NL2025592B1

NL2025592B1 - Drink composition comprising microalgae

Info

Publication number: NL2025592B1
Application number: NL2025592A
Authority: NL
Inventors: Mik Ivana; Streuli Julia; Prat Cristina; Guaglio Sara
Original assignee: Ful Foods B V
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2021-11-30

Abstract

The current invention relates to a method of producing a microalgae comprising drink, a method for producing a microalgae dried product and to such drink and dried product. Preferably fresh microalga is used in the methods disclosed. The obtained drink and dried product are characterized by excellent taste, smell and texture and can be used in food and feed products. The invention includes a step of fermentation using lactic acid producing bacteria.

Description

Title: Drink composition comprising microalgae Background of the invention The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Microalgae have been part of the human diet since thousands of years and microalgae have been used as a nutrient-dense food source. Historical records suggest that microalgae such as Spirulina platensis were consumed by tribes in Africa and by the Aztecs in Mexico.

During the last decades there has been increasing interest in the commercial production of food-grade microalgae for human consumption and/or as feed for livestock. One of the reasons is that microalgae have demonstrated potential to meet the population's need for a more sustainable food supply, specifically with respect to protein demand. Additionally, microalgae produce several bioactive compounds with potential benefits for human health.

Microalgae as a sustainable source of proteins is a rather new idea and could significantly contribute to meet the population's need for protein, with several advantages over other currently used protein sources. Microalgae-based proteins have low land requirements compared to animal-based proteins and are also lower than for some other plant-based proteins.

Among the various microalgae that have been explored for their commercial potential Arthrospira species (generally referred to as Spirulina) and Chlorella species are two major types of microalgae that have been successfully produced and are in widespread use. Both Arthrospira and Chlorella can provide high-quality proteins with a well-balanced amino acid profile.

In addition, microalgae as part of the diet may provide potential benefits for health due to the presence of bioactive compounds in the microalgae (for example, antioxidative, antihypertensive, immunomodulatory, anticancerogenic, hepato- protective, and anticoagulant activities have been attributed to microalgae-derived peptides).

Despite the fact that microalgae clearly show potential as part of sustainable food solutions, utilization of microalgae or microalgae-derived products in food products is limited. This is in part due because of the underdeveloped technologies and processes currently available for microalgae processing towards high quality and palatable food products.

Microalgae have, for example been incorporated in such products as cookies, biscuits, bread and past as these products allow for reasonable acceptance of taste, texture, and appearance, but most microalgae is still presented in the form of food supplements and tablets.

Indeed the incorporation of microalgae into traditional products has been found inconvenient because of its color, fishy taste, and odor, as well as its powdery consistency and appearance, all adversely affecting consumers’ perception about taste and quality. All these aspects constitute main areas for improvement. For example, it has not yet been possible to sufficiently disguise the taste and odor of microalgae, therefore, limiting the amount to be used in products.

Another issue is the fact that freshly harvested microalgae can only be preserved for up to a few days. Microalgae are therefore commercialized, mostly, as dried powders to facilitate their use as food ingredients and to allow easy transportation and long-term stability. The drying process of course increases energy need, reducing the overall sustainability of the product. In addition, these dried or dehydrated microalgae powders are characterized by loss of nutritional value and a fishy smell.

In light of this, new microalgae-based food products, compositions, methods and uses would be highly desirable. In particular, there is a clear need in the art for sustainable microalgae comprising products and methods for producing such products, wherein the products meet nowadays standards of sustainability, taste, odor and nutritional value and quality. Accordingly, the technical problem underlying the present invention can be seen in the provision of such products, compositions, and methods for complying with any of the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below. Description Drawings Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1: Increase in solubility using enzymes, fermentation and combinations thereof.

Definitions A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

For purposes of the present invention, the following terms are defined below.

As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a bacterium” includes a combination of two or more individual bacteria, and the like.

As used herein, “about” and “approximately”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 120% or £10%, more preferably 5%, even more preferably +1%, and still more preferably £0.1% from the specified value, as such variations are appropriate to perform the disclosed invention.

As used herein, “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, “at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6,7,8,9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term "at most” a particular value means that particular value or less. For example, "at most 5” is understood to be the same as "5 or less" i.e, 5, 4, 3,-10, -11, etc.

As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”.

As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art. and are discussed, in various handbooks and literature references.

As used herein, “anaerobic fermentation”, refers to a metabolic process (the breakdown of molecules to obtain energy) occurring in the absence of oxygen or in the presence of reduced oxygen and by which sugars are converted into cellular energy and other metabolites. In case of lactic acid fermentation, a type of anaerobic fermentation, sugars are converted into cellular energy and lactate (lactic acid in solution). The term included facultative anaerobic fermentation. Anaerobic fermentation incudes, for example, fermentation in de-aerated water (water with reduced oxygen content), as is known to the skilled person.

As used herein, “Arthrospira maxima”, refers to the species Arthrospira maxima of the genus Arthrospira, a free-floating filamentous cyanobacteria (oxygenic photosynthetic) characterized by cylindrical, multicellular trichomes (outgrowths) in an open left-hand helix. Arthrospira spp.. belong to the cyanobacteria, also known as blue-green algae, blue-green bacteria or Cyanophyta.

