Field of the invention

The present invention relates to the encapsulation of an active agent which comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said saccharide and said saccharides are obtained from an in vitro and/or ex vivo culture of cells. More specifically, the present invention relates to the encapsulation of an active agent which comprises a milk oligosaccharide or a mixture of at least two different milk oligosaccharides, wherein said milk oligosaccharide and said milk oligosaccharides are obtained from an in vitro and/or ex vivo culture of cells.

Background of the invention

To date saccharides and especially oligosaccharides are gaining more and more attention. This molecule group is very diverse in chemical structure, and are composed out of a diverse number of monosaccharides, such as glucose, galactose, N-acetylglucosamine, xylose, rhamnose, N- acetylneuraminic acid, N-acetylgalactosamine, galactosamine, glucosamine, glucuronic acid, galacturonic acid,... These oligosaccharides or glycans are macromolecules in nature with a range of important biological activities and widely distributed in all living organisms. These oligosaccharides or glycans play important roles in a variety of normal physiological and pathological processes, such as cell metastasis, signal transduction, intercellular adhesion, inflammation and immune response.

An example of such saccharides are milk saccharides (Urashima T. et al., 2011, Milk Oligosaccharides, Nova Biomedical Books, New York ISBN 978-1-61122-831-1; Coppa et al, 2013, Ital. J. Pediatr. 2013, 39(2)), in particular milk oligosaccharides (MOs), i.e. (oligo)saccharides which are found in milk of animals such as mammals and humans (Urashima et al, 2011; Coppa et al, 2013). A replete amount of milk saccharide structures have been elucidated so far. The majority of milk oligosaccharides found in animals such as mammals and humans comprise lactose at the reducing end (Urashima et al, 2011). Other milk oligosaccharides comprise N-acetyllactosamine (Gal-pi,4-GlcNAc) or lacto-N-biose (Gal-pi,3-GlcNAc) at the reducing end (Urashima et al, 2011; Wrigglesworth et al, 2020, PLoS ONE 15(12); Urashima et al, 2013, Biosci. Biotechnol. Biochem 77(3): p. 455-466; Wei et al, 2018, Sci. Rep. 8:4688). Examples hereof are 3- FLN (Gal-pi,4-(Fuc-al,3-)GlcNAc; also known as Lewis x antigen), 3'-SLN (Neu5Ac-a2,3-Gal-pi,4-GlcNAc), 6'-SLN (Neu5Ac-a2,6-Gal-pi,4-GlcNAc) (Urashima et al, 2011; Wrigglesworth et al, 2020; Wei et al, 2018). Further, milk saccharides comprise milk glycosaminoglycans (GAGs; Coppa et al, 2013; Rai et al, 2021, Int. J. Biol. Macromolecules, 193(A): p. 137-144). Antigens of the human ABO blood group system such as the A determinant (GalNAc-alphal,3(Fuc-alphal,2)-Gal), B determinant (Gal-alphal,3(Fuc-alphal,2)-Gal) and H-determinant (Fuc-alphal,2-Gal) have been shown to be present in saccharides of the milk of mammals (e.g. A tetrasaccharide in a lion and leopard; Wrigglesworth et al, 2020). Such milk, more specifically, human milk is to date considered as the best food for newborns and infants. It is composed of several fractions of which milk oligosaccharides are the fourth largest fraction. Besides lactose, human milk, as well as milk of other mammals, contains various structurally diverse oligosaccharides which are also known as human milk oligosaccharides (HMOs) or mammalian milk oligosaccharides (MMOs), respectively (Urashima T. et al., 2011). The importance of MOs for mammalian and human infant nutrition is directly linked to their biological activities including protection of the neonate from pathogens, supporting development of the infant's immune system and cognitive abilities. HMOs and MMOs are further known to act as decoys to reduce the risk of infections by bacterial and viral pathogens which adhere to human cells by binding to these cells' surface glycoproteins. Additionally, various HMOs and MMOs possess an anti-inflammatory effect and act as immunomodulators (e.g. reducing the risk of developing food allergies). Altogether, these beneficial effects make milk oligosaccharides, especially mammalian (MMOs) and human milk oligosaccharides (HMOs), promising candidates to incorporate into food and feed.

Saccharides, especially oligosaccharides such as mammalian and human milk oligosaccharides, are in most cases chemically reactive molecules and/or labile under certain conditions (e.g. high temperature, low or high pH, and/or high moisture content). They are redox- and also biologically active, sensitive to mechanical stress (e.g. excessive shear) and in addition temperature sensitive. Furthermore, dissolved oligosaccharides can react chemically by oxidative or reductive conditions. Moreover, said oligosaccharides can undergo intra- and intermolecular substitutions and hydrolysis reactions rendering the oligosaccharides labile, which in extreme cases may even lead to isomerization or disintegration and coloration of the end product, by for example, the formation of an isomer form at the reducing end of the oligosaccharide or the formation of hydroxymethylfurfural (HMF), which is a well-known sugar decomposition product in literature, occurring when applying too basic, too acidic and/or too hot conditions (Cammerer et al., 1999, Eur. Food Res. Technol. 1999, 209(3-4), 261-265; Fagerson, 1969, J. Agric. Food Chem. 17(4), 747-750; Wilson, K. et al., 2014, Chapter 19, Bio-based chemicals from biorefining: carbohydrate conversion and utilization, 624-658, In Waldron, K. (Editor), Advances in Biorefineries: Biomass and Waste Supply Chain Exploitation, ISBN: 978-0-85709-521-3; Van der Fels-Klerx, H. J. et al., 2014, Food Res. Int. 57, 210-217).

Although the incorporation of milk saccharides, especially milk oligosaccharides such as MMOs and HMOs into food and feed might be promising, conditions relating to the manufacturing or storing of food and feed may have a negative impact on the oligosaccharides considering their properties as outlined earlier. Such conditions typically entail a high pressure (e.g. a pressure of above 10 bar during extrusion), a high temperature (e.g. a temperature above 100°C during extrusion and baking), a pH lower than 5, a pH higher than 8, and/or a high moisture content. Moreover, other molecules within food or feed such as proteins could chemically interact with MMOs and HMOs especially under the harsh conditions during food/feed manufacturing. There is hence a significant need for protecting saccharides, especially oligosaccharides such as MMOs and HMOs, during food/feed manufacture and storage to ensure their retention in the final food and feed.

Furthermore, protecting saccharides, especially oligosaccharides such as MMOs and HMOs, is also of particular importance in feeding conditions, for example if they are incorporated into aquaculture feed as the feed remains in contact with water for a prolonged time (few minutes for instant feeders such as trout and catfish, up to a few hours for shrimp for example) until consumed by the animal. Not only the physical integrity of the aquaculture feed while in contact with water is crucial, but also of paramount importance is to reduce leaching of incorporated substances such as said saccharides. Also here there is a significant need for protecting saccharides, especially oligosaccharides such as MMOs and HMOs, from contact with a liquid such as water, e.g. protecting saccharides, especially oligosaccharides such as MMOs and HMOs, in aquaculture feed to reduce leaching into the water and hence obtain an aquaculture feed with a high pellet water stability.

Summary of the invention

It was surprisingly found in the present invention that encapsulating milk saccharides serves a great strategy to overcome aforementioned issues by reducing and/or avoiding contact with other components and retaining the milk saccharides despite exposure to conditions which would otherwise affect the milk saccharides such as a high pressure, high temperature, high moisture content (e.g. contact with water), pH which is lower than 5 or higher than 8, etc. as described further herein.

In a first aspect, the invention provides an encapsulate comprising (i) an active agent comprising a milk saccharide or a mixture of at least two different milk saccharides, and (ii) a first carrier material, wherein said saccharide and said saccharides are obtained from an in vitro and/or ex vivo culture of cells.

In a second aspect, the invention provides a method for the production of an encapsulated active agent comprising a milk saccharide or a mixture of at least two different milk saccharides, wherein said saccharide and said saccharides are obtained from an in vitro and/or ex vivo culture of cells.

In a third aspect, the invention provides a composition, preferably a nutritional composition, comprising an encapsulate according to the first aspect or comprising an encapsulated active agent obtainable by a method according to the second aspect.

In a fourth aspect, the invention provides a pharmaceutical composition comprising an encapsulate according to the first aspect or comprising an encapsulated active agent obtainable by a method according to the second aspect. In a fifth aspect, the invention provides an encapsulate (first aspect), encapsulated active agent (second aspect), nutritional composition (third aspect) or pharmaceutical composition (fourth aspect) for use as a medicament.

In a sixth aspect, the invention provides a method for the production of a composition, preferably a nutritional composition and/or a pharmaceutical composition.

In a seventh aspect, the invention provides the use of an encapsulate (first aspect) or an encapsulated active agent (second aspect) in (i) the manufacturing of a nutritional composition or (ii) feeding an animal.

Detailed description of the invention

Encapsulate

In a first aspect, the invention provides an encapsulate comprising: an active agent comprising a milk saccharide or a mixture of at least two different milk saccharides, preferably said active agent comprising a milk oligosaccharide or a mixture of at least two different milk oligosaccharides, wherein said saccharide, preferably oligosaccharide, and said saccharides, preferably oligosaccharides, are obtained from an in vitro and/or ex vivo culture of cells, and a first carrier material which forms (i) a shell around said active agent as to form an encapsulate of the core-shell type or (ii) a matrix wherein said active agent is distributed as to form an encapsulate of the matrix type, preferably said first carrier material forms a shell around said active agent as to form an encapsulate of the core-shell type.

In the context of the present invention, an "encapsulate" comprises a substance that is encapsulated, i.e. said substance is coated and/or trapped in a carrier material. In the field of (micro)encapsulation, the substance that is encapsulated is typically called an "active agent", "internal phase" or "payload phase", these terms are hence used interchangeably herein. The substance that is encapsulating the active agent is typically called "carrier material", "wall material", "external phase", "encapsulant" or "matrix", these terms are hence used interchangeable herein.

As known in the field of (micro)encapsulation, there are generally two main types of encapsulates.

A first type of encapsulate contains a shell around the active agent (i.e. the core of the encapsulate), i.e. the carrier material forms a shell around the active agent. This type of encapsulate is generally referred to as "core-shell type", "(micro)capsule", "single-core" or "mono-core", said terms are used interchangeably herein. The core can be either liquid or solid. As soon as the carrier material (i.e. the shell) partially encapsulates the active agent (i.e. the core), it is considered to be a core-shell-type encapsulate in the context of the present invention and hence within the scope of the present invention. It is however a preferred embodiment of the present invention that the carrier material completely encapsulates the active agent.

A second type of encapsulate is generally referred to as "matrix type" or "microsphere", said terms are used interchangeably herein. Herein, the active agent is dispersed in a continuous network of the carrier material. The active agent can be present for example as small microparticles or droplets, or more homogeneously distributed over the encapsulate. An encapsulate of the matrix type is a solid matrix particle, wherein the active agent is typically dissolved/dispersed or melted in it.

The skilled person will readily understand that an encapsulate of the matrix type can be further coated as to form a so-called "coated matrix type", which is essentially a core-shell type of encapsulate wherein the solid core consists of the active agent dissolved/dispersed into carrier material. In the context of the invention, a coated matrix type of encapsulate is considered to be a core-shell type of encapsulate, unless specifically stated otherwise.

In the context of the present invention, "a matrix wherein said active agent is distributed as to form an encapsulate of the matrix type" refers to the situation wherein the active agent is dissolved or dispersed into the carrier material, either heterogeneously or homogeneously (homogeneously is preferred but not essential in the context of the present invention). The term "distributed" can hence be interchangeably used with the term "dispersed". The term "dissolved" refers to the situation wherein the active agent is incorporated into a liquid or melt so as to form a solution and therefore represents an example of dispersion. The skilled person will readily understand that the term "dispersion" not only encompasses the situation wherein the active agent is dissolved into a carrier material, but also the situation wherein the active agent and carrier material form a suspension.

In a preferred embodiment, the encapsulate according to the invention is a microparticle, preferably a spherical microparticle. Preferably, said encapsulate has an aspect ratio of 0.8-1.2, more preferably an aspect ratio of 0.9-1.1. In the context of the present invention, the term "aspect ratio" preferably means the ratio between the length and width of a particle. The encapsulates according to the invention preferably have a regular symmetry, i.e. having an aspect ratio which lies in the range of 0.8-1.2, preferably 0.9-1.1. A "microparticle" is a particle having a physical dimension of 1 to 1000 micrometer, preferably < 750 pm, more preferably < 600 pm. A microparticle may or may not be spherical in shape.

In an additional and/or alternative preferred embodiment, the encapsulate according to the invention has a median diameter (D50 also known as D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 175 pm; and/or said encapsulate has a median diameter (D50 also known as D (v, 0.5)) of < 400 pm, preferably < 350 pm, more preferably < 300 pm. Throughout the description and claims, the terms "D50" and "D(v,0.5)" can be interchangeably used. In the context of the present invention, "D(v,0.5)" also known as the volume median diameter is the diameter of a particle where 50 % of the distribution is above and 50% is below. Several techniques are available to determine the size of a particle such as laser diffraction, dynamic light scattering, automated imaging, sedimentation, electrozone sensing and sieving. Each technique has a particle size range wherein it can be applied. In the context of the present invention, laser diffraction is preferred to assess the size of a particle and hence to determine the D(v,0.1), D(v,0.5), D(v,0.9) and D(4,3) of an encapsulate/particle as defined herein. A particularly suitable system for laser diffraction is a MasterSizer S apparatus from Malvern Instruments Ltd.

When an encapsulate according to the invention is of the core-shell type, it is a preferred embodiment that the core (preferably an encapsulate of the matrix type) has a median diameter (D50 also known as D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 165 pm. Preferably, said median diameter is 75-250 pm, more preferably 100-250 pm, even more preferably 125- 250 pm, even more preferably 150-250 pm, even more preferably 165-250 pm, most preferably 165-225 pm. In an additional and/or alternative preferred embodiment in this context (i.e. when said encapsulate is of the core-shell type), the core-shell type encapsulate has a median diameter (D50 also known as D(v, 0.5)) which is 10-100 pm, preferably 20-100 pm, more preferably 20-75 pm, even more preferably 35-75 pm, longer than the median diameter of the core (preferably an encapsulate of the matrix type). In an additional and/or alternative preferred embodiment in this context (i.e. when said encapsulate is of the core-shell type), the core-shell type encapsulate has a median diameter (D50 also known as D(v, 0.5)) which is 10%-40%, preferably 10%-35%, more preferably 10%-30%, even more preferably 15%-30%, most preferably 20%-30%, longer than the median diameter of the core (preferably an encapsulate of the matrix type).

In an additional and/or alternative preferred embodiment, the encapsulate according to the invention has a D(4,3) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 175 pm; and/or said encapsulate has a D(4,3) of < 400 pm, preferably < 350 pm, more preferably < 300 pm. throughout the description and claims, the terms "D(4,3)" and "De Brouckere mean diameter" can be interchangeably used. In the context of the present invention, "D(4,3)" refers to the mean of a particle size distribution weighted by the volume and is also known as the volume-weighted mean diameter.

In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a particle size distribution characterized by:

D(v, 0.1) ranging from 35 to 200 pm, preferably from 50 to 200 pm, more preferably 75 to 200 pm; and/or D(v, 0.5) ranging from 50 to 400 pm, preferably from 50 to 350 pm, more preferably from 100 to 350 pm, even more preferably from 125 to 350 pm, even more preferably form 150 pm to 350 pm, even more preferably from 175 to 300 pm; and/or

D(v, 0.9) ranging from 100 to 600 pm, preferably from 100 to 500 pm, more preferably from 150 to 500 pm, even more preferably from 200 to 500 pm, even more preferably from 250 to 500 pm, most preferably from 300 to 500 pm; and/or

D(4,3) ranging from 50 to 400 pm, preferably from 50 to 350 pm, more preferably from 100 to 350 pm, even more preferably from 125 to 350 pm, even more preferably form 150 pm to 350 pm, even more preferably from 175 to 325 pm; and/or a span of 0.75-1.3, preferably 0.75-1.25, more preferably 0.85-1.25, even more preferably 0.9-1.25, most preferably 0.9-1.2, wherein said span is calculated as (D(v,0.9) - D(v,0.1)) / D(v,0.5).

In the context of the present invention, "D(v,0.1)" refers to the diameter of a particle where 10 % of the volume distribution is below; "D(v,0.9)" refers to the diameter of a particle where 90 % of the volume distribution is below. "Span" refers to the width of the particle size distribution based on the 10 %, 50 % and 90 % quantile, i.e. span = (D(v,0.9) - D(v,0.1)) / D(v,0.5).

Advantageously, the encapsulates according to the invention have a homogeneous size distribution as the span is in the range of 0.9-1.2.

In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a moisture content, preferably water content, of < 15 % (VJ/VJ), preferably < 10 % (VJ/VJ), more preferably < 9 % (VJ/VJ), even more preferably < 8 % (VJ/VJ), even more preferably < 7 % (VJ/VJ), even more preferably < 5 % (VJ/VJ), even more preferably < 4 % VJ/VJ), even more preferably < 3 % (VJ/VJ), most preferably < 2 % (VJ/VJ), of the total weight of the composition. In other words, the encapsulate according to the invention has a dry extract (% (w/w)) of at least 85 %, preferably at least 90 %, more preferably at least 91 %, even more preferably at least 92 %, even more preferably at least 93 %, even more preferably at least 95 %, even more preferably at least 96 %, even more preferably at least 97 %, most preferably at least 98%, preferably as determined by a thermogravimetric analysis or Karl Fisher titration analysis, more preferably as determined by a thermogravimetric analysis. The moisture content (preferably water content) can be determined gravimetrically as routinely applied in the field by drying the sample (i.e. encapsulate according to the invention) at 105°C up to a constant weight is obtained, preferably said sample is dried for 24 hours at 105°C (e.g. Hernandez-Lopez et al, 2018, LWT 2018, 95: p. 216-222; Pang et al, 2017, J. Food Quality, article id 1612708). Hence, said % dry extract is preferably calculated as (mass of encapsulate after 24 hours at 105°C / initial mass of the encapsulate) x 100.

In this context of the invention, the term "moisture content" can be preferably replaced with "liquid content" and vice versa, unless explicitly stated otherwise. In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a water activity (A _w ) which is higher than the water activity of the non-encapsulated saccharide or nonencapsulated mixture of saccharides, preferably the non-encapsulated active agent. In other words, the water activity of the saccharide or mixture of at least two saccharides as disclosed herein (nonencapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), preferably the active agent as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), is lower than the water activity of the encapsulate according to the invention which comprises an identical saccharide or mixture of saccharides, preferably an identical active agent.

In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a water activity (A _w ) which is at least 0.05, preferably at least 0.075, more preferably at least 0.10, even more preferably at least 0.15, most preferably which is at least 0.16 and/or wherein said encapsulate has a water activity of < 0.50, preferably < 0.45, more preferably < 0.40, even more preferably < 0.35, most preferably < 0.30. Preferably, the encapsulate according to the invention has a water activity (A _w ) which is 0.05-0.50, preferably 0.05-0.45, more preferably 0.05-0.40, even more preferably 0.05-0.35, even more preferably 0.05-0.30, even more preferably 0.10-0.30, even more preferably 0.15-0.30, most preferably 0.16-0.30. The skilled person will readily understand that the water activity has to be determined using an identical (or at least substantially the same) method and under identical (or at least substantially the same) conditions. In the context of the present invention, the term "water activity (A _w )" refers to the dimensionless ratio between the vapor pressure of water in the encapsulate and the vapor pressure of pure water at the same temperature. Hence, the activity water level reflects the thermodynamic energy status of the water in the encapsulate, ranging from Oto 1.00. Preferably, said water activity is determined at ambient temperature, more preferably at a temperature of 24.5-24.9°C. Preferably, said water activity is determined by a LabSwift-aw system (preferably from Novasina).

In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a flowability which is higher than the flowability of the non-encapsulated saccharide or non-encapsulated mixture of saccharides, preferably the non-encapsulated active agent. In other words, the flowability of the saccharide or mixture of at least two saccharides as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), preferably the flowability of the active agent as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), is lower than the flowability of the encapsulate according to the invention which comprises an identical saccharide or mixture of saccharides, preferably which comprises an identical active agent. The skilled person will readily understand that the flowability has to be determined using an identical (or at least substantially the same) method and under identical (or at least substantially the same) conditions. In the context of the present invention, the term "flowability" refers to the relative movement of a bulk of particles among neighboring particles or along the container wall surface. Flowability cannot be determined by a simple test method. Good indicators of flowability of particles are the Hausner Ratio (Rh) and Carr index (l _c ), both indicators assume that the compressibility of a particle/solid relates to its flowability. The "Hausner Ratio (Rh)" can be calculated by taking the ratio between the tapped density (p _t ) and bulk density (pb), i.e. Rh = pt / Pb- The "Carr index (l _c )" can be calculated as follows: lc = ((p _t - pb) / Pb) x 100. The lower the Carr Index or Hausner Ratio of an encapsulate/particle/solid is, the more flowable said encapsulate/particle/solid is. As used herein, the term "bulk density" is the weight of the particles of a particulate solid (such as a powder) in a given volume, and is expressed in grams per liter (g/L). The total volume that the particles of a particulate solid occupy depends on the size of the particles themselves and the volume of the spaces between the particles. Entrapped air between and inside the particles also can affect the bulk density. Thus a particulate solid consisting of large, porous particles with large inter-particulate spaces will have a lower bulk density than a particulate solid consisting of small, non-porous particles that compact closely together. Bulk density can be expressed in two forms: loose bulk density and tapped bulk density. "Loose bulk density" (also known in the art as "freely settled" or "poured" bulk density) is the weight of a particulate solid divided by its volume where the particulate solid has been allowed to settle into that volume of its own accord (e.g. a powder poured into a container).

Closer compaction of a particulate solid within a container may be achieved by tapping the container and allowing the particles to settle more closely together, thereby reducing volume while weight remains the same. Tapping therefore increases bulk density. "Tapped bulk density" (also known in the art as "tamped" bulk density) is the weight of a particulate solid divided by its volume where the particulate solid has been tapped and allowed to settle into the volume a precise number of times. The number of times the particulate solid has been tapped is typically when stating the tapped bulk density. For example, "lOOx tapped bulk density" refers to the bulk density of the particulate solid after it has been tapped 100 times. Techniques for measuring bulk density are well known in the art. Loose bulk density may be measured using a measuring cylinder and weighing scales: the particulate solid is poured into the measuring cylinder and the weight and volume of the particulate solid; weight divided by volume gives the loose bulk density. Tapped bulk density can be measured using the same technique, with the addition of tapping the measuring cylinder a set number of times before measuring weight and volume. Automation of tapping ensures the number, timing and pressure of individual taps is accurate and consistent. Automatic tapping devices are readily available, an example being the Jolting Stampfvolumeter (STAV 203) from J. Englesmann AG.

Advantageously, the encapsulates according to the invention as described herein have a good to excellent flowability, whereas the flowability of the non-encapsulated saccharide or non-encapsulated mixture of saccharides, preferably non-encapsulated active agent, is poor. A poor, good or excellent flowability can be characterized by a Carr index and/or Hausner ratio as depicted in Table 1.

Table 1 - Flowability scoring

A predictable and good/excellent flowability is of great importance to achieve better manufacturing efficiency and product quality. An irregular and/or poor flowability can result in capacity shortfall and production interruption. For example, the food industry makes extensive use of vertical bins/silos for powder storage. Irregular and/or poor flow of powders out of those is a major cause of loss in production capacity and product deterioration. Also in the pharmaceutical industry, an irregular and/or poor flowability can affect manufacturing efficiency and hence affect the weight and content uniformity (i.e. quality) of the product.

In this regard, an encapsulate according to the invention wherein the carrier material comprises a wax (it is referred to the section entitled "Carrier material"), preferably selected from Candelilla wax, Carnauba wax, beeswax, rice bran wax, paraffin wax, jojoba wax, microcrystalline wax and japan wax, more preferably said wax is Candelilla wax, is particularly preferred as these encapsulates exert an excellent flowability. Such an encapsulate can be of the matrix type or core-shell type. As already depicted herein, an encapsulate of the so-called coated matrix type (in the present context, at least the carrier material of the shell comprises said wax, optionally the carrier material of the core comprises said wax) is also considered to be a core-shell type of encapsulate.

In additional and/or alternative preferred embodiment, the encapsulate according to the invention has a Hausner ratio (Rh) which is < 1.34, preferably < 1.25, more preferably < 1.18. In an additional and/or alternative preferred embodiment, the encapsulate according to the invention has a Carr index (l _c ) which is < 25, preferably < 20, more preferably < 15.

In additional and/or alternative preferred embodiment, the quantity and/or quality of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, of an encapsulate according to the invention is less affected upon (i.e. after) short and/or prolonged exposure to conditions compared to non-encapsulated milk saccharide or non-encapsulated mixture of at least two different milk saccharides, preferably compared to the non-encapsulated active agent. In other words, the quantity and/or quality of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), is more prone to said conditions than that of the encapsulate according to the invention which comprises an identical milk saccharide or mixture of milk saccharides, preferably an identical active agent. In the context of the present invention, the expressions "is more prone" and "is more affected" are preferably interchangeably used, unless specifically stated otherwise. In the context of the present invention, the expressions "is less prone" and "is less affected" are preferably interchangeably used, unless specifically stated otherwise.

In the context of the present invention, said "conditions" are preferably conditions which result in degradation and/or modification of the milk saccharide or mixture of milk saccharides (preferably the active agent) or a fraction thereof upon short and/or prolonged exposure to said conditions. The skilled person will readily understand that the quantity/quality of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) has to be determined using an identical (or at least substantially the same) method and under identical (or at least substantially the same) conditions. Several techniques/methods can be used to assess which saccharide or saccharides are present and in what quantity. Preferred in the context of the present invention is the use of high-performance liquid chromatography (HPLC) or ultra-performance liquid chromatography (UPLC), preferably with refractive index detection, to determine the quantity and/or quality of a saccharide or mixture of at least two different saccharides. As an example, it is referred to the method as described in Christensen et al, 2020, J. Food Sci. 2020, 85(2), p. 332-339 (incorporated by reference) wherein 2'-fucosyllactose and 3- fucosyllactose are quantified. Alternatively, and especially for saccharides at low concentration (below 50 mg/L), it is preferred in the context of the present invention to use high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Such a method is described in the Examples section of the present application.

Preferably, said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; gastro-intestinal conditions of an animal, preferably stomach conditions of an animal; presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source; and/or pH which is lower than 5 or higher than 8.

Said moisture content is preferably determined by a thermogravimetric analysis (preferably as described herein) or a Karl Fisher titration analysis (preferably as described herein).

More preferably, said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source; and/or pH which is lower than 5 or higher than 8.

Even more preferably, said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; and/or presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source.

