Field of the invention

The present invention relates to a heated nutritional composition comprising a milk saccharide or a mixture of at least two different milk saccharides. More specifically, the present invention relates to a heated nutritional composition comprising a mammalian milk saccharide or a mixture of at least two different mammalian milk saccharides. Even more specifically, the present invention relates to a heated nutritional composition comprising a mammalian milk oligosaccharide or a mixture of at least two different mammalian milk oligosaccharides Even more specifically, the present invention relates to a heated nutritional composition comprising a human milk oligosaccharide or a mixture of at least two different human milk oligosaccharides.

Background of the invention

Milk saccharides, i.e. saccharides which are found in milk of animals such as mammals and humans, in particular mammalian milk oligosaccharides (MMOs) and human milk oligosaccharides (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): p. Datsenko and Wanner, PNAS 2000, 97(12): p. 6640-6645), are gaining more and more attention as a replete amount of beneficial effects have been demonstrated. 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, PLoS ONE 15(12)).

The importance of milk saccharides, in particular milk oligosaccharides such as MMOs and HMOs, 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 saccharides, in particular mammalian (MMOs) and human milk oligosaccharides (HMOs), promising candidates to incorporate into for example food and feed.

However, it is well-known that (oligo)saccharides, in particular sialylated mammalian/human milk (oligo)saccharides, in the presence of other constituents such as proteins, amino acids etc, which are present in common food and feed products, could react and/or are labile (e.g. sialylated oligosaccharides in an acidic environment), especially under conditions of high temperature (i.e. at a temperature of above 100°C, in particular above 150°C) and/or in the presence of moisture (e.g. water, milk). A well-known example hereof is the so-called Maillard reaction. Such reactions are unwanted as they have a negative impact on the concentration of MMOs/HMOs, in particular sialylated MMOs/HMOs, in the final food or feed.

Summary of the invention

The invention is directed to the surprising finding that milk saccharides, even sialylated milk saccharides which are known to be even more sensitive to the environmental conditions, retain more than 85.0 % of their concentration (expressed as % of the total weight of dry matter) in a nutritional composition upon being heated at temperature of 100°C or higher, even up to 225°C.

In a first aspect, the invention provides a nutritional composition comprising a milk saccharide or a mixture of at least two different milk saccharides, wherein said nutritional composition was heated at a temperature of 100°C or higher.

In a second aspect, the invention provides a method for the production of a heated nutritional composition, preferably a baked nutritional composition.

Detailed description of the invention

Nutritional composition

In a first aspect, the invention provides a nutritional composition comprising a milk saccharide or a mixture of at least two different milk saccharides, preferably a milk oligosaccharide or a mixture of at least two different milk oligosaccharides, wherein said nutritional composition was heated at a temperature of 100°C or higher. In a preferred embodiment, the nutritional composition according to the invention is for human and/or animal consumption, preferably for human consumption, more preferably for animal consumption. In a more preferred embodiment, said nutritional composition is an animal food or animal feed. In an even more preferred embodiment, said nutritional composition is a pet food or pet feed. In a most preferred embodiment, said nutritional composition is a companion animal food or companion animal feed. Throughout the application and claims, unless specifically stated otherwise, the term "food" is preferably replaced with "feed". The term "companion animal" throughout the application and claims preferably refers to a domestic animal, more preferably to a dog or cat.

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", more 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.

In an additional and/or alternative preferred embodiment, the nutritional composition according to the invention is edible.

In an additional and/or alternative preferred embodiment, the nutritional composition according to the invention is not a liquid product (i.e. is not a liquid nutritional composition).

In an additional and/or alternative preferred embodiment, the nutritional composition according to the invention is not a powder (i.e. is not a powdered nutritional composition). Such a powdered nutritional composition can be obtained by various drying techniques as known in the art (spray drying and freeze drying are widely used examples hereof).

In an additional and/or alternative preferred embodiment, the nutritional composition according to the invention is not a milk replacer. As understood by the skilled person, the term "milk replacer" refers to a nutritional composition which serves as a substitute for mother's milk.

In an additional and/or alternative preferred embodiment, the nutritional composition according to the invention is a baked nutritional composition. 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. Further, as the skilled person readily understands, "baking" does not encompass (hot) extrusion. The latter is for example a typical high- temperature-short-time (HTST) method in contrast to baking.

In an additional and/or alternative preferred embodiment, said nutritional composition according to the invention, preferably said baked nutritional composition according to the invention, is selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a bread, a cake, a pie, a tart, a pastry, a candy bar, an energy bar, granola, a granola bar, a quiche, cereal, a pizza, a corn chip, a tortilla chip, a potato chip and a baked cracker; preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a bread, a cake, a pie, a tart, a pastry, a candy bar, an energy bar, granola, a granola bar and cereal; more preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a pastry, a candy bar, an energy bar, granola, a granola bar and cereal; even more preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a candy bar, an energy bar, granola, a granola bar and cereal; even more preferably selected from a cookie, pet food (preferably dry pet food), a pet treat, an energy bar, granola, and a granola bar; even more preferably a cookie, pet food (preferably dry pet food) and pet treat, most preferably a cookie or pet food (preferably dry pet food). Said cookie is for human and/or animal consumption, preferably for animal consumption as described herein. Said pet food is preferably food for a companion animal. A particular preferred example of food for a companion animal is a treat for a companion animal (such as kibbles and biscuits).

In an additional and/or alternative preferred embodiment, the nutritional composition is a treat or toy for a companion animal or is part of a treat or toy for a companion animal. In this context, a treat or toy for the treatment of dental problems, such as inhibition of plaque formation and/or removal of tartar, is particularly preferred.

In a more preferred embodiment, said nutritional composition according to the invention, preferably said baked nutritional composition according to the invention, is a pet food, more preferably a dry pet food, even more preferably a dry pet food for a companion animal (i.e. dry companion animal food). A dry pet food typically contains at least 85.0 % (w/w), preferably at least 90.0 % (w/w), of dry matter (in other words, the moisture content is 15.0 % (w/w) or less, preferably 10.0 % (w/w) or less). A dry pet food comprises kibbles, biscuits, meals or extruded product. A dry pet food is preferably selected from kibble, biscuit, meal and an extruded product, more preferably selected from kibble, biscuit and meal, even more preferably selected from kibble and biscuit. Kibbles and biscuits are usually prepared by mixing all the ingredients into a homogeneous dough which is then baked. For the production of biscuits, the dough is formed into the desired shape before baking (which is similar to the baking of cookies and crackers). For the production of kibbles, the dough is usually spread onto large sheets before baking. After cooling, the sheets are broken into bite-size pieces. A meal is produced by mixing dried, flaked or granular ingredients. Extruded products have become more and more popular wherein the ingredients are mixed together as to form a dough which is then cooked/baked under conditions of high pressure and temperature (100°C or above). Extrusion results in rapid cooking/baking, resulting in an increased digestibility and palatability. Extrusion is nowadays frequently used to make biscuits and kibbles for example.

A total moisture content of 10% (w/w) or less can be achieved through for example hot-air drying as known in the art.