As used herein, “Arthrospira platensis”, refers to the species Arthrospira platensis of the genus Arthrospira, a free-floating filamentous cyanobacteria (oxygenic photosynthetic) characterized by cylindrical, multicellular trichomes (outgrowths) in an open left-hand helix. Arthrospira spp.. belong to the cyanobacteria, also known as blue-green algae, blue-green bacteria or Cyanophyta.

As used herein, “bacteria inactivation”, refers to killing bacteria and/or to preventing cellular replication of bacteria.

As used herein, “Bifidobacteria”, refers to the major genera of bacteria that make up the gastrointestinal tract microbiota in mammals, including humans. Some

Bifidobacteria are used as probiotics. The genus Bifidobacterium refers to the genus of gram-positive, non-motile, often branched anaerobic bacteria.

As used herein, “Chlorella sorokiniana”, refers to species Chlorella sorokiniana of the genus Chlorella, a freshwater green microalga with a characteristic emerald- 5 green color and pleasant grass odor.

As used herein, “Chlorella spp.”, refers to one or more species of the genus Chlorella.

As used herein, “fermentation”, refers to a metabolic process (the breakdown of molecules to obtain energy) by which sugars are converted into cellular energy and other metabolites, such as lactic acid.

As used herein, “fresh microalga”, refers to (living) microalga that have not been dried or dehydrated. The term is intended to mean a non-dried, non-dehydrated microalga harvested less than 48 hours previously, but also included microalga that after harvested as immediately be cooled or frozen, without drying or dehydration.

As used herein, “inoculating”, refers to the introduction of bacteria or microalgae to a medium.

As used herein, “lactic acid producing bacterium”, refers to any bacterium that mainly or predominantly produces lactic acid during fermentation. A homofermentative lactic acid producing bacterium converts sugars to lactic acids as the only or major end-product, while a heterofermentative lactic acid producing bacterium may produce lactic acid and additional metabolites such as ethanol, acetic acid and carbon dioxide.

As used herein, “Lactobacilli”, refers to bacteria of the genus Lactobacillus of Gram-positive, facultative anaerobic, rod-shaped, non-spore-forming bacteria. They are a major part of lactic acid producing bacteria, converting sugars into lactic acid.

As used herein, “Lactobacillus plantarum”, refers to species Lactobacillus plantarum of the genus Lactobacillus of Gram-positive, facultative anaerobic, rod- shaped, non-spore-forming bacteria. They are a major part of lactic acid producing bacteria, converting sugars into lactic acid.

As used herein, “lysozyme”, refers to any enzyme that functions as antimicrobial agents by cleaving the peptidoglycan component of bacterial cell walls, leading to cell death.

As used herein, “microalga”, refers to a microscopic alga, including cyanobacteria, typically found in freshwater and marine systems, living in both the water column and sediment. They are unicellular organisms, existing individually or in chains or groups. Depending on the species, the size can range from a few to a few hundred micrometers.

Among the microalga genera largely employed for human consumption, and which may be used in the current invention are Arthrospira, Chlorella and Aphanizomenon and Dunaliella and Haematococcus.

As used herein, “sugar”, refers to a simple sugars being monosaccharides and disaccharides.

Examples are glucose, fructose and galactose, lactose, sucrose and mannose.

Detailed Description It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

Embodiments discussed in the context of methods and/or compositions of the invention may be employed with respect to any other method or composition described herein.

Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.

As embodied and broadly described herein, the present invention is directed to the surprising finding that with the method according to the invention a drinkable composition characterized by relative high amount of microalgae and high quality proteins from microalgae can be obtained and wherein the product is devoid of the fishy taste and odor present in products in the prior art.

In addition, it was found that during the processing of the microalgae with the method of the invention at certain stages volatile components with a sulfuric (rotten egg; hydrogen sulfide, H2S) odor were released and removed from the product.

Experiments showed that these steps dramatically improved overall taste, smell and palatability of the obtained microalgae comprising drink.

In addition, it was found that the method according to the invention allows for the efficient disruption of the cell wall of the microalgae, thereby allowing release of internal content of the microalgae in the drink, increasing overall solubility of the microalgae in the drink formulation {allowing to increase the amount of microalgae used), increasing bio-assimilation and digestibility of the product as well as improved stability of the product while improving texture and appearance of the drink formulation.

Therefore, overall the inventors have found a new and inventive, yet simple, method for producing a microalgae comprising food product, more particularly a microalgae comprising drink formulation.

Test panel evaluation showed that the obtained microalgae-based drink is characterized by a taste, odor, texture and palatability that is highly appreciated by the consumer. Furthermore, the method provides for an effective disruption of the microalgae, causing release of intracellular content, improved solubility and improved texture (mouthfeel) of the drink formulation.

In addition, the product has good shelf life, effectively providing an environmental friendly alternative to drying of fresh algae as a means to preserve fresh microalgae. Furthermore, it was found that the drinkable composition is a healthy alternative "food to go", a food product that is ready to drink and can easily be transported to the place of consumption. The drink may be consumed at different temperatures without any loss of taste.