Even more preferably, said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); and/or feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water. Even more preferably, said conditions are any one or both of the following: food/feed manufacturing or storage conditions; and/or feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water.

Even more preferably, said conditions are food/feed manufacturing or storage conditions. Most preferably, said conditions are food/feed manufacturing conditions.

In this context of the invention, it is preferred that said conditions are not stomach conditions of an animal. More preferably, said conditions are not gastro-intestinal conditions of an animal.

In the context of the invention, the term "food/feed manufacturing or storage conditions" refers to conditions used during the preparation or storage of food and/or feed. Various methods and techniques are known to the skilled person for preparing food and feed as described thoroughly in the book entitled "Food processing technology: principles and practice" (Fellows; second edition; Woodhead Publishing Limited; 2000) which is incorporated by reference. A preferred preparation method comprises any one or more of baking, roasting, frying, heating (dielectric, ohmic or infrared), blanching, pasteurization, heat sterilization, fermentation, irradiation, processing using pulsed electric field, high pressure and extrusion (dry or wet), preferably any one or more of baking, heating (dielectric, ohmic or infrared), heat sterilization, processing using pulsed electric field or high pressure, and extrusion (dry or wet), more preferably any one or more of baking, heating (dielectric, ohmic or infrared), heat sterilization and extrusion (dry or wet), even more preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared) and extrusion (dry or wet), even more preferably any one or more of baking and extrusion (dry or wet), most preferably comprises extrusion (dry or wet). A preferred preparation method is selected from the list consisting of baking, roasting, frying, heating (dielectric, ohmic or infrared), blanching, pasteurization, heat sterilization, fermentation, irradiation, processing using pulsed electric field, high pressure and extrusion (dry or wet). A more preferred preparation method is selected from a list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization, processing using pulsed electric field or high pressure, and extrusion (dry or wet). An even more preferred preparation method is selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization and extrusion (dry or wet). An even more preferred preparation method is selected from the list consisting of baking, heating (dielectric, ohmic or infrared) and extrusion (dry or wet). An even more preferred preparation method is baking or extrusion (dry or wet). Most preferred preparation method comprises extrusion (dry or wet). The latter is preferably as defined in aspect six of the present invention (it is referred to the section entitled "Method for production of a composition" as described herein). Another most preferred preparation method comprises baking. In this regard, it is preferred that the food/feed that is baked has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

It is preferred that a food or feed as disclosed herein has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w). Said moisture content is preferably determined by a thermogravimetric analysis (preferably as described herein) or a Karl Fisher titration analysis (preferably as described herein). For example, it is preferred according to the invention that a dough or batter before baking has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w). Such a dough or batter, when baked, results in a food/feed having a moisture content of 3-50 % w/w), preferably 5-50% (w/w), more preferably 10-50% (w/w), even more preferably 15-50% (w/w). For example, the moisture content (% w/w) of a cake is 15-30% (w/w)) and of a bread is 35-45% (w/w), while biscuits/cookies typically have a moisture content of 5-10% (w/w), pastries have a moisture content of < 15% (w/w) (Figoni, 2011).

Various methods and techniques are known to the skilled person for storing food and feed as described in said book which is incorporated by reference. Preferred storage methods comprise chilling, atmosphere storage (modified or controlled) and drying (freeze drying, spray drying, granulation, lyophilization, belt drying, drum drying, agitated thin film drying). More preferred storage methods comprise atmosphere storage (modified or controlled) and drying (freeze drying, spray drying, granulation, lyophilization, belt drying, drum drying, agitated thin film drying). Even more preferred storage methods comprise atmosphere storage (modified or controlled), granulation, lyophilization and freeze drying. Reducing and/or preventing contact between the milk saccharide or mixture of milk saccharides (preferably active agent) and a liquid as described further herein, is a preferred example of said storage conditions as described herein.

In the context of the present invention, the term "feeding conditions" refers to conditions to which feed/food is exposed to during the feeding of a human or an animal (preferably as described herein). Preferably said feeding conditions comprise or consist of contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water. Throughout the application and claims, unless specifically stated otherwise, the expression "feeding conditions" is preferably replaced with "contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water". Said liquid may be in direct contact with the encapsulate or might become in contact with the encapsulate after a short period of time, preferably at least 1 min, more preferably at least 5 min, even more preferably at least 10 min, even more preferably at least 30 min, even more preferably at least 1 hour, even more preferably at least 2 hours and/or an exposure time of < 1 day (i.e. 24 hours), preferably < 16 hours, more preferably < 12 hours, even more preferably < 8 hours, even more preferably < 4 hours, most preferably < 1 hour; or after a prolonged period of time, preferably at least 1 day (i.e. 24 hours), more preferably at least 2 days, even more preferably at least 1 week, even more preferably at least 1 month, even more preferably at least 3 months, even more preferably at least 6 months, most preferably at least 1 year. A particular preferred example in this regard is the incorporation of an encapsulate according to the invention into aquaculture feed (preferably for aquatic animals, more preferably for a water breathing animal, even more preferably for a fish or shrimp). This feed remains in contact with water until consumed by the animal. Contact with the water can take a few minutes for instant feeders (e.g. trout and catfish) or a few hours (e.g. shrimp).

In the context of the invention, "gastro-intestinal conditions of an animal" comprise conditions reflecting those of the stomach (i.e. stomach conditions) and conditions reflecting those of the intestines (i.e. intestinal conditions). Stomach conditions of a monogastric animal typically comprise a pH which is lower than 5.0 (i.e. pH of 1.0 to 5.0) and the presence of enzymes as known to the skilled person. The stomach of a ruminant contains four compartments: rumen (pH 5.5-6.5), reticulum (pH 5.5-6.5), omasum (pH 5.5- 6.5) and abomasum (pH 1-3). Intestinal conditions typically comprise a pH of 6.0 to 8.0, the presence of enzymes and bile salt as known to the skilled person. In the context of the invention, gastro-intestinal conditions of an animal preferably refers to a pH which is lower than 6.0 (i.e. pH of 1.0 to 6.0), preferably lower than 5.0 (i.e. pH of 1.0 to 5.0), and the presence of stomach enzymes as known by the skilled person. In the context of the present invention, "short and/or prolonged exposure to conditions" refers to the duration of exposure to said conditions as described herein. As understood by the skilled person, the duration depends on the intended application (e.g. method for preparing the food/feed or method for storing the food/feed or residence time of aquaculture feed before taken by the animal as described herein). For example, for the manufacture of food/feed using extrusion, the duration of exposure of said conditions is at least the length of the extrusion protocol (kneading, heating, squeezing, cutting) including pre-extrusion (i.e. homogenizing active agent or encapsulate of the invention with the other food/feed ingredients of interest) and post-extrusion (e.g. drying of obtained food/feed in an oven). As to the storage of food/feed, the exposure to said conditions is typically much longer (i.e. at least a day to several days, weeks or months) than is the case for food/feed manufacturing (i.e. half an hour to several hours). "Short exposure to conditions" hence preferably refers to an exposure time of at least 1 min, preferably at least 5 min, more preferably at least 10 min, even more preferably at least 30 min, even more preferably at least 1 hour, even more preferably at least 2 hours and/or an exposure time of < 1 day (i.e. 24 hours), preferably < 16 hours, more preferably < 12 hours, even more preferably < 8 hours, even more preferably < 4 hours, most preferably < 1 hour. "Prolonged exposure to conditions" hence preferably refers to an exposure time of at least 1 day (i.e. 24 hours), preferably at least 2 days, more preferably at least 1 week, even more preferably at least 1 month, even more preferably at least 3 months, even more preferably at least 6 months, most preferably at least 1 year. In an additional and/or alternative preferred embodiment, an encapsulate according to the invention reduces and/or prevents contact between the milk saccharide or mixture of at least two different milk saccharides (preferably active agent) and a liquid, preferably wherein said liquid comprises water, more preferably wherein said liquid is water. Said liquid may be in direct contact with the encapsulate or might become in contact with the encapsulate after a short period of time, preferably at least 1 min, more preferably at least 5 min, even more preferably at least 10 min, even more preferably at least 30 min, even more preferably at least 1 hour, even more preferably at least 2 hours and/or an exposure time of < 1 day (i.e. 24 hours), preferably < 16 hours, more preferably < 12 hours, even more preferably < 8 hours, even more preferably < 4 hours, most preferably < 1 hour; or after a prolonged period of time, preferably at least 1 day (i.e. 24 hours), more preferably at least 2 days, even more preferably at least 1 week, even more preferably at least 1 month, even more preferably at least 3 months, even more preferably at least 6 months, most preferably at least 1 year.

Hence, reducing and/or preventing contact between the milk saccharide or mixture of milk saccharides (preferably active agent) and a liquid as described herein, is a preferred example of said storage conditions as described herein.

Moreover, an encapsulate according to the invention is particularly useful to be incorporated into feed which is in contact (direct or indirect, preferably direct contact) with an environment having a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40%, even more preferably at least 50% (w/w), most preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water. Particularly preferred is the incorporation of an encapsulate according to the invention into aquaculture feed (preferably for aquatic animals, more preferably for a water breathing animal, even more preferably for a fish or shrimp). This feed remains in contact with water until consumed by the animal. Contact with the water can take a few minutes for instant feeders (e.g. trout and catfish) or a few hours (e.g. shrimp). An aquaculture feed with a high pellet water stability (preferably at least 70 %, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%), i.e. the retention of pellet physical integrity with minimal disintegration and nutrient leaching (e.g. protein, lipid) while in the water until consumed by the animal (Obaldo et al, 2002, Aquaculture Research 33 : p. 369-377), is desired. In this context of the invention, an encapsulate according to the invention incorporated into aquaculture feed significantly reduces and/or prevents leaching of the encapsulated milk saccharide(s) according to the invention into the liquid (preferably comprising water, more preferably said liquid is water) compared to when non-encapsulated saccharide(s) (in an identical or similar amount) are incorporated in the aquaculture feed. In other words, the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), is more prone to leaching from the aquaculture feed than the encapsulate according to the invention which comprises an identical milk saccharide or mixture of milk saccharides, preferably an identical active agent. In other words, the quantity and/or quality, preferably the quantity, of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as disclosed herein (non-encapsulated, i.e. in the absence of any carrier material, and preferably provided as powder), is more prone to contact with a liquid (preferably liquid comprising water, more preferably said liquid is water) than the encapsulate according to the invention which comprises an identical milk saccharide or mixture of milk saccharides, preferably which comprises an identical active agent. It is within the context of the invention that the encapsulate of the invention further comprises one or more nutrients of the aquaculture feed in order to reduce and/or prevent leaching of said nutrient(s) from the aquaculture feed into the liquid. Methods to produce aquaculture feed (preferably made by pelleting or extrusion, more preferably by extrusion), to assess water stability of aquaculture feed and to assess the leaching of nutrients (e.g. lipid, protein) are well- known and routinely applied by the skilled person. It is for example referred to Obaldo et al, 2002; Arevalo et al, 2018, Journal of Food, Agriculture & Environment 16(3/4): p. 10-17 and Ighwela et al, 2013, global Veterinaria 2013, 10(6): p. 638-642, all of which are incorporated by reference. Leaching of saccharide(s) from aquaculture feed can be assessed by periodically measuring the amount of each saccharide in the water (wherein the aquaculture feed is put preferably at room temperature, more preferably at 25°C) by using routine available methods such as HPLC, UPLC, mass spectrometry and/or a HPAEC system with pulsed amperometric detection. Preferred analysis methods are described in the Examples of the present application. The amount detected in the water can then be compared with the amount of saccharide(s) originally present in the aquaculture feed.

In a preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consist of, at least one of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar; a pH which is lower than 5 or higher than 8, preferably a pH which is lower than 5; sheering including extrusion, mixing and compressing; interaction with an enzyme which can degrade and/or modify said milk saccharide or mixture of at least two different milk saccharides; contact with a food/feed component that can result in oxidation, reduction, hydrolysis, consumption and/or conversion of said milk saccharide or mixture of at least two different milk saccharides; and/or contact of said saccharide or mixture of at least two different saccharides with a food/feed component that can result in a Maillard reaction and/or glycation of said food/feed component.

In a more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consists of, at least one of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar; a pH which is lower than 5 or higher than 8, preferably a pH which is lower than 5; and/or contact of said saccharide or mixture of at least two different saccharides with a food/feed component that can result in a Maillard reaction and/or glycation of said food/feed component.

In an even more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consists of, at least one of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; and/or a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar.

In an even more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consists of, at least one of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; and/or a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40%; and/or direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water.

In an even more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

In an additional and/or alternative even more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consist of, a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher. In an additional and/or alternative even more preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise direct or indirect contact with a liquid, preferably said liquid comprises water, more preferably said liquid is water. An example hereof is aquaculture feed as described herein.

In a most preferred embodiment of the invention, said food/feed manufacturing or storage conditions comprise, preferably consist of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; and a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

One or more of above food/feed manufacturing or storage conditions apply to the preparation or storage methods described herein. For example, extrusion typically occurs at a pressure higher than 1013 mbar, preferably higher than 5 bar, more preferably higher than 10 bar. Further, extrusion typically occurs at a temperature of 50°C or higher, preferably 75°C or higher, more preferably at 100°C or higher, even more preferably at 115°C or higher. Moreover, sheering occurs during extrusion (as further elaborated in the sixth aspect of the invention as described herein). And in particular in wet extrusion, a high moisture content (by weight, at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w)) is present. Another example relates to baking of a dough or batter which typically occurs at a temperature of 100°C or higher, preferably 115°C or higher, more preferably 125°C or higher, even more preferably 150°C or higher, even more preferably 150°C to 225°C, even more preferably 180°C to 225°C. Moreover, dough/batter can comprise a moisture content of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

In the context of the present invention, "quantity is less affected" means that in an identical (or at least substantially the same) method it is established that the amount of non-encapsulated (i.e. in the absence of any carrier material, and preferably provided as powder) milk saccharide or non-encapsulated mixture of at least two different milk saccharides (preferably non-encapsulated active agent) upon exposure to identical conditions is lower than the amount of an identical non-encapsulated milk saccharide or nonencapsulated mixture of at least two different milk saccharides (preferably an identical non-encapsulated active agent) when comprised in an encapsulate according to the invention. The skilled person will readily understand that two active agents (or two milk saccharides or two mixtures of milk saccharides) are identical if they consist of the same components and that each component is present in the same quantity in both active agents (or both milk saccharides or both mixtures of milk saccharides). The skilled person will further understand that the exposure time should be identical (or at least substantially the same).

Preferably, the quantity of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) when comprised in an encapsulate according to the invention is at least 5.0 %, preferably at least 10.0 %, more preferably at least 15.0 %, higher than the quantity of the nonencapsulated and identical milk saccharide or non-encapsulated and identical mixture of at least two different milk saccharides (preferably non-encapsulated and identical active agent) upon exposure to said conditions.

Preferably, the quantity of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) comprised in an encapsulate according to the invention upon exposure to said conditions as described herein is at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of the quantity of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) comprised in the encapsulate before exposure to said conditions. In other words, upon exposure to said conditions as described herein at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) is retained in the encapsulate upon exposure to said conditions. For the sake of clarity and as an example, if 20 mg of a saccharide is present in an encapsulate according to the invention, then 20 mg of the same saccharide (i.e. non-encapsulated active agent) is exposed to the same conditions as described herein (e.g. wet extrusion process). The amount of the saccharide is determined in the encapsulated (e.g. 19 mg) and non-encapsulated sample (e.g. 16.5 mg). It can here be concluded that (i) the quantity of the saccharide in the encapsulate is at least 15.0 % higher than in the non-encapsulated sample upon exposure to said conditions and (ii) 95.0 % of the saccharide is retained in the encapsulate upon exposure to said conditions.

In the context of the present invention, "quality is less affected" means that in an identical (or at least substantially the same) method it is established that the extent of degradation and/or modification of non-encapsulated (i.e. in the absence of any carrier material, and preferably provided as powder) nonencapsulated milk saccharide or non-encapsulated mixture of at least two different milk saccharides (preferably non-encapsulated active agent) upon exposure to identical conditions is higher than the extent of degradation and/or modification of an identical non-encapsulated milk saccharide or nonencapsulated mixture of at least two different milk saccharides (preferably an identical non-encapsulated active agent) when comprised in an encapsulate according to the invention. The skilled person will readily understand that two active agents (or two milk saccharides or two mixtures of milk saccharides) are identical if they consist of the same components and that each component is present in the same quantity in both active agents (or both saccharides or both mixtures of saccharides). The skilled person will further understand that the exposure time should be identical (or at least substantially the same).

Preferably, the quantity of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) which is degraded and/or modified when comprised in an encapsulate according to the invention is at least 5.0 %, preferably at least 10 %, more preferably at least 15 %, lower than the quantity of the non-encapsulated and identical milk saccharide or non-encapsulated and identical mixture of at least two different milk saccharides (preferably non-encapsulated and identical active agent) which is degraded and/or modified upon exposure to said conditions.

Preferably, the quantity of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) which is not degraded and/or not modified when comprised in an encapsulate according to the invention upon exposure to said conditions as described herein is at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of the quantity of said milk saccharide or mixture of milk saccharides (preferably said active agent) comprised in the encapsulate before exposure to said conditions. In other words, upon exposure to said conditions as described herein at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of said milk saccharide or mixture of at least two different milk saccharides (preferably said active agent) comprised in the encapsulate is not degraded and/or not modified upon exposure of the encapsulate to said conditions.

In this regard, an encapsulate according to the invention wherein the carrier material comprises a wax (it is referred to the section entitled "Carrier material"), preferably selected from Candelilla wax, Carnauba wax, beeswax, rice bran wax, paraffin wax, jojoba wax, microcrystalline wax and japan wax, more preferably said wax is Candelilla wax, is particularly preferred as the milk saccharide or mixture of at least two different milk saccharides according to the invention (preferably the active agent according to the invention) is less affected (it is referred to the quantity and/or quality of said active agent as described herein) upon short and/or prolonged exposure to conditions (as described herein) compared to the nonencapsulated milk saccharide or non-encapsulated mixture of at least two different milk saccharides (preferably compared to the non-encapsulated active agent). Such an encapsulate can be of the matrix type or core-shell type. If core-shell type, than it is preferred that at least the carrier material of the outer layer (i.e. shell) comprises said wax.

Even better results in this regard are obtained using an encapsulate according to the invention wherein the carrier material comprises a resin (it is referred to the section entitled "Carrier material"), preferably said resin is a gum, more preferably wherein said resin is a gum selected from the list consisting of Shellac gum, Guar gum, Locust bean gum, Konjac gum, Arabic gum, Xanthan gum, Gellan gum, Cassia gum, cellulose gum, modified Arabic gum and Tara gum (from this list Shellac gum, Arabic gum and modified Arabic gum are preferred; shellac gum and Arabic gum are more preferred; Shellac gum is most preferred). Such an encapsulate can be of the matrix type or core-shell type. If core-shell type, than it is preferred that at least the carrier material of the outer layer (i.e. shell) comprises said resin, preferably said gum; if core shell-type, than it is more preferred that only the carrier material of the outer layer (i.e. shell) comprises said resin, preferably said gum. It is preferred that the carrier material further comprises a polysaccharide, preferably a polysaccharide selected from the list consisting of starch, pectin, maltodextrine, alginate, agarose, chitosan, hyaluronic acid, dextran, galactomannan, pullulan, carrageenan, fructan and fructo-oligosaccharide, more preferably a polysaccharide selected from the list consisting of alginate, pectin, starch and maltodextrine, even more preferably said polysaccharide is alginate or starch, most preferably said polysaccharide is starch. In the context of the present invention, it is preferred that said polysaccharide is not alginate, more preferably that said polysaccharide does not comprise alginate. Preferably said polysaccharide constitutes at least 1.0 % (VJ/VJ), 2.0% (VJ/VJ), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w), of the carrier material. Preferably said polysaccharide constitutes less than 15% (w/w), more preferably less than 10% (w/w), of the carrier material. Such an encapsulate can be of the matrix type or core-shell type. As already depicted herein, an encapsulate of the so-called coated matrix type (in the present context, at least the carrier material of the shell comprises said wax or said resin, optionally the carrier material of the core comprises said wax or said resin) is also considered to be a core-shell type of encapsulate.

An encapsulate of the core-shell type is most preferred in the context of the present invention, as the saccharide or mixture of at least two different saccharides according to the invention (preferably the active agent) is least affected (it is referred to the quantity and/or quality of said active agent as described herein) upon short and/or prolonged exposure to conditions (as described herein) compared to the nonencapsulated milk saccharide or non-encapsulated mixture of at least two different milk saccharides (preferably the non-encapsulated active agent).

Active agent

In an embodiment of the first aspect of the invention, said active agent comprises a milk saccharide or a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides. Throughout the application and claims, said milk saccharide (preferably said milk oligosaccharide) and said milk saccharides (preferably said milk oligosaccharides) according to the invention are obtained from an in vitro and/or ex vivo culture of cells as described herein.

In the context of the present invention, the term "saccharide" refers to a molecule comprising at least one monosaccharide. Preferably, "saccharide" refers to a molecule that comprises at least one monosaccharide and does not comprise a moiety which is different from a sugar such as a nucleobase, a nucleotide or an amino acid. More preferably, "saccharide" refers to a molecule consisting of one or more monosaccharides, i.e. "saccharide" refers to a monosaccharide, disaccharide, oligosaccharide and polysaccharide, preferably as defined herein. Even more preferably, "saccharide" refers to an oligosaccharide and polysaccharide, preferably as defined herein. Most preferably, "saccharide" refers to an oligosaccharide, preferably as defined herein. The term "oligosaccharide" as used in the context of the present invention preferably refers to a saccharide containing 2 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 2-20. More preferably, said oligosaccharide according to the invention contains at least 3 monosaccharides, i.e. the degree of polymerization is preferably 3-20. Even more preferably, said oligosaccharide according to the invention consists of 3-9, preferably 3-8, more preferably 3-7 monosaccharides. Throughout the application and claims, it is preferred that an oligosaccharide according to the invention (i.e. milk oligosaccharide, preferably mammalian milk oligosaccharide, more preferably human milk oligosaccharide) is not lactose. Likewise, in the case of a mixture of at least two (preferably at least three, more preferably at least 4, most preferably at least 5) different oligosaccharides according to the invention, it is preferred that at least one, preferably at least two, more preferably at least three, even more preferably at least 4, even more preferably at least 5 ,most preferably all, of said oligosaccharides according to the invention is/are not lactose. In the context of the present invention, the term "milk saccharide" refers to a saccharide which is found in milk of an animal, preferably mammal and/or human.

In a preferred embodiment, said milk saccharide and said milk saccharides are a milk oligosaccharide and milk oligosaccharides, respectively. In other words, in a preferred embodiment, said active agent comprises a milk oligosaccharide or a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk oligosaccharides. Throughout the application and claims, unless specifically stated otherwise, the terms "saccharide" and "saccharides" are preferably replaced with the terms "oligosaccharide" and "oligosaccharides", respectively. In the context of the present invention, the term "different" saccharides/oligosaccharides preferably means "structurally different" or "structurally distinct". These terms are hence preferably interchangeably used in the context of the present invention.

In a more preferred embodiment of the invention, said milk saccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of said milk saccharides in said mixture is/are a mammalian milk saccharide/mammalian milk oligosaccharide (MMO). Preferably, said milk saccharide/milk oligosaccharide is a human milk saccharide/human milk oligosaccharide (HMO). Mammalian milk (oligo)saccharides comprise (oligo)saccharides present in milk found in any phase during lactation including colostrum milk from humans (i.e. human milk (oligo)saccharides) and mammals including but not limited to cows (Bos Taurus), sheep (Ovis aries), goats (Capra aegagrus hircus), bactrian camels (Camelus bactrianus), horses (Eguus ferus caballus), pigs (Sus scropha), dogs (Canis lupus familiaris), ezo brown bears (Ursus arctos yesoensis), polar bear (Ursus maritimus), Japanese black bears (Ursus thibetanus japonicus), striped skunks (Mephitis mephitis), hooded seals (Cystophora cristata), Asian elephants (Elephas maximus), African elephant (Loxodonta africana), giant anteater (Myrmecophaga tridactyla), common bottlenose dolphins (Tursiops truncates), northern minke whales (Balaenoptera acutorostrata), tammar wallabies (Macropus eugenii), red kangaroos (Macropus rufus), common brushtail possum (Trichosurus Vulpecula), koalas (Phascolarctos cinereus), eastern quolls (Dasyurus viverrinus), platypus (Ornithorhynchus anatinus).

Throughout the application and claims, the term "milk saccharide" is preferably replaced with the term "mammalian milk saccharide", even more preferably replaced with the term "human milk saccharide". Likewise, throughout the application and claims, the term "milk oligosaccharide" is preferably replaced with the term "mammalian milk oligosaccharide", even more preferably replaced with the term "human milk oligosaccharide".

Preferably, said milk saccharide/oligosaccharide is selected from the list consisting of (i) oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end; (ii) oligosaccharide comprising N-acetyllactosamine (LacNAc) at its reducing end; (iii) oligosaccharide comprising lacto-N-biose (LNB) at its reducing end; (iv) milk glycosaminoglycan; (v) antigen of the human ABO blood group system, optionally bound to a monosaccharide selected from the list consisting of betal,3-GlcNAc, betal,4-GlcNAc, betal,3-GalNAc and -betal,4-Glc; and (vi) Lewis-type antigen oligosaccharide.

More preferably, said milk saccharide/oligosaccharide is selected from the list consisting of (i) oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end; (ii) oligosaccharide comprising N- acetyllactosamine (LacNAc) at its reducing end; (iii) oligosaccharide comprising lacto-N-biose (LNB) at its reducing end; and (iv) antigen of the human ABO blood group system, optionally bound to a monosaccharide selected from the list consisting of betal,3-GlcNAc, betal,4-GlcNAc, betal,3-GalNAc and -betal,4-Glc.

Even more preferably, said milk saccharide/oligosaccharide is selected from the list consisting of (i) oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end; (ii) oligosaccharide comprising N- acetyllactosamine (LacNAc) at its reducing end; and (iii) oligosaccharide comprising lacto-N-biose (LNB) at its reducing end.

Most preferably, said milk saccharide/oligosaccharide is an oligosaccharide comprising lactose (Gal-bl,4- Glc) at its reducing end.