In an additional and/or alternative more preferred embodiment, said nutritional composition comprises at least 85.0 %, preferably at least 87.0 %, more preferably at least 90.0 %, most preferably at least 92.0 % dry matter. In other words, said nutritional composition comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less moisture. Preferably wherein said moisture is liquid, more preferably is water and/or milk, most preferably is water. The % of dry matter (i.e. dry extract) can be determined by a thermogravimetric analysis or a Karl Fisher titration analysis, preferably by a Karl Fisher titration analysis (preferably as described herein); preferably wherein said % dry extract is calculated as (mass of nutritional composition after 24 hours at 105°C / initial mass of the nutritional composition) x 100. In this context of the invention, the term "moisture" can be preferably replaced with "liquid" and vice versa, unless explicitly stated otherwise. As the skilled person is aware, 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, How baking works : exploring the fundamentals of baking science, 3th edition, John Wiley & Sons Inc).

In an additional and/or alternative more preferred embodiment, said nutritional composition before being heated at a temperature of 100°C or higher according to the invention (i.e. unheated nutritional composition, preferably unbaked nutritional composition), is a dough or a batter, preferably a dough. Preferably, said unheated nutritional composition comprises 3.0-70.0 % w/w, preferably 3.0-60.0 % w/w, more preferably 3.0-50.0 % w/w, even more preferably 5.0-50.0 % w/w, most preferably 10.0-50.0 % w/w, moisture; preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water. In the context of the present invention, throughout the application and claims, a product that comprises a-b % of a component A means that the product can contain further components besides component A but component A can only be present in an amount falling in the range a-b. In an additional and/or alternative more preferred embodiment, said nutritional composition before being heated at a temperature of 100°C or higher according to the invention (i.e. unheated nutritional composition, preferably unbaked nutritional composition), is a dough or a batter, preferably a dough. Preferably, said unheated nutritional composition comprises 10.0-70.0 % w/w, preferably 15.0-70.0 % w/w, more preferably 20.0-70.0 % w/w, even more preferably 25.0-70.0 % w/w, even more preferably 30.0-70.0 % w/w, even more preferably 35.0-70.0 % w/w, even more preferably 35.0-60.0 % w/w, most preferably 40.0-60.0 % w/w, moisture; preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

In the context of the present invention, the term "dough" preferably refers to a mixture of (i) flour and/or meal and (ii) other ingredients such that said mixture is firm enough to knead or roll. Dough may be fresh or frozen, preferably fresh. The preparation of frozen dough is described in the book entitled "Frozen and Refrigerated doughs and batters" (Kulp et al, 1995, Frozen and refrigerated doughs and batters, Amer Assn of Cereal Chemists) which is incorporated by reference. The term "batter" preferably refers to a mixture of (i) flour and/or meal and (ii) other ingredients such that said mixture is thinner and more liquid than dough. Batter is consequently not firm enough to knead (mixing is usually done by a hand mixer or electric mixer), in other words batter it is not malleable in contrast to dough.

In an additional and/or alternative more preferred embodiment, said unheated nutritional composition has a pH of 2.0-8.0, preferably 2.5-7.5, more preferably 2.5-7.0, even more preferably 3.0-7.0, even more preferably 3.0-6.5, most preferably 3.0-6.0.

In an even more preferred embodiment, said nutritional composition is a synthetic nutritional composition. In the context of the present invention, a "synthetic nutritional composition" refers to a composition which is artificially prepared and preferably refers to a nutritional 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 nutritional composition.

Nutritional composition ingredients

In a preferred embodiment of the first aspect of the invention, said nutritional composition according to the invention comprises flour and/or meal, preferably flour. "Flour" as understood by the skilled person is a powder which is obtained by grinding grains, roots, beans, nuts or seeds. "Meal" as understood by the skilled person is identical to flour, except that the particle size is slightly more coarse than that of flour. In a more preferred embodiment, said nutritional composition according to the invention comprises flour obtained from any one or more selected from the list consisting of wheat, buckwheat, barley, oat, spelt, rye, sorghum, maize, triticale, millet, teff, sunflower, sesame, linseed, amaranth, flaxseed, pumpkin, rapeseed, soybean, farro, kamut, quinoa, beans, peas, chick peas, nuts (e.g. cashew, almond, hazelnut and peanut) and rice; preferably any one or more selected from the list consisting of wheat, barley, rye, sorghum, sesame, millet, spelt, teff, maize, sunflower and rice; more preferably any one or more selected from the list consisting of wheat, barley, rye, sorghum, sesame and sunflower; even more preferably any one or more selected from the list consisting of wheat, barley and rye; even more preferably wheat and/or barley; most preferably wheat.

In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises a lipid ingredient, preferably any one or more selected from a list consisting of an oil, a fat and a fat substitute, more preferably an oil and/or a fat. Preferably, said oil is selected from the list consisting of sunflower oil, olive oil, corn oil, peanut oil, coconut oil, soy oil, safflower oil, palm oil, fish oil and rapeseed oil. Preferably, said fat is butter, margarine or shortening, more preferably said fat is butter or margarine, even more preferably said fat is butter.

In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises any one or more selected from the list consisting of an egg, egg white, egg yolk and egg replacer.

In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises a liquid, preferably milk and/or water, more preferably water. It is particularly preferred that said liquid is not milk. As described in the section "Nutritional composition", said nutritional composition preferably comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of said liquid. As described in the section "Nutritional composition", said nutritional composition before being heated at a temperature of 100°C or higher according to the invention (i.e. unheated nutritional composition, preferably unbaked nutritional composition), comprises 3.0-70.0 % w/w, preferably 3.0-60.0 % w/w, more preferably 3.0-50.0 % w/w, even more preferably 5.0-50.0 % w/w, most preferably 10.0-50.0 % w/w, of said liquid.

In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises sugar, preferably sugar which is different from the milk saccharide(s), preferably milk oligosaccharide(s), according to the invention, and/or a sweetener. In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises one or more food and/or feed ingredients, preferably one or more functional ingredients, more preferably selected from the list consisting of salt, salty-like ingredient, sugar, sweetener, ethanol, bicarbonate, baking powder, diacetyl, natural aromas ingredient, synthetic aromas ingredient, flavor ingredient, flavor improvement ingredient, enzyme, oxidizing agent, reducing agent, emulsifier, vitamin, coloring agent, antimicrobial agent and food-grade microorganism.

In the context of the present invention, a "functional ingredient" refers to an ingredient which provides a desired function to the nutritional composition according to the invention. Such function can be taste (e.g. addition of salt, salty-like ingredient, sugar, sweetener, natural/synthetic aromas ingredient, diacetyl, flavor ingredient and flavor improvement ingredient), appearance (e.g. coloring agent), retention of flavor (natural/synthetic aromas ingredient, flavor ingredient and especially flavor improvement ingredient), shelf life (e.g. antimicrobial agent), stability (e.g. enzyme), structure/texture (e.g. bicarbonate, emulsifier, food-grade microorganism, enzyme), leavening (e.g. food-grade microorganism) and health (e.g. foodgrade microorganism) among other functions as known by the skilled person.

Throughout the application and claims, unless specifically stated otherwise, the term "food and/or feed ingredients" is preferably replaced with the term "functional ingredients".

In an additional and/or alternative more preferred embodiment, said nutritional composition according to the invention comprises a leavening agent, preferably chosen from the list consisting of baking soda, baking powder, whipped egg white, cream and a food-grade microorganism, preferably chosen from the list consisting of baking soda, baking powder and a food-grade microorganism, more preferably a foodgrade microorganism. Preferably, throughout the application and claims, said food-grade microorganism is a yeast.