In a first aspect the present invention relates to a method of producing a drinkable composition the method comprising the steps of: a) providing an aqueous mixture wherein the aqueous mixture comprises water, at least one microalga and a sugar; b) inoculating the aqueous mixture with a lactic acid producing bacterium; C) allowing fermentation of the inoculated aqueous mixture of step b) by the lactic acid producing bacterium.

In the method according to the invention a drinkable composition with the aforementioned advantages and characteristics is obtained by fermentation of an aqueous mixture comprising, next to water, at least one microalga (species of), and an additional source of sugar.

In the method according to the invention fermentation is performed by a lactic acid producing bacterium. The lactic acid producing bacterium uses the sugar present in the aqueous mixture as energy source, thereby consuming the sugar, while at the same time producing lactic acid and fermenting the microalga present in the aqueous mixture.

After the fermentation a drinkable composition is obtained wherein the composition is characterized by a well-accepted taste, odor, and texture and is received as highly palatable.

In a preferred embodiment the aqueous mixture is treated, most preferably during fermentation with enzymes, such as a peptidase, protease, lysozyme and/or glycosidase.

More specifically, in a first step, an aqueous mixture in prepared or obtained comprising, next to water, at least on species of a microalga and an additional source of sugar, i.e. a source of sugar other than the sugars that may naturally be present in the microalga. The aqueous mixture may for example be prepared by first mixing the microalga and the additional source of sugar and then adding water while steering.

Although not in particular limited to a specific temperature, it was found that preferably the temperature of the water used for preparing the aqueous mixture has a temperature of between 4 — 25 °C, preferably between 10 — 25 °C.

The microalga used in preparing the aqueous mixture may be any microalga that may suitable be included in a food product, i.e. which can safely be consumed by a person, and at least in the amounts provided in the end product. The microalga may be provided as dried microalga as most of the microalga present as raw material in de market is available a dried/dehydrated microalga, in particular in the form of microalga powder.

However, and importantly, in a preferred embodiment, the microalga that used in the aqueous mixture, and therefor present in the drink composition according to the invention is fresh microalga, i.e. microalga that has not been extensively dried after harvesting. Normally fresh microalga are harvested less than 48 hours, 24 hours or even less than 12 hours previously, depending on the microalga. The fresh microalga may after harvest also be cooled or frozen and used in the method of the invention. It was found that although dried microalga may be used in the method according to the invention, and therefore in the drinkable composition of the invention, fresh microalga provides for a much more pleasant and palatable end product. For example, in performing the method according to the invention, it was found that unpleasant odor was released more efficiently when using fresh microalga when compared to dried microalga. Indeed overall, a better quality product was obtained when using fresh microalga instead of dried microalga. Better quality is for example seen in higher solubility of e.g. the microalga as well as stability of the product, bioavailability, and digestibility of the nutrients. Despite the fact that the use of fresh form of microalga is not widespread owing to the fact that it rapidly rots shortly after harvesting, it was found that in the context of the current invention fresh microalga are preferred.

Therefore, although dried microalga may be used in the current invention, preferably fresh microalga are used. Of course, in some embodiment, a combination of both fresh and dried microalga may be used.

In some embodiments according to the invention, only one type (species) of microalga is used. In some embodiments more than one type (species) of microalga, i.e. a combination of microalgae is used.

In some embodiments according to the invention, between 1 — 25 wt.% fresh microalga is used in the method or the product of the invention, preferably 1 — 20 wt. %, 1-15 wt.%, or 1 — 10 wt.%, in particular Arthrospira.

The aqueous mixture further comprises sugar. The sugar is added to the aqueous mixture in addition to the microalga. The sugar may be added in pure form (e.g. glucose or sucrose) or may be provided to the aqueous mixture as part of a composition comprising other components. In some embodiments one type of sugar is provided (e.g. sucrose). In some embodiments a mixture of sugars are provided (e.g. sucrose, lactose and glucose, of fructose and galactose). In a preferred embodiment, the sugar is provided in the form of a (concentrated) juice, for example apple juice or concentrated apple juice. The skilled person will understand there is no limitation with respect to the type of sugar or combination of sugars that are used in the context of the invention as long as at least one of the sugars may be utilized as an energy source by the lactic acid producing bacterium in the subsequent fermentation step.

Although not in particular limited to any specific pH value, in a preferred embodiment the pH value of the aqueous mixture is set to a pH that is below pH 6.0, preferably below pH 5.8, and preferably above pH 4.0 at 20°C, for example between pH 4.0 and pH 6.0. It was found that with pH values between these limits the best drinkable compositions were obtained by the method of the invention. In some embodiments the pH value is set by using strong or weak acids or bases. In some embodiments, the acids or bases are provided to the aqueous composition as pure compounds. In some embodiments natural sources, for example juices comprising acids such as lemon or lime juice are used to set the pH value of the aqueous mixture.

Although not in particular limited, preferably the aqueous mixture is prepared in or provided to a sterile or non-sterile mashing tank.

In a next step of the method of the invention, the aqueous mixture is inoculated with a living lactic acid producing bacterium, for example of the genus Lactobacillus.

The lactic acid producing bacterium may be any kind of bacterium that may safely be used in the preparation of food products. The skilled person is well aware of the various lactic acid producing bacteria that are available to him for use in the method of the invention.