Said oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end refers to a replete amount of oligosaccharide structures as found in the milk of animals such as mammals and humans (e.g. Table 2 of

Urashima et al, 2011). Examples include: neutral, fucosylated oligosaccharides: e.g. 2'-fucosyllactose (2'FL), 3-fucosyllactose (3-FL), difucosyllactose (diFL), Lacto-N-fucopentaose I (LNFP-I; Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal- bl,4-Glc), GalNAc-LNFP-l (GalNAc-al,3-(Fuc-al,2)-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc), Lacto-

N-fucopentaose (LNFP-II; Gal-bl,3-(Fuc-al,4)-GlcNAc-bl,3-Gal-bl,4-Glc), Lacto-N- fucopentaose III (LNFP III; Gal-bl,4-(Fuc-al,3)-GlcNAc-bl,3-Gal-bl,4-Glc), Lacto-N- fucopentaose V (LNFP-V; Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc), Lacto-N- fucopentaose VI (LNFP-VI; Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc), lacto-N- neofucopentaose (LNnFP Fuc-al,2-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc), lacto-N- difucohexaose I (LNDFH I; Fuc-al,2-Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-Gal-bl,4-Glc), lacto-N- difucohexaose II (LNDFH II; Fuc-al,4-(Gal-bl,3)-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc),

Monofucosyllacto-N-hexaose III, Difucosyllacto-N-hexaose, difucosyl-lacto-N-neohexaose,

LNnDFH (Gal-bl,4-(Fuc-al,3)-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc), A-tetrasaccharide

(GalNAc-al,3-(Fuc-al,2)-Gal-bl,4-Glc),... neutral, non-fucosylated oligosaccharides: e.g. Lacto-N-triose II (LN3), Lacto-N-neotetraose (LNnT), Lacto-N-tetraose (LNT), para-Lacto-N-neopentaose, para-Lacto-N-pentaose, para- Lacto-N-neohexaose, para-Lacto-N-hexaose, GalNAc-bl,3-Gal-al,4-Gal-bl,4-Glc (globo-N- tetraose),... charged oligosaccharides: e.g. 3'-sialyllactose, 6'-sialyllactose, 3,6-disialyllactose, sialylated lacto-N-triose, sialylated lacto-N-tetraose comprising LSTa and LSTb, sialyllacto-N- neotetraose comprising LSTc and LSTd, monosialyllacto-N-hexaose, disialyllacto-N-hexaose I, monosialyllacto-N-neohexaose I, monosialyllacto-N-neohexaose ll ₇ disialyllacto-N- neohexaose, disialyllacto-N-tetraose, disialyllacto-N-hexaose II, sialyllacto-N-tetraose a, disialyllacto-N-hexaose I, sialyllacto-N-tetraose b, 3'-sialyl-3-fucosyllactose (3'S-3-FL), disialomonofucosyllacto-N-neohexaose, sialyllacto-N-fucohexaose II, disialyllacto-N- fucopentaose II, monofucosyldisialyllacto-N-tetraose,...

Said oligosaccharide comprising N-acetyllactosamine (LacNAc) at its reducing end refers to oligosaccharides such as but not limited to 3'-sialyllactosamine (3'SLacNAc; 3'-SLN; Neu5Ac-a2,3-Gal- pi,4-GlcNAc), 6'-sialyllactosamine (6'SLacNAc; 6'-SLN; Neu5Ac-a2,6-Gal-pi,4-GlcNAc), sialyl Lex (sialyl Lewis x; Neu5Ac-a2,3-Gal-pi,4-[Fuc-al,3]-GlcNAc), Neu5Gc-a2,3-Gal-bl,4-GlcNAc, 2'FLacNAc, 3-FLacNAc (lewis x; 3-FLN; Gaip-l,4-[Fuc-al,3]-GlcNAc), Ley (Lewis y; Fuc-al,2-Gal-bl,4-(Fuc-al,3)-GlcNAc), ....

Said oligosaccharide comprising lacto-N-biose (LNB) at its reducing end refers to oligosaccharides such as but not limited to 3'-sialyllacto-N-biose (3'SLNB), 6'-sialyllacto-N-biose (6'SLNB), sialyl Lea (sialyl Lewis a; Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc), 2'FLNB, 4-FLNB, Leb (Lewis b; Fuc-al,2-Gal-bl,3-(Fuc-al,4)- GIcNAc),... .

Said glycosaminoglycan is preferably selected from the list consisting of chondroitin sulfate, heparan sulfate, hyaluronic acid, dermatan sulfate and heparin.

Said antigen of the human ABO blood group system refers to A determinant (GalNAc-alphal,3(Fuc- alphal,2)-Gal), B determinant (Gal-alphal,3(Fuc-alphal,2)-Gal) and H-determinant (Fuc-alphal,2-Gal), which can optionally be bound to a monosaccharide selected from the list consisting of betal,3-GlcNAc, betal,4-GlcNAc, betal,3-GalNAc and -betal,4-Glc.

Said Lewis-type antigen oligosaccharide preferably refers to Hl antigen, which is Fuc-al,2-Gal-pi,3- GIcNAc, or in short 2'FLNB; Lewisa (or Lea), which is the trisaccharide Gal-pi,3-[Fuc-al,4]-GlcNAc, or in short 4-FLNB; Lewisb (or Leb), which is the tetrasaccharide Fuc-al,2-Gal-pi,3-[Fuc-al,4]-GlcNAc, or in short DiF-LNB; sialyl Lewisa (or sialyl Lea) which is 5-acetylneuraminyl-(2-3)-galactosyl-(l-3)- (fucopyranosyl-(l-4))-N-acetylglucosamine, or written in short Neu5Ac-a2,3-Gal-pi,3-[Fuc-al,4]-GlcNAc; H2 antigen, which is Fuc-al,2-Gal-pi,4GlcNAc, or otherwise stated 2'fucosyl-N-acetyl-lactosamine, in short 2'FLacNAc; Lewisx (or Lex), which is the trisaccharide Gal-pi,4-[Fuc-al,3]-GlcNAc, or otherwise known as 3-Fucosyl-N-acetyl-lactosamine, in short 3-FLacNAc, Lewisy (or Ley), which is the tetrasaccharide Fuc-al,2-Gal-pi,4-[Fuc-al,3]-GlcNAc and sialyl Lewisx (or sialyl Lex) which is 5-acetylneuraminyl-(2-3)- galactosyl-(l-4)-(fucopyranosyl-(l-3))-N-acetylglucosamine, or written in short Neu5Ac-a2,3-Gal-pi,4- [Fuc-al,3]GlcNAc. In an additional and/or alternative more preferred embodiment of the invention, said saccharide (preferably said oligosaccharide) is not lactose and none of the saccharides (preferably said oligosaccharides) of said mixture according to the invention are lactose. Preferably, said saccharide (preferably oligosaccharide) is not a disaccharide and none of the saccharides (preferably oligosaccharides) of said mixture according to the invention are a disaccharide.

In a preferred embodiment of the first aspect of the invention, said milk saccharide (preferably said milk oligosaccharide) or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of said milk saccharides (preferably said milk oligosaccharides) in said mixture according to the invention is/are obtained from an in vitro and/or ex vivo culture of cells as described herein. Preferably, said cells are chosen from the list consisting of a microorganism, said microorganism is preferably a bacterium, a yeast or a fungus; a plant cell; an animal cell and a protozoan cell, more preferably wherein said cells are a microorganism, preferably a bacterium, a yeast or a fungus. The latter bacterium preferably is an Escherichia coli strain, more preferably an Escherichia coli strain which is a K-12 strain, even more preferably the Escherichia coli K-12 strain is E. coli MG1655. The latter yeast is preferably selected from Pichia pastoris, Yarrowia lipolitica, Saccharomyces cerevisiae and Kluyveromyces lactis. The latter fungus belongs preferably to the genus Rhizopus, Dictyostelium, Penicillium, Mucor or Aspergillus. The latter plant cell includes cells of flowering and non-flowering plants, as well as algal cells. Preferably, said plant cell is a tobacco, alfalfa, rice, cotton, rapeseed, tomato, corn, maize or soybean cell. The latter animal cell is preferably derived from a non-human animal or is a genetically modified cell line derived from human cells excluding embryonic stem cells. The latter protozoan cell preferably is a Leishmania tarentolae cell.

In the context of the present invention, the active agent of an encapsulate according to the invention may comprise additional (oligo)saccharides than the milk (oligo)saccharide or mixture of milk (oligosaccharides according to the invention. These additional (oligo)saccharides may be isolated from any source such as microbial cultivation or fermentation, cell culture, enzymatic reaction and chemical reaction.

In the context of the invention, it is also within the scope of the present invention that two or more different cells (preferably as defined herein), produce the saccharides of the mixture according to the invention, wherein each cell produces a different saccharide and/or a different mixture of saccharides.

In a more preferred embodiment of the first aspect of the invention, said milk saccharide (preferably said milk oligosaccharide) or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of said milk saccharides (preferably said milk oligosaccharides) in said mixture according to the invention is obtained from an in vitro and/or ex vivo culture of microorganism cells, preferably said microorganism is a bacterium or a yeast, more preferably said microorganism is a bacterium, even more preferably said microorganism is Escherichia coli. In other words, said milk saccharide/oligosaccharide or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of said milk saccharides/oligosaccharides in said mixture according to the invention is/are isolated from a microbial cultivation or fermentation, preferably said microorganism is a bacterium or a yeast, more preferably said microorganism is a bacterium, even more preferably said microorganism is Escherichia coli.

In an additional and/or alternative more preferred embodiment of the first aspect of the invention, said milk saccharide (preferably said milk oligosaccharide) or any one, preferably at least two, more preferably at least three, even more preferably at least four, most preferably all, of said milk saccharides (preferably said milk oligosaccharides) in said mixture according to the invention is/are obtained from an in vitro and/or ex vivo culture of mammary epithelial cells, mammary myoepithelial cells and/or mammary progenitor cells, preferably wherein said cells are generated from non-mammary adult stem cells, more preferably wherein said cells are generated from mesenchymal stem cells. Such cells are well-known to the skilled person, it is in this regard referred to for example WO2021/067641 and WO2021/242866 (mammary epithelial cells derived from non-mammary adult stem cells, preferably from mesenchymal stem cells) and WO2021/142241 (mammary epithelial cells, mammary myoepithelial cells, mammary progenitor cells).

Throughout the application and claims, it is preferred that said "in vitro and/or ex vivo culture of cells" produces the saccharide(s) according to the invention intracellularly, i.e. the saccharide(s) is/are produced inside the cell, wherein optionally one or more saccharides are exported outside the cell. In other words, the situation wherein cells are added to a reaction medium comprising for example an acceptor saccharide, wherein the cells are subsequently lysed in order to release the required enzymes and reagents for glycosylating the acceptor saccharide, is not encompassed by "intracellular production of a saccharide by an in vitro and/or ex vivo culture of cells".

Throughout the application and claims, the term "obtained" is preferably replaced with "isolated".

In an embodiment of the first aspect of the invention, said milk saccharide (preferably said milk oligosaccharide) or said mixture of milk saccharides (preferably said milk oligosaccharides) is provided as a solution or as a powder, preferably as a powder, before combining with said first carrier material as to form an encapsulate according to the invention. Preferably, said solution is a slurry, i.e. a semi-liquid mixture. Preferably, said saccharide or said mixture of saccharides is present in said solution/slurry in an amount of at least 1.0 % (w/v), 2.0 % (w/v), 5.0 % (w/v), 10 % (w/v), 15 % (w/v), 20 % (w/v), 25 % (w/v), 30 % (w/v), 35 % (w/v), 40 % (w/v), 45 % (w/v), 50% (w/v), 55 % (w/v) or 60 % (w/v), preferably at least 10 % (w/v), more preferably at least 20 % (w/v), even more preferably at least 30 % (w/v), most preferably at least 40 % (w/v). Different techniques can be used to assess the oligosaccharide % (w/v) within a solution. For example, dissolution of sugar in an aqueous solution changes the refractive index of the solution. Accordingly, an appropriately calibrated refractometer can be used to measure the oligosaccharide % (VJ/V). Alternatively, the density of a solution may be measured and converted to the oligosaccharide % (VJ/V). A digital density meter can perform this measurement and conversion automatically, or a hydrometer or pycnometer may be used.

Saccharide or mixture of saccharides according to the invention is typically isolated from an in vitro and/or ex vivo culture of cells as described herein, resulting in a solution containing said milk saccharide or mixture of milk saccharides.

Such a solution can for example be obtained by a method comprising the steps of:

(a) cultivating at least one cell as defined herein, preferably a single cell, that is capable to produce said milk saccharide or said mixture of at least two milk saccharides in a suitable cultivation medium to form a cultivation broth, preferably wherein said cell is metabolically engineered for the production of said saccharide or said mixture, and

(b) purifying said milk saccharide or said mixture of milk saccharides from the cultivation broth by:

(i) clarifying the cultivation broth, and

(ii) removing salts and/or medium components form said clarified cultivation broth, and/or

(iii) concentrating said milk saccharide or said mixture of milk saccharides in said clarified cultivation broth, thereby providing a solution comprising a purified milk saccharide or a purified mixture of at least 2 different milk saccharides.

Several drying techniques are known to the skilled person which can be used to obtain powder from a solution containing said milk saccharide or said mixture of at least two different milk saccharides as described herein. The slurry and powder according to the invention are preferably obtained by spray drying, freeze drying, spray freeze-drying, crystallization, lyophilization, band or belt drying, drum or roller drying, and/or agitated thin film drying, preferably by spray drying, drum or roller drying, or agitated thin film drying, of a solution containing said milk saccharide or said mixture of milk saccharides.

Spray drying is a technique wherein a liquid or slurry containing the molecule(s) of interest are brought directly into contact with a hot gas. In drum drying (also known as roller drying), the product is applied continuously as a thin film on the underside or top of the drum, while the drum is heated on the inside. An agitated thin film dryer essentially consists of two major elements, a cylindrical drying chamber with a heating jacket, and a rotor with fixed blades. The liquid feed is applied to the inside of the chamber (which is heated from the outside) where the rotating blades agitate the liquid feed, resulting in a thin film on the inside of the chamber. The liquid feed will transform into a viscous liquid, then into a paste and subsequently into a solid which is removed (i.e. scraped) from the chamber by the action of the blades.

Any technique that can be suitably used to dry a solution containing a milk saccharide or mixture of milk saccharides according to the invention is encompassed within the scope of the present invention as the obtained powder can be encapsulated by one or more known encapsulation techniques as described herein.

Further, the preferred drying techniques as described herein, i.e. spray drying, freeze drying, spray freeze- drying, crystallization, lyophilization, band or belt drying, drum or roller drying, and agitated thin film drying, result in a powder having a median diameter (D50) in the pm range which can be encapsulated using any known encapsulation technique, especially the preferred encapsulation techniques as described herein, i.e. prilling, spray chilling, spray cooling, fluidized air bed coating, granulation, spray drying and freeze drying, and the more preferred encapsulation techniques as described herein: prilling, spray chilling, spray cooling, spray drying, freeze drying and fluidized air bed coating.

Preferably, said obtained powder contains < 15 wt. %, preferably < 10 wt. %, more preferably < 9 wt. %, more preferably < 8 wt. %, more preferably < 7 wt. %, even more preferably < 5 wt. %, even more preferably < 4 wt. % of liquid, even more preferably < 3 wt. % of liquid, even more preferably < 2 wt. % of liquid, most preferably < 1 wt. %, preferably wherein said liquid is water. The liquid content is preferably determined by a thermogravimetric analysis or Karl Fisher titration analysis, more preferably by Karl Fisher titration analysis.

In an additional and/or alternative embodiment of the first aspect of the invention, said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes < 60% (w/w), preferably < 55% (w/w), more preferably < 50% (w/w), even more preferably < 45% (w/w), most preferably < 40% (w/w) of the total weight of the encapsulate of the invention.

In an additional and/or alternative embodiment, said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes at least 5% (w/w), preferably at least 10% (w/w), more preferably at least 15% (w/w), even more preferably at least 20% (w/w), even more preferably at least 25% (w/w) of the total weight of the encapsulate of the invention.

In an additional and/or alternative embodiment, said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes 5-50% (w/w), preferably 10-40 % (w/w), more preferably 20-40% (w/w), even more preferably 20-35% (w/w), of the total weight of the encapsulate of the invention.

In an additional and/or alternative embodiment, said active agent according to the invention further comprises: a lipid, preferably one or more selected from the list consisting of an oil, fat, ester, monoglyceride, diglyceride, triglyceride and free fatty acid; a vitamin, preferably one or more selected from the list consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin H, or a derivate thereof; an amino acid compound; a trace element; a mineral, preferably one or more selected from the list consisting of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, selenium and chloride; an antioxidant; a prebiotic agent, preferably one or more selected from the list consisting of GOS (galactooligosaccharide), FOS (fructo-oligosaccharide), inulin and resistant starch; an antimicrobial agent; a protein, preferably an enzyme or antibody, more preferably an enzyme; and/or a feed ingredient.

In an additional and/or alternative embodiment, said active agent according to the invention further comprises a microbial strain, preferably a bacterial or yeast strain. Preferably, said microbial strain does not use (preferably is not capable to use) said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, as a carbon source. In this regard, it is noted that an encapsulate according to the invention preferably has a low moisture content as described herein such that said microbial strain is not able to use said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, as a carbon source. In the context of the invention, an active agent of an encapsulate according to the present invention preferably does not comprise a Bifidobacteria strain. In an alternative embodiment, an active agent of an encapsulate according to the present invention preferably does not comprise a bacterial strain, preferably does not comprise a bacterial strain or yeast strain, more preferably does not comprise a microbial strain, even more preferably does not comprise a probiotic agent. ion

In an embodiment of the first aspect of the invention, said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; and applying one or more encapsulation steps, wherein said encapsulation step can be any encapsulation method known by the skilled person such as described in Garti and McClements, 2012, Encapsulation technologies and delivery systems for food ingredients and nutraceuticals, 2012, Woodhead publishing series in food science, technology and nutrition (part II: "Processing technology approaches to produce encapsulation and delivery systems, in particular chapters 4, 5 and 7), Farheen et al, 2017; Int. J. Pharma Res. Health Sci. 5(5): p. 1823-1830; Lengyel et al, 2019, Scientia Pharmaceutica 87(20) : p. 1-31; Nedal Abu-Thabit, 2021, Nano- and microencapsulation, IntechOpen and Gibbs et al, 1999, Int. J. Food Sci. Nutr. 50(3): p. 213-224 (all incorporated herein by reference), in particular the encapsulation methods which are generally employed in the encapsulation of solid particles such as but not limited to prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating (FAB). All of these encapsulation methods are well-known by the skilled person and are described in for example aforementioned references which are incorporated by reference and are further described in the section entitled "Method for production of encapsulated active agent", i.e. the second aspect of the present invention.

Preferably, said encapsulation step does not comprise extrusion and/or coacervation.

In a preferred embodiment of the first aspect of the invention, said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; and applying one or more encapsulation steps, chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying.

Preferably, an encapsulate of the matrix type according to the invention is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; and applying one or more encapsulation steps, chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying and granulation, more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling.

Preferably, an encapsulate of the core-shell type according to the invention is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; applying one or more encapsulation steps to obtain an encapsulate of the matrix type, preferably chosen from the list consisting prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, even more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying and granulation, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and applying one or more encapsulation steps selected from the list consisting of fluidized air bed coating, prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, preferably chosen from the list consisting of fluidized air bed coating, prilling, spray chilling and spray cooling, more preferably fluidized air bed coating.

Fluidized air bed coating (FAB) is generally considered as the golden standard in the field for coating a matrix type encapsulate in order to obtain a core-shell type encapsulate (Abu-Thabit, 2021; Teunou et al, 2002, J. Food Engineering 53: p. 325-340).

In an even more preferred embodiment, said encapsulate according to the invention is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; applying a prilling, spray cooling or spray chilling step, preferably a prilling step; optionally followed by applying a fluidized air bed coating step.

In a most preferred embodiment, said encapsulate according to the invention is obtainable by a method according to the second aspect of the invention.

Carrier material

In an embodiment of the first aspect of the invention, said encapsulate comprises a first carrier material which forms (i) a shell around said active agent as to form an encapsulate of the core-shell type or (ii) a matrix wherein said active agent is distributed as to form an encapsulate of the matrix type.

In an additional and/or alternative embodiment, said encapsulate according to the invention further comprises a second carrier material which at least partially, preferably completely, encapsulates said first carrier material and said active agent as to form an encapsulate of the core-shell type, wherein said active agent and said first carrier material form a core component which is surrounded by a shell formed by the second carrier material. In this context of the invention, said core component comprising the active agent and first carrier material can be an encapsulate of the matrix type or of the core-shell type.

In an additional and/or alternative embodiment, the composition of said first carrier material is different from the composition of said second carrier material. In the context of the present invention, a first carrier material is different from a second carrier material if: (i) one or more components are not the same (e.g. component A is present in the first carrier material whereas component B is present in the second carrier material), i.e. qualitative composition of the first carrier material is different from that of the second carrier material; and/or

(ii) the relative abundance of one or more components is not the same (e.g. the first carrier material comprises 20 % of component A, whereas the second carrier material comprises 35 % of component A), i.e. quantitative composition of the first carrier material is different from that of the second carrier material.

It is a preferred embodiment of the present invention, that the quantitative and/or qualitative, preferably qualitative, composition of said first carrier material differs from that of the second carrier material.

In an alternative preferred embodiment, the qualitative composition of said first carrier material is identical to the qualitative composition of said second carrier material. In another alternative preferred embodiment, the quantitative composition of said first carrier material is identical to the quantitative composition of said second carrier material.

In an additional and/or alternative embodiment, said first carrier material of an encapsulate according to the invention comprises a hot melt material and/or a polymer. In another embodiment, said first carrier material comprises two or more different hot melt materials, optionally a polymer. In another embodiment, said first carrier material comprises two or more different polymers, optionally a hot melt material. In another embodiment, said first carrier material comprises two or more different hot melt materials and/or two or more different polymers.

In an additional and/or alternative embodiment, said second carrier material of an encapsulate according to the invention comprises a hot melt material and/or a polymer. In another embodiment, said second carrier material comprises two or more different hot melt materials, optionally a polymer. In another embodiment, said second carrier material comprises two or more different polymers, optionally a hot melt material. In another embodiment, said second carrier material comprises two or more different hot melt materials and/or two or more different polymers.

In an additional and/or alternative embodiment of the invention, said second carrier material comprises a hot melt material and/or a polymer, preferably a polymer, optionally further comprising starch. Preferably, said hot melt material is an oil and/or a wax, more preferably a hydrogenated oil and/or a wax. Preferably, said polymer is a resin, more preferably said polymer is a gum. When starch is present in the second carrier material, then it is preferred that said starch constitutes less than 15% (w/w) of said second carrier material, more preferably said starch constitutes less than 10% (w/w) of said second carrier material. Preferably, said starch constitutes at least 1.0 % (VJ/VJ), 2.0% (VJ/VJ), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w) of said second carrier material. Said hot melt material, oil, wax, resin and gum are as described herein.

In an additional and/or alternative embodiment of the invention, said first carrier material and/or said second carrier material does not comprise 2'FL. Preferably, said first and said second carrier material do not comprise 2'FL. In the context of the present invention, "a carrier material does not comprise 2'FL" means that said 2'FL is not an encapsulant or a co-encapsulant. In the situation wherein the encapsulate according to the invention comprises a third (and optionally a fourth etc) carrier material, it is preferred that none of the carrier materials comprise 2'FL. More preferably, said first carrier material and/or said second carrier material does not comprise a saccharide or a mixture of at least two different saccharides according to the invention (it is referred to the section "Active agent"). Even more preferably, said first and said second carrier material do not comprise a saccharide or a mixture of at least two different saccharides according to the invention (it is referred to the section "Active agent"). In the context of the present invention, "a carrier material does not comprise a saccharide or a mixture of at least two different saccharides according to the invention" means that said saccharide or a mixture of at least two different saccharides according to the invention is not an encapsulant or a co-encapsulant. In the situation wherein the encapsulate according to the invention comprises a third (and optionally a fourth etc) carrier material, it is preferred that none of the carrier materials comprise a saccharide or a mixture of at least two different saccharides according to the invention.

In a more preferred embodiment, said hot melt material is a lipophilic material, preferably a lipid, more preferably selected from the list consisting of an oil, a wax, a glyceride and a fatty acid, some typically used as food and feed additives and listed as an E number in the ingredients list and found in food additive regulations of EFSA (incorporated by reference). In the context of the present invention, said oil, a wax, a glyceride and/or a fatty acid are preferably a food/feed additive which is listed as an E number in the ingredients list and found in food additive regulations of EFSA (incorporated by reference).

Preferably, said lipid is palm stearin. More preferably said lipid is hydrogenated palm stearin.

Preferably, said oil is a hydrogenated oil. As understood by the skilled person, a hydrogenated oil is an oil that has been treated with hydrogen, resulting in an oil with a lower level of unsaturated bonds (i.e. a lower Iodine value) compared to the untreated oil. Preferably, said oil is a vegetable oil. More preferably, said oil is selected from the list consisting of palm oil, sunflower oil, soybean oil, rapeseed oil, coconut oil, babassu oil, palm kernel oil, maize oil, sesame oil and cottonseed oil. Even more preferably said oil is selected from the list consisting of palm oil, sunflower oil, soybean oil and rapeseed oil. Even more preferably, said oil is palm oil. Most preferably said oil is hydrogenated palm oil. Preferably, said wax is selected from the list consisting of Candelilla wax (e.g. E902), Carnauba wax (e.g. E903), beeswax (e.g. E901), rice bran wax, paraffin wax, jojoba wax, microcrystalline wax (e.g. E905) and japan wax. More preferably, said wax is Candelilla wax (e.g. E902) or Carnauba wax (e.g. E903). Most preferably, said wax is Candelilla wax (e.g. E902).

Preferably, said glyceride is a monoglyceride, a diglyceride or a triglyceride. More preferably, said glyceride is a monoglyceride, for example glycerol monostearate. More preferably, said glyceride is a diglyceride, for example glyceryl dibehenate. More preferably, said glyceride is a triglyceride.

In an additional and/or alternative more preferred embodiment, said hot melt material has a melting point of at least 30°C, preferably at least 45°, more preferably at least 50°C.

In an additional and/or alternative preferred embodiment, said hot melt material has a melting point which is lower than 100°C, preferably lower than 90°C, more preferably lower than 80°C, even more preferably lower than 76°C.

In an additional and/or alternative more preferred embodiment, said hot melt material has an Iodine value which is lower than 100, preferably lower than 90, more preferably lower than 80, even more preferably lower than 70, even more preferably lower than 60, even more preferably lower than 50, even more preferably lower than 40, even more preferably lower than 30, even more preferably lower than 20, even more preferably lower than 10, most preferably lower than 5.

In an additional and/or alternative more preferred embodiment, said hot melt material contains unsaponifiable matter no greater than 2.0% (w/w), preferably no greater than 1.5% (w/w).

In an additional and/or alternative more preferred embodiment, said hot melt material comprises an oil as described herein, preferably a hydrogenated oil, and/or a wax as described herein.