The nutritional composition according to the invention comprises (i) 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 (it is referred to the section "milk saccharide"), preferably 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, and (ii) one or more conventional ingredients, for example the ingredients described in the present section. The skilled person understands that the choice of conventional ingredients (and their (relative) quantities) depends on and varies between the desired nutritional composition. Depending on the desired nutritional composition and its properties, the skilled person will be able to routinely determine the appropriate ingredients to obtain said nutritional composition.

Milk saccharide

In an embodiment of the first aspect of the invention, said nutritional composition 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. Preferably, said nutritional composition 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.

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.

In the context of the present invention, the term "milk saccharide" refers to a saccharide which is found in milk of an animal, preferably a 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 nutritional composition 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.

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. Preferably, said oligosaccharide according to the invention is not lacto-N-triose II. 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).

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, more preferably a mammalian milk oligosaccharide (MMO). Preferably, said milk saccharide (preferably 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 milk oligosaccharides) in said mixture is/are a human milk saccharide (preferably a human milk oligosaccharide (HMO)). Hence, it is particularly preferred that said nutritional composition according to the invention comprises a human milk oligosaccharide or a mixture of at least two , preferably at least three, more preferably at least 4, most preferably at least 5, different human milk oligosaccharides. 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".

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).

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 II, 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 milk saccharide (preferably said milk oligosaccharide) is not lactose and none of the milk saccharides (preferably said milk oligosaccharides) of said mixture according to the invention are lactose. Preferably, said milk saccharide (preferably oligosaccharide) is not a disaccharide and none of the milk saccharides (preferably oligosaccharides) of said mixture according to the invention are a disaccharide.

In an even more preferred embodiment of the first aspect of the invention, said milk saccharide (preferably 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 milk oligosaccharides) of said mixture according to the invention is/are (has/have been) isolated from a microbial cultivation or fermentation, cell culture, enzymatic reaction or chemical reaction, preferably isolated from a microbial cultivation or fermentation, or cell culture, more preferably isolated from a microbial cultivation or fermentation, most preferably isolated from a microbial fermentation.

In an additional and/or alternative even more preferred embodiment of the first aspect of the invention, said milk saccharide (preferably 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 milk oligosaccharides) of said mixture according to the invention is/are obtained from an in vitro and/or ex vivo culture of cells, wherein said cells are preferably 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 or a protozoan cell.

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 invention, it is also within the scope of the present invention that two or more different cells (preferably as defined herein), produce the milk saccharides of the mixture according to the invention, wherein each cell produces a different milk saccharide and/or a different mixture of milk saccharides. In an additional and/or alternative even more preferred embodiment of the first aspect of the invention, said milk saccharide (preferably 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 milk oligosaccharides) of 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).

In another additional and/or alternative more preferred embodiment, said milk saccharide (preferably 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 milk oligosaccharides) of 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 an even more preferred embodiment of the first aspect of the invention, said nutritional composition comprises a mammalian milk oligosaccharide or a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different mammalian milk oligosaccharides, wherein said mammalian milk oligosaccharide(s) or wherein at least one, preferably at least two, more preferably at least three, most preferably all, oligosaccharides of said mixture is/are selected from a list consisting of a neutral MMO and a sialylated MMO, preferably a sialylated MMO. Preferably, wherein said mammalian milk oligosaccharide(s) or wherein at least one, preferably at least two, more preferably at least three, most preferably all, oligosaccharides of said mixture is/are selected from a list consisting of lacto-N-triose II, 3-fucosyllactose, 2'-fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4- difucosyllactose, 6'-sialyllactose, 3'-sialyllactose, 3,6-disialyllactose, 6,6'-disialyllactose, 8,3- disialyllactose, 3,6-disialyllacto-N-tetraose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto- N-fucopentaose VI, sialyllacto-N-neotetraose d, sialyllacto-N-neotetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N-difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N- neohexaose, para-lacto-N-hexaose, monofucosylmonosialyllacto-N-neotetraose c, monofucosyl para- lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose III, isomeric fucosylated lacto-N-hexaose I, sialyllacto-N-hexaose, sialyllacto-N-neohexaose II, difucosyl-para-lacto-N- hexaose, difucosyllacto-N-hexaose, difucosyllacto-N-hexaose a and difucosyllacto-N-hexaose c; more preferably selected from a list consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'- sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N- fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, sialyllacto-N- neotetraose d, sialyllacto-N-neotetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, lacto-N- hexaose, lacto-N-neohexaose and para-lacto-N-hexaose; even more preferably selected from a list consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N- fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose and para-lacto-N-hexaose; even more preferably selected from a list consisting of 2'-fucosyllactose, 3- fucosyllactose, 2',3-difucosyllactose, 3'-sialyllactose, 6'-sialyllactose, lacto-N-tetraose and lacto-N- neotetraose; even more preferably selected from a list consisting of 2'-fucosyllactose, 3-fucosyllactose, 3'-sialyllactose and 6'-sialyllactose; most preferably selected from 3'-sialyllactose and 6'-sialyllactose.

In the context of the invention, a "neutral oligosaccharide" as used herein and as generally understood in the state of the art is an oligosaccharide that has no negative charge originating from a carboxylic acid group. A neutral MMO is preferably selected from a list consisting of lacto-N-triose II, 3-fucosyllactose, 2'- fucosyllactose, 6-fucosyllactose, 2',3-difucosyllactose, 2',2-difucosyllactose, 3,4-difucosyllactose, lactodifucotetraose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N- fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N- difucohexaose I, lacto-N-difucohexaose II, lacto-N-hexaose, lacto-N-neohexaose, para-lacto-N-hexaose, monofucosyl para-lacto-N-hexaose, monofucosyllacto-N-hexaose III, isomeric fucosylated lacto-N- hexaose III, isomeric fucosylated lacto-N-hexaose I, difucosyl-para-lacto-N-hexaose, difucosyllacto-N- hexaose, difucosyllacto-N-hexaose a and difucosyllacto-N-hexaose c; more preferably selected from a list consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-tetraose, lacto-N- neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N- fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N-neohexaose and para-lacto-N- hexaose; even more preferably selected from a list consisting of 2'-fucosyllactose, 3-fucosyllactose, 2',3- difucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose I, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-fucopentaose VI, lacto-N-hexaose, lacto-N- neohexaose and para-lacto-N-hexaose; even more preferably selected from a list consisting of 2'- fucosyllactose, 3-fucosyllactose, 2',3-difucosyllactose, lacto-N-tetraose and lacto-N-neotetraose; most preferably selected from a list consisting of 2'-fucosyllactose and 3-fucosyllactose.

A sialylated IVIMO is preferably selected from a list consisting of 6'-sialyllactose, 3'-sialyllactose, 3,6- disialyllactose, 6,6'-disialyllactose, 8,3-disialyllactose, 3,6-disialyllacto-N-tetraose, sialyllacto-N- neotetraose d, sialyllacto-N-neotetraose c, sialyllacto-N-tetraose b, sialyllacto-N-tetraose a, monofucosylmonosialyllacto-N-neotetraose c, sialyllacto-N-hexaose and sialyllacto-N-neohexaose II; more preferably selected from a list consisting of 3'-sialyllactose, 6'-sialyllactose, sialyllacto-N- neotetraose d, sialyllacto-N-neotetraose c, sialyllacto-N-tetraose b and sialyllacto-N-tetraose a; most preferably selected from 3'-sialyllactose and 6'-sialyllactose.