The lactic acid producing bacterium may be provided to the aqueous mixture by any mean known to the skilled person. In some (preferred) embodiments the lactic acid producing bacterium is provided in the form of a starter culture (or fermentation starter — comprising metabolic active bacteria in a suitable medium) and may already be metabolic active. In other embodiments, the lactic acid bacterium is provided as freeze dried material. Preferably one type of lactic acid producing bacterium is used, although in some embodiment a combination of different lactic acid bacteria may be used.

In a next step the inoculated aqueous mixture is allowed to ferment. The skilled person is well aware of suitable conditions to allow fermentation of the inoculated aqueous mixture, for example with respect to temperature, pH (control), stirring, oxygen tension and duration. For example, during fermentation pH, temperature and the like may be monitored and if so desired adjusted in order to allow for optimal fermentation of the inoculated aqueous mixture comprising the at least one microalga.

The sugars in the inoculated aqueous mixture may serve as an energy source for the lactic acid producing bacterium and during fermentation of the aqueous mixture comprising the microalga, the bacterium will consume these sugars and produce lactic acid. For example, by following the pH value fermentation may be followed. It may for example be decided to stop fermentation at a certain pH value or after a certain amount of time. Alternatively pH may be adjusted during fermentation and monitored as well. Is some embodiments fermentation is stopped when most of the sugars that were added to the aqueous mixture have been consumed, for example when 50%, 60%, 70%, 80%, 90%, 95% of the sugars initially provided in the aqueous mixture have been consumed during fermentation.

As will be understood by the skilled person, allowing large part of the sugars to be fermented in this step can provide for a drinkable composition that is characterized by low sugar content, or can be marketed as sugar-free.

Preferably the fermentation is performed at a temperature that allows good to optimal metabolic activity of the lactic acid producing bacterium. Preferably fermentation is at a pH that allows for good to optimal metabolic activity of the lactic acid producing bacterium. Depending on the lactic acid bacterium (or combination of lactic acid bacteria) used in the fermentation, the fermentation may be under aerobic or under anaerobic conditions. Preferably the fermentation is under anaerobic conditions (i.e. conditions where no oxygen is provided to the fermentation). Preferably the fermentation is performed under conditions wherein the lactic acid bacterium is homefermentative, i.e. wholly or principally producing lactic acid from the sugars.

It was found that, within the context of the invention, the most desirable results were obtained for the drinkable product according to the invention when indeed large part of the sugars, for example more than 80%, 90%, or 95% of the sugars, are consumed during homefermentative fermentation, thereby providing a drinkable composition according to the invention.

In preferred embodiments of the method according to the invention, the at least one microalga is selected from the group consisting of Arthrospira spp.., Arthrospira platensis, Arthrospira maxima, Chlorella spp.., Chlorella sorokiniana and combinations thereof.

These microalgae are well-known to the skilled person and have as such extensively been analyzed and characterized.

As described herein, it is contemplated that more than one of the above microalgae are combined in the method and product of the invention.

For example, a combination of one or more Arthrospira and one or more Chlorella, and in any suitable ratio, may be used.

In a preferred embodiment only Arthrospira is used, for example Arthrospira platensis, Arthrospira maxima, or a combination thereof, in the method and drinkable composition according to the invention.

Preferably fresh Arthrospira spp., Arthrospira platensis, Arthrospira maxima, Chlorella spp., Chlorella sorokiniana and combinations thereof is used.

In preferred embodiments a method according to the invention is provided wherein between step a) and step b) the aqueous mixture of step a) is heated at a temperature of at least 60°C, preferably at least 70°C, preferably for a period of between 30 seconds and 30 minutes.

It was found that if the aqueous mixture is heated until a temperature of above 60°C, preferably above 70°C, and kept at this temperature for a period of between 20 seconds and 45 minutes, this may further improve the quality (taste, odor, texture, palatability) of the drinkable composition according to the invention.

In some embodiment the temperature is between 60°C and 90°C, for example between 70°C and 80°C.

In some embodiment the period is between 20 seconds and 10 minutes or between 45 seconds and 5 minutes.

Without being bound by theory it was observed by the inventors that, in particular when fresh alga is used, during this step it appears that undesirable components, for example have bad odor (for example H2S from sulfur compounds that are present in the microalgae) are released and that therefor this step, and before inoculation and fermentation is started, therewith contributes to the quality of the product according to the invention, in particular with respect to taste, odor, but possibly also texture and mouthfeel, and therewith to palatability of the drinkable composition.

The skilled person understand that the heating step described above may be performed using any suitable means, for example by using a boiling kettle or the like.

In preferred embodiments a method according to the invention is provided wherein after the aqueous mixture of step a) is heated, as described above,, the aqueous mixture is cooled down, preferably to a temperature below 45°C and preferably within a time period of 0.1 — 15 minutes, preferably wherein the aqueous mixture is cooled down by addition of water to the aqueous mixture, preferably wherein the volume ratio aqueous mixture: added water is from 1:3 to 3:1.

Without being bound by theory, a relevant aspect in providing good quality drinkable compositions comprising (fresh) microalga is believed to reside in the need to, at least partially, disrupt the microalga in the aqueous composition, preferably before commencing fermentation.