In an additional and/or alternative more preferred embodiment of the invention, said polymer is a natural or a synthetic polymer. Some polymers are typically used as food and feed additives and listed as an E number in the ingredients list and found in food additive regulations of EFSA (incorporated by reference). In the context of the present invention, said polymer is preferably a food/feed additive which is listed as an E number in the ingredients list and found in food additive regulations of EFSA (incorporated by reference).

Preferably, said polymer is selected from a list consisting of a polysaccharide, a resin, a cellulose or cellulose-derivative, a protein and/or a synthetic polymer. More preferably, said polymer is selected from the list consisting of a polysaccharide, a resin, a cellulose or cellulose-derivative. Even more preferably, said polymer is a polysaccharide, a resin or a cellulose-derivative. Most preferably, said polymer is a resin. In the context of the present invention, it is preferred that the first carrier material of an encapsulate according to the invention does not comprise a protein. More preferably, each carrier material of an encapsulate according to the invention (i.e. first carrier material as well as the second, third,., carrier material if present) does not comprise a protein. In the context of the present invention, it is preferred that said synthetic polymer is not poly-L-lysine, more preferably that said synthetic polymer does not comprise poly-L-lysine.

In the context of the present invention, derivatives of the polymers described herein are also preferably encompassed by the scope of the present invention.

Preferably, said polysaccharide is selected from the list consisting of starch, pectin, maltodextrine, alginate, agarose, chitosan, hyaluronic acid, dextran, galactomannan, pullulan, carrageenan, fructan and fructo-oligosaccharide. Starch can be obtained from for example wheat, corn, potato, rice and tapioca. More preferably, said polysaccharide is selected from the list consisting of alginate, pectin, starch and maltodextrine. Even more preferably, said polysaccharide is alginate or starch. Most preferably said polysaccharide is starch. In the context of the present invention, it is preferred that said polysaccharide is not alginate, more preferably that said polysaccharide does not comprise alginate. Preferably, said polysaccharide constitutes at least 1.0 % (w/w), 2.0% (w/w), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said polysaccharide constitutes at least 5.0% (w/w), of said carrier material. Preferably said polysaccharide constitutes less than 15% (w/w), more preferably less than 10% (w/w), of the carrier material. Such an encapsulate can be of the matrix type or core-shell type.

Preferably, said resin is a gum. More preferably, said resin is selected from the list consisting of Shellac gum (e.g. E904), Guar gum (e.g. E412), Locust bean gum (E410), Konjac gum (E425), Arabic gum (e.g. E414), Xanthan gum (e.g. E415), Gellan gum (e.g. E418), Cassia gum (e.g. E427), cellulose gum (e.g. E466), modified Arabic gum (e.g. E423) and Tara gum (e.g. E417). More preferably, said resin is selected from Shellac gum (e.g. E904), Arabic gum (e.g. E414) and modified Arabic gum (e.g. E423). Even more preferably, said resin is selected from Shellac gum (e.g. E904) and Arabic gum (e.g. E414). Most preferably, said resin is Shellac gum (e.g. E904).

Preferably, said cellulose-derivative is selected from the list consisting of carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropoylcellulose, methylcellulose and ethylcellulose. More preferably, said cellulose-derivative is selected from hydroxypropylmethylcellulose and carboxymethylcellulose.

Preferably, said protein is selected from the list consisting of Zein, albumin, whey protein and soy protein. Preferably, said synthetic polymer is selected from the list consisting of poly-L-lysine, polylactic acid- glycolic acid copolymer, polyacrylic acid, polymethylacrylates, polyethylene glycols and fumaryl diketopiperazine. Even more preferably, said synthetic polymer is selected from the list consisting of polylactic acid-glycolic acid copolymer, polyacrylic acid, polymethylacrylates, polyethylene glycols and fumaryl diketopiperazine. In an additional and/or alternative more preferred embodiment, said first carrier material comprises starch, preferably said starch constitutes less than 15% (w/w) of said first carrier material, more preferably said starch constitutes less than 10% (w/w) of said first carrier material. Preferably, said first carrier material comprises starch, wherein said starch constitutes at least 1.0 % (w/w), 2.0% (w/w), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w) of said first carrier material.

In an additional and/or alternative more preferred embodiment, said second carrier material comprises starch, preferably said starch constitutes less than 15% (w/w) of said second carrier material, more preferably said starch constitutes less than 10% (w/w) of said second carrier material. Preferably, said second carrier material comprises starch, wherein said starch constitutes at least 1.0 % (w/w), 2.0% (w/w), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w) of said second carrier material.

In an additional and/or alternative more preferred embodiment, said first carrier material and/or said second carrier material comprises an additive. Preferably, said additive is glycerol or a derivative thereof, more preferably, said additive is glycerol.

In an even more preferred embodiment of the invention, wherein said encapsulate is of the matrix type, said first carrier material comprises a hot melt material, preferably said first carrier material comprises an oil and/or a wax, more preferably said first carrier material comprises a hydrogenated oil and/or a wax. Optionally, said carrier material further comprises starch. Said hot melt material, oil, wax and starch are as described herein.

In another even more preferred embodiment of the invention, wherein said encapsulate is of the coreshell type, said first carrier material comprises a hot melt material and/or a polymer, preferably a hot melt material, optionally further comprising starch. Preferably, said hot melt material is an oil and/or a wax, more preferably a hydrogenated oil and/or a wax. Preferably, said polymer is a resin, more preferably said polymer is a gum. When starch is present in the first carrier material, then it is preferred that said starch constitutes less than 15% (w/w) of said first carrier material, more preferably said starch constitutes less than 10% (w/w) of said first carrier material. Preferably, said starch constitutes at least 1.0 % (w/w), 2.0% (w/w), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w) of said first carrier material. Said hot melt material, oil, wax, resin and gum are as described herein. When said encapsulate of the core-shell type comprises a second carrier material, said second carrier material comprises a hot melt material and/or a polymer, preferably a polymer, optionally further comprising starch. Preferably, said hot melt material is an oil and/or a wax, more preferably a hydrogenated oil and/or a wax. Preferably, said polymer is a resin, more preferably said polymer is a gum. When starch is present in the second carrier material, then it is preferred that said starch constitutes less than 15% (w/w) of said second carrier material, more preferably said starch constitutes less than 10% (w/w) of said second carrier material. Preferably, said starch constitutes at least 1.0 % (w/w), 2.0% (w/w), 3.0% (w/w), 4.0% (w/w), 5.0% (w/w), more preferably said starch constitutes at least 5.0% (w/w) of said second carrier material. Said hot melt material, oil, wax, resin and gum are as described herein.

Method for production of encapsulated active agent

In a second aspect, the invention provides a method for the production of an encapsulated active agent, said method comprising the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps.

Said method results in the production of an encapsulate of the core-shell type or matrix type, preferably core-shell type.

Hence, the invention provides a method for the production of an encapsulated active agent, said method comprising the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps

Said method results in the production of an encapsulate of the core-shell type or matrix type, preferably core-shell type.

In the context of the present invention, said "active agent", "slurry", "powder", "saccharide", "mixture of at least two different saccharides", "first carrier material", "second carrier material", "core-shell type", "matrix type" and "encapsulate" as described throughout the second aspect of the present invention and claims are preferably as described in the first aspect of the invention.

When an encapsulate obtained by a method according to the invention is of the core-shell type, it is a preferred embodiment that the core (preferably an encapsulate of the matrix type) has a median diameter (D50 also known as D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 165 pm. Preferably, said median diameter is 75-250 pm, more preferably 100-250 pm, even more preferably 125-250 pm, even more preferably 150-250 pm, even more preferably 165-250 pm, most preferably 165-225 pm. In an additional and/or alternative preferred embodiment in this context (i.e. when said encapsulate is of the core-shell type), the core-shell type encapsulate has a median diameter (D50 also known as D(v, 0.5)) which is 10-100 pm, preferably 20-100 pm, more preferably 20-75 pm, even more preferably 35-75 pm, longer than the median diameter of the core (preferably an encapsulate of the matrix type). In an additional and/or alternative preferred embodiment in this context (i.e. when said encapsulate is of the core-shell type), the core-shell type encapsulate has a median diameter (D50 also known as D(v, 0.5)) which is 10%-40%, preferably 10%-35%, more preferably 10%- 30%, even more preferably 15%-30%, most preferably 20%-30%, longer than the median diameter of the core (preferably an encapsulate of the matrix type).

Throughout the description and claims, the feature "active agent" and "active agent comprising a milk saccharide or mixture of at least two milk saccharides" is preferably replaced with "milk saccharide or mixture of at least two milk saccharides", wherein said milk saccharide/saccharides are preferably as described in the first aspect of the invention. In a preferred embodiment of the second aspect of the invention, a method for the production of an encapsulated milk saccharide or encapsulated mixture of at least two different milk saccharides is provided, said method comprising the steps of: providing a milk saccharide or a mixture of at least two different milk saccharides in the form of a slurry or a powder, preferably a powder, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating, granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps.

Hence, a preferred method for the production of an encapsulated milk saccharide or encapsulated mixture of at least two different milk saccharides is provided, said method comprising the steps of: providing a milk saccharide or a mixture of at least two different milk saccharides in the form of a slurry or a powder, preferably a powder, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said saccharide or mixture of saccharides into said first carrier material; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating, granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps.

In a more preferred embodiment, said one or more encapsulation steps are chosen from: for an encapsulate of the matrix type: prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, even more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying and granulation, even more preferably chosen from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably chosen from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; for an encapsulate of the core-shell type: fluidized air bed coating, prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably chosen from the list consisting of fluidized air bed coating, prilling, spray chilling and spray cooling, even more preferably prilling and/or fluidized air bed coating, most preferably fluidized air bed coating. It is particularly preferred in this context to (i) apply one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying and granulation, even more preferably chosen from the list consisting of prilling, spray chilling, spray cooling and granulation, more preferably chosen from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling, to obtain an encapsulate of the matrix type; and to (ii) apply one or more encapsulation steps to the obtained encapsulate of the matrix type, wherein said one or more encapsulation steps are chosen from the list consisting of fluidized air bed coating, prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, more preferably one or more fluidized air bed coating steps.

In an even more preferred additional and/or alternative embodiment, said method comprises the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said saccharide and said saccharides are obtained from an in vitro and/or ex vivo culture of cells; applying a prilling, spray cooling or spray chilling step, preferably a prilling step, as to obtain an encapsulate of the matrix type; optionally followed by applying a fluidized air bed coating step as to obtain an encapsulate of the core shell type.

Hence, an even more preferred method according to the invention comprises the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material; applying a prilling, spray cooling or spray chilling step, preferably a prilling step, as to obtain an encapsulate of the matrix type; optionally followed by applying a fluidized air bed coating step as to obtain an encapsulate of the core shell type.

In a most preferred embodiment, the method according to the second aspect of the invention comprises the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material as to obtain a mixture (also referred to as feed liquor); atomizing the obtained mixture, preferably through a nozzle or a rotating/spinning disk, as to form droplets; cooling the droplets by an ambient (preferably a temperature of 24-25°C) air stream or cold gas/air (preferably a temperature below 25°C, more preferably a temperature below 24°C) as to form solidified encapsulates.

Said method provides an encapsulate of the matrix type. Optionally, said method further comprises the steps of: providing a second carrier material, optionally forming a melt or liquid of said second carrier material by increasing the temperature above the melting temperature of said second carrier material; coating, preferably spraying, more preferably fluidized air bed coating, said second carrier material on the surface of said solidified encapsulates resulting in at least one layer of the second carrier material surrounding said solidified encapsulates; and optionally repeating the spraying step as to obtain a thicker layer and/or more layers of said second carrier material.

Said method provides an encapsulate of the core-shell type.

Prilling as disclose herein (Garti and McClements, 2012, chapter 5; Jannin and Cuppok, 2012; all incorporated by reference) is an encapsulation technique wherein uniform spherical particles are produced from molten carrier material, strong solutions or slurries, i.e. so-called feed liquor (i.e. mixture comprising active agent dispersed/dissolved into a first carrier material in the context of the present invention). In this context, a hot melt material and/or a polymer, preferably a hot melt material, as described in section "carrier material" of the first aspect of the invention is particularly suitable for prilling (i.e. for prilling, the carrier material preferably comprises a hot melt material and/or polymer, more preferably a hot melt material). Liquid drops are generated (i.e. atomizing) which are subsequently solidified individually by cooling as they fall through an ambient air stream (preferably a temperature of 24-25°C) or cold gas/air (preferably a temperature below 25°C, more preferably a temperature below 24°C). As understood by the skilled person, encapsulation by prilling comprises the following steps: (i) preparation of a feed liquor by dispersing/dissolving the active agent (as described in the first aspect of the invention) into a first carrier material as described herein, preferably a hot melt material and/or a polymer, preferably a hot melt material, as described in section "carrier material" of the first aspect of the invention; (ii) atomizing the feed liquor as to form droplets; (iii) cooling the droplets by an ambient air stream or cold gas as to form solidified encapsulates. Throughout the application and claims, unless specifically stated otherwise, the terms "encapsulate" and "encapsulates" are preferably replaced with "particle" and "particles", respectively, more preferably replaced with "microparticle" and "microparticles", respectively. In the context of the present invention, the feature "atomizing" refers to the disintegration of the liquid melt (i.e. so-called feed liquor) into fine spherical droplets. The feature "solidification" refers to the transformation of the liquid melt into a solid by cooling.

As there is no agglomeration, the size distribution of the droplets determines that of the final particles. The production of liquid drops can be obtained through the use of nozzles. A high pressure nozzle, a fountain nozzle or a vibrating nozzle can be used for example. The formation of droplets of the desired sized can be improved through the use of fractionation means. For example at least one cutting jet, i.e. a continuous jet of liquid or a rotating cutting disc which strikes the feed liquor intermittently to form successive droplets (it is referred to FR2997869 and Lopez-Iglesias et al, 2020, which are incorporated by reference). Another example is to apply an electrostatic potential to a forming droplet in order to reduce its size. Alternatively, spinning discs or spinning perforated baskets are used, wherein the feed liquor is dispersed into liquid droplets by centrifugal force. Said discs and baskets are advantageously used when the feed liquor is more difficult to handle (e.g. higher viscosity), which is for example the case if it is a slurry. It is hence a preferred embodiment of the present invention, to use a prilling device equipped with a rotating cutting disc, a spinning disc or a spinning perforated basket, preferably a spinning disc (also known as rotating disc). Further, suitable types of equipment for prilling comprise a fluid bed coater equipped for top-spray or tangential-spray (top-spray is preferred) and a turbo jet (Jannin and Cuppok, 2012). For large scale production, a prilling tower is used in the field.

In a preferred embodiment, the temperature of the feed liquor is 7 to 12 degrees (°C), preferably 8 to 10 degrees (°C), more preferably one to five degrees (°C), even more preferably one or two degrees (°C), higher than the solidification point of said feed liquor. In other words, it is preferred that the mixture obtained by dispersing the active agent into the first carrier material has a temperature which is 5-12 °C, preferably 7-12 °C, more preferably 8-10 °C, even more preferably 1-5 °C, even more preferably 1-2 °C, higher than the solidification point / melting temperature of said mixture.

In an additional and/or alternative preferred embodiment, the viscosity of the feed liquor is 20 - 800 millipascal second (mPa.s), preferably 20 - 650 mPa.s, more preferably 20 - 500 mPa.s, even more preferably 20 - 300 mPa.s, most preferably 50 - 200 mPa.s.

In an additional and/or alternative preferred embodiment, the atomizing pressure applied to generate droplets in the prilling process is at least 3 bar, preferably at least 4 bar. Preferably, said atomizing pressure is 4-6 bar.

In an additional and/or alternative preferred embodiment, the contact between the hot melt material (it is referred to the section "carrier material" of the first aspect of the invention) and the active agent (it is referred to the section "active agent" of the first aspect of the invention) is conducted for 1-15 minutes. In an additional and/or alternative preferred embodiment, the feed liquor comprises at least 5% (w/w), preferably at least 10% (w/w), more preferably at least 15% (w/w), even more preferably at least 20% (w/w), even more preferably at least 25% (w/w), most preferably at least 30% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the feed liquor comprises < 60% (w/w), preferably < 55% (w/w), more preferably < 50% (w/w), even more preferably < 45% (w/w), most preferably < 40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the feed liquor comprises 5-60% (w/w), preferably 10-50% (w/w), more preferably 15-50% (w/w), even more preferably 20-50% (w/w), even more preferably 25-50% (w/w), even more preferably 25-45% (w/w), even more preferably 25-40% (w/w), most preferably 30-40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention.

In an additional and/or alternative preferred embodiment, the feed liquor comprises 5-80 % (w/w), preferably 5-75 % (w/w), more preferably 5-70 % (w/w), even more preferably 10-70 % (w/w), even more preferably 20-70 % (w/w), even more preferably 30-70 % (w/w), even more preferably 50-70 % (w/w), most preferably 60-70 % (w/w), carrier material.

Prilling generally results in encapsulates with a median diameter of 50 pm-1000 pm. For most materials (including hot melt material as described in the section "carrier material' of the first aspect of the invention). Hence, prilling is a preferred encapsulation technique to obtain encapsulates with a median diameter which is longer than 100 pm and/or < 300 pm.

In the field of the present invention, "spray cooling", "spray chilling", "prilling" and "spray congealing" are interchangeable used (Favaro-Trindade et al, 2015, Encapsulation via Spray Chilling/Cooling/Congealing. In: Mishra, M., Ed., Handbook of Encapsulation and Controlled Release, CRC Press, Boca Raton, p. 71-88; Garti and McClements, 2012, chapter 5.1). In the context of the present invention, these terms can be interchangeably used, unless explicitly stated otherwise, and each embodiment disclosed in the context of prilling is considered to be disclosed as well in the context of spray cooling, spray chilling and spray congealing and vice versa. Importantly, spray cooling and spray chilling refer to the same encapsulation principle (i.e. prilling), however, in spray cooling solidification occurs upon contact with ambient air (preferably a temperature of 24-25°C), whereas in spray chilling solidification occurs upon contact with a cooled gas or air (preferably a temperature below 25°C, more preferably a temperature below 24°C). Spray cooling is consequently typically used in conjunction with a feed liquor (i.e. the mixture obtained by dispersing the active agent into the first carrier material according to the invention) which has a melting point lying in the range of 27-42°c, preferably 34-42°C, whereas spray chilling is typically used in conjunction with said feed liquor which has a melting point of > 42°C (and preferably lower than 122°C).

Granulation as disclosed herein is an encapsulation technique which is similar to prilling, with the important difference that not only the feed liquor as disclosed above in the context of prilling is provided, but also a binding agent. The latter allows to bind together small particles as to form granules. Encapsulates obtained by granulation have hence a larger median diameter. Size of the encapsulates is mainly determined by the residence time in the granulator (the longer the layers are allowed to build up, the larger the encapsulates become). Different granulation approaches have been developed which are suitable for encapsulating an active agent according to the invention. Preferably, said granulation is selected from the list consisting of high shear wet granulation, high shear melt granulation, fluid bed wet granulation, continuous wet granulation and continuous melt granulation, more preferably selected from the list consisting of fluid bed wet granulation, continuous wet granulation and continuous melt granulation, most preferably fluid bed wet granulation and continuous wet granulation.

Each embodiment disclosed in the context of prilling is also disclosed in the context of granulation. In a preferred embodiment, said binding agent is selected from the list consisting of starch, liquid glucose, povidone, cellulose, methyl cellulose, ethyl cellulose, polyvinyl pyrrolidone, gelatin, polyethylene glycol, sucrose, acacia, tragacanth and a gum. Preferably, a gum as disclosed in the present application.

Granulation generally results in encapsulates with a median diameter of at least 50 pm to hundreds of micrometer and is particularly preferred if larger encapsulates are required than for example can be obtained with prilling.

Spray drying as disclosed herein (Garti and McClements, 2012, chapter 4; Pinon-Balderrama et al, 2020, Processes 8(889); all incorporated by reference) is an encapsulation technique which is similar to prilling, i.e. spherical particles are produced from molten carrier material, strong solutions or slurries, i.e. so-called feed liquor (i.e. mixture comprising active agent dispersed/dissolved into a first carrier material in the context of the present invention). In this context, a hot melt material and/or a polymer, preferably a hot melt material, as described in section "carrier material" of the first aspect of the invention is particularly suitable for spray drying (i.e. for spray drying, the carrier material preferably comprises a hot melt material and/or polymer, more preferably a hot melt material). In contrast to prilling, liquid drops that are generated (i.e. atomizing) are subsequently solidified individually by rapid evaporation of the solvent in which the carrier material is dissolved. Usually air, preferably heated (i.e. temperature higher than ambient temperature as defined earlier herein), supplies the latent heat of vaporization required to remove the solvent from the coating material to form the encapsulates. Contact between the spray droplets and the air can be done in three ways: (i) co-current drying (air and particles move in same direction through drying chamber), counter-current drying (air and particles move in opposite directions through drying chamber) and (iii) mixed-flow (both co-current and counter-current phases are present) (Farheen et al, 2017). In the context of the present invention, co-current drying is preferred. A spray dryer includes an atomizer (preferably a nozzle or spinning wheel), a drying chamber supplied with air (preferably heated air), air heater, blower or fan (movement of air), product collector and a cyclone or bag-filter (separation of dried particles from humid air). As understood by the skilled person, encapsulation by spray drying comprises the following steps: (i) preparation of a feed liquor by dispersing/dissolving the active agent (as described in the first aspect of the invention) into a first carrier material as described herein, preferably a hot melt material and/or a polymer, preferably a hot melt material, as described in section "carrier material" of the first aspect of the invention; (ii) atomizing the feed liquor as to form droplets; (iii) drying the droplets, preferably by an air stream, more preferably by a heated air stream (i.e. temperature higher than ambient temperature as defined earlier herein) as to form solidified encapsulates. In the context of the present invention, the feature "atomizing" refers to the disintegration of the liquid melt (i.e. so-called feed liquor) into fine spherical droplets. The feature "solidification" refers to the transformation of the liquid melt into a solid by cooling.

In a preferred embodiment, the inlet temperature, i.e. the temperature of the drying medium (gas stream), is 140-220°C, preferably 150-200°C, more preferably 160-200°C, most preferably 160-190°C.

In an additional and/or alternative preferred embodiment, the feed flow rate is chosen such that the median diameter of the produced encapsulates is as described in the section "Encapsulate" of the first aspect of the invention. A higher feed flow rate generally results in a larger median diameter of the obtained encapsulates. Preferably, the feed flow rate is 0.5-100 mL/min, more preferably 0.5-50 mL/min, even more preferably 0.5-25 mL/min, even more preferably 0.5-15 mL/min, most preferably 1.0-10.0 mL/min.

In an additional and/or alternative preferred embodiment, the residence time, i.e. time elapsed from the entrance of the atomized liquid into the drying chamber until it leaves the dryer in the form of encapsulates, is preferably 2-35 seconds, more preferably 5-25 seconds, even more preferably 5-15 seconds, most preferably 10-15 seconds.

In an additional and/or alternative preferred embodiment, the viscosity of the feed liquor is 20 - 800 millipascal second (mPa.s), preferably 20 - 650 mPa.s, more preferably 20 - 500 mPa.s, even more preferably 20 - 300 mPa.s, most preferably 50 - 200 mPa.s.

In an additional and/or alternative preferred embodiment, the feed liquor constitutes at least 5% (w/w), preferably at least 10% (w/w), more preferably at least 15% (w/w), even more preferably at least 20% (w/w), even more preferably at least 25% (w/w), most preferably at least 30% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the feed liquor constitutes < 60% (w/w), preferably < 55% (w/w), more preferably < 50% (w/w), even more preferably < 45% (w/w), most preferably < 40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the feed liquor constitutes 5-60% (w/w), preferably 10-50% (w/w), more preferably 15-50% (w/w), even more preferably 20-50% (w/w), even more preferably 25-50% (w/w), even more preferably 25-45% (w/w), even more preferably 25-40% (w/w), most preferably 30-40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention.

In an additional and/or alternative preferred embodiment, the feed liquor comprises 5-80 % (w/w), preferably 5-75 % (w/w), more preferably 5-70 % (w/w), even more preferably 10-70 % (w/w), even more preferably 20-70 % (w/w), even more preferably 30-70 % (w/w), even more preferably 50-70 % (w/w), most preferably 60-70 % (w/w), carrier material.

Spray drying generally results in encapsulates with a median diameter of 1 pm-100 pm. Hence, spray drying is a preferred encapsulation technique to obtain encapsulates with a median diameter which is not longer than 100 pm.

Freeze drying as disclosed herein, also known as lyophilization and cryodesiccation, refers to an encapsulation technique wherein particles are produced (Garti and McClements, 2012, chapter 4; incorporated by reference) based on the principle of dehydration with sublimation, i.e. frozen unbound liquid (usually water) sublimates from solid state into gas state when adequate amount of heat is applied under very low environment pressure by a high vacuum condition. As understood by the skilled person, encapsulation by freeze drying is a multi-stage process comprising the following steps: (i) preparation of a mixture by dispersing/dissolving the active agent (as described in the first aspect of the invention) and a first carrier material as described herein, preferably a polymer, more preferably selected from a polysaccharide, a resin, a cellulose, a cellulose-derivative and a protein, as described in section "carrier material" of the first aspect of the invention, in a liquid, preferably water; (ii) cooling said mixture so that said liquid crystallizes, in other words freezing said mixture, preferably at a temperate of < -20°C, more preferably < -30°C, even more preferably < -40°C, even more preferably at a temperature of -20°C to - 80°C, even more preferably -20°C to -60°C, most preferably -30°C to -60°C; (iii) drying said mixture under low environmental pressure, preferably said pressure is constant, as to remove frozen free moisture (known as primary drying step); and (iv) further drying said mixture at a temperature which is higher than in step, whereas pressure is preferably kept constant (iii), this step is known as secondary drying step, as to form encapsulates. A freeze dryer typically comprises a product chamber (e.g. for vials or for trays), a condenser (attracting the vapors being sublimed off the frozen mixture) and a vacuum pomp.

In a preferred embodiment, the temperature during the primary drying step is below the collapse temperature of the frozen mixture. The collapse temperature can be routinely determined by the skilled person, crystalline products have a well-defined freezing/melting point (i.e. collapse temperature) whereas the collapse temperature of amorphous products is typically a few degrees warmer than the glass transition temperature of said amorphous products. Preferably, the temperature during the primary drying step (i.e. the applied temperature) is 3-10°C, preferably 5-10°C, more preferably 5-7°C, lower than the collapse temperature of the frozen mixture. In an additional and/or alternative preferred embodiment, the temperature during the primary drying step is -40°C to 20°C, preferably -40°C to 0°C, more preferably -40°C to -10°C, even more preferably -40°C to -20°C.