In an even more preferred embodiment, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to 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), even more preferably 0.001 to 1.0 % (w/w), most preferably 0.010 to 1.0 % (w/w), of the total weight, i.e. % (w/w), of said nutritional composition before being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to 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), even more preferably 0.001 to 1.0 % (w/w), most preferably 0.010 to 1.0% (w/w), of the total weight of the unheated nutritional composition (preferably unbaked nutritional composition). In other words, the nutritional composition before being heated according to the invention (i.e. unheated nutritional composition) comprises 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), even more preferably 0.001 to 1.0 % (w/w), most preferably 0.010 to 1.0 % (w/w), i.e. % (w/w), of said milk saccharide or said mixture of milk saccharides.

Preferably, said unheated nutritional composition is a dough or batter, preferably a dough.

Preferably, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 0.05 to 15.0 % (w/w), preferably 0.05 to 10.0 % (w/w), more preferably 0.05 to 5.0 % (w/w), even more preferably 0.05 to 3.0 % (w/w), even more preferably 0.05 to 1.0 % (w/w), most preferably 0.10 to 1.0 % (w/w), of the total weight, i.e. % (w/w), of said nutritional composition before being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 0.05 to 15.0 % (w/w), preferably 0.05 to 10.0 % (w/w), more preferably 0.05 to 5.0 % (w/w), even more preferably 0.05 to 3.0 % (w/w), even more preferably 0.05 to 1.0 % (w/w), most preferably 0.10 to 1.0 % (w/w), of the total weight of the unheated nutritional composition (preferably unbaked nutritional composition). In other words, the nutritional composition before being heated according to the invention (i.e. unheated nutritional composition) comprises 0.05 to 15.0 % (w/w), preferably 0.05 to 10.0 % (w/w), more preferably 0.05 to 5.0 % (w/w), even more preferably 0.05 to 3.0 % (w/w), even more preferably 0.05 to 1.0% (w/w), most preferably 0.10 to 1.0% (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative even more preferred embodiment, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 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), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of said nutritional composition before being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 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), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of the unheated nutritional composition (preferably unbaked nutritional composition). In other words, the total weight of dry matter of the nutritional composition before being heated according to the invention (i.e. unheated nutritional composition) comprises 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

Preferably, said unheated nutritional composition is a dough or batter, preferably a dough.

Preferably, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of the total weight of dry matter of said nutritional composition before being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 0.1 to 40.0% (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0% (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of the total weight of dry matter of the unheated nutritional composition (preferably unbaked nutritional composition). In other words, the total weight of dry matter of the nutritional composition before being heated according to the invention (i.e. unheated nutritional composition) comprises 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative even more preferred embodiment, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides constitutes 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), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of said nutritional composition after being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 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), most preferably 10.0 to 40.0 % (w/w), of the total weight of dry matter of the heated nutritional composition, preferably baked nutritional composition as described herein. In other words, the total weight of dry matter of the nutritional composition after being heated according to the invention (i.e. heated nutritional composition) comprises 0.01 to 60.0 %, 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

Preferably, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides constitutes 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of the total weight of dry matter of said nutritional composition after being heated at a temperature of 100°C or higher according to the invention as described herein. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) according to the invention, constitutes 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of the total weight of dry matter of the heated nutritional composition, preferably baked nutritional composition as described herein. In other words, the total weight of dry matter of the nutritional composition after being heated according to the invention (i.e. heated nutritional composition) comprises 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative even more preferred embodiment, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) in said nutritional composition after being heated at a temperature of 100°C or higher according to the invention, constitutes at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said milk oligosaccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) in said nutritional composition before being heated at said temperature of 100°C or higher according to the invention. In other words, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) in said heated nutritional composition according to the invention, comprises at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) in the unheated nutritional composition (preferably unbaked nutritional composition). Preferably, said unheated nutritional composition is a dough or batter, preferably a dough.

Heating

In an embodiment of the first aspect of the invention, said nutritional composition 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, wherein said nutritional composition was heated at a temperature of 100°C or higher. Said "milk saccharide" and "milk saccharides" are as described in the section "milk saccharide".

Pasteurization is a treatment using mild heat in order to eliminate pathogens and hence extend the shelf life of a product. As pasteurization typically occurs at a temperature which is lower than 100°C, it is not encompassed by the scope of the present invention. Hence, in a preferred embodiment of the present invention, said heating at a temperature of 100°C or higher does not encompass pasteurization.

In a preferred embodiment of the invention, said nutritional composition was heated at a temperature of at least 100°C, preferably at least 125°C, more preferably at least 135°C, even more preferably at least 150°C, even more preferably at least 160°C, even more preferably at least 170°C, most preferably at least 180°C.

In an additional and/or alternative preferred embodiment, said nutritional composition was heated at a temperature of 225°C or lower, preferably at 220°C or lower.

In an additional and/or alternative preferred embodiment of the invention, said nutritional composition was heated at a temperature of 100-225°C, preferably 125-225°C, more preferably 135-225°C, even more preferably 150-225°C, even more preferably 160-225°C, even more preferably 170-225°C, even more preferably 180-225°C, most preferably 180-220°C.

Various methods and techniques are known to the skilled person for heating a nutritional composition as described thoroughly in the book entitled "Food processing technology: principles and practice" (Fellows; second edition; Woodhead Publishing Limited; 2000). It is particularly referred to part III - "Processing by application of heat" (p. 229-384) hereof, which is incorporated by reference. A preferred heating method comprises baking and/or extrusion. Said extrusion can be dry extrusion or wet extrusion (wherein steam injection is applied), preferably said extrusion is dry extrusion. Preferably, said extrusion is not wet extrusion. A more preferred heating method comprises baking. As understood by the skilled person, baking of a nutritional composition can be achieved by subjecting it to heat (e.g. hot air), preferably dry heat. Alternatively, the nutritional composition is steam-heated. In the context of the present invention, it is preferred that said nutritional composition according to the invention was heated by baking, more preferably by subjecting the nutritional composition to heat, preferably dry heat.

It is preferred to heat the nutritional composition according to the invention 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. As the skilled person understands, the feature "oven" as applied in the field and the present application, does not encompass a barrel (which can be heated/cooled) used during extrusion.

In an additional and/or alternative preferred embodiment of the invention, said nutritional composition was heated 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. In an additional and/or alternative preferred embodiment of the invention, said nutritional composition was heated 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 skilled person understands that the preferred heat and duration will vary depending upon the particular nutritional composition, preferably baked 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 heated until the desired doneness has been reached, more preferably heated until said nutritional composition is cooked.

In a more preferred embodiment of the invention, said nutritional composition according to the invention is obtainable by a method according to the second aspect of the invention.

Method

In a second aspect, the invention provides a method for the production of a heated nutritional composition, said method comprising the steps of: providing a solution or a powder, preferably a powder, comprising a milk saccharide (preferably a milk oligosaccharide) or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides (preferably milk oligosaccharides), providing one or more food and/or feed ingredients, preferably one or more functional ingredients, mixing said solution or powder with said food and/or feed ingredient(s) to obtain a mixture, preferably a homogenous mixture, and heating said obtained mixture at a temperature of 100°C or higher, preferably until said mixture has reached the desired doneness, more preferably until said mixture is cooked.

Preferably, said nutritional composition is according to the first aspect of the present invention (it is referred to the section "Nutritional composition"). Throughout the application and claims, the term "cooked" is preferably replaced with "baked".

In the context of the present invention, said "milk saccharide", "milk oligosaccharide", "milk saccharides", "milk oligosaccharides", "food ingredient", "feed ingredient", "functional ingredient", "heating" as described throughout the second aspect of the invention and claims are preferably as described in the first aspect of the invention.