It was surprisingly found that cooling the aqueous mixture from a temperature of between 60°C and 90°C, after the heating step as described above, to a temperature below 45°C and within a short period of time, improves the quality of the end product, the drinkable composition according to the invention.

It was observed that during this quick cooling step the microalga appear to be disrupted as was witnessed by the release of the blue pigment phycocyanin from Arthrospira, for example Arthrospira platensis. Phycocyanin is a pigment-protein complex normally adhered to the membrane of the microalga and released upon rupturing of the cell wall. Phycocyanin is used as a pigment in various applications and is normally released from the microalga using mechanical forces like freeze-thawing.

The cooling of the aqueous mixture may be performed using any suitable means known to the skilled person. In a preferred embodiment quick cooling down is achieved by the addition of (cold) water to the heated aqueous mixture. It was found that by adding water to the aqueous mixture, temperature can be reduced to below 45°C and within a short period of time, whereas it may also be that the addition of water may additional cause an osmotic shock.

In a preferred embodiment the cooling down to a temperature below 45°C is within atime period of 0.1 — 10 minutes, 0.1 — 5 minutes, or even within less than one minute.

In a preferred embodiment the aqueous mixture is cooled down to a temperature of less than 40°C, less than 38°C, or less than 37°C. Preferably the aqueous mixture is cooled down, preferably by the addition of water, to a temperature that is suitable for performing the fermentation as described above. Preferably the aqueous mixture is cooled down to a temperature no lower than 30 °C.

Preferably, when (cold) water is added to the aqueous mixture for cooling down, in order to quickly cool down, the volume ratio aqueous mixture: added water is from 1:3to 3:1.

As will be understood by the skilled person, in a preferred embodiment the above-described heating and cooling step are performed, and preferably performed immediately after each other. In other words, in a preferred embodiment of the method according to the invention, the aqueous mixture is for heated at a temperature and for a period described above, and immediately after said heating step, for example, within a period 0 — 5 minutes the aqueous mixture is cooled down, preferably by the addition of (cold) water.

In preferred embodiments a method according to the invention is provided wherein the aqueous mixture that is inoculated with the lactic acid producing bacterium in step b) comprises between 0.5 — 4 wt.% sugar, preferably wherein the sugar is selected from the group consisting of glucose, fructose, galactose, lactose, sucrose, and mannose and combinations thereof.

Although the invention is not in particular limited to an lower or upper limit with respect to the sugar content in the aqueous mixture, the skilled person will understand that too low sugar will hamper efficient fermentation whereas to high levels of sugar may likewise not be beneficial for an efficient fermentation, for example because only a small part of total sugar may be consumed during the period of fermentation, or too much lactic acid producing bacterium may end up in the product, or the products becomes too acidic.

After having performed various test with different concentrations of sugars it was found that preferably the aqueous mixture comprises between 0.5 — 4 wt.% sugar before the fermentation is started, preferably between 1.0 and 2.5 wt.% sugar.

Preferably the sugar is selected from the group consisting of monosaccharides and disaccharides, in particular glucose, fructose, galactose, lactose, sucrose, and mannose and combinations thereof. As discussed above, the sugars may be provided as pure compounds, alone or in combination, and/or are may be provided as part of a composition, for example as comprised in particular fruit juices such as apple juice (comprising, for example glucose, fructose, and sucrose)./nlp

In preferred embodiments a method according to the invention is provided wherein the lactic acid bacterium is selected from Bifidobacteria and/or Lactobacilli, more preferably wherein the bacterium is Lactobacillus plantarum. The inventors performed test with a wide variety of bacteria, including single species as well as combination of different species, and although acceptable results were obtained with various different types, best results were obtained with lactic acid producing bacteria, in particular, Bifidobacteria and/or Lactobacilli, including various Lactobacillus species such as Lactobacillus delbruecki, Lactobacillus brevis and Lactobacillus plantarum, with the latter being most preferred.

In preferred embodiments a method according to the invention is provided wherein between 0.1 - 5x10A10 CFU/L, preferably between 1 — 3x10410 CFU/L of the bacterium is inoculated. Although the skilled person understand that different amounts (CFU) may be used, it was found that the combination of the microalga, the preferred amount of sugar in the aqueous mixture and the preferred amount of bacteria, provide a very good results with respect to the end product, the drinkable composition according to the invention.

In preferred embodiments a method according to the invention is provided wherein the aqueous mixture that is inoculated with the lactic acid producing bacterium in step b) has a pH of 4.0 — 6.0, preferably 4.5 — 5.4, and/or a temperature between 30 — 40°C.

It was found that, in combination with the other parameters disclosed herein, such starting pH and/or temperature are optimal for producing the drinkable composition according to the invention.

In preferred embodiments a method according to the invention is provided wherein fermentation in step c) is at a temperature of 30 — 40°C, and/or wherein fermentation is for a period of between 2 — 24 hours, preferably wherein fermentation is between 2 — 8 hours at a temperature between 32 - 38°C, preferably wherein the fermentation is anaerobic fermentation.