In an additional and/or alternative preferred embodiment, the pressure during the primary drying step is constant. In an additional and/or alternative preferred embodiment, the pressure during the primary drying step is < 1 mbar, preferably < 0.5 mbar, more preferably < 0.2 mbar. Preferably, the pressure during the primary drying step is 0.1-0.5 mbar, more preferably 0.1-0.25 mbar, most preferably 0.1-0.2 mbar.

In an additional and/or alternative preferred embodiment, the primary drying step is conducted until at least one of the following occurs: temperature of the mixture is similar or equal to the applied temperature; condenser temperature has returned to its original temperature (i.e. condenser does not trap high loads of vapor); pressure in the system has returned to its original low value.

In an additional and/or alternative preferred embodiment, the temperature during the secondary drying step is higher than the temperature during the primary drying step, preferably the temperature is higher than the ambient temperature, more preferably the temperature is at least 20°C, even more preferably the temperature is 25°C-50°C, most preferably 25°C-40°C.

In an additional and/or alternative preferred embodiment, the pressure during the secondary drying step is constant. In an additional and/or alternative preferred embodiment, the pressure during the secondary drying step is lower than the pressure during the primary drying step. Preferably, the pressure during the secondary drying step is < 1 mbar, preferably < 0.5 mbar, more preferably < 0.2 mbar, even more preferably < 0.1 mbar.

In an additional and/or alternative preferred embodiment, the mixture constitutes at least 5% (w/w), preferably at least 10% (w/w), more preferably at least 15% (w/w), even more preferably at least 20% (w/w), even more preferably at least 25% (w/w), most preferably at least 30% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the mixture constitutes < 60% (w/w), preferably < 55% (w/w), more preferably < 50% (w/w), even more preferably < 45% (w/w), most preferably < 40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention. In an additional and/or alternative preferred embodiment, the mixture constitutes 5-60% (w/w), preferably 10-50% (w/w), more preferably 15-50% (w/w), even more preferably 20-50% (w/w), even more preferably 25- 50% (w/w), even more preferably 25-45% (w/w), even more preferably 25-40% (w/w), most preferably 30-40% (w/w), of the milk saccharide or mixture of at least two different milk saccharides (preferably the active agent) as described in the first aspect of the invention.

In an additional and/or alternative preferred embodiment, the mixture (before freezing) constitutes 5-80 % (w/w), preferably 5-75 % (w/w), more preferably 5-70 % (w/w), carrier material.

Freeze drying generally results in encapsulates with a median diameter of 1 pm-100 pm. Hence, freeze drying is a preferred encapsulation technique to obtain encapsulates with a median diameter which is not longer than 100 pm.

Fluidized air bed coating (FAB; also referred to as spray coating and air suspension coating ; sometimes also referred to as Wuster coating as Wuster invented the first version of FAB, however this is a specific version of FAB and since then several modified versions have been developed, all of which are fluidized air bed coating in the context of the present invention) as disclosed herein refers to an encapsulation technique wherein a carrier material is applied onto particles (i.e. spraying in the context of the present invention) in a batch process or a continuous set-up (Garti and McClements, 2012, chapter 7; Subasi et al, 2021, Plant Antioxidants and Health, p. 1 to 37, section 3.1; Ronsse, 2006; "Modelling heat and mass transfer in fluidized bed coating processes, Ph.D. thesis, Ghent University, ISBN 90-5989-120-1; all incorporated by reference). Herein, encapsulates (preferably of the matrix-type; preferably obtained by an encapsulation method as described herein) are suspended by a gas stream (preferably an air stream) and sprayed with an atomized carrier material. In this context, a hot melt material and/or a polymer, preferably a polymer, as described in section "carrier material" of the first aspect of the invention, is particularly suitable for fluidized air bed coating (i.e. for FAB, the carrier material preferably comprises a hot melt material and/or polymer, more preferably a polymer). Fluidization arises when an upward moving gas (preferably air) drawn to a bed of encapsulates reaches a sufficient velocity to suspend said encapsulates. This fluidizing gas/air can also provide the evaporative capacity to dry the coating material on the particles.

The carrier material (i.e. coating) is applied through a spray, i.e. liquid drops are generated (e.g. atomization through the use of a nozzle) and depending on the spray direction from the nozzle, said spray can be a top spray, a bottom spray or a tangential spray (Ronsse, 2006, sections 1.3.3.1 to 1.3.3.3). In the context of the present invention, the carrier material (i.e. coating) is preferably applied (i.e. sprayed) at the bottom (e.g. bottom spray) or through a tangential spray, more preferably through a bottom spray, both have the advantage of reducing the distance between the particles and the drops of coating solution, thereby reducing the risk of premature drying of the coating. Moreover, a more uniform coating is obtained. As understood by the skilled person, encapsulation by FAB comprises the following steps: (i) providing encapsulates, preferably as described according to the first aspect or obtained by a method according to the second aspect, more preferably encapsulates of the matrix type, as a fluidized bed; (ii) providing a carrier material (i.e. a coating), preferably a hot melt material and/or a polymer, more preferably a polymer, as described in the first aspect of the invention; and (iii) atomizing said carrier material, preferably through the use of a nozzle, as to form a spray of droplets that coat said encapsulates (i.e. resulting in coated encapsulates).

A fluidized bed instrument generally comprises a product container, an expansion chamber, an gas/air handling system and an exhaust system (for retention of particles within the processing chamber) (Garti and McClements, 2012, chapter 7.3.3).

In a preferred embodiment, the inlet air temperature is 20°C-120°C, preferably 30°C-90°C, more preferably 40°C-90°C, even more preferably 40°C-80°C, most preferably 40°C-70°C.

In an additional and/or alternative preferred embodiment, the atomization gas/air temperature is 20°C- 120°C, preferably 30°C-90°C, more preferably 40°C-90°C, even more preferably 40°C-80°C, most preferably 40°C-70°C.

In an additional and/or alternative preferred embodiment, the atomization gas/air pressure ranges from 0.5 to 5.0, preferably 1.5 to 4.0 bar, more preferably 2.0 to 3.5 bar, most preferably 2.5 to 3.5 bar.

In an additional and/or alternative preferred embodiment, the atomization gas/air relative humidity is < 60 %, preferably < 50 %, more preferably < 40 %, most preferably < 30 %.

In an additional and/or alternative preferred embodiment, the air flow rate is chosen as such that the encapsulates are brought into suspension, preferably the air flow rate is 80% in the center and 20% in the periphery (in order to increase the drying rate while reducing agglomeration).

In an additional and/or alternative preferred embodiment, the atomized carrier material has a droplet size ranging from 10 to 40 micrometer.

In an additional and/or alternative preferred embodiment, the spraying rate is 0.01 - 1.0 g/s, preferably 0.01 - 0.5 g/s, more preferably 0.05 - 0.5 g/s, most preferably 0.05 - 0.25 g/s.

In an additional and/or alternative preferred embodiment, the storage vessel (from which the molten carrier material is pumped), the nozzle and atomizing air are heated in order to prevent solidification of the carrier material (i.e. coating).

In an additional and/or alternative preferred embodiment, the viscosity of the coating (i.e. carrier material as disclosed herein) is 20 - 800 millipascal second (mPa.s), preferably 20 - 650 mPa.s, more preferably 20 - 500 mPa.s, even more preferably 20 - 300 mPa.s; most preferably 50 - 200 mPa.s.

In an additional and/or alternative preferred embodiment, the temperature of the coating (i.e. carrier material as disclosed herein) is 1-25°C, preferably l-20°C, more preferably 5-20°C, more preferably 5- 15°C, most preferably 5-10°C, higher than the solidification point / melting temperature of said coating.

In an additional and/or alternative preferred embodiment, the coating (i.e. carrier material) applied by the fluidized air bed coating step constitutes 5-80 % (VJ/VJ), preferably 10-70 % (VJ/VJ), more preferably 20-60 % (VJ/VJ), even more preferably 30-60 % (VJ/VJ), even more preferably 35-55 % (VJ/VJ), most preferably 35-50 % (VJ/VJ), of the total weight of the obtained encapsulate.

In a more preferred embodiment, the encapsulates which are fluidized to be coated by FAB have a median diameter (D50 also known as D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 165 pm. Preferably, said median diameter is 75-250 pm, more preferably 100-250 pm, even more preferably 125-250 pm, even more preferably 150-250 pm, even more preferably 165-250 pm, most preferably 165-225 pm.

In an additional and/or alternative more preferred embodiment, the coated encapsulates (i.e. encapsulates to which a coating layer has been applied to by FAB) have a median diameter (D50 also known as D(v, 0.5)) which is 10-100 pm, preferably 20-100 pm, more preferably 20-75 pm, even more preferably 35-75 pm, longer than the median diameter of the uncoated encapsulates.

In an additional and/or alternative more preferred embodiment, the coated encapsulates (i.e. encapsulates to which a coating layer has been applied to by FAB) have a median diameter (D50 also known as D(v, 0.5)) which is 10%-40%, preferably 10%-35%, more preferably 10%-30%, even more preferably 15%-30%, most preferably 20%-30%, longer than the median diameter of the uncoated encapsulates.

Nutritional composition

In a third aspect, the invention provides a composition, preferably a nutritional composition, comprising an encapsulate according to the first aspect of the invention or comprising an encapsulated active agent (i.e. encapsulate according to the invention) obtained by a method according to the second aspect of the invention. Preferably, said composition, preferably said nutritional composition, is obtained by a method according to the sixth aspect of the invention. Throughout the application and claims, the term "composition" is preferably replaced with the term "nutritional composition", unless explicitly stated otherwise.

In a preferred embodiment, said composition, preferably said nutritional composition, further comprises a feed ingredient and/or a food ingredient, wherein said feed/food ingredient is preferably chosen from the list consisting of: a lipid, preferably one or more selected from the list consisting of an oil, fat, ester, monoglyceride, diglyceride, triglyceride and free fatty acid; a vitamin, preferably one or more selected from the list consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin H, or a derivate thereof; an amino acid compound; a trace element; a mineral, preferably one or more selected from the list consisting of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, selenium and chloride; an antioxidant; a prebiotic agent , preferably one or more selected from the list consisting of GOS (galactooligosaccharide), FOS (fructo-oligosaccharide), inulin and resistant starch; a carbohydrate; an antimicrobial agent; and/or a protein.

Generally, any source of protein may be used so long as it is suitable for nutritional compositions and is otherwise compatible with any other selected ingredients or features in the (nutritional) composition. Non-limiting examples of suitable proteins (and sources thereof) suitable for use in the composition (preferably the nutritional composition) according to the invention include, but are not limited to, intact, hydrolyzed, or partially hydrolyzed protein, which may be derived from any known or otherwise suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn, wheat), vegetable (e.g., soy, pea, potato, bean), and combinations thereof. The protein may also include a mixture of amino acids (often described as free amino acids) known for use in nutritional products or a combination of such amino acids with the intact, hydrolyzed, or partially hydrolyzed proteins described herein. The amino acids may be naturally occurring or synthetic amino acids. More particular examples of suitable protein (or sources thereof) used in a composition, preferably a nutritional composition, according to the invention include, but are not limited to, whole cow's milk, partially or completely defatted milk, milk protein concentrates, milk protein isolates, nonfat dry milk, condensed skim milk, whey protein concentrates, whey protein isolates, acid caseins, sodium caseinates, calcium caseinates, potassium caseinates, legume protein, soy protein concentrates, soy protein isolates, pea protein concentrates, pea protein isolates, collagen proteins, potato proteins, rice proteins, wheat proteins, canola proteins, quinoa, insect proteins, earthworm proteins, fungal (e.g., mushroom) proteins, hydrolyzed yeast, gelatin, bovine colostrum, human colostrum, glycol macropeptides, mycoproteins, proteins expressed by microorganisms (e.g., bacteria and algae), and combinations thereof. A composition, preferably a nutritional composition, according to the invention may include any individual source of protein or combination of the various sources of protein listed above. In addition, the proteins for use herein can also include, or be entirely or partially replaced by, free amino acids known for use in nutritional products, non-limiting examples of which include L-tryptophan, L-glutamine, L-tyrosine, L-methionine, L-cysteine, taurine, L-arginine, L- carnitine, and combinations thereof.

The carbohydrate or source of carbohydrate suitable for use in a composition, preferably a nutritional composition, according to the invention may be simple, complex, or variations or combinations thereof. Generally, the carbohydrate may include any carbohydrate or carbohydrate source that is suitable for use in nutritional compositions and is otherwise compatible with any other selected ingredients or features in the (nutritional) composition. Non-limiting examples of carbohydrates suitable for use in the composition, preferably the nutritional composition, according to the invention, but are not limited to, polydextrose, maltodextrin; hydrolyzed or modified starch or cornstarch; glucose polymers; corn syrup; corn syrup solids; sucrose; glucose; fructose; lactose; high fructose corn syrup; honey; sugar alcohols (e.g., maltitol, erythritol, sorbitol); isomaltulose; sucromalt; pullulan; potato starch; and other slowly-digested carbohydrates; dietary fibers including, but not limited to, fructooligosaccharides (FOS), galactooligosaccharides (GOS), oat fiber, soy fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, gellan gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinogalactans, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans (e.g., oat beta-glucan, barley beta-glucan), carrageenan and psyllium, soluble and insoluble fibers derived from fruits or vegetables; other resistant starches; and combinations thereof. A composition, preferably a nutritional composition, according to the invention may include any individual source of carbohydrate or combination of the various sources of carbohydrate listed above.

The fat or source of fat suitable for use in a composition, preferably a nutritional composition, according to the invention may be derived from various sources including, but not limited to, plants, animals, and combinations thereof. Generally, the fat may include any fat or fat source that is suitable for use in a composition, preferably a nutritional composition, according to the invention and is otherwise compatible with any other selected ingredients or features in the (nutritional) composition. Non-limiting examples of suitable fat (or sources thereof) for use in a composition, preferably a nutritional composition, according to the invention include coconut oil, fractionated coconut oil, soy oil, high oleic soy oil, corn oil, olive oil, safflower oil, high oleic safflower oil, medium chain triglyceride oil (MCT oil), high gamma linolenic (GLA) safflower oil, sunflower oil, high oleic sunflower oil, palm oil, palm kernel oil, palm olein, canola oil, high oleic canola oil, marine oils, fish oils, algal oils, borage oil, cottonseed oil, fungal oils, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA), conjugated linoleic acid (CLA), alphalinolenic acid, rice bran oil, wheat bran oil, interesterified oils, transesterified oils, structured lipids, and combinations thereof. Generally, the fats used in a nutritional composition for formulating infant formulas and pediatric formulas provide fatty acids needed both as an energy source and for the healthy development of the infant, toddler, or child. These fats typically comprise triglycerides, although the fats may also comprise diglycerides, monoglycerides, and free fatty acids. Fatty acids provided by the fats in the nutritional composition include, but are not limited to, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, ARA, EPA, and DHA. The nutritional composition can include any individual source of fat or combination of the various sources of fat listed above. Preferably, the fat is a mixture of vegetable fat and milk fat such as obtained from milk from a mammal like cow, sheep, goat, mare, or camel. More preferably, wherein the milk fat is bovine milk fat. Mixtures of different types of fat are preferred because they help to provide different fatty acids and better resemble the type of linkage between the glycerol moiety and the fatty acid moiety in the fat, when compared to human mother's milk.

When one or more prebiotics is/are present, then it is preferred that the weight ratio between the saccharide(s) according to the invention and said prebiotic(s) is in the range of from 0.5:10 to 10:0.5.

In an additional and/or alternative preferred embodiment, said composition, preferably said nutritional composition, according to the invention comprises one or more probiotics for its beneficial effect on the subject's gut microbiome. Examples of suitable probiotics include Bifidobacterium, Lactobacillus and Saccharomyces boulardii.

In an additional and/or alternative preferred embodiment, said composition, preferably said nutritional composition, is a synthetic nutritional composition. In the context of the present invention, a "synthetic composition" or a "synthetic nutritional composition" refers to a composition which is artificially prepared and preferably refers to a composition comprising at least one component that is produced ex vivo, either chemically and/or biologically, e.g. by means of chemical reaction, enzymatic reaction or recombinantly, or purified by humans. It is preferred that a synthetic nutritional composition of the invention is not identical with a naturally occurring composition.

In a more preferred embodiment, said composition, preferably said nutritional composition, is formed as a mash mixture, granules, particles, kibbles or pellets, preferably formed as granules, particles, kibbles or pellets. Such a nutritional composition is preferably prepared by a method comprising an extrusion step (it is referred to the sixth aspect of the present invention). In an additional and/or alternative more preferred embodiment, said composition, preferably said nutritional composition, is formed from a dough or batter comprising an encapsulate according to the invention. Such a (nutritional) composition is preferably prepared by a method comprising a baking, frying, heating and/or a heat sterilization step, more preferably prepared by a method comprising a baking and/or a frying step, even more preferably prepared by a method comprising a baking step (it is referred to the sixth aspect of the present invention). Baking of a dough or batter preferably occurs at a temperature of 100°C or higher, more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, even more preferably 150°C to 225°C, even more preferably 180°C to 225°C. Preferably, said nutritional composition is a bakery product. More preferably, said nutritional composition is a bread, a cake, a biscuit/cookie or a pastry. More preferably, said nutritional composition is a bread, a cake or a biscuit/cookie. Even more preferably, said nutritional composition is a bread or a cake. In the context of the present invention, it is preferred that said dough or batter has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

In an additional and/or alternative more preferred embodiment, said composition, preferably said nutritional composition, is a food product, preferably wherein said food product is selected from a list consisting of dairy product, bar, liquid product, savory snack, savory biscuit, bakery product, pasta and food supplement, more preferably wherein said food product is a dairy product or a food supplement. Preferably, said nutritional composition according to the invention is not a liquid product.

In an additional and/or alternative more preferred embodiment, said composition, preferably said nutritional composition, is selected from a list consisting of infant formula, baby food, infant cereal composition, growing-up milk, milk replacer, creep feed, pet food, dry feed, frozen feed, prestarter diet, pre weaning feed, weaning feed and post weaning feed, preferably selected from a list consisting of dry feed, frozen feed, milk replacer, creep feed, pet food, prestarter diet, pre weaning feed, weaning feed and post weaning feed, more preferably selected from a list consisting of dry feed, frozen feed, milk replacer, creep feed, post weaning feed and pet food, even more preferably wherein said nutritional composition is dry feed, frozen feed, creep feed, post weaning feed or pet food, even more preferably wherein said nutritional composition is pet food or creep feed, most preferably wherein said (nutritional) composition is a pet food.

In the context of the present invention, an "infant formula" refers to to a nutritional composition that has the proper balance of macronutrients, micro-nutrients, and calories to provide sole or supplemental nourishment for and generally maintain or improve the health of infants, toddlers, or both. Infant formulas preferably comprise nutrients in accordance with the relevant infant formula guidelines for the targeted consumer or user population, an example of which would be the Infant Formula Act, 21 U.S.C. Section 350(a). Another example with guidelines for nutrients of an infant formula, in particular for a person of 0-12 months of age and for children up to 36 months old, may be found in the CODEX Alimentarius (CODEX STAN 72-1981), further referred to as the CODEX). Nutritional compositions for infants are commonly referred to as infant formula. When used as infant formula, the nutritional composition according to the invention should contain the ingredients in the amounts as prescribed by the CODEX and, if needed, as prescribed by additional regulations of individual countries. The protein component is typically present in an amount of from 5% to 35% (w/w) by weight of the infant formula (i.e., the dry weight), including from 10% to 30% (w/w), from 10% to 25% (w/w), from 15% to 25% (w/w), from 20% to 30% (w/w), from 15% to 20% (w/w), and also including from 10% to 16% (w/w) by weight of the infant formula (i.e., the dry weight). The carbohydrate component is typically present in an amount of from 40% to 75% (w/w) by weight of the infant formula (i.e., the dry weight), including from 45% to 75% (w/w), from 45% to 70% (w/w), from 50% to 70% (w/w), from 50% to 65% (w/w), from 50% to 60% (w/w), from 60% to 75% (w/w), from 55% to 65% (w/w), and also including from 65% to 70% (w/w) by weight of the infant formula (i.e., the dry weight). The fat component is typically present in an amount of from 10% to 40% by weight of the infant formula (i.e., the dry weight), including from 15% to 40% (w/w), from 20% to 35% (w/w), from 20% to 30% (w/w), from 25% to 35% (w/w), and also including from 25% to 30% (w/w) by weight of the infant formula (i.e. the dry weight).

In the context of the present invention, "growing-up milk" refers to a milk-based beverage adapted for the specific nutritional needs of young children.

In the context of the invention, the term "weaning" or "weaning period" refers to the period during which the mother's milk is substituted by other food in the diet.

In the context of the invention, the term "creep feed" refers to a nutritional composition which is used to supplement a pre-weaned subject with a solid diet while said subject is suckling. For example, a piglet receives creep feed to ease the transition from sow's milk to solid pig starter feed. The creep feed stimulates the digestive system of the piglet to produce e.g. amylase which digests carbohydrates in dry feed. If a pig is better able to digest dry feed, he can start eating quickly post-weaning for better performance.

In the context of the invention, the term "milk replacer" refers to a nutritional composition which serves as a substitute for mother's milk. A milk replacer comprises milk proteins, fat, carbohydrates, vitamins and minerals. Preferably, a milk replacer comprises 20 wt. % to 30 wt. % water, 18 wt. % to 24 wt. % of at least one protein, 15 wt. % to 28 wt. % (preferably 20 wt. % to 25 wt. %) of at least one fat and lactose at < 50 wt. %.

In an additional and/or alternative more preferred embodiment, said composition, preferably said nutritional composition, is for feeding a human, preferably an infant, a child, an adolescent and/or an adult, more preferably an infant, a child and/or an adolescent, even more preferably an infant and/or a child. In the context of the present invention, "an infant" has an age of 0-1 year, "a child" has an age of 1- 9 years, preferably 1-6 years, more preferably 1-4 years, "an adolescent young adult" has an age of 10-18 years.

In an additional and/or alternative more preferred embodiment, said composition, preferably said nutritional composition, is for feeding an infant animal, a young animal and/or an adult animal, preferably a non-adult animal or adult animal, more preferably a non-adult animal. Preferably, said animal is selected from a companion animal, an aquatic animal (preferably a water breathing animal), a farm animal, a bird, a reptile and an animal grown for food consumption. In a more preferred embodiment said animal is a companion animal, preferably selected from the list consisting of a dog, a cat, a rabbit, a guinea pig, a hamster, a mouse, a rat, a gerbil, a bird, a chinchilla, a turtle, a ferret and a horse, more preferably a dog or a cat. In another more preferred embodiment said animal is an aquatic animal, preferably a water breathing animal, more preferably selected from a list consisting of a fish, a crustacean, a water turtle and an amphibian, even more preferably a fish or a crustacean (preferably a shrimp). In another more preferred embodiment said animal is a farm animal, preferably selected from the list consisting of a cow, a horse, a pig, a chicken, a goat, a sheep, a llama, an alpaca, a donkey, a mule and poultry, more preferably a cow, a pig or a horse. In another more preferred embodiment said animal is an animal grown for food consumption, preferably selected from the list consisting of a cow, a pig, a horse, a chicken, a goat, a sheep, a turkey, a goose, a rabbit and a duck, more preferably a cow or a pig. In another more preferred embodiment said animal is a bird, preferably selected from the list consisting of a chicken, a pigeon, an ostrich, a goose, a duck, a canary, a finch, a guinea fowl, a turkey, an owl and a falcon, more preferably poultry, even more preferably a chicken.

As understood by the skilled person, a "farmed animal" refers to an animal that is reared in an agricultural setting in order to produce various commodities such as food (meat, organs, eggs, dairy products) and/or hair or wool. Further, a "companion animal" preferably refers to a domestic animal. Further, an "aquatic animal" is an animal that lives in water for most or all of its lifetime, said animal can be an invertebrate or a vertebrate. Further, a "water breathing animal" or "water breathing aquatic animal" refers to an aquatic animal which is able to breath under water. Throughout the application, unless specifically stated otherwise, a "non-adult animal" can be replaced with "juvenile animal" and vice versa.

In an even more preferred embodiment, said composition, preferably said nutritional composition, according to the invention is for feeding an aquatic animal as described herein. Hence, a particularly preferred nutritional composition is an aquaculture feed. Preferably said nutritional composition is a dry feed or frozen feed, more preferably a dry feed. As the skilled person is aware, such a feed can be pellet feed, flake feed or powdered feed. An nutritional composition for feeding an aquatic animal (preferably as described herein), preferably an aquaculture feed, comprises proteins, fat, carbohydrates, vitamins and minerals. Preferably, said nutritional composition comprises 10 wt. % to 20 wt. % fat and 15 wt. % to 35 wt. % carbohydrates. Preferably, said vitamin is any one or more of vitamin A, Bl, B2, B3, B5, B6, B12, biotin, C, choline, D3, E, folacin, inositol and K. Preferably, said mineral is any one or more of calcium, phosphorus, sodium, magnesium, iron, iodine, chloride, copper, potassium, sulfur and zinc, more preferably said mineral is calcium and/or phosphorus. Optionally, a binding agent for providing water stability to the feed is present. Preferably, said binding agent constitutes < 1.0 wt. % of the nutritional composition. Examples of suitable binding agents comprise guar gum, carboxy methyl cellulose and maltose, preferably guar gum and/or carboxy methyl cellulose. In the context of the invention, it is preferred that the nutritional composition does not comprise maltose. Optionally, a preservative is present. Preferably, said preservative is an antioxidant and/or an anti-microbial. More preferably, said preservative is an antioxidant, preferably selected from a list consisting of vitamin E, butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquine. More preferably, said preservative is an antimicrobial, preferably selected from a list consisting of sodium, benzoic and sorbic acid. It is preferred that said preservative constitutes < 0.1 wt. % of the nutritional composition. Optionally, an attractive is present. Its function is to make it more palatable. It is preferred that said attract constitutes < 5.0 wt. % of the nutritional composition. Common sources of attractants are hydrolysates and condensed fish solubles. Optionally, a color enhancer is present. Its function is to obtain a vibrant color. Typically, carotenoids are added as color enhancer (e.g. astaxanthin and canthaxanthin are used for salmon feed). In the context of the invention, "pellet feed" refers to feed that is obtained by grinding up the feed components, extruding it with heat and pressure before producing the pellets in the desired size. Some pellet feed are designed to float on water, while others are designed to sink. "Flake feed" refers to feed that will float for a certain amount of time before slowly sinking to the bottom. "Powdered feed" refers to feed that is either directly fed or is mixed in water before feeding it. "Powdered feed" is often referred to as "fry feed".