Preferably, said solution comprising a milk saccharide (preferably a milk oligosaccharide) or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides (preferably milk oligosaccharides) is not milk. Preferably, said solution is water.

Preferably, said powder comprising a milk saccharide (preferably a milk oligosaccharide) or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides (preferably milk oligosaccharides) is not milk powder.

Preferably, the mixture obtained by mixing said solution or powder with said food and/or feed ingredients is a dough or batter, preferably a dough. In case of a dough, said mixing is preferably done by kneading.

Optionally, said method according to the invention further comprises a step wherein the obtained mixture, preferably said homogenous mixture, is provided a desired shape. For example, the obtained dough can optionally be divided in small portions and each small portion is subsequently formed into the shape of a biscuit.

Optionally, said method according to the invention further comprises a leavening step, said step is preferably conducted after obtaining said mixture, preferably said homogenous mixture. In this context, a leavening agent (preferably as described in the first aspect of the invention) is mixed together with the solution or powder comprising said saccharide(s) and one or more food and/or feed ingredients. As understood by the skilled person, leavening causes the mixture (preferably homogenous mixture) to expand by gas which is released when a leavening agent comes into contact with a liquid, acid or heat.

Optionally, said method according to the invention further comprises a drying step, preferably at a temperature > 50°C and/or preferably < 100°C, more preferably < 80°C, even more preferably < 70 °C, most preferably < 60°C. Preferably, said drying step is conducted in an oven. In an embodiment, said drying step occurs after obtaining said heated mixture. In an additional and/or alternative embodiment, said drying step occurs before heating said mixture.

In a preferred embodiment of the invention, said heating is as described in the section "Heating" of the first aspect of the invention. Preferably, heating said mixture comprises baking and/or extrusion. Said extrusion can be dry extrusion or wet extrusion (wherein steam injection is applied), preferably said extrusion is dry extrusion. More preferably, heating said mixture comprises baking, preferably in an oven. Accordingly, such a method results in the production of a baked nutritional composition, preferably a baked nutritional composition as described in the first aspect of the present invention (it is referred to the section "Nutritional composition").

In an additional and/or alternative preferred embodiment of the invention, said food and/or feed ingredient(s) is as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

In an additional and/or alternative preferred embodiment of the invention, said mixture obtained by mixing said solution or powder with said food and/or feed ingredient(s) comprises 3.0-70.0 % w/w, preferably 3.0-60.0 % w/w, more preferably 3.0-50.0 % w/w, even more preferably 5.0-50.0 % w/w, of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water. Preferably, said mixture obtained by mixing said solution or powder with said food and/or feed ingredient(s) comprises 10.0-70.0 % w/w, preferably 15.0-70.0 % w/w, more preferably 20.0-70.0 % w/w, even more preferably 25.0-70.0 % w/w, even more preferably 30.0- 70.0 % w/w, even more preferably 35.0-70.0 % w/w, even more preferably 35.0-60.0 % w/w, most preferably 40.0-60.0 % w/w, moisture; preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

In an additional and/or alternative preferred embodiment of the invention, said heated nutritional composition according to the invention comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

In a more preferred embodiment of the invention, the method for the production of a heated nutritional composition, preferably a baked nutritional composition, comprises a step of providing flour and/or meal, preferably flour. As understood by the skilled person, said flour and/or meal is provided before mixing all ingredients to obtain a mixture. Said flour and said meal are preferably as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

In an additional and/or alternative more preferred embodiment of the invention, the method for the production of a heated nutritional composition, preferably a baked nutritional composition, comprises a step of providing a liquid. As understood by the skilled person, said liquid is provided before mixing all ingredients to obtain a mixture. Said liquid is preferably as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

In an additional and/or alternative more preferred embodiment of the invention, the method for the production of a heated nutritional composition, preferably a baked nutritional composition, comprises a step of providing one or more functional ingredients. As understood by the skilled person, said functional ingredient is provided before mixing all ingredients to obtain a mixture. Said functional ingredient is preferably as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

In an additional and/or alternative more preferred embodiment of the invention, the method for the production of a heated nutritional composition, preferably a baked nutritional composition, comprises a leavening step. Said step is preferably conducted after obtaining said mixture, preferably said homogenous mixture. In this context, a leavening agent (preferably as described in the first aspect of the invention) is mixed together with the solution or powder comprising said saccharide(s) and one or more food and/or feed ingredients. In an even more preferred embodiment, the invention provides a method for the production of a heated nutritional composition, said method comprising the steps of: providing a solution or a powder, preferably a powder, comprising a milk saccharide (preferably milk oligosaccharide) or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides (preferably milk oligosaccharides), providing flour and/or meal, preferably flour, providing a liquid, providing one or more food and/or feed ingredients, preferably one or more functional ingredients, mixing said solution or powder with all ingredients to obtain a mixture, preferably a homogenous mixture, optionally leaven said mixture, optionally providing a desired shape to said mixture, optionally leaven said mixture, and heating said obtained mixture at a temperature of 100°C or higher, preferably until said mixture has reached the desired doneness, more preferably until said mixture is cooked.

Optionally, said method according to the invention further comprises a drying step, preferably at a temperature > 50°C and/or preferably < 100°C, more preferably < 80°C, even more preferably < 70 °C, most preferably <60°C ). Preferably, said drying step is conducted in an oven. In an embodiment, said drying step occurs after obtaining said heated mixture. In an additional and/or alternative embodiment, said drying step occurs before heating said mixture.

Throughout the application and claims, unless specifically stated otherwise, the step of "providing one or more food and/or feed ingredients" is optional in the disclosed methods (in other words, said step can be omitted) if said methods comprise the step of "providing flour and/or meal, preferably flour" and/or the step of "providing a liquid".

Preferably, said nutritional composition is according to the first aspect of the present invention (it is referred to the section "Nutritional composition"). Preferably, said food and/or feed ingredients is as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

Preferably, said flour and said meal are as described in the section "Nutritional composition ingredients" of the first aspect of the invention. Preferably, said liquid is as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

In an even more preferred embodiment, the invention provides a method for the production of a baked nutritional composition, said method comprising the steps of: providing a solution or a powder, preferably a powder, comprising a mammalian milk oligosaccharide or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different mammalian milk oligosaccharides, providing flour and/or meal, preferably flour, providing a liquid, providing one or more food and/or feed ingredients, preferably one or more functional ingredients, mixing said solution or powder with all ingredients to obtain a mixture, preferably a homogenous mixture, optionally leaven said mixture, optionally providing a desired shape to said mixture, optionally leaven said mixture, and baking said obtained mixture at a temperature of 100°C or higher, preferably until said mixture has reached the desired doneness, more preferably until said mixture is baked.

Optionally, said method according to the invention further comprises a drying step, preferably at a temperature > 50°C and/or preferably < 100°C, more preferably < 80°C, even more preferably < 70 °C, most preferably < 60°C ). Preferably, said drying step is conducted in an oven. In an embodiment, said drying step occurs after obtaining said heated mixture. In an additional and/or alternative embodiment, said drying step occurs before heating said mixture.

Preferably, said nutritional composition is according to the first aspect of the present invention (it is referred to the section "Nutritional composition"). Preferably, said food and/or feed ingredients is as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

Preferably, said flour and said meal are as described in the section "Nutritional composition ingredients" of the first aspect of the invention. Preferably, said liquid is as described in the section "Nutritional composition ingredients" of the first aspect of the invention.