It was found that by optimizing the various parameters as disclosed herein fermentation may be as short as only 2 or 4 hours, and may be as long as 24 hours. It was found that within these broad time range good quality end products, i.e. drinkable compositions according to the invention are obtained. It was for example found that it is possible to obtain end product with very low sugar content, indication that almost all sugars originally present in the aqueous mixture before fermentation have been consumed by the lactic acid producing bacteria, and such products were found to have very good taste, smell and texture. In preferred embodiments a method according to the invention is provided wherein before step c), during step c) and/or after step c), preferably at least during step ¢), the (fermented) aqueous mixture is treated with a glycosidase, preferably wherein the glycosidase is selected from the group consisting of lysozyme, beta-glucanase, xylanase, pentosanase, cellulase, alfa-amylase, arabanase, and/or hemicellulase, a protease and/or lipase. Preferably treatment comprises at least the use of lysozyme, and/or a protease.

It was surprisingly found, in particular during fermentation, the further addition of these enzymes dramatically improves solubility of the various ingredients, in particular of the microalga in the aqueous mixture. Thus where fermentation as such already improved solubility of the microalga in the aqueous mixture, the additional, preferable simultaneous, treatment of the aqueous mixture with one or more of the above enzymes further improves solubility of the microalga. The skilled person is aware of the above-mentioned enzymes and how to use these in the context of the current invention. The enzymes are each alone, of in combination available from various commercial sources.

In preferred embodiments a method according to the invention is provided wherein after step c) the bacteria are inactivated, preferably heat-inactivated. As will be understood by the skilled person, preferably, fermentation is stopped after step ¢) and for this reason the bacteria present in the aqueous mixture may be inactivated and/or killed, for example using techniques known to the skilled person, including sterilization and/or pasteurization.

In preferred embodiments a method according to the invention is provided wherein after step c) further ingredients are provided to the fermented aqueous mixture, preferably wherein the further ingredients are selected from the group consisting of sweeteners, flavors, antioxidants, emulsifiers, preservatives, colorants, acidity regulators, gellants and any combination thereof.

It will be understood by the skilled person that all kinds of sweeteners, flavors, antioxidants, emulsifiers, preservatives, colorants, acidity regulators, gellants and any combination thereof, may be added to the composition according to the invention.

In preferred embodiments a method according to the invention is provided wherein the fermented aqueous mixture comprises between 0.2 — 4.0 wt.%, preferably 0.2 -

1.5 wt. % protein and/or between 0 and 2.5 wt. % sugar. In some embodiments, the composition comprises at least 0.2, 0.3, 0.4, 0.6, 1.0, 1.3 or 1.4 wt. % protein.

In some embodiments, the composition comprises less than 2.4, 2.2, 2.1, 1.5, 1.0 or 0.5 wt. % sugar.

In preferred embodiments a method according to the invention is provided wherein after step c) non-dispersed material is removed from the fermented aqueous mixture.

Although not necessary for the method and/or the product according to the invention, in some embodiments non-dispersed material may be removed, for example by centrifugation and/or filtering.

In preferred embodiments a method according to the invention is provided wherein the aqueous mixture that is inoculated with the lactic acid producing bacterium in step b) is prepared by mixing: a) between 60 — 95 wt.% water; b) microalga between 1 — 25 wt.% fresh microalga and/or between 0.2 — 5 wt.% dried or dehydrated microalga; c) between 0.5 — 4.0 wt.% sugar, preferably wherein the sugar is provided by a juice, preferably apple juice, preferably by 5 — 30 wt.% apple juice; d) optionally 0.1 — 2.5 wt.% lemon juice or lime juice; and e) optionally further ingredients.

In preferred embodiments a method according to the invention is provided wherein, after fermentation, the obtained fermented aqueous mixture is dried to remove water and obtain the fermented micro-alga as dried material.

The dried material may contain up to 10 wt.% water, preferably up to 5 wt.% water.

The skilled person understands that drying may be by any suitable method available in the prior art.

The thus obtained dried product comprising the microalga was found to be very stable and suitable for use in various food and feed products.

Like the drinkable formula obtained with the method of the invention, also the dried product that can be obtained with the method of the invention has good taste, flavor and smell, making it suitable for use in a wide range of products.

The skilled person will understand that when dried product is to be obtained with the method of the invention preferably the weight percentage of microalga in the initial aqueous mixture is at the high end (e.g. 10 — 25 wt. %). According to another aspect of the invention there is provided for a drinkable composition obtainable with the any of the method of the invention as described above.

According to another aspect of the invention there is provided for a drinkable composition as described below.

It will be understood by the skilled person that all features, preferences and characteristics already presented herein within the context of the method according to the invention likewise applies to the drinkable composition according to the invention, and such features, preferences and characteristics will therefore not be repeated below.

According to another aspect of the invention there is provided for a drinkable composition comprising a) between 60 — 95 wt. % water b) between 1 — 25 wt. % fresh microalga and/or between 0.2 — 5 wt. % dried or dehydrated microalga; © juice, preferably apple juice, preferably by 5 — 30 wt. % apple juice; d) optionally 0.1 — 2.5 wt. % lemon juice or lime juice; and e) optionally further ingredients.