In an even more preferred embodiment, the amount of said milk saccharide or mixture of at least two different milk saccharides (as described in the first aspect of the invention), preferably said active agent (as described in the first aspect of the invention), constitutes 0.001 to 15.0 % (w/w), preferably 0.001 to 10.0 % (w/w), more preferably 0.001 to 5.0 % (w/w), even more preferably 0.001 to 3.0 % (w/w), most preferably 0.001 to 1.0 % (w/w), of the total weight of said composition, preferably said nutritional composition. In other words, the composition, preferably the nutritional composition, according to the invention preferably comprises, more preferably consists of, 0.001 to 15.0 % (w/w), preferably 0.001 to 10.0 % (w/w), more preferably 0.001 to 5.0 % (w/w), even more preferably 0.001 to 3.0 % (w/w), most preferably 0.001 to 1.0 % (w/w), of said milk saccharide or said mixture of milk saccharides, preferably said active agent. In an even more preferred embodiment, the amount of said milk saccharide or mixture of at least two different milk saccharides (as described in the first aspect of the invention), preferably said active agent (as described in the first aspect of the invention), constitutes 0.01 to 15.0 % (VJ/VJ), preferably 0.01 to 10.0 % (VJ/VJ), more preferably 0.01 to 5.0 % (VJ/VJ), even more preferably 0.01 to 3.0 % (VJ/VJ), most preferably 0.01 to 1.0 % (VJ/VJ), of the total weight of said composition, preferably said nutritional composition. In other words, the composition, preferably the nutritional composition, according to the invention preferably comprises, more preferably consists of, 0.01 to 15.0 % (VJ/VJ), preferably 0.01 to 10.0 % (VJ/VJ), more preferably 0.01 to 5.0 % (VJ/VJ), even more preferably 0.01 to 3.0 % (VJ/VJ), most preferably 0.01 to 1.0 % (VJ/VJ), of said milk saccharide or said mixture of milk saccharides, preferably said active agent.

In an even more preferred embodiment, the amount of said milk saccharide or mixture of at least two different milk saccharides (as described in the first aspect of the invention), preferably said active agent (as described in the first aspect of the invention), constitutes 0.1 to 15.0 % (VJ/VJ), preferably 0.1 to 10.0 % (VJ/VJ), more preferably 0.1 to 5.0 % (VJ/VJ), even more preferably 0.1 to 3.0 % (VJ/VJ), most preferably 0.1 to 1.0 % (VJ/VJ), of the total weight of said composition, preferably said nutritional composition. In other words, the composition, preferably the nutritional composition, according to the invention preferably comprises, more preferably consists of, 0.1 to 15.0 % (VJ/VJ), preferably 0.1 to 10.0 % (VJ/VJ), more preferably 0.1 to 5.0 % (VJ/VJ), even more preferably 0.1 to 3.0 % (VJ/VJ), most preferably 0.1 to 1.0 % (VJ/VJ), of said milk saccharide or said mixture of milk saccharides, preferably said active agent.

It is particularly preferred that the amount of said milk saccharide or mixture of at least two different milk saccharides (as described in the first aspect of the invention), preferably said active agent (as described in the first aspect of the invention, constitutes 0.01 to 60.0 % (VJ/VJ), preferably 0. 1 to 60.0 % (VJ/VJ), more preferably 1.0 to 60.0 % (VJ/VJ), even more preferably 1.0 to 50.0 % (VJ/VJ), even more preferably 5.0 to 50.0 % (w/w), even more preferably 10.0 to 50.0 % (w/w), even more preferably 10.0 to 40.0 % (w/w), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of said composition, preferably said nutritional composition. In other words, the total weight of dry matter of the composition, preferably the nutritional composition, according to the invention preferably comprises, more preferably consists of, 0.01 to 60.0 % (w/w), preferably 0. 1 to 60.0 % (w/w), more preferably 1.0 to 60.0 % (w/w), even more preferably 1.0 to 50.0 % (w/w), even more preferably 5.0 to 50.0 % (w/w), even more preferably 10.0 to 50.0 % (w/w), even more preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides, preferably said active agent.

Pharmaceutical

In a fourth aspect, the invention provides a pharmaceutical composition comprising an encapsulate according to the first aspect of the invention or comprising an encapsulated active agent (i.e. an encapsulate according to the invention) obtained by a method according to the second aspect of the invention or comprising an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention. Preferably, said pharmaceutical composition is obtained by a method according to the sixth aspect of the invention.

In a preferred embodiment, said pharmaceutical composition further comprises a pharmaceutical ingredient, preferably a pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, excipient, salt, adjuvant and/or solvent. Such pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, salt, adjuvant, solvent and/or excipient may for instance be found in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

Each embodiment disclosed in the third aspect of the present invention (i.e. "Nutritional composition") is also disclosed in the context of the present aspect of the present invention, i.e. a pharmaceutical composition. In other words, the terms "composition" and "nutritional composition" as disclosed in the third aspect of the invention is preferably replaced with "pharmaceutical composition" in the context of the fourth aspect of the invention, i.e. a pharmaceutical composition.

Further, the skilled person is well-aware that a composition can be both a nutritional composition and a pharmaceutical composition. Such a composition is hence considered to be a nutritional composition according to the invention, but also a pharmaceutical composition according to the invention.

Medical use

In a fifth aspect, the invention provides an encapsulate (as described in the first aspect of the invention), an encapsulated active agent (i.e. encapsulate according to the invention) obtained by a method according to the second aspect of the invention, an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention, a nutritional composition (as described in the third aspect to the invention) or a pharmaceutical composition (as described in the fourth aspect of the invention) for use as a medicament.

In a preferred embodiment, said encapsulate, encapsulated active agent, encapsulated saccharide/saccharide mixture, nutritional composition or pharmaceutical composition is for use in preventing, delaying, curing, ameliorating and/or treating gastro-intestinal disease in a human (preferably as described in the present application) or an animal (preferably as described in the present application), preferably an animal (preferably as described in the present application).

In another preferred embodiment, said encapsulate, encapsulated active agent, encapsulated saccharide/saccharide mixture, nutritional composition or pharmaceutical composition is for use in preventing, delaying, curing, ameliorating and/or treating allergic disease in a human (preferably as described in the present application) or an animal (preferably as described in the present application), preferably an animal (preferably as described in the present application).

In a fifth aspect, the invention provides the use of an encapsulate (as described in the first aspect of the invention), an encapsulated active agent obtained by a method according to the second aspect of the invention, an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention, a nutritional composition (as described in the third aspect to the invention) or a pharmaceutical composition (as described in the fourth aspect of the invention) in the manufacture of a medicament. In a preferred embodiment, said use is for preventing, delaying, curing, ameliorating and/or treating gastro-intestinal disease in a human (preferably as described in the present application) or an animal (preferably as described in the present application), preferably an animal (preferably as described in the present application). In another preferred embodiment, said use is for preventing, delaying, curing, ameliorating and/or treating allergic disease in a human (preferably as described in the present application) or an animal (preferably as described in the present application), preferably an animal (preferably as described in the present application).

In a fifth aspect, the invention provides a method for preventing, delaying, curing, ameliorating and/or treating gastro-intestinal disease in a human (preferably as described in the present application) or an animal (preferably as described in the present application), preferably an animal (preferably as described in the present application). The method comprises administering said encapsulate encapsulated active agent, encapsulated saccharide/saccharide mixture, nutritional composition or pharmaceutical composition of the invention as described herein to said human (preferably as described in the present application) or animal (preferably as described in the present application), preferably to said animal (preferably as described in the present application).

Method for production of a composition

In a sixth aspect, the invention provides a method for the production of a composition, said method comprising the steps of: providing an encapsulate according to the first aspect of the invention or an encapsulated active agent (i.e. encapsulate according to the invention) obtained by a method according to the second aspect of the invention or an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; and mixing said encapsulate or encapsulated active agent or encapsulated saccharide/saccharide mixture with said ingredient to obtain a mixture, preferably a homogeneous mixture. Preferably, said composition is according to the third aspect of the present invention.

Said "food and/or feed ingredient" is preferably as described in the third aspect of the present invention entitled "nutritional composition".

Said "pharmaceutical ingredient" is preferably as described in the fourth aspect of the present invention entitled "pharmaceutical composition".

In a preferred embodiment, said method is for the production of a nutritional composition. Preferably, said nutritional composition is according to the third aspect of the present invention.

In an additional and/or alternative preferred embodiment, said method is for the production of a pharmaceutical composition. Preferably, said pharmaceutical composition is according to the fourth aspect of the present invention.

Further, the skilled person is well-aware that a composition can be both a nutritional composition and a pharmaceutical composition. Such a composition is hence considered to be a nutritional composition according to the invention, but also a pharmaceutical composition according to the invention.

Optionally, said method comprises a drying step, preferably in an oven (preferably at a temperature > 50°C and/or preferably < 100°C). In an embodiment, said drying step occurs after obtaining said mixture.

In an additional and/or alternative preferred embodiment of the sixth aspect of the invention, said obtained mixture has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

In an additional and/or alternative preferred method, said method further comprises a step for preparing food and/or feed. Throughout the application and claims, the expression "food and/or feed" and "food/feed" is preferably replaced with "food" or is preferably replaced with "feed". In the context of the present invention, "food" preferably refers to a nutritional composition for human consumption, whereas "feed" preferably refers to a nutritional composition for animal consumption. A step for preparing food and/or feed is well-known to the skilled person as described thoroughly in the book entitled "Food processing technology: principles and practice" (Fellows; second edition; Woodhead Publishing Limited; 2000) which is incorporated by reference.

Preferably, said step for preparing food and/or feed is selected from the list consisting of baking, roasting, frying, heating (dielectric, ohmic or infrared), blanching, pasteurization, heat sterilization, fermentation, irradiation, processing using pulsed electric field, high pressure and extrusion (dry or wet), preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization, processing using pulsed electric field or high pressure, and extrusion (dry or wet), more preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization and extrusion (dry or wet), even more preferably baking or extrusion (dry or wet), most preferably extrusion (dry or wet). Another most preferred step for preparing food and/or feed is baking. In the context of the present invention, said method can comprise two or more steps for preparing food and/or feed, preferably wherein each step is different.

In a more preferred embodiment, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) wherein said step comprises, preferably consists of, one or more food/feed manufacturing conditions as described in the first aspect of the invention.

Preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of, one or more of the following conditions: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar; a pH which is lower than 5 or higher than 8, preferably a pH which is lower than 5; sheering including extrusion, mixing and compressing; interaction with an enzyme which can degrade and/or modify said milk saccharide or mixture of at least two different milk saccharides; contact with a food/feed component that can result in oxidation, reduction, hydrolysis, consumption and/or conversion of said milk saccharide or mixture of at least two different milk saccharides; contact of said saccharide or mixture of at least two different saccharides with a food/feed component that can result in a Maillard reaction and/or glycation of said food/feed component.

More preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of, one or more of the following conditions: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than

5 bar, even more preferably higher than 10 bar; a pH which is lower than 5 or higher than 8, preferably a pH which is lower than 5; contact of said saccharide or mixture of at least two different saccharides with a food/feed component that can result in a Maillard reaction and/or glycation of said food/feed component.

Even more preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of, one or more of the following conditions: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar.

Even more preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of, one or more of the following conditions: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40%; direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water.

Even more preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of, one or more of the following conditions: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40%.

Most preferably, said method further comprises a step for preparing food and/or feed (preferably as described earlier herein) at conditions comprising, preferably consisting of a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; optionally a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40%.

In an even more preferred embodiment, the invention provides a method for the production of a composition, said method comprising the steps of: providing an encapsulate according to the first aspect of the invention or an encapsulated active agent (i.e. encapsulate according to the invention) obtained by a method according to the second aspect of the invention or an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated saccharide/saccharide mixture with said ingredient to obtain a mixture, preferably a homogeneous mixture; and a step of preparing food and/or feed at conditions comprising, preferably consisting of, a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; and optionally a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40%.

Preferably, said composition is according to the third aspect of the present invention.

Said "food and/or feed ingredient" is preferably as described in the third aspect of the present invention entitled "nutritional composition".

Said "pharmaceutical ingredient" is preferably as described in the fourth aspect of the present invention entitled "pharmaceutical composition". Throughout the application and claims, a step of preparing food and/or feed is selected from the list consisting of baking, roasting, frying, heating (dielectric, ohmic or infrared), blanching, pasteurization, heat sterilization, fermentation, irradiation, processing using pulsed electric field, high pressure and extrusion (dry or wet), preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization, processing using pulsed electric field or high pressure, and extrusion (dry or wet), more preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization and extrusion (dry or wet), even more preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared) and extrusion (dry or wet), even more preferably baking or extrusion (dry or wet), most preferably extrusion (dry or wet). Another most preferred step for preparing food and/or feed is baking. In the context of the present invention, said method can comprise two or more steps for preparing food and/or feed, preferably wherein each step is different.

In an even more preferred embodiment, the invention provides a method for the production of a composition, said method comprising the steps of: providing an encapsulate according to the first aspect of the invention or an encapsulated active agent (i.e. encapsulate according to the invention) obtained by a method according to the second aspect of the invention or an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated saccharide/saccharide mixture with said ingredient to obtain a mixture, preferably a homogeneous mixture; and extruding said mixture to obtain a desired shape, preferably selected from mash mixture, granules, pellets, kibbles and particles, more preferably selected from granules, pellets, kibbles and particles.

Preferably, said composition is according to the third or fourth aspect of the present invention, more preferably according to the third aspect of the present invention. Preferred examples of nutritional compositions obtained by latter method comprise those described according to the third aspect of the present invention, more preferably pet food, aquaculture feed, dried feed and frozen feed, even more preferably pet food, aquaculture feed and dried feed. Preferred examples of pharmaceutical compositions obtained by latter method comprise a solid dosage unit, preferably a capsule, tablet, a dragee or a lozenge, more preferably a tablet, a dragee or a lozenge, even more preferably a tablet or a lozenge.

Optionally, a step of conditioning the mixture with steam precedes said extrusion step. Preferably, said steam conditioning includes providing steam at about 175 pounds per square inch for about 30 seconds. Optionally, said method comprises a drying step, preferably in an oven (preferably at a temperature > 50°C and/or preferably < 100°C). In an embodiment, said drying step occurs after obtaining said mixture. In another embodiment said drying step occurs after said extrusion step.

In the context of the present invention, "extrusion"/"extruding" preferably refer to the process of kneading, heating at high temperature and moisture, and squeezing under high pressure said obtained mixture through a die to obtain a desired shape of the nutritional composition. Said high temperature is a direct result of friction (dry extrusion) or preconditioning and steam injection (wet extrusion).

In the process of dry extrusion, it adopts the heat from friction for warming materials, forces materials to pass through a die and gets certain pressure simultaneously under the action of extrusion (e.g. screw extrusion). After extrusion, materials pressure will decrease sharply and moisture will evaporate so as to achieve the goal of extrusion. Moisture content (preferably water content) during dry extrusion is < 20 %. The principle of wet extrusion is similar to that of dry extrusion, but requires the addition of water and vapor to obtain a moisture content (preferably water content) of > 20 %, preferably > 25 %, and sometimes even > 30 %, as the rising of materials temperature depends on the addition of vapor.

In the context of the present invention, said "extruding" refers to dry extrusion and wet extrusion, preferably wet extrusion.

In an embodiment, said extrusion occurs at a pressure higher than 1013 mbar, preferably higher than 5 bar, more preferably higher than 10 bar.

In an additional and/or alternative embodiment, said extrusion occurs at a temperature of 50°C or higher, preferably 75°C or higher, more preferably at 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher. In an additional and/or alternative embodiment, said extrusion occurs at different temperatures, preferably at a temperature of 50°C or higher, at a temperature of 75°C or higher and/or at a temperature of 100°C or higher.

When extrusion occurs at different temperatures, it is particularly preferred that the highest temperature is 75°C or higher, more preferably 100°C or higher, even more preferably 115°C or higher, even more preferably 125°C or higher.

When extrusion occurs at different temperature, it is particularly preferred that the lowest temperature is 50°C or higher, more preferably 75°C or higher.

In another more preferred embodiment, the invention provides a method for the production of a composition, preferably a nutritional composition, said method comprising the steps of: providing an encapsulate according to the first aspect of the invention or an encapsulated active agent obtained by a method according to the second aspect of the invention or an encapsulated saccharide or mixture of at least two different saccharides obtained by a method according to the second aspect of the invention; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated saccharide/saccharide mixture with said ingredient to obtain a mixture, preferably a homogeneous mixture; and baking said mixture, preferably at a temperature of 100°C or higher, more preferably 115°C or higher, even more preferably 125°C or higher, even more preferably 150°C or higher, even more preferably 150°C to 225°C, even more preferably 180°C to 225°C.

Preferably, said composition is according to the third or fourth aspect of the present invention, more preferably according to the third aspect of the present invention. Preferred examples of nutritional compositions obtained by latter method comprise those described according to the third aspect of the present invention, more preferably a bakery product, even more preferably a bread, a cake, a biscuit/cookie or a pastry, even more preferably a bread or a cake.

Preferably, said mixture has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

Preferably, said mixture is a dough or batter, more preferably a dough or batter with a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

The skilled person is familiar with choosing the appropriate baking temperature and time depending on the intended nutritional composition. Depending on the properties of the nutritional composition, thickness of said composition and desired doneness, the skilled person will be able to routinely determine the appropriate temperature and duration of the baking process. Further, it is a well-known principle that one can achieve similar results by either baking for a short time at a higher temperature or baking for a longer time at a lower temperature. Preferably, said nutritional composition is baked until the desired doneness has been reached, more preferably heated until said nutritional composition is cooked.

Preferably, throughout the application and claims, said baking is for at least 2 minutes, preferably at least 3 minutes, more preferably at least 5 minutes, even more preferably at least 7 minutes, most preferably at least 10 minutes. Additionally and/or preferably, said baking is for 60 minutes or less, preferably 45 minutes or less, more preferably 30 minutes or less, even more preferably 25 minutes or less, most 20 minutes or less.

The terms "bake", "baking" and "baked" are well-understood by the skilled person (it is referred to the book entitled "Food processing technology: principles and practice" (Fellows; second edition; Woodhead Publishing Limited; 2000), chapter 16, i.e. p. 341-352, which is incorporated by reference). Throughout the application and claims, the terms "bake", "baking" and "baked" preferably refer to the process of cooking of a product, preferably a food/feed by heat, preferably dry heat, without direct exposure of said product, preferably food/feed, to a flame, preferably in an oven. The skilled person understands that during baking, heat is transferred into the product, preferably food/feed, (from e.g. hot surface and/or hot air) while moisture is transferred from the product, preferably food/feed, to the air that surrounds said food/feed.

Throughout the application and claims, it is preferred that said baking is done in an oven. Said oven can be a direct heating oven or an indirect heating oven. Said oven can be a continuous oven or a semi- continuous oven. Such types of ovens are known to the skilled person. Examples can be found in said book entitled ""Food processing technology: principles and practice" on p. 343-348.

Use

In a seventh aspect, the invention provides the use of an encapsulate according to the first aspect of the invention in the manufacturing of a nutritional composition, preferably a feed or a food. Preferably, a nutritional composition according to the third aspect of the invention.

In a seventh aspect, the invention provides the use of an encapsulated active agent (i.e. an encapsulate according to the invention), obtained by a method according to the second aspect of the invention, in the manufacturing of a nutritional composition, preferably a feed or a food. Preferably, a nutritional composition according to the third aspect of the invention.

In a seventh aspect, the invention provides the use of an encapsulated saccharide or mixture of at least two different saccharides, obtained by a method according to the second aspect of the invention, in the manufacturing of a nutritional composition, preferably a feed or a food. Preferably, a nutritional composition according to the third aspect of the invention.

In a seventh aspect, the invention provides the use of an encapsulate according to the first aspect of the invention in feeding a human or an animal, preferably an animal. Preferably, an animal and human as described herein.

In a seventh aspect, the invention provides the use of an encapsulated active agent (i.e. an encapsulate according to the invention), obtained by a method according to the second aspect of the invention, in feeding a human or an animal, preferably an animal. Preferably, an animal and human as described herein.

In a seventh aspect, the invention provides the use of an encapsulated saccharide or mixture of at least two different saccharides, obtained by a method according to the second aspect of the invention, in feeding a human or an animal, preferably an animal. Preferably, an animal and human as described herein.

In the context of the present invention, said "active agent", "powder", "saccharide", "mixture of at least two different saccharides", "first carrier material", "second carrier material" and "encapsulate" as described throughout the seventh aspect of the present invention and claims are preferably as described in the first aspect of the invention. In the context of the present invention, said "manufacturing of a nutritional composition" is preferably as described in the sixth aspect of the present invention.

Specific embodiments

The present invention preferably relates to the following specific embodiments: . An encapsulate comprising: an active agent comprising a milk saccharide or a mixture of at least two different milk saccharides, preferably said active agent comprising a milk oligosaccharide or a mixture of at least two different milk oligosaccharides, wherein said saccharide, preferably oligosaccharide, and said saccharides, preferably oligosaccharides, are obtained from an in vitro and/or ex vivo culture of cells, and a first carrier material which forms (i) a shell around said active agent as to form an encapsulate of the core-shell type or (ii) a matrix wherein said active agent is distributed as to form an encapsulate of the matrix type, preferably said first carrier material forms a shell around said active agent as to form an encapsulate of the core-shell type. . An encapsulate according to embodiment 1, wherein said milk saccharide or at least one, preferably at least two, more preferably all, of the milk saccharides in said mixture is selected from the list consisting of (i) oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end; (ii) oligosaccharide comprising N-acetyllactosamine (LacNAc) at its reducing end; (iii) oligosaccharide comprising lacto-N-biose (LNB) at its reducing end; (iv) milk glycosaminoglycan; (v) antigen of the human ABO blood group system, optionally bound to a monosaccharide selected from the list consisting of betal,3-GlcNAc, betal,4-GlcNAc, betal,3-GalNAc and -betal,4-Glc; and (vi) Lewis-type antigen oligosaccharide. . An encapsulate according to embodiment 1 or 2, wherein said milk saccharide or said mixture of milk saccharides, preferably said active agent, is provided as a solution, preferably as a slurry, or as a powder, preferably as a powder, more preferably wherein said slurry/powder is obtained by spray drying, freeze drying, spray freeze-drying, crystallization, lyophilization, band or belt drying, drum or roller drying, and/or agitated thin film drying, preferably by spray drying, drum or roller drying, or agitated thin film drying. . An encapsulate according to any one of embodiments 1 to 3, wherein said encapsulate further comprises a second carrier material which at least partially, preferably completely, encapsulates said first carrier material and said active agent as to form an encapsulate of the core-shell type, wherein said active agent and said first carrier material form a core component which is surrounded by a shell formed by the second carrier material. 5. An encapsulate according to any one of embodiments 1 to 4, wherein said first carrier material and/or second carrier material comprises a hot melt material and/or a polymer.

6. An encapsulate according to any one of embodiments 1 to 5, wherein said first carrier material and/or second carrier material comprises two or more different hot melt materials and/or two or more different polymers.

7. An encapsulate according to any one of embodiments 4 to 6, wherein the quantitative and/or qualitative, preferably qualitative, composition of said first carrier material differs from that of the second carrier material.

8. An encapsulate according to any one of embodiments 5 to 7, wherein said hot melt material is a lipophilic material, preferably a lipid, more preferably selected from the list consisting of an oil, a wax, a glyceride and a fatty acid, preferably wherein: said oil is a hydrogenated oil and/or a vegetable oil, preferably said oil is selected from palm oil, sunflower oil, soybean oil, rapeseed oil, coconut oil, babassu oil, palm kernel oil, maize oil, sesame oil and cottonseed oil, more preferably said oil is selected from the list consisting of palm oil, sunflower oil, soybean oil and rapeseed oil, even more preferably said oil is palm oil, most preferably said oil is hydrogenated palm oil; said wax is selected from Candelilla wax, Carnauba wax, beeswax, rice bran wax, paraffin wax, jojoba wax, microcrystalline wax and japan wax, more preferably said wax is Candelilla wax or Carnauba wax, most preferably said wax is Candelilla wax; and/or said glyceride is a monoglyceride, a diglyceride or a triglyceride.

9. An encapsulate according to any one of embodiments 5 to 8, wherein said hot melt material has a melting point of at least 30°C, preferably at least 45°, more preferably at least 50°C.

10. An encapsulate according to any one of embodiments 5 to 9, wherein said hot melt material has a melting point which is lower than 100°C, preferably lower than 90°C, more preferably lower than 80°C, even more preferably lower than 76°C.

11. An encapsulate according to any one of embodiments 5 to 10, wherein said hot melt material has an Iodine value which is lower than 100, preferably lower than 90, more preferably lower than 80, even more preferably lower than 70, even more preferably lower than 60, even more preferably lower than 50, even more preferably lower than 40, even more preferably lower than 30, even more preferably lower than 20, even more preferably lower than 10, most preferably lower than 5.

12. An encapsulate according to any one of embodiments 5 to 11, wherein said hot melt material contains unsaponifiable matter no greater than 2.0% (w/w), preferably no greater than 1.5% (w/w).

13. An encapsulate according to any one of embodiments 5 to 12, wherein said hot melt material comprises an oil, preferably a hydrogenated oil, and/or a wax.

14. An encapsulate according to any one of embodiments 5 to 7, wherein said polymer is a natural or synthetic polymer. 15. An encapsulate according to embodiment 14, wherein said polymer is selected from a list consisting of a polysaccharide, a resin, a cellulose or cellulose-derivative, a protein and/or a synthetic polymer, preferably wherein: said polysaccharide is selected from the list consisting of starch, pectin, maltodextrine, alginate, agarose, chitosan, hyaluronic acid, dextran, galactomannan, pullulan, fructan and fructooligosaccharide, preferably said polysaccharide is selected from the list consisting of alginate, pectin, starch and maltodextrine, more preferably said polysaccharide is alginate or starch, most preferably said polysaccharide is starch; said resin is a gum, more preferably selected from the list consisting of Shellac gum, Guar gum, Locust bean gum, Konjac gum, Arabic gum, Xanthan gum, Gellan gum, Cassia gum, cellulose gum, modified Arabic gum and Tara gum, even more preferably selected from Shellac gum, Arabic gum and modified Arabic gum, even more preferably selected from Shellac gum and Arabic gum, most preferably said resin is Shellac gum; said cellulose-derivative is selected from the list consisting of carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxypropoylcellulose, methylcellulose and ethylcellulose, preferably said cellulose-derivate is selected from hydroxypropylmethylcellulose and carboxymethylcellulose; said protein is selected from the list consisting of Zein, albumin, whey protein and soy protein; and/or said synthetic polymer is selected from the list consisting of poly-L-lysine, polylactic acid-glycolic acid copolymer, polyacrylic acid, polymethyacrylates, polyethylene glycols and fumaryl diketopiperazine, preferably selected from the list consisting of polylactic acid-glycolic acid copolymer, polyacrylic acid, polymethylacrylates, polyethylene glycols and fumaryl diketopiperazine.