Preferably, the mixture obtained by mixing said solution or powder with the other ingredients (said flour and/or meal; said liquid and said food and/or feed ingredients) is a dough or batter, preferably a dough. In case of a dough, said mixing is preferably done by kneading.

Preferably, said heating is as described in the section "Heating" of the first aspect of the invention. Preferably, heating said mixture comprises baking and/or extrusion. Said extrusion can be dry extrusion or wet extrusion (wherein steam injection is applied), preferably said extrusion is dry extrusion. More preferably, heating said mixture comprises baking, preferably in an oven. Accordingly, such a method results in the production of a baked nutritional composition, preferably a baked nutritional composition as described in the first aspect of the present invention (it is referred to the section "Nutritional composition").

It is a preferred embodiment in the context of the second aspect of the invention that said obtained mixture (before heating/baking) comprises 0.001 to 15.0 %, preferably 0.001 to 10.0 %, more preferably 0.001 to 5.0 %, even more preferably 0.001 to 3.0 %, even more preferably 0.001 to 1.0 %, most preferably 0.010 to 1.0 %, i.e. % (w/w), of said milk saccharide or said mixture of milk saccharides. More preferably, said obtained mixture (before heating/baking) comprises 0.05 to 15.0 % (w/w), preferably 0.05 to 10.0 % (w/w), more preferably 0.05 to 5.0 % (w/w), even more preferably 0.05 to 3.0 % (w/w), even more preferably 0.05 to 1.0 % (w/w), most preferably 0.10 to 1.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative preferred embodiment, the total weight of dry matter of said obtained mixture (before heating/baking) comprises 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides. More preferably, the total weight of dry matter of said obtained mixture (before heating/baking) comprises 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative preferred embodiment, the total weight of dry matter of said heated (preferably baked) nutritional composition comprises 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides. More preferably, the total weight of dry matter of said heated (preferably baked) nutritional composition comprises 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

In an additional and/or alternative preferred embodiment, the amount of said milk saccharide (preferably milk oligosaccharide) or the amount of said mixture of at least two different milk saccharides (preferably milk oligosaccharides) in the obtained heated nutritional composition is at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said mammalian milk oligosaccharide or the amount of said mixture of at least two different mammalian milk oligosaccharides in the mixture before heating said mixture.

In an additional and/or alternative preferred embodiment, said mixture obtained by mixing said solution or powder with the other ingredients, comprises 3.0-70.0 % w/w, preferably 3.0-60.0 % w/w, more preferably 3.0-50.0 % w/w, even more preferably 5.0-50.0 % w/w, most preferably 10.0-50.0 % w/w, of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water. More preferably, said mixture obtained by mixing said solution or powder with the other ingredients, comprises 10.0-70.0 % w/w, preferably 15.0-70.0 % w/w, more preferably 20.0-70.0 % w/w, even more preferably 25.0-70.0 % w/w, even more preferably 30.0- 70.0 % w/w, even more preferably 35.0-70.0 % w/w, even more preferably 35.0-60.0 % w/w, most preferably 40.0-60.0 % w/w, of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

In an additional and/or alternative preferred embodiment, said heated nutritional composition comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

Solution or powder

In an embodiment of the second aspect of the invention, a solution or a powder, preferably a powder, comprising a milk saccharide (preferably 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 saccharides (preferably milk oligosaccharides) is provided.

In a preferred embodiment, said solution or powder comprising a milk saccharide (preferably 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 saccharides (preferably milk oligosaccharides), is typically isolated (preferably obtained) from a microbial cultivation or fermentation, cell culture, enzymatic reaction or chemical reaction, preferably isolated from a microbial cultivation or fermentation, or cell culture, more preferably isolated from a microbial cultivation or fermentation, most preferably isolated from a microbial fermentation, as described in the present application, resulting in a solution containing said milk saccharide or said mixture of different 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 different milk saccharides in a suitable cultivation medium to form a cultivation broth, preferably wherein said cell is metabolically engineered for the production of said milk saccharide or said mixture of milk saccharides, 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. In an additional and/or alternative preferred embodiment, said solution or powder comprising a milk saccharide (preferably 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 saccharides (preferably milk oligosaccharides), is obtained from an in vitro and/or ex vivo culture of cells, wherein said cells are preferably 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 or a protozoan cell.

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

In an additional and/or alternative preferred embodiment, said solution or powder comprising a milk saccharide (preferably 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 saccharides (preferably milk oligosaccharides), is 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).

In an additional and/or alternative preferred embodiment, said solution or powder comprising a milk saccharide (preferably 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 saccharides (preferably milk oligosaccharides), 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.

Several drying techniques are known to the skilled person which can be used to obtain a slurry (i.e. concentrated solution) or powder from a solution containing said milk saccharide or said mixture of at least two different milk saccharides. 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.

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 (preferably as described herein).

In another preferred embodiment, said milk saccharide and said mixture of different milk saccharides is as described in the section "Milk saccharide" of the first aspect of the invention.

Specific embodiments

The present invention preferably relates to the following specific embodiments:

1. A method for the production of a heated nutritional composition, preferably a baked nutritional composition, said method comprising the steps of: providing a solution or a powder, preferably a powder, comprising a milk saccharide, preferably milk oligosaccharide, or comprising a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different milk saccharides, preferably milk oligosaccharides, providing one or more food and/or feed ingredients, preferably one or more functional ingredients, mixing said solution or powder with said food and/or feed ingredients to obtain a mixture, preferably a homogenous mixture, and preferably wherein said mixture is a dough or batter, more preferably a dough, optionally leaven said mixture, optionally providing a desired shape to said mixture, optionally leaven said mixture, and heating, preferably comprising baking and/or extrusion, more preferably comprising baking, said mixture at a temperature of 100°C or higher preferably until said mixture has reached the desired doneness, more preferably until said mixture is cooked.

2. A method according to embodiment 1, wherein said milk saccharide or at least 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 has been isolated from a microbial cultivation or fermentation, cell culture, enzymatic reaction or chemical reaction, preferably has been isolated from a microbial cultivation or fermentation.

3. A method according to embodiment 1 or 2, wherein said milk saccharide or at least 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 obtained from an in vitro and/or ex vivo culture of cells, wherein said cells are preferably chosen from the list consisting of a microorganism, a plant cell, an animal cell or a protozoan cell, more preferably wherein said cells are a bacterium, a yeast or a fungus.

4. A method according to any one of embodiments 1 to 3, said method further comprises a drying step, preferably at a temperature > 50°C and/or preferably < 100°C, more preferably < 80°C, even more preferably < 70 °C, most preferably < 60°C.

5. A method according to any one of embodiments 1 to 4, wherein said heating is at a temperature of at least 125°C, preferably at least 135°C, more preferably at least 150°C, even more preferably at least 160°C, even more preferably at least 170°C, most preferably at least 180°C; and/or at a temperature of 225°C or lower, preferably at 220°C or lower.

6. A method according to any one of embodiments 1 to 5, wherein said heating comprises baking and/or extrusion, preferably baking and/or dry extrusion, more preferably baking.

7. A method according to any one of embodiments 1 to 6, wherein said heating 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 and/or 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.

8. A method according to any one of embodiments 1 to 7, wherein said method further comprises: providing flour and/or meal, preferably flour, providing a liquid, preferably water and/or milk, more preferably water, and/or providing one or more functional ingredients.