In preferred embodiments a drinkable composition according to the invention is provided wherein the drinkable composition further comprises non-replicating lactic acid producing bacterium, preferably wherein the bacterium is selected from Bifidobacteria and/or Lactobacilli, more preferably wherein the bacterium is Lactobacillus plantarum.

In preferred embodiments a drinkable composition according to the invention is provided wherein the at least one microalga is selected from the group consisting of Arthrospira platensis, Arthrospira maxima, Chlorella spp., Chlorella sorokiniana and combinations thereof.

In preferred embodiments a drinkable composition according to the invention is provided wherein the drinkable composition comprises between 0.2 — 4.0 wt. %, preferably 0.2 - 1.5 wt. % protein and/or between 0 and 2.5 wt. % sugar.

In preferred embodiments a drinkable composition according to the invention is provided wherein the drinkable composition further comprises sweeteners, flavors, antioxidants, emulsifiers, preservatives, colorants, acidity regulators, gellants and any combination thereof.

In a preferred embodiment the drinkable composition according to the invention is dried, therewith obtaining a dried product comprising fermented microalga. The thus obtained dried product comprising the microalga was found to be very stable and suitable for use in various food and feed products. Like the drinkable formula obtained with the method of the invention, also the dried product that can be obtained with the method of the invention has good taste, flavor and smell, making it suitable for use in a wide range of products. Therefore in a further aspect of the invention there is provided for a dried product obtainable by the method of the invention. In an embodiment, the dried product comprises: - between 0.01 — 0.1 wt.% lactic acid - between 5 — 20 wt.% carbohydrates - between 20 — 60 wt.% proteins - between 5-10 wt.% fat; and - inactivated lactic acid producing bacteria. The proteins and fat are derived from/obtained from, at least, the microalga used in the method of the invention (protein from microalga, fat from microalga). The skilled person understands that the term protein also comprises amino acids, peptides and the like. The skilled person understands that the term fat also included lipids, diacylglycerols, monoacylglycerols and fatty acids.

Finally also provided is for a bottle or container comprising a drinkable composition according to the invention.

It will be understood that all details, embodiments and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments and preferences for all aspect separately.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Examples Example 1: A drinkable composition was produced according to the method of the invention, using a 5 hour fermentation step.

The product comprised between 84 wt. % water, 4 wt.% fresh microalga, 10 wt.% apple juice and 2 wt.% lemon juice and non-replicating Lactobacillus plantarum. The product comprised about 1.0 wt. % protein and less than 0.2 wt. % sugar (as defined herein i.e. mono- and or disaccharides as disclosed herein).

The final product has a dark green color, it has a fresh tongue-tickling taste, reminding of rhubarb. Slightly sour (acidic), naturally sweet. Added aroma may give a citrusy smell and fizzy aftertaste. It was rated by a test panel as refreshing, delicious and energizing. No fishy taste or smell was reported.

Example 2: In accordance with the method of the invention, several aqueous mixtures of algae and sugars were prepared (as in step a) of the method of the invention). In order to test the effect of enzymes (glycosidase, lysozyme, protease) the aqueous mixture were kept at 60 degrees for 30 minutes in the presence or absence of these enzymes (see samples below), and cooled down and Lactobacillus was added. Mixtures were fermented at 35 degrees for 10 hours. Solubility of supernatant was measured before and after treatment.

Treatments: - 1 sample with enzymes at 60 degrees, no fermentation.

- 1 sample that was treated with enzymes and subsequently fermented.

- 1 sample did not get any enzymes, but was fermented.

- 1 sample did not have any enzymes during 60 degrees treatment, but got enzymes at the same time with lactobacillus.

- 1 with no treatment Solubility was measured by comparing before and after treatment.

Results are reported for Arthrospira platensis. The data showed that both fermentation and enzyme treatment increase solubility by up to 33 % (fermentation) and 32 % (enzymes) relative to no enzyme and no fermentation. The combination of enzyme treatment followed by fermentation further improved solubility by 100% and the combination of enzymes and fermentation at the same time showed a remarkable increase is solubility of more than 125%.

Example 3: Process for powder production:

1. Mix ingredients a. Water: 66% b. Fresh spirulina: 16% c. Lemon Juice: 6% d. Apple Juice: 13%

2. Heat up to 70C

3. Flash Cooling: Adding equal amount of water to cool down.

4. Fermentation + enzymatic treatment (same as the drink)

5. Drying step Final product after drying per 100g: Lactic acid (between 0.01 and 0,1 g of lactic acid}, carbohydrates (between 5-20g), protein (between 20-36 g), fat between (5-109), vitamins and minerals, bacteria and enzymes are inactivated.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention.

Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Claims

CONCLUSIONS

A method for producing a drinkable composition, the method comprising the steps of: a. providing an aqueous mixture wherein the aqueous mixture comprises water, at least a microalgae and a sugar; b. inoculating the aqueous mixture with a lactic acid producing bacterium; c. allowing fermentation of the inoculated aqueous mixture of step b) by the lactic acid producing bacterium.

The method of claim 1, wherein the at least one microalga is selected from the group consisting of Arthrospira platensis, Arthrospira maxima, Chlorella spp, Chlorella sorokiniana, and combinations thereof.