16. An encapsulate according to any one of embodiments 4 to 15, wherein said first carrier material and/or second carrier material comprises starch, preferably said starch constitutes less than 15% (w/w) of said first and/or second carrier material, more preferably said starch constitutes less than 10% (w/w) of said first and/or second carrier material.

17. An encapsulate according to any one of embodiments 4 to 16, wherein said first carrier material and/or second carrier material comprises an additive, preferably wherein said additive is glycerol or a derivative thereof.

18. An encapsulate according to any one of embodiments 1 to 17, wherein said first carrier material comprises: encapsulate of the matrix type: a hot melt material, preferably an oil and/or a wax, more preferably a hydrogenated oil and/or a wax, optionally further comprising starch, preferably wherein said starch constitutes less than 15% (w/w) of said first carrier material, more preferably said starch constitutes less than 10% (w/w) of said first carrier material; encapsulate of the core-shell type: a hot melt material and/or a polymer, preferably a hot melt material, optionally further comprising starch, preferably wherein said starch constitutes less than 15% (w/w) of said first carrier material, more preferably said starch constitutes less than 10% (w/w) of said first carrier material, preferably wherein said hot melt material is an oil and/or a wax, more preferably a hydrogenated oil and/or a wax, preferably wherein said polymer is a resin, more preferably a gum.

19. An encapsulate according to any one of embodiments 4 to 18, wherein said second carrier material comprises a hot melt material and/or a polymer, preferably a polymer, optionally further comprising starch, preferably wherein said starch constitutes less than 15% (w/w) of said second carrier material, more preferably said starch constitutes less than 10% (w/w) of said second carrier material, preferably wherein said hot melt material is an oil and/or a wax, more preferably a hydrogenated oil and/or a wax, preferably wherein said polymer is a resin, more preferably a gum.

20. An encapsulate according to any one of embodiments 1 to 19, wherein said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes < 60% (w/w), preferably < 55% (w/w), more preferably < 50% (w/w), even more preferably < 45% (w/w), most preferably < 40% (w/w), of the total weight of the encapsulate.

21. An encapsulate according to any one of embodiments 1 to 20, wherein said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes at least 5% (w/w), preferably at least 10% (w/w), more preferably at least 15% (w/w), even more preferably at least 20% (w/w), even more preferably at least 25% (w/w), of the total weight of the encapsulate.

22. An encapsulate according to any one of embodiments 1 to 21, wherein said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, constitutes 5-50% (w/w), preferably 10-40 % (w/w), more preferably 20-40% (w/w), even more preferably 20-35% (w/w), of the total weight of the encapsulate.

23. An encapsulate according to any one of embodiments 1 to 22, wherein said encapsulate is a microparticle, preferably a spherical microparticle, more preferably wherein the aspect ratio is 0.8 - 1.2, even more preferably wherein the aspect ratio is 0.9 - 1.1.

24. An encapsulate according to any one of embodiments 1 to 23, wherein said encapsulate has a median diameter (D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 175 pm; and/or said encapsulate has a median diameter (D(v, 0.5)) of < 400 pm, preferably < 350 pm, more preferably < 300 pm.

25. An encapsulate according to any one of embodiments 1 to 24, wherein said encapsulate has a particle size distribution characterized by:

D(v, 0.1) ranging from 35 to 200 pm, preferably from 50 to 200 pm, more preferably 75 to 200 pm; and/or

D(v, 0.5) ranging from 50 to 400 pm, preferably from 50 to 350 pm, more preferably from 100 to 350 pm, even more preferably from 125 to 350 pm, even more preferably form 150 pm to 350 pm, even more preferably from 175 to 300 pm; and/or

D(v, 0.9) ranging from 100 to 600 pm, preferably from 100 to 500 pm, more preferably from 150 to 500 pm, even more preferably from 200 to 500 pm, even more preferably from 250 to 500 pm, most preferably from 300 to 500 pm; and/or

D(4,3) ranging from 50 to 400 pm, preferably from 50 to 350 pm, more preferably from 100 to 350 pm, even more preferably from 125 to 350 pm, even more preferably form 150 pm to 350 pm, even more preferably from 175 to 325 pm; and/or a span of 0.75-1.3, preferably 0.75-1.25, more preferably 0.85-1.25, even more preferably 0.9- 1.25, most preferably 0.9-1.2, wherein said span is calculated as (D(v,0.9) - D(v,0.1)) / D(v,0.5).

26. An encapsulate according to any one of embodiments 1 to 25, wherein the moisture content, preferably water content, is < 15 % (VJ/VJ), preferably < 10 % (VJ/VJ), more preferably < 9 % (VJ/VJ), even more preferably < 8 % (VJ/VJ), even more preferably < 7 % (VJ/VJ), even more preferably < 5 % (VJ/VJ), even more preferably < 4 % VJ/VJ), even more preferably < 3 % (VJ/VJ), most preferably < 2 % (VJ/VJ), of the total weight of the encapsulate.

27. An encapsulate according to any one of embodiments 1 to 26, wherein the flowability and/or water activity of said encapsulate is higher than that of the non-encapsulated saccharide or nonencapsulated mixture of at least two different saccharides, preferably the non-encapsulated active agent.

28. An encapsulate according to any one of embodiments 1 to 27, wherein said encapsulate has: a Hausner ratio (Rh) which is < 1.34, preferably < 1.25, more preferably < 1.18, wherein said Rh is the ratio between the tapped density p _t and the bulk density pb (Rh = Pt / Pb); and/or a Carr index (l _c ) which is < 25, preferably < 20, more preferably < 15, wherein said Ic is calculated as 100 x (p _t - pb) / Pb, wherein p _t is the tapped density and wherein pb is the bulk density.

29. An encapsulate according to any one of embodiments 1 to 28, wherein said encapsulate has a water activity (A _w ) which is at least 0.05, preferably at least 0.075, more preferably at least 0.10, even more preferably at least 0.15, most preferably which is at least 0.16 and/or wherein said encapsulate has a water activity (A _w ) which is < 0.50, preferably < 0.45, more preferably < 0.40, even more preferably < 0.35, most preferably < 0.30.

30. An encapsulate according to any one of embodiments 1 to 29, wherein the quantity and/or quality of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, is less affected upon short and/or prolonged exposure to conditions compared to said milk saccharide or mixture of at least two different milk saccharides, preferably compared to said non-encapsulated active agent. An encapsulate according to embodiment 30, wherein said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water ; gastro-intestinal conditions of an animal, preferably stomach conditions of animal; presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source; and/or pH which is lower than 5 or higher than 8, preferably wherein said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source; and/or pH which is lower than 5 or higher than 8, more preferably, wherein said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; and/or presence of a microbial strain which can use said milk saccharide or mixture of at least two milk saccharides, preferably said active agent, as a carbon source, even more preferably, wherein said conditions are any one or more of the following: food/feed manufacturing or storage conditions; moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), even more preferably at least 40% (w/w), most preferably at least 50% (w/w); and/or feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water, even more preferably, wherein said conditions are any one or both of the following: food/feed manufacturing or storage conditions; and/or feeding conditions, preferably contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water, most preferably, wherein said conditions are food/feed manufacturing or storage conditions. An encapsulate according to embodiment 31, wherein said food/feed manufacturing or storage conditions comprise, preferably consist of, at least one of: a temperature of 50°C or higher, preferably 75°C or higher, more preferably 100°C or higher, even more preferably 150°C or higher, most preferably 180°C or higher; a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w); direct or indirect contact with a liquid, wherein said liquid preferably comprises water, more preferably wherein said liquid is water; a pressure higher than 1013 mbar, preferably higher than 1 bar, more preferably higher than 5 bar, even more preferably higher than 10 bar; a pH which is lower than 5 or higher than 8, preferably a pH which is lower than 5; sheering including extrusion, mixing, compressing; interaction with an enzyme which can degrade and/or modify said saccharide or mixture of at least two different saccharides; contact with a food/feed component that can result in oxidation, reduction, hydrolysis, consumption and/or conversion of said saccharide or mixture of at least two different saccharides; and/or contact of said saccharide or mixture of at least two different saccharides with a food/feed component that can result in a Maillard reaction and/or glycation of said food/feed component. An encapsulate according to any one of embodiments 30 to 32, wherein the quantity and/or quality, preferably the quantity, of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, is less affected upon short and/or prolonged exposure to contact with a liquid, compared to said non-encapsulated milk saccharide or non-encapsulated mixture of at least two different milk saccharides, preferably compared to said non-encapsulated active agent.

34. An encapsulate according to any one of embodiments 30 to 33, wherein said encapsulate reduces and/or prevents contact between said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, and a liquid.

35. An encapsulate according to any one of embodiments 30 to 34, wherein the quantity of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, is at least 5.0 %, preferably at least 10.0 %, more preferably at least 15.0 %, higher than the quantity of the nonencapsulated and identical milk saccharide or non-encapsulated and identical mixture of at least two different milk saccharides, preferably the non-encapsulated and identical active agent, upon exposure to said conditions.

36. An encapsulate according to any one of embodiments 30 to 35, wherein the quantity of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, comprised in said encapsulate upon exposure to said conditions is at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of the quantity of said milk saccharide or mixture of at least two different milk saccharides, preferably said active agent, comprised in the encapsulate before exposure to said conditions.

37. An encapsulate according to any one of embodiments 1 to 36, wherein said active agent further comprises a microbial strain, preferably a bacterial or yeast strain, more preferably wherein said microbial strain does not use said saccharide or mixture of at least two saccharides, preferably said active agent, as a carbon source.

38. An encapsulate according to any one of embodiments 1 to 37, wherein said active agent further comprises: a lipid, preferably one or more selected from the list consisting of an oil, fat, ester, monoglyceride, diglyceride, triglyceride and free fatty acid; a vitamin, preferably one or more selected from the list consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin H, or a derivate thereof; an amino acid compound; a trace element; a mineral, preferably one or more selected from the list consisting of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, selenium and chloride; an antioxidant; a prebiotic agent, preferably one or more selected from the list consisting of GOS (galactooligosaccharide), FOS (fructo-oligosaccharide), inulin and resistant starch; an antimicrobial agent; a protein, preferably an enzyme or antibody, more preferably an enzyme; and/or a feed ingredient. An encapsulate according to any one of embodiments 1 to 38, wherein said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or powder, preferably powder; and applying one or more encapsulation steps preferably chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying, more preferably chosen from: o for encapsulate of the matrix type: prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying and granulation, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; o for encapsulate of the core-shell type:

■ applying one or more encapsulation steps to obtain an encapsulate of the matrix type, preferably chosen from the list consisting prilling, spray chilling, spray cooling, spray drying, freeze drying, granulation and fluidized air bed coating, more preferably chosen from the list consisting of prilling, spray chilling, spray cooling, spray drying, freeze drying and granulation, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and

■ applying one or more encapsulation steps selected from the list consisting of fluidized air bed coating, prilling, spray chilling, spray cooling, spray drying, granulation and freeze drying, preferably chosen from the list consisting of fluidized air bed coating, prilling, spray chilling and spray cooling, more preferably fluidized air bed coating. optionally followed by a fluidized air bed coating step. 40. An encapsulate according to any one of claims 1 to 38, wherein said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or powder, preferably powder; and applying a prilling, spray cooling or spray chilling step, preferably a prilling step; optionally followed by applying a fluidized air bed coating step.

41. An encapsulate according to any one of claims 1 to 38, wherein said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; applying a prilling, spray cooling or spray chilling step, preferably a prilling step, as to obtain an encapsulate of the matrix type; optionally followed by applying a fluidized air bed coating step as to obtain an encapsulate of the core shell type.

42. An encapsulate according to any one of claims 1 to 38, wherein said encapsulate is obtainable by a method comprising the steps of: providing said active agent in the form of a slurry or a powder, preferably a powder; providing said first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material as to obtain a mixture; atomizing the obtained mixture, preferably through a nozzle or a rotating/spinning disk, as to form droplets; cooling the droplets by an ambient (preferably a temperature of 24-25°C) air stream or cold gas/air (preferably a temperature below 25°C, more preferably a temperature below 24°C) as to form solidified encapsulates.

43. An encapsulate according to any one of claims 1 to 38, wherein said method further comprises the steps of: providing a second carrier material, optionally forming a melt or liquid of said second carrier material by increasing the temperature above the melting temperature of said second carrier material; coating, preferably spraying, more preferably fluidized air bed coating, said second carrier material on the surface of said solidified encapsulates resulting in at least one layer of the second carrier material surrounding said solidified encapsulates; and optionally repeating the spraying step as to obtain a thicker layer and/or more layers of said second carrier material.

44. A method for the production of an encapsulated active agent, said method comprising the steps of: providing an active agent in the form of a slurry or powder, preferably powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps. according to claim 44, said method comprising the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material; applying a prilling, spray cooling or spray chilling step, preferably a prilling step, as to obtain an encapsulate of the matrix type; optionally followed by applying a fluidized air bed coating step as to obtain an encapsulate of the core shell type. according to claim 45, said method comprises the steps of: providing an active agent in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said active agent into said first carrier material as to obtain a mixture; atomizing the obtained mixture, preferably through a nozzle or a rotating/spinning disk, as to form droplets; cooling the droplets by an ambient (preferably a temperature of 24-25°C) air stream or cold gas/air (preferably a temperature below 25°C, more preferably a temperature below 24°C) as to form solidified encapsulates. A method according to claim 46, said method further comprises the steps of: providing a second carrier material, optionally forming a melt or liquid of said second carrier material by increasing the temperature above the melting temperature of said second carrier material; coating, preferably spraying, more preferably fluidized air bed coating, said second carrier material on the surface of said solidified encapsulates resulting in at least one layer of the second carrier material surrounding said solidified encapsulates; and optionally repeating the spraying step as to obtain a thicker layer and/or more layers of said second carrier material. A method for the production of an encapsulated saccharide or encapsulated mixture of at least two different saccharides, said method comprising the steps of: providing a milk saccharide or a mixture of at least two different milk saccharides in the form of a slurry or powder, preferably powder, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; applying one or more encapsulation steps chosen from the list consisting of prilling, spray chilling, spray cooling, fluidized air bed coating (FAB), granulation, spray drying and freeze drying, preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation, spray drying and freeze drying, more preferably selected from the list consisting of prilling, spray chilling, spray cooling, granulation and spray drying, even more preferably selected from the list consisting of prilling, spray chilling, spray cooling and granulation, even more preferably selected from the list consisting of prilling, spray chilling and spray cooling, most preferably prilling; and optionally further applying one or more fluidized air bed coating steps. A method according to claim 48, said method comprising the steps of: providing a milk saccharide or a mixture of at least two different milk saccharides in the form of a slurry or a powder, preferably a powder, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said saccharide or mixture of saccharides into said first carrier material; applying a prilling, spray cooling or spray chilling step, preferably a prilling step, as to obtain an encapsulate of the matrix type; optionally followed by applying a fluidized air bed coating step as to obtain an encapsulate of the core shell type. A method according to claim 49, said method comprises the steps of: providing a milk saccharide or a mixture of at least two different milk saccharides in the form of a slurry or a powder, preferably a powder, wherein said active agent comprises a milk saccharide or a mixture of at least two different milk saccharides, wherein said milk saccharide and said milk saccharides are obtained from an in vitro and/or ex vivo culture of cells; providing a first carrier material, optionally forming a melt or liquid of said first carrier material by increasing the temperature above the melting temperature of said first carrier material; dispersing/dissolving said saccharide or mixture of saccharides into said first carrier material as to obtain a mixture; atomizing the obtained mixture, preferably through a nozzle or a rotating/spinning disk, as to form droplets; cooling the droplets by an ambient (preferably a temperature of 24-25°C) air stream or cold gas/air (preferably a temperature below 25°C, more preferably a temperature below 24°C) as to form solidified encapsulates. A method according to claim 50, said method further comprises the steps of: providing a second carrier material, optionally forming a melt or liquid of said second carrier material by increasing the temperature above the melting temperature of said second carrier material; coating, preferably spraying, more preferably fluidized air bed coating, said second carrier material on the surface of said solidified encapsulates resulting in at least one layer of the second carrier material surrounding said solidified encapsulates; and optionally repeating the spraying step as to obtain a thicker layer and/or more layers of said second carrier material. A method according to any one of embodiments 44 to 51, wherein said milk saccharide or at least one, preferably at least two, more preferably all, of the milk saccharides in said mixture is selected from the list consisting of (i) oligosaccharide comprising lactose (Gal-bl,4-Glc) at its reducing end; (ii) oligosaccharide comprising N-acetyllactosamine (LacNAc) at its reducing end; (iii) oligosaccharide comprising lacto-N-biose (LNB) at its reducing end; (iv) milk glycosaminoglycan; (v) antigen of the human ABO blood group system, optionally bound to a monosaccharide selected from the list consisting of betal,3-GlcNAc, betal,4-GlcNAc, betal,3-GalNAc and -betal,4-Glc; and (vi) Lewis-type antigen oligosaccharide. A composition, preferably a nutritional and/or pharmaceutical composition, more preferably a nutritional composition, comprising an encapsulate according to any one of embodiments 1 to 43 or comprising an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or comprising an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52.

54. A composition according to embodiment 53 further comprising a feed ingredient and/or a food ingredient, wherein said feed/food ingredient is preferably chosen from the list consisting of: a lipid, preferably one or more selected from the list consisting of an oil, fat, ester, monoglyceride, diglyceride, triglyceride and free fatty acid; a vitamin, preferably one or more selected from the list consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin H, or a derivate thereof; an amino acid compound; a trace element; a mineral, preferably one or more selected from the list consisting of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium, potassium, molybdenum, chromium, selenium and chloride; an antioxidant; a prebiotic agent, preferably one or more selected from the list consisting of GOS (galactooligosaccharide), FOS (fructo-oligosaccharide), inulin and resistant starch; a carbohydrate; an antimicrobial agent; and/or a protein.

55. A composition according to embodiment 53 or 54, wherein said composition comprises one or more probiotics.

56. A composition according to any one of embodiments 53 to 55, wherein said composition is a food product, preferably wherein said food product is selected from a list consisting of dairy product, bar, liquid product, savory snack, savory biscuit, bakery product, pasta and food supplement, more preferably wherein said food product is a dairy product or a food supplement.

57. A composition according to any one of embodiments 53 to 56, wherein said composition is a nutritional composition selected from a list consisting of infant formula, baby food, infant cereal composition, growing-up milk, milk replacer, creep feed, pet food, dry feed, frozen feed, prestarter diet, pre weaning feed, weaning feed and post weaning feed, preferably selected from a list consisting of dry feed, frozen feed, milk replacer, creep feed, pet food, prestarter diet, pre weaning feed, weaning feed and post weaning feed, more preferably selected from a list consisting of dry feed, frozen feed, milk replacer, creep feed, post weaning feed and pet food, even more preferably wherein said nutritional composition is dry feed, frozen feed, creep feed, post weaning feed or pet food, even more preferably wherein said nutritional composition is pet food or creep feed, most preferably wherein said nutritional composition is a pet food.

58. A composition according to any one of embodiments 53 to 47, wherein said composition is for feeding a human, preferably an infant, a child, an adolescent and/or an adult, more preferably an infant, a child and/or an adolescent, even more preferably an infant and/or a child.

59. A composition according to any one of embodiments 53 to 57, wherein said composition is for feeding an infant animal, a young animal and/or an adult animal, preferably a non-adult animal or adult animal, more preferably a non-adult animal, wherein said animal is preferably selected from a companion animal, an aquatic animal, a farm animal, a bird, a reptile and an animal grown for food consumption, preferably wherein: a companion animal is selected from the list consisting of a dog, a cat, a rabbit, a guinea pig, a hamster, a mouse, a rat, a gerbil, a bird, a chinchilla, a turtle, a ferret and a horse, more preferably a dog or cat; an aquatic animal is a water breathing animal, preferably selected from the list consisting of a fish, a crustacean, a water turtle and an amphibian, more preferably a fish or a crustacean; a farm animal is selected from the list consisting of a cow, a horse, a pig, a chicken, a goat, a sheep, a llama, an alpaca, a donkey, a mule and poultry, preferably a cow, a pig or a horse; a bird is selected from the list consisting of a chicken, a pigeon, an ostrich, a goose, a duck, a canary, a finch, a guinea fowl, a turkey, an owl and a falcon, preferably poultry, more preferably a chicken; and/or an animal grown for food consumption is selected from the list consisting of a cow, a pig, a horse, a chicken, a goat, a sheep, a turkey, a goose, a rabbit and a duck, preferably a cow or a pig.

60. A composition according to embodiment 59, wherein said composition is for feeding an aquatic animal, preferably a water breathing animal, more preferably selected from the list consisting of a fish, a crustacean, a water turtle and an amphibian, even more preferably a fish or a crustacean, wherein said composition is preferably an aquaculture feed.

61. A composition according to any one of embodiments 53 to 60, wherein said composition is formed as a mash mixture, granules, particles, kibbles or pellets, preferably formed as granules, particles, kibbles or pellets.

62. A composition according to any one of embodiments 53 to 61, wherein the amount of said saccharide or said mixture of at least two different saccharides, preferably said active agent, constitutes 0.01 to 60.0 % (VJ/VJ), preferably 0. 1 to 60.0 % (VJ/VJ), more preferably 1.0 to 60.0 % (VJ/VJ), even more preferably 1.0 to 50.0 % (VJ/VJ), even more preferably 5.0 to 50.0 % (VJ/VJ), even more preferably 10.0 to 50.0 % (w/w), even more preferably 10.0 to 40.0 % (w/w), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of said composition.

63. A composition according to any one of embodiments 53 to 62, wherein the amount of said saccharide or said mixture of at least two different saccharides, preferably said active agent, constitutes 0.001 to 15.0 % (w/w), preferably 0.001 to 10.0 % (w/w), more preferably 0.001 to 5.0 % (w/w), even more preferably 0.001 to 3.0 % (w/w), most preferably 0.001 to 1.0 % (w/w), of the total weight of said composition.

64. A composition according to any one of embodiments 53 to 63 which is a pharmaceutical composition.

65. A pharmaceutical composition according to embodiment 64, further comprising a pharmaceutical ingredient, preferably a pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, excipient, salt, adjuvant and/or solvent.

66. An encapsulate according to any one of embodiments 1 to 43, an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52, an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52, a nutritional composition according to any one of embodiments 53 to 63 or a pharmaceutical composition according to embodiment 64 or 65, for use as a medicament, preferably for use in preventing, delaying, curing, ameliorating and/or treating gastro-intestinal disease in a human or an animal, preferably an animal, or for use in preventing, delaying, curing, ameliorating and/or treating allergic disease in a human or an animal, preferably an animal.

67. A method for the production of a composition, preferably a nutritional and/or pharmaceutical composition, more preferably a nutritional composition, said method comprising the steps of: providing an encapsulate according to any one of embodiments 1 to 43 or an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; and mixing said encapsulate or encapsulated active agent or encapsulated milk saccharide/milk saccharide mixture with said food and/or feed ingredient to obtain a mixture, preferably a homogeneous mixture.

68. A method according to embodiment 67, wherein said mixture has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

69. A method according to embodiment 67 or 68, wherein said method further comprises a drying step, preferably in an oven and preferably at a temperature of > 50°C and/or < 100°C.

70. A method according to any one of embodiments 67 to 69, wherein said method further comprises a step of preparing food and/or feed.

71. A method according to embodiment 70, wherein said step of preparing food and of feed is selected from the list consisting of baking, roasting, frying, heating (dielectric, ohmic or infrared), blanching, pasteurization, heat sterilization, fermentation, irradiation, processing using pulsed electric field, high pressure and extrusion (dry or wet), preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization, processing using pulsed electric field or high pressure, and extrusion (dry or wet), more preferably selected from the list consisting of baking, heating (dielectric, ohmic or infrared), heat sterilization and extrusion (dry or wet), even more preferably baking or extrusion (dry or wet).

72. A method according to any one of embodiments 67 to 77, wherein said method comprises the steps of: providing an encapsulate according to any one of embodiments 1 to 43 or an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated milk saccharide/milk saccharide mixture with ingredient to obtain a mixture, preferably a homogeneous mixture; and extruding said mixture to obtain a desired shape, preferably selected from mash mixture, granules, pellets, kibbles and particles, more preferably selected from granules, pellets, kibbles and particles.

73. A method according to any one of embodiments 67 to 71, wherein said method comprises the steps of: providing an encapsulate according to any one of embodiments 1 to 43 or an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52; providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated milk saccharide/milk saccharide mixture with said ingredient to obtain a mixture, preferably a homogeneous mixture; and baking said mixture, preferably at a temperature of 100°C or higher, more preferably 150°C or higher, even more preferably 150°C to 225°C, even more preferably 180°C to 225°C.

74. Use of an encapsulate according to any one of embodiments 1 to 43 or an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52 in the manufacturing of a nutritional composition, preferably a feed or a food.

75. Use of an encapsulate according to any one of embodiments 1 to 43 or an encapsulated active agent obtained by a method according to any one of embodiments 44 to 47 and 52 or an encapsulated milk saccharide or mixture of at least two different milk saccharides obtained by a method according to any one of embodiments 48 to 52 in feeding a human or an animal, preferably an animal.

Moreover, the invention relates to the following preferred specific embodiments:

1. A method for the production of a composition, said method comprising the steps of: providing an encapsulate comprising: o an active agent comprising a milk oligosaccharide or a mixture of at least two different milk oligosaccharides, and o a first carrier material which forms (i) a shell around said active agent as to form an encapsulate of the core-shell type or (ii) a matrix wherein said active agent is distributed as to form an encapsulate of the matrix type providing an ingredient, preferably (i) a food and/or feed ingredient and/or (ii) a pharmaceutical ingredient, more preferably providing a food and/or feed ingredient; mixing said encapsulate or encapsulated active agent or encapsulated saccharide/saccharide mixture with said food and/or feed ingredient to obtain a mixture, preferably a homogeneous mixture; a step of preparing food and/or feed from said obtained mixture at conditions comprising a temperature of 100°C or more.

2. A method according to embodiment 1, wherein said milk oligosaccharide and at least one of said at least two different milk oligosaccharides have a degree of polymerization of 3-20, preferably 3-9.

3. A Method according to embodiment 1 or 2, wherein said obtained mixture has a moisture content (by weight) of at least 15% (w/w), preferably at least 20% (w/w), more preferably at least 25% (w/w), even more preferably at least 30% (w/w), even more preferably at least 35% (w/w), most preferably at least 40% (w/w).

4. A method according to any one of embodiments 1 to 3, wherein said step of preparing food and/or feed is selected from the list consisting of baking, heating and extrusion.