9. A method according to any one of embodiments 1 to 8, wherein said milk saccharide or at least 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 a milk oligosaccharide, preferably having a degree of polymerization of 2-20, more preferably 3-20, even more preferably 3-9, even more preferably 3-8, most preferably 3-7.

10. A method according to any one of embodiments 1 to 9, wherein said milk saccharide or at least 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 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- GIcNAc, betal,3-GalNAc and -betal,4-Glc; and (vi) Lewis-type antigen oligosaccharide.

11. A method according to any one of embodiments 1 to 10, wherein (i) said milk saccharide is not lactose, preferably not a disaccharide, and (ii) none of the milk saccharides of said mixture are lactose, preferably none of the milk saccharides of said mixture are a disaccharide.

12. A method according to any one of embodiments 1 to 11, wherein said milk saccharide and at least one, preferably at least two, more preferably at least three, most preferably all, oligosaccharides of said mixture is selected from a list consisting of a neutral mammalian milk oligosaccharide and a sialylated mammalian milk oligosaccharide, preferably a sialylated mammalian milk oligosaccharide.

13. A method according to any one of embodiments 1 to 12, wherein said obtained mixture comprises, preferably consists of, 3.0-70.0 % w/w, preferably 3.0-60.0 % w/w, more preferably 3.0-50.0 % w/w, even more preferably 5.0-50.0 % w/w, most preferably 10.0-50.0 % w/w, of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

14. A method according to any one of embodiments 1 to 13, wherein said obtained mixture has a pH of 2.0-8.0, preferably 2.5-7.5, more preferably 2.5-7.0, even more preferably 3.0-7.0, even more preferably 3.0-6.5, most preferably 3.0-6.0.

15. A method according to any one of embodiments 1 to 14, wherein said obtained mixture comprises, preferably consists of, 0.001 to 15.0 %, preferably 0.001 to 10.0 %, more preferably 0.001 to 5.0 %, even more preferably 0.001 to 3.0 %, even more preferably 0.001 to 1.0 %, most preferably 0.010 to 1.0 %, i.e. % (w/w), of said milk saccharide or said mixture of milk saccharides.

16. A method according to any one of embodiments 1 to 15, wherein the total weight of dry matter of said obtained mixture comprises, 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

17. A method according to any one of embodiments 1 to 16, wherein said heated nutritional composition comprises, preferably consists of, 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

18. A method according to any one of embodiments 1 to 17, wherein the total weight of dry matter of said heated nutritional composition comprises, 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

19. A method according to any one of embodiments 1 to 18, wherein the amount of said milk saccharide or the amount of said mixture of milk saccharides in said heated nutritional composition is at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said mammalian milk oligosaccharide or the amount of said mixture of milk saccharides in said obtained mixture.

20. A nutritional composition comprising a milk saccharide or mixture of at least two different milk saccharides, preferably a milk oligosaccharide or mixture of at least two different milk oligosaccharides, wherein said nutritional composition was heated at a temperature of 100°C or higher.

21. A nutritional composition according to embodiment 20 which is obtainable by a method according to any one of embodiments 1 to 19.

22. A nutritional composition according to embodiment 20 or 21, which is for human and/or animal consumption, preferably animal consumption.

23. A nutritional composition according to any one of embodiments 20 to 22, which is an animal food or animal feed, preferably a pet food or pet feed, more preferably a companion animal food or companion animal feed.

24. A nutritional composition according to any one of embodiments 20 to 23, wherein said nutritional composition is selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a bread, a cake, a pie, a tart, a pastry, a candy bar, an energy bar, granola, a granola bar, a quiche, cereal, a pizza, a corn chip, a tortilla chip, a potato chip and a baked cracker; preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a bread, a cake, a pie, a tart, a pastry, a candy bar, an energy bar, granola, a granola bar and cereal; more preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a pastry, a candy bar, an energy bar, granola, a granola bar and cereal; even more preferably selected from a list consisting of a cookie, pet food (preferably dry pet food), a pet treat, a candy bar, an energy bar, granola, a granola bar and cereal; even more preferably selected from a cookie, pet food (preferably dry pet food), a pet treat, an energy bar, granola, and a granola bar; even more preferably a cookie, pet food (preferably dry pet food) and pet treat, most preferably a cookie or pet food (preferably dry pet food).

25. A nutritional composition according to embodiment 24, wherein said nutritional composition is a pet food, preferably a dry pet food, more preferably a dry pet food for a companion animal.

26. A nutritional composition according to embodiment 25, wherein said pet food is selected from the list consisting of kibble, biscuit, meal and an extruded product, more preferably selected from kibble, biscuit and meal, even more preferably selected from kibble and biscuit.

27. A nutritional composition according to any one of embodiments 20 to 22, which is a treat or toy for a companion animal or is part of a treat or toy for a companion animal.

28. A nutritional composition according to any one of embodiments 20 to 27, wherein said nutritional composition is a baked nutritional composition.

29. A nutritional composition according to any one of embodiments 20 to 28, wherein said nutritional composition is not a liquid product.

30. A nutritional composition according to any one of embodiments 20 to 29, wherein said nutritional composition is not a powder.

31. A nutritional composition according to any one of embodiments 20 to 30, wherein said nutritional composition is not a milk replacer.

32. A nutritional composition according to any one of embodiments 20 to 31, wherein said nutritional composition comprises, preferably consists of, 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture, preferably wherein said moisture is liquid, more preferably said moisture is milk and/or water, even more preferably said moisture is water.

33. A nutritional composition according to any one of embodiments 20 to 32, wherein the total weight of dry matter of said nutritional composition comprises, 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), most preferably 10.0 to 40.0 % (w/w), of said milk saccharide or said mixture of milk saccharides.

34. A nutritional composition according to any one of embodiments 20 to 33, wherein the amount of said milk saccharide or the amount of said mixture of milk saccharides in said nutritional composition is at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said mammalian milk oligosaccharide or the amount of said mixture of milk saccharides in the nutritional composition before being heated.

35. A nutritional composition according to any one of embodiments 20 to 34, wherein said nutritional composition comprises: flour and/or meal, preferably flour, more preferably wherein said flour is obtained from any one or more selected from the list consisting of wheat, buckwheat, barley, oat, spelt, rye, sorghum, maize, triticale, millet, teff, sunflower, sesame, linseed, amaranth, flaxseed, pumpkin, rapeseed, soybean, farro, kamut, quinoa, beans, peas, chick peas, nuts (e.g. cashew, almond, hazelnut and peanut) and rice; even more preferably any one or more selected from the list consisting of wheat, barley, rye, sorghum, sesame, millet, spelt, teff, maize, sunflower and rice; even more preferably any one or more selected from the list consisting of wheat, barley, rye, sorghum, sesame and sunflower; even more preferably any one or more selected from the list consisting of wheat, barley and rye; even more preferably wheat and/or barley; most preferably wheat; a lipid ingredient, preferably any one or more selected from a list consisting of an oil, a fat and a fat substitute, more preferably an oil and/or a fat; any one or more selected from the list consisting of an egg, egg white, egg yolk and egg replacer; a liquid, preferably milk and/or water, more preferably water; a leavening agent, preferably selected from the list consisting of baking soda, baking powder, whipped egg white, cream and a food-grade microorganism, preferably selected from the list consisting of baking soda, baking powder and a food-grade microorganism, more preferably a food-grade microorganism, most preferably a yeast, and/or one or more functional ingredients, preferably selected from the list consisting of salt, salty- like ingredient, sugar, sweetener, ethanol, bicarbonate, baking powder, diacetyl, natural aromas ingredient, synthetic aromas ingredient, flavor ingredient, flavor improvement ingredient, enzyme, oxidizing agent, reducing agent, emulsifier, vitamin, coloring agent, antimicrobial agent and food-grade microorganism (preferably yeast).