The method according to any one of the preceding claims, wherein between step a) and step b) the aqueous mixture of step a) is heated to a temperature of at least 60°C, preferably at least 70°C, preferably for a period of time between 20 seconds and 45 minutes.

The method according to claim 3) wherein after the aqueous mixture of step a) has been heated, the aqueous mixture is cooled, preferably to a temperature below 45 °C and preferably within a time period of 0.1 - 15 minutes, at preferably wherein the aqueous mixture is cooled by adding water to the aqueous mixture, preferably wherein the volume ratio aqueous mixture : added water is 1:3 to 3:1.

The method according to any one of the preceding claims, wherein the aqueous mixture inoculated with the lactic acid producing bacteria in step b) comprises between 0.5 - 4% by weight of sugar, preferably wherein the sugar is selected from the group consisting of from glucose, fructose, galactose, lactose, sucrose, mannose and combinations thereof.

The method according to any one of the preceding claims, wherein the lactic acid producing bacterium is selected from Bifidobacteria and/or Lactobaccilli, more preferably wherein the bacterium is Lactobacillus plantarum.

The method according to any one of the preceding claims, wherein between 0.2 g/L of 10411 cfu/gm, therefore 2x10 10 CFU/L is inoculated.

The method according to any one of the preceding claims, wherein the aqueous mixture inoculated with the lactic acid producing bacteria in step b) has a pH of 4.0 - 6.0 and/or a temperature between 30 - 40°C.

The method according to any one of the preceding claims, wherein the fermentation in step c) takes place at a temperature of 30 - 40 °C, and/or wherein the fermentation takes place over a period of 2 - 24 hours, preferably wherein the fermentation between 2-8 hours at a temperature between 32-38°C, preferably wherein the fermentation is anaerobic fermentation.

The method according to any one of the preceding claims, wherein before step ¢), during step c) and/or after step c), preferably at least during step c}, the (fermented) aqueous mixture is treated with a glycosidase, preferably wherein the glycosidase is selected from the group consisting of lysozyme, beta-glucanase, xylanase, pentosanase, cellulase, alpha-amylase, arabanase and/or hemicellulose, a protease and or a lipase.

The method according to any one of the preceding claims, wherein after step c) the bacteria are inactivated, preferably heat inactivated.

The method according to any one of the preceding claims, wherein after step c) further ingredients are provided to the fermented aqueous mixture, preferably wherein the further ingredients are selected from the group consisting of sweeteners, flavors, antioxidants, emulsifiers, preservatives, colorants , acidity regulators , gelling agents and any combination thereof.

The method according to any one of the preceding claims, wherein the fermented aqueous mixture comprises between 0.2-1.5% by weight protein and/or between 0 and 2.5% by weight sugar.

The method according to any one of the preceding claims, wherein after step c) undissolved material is removed from the fermented aqueous mixture.

The method according to any one of the preceding claims, wherein the aqueous mixture inoculated with the lactic acid producing bacteria in step b) is prepared by mixing: a. between 60-95% by weight water; b. between 1 - 25 wt.% fresh microalgae and/or between 0.2 - 5 wt.% dried microalgae; c. between 0.5-5.0 wt% sugar, preferably wherein the sugar is provided by a juice, preferably apple juice, preferably from 5-30 wt% apple juice; d. optional 0.1-2.5% by weight lemon juice or lime juice; and e. optional other ingredients.

A method according to any preceding claim wherein the fermented aqueous solution is dried to remove water.

A drinkable composition obtainable by any of the method claims 1)-15), or a dried composition obtainable according to claim 16).

18. A drinkable composition comprising a. between 60-95% by weight water b. between 1 - 25% by weight fresh microalgae and/or between 0.2% and 5% dried algae c. juice, preferably apple juice, preferably with 5-30% by weight apple juice; d. optional 0.1-2.5% by weight lemon juice or lime juice; and e. optional other ingredients.

The drinkable composition according to claim 18) wherein the drinkable composition further comprises non-replicating lactic acid producing bacteria, preferably wherein the lactic acid producing bacteria is selected from Bifidobacteria and/or Lactobacilli, more preferably wherein the bacteria is Lactobacillus plantarum.

The drinkable composition according to any one of claims 18)-19) wherein the at least one microalgae is selected from the group consisting of Arthrospira platensis, Arthrospira maxima, Chlorella spp, Chlorella sorokiniana and combinations thereof.

The drinkable composition according to any one of claims 18) - 20) wherein the drinkable composition comprises between 0.2 - 1.5 wt% protein and/or between 0 and 2.0 wt% sugar.

The drinkable composition according to any one of claims 18) to 21) wherein the drinkable composition further comprises sweeteners, flavors, antioxidants, emulsifiers, preservatives, colorants, acidity regulators, gelling agents and any combination thereof.

23. A dry composition obtainable by the method of claim 16.

24. A dry composition comprising a. between 0.01 - 0.1 wt.% lactic acid b. between 5 - 20 wt.% carbohydrates c. between 20-60 wt.% proteins d. between 5 - 10 wt.% fat; and e. inactivated lactic acid producing bacteria, wherein preferably the proteins come at least partly from microalgae.

A bottle or container comprising a drinkable composition according to any one of claims 18)) - 22).