 7.5.

18. A nutrient medium according to any of the claims 14 - 17.

19. An edible composition obtainable by the method according to any of the claims 2 - 17, comprising at least 25- weight% DHA.

20. A method for micro- or nanoencapsulating the edible composition according to claim 19, said method comprises preparing core particles of the edible composition according to claim 19 and coating the core particles with a coating polymer.

21. The method according to claim 20, wherein the coating polymer is alginate.

22. Micro-or nanocapsules obtained by the method according to any of the claims 20 or 21.

23. A dietary supplement comprising the micro-or nanocapsules according to claim 22 or the composition according to claim 19.

24. A pharmaceutical composition comprising the micro-or nanocapsules according to claim 22 or the composition according to claim 19.

25. Use of the pharmaceutical composition according to claim 24 for reduction of cardiovascular and inflammatory diseases or reduction of depression or increasing length of gestation in the third trimester or and inhibiting tumor growth.