5. A method according to any one of embodiments 1 to 3, wherein said step of preparing food and/or feed is extruding said obtained mixture to obtain a desired shape.

6. A method according to any one of embodiments 1 to 3, wherein said step of preparing food and/or feed is baking said obtained mixture. 7. A method according to any one of embodiments 1 to 6, wherein the amount of said milk oligosaccharide or said mixture of at least two different oligosaccharides constitutes 0.01 to 60.0 % (VJ/VJ), preferably 0. 1 to 60.0 % (VJ/VJ), more preferably 1.0 to 60.0 % (VJ/VJ), even more preferably 1.0 to 50.0 % (VJ/VJ), even more preferably 5.0 to 50.0 % (VJ/VJ), even more preferably 10.0 to 50.0 % (VJ/VJ), even more preferably 10.0 to 40.0 % (VJ/VJ), most preferably 10.0 to 40.0 % (VJ/VJ), of the total weight of dry matter of said composition.

8. A method according to any one of embodiments 1 to 7 , wherein the amount of said milk oligosaccharide or said mixture of at least two different oligosaccharides constitutes 0.001 to 15.0 % (VJ/VJ), preferably 0.001 to 10.0 % (VJ/VJ), more preferably 0.001 to 5.0 % (VJ/VJ), even more preferably 0.001 to 3.0 % (VJ/VJ), most preferably 0.001 to 1.0 % (VJ/VJ), of the total weight of said composition.

9. A method according to any one of embodiments 1 to 8, wherein said method further comprises a drying step, preferably in an oven and preferably at a temperature of > 50°C and/or < 100°C.

10. A method according to any one of embodiments 1 to 9, wherein said composition is a nutritional composition.

11. A method according to any one of embodiments 1 to 10, wherein said composition is an aquaculture feed.

12. A method according to any one of embodiments 1 to 11, wherein said first carrier material comprises an oil and/or a wax, optionally further comprising starch.

13. A method according to any one of embodiments 1 to 11, wherein said first carrier material comprises a wax, optionally further comprising starch.

14. A method according to any one of embodiments 1 to 11, wherein said encapsulate further comprises a second carrier material which at least partially, preferably completely, encapsulates said first carrier material and said active agent as to form an encapsulate of the core-shell type, wherein said active agent and said first carrier material form a core component which is surrounded by a shell formed by the second carrier material.

15. A method according to embodiment 14, wherein said second carrier material comprises a wax, optionally further comprising starch.

16. A method according to embodiment 13 or 15, wherein said wax is selected from the list consisting of candelilla wax, carnauba wax, beeswax, rice bran wax, paraffin wax, jojoba wax, microcrystalline wax and japan wax.

17. A method according to embodiment 14, wherein said second carrier material comprises a polymer, optionally further comprising starch.

18. A method according to embodiment 17, wherein said polymer is a gum, optionally further comprising starch.

19. A method according to embodiment 18, wherein said gum is selected from the list consisting of shellac gum, guar gum, locust bean gum, konjac gum, arabic gum, xanthan gum, gellan gum, cassia gum, cellulose gum, modified arabic gum and tara gum. 0. A method according to any one of embodiments 1 to 19, wherein said encapsulate has a median diameter (D(v, 0.5)) of at least 50 pm, preferably at least 75 pm, more preferably at least 100 pm, even more preferably at least 125 pm, even more preferably at least 150 pm, most preferably at least 175 pm; and/or said encapsulate has a median diameter (D(v, 0.5)) of < 400 pm, preferably < 350 pm, more preferably < 300 pm. 1. A method according to any one of embodiments 1 to 20, wherein the quantity of said milk oligosaccharide or mixture of at least two different milk oligosaccharides comprised in said encapsulate upon exposure to said conditions of said step of preparing food and/or feed is at least 80.0 %, preferably at least 85.0 %, more preferably at least 87.5 %, even more preferably at least 90.0 %, even more preferably at least 92.5 %, most preferably at least 95.0 %, of the quantity of said milk oligosaccharide or mixture of at least two different milk oligosaccharides comprised in the encapsulate before exposure to said conditions of said step of preparing food and/or feed.

Definitions

The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus, if an element can be understood in the context of this specification as including more than one meaning, then its use in a claim must be understood as being generic to all possible meanings supported by the specification and by the word itself.

The various aspects and embodiments of the invention disclosed herein are to be understood not only in the order and context specifically described in this specification, but to include any order and any combination thereof. Each embodiment as identified herein may be combined together unless otherwise indicated. All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Unless specifically stated otherwise, all words used in the singular number shall be deemed to include the plural and vice versa. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization described herein are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications.

In the drawings and specification, there have been disclosed embodiments of the invention, and although specific terms are employed, the terms are used in a descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the following claims. It must be understood that the illustrated embodiments have been set forth only for the purposes of example and that it should not be taken as limiting the invention. It will be apparent to those skilled in the art that alterations, other embodiments, improvements, details and uses can be made consistent with the letter and spirit of the invention herein and within the scope of this invention, which is limited only by the claims, construed in accordance with the patent law, including the doctrine of equivalents. In the claims which follow, reference characters used to designate claim steps are provided for convenience of description only, and are not intended to imply any particular order for performing the steps (unless specifically stated otherwise).

In this document and in its claims, the verbs "to comprise", "to have" and "to contain", and their conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The verb "to consist essentially of" means that a solution as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention. Throughout the document and claims, unless specifically stated otherwise, the verbs "to comprise", "to have" and "to contain", and their conjugations, may be preferably replaced by "to consist" (and its conjugations) or "to consist essentially of" (and its conjugations) and vice versa. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word "about" or "approximately" or "around" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10%, preferably 5%, more preferably 1% of the value.

The term "monosaccharide" as used herein refers to a sugar that is not decomposable into simpler sugars by hydrolysis, is classed either an aldose or ketose, and contains one or more hydroxyl groups per molecule. Monosaccharides are saccharides containing only one simple sugar.

The term "oligosaccharide" as used in the context of the present invention preferably refers to a saccharide containing 2 up to and including 20 monosaccharides, i.e. the degree of polymerization (DP) is 2-20. Preferably, said oligosaccharide according to the invention contains at least 3 monosaccharides, i.e. the degree of polymerization is preferably 3-20. More preferably, said oligosaccharide according to the invention consists of 3-9, preferably 3-8, more preferably 3-7 monosaccharides.

Preferably, said oligosaccharide according to the invention is not lactose. An oligosaccharide can be a linear structure or can include branches. The linkage (e.g. glycosidic linkage, galactosidic linkage, glucosidic linkage, etc.) between two sugar units can be expressed, for example, as 1,4, l->4, or (1-4), used interchangeably herein. Each monosaccharide can be in the cyclic form (e.g. pyranose or furanose form). An oligosaccharide can contain both alpha- and beta-glycosidic bonds or can contain only beta- glycosidic bonds.

The term "polysaccharide" as used in the context of the present invention refers to a saccharide containing a plurality of repeating units comprised of simple sugars. In the context of the invention, said polysaccharide preferably has a degree of polymerization which is at least 40 (and preferably < 3000).

The terms "LNT 11", "LNT-II", "LN3", "lacto-N-triose II", "lacto-N-triose II", "lacto-N-triose", "lacto-N-triose" or "GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably.

The terms "LNT", "lacto-N-tetraose", "lacto-/V-tetraose" or "Gaipi-3GlcNAcpi-3Gaipi-4Glc" as used in the present invention, are used interchangeably.

The terms "LNnT", "lacto-N-neotetraose", "lacto-/V-neotetraose", "neo-LNT" or "Gaipi-4GlcNAcpi- 3Gaipi-4Glc" as used in the present invention, are used interchangeably.

The terms "2' fucosyllactose", "2'-fucosyllactose", "alpha-1, 2-fucosyllactose", "alpha 1,2 fucosyllactose", "a-l,2-fucosyllactose", "a 1,2 fucosyllactose", "Gaip-4(Fucal-2)Glc", 2FL" and "2'FL" are used interchangeably.

The terms "3-fucosyllactose", "alpha-1, 3-fucosyllactose", "alpha 1,3 fucosyllactose", "a-1,3- fucosyllactose", "a 1,3 fucosyllactose", "Gaip-4(Fucal-3)Glc", 3FL" and "3-FL" are used interchangeably.

The terms "difucosyllactose", "di-fucosyllactose", "lactodifucotetraose", "2',3-difucosyllactose", "2', 3 difucosyllactose", "a-2', 3-fucosyllactose", "a 2', 3 fucosyllactose, "Fucal-2Gaipi-4(Fucal-3)Glc", "DFLac", 2', 3 diFL", "DFL", "DiFL" and "diFL" are used interchangeably.

The terms "6' sialyllactose", "6'-sialyllactose", "alpha-2, 6-sialyllactose", "alpha 2,6 sialyllactose", "a-2,6- sialyllactose", "a 2,6 sialyllactose", "6SL", "Siaa2-6Gaipi-4Glc" and "6'SL" as used in the present invention, are used interchangeably.

The terms "3' sialyllactose", "3' -sialyllactose", "alpha-2, 3-sialyllactose", "alpha 2,3 sialyllactose", "a-2,3- sialyllactose", "a 2,3 sialyllactose", "3SL", "Siaa2-3Gaipi-4Glc" and "3'SL" as used in the present invention, are used interchangeably.

The terms "LSTa", "LS-Tetrasaccharide a", "Sialyl-lacto-N-tetraose a", "sialyllacto-N-tetraose a" or "Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably.

The terms "LSTb", "LS-Tetrasaccharide b", "Sialyl-lacto-N-tetraose b", "sialyllacto-N-tetraose b" or "Gal- bl,3-(Neu5Ac-a2,6)-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably.

The terms "LSTc", "LS-Tetrasaccharide c", "Sialyl-lacto-N-tetraose c", "sialyllacto-N-tetraose c", "sialyllacto-N-neotetraose c" or "Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc" as used in the present invention, are used interchangeably.

The terms "LSTd", "LS-Tetrasaccharide d", "Sialyl-lacto-N-tetraose d", "sialyllacto-N-tetraose d", "sialyllacto-N-neotetraose d" or "Neu5Ac-a2,3-Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc".

The terms "DSLNnT" and "Disialyllacto-N-neotetraose" are used interchangeably and refer to Neu5Ac- a2,6-[Neu5Ac-a2,6-Gal-bl,4-GlcNAc-bl,3]-Gal-bl,4-Glc.

The terms "DSLNT" and "Disialyllacto-N-tetraose" are used interchangeably and refer to Neu5Ac-a2,6- [Neu5Ac-a2,3-Gal-bl,3-GlcNAc-bl,3]-Gal-bl,4-Glc.The terms "LNFP-I", "lacto-N-fucopentaose I", "LNFP I", "LNF I OH type I determinant", "LNF I", "LNF1", "LNF 1" and "Blood group H antigen pentaose type 1" are used interchangeably and refer to Fuc-al,2-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc.

The terms "GalNAc-LNFP-l" and "blood group A antigen hexaose type I" are used interchangeably and refer to GalNAc-al,3-(Fuc-al,2)-Gal-bl,3-GlcNAc-bl,3-Gal-bl,4-Glc.

The terms "LNFP-II" and "lacto-N-fucopentaose II" are used interchangeably and refer to Gal-bl,3-(Fuc- al,4)-GlcNAc-bl,3-Gal-bl,4-Glc.

The terms "LNFP-III" and "lacto-N-fucopentaose III" are used interchangeably and refer to Gal-bl,4-(Fuc- al,3)-GlcNAc-bl,3-Gal-bl,4-Glc.

The terms "LNFP-V" and "lacto-N-fucopentaose V" are used interchangeably and refer to Gal-bl,3- GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc.

The terms "LNFP-VI", "LNnFP V" and "lacto-N-neofucopentaose V" are used interchangeably and refer to Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc.

The terms "LNnFP I" and "Lacto-N-neofucopentaose I" are used interchangeably and refer to Fuc-al,2- Gal-bl,4-GlcNAc-bl,3-Gal-bl,4-Glc.

The terms "LNDFH I", "Lacto-N-difucohexaose I", "LNDFH-I", "LDFH I", "Le ^b -lactose" and "Lewis-b hexasaccharide" are used interchangeably and refer to Fuc-al,2-Gal-bl,3-[Fuc-al,4]-GlcNAc-bl,3-Gal- bl,4-Glc.

The terms "LNDFH II", "Lacto-N-difucohexaose II", "Lewis a-Lewis x" and "LDFH II" are used interchangeably and refer to Fuc-al,4-(Gal-bl,3)-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc.

The terms "LNnDFH", "Lacto-N-neoDiFucohexaose" and "Lewis x hexaose" are used interchangeably and refer to Gal-bl,4-(Fuc-al,3)-GlcNAc-bl,3-Gal-bl,4-(Fuc-al,3)-Glc.

The terms "alpha-tetrasaccharide" and "A-tetrasaccharide" are used interchangeably and refer to Gal N Acai, 3-(Fuc-al,2)-Gal-bl,4-Glc.

The term "cultivation" refers to the culture medium wherein the cell is cultivated or fermented, the cell itself, and the saccharide(s) that is/are produced by the cell in whole broth, i.e. inside (intracellularly) as well as outside (extracellularly) of the cell. Examples

The invention will be described in more detail in the examples. The following examples will serve as further illustration and clarification of the present invention and are not intended to be limiting in any way.

Example 1: Materials and Methods (oligosaccharides)

3'SL and 6'SL

3'sialyllactose (3'SL) and 6'sialyllactose (6'SL) were recombinantly produced in E. coli and purified as described in WO 2022/034079 (Examples 11, 13 and 14; the E. coli strains are described in Example 3 of WO 2018/122225 for 6'SL and Example 7 of WO 2018/122225 for 3'SL wherein an alpha-2, 3- sialyltransferase from Pasteurella multocida, i.e. amino acids 1-268 of Uniprot ID Q9CLP3 sequence vl, was used) and subsequently spray dried as described in Example 21 of WO 2022/034079 to obtain 3'SL powder (purity 98.4 %) and 6'SL powder (purity 98.6 %).

Analytical analysis

Standards such as but not limited to sucrose, lactose, N-acetyllactosamine (LacNAc, Gal-bl,4-GlcNAc), lacto-N-biose (LNB, Gal-bl,3-GlcNAc), fucosylated LacNAc (2'FLacNAc, 3-FLacNAc), sialylated LacNAc, (3'SLacNAc, 6'SLacNAc), fucosylated LNB (2'FLNB, 4'FLNB), lacto-/V-triose II (LN3), lacto-/V-tetraose (LNT), lacto-/V-neo-tetraose (LNnT), LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LSTc and LSTd were purchased from Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house made standards (e.g. 3'SL and 6'SL).

The oligosaccharide composition in pre-extrusion and post-extrusion samples were determined by mixing 4 g of said sample into 20 ml of citrate buffer (0.02M, pH 5.0). An internal standard (galactoronic acid) was added until a final concentration of 3 mg/ml. The mixture was incubated at 70°C for 10 min before it was blended at 20000 rpm for 5 min using an ultra turrax (IKA T 18), followed by shaking a 37°C for 30 min. Protein precipitation was performed using MeOH at the ratio of 2:1 volume of MeOH : sample. The mixture was kept at -20°C for lh, followed by centrifugation at 14000 rpm for 5 min. The supernatant was filtrated using PTFE filter 0.2 pm. 3'SL and 6'SL in the flowthrough was quantified using HPAEC-PAD on a Dionex ICS5000 ⁺ system (Thermo Fisher). A volume of 5 pL of sample was injected on a Dionex CarboPac PA200 column 4 x 250 mm with a Dionex CarboPac PA200 guard column 4 x 50 mm. The column temperature was set to 30 °C. A gradient was used wherein eluent A was deionized water, wherein eluent B was 200 mM Sodium hydroxide and wherein eluent C was 500 mM Sodium acetate. The oligosaccharides were separated in 60 min while maintaining a constant ratio of 25 % of eluent B using the following gradient: an initial isocratic step maintained for 10 min of 75 % of eluent A, an initial increase from 0 to 4 % of eluent C over 8 min, a second isocratic step maintained for 6 min of 71 % of eluent A and 4 % of eluent C, a second increase from 4 to 12 % of eluent C over 2.6 min, a third isocratic step maintained for 3.4 min of 63 % of eluent A and 12 % of eluent C and a third increase from 12 to 48 % of eluent C over 5 min. As a washing step 48 % of eluent C was used for 3 min. For column equilibration, the initial condition of 75 % of eluent A and 0 % of eluent C was restored in 1 min and maintained for 11 min. The applied flow was 0.5 mL/min. The actual concentration of 3'SL and 6'SL in the pre-extrusion and post-extrusion samples (mg oligosaccharide per g dried matter) were computed based on their concentrations measured by the Dionex system and the dried matter of dog food after subtracting the water content.

Oligosaccharide composition of an encapsulate

The composition of 3'SL and 6'SL in microcapsules were determined as follows. First, 20 ml of citrate buffer (0.02M, pH 5.0) was added into 50 mg of oligosaccharide microcapsules. Internal standard (galactoronic acid) was added until a final concentration of 3 mg/ml. The mixture was incubated at 70°C for 10 min before it was blended at 20000 rpm for 5 min using an ultra turrax (IKAT 18), followed by shaking a 37°C for 30 min. Protein precipitation was performed using MeOH at the ratio of 2:1 volume of MeOH : sample. The mixture was kept at -20°C for lh, followed by centrifugation at 14000 rpm for 5 min. The supernatant was filtrated using PTFE filter 0.2 pm. 3'SL and 6'SL in the flowthrough was quantified using HPAEC-PAD on a Dionex ICS5000 ⁺ system (Thermo Fisher) according to Example 1.

Example 2: Encapsulation

A mixture of the 3'sialyllactose (3'SL) powder and the 6'sialyllactose (6'SL) powder was made in a ratio of 9 (3'SL):1 (6'SL). The obtained mixture was prilled at a concentration of 35% (w/w) in (1) hydrogenated palm oil (GV60, Archer Daniels Midland (ADM)) or (2) Candelilla wax (Interchimie). Microparticles were formed upon contacting ambient air (microparticles "A" = hydrogenated palm oil; microparticles "B" = Candelilla wax). Characteristics of the obtained microparticles are provided in Table 3.

A fluidized bed coater (e.g. Glatt; Wurster geometry, i.e. bottom spray) was used to apply a coating to microparticles "A" and "B" as to obtain microparticles of the core-shell type. A high velocity air stream is applied through a cylinder that is centrally placed just above the air inlet at the narrow bottom of a conus shaped chamber. Microparticles "A" or "B" were introduced into the chamber of the coating apparatus as to form a fluidized bed. The coating material was molten and fed through heated tubes towards the nozzle, wherein the coating material was selected from:

Hydrogenated palm oil (GV60, Archer Daniels Midland (ADM),

Candelilla wax (Interchimie),

Shellac gum (Aquagold; Stroever Shellack Bremen) mixed with starch (Amioca powder TF; Ingredion) at the ratio 95:5 (w/w), respectively. The coating was applied onto the microparticles "A" or "B" by spraying it from the nozzle onto the fluidized microparticles within the cylinder. During the coating process, microparticles left the top of the cylinder and descended back into the fluidized bed. Cool air congealed the coating during the descent. The coating applied during the FAB step represented up to 40% weight of the finally obtained coated microparticles. The coated microparticles each consisted of approximately 25% (w/w) oligosaccharides (i.e. said mixture of 3'SL and 6'SL) as outlined in Table 2. Hence, the microparticles were obtained as follows:

Microparticles "C": microparticles A + hydrogenated palm oil coating

Microparticles "D": microparticles A + Candelilla wax coating

Microparticles "E": microparticles A + Shellac gum with starch coating

Microparticles "F": microparticles B + hydrogenated palm oil coating Microparticles "G": microparticles B + Candelilla wax coating Microparticles "H": microparticles B + Shellac gum with starch coating

Table 2 - Microcapsule composition

Microscopic analysis (optical microscope at lOOx magnification; stereomicroscope at 50x magnification) showed that the microparticles "A" to "H" are beige to slightly marron, rather spherical and rather homogeneous. The coating membrane of microparticles "C" to "H" is thin but completely surrounds the prills.

The water activity, % dry extract (i.e. (mass of the sample after 24h at 105°C / initial mass of the sample) x 100), particle size distribution parameters and flowability properties were determined for (i) microparticles "A" to "H" and (ii) the mixture of dried 3'SL powder and dried 6'SL powder (referred to as "oligosaccharide mixture", i.e. not encapsulated). An overview of the results can be found in Table 3: Table 3 - Overview microparticles

As shown in Table 3, all microparticles "A" to "H" have a water activity (Aw) which is higher than that of the non-encapsulated active agent (mixture of dried 3'SL and dried 6'SL = "oligosaccharide mixture"). Further, the flowability of the microparticles is improved from poor to good or even excellent compared to the oligosaccharide mixture. In particular, microcapsules "B", "D" and "G", wherein the complete particle is made of Candelilla wax (microcapsule "B") or wherein the shell is made of Candelilla wax (microcapsules "D" and "G") are characterized by an excellent flowability.

In addition, it was noted that each microparticle contain a high dry extract (> 96.6 % and exceeding that of the oligosaccharide mixture) and show a particle size distribution which is homogeneous with a span score around 1.

Water activity (Aw)

Water activity (Aw) values were obtained with a LabSwift-aw (Novasina) at ambient temperature between 24.4-24.9 °C. The values were recorded after a stabilization period (time varying according to the sample tested). Dry extract determination

Dry extracts were obtained by a thermogravimetric analysis. Samples of each powder were incubated in a stove at 105°C for 24h. The measures were performed in triplicate for each sample. The dry extract proportion contained in the sample was obtained using the following equation : % dry extract = (mass of the sample after 24h at 105°C / initial mass of the sample) x 100.

Particle size distribution

Particle size distribution was obtained by the laser diffraction technique using a MasterSizer S apparatus

(Malvern Instruments Ltd), 5000 counts.

Flowability

Carr index l _c (Compressivity index) and Hausner ratio Rh were determined by measuring the bulk density (pb) and the tapped density (p _t ) of the powders, pb and p _t were obtained with a 100 ml measuring cylinder, pb is the initial density and p _t is obtained after tapping the measuring cylinder on a flat surface, 1 tap / s, 180 taps. Below are equations to calculate lc and Rh: lc = 100 x (p _t - p _b ) / p _t Rh = p _t / Pb

The flow character was then scored as depicted in Table 1.

Example 3: Materials and Methods (production of dog food kibbles)

Extrusion procedure

Microparticles "D", "E" or "H" as prepared in Example 2 was mixed into 5 kg dog food (major components shown in Table 4) at the inclusion ratio of 48 g microcapsules per kg dog food. A control batch was prepared by mixing dog food with the same amount of the non-encapsulated oligosaccharide mixture (3'SL+6'SL). Before subjecting each dog food batch to the extrusion process, each batch was homogenized for 15 min.

A portion of 100 g of each prepared batch was preserved at -80°C for analytics ("pre-extrusion sample").

Table 4 - Major components of dog food

Each batch of the homogenized dog food (5kg) was processed using the extruder type BC 45 (Clextral).

Extruder parameters are summarized in table 5. The kibbles at the outcome of the dies were dried in the oven for 50 min at 60°C. A portion of 100 g was preserved at -80°C for analytics ("post-extrusion sample").

Table 5 - Extrusion parameters

Water content

Water content of pre-extrusion and post-extrusion samples were determined using the Karl-Fisher method (Laboratorium ECCA N.V., Belgium).

Example 4: Production of dog food kibbles

Dog food kibbles containing an equal amount of microparticles "D", "E" or "H" were prepared according to the extrusion procedure described in Example 3. As a control, dog food kibbles containing spray-dried 3'SL and spray-dried 6'SL (in an amount which is similar to the microparticle containing kibbles) were prepared using the same extrusion procedure ("non-encapsulated oligosaccharide mixture"). The concentration of 3'SL and 6'SL were determined for each dog food batch before extrusion ("preextrusion") and after extrusion ("post-extrusion") and expressed as mg/g dried matter (it is referred to Example 1) as shown in Table 6. Table 6 - Oligosaccharide concentration in pre- and post-extruded dog food kibbles (mg/g dried matter)

As shown in Table 6, the retention of 3'SL and 6'SL within the dog food upon exposure to the extrusion process is greatly improved when the oligosaccharides are encapsulated compared to the control. These data hence demonstrate that encapsulating oligosaccharides according to the invention provides a good to excellent protection against degradation during dog food extrusion (hallmarked by conditions such as a high temperature, high pressure and high moisture content during wet extrusion). Example 5: Materials and Methods (production of dog food kibbles at very high temperature)

Extrusion procedure

Microparticles "E" as prepared in Example 2 was mixed into 5 kg dog food (major components shown in Table 4) at the inclusion ratio of 48 g microcapsules per kg dog food. A control batch was prepared by mixing dog food with the same amount of the non-encapsulated oligosaccharide mixture (3'SL+6'SL). Before subjecting each dog food batch to the extrusion process, each batch was homogenized for 15 min. A portion of 100 g of each prepared batch was preserved at -80°C for analytics ("pre-extrusion sample").

Each batch of the homogenized dog food (5kg) was processed using the extruder type BC 45 (Clextral). Extruder parameters are summarized in Table 7 (which are different from those summarized in Table 5 of Example 3). The kibbles at the outcome of the dies were dried in the oven for 50 min at 60°C. A portion of

100 g was preserved at -80°C for analytics ("post-extrusion sample").

Table 7 - Extrusion parameters

Water content

Water content of pre-extrusion and post-extrusion samples were determined using the Karl-Fisher method (Laboratorium ECCA N.V., Belgium).

Example 6: Production of dog food kibbles at very high temperature

Dog food kibbles containing microparticles "E" were prepared according to the extrusion procedure described in Example 5. As a control, dog food kibbles containing spray-dried 3'SL and spray-dried 6'SL (in an amount which is similar to the microparticle containing kibbles) were prepared using the same extrusion procedure ("non-encapsulated oligosaccharide mixture"). The concentration of 3'SL and 6'SL were determined for each dog food batch before extrusion ("pre-extrusion") and after extrusion ("postextrusion") and expressed as mg/g dried matter (it is referred to Example 1) as shown in Table 8.

Table 8 - Oligosaccharide concentration in pre- and post-extruded dog food kibbles (mg/g dried matter)

As shown in Table 8, the retention of 3'SL and 6'SL within the dog food upon exposure to the extrusion process is greatly improved when the oligosaccharides are encapsulated compared to the control, even under the extremely high temperatures used during the extrusion process (see Table 7). These data hence demonstrate that encapsulating oligosaccharides according to the invention provides an excellent protection against degradation during dog food extrusion even at very high temperatures (further hallmarked by conditions such as a high pressure and high moisture content during wet extrusion).