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

1. A method for the production of a baked nutritional composition, said method comprising the steps of: providing a solution or a powder, preferably a powder, comprising a human milk oligosaccharide (HMO) or a mixture of at least two, preferably at least three, more preferably at least 4, most preferably at least 5, different human milk oligosaccharides (HMOs), providing one or more food and/or feed ingredients, preferably one or more functional ingredients, mixing said solution or powder with said food and/or feed ingredients to obtain a mixture, preferably a homogenous mixture, and preferably wherein said mixture is a dough or batter, more preferably a dough, optionally leaven said mixture, optionally providing a desired shape to said mixture, optionally leaven said mixture, and baking said obtained mixture at a temperature of at least 150°C until said mixture has reached the desired doneness, preferably until said mixture is cooked.

2. A method according to embodiment 1, wherein said solution is not milk and wherein said powder is not milk powder.

3. A method according to embodiment 1 or 2, wherein said solution or powder is obtained from an in vitro and/or ex vivo culture of cells, wherein said cells are preferably chosen from the list consisting of a microorganism, a plant cell, an animal cell or a protozoan cell, more preferably wherein said cells are a bacterium, a yeast or a fungus.

4. A method according to any one of embodiments 1 to 3, wherein said obtained mixture comprises 0.05 to 15.0 % (w/w), preferably 0.05 to 10.0 % (w/w), more preferably 0.05 to 5.0 % (w/w), even more preferably 0.05 to 3.0 % (w/w), even more preferably 0.05 to 1.0 % (w/w), most preferably 0.10 to 1.0 % (w/w), of said HMO or said mixture of HMOs.

5. A method according to any one of embodiments 1 to 4, wherein the total weight of dry matter of said obtained mixture comprises 0.1 to 40.0% (w/w), preferably 0.1 to 30.0% (w/w), more preferably 0.1 to 20.0 % (w/w), even more preferably 0.1 to 10.0 % (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said HMO or said mixture of HMOs.

6. A method according to any one of embodiments 1 to 5, wherein said baking is in an oven.

7. A method according to any one of embodiments 1 to 6, wherein said human milk oligosaccharide(s) has/have a degree of polymerization of 3-9.

8. A method according to any one of embodiments 1 to 7, wherein said obtained mixture comprises 15.0-70.0 % w/w, preferably 20.0-70.0 % w/w, more preferably 25.0-70.0 % w/w, even more preferably 30.0-70.0 % w/w, even more preferably 35.0-70.0 % w/w, even more preferably 35.0-60.0 % w/w, most preferably 40.0-60.0 % w/w, of moisture.

9. A method according to any one of embodiments 1 to 8, wherein said baked nutritional composition comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture.

10. A method according to any one of embodiments 1 to 9, said method further comprises a drying step, preferably at a temperature > 50°C and/or preferably < 100°C, more preferably < 80°C, even more preferably < 70 °C, most preferably < 60°C.

11. A method according to any one of embodiments 1 to 10, wherein 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 and/or 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.

12. A method according to any one of embodiments 1 to 11, wherein said method further comprises: providing flour and/or meal, preferably flour, providing a liquid, preferably water and/or milk, more preferably water, and/or providing one or more functional ingredients.

13. A method according to any one of embodiments 1 to 12, wherein said HMO and at least one, preferably at least two, more preferably at least three, most preferably all, HMOs of said mixture is selected from a list consisting of a neutral HMO and a sialylated HMO, preferably a sialylated HMO.

14. A method according to any one of embodiments 1 to 13, wherein said obtained mixture has a pH of 2.0-8.0, preferably 2.5-7.5, more preferably 2.5-7.0, even more preferably 3.0-7.0, even more preferably 3.0-6.5, most preferably 3.0-6.0.

15. A method according to any one of embodiments 1 to 17, wherein the total weight of dry matter of said baked nutritional composition comprises 0.1 to 40.0 % (w/w), preferably 0.1 to 30.0 % (w/w), more preferably 0.1 to 20.0% (w/w), even more preferably 0.1 to 10.0% (w/w), even more preferably 0.1 to 5.0 % (w/w), most preferably 0.2 to 5.0 % (w/w), of said milk HMO or said mixture of HMOs.

16. A method according to any one of embodiments 1 to 18, wherein the amount of said HMO or the amount of said mixture of HMOs in said heated nutritional composition is at least 85.0%, preferably at least 87.5%, more preferably at least 90.0%, even more preferably at least 92.0%, of the amount of said HMO or the amount of said mixture of HMOs in said obtained mixture.

17. A nutritional composition obtainable by a method according to any one of embodiments 1 to 16.

18. A nutritional composition according to embodiment 17, wherein said nutritional composition comprises 15.0 % (w/w) or less, preferably 13.0 % (w/w) or less, more preferably 10.0 % (w/w) or less, most preferably 8.0 % (w/w) or less of moisture.

19. A nutritional composition according to embodiment 17 or 18, which is an animal food or animal feed, preferably a pet food or pet feed, more preferably a companion animal food or companion animal feed.

20. A nutritional composition according to embodiment 17 or 18, which is a treat or toy for a companion animal or is part of a treat or toy for a companion animal.

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 terms "LNT II", "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. 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 water content of pre-baked and post-baked dog cookies/biscuits were determined using the Karl- Fisher method (Laboratorium ECCA N.V., Belgium).

The milk saccharide composition in pre-baking and post-baking dog cookies/biscuits 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 saccharides 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-baking and post-baking 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.

Example 2: Materials and Methods (production of dog cookies/biscuits by baking)

Dough batch and baking procedure

Oligosaccharides 3'SL and 6'SL (as prepared in Example 1) were mixed into 1 kg of dog food meal (moisture content of 5.5 % w/w) from United Petfood (major components shown in Table 1) at the inclusion ratio of 12 g oligosaccharides per kg dog food. Before subjecting the mixture to the baking process, said mixture was first homogenized for 15 min. A portion of 100 g of the homogenized mixture was preserved at -80°C for analytics ("pre-baked sample").

Table 1- Major components of dog food (United Petfood)

The homogenized dog food (860 g) was added to 640 mL water and mixed by hand to obtain a dough. The dough was shaped manually (approximately 10 g per cookie/biscuit) before baking for 11 min at 220°C in a pre-heated oven. Subsequently, the baked cookies/biscuits were dried in the oven for 50 min at 60°C. A portion of 100 g was preserved at -80°C for analytics ("post-baked sample").

Example 3: Production of dog cookies/biscuits by baking

Dog food cookies/biscuits (water content < 10.0 % w/w) containing spray-dried 3'SL and spray-dried 6'SL were prepared according to the baking procedure described in Example 2. The concentrations of 3'SL and 6'SL were determined before baking ("pre-baking") and after baking ("post-baking") and expressed as mg/g dried matter (it is referred to Example 1) as shown in Table 2.

Table 2 - Oligosaccharide concentration in pre- and post-baked dog food cookies/biscuits (mg/g dried matter)

As shown in Table 2, the retention of 3'SL and 6'SL, both being sialylated saccharides, within the dog food upon exposure to a temperature of 220°C during baking (while the moisture content before baking is high, i.e. 48.1 % w/w) is surprisingly more than 92 %.