Means and methods for regulating behavior

Abstract

The present invention relates to methods for using oligosaccharide preparations to modulate animal behavior, improve animal welfare, and increase the production of gamma-aminobutyric acid, kynurenine and / or serotonin to improve animal performance.

Description

Detailed Description of the Invention

[0001] The present invention relates to methods for using oligosaccharide preparations to modulate animal behavior, improve animal welfare, and increase the production of gamma-aminobutyric acid, kynurenine and / or serotonin to improve animal performance.

[0002]

[0002] Secondary metabolites are molecules produced by living organisms that exert their functions in the interaction of the producing organism with its environment. Some well-known secondary metabolites include caffeine, taxol, nicotine, artemisinin, morphine, and botulinum toxin. In particular, many secondary metabolites can serve multiple purposes. For example, caffeine is thought to be produced by plants as a pesticide, while humans consume it to increase alertness. Other secondary metabolites produced by one organism can act as neurotransmitters in another organism. Neurotransmitters are signaling molecules that enable communication between cells in an organism. Receptors for neurotransmitters can be present, for example, on muscle cells or neurons. Gamma-aminobutyric acid (GABA; CAS number 56-12-2; also known as 5-aminobutanoic acid) is known to be the major inhibitory neurotransmitter in the brain and spinal cord. GABA slows or blocks certain nerve signals in the brain, thereby regulating the levels of anxiety, fear, and / or stress perceived by humans and animals. Thus, imbalanced GABA activity in the body can lead to related disorders, such as anxiety disorders.

[0003]

[0003] Kynurenine (KYN) is known as a neuromodulator of stress. In particular, the ratio of KYN to tryptophan (TRP) has been found to be related to aggressive behaviors, such as feather pecking, in particular, and social impairment in laying hens in general. A lower KYN / TRP ratio has been found to be associated with a higher social impairment profile. It is hypothesized that an increase in the kynurenine / tryptophan ratio is involved in the reduction of socially impaired behaviors and, therefore, the improvement of animal welfare.

[0004]

[0004] Serotonin in the central nervous system cannot cross the blood-brain barrier, but tryptophan can. Therefore, higher intestinal tryptophan levels mean that more tryptophan can cross the blood-brain barrier and be converted to central serotonin there. Serotonin is a precursor to melatonin. Therefore, increased serotonin levels will cause increased melatonin levels. It is known that melatonin and its precursor, serotonin, can affect the production of insulin and glucagon. Increased melatonin concentrations can increase insulin and glucagon levels in animals. Increased insulin and glucagon levels are also known to increase fat synthesis. Both insulin and melatonin are also involved in controlling circadian rhythms. Changes in the light cycle affect the levels of insulin and melatonin produced by animals. Changes in insulin and melatonin levels in animals then control the animal's physiological response to changes in the light cycle. Poultry production in general, and broiler rearing processes, are now moving towards longer photoperiods of up to 23 hours per day. This lighting regimen has a strong impact on production performance, such as faster fat gain, but is detrimental to animal welfare.

[0005]

[0005] Tail biting (TB) is an abnormal pathological behavior frequently observed during the weaning and growing-finishing stages in commercial pig production and is therefore a major health and welfare problem due to the risk of infection, pain, and stress experienced by the bitten animal, as well as the additional stress experienced by the entire herd. Previous studies have shown that tail-biting pigs have different concentrations of neurotransmitters than pigs that do not exhibit this aggressive behavior. It is increasingly recognized that neurochemicals or neurotransmitters produced by either or both animals and / or their microbiome represent a common language for host-microbe communication. Microbial endocrinology has been described to play a role in animal behavior via the microbiota-gut-brain axis (MGBA) (Lyte et al. 2019. Animal. 13(11):2689-2698). The gut microbiota is known to be capable of synthesizing neuroactive molecules structurally similar to host-derived neurotransmitters, including gamma-aminobutyric acid (GABA), indole derivatives, and catecholamines such as dopamine and norepinephrine. Many of these co-synthesized neurotransmitters are involved in the regulation of host mood, behavior, and cognition.

[0006] The GABAergic system is widely distributed in the hippocampus of piglets and exerts an inhibitory effect on hippocampal neurons. Generally, GABA is synthesized by the decarboxylation of glutamate catalyzed by glutamic acid decarboxylase (GAD), is widely distributed in the nervous system, and reduces neuronal activity by binding to GABA receptors. Brain levels of GABA are low in mice and rats exhibiting aggressive behavior, which is interpreted as consistent with the proposed inhibitory role of GABA in aggression (Clement et al. 1987. Pharmacol Biochem Behav. 26(1):83-8). Furthermore, direct GABA supplementation can control stress, which is reflected in reduced aggressive behavior and improved growth performance. Furthermore, GABA supplementation can ameliorate transport stress in growing-fattening pigs (Bi et al. 2020. J Anim Physiol Anim Nutr. 104:590-596). However, direct exogenous delivery of GABA, in particular, has yielded conflicting results and has thus far been of limited efficacy, and the short half-life of GABA in the body (e.g., less than 17 minutes in mice) further reduces the efficacy and efficiency of direct GABA administration.

[0007]

[0007] Despite the clear relevance of such secondary metabolites to human and animal happiness, welfare, and performance, convenient and reliable means and methods for regulating the levels of these secondary metabolites in the body to regulate behavior and improve welfare, performance, and well-being have not yet been identified.

[0008]

[0008] In view of the prior art outlined above, the object of the present invention is to provide means and methods for regulating animal behavior, improving animal welfare, and improving animal performance in both non-stressed and stressed animals.

[0009] Surprisingly, the object is to provide a method for: i) modulating animal behaviour, in particular reducing aggressive behaviour; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing the cortisol concentration in the plasma, saliva and / or hair of animals experiencing stress, comprising: 1) a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or a) providing an oligosaccharide preparation produced or producible by condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; 2) feeding the oligosaccharide preparation to an animal; and 3) increasing the levels of gamma-aminobutyric acid, kynurenine, and / or serotonin in the animal's body, particularly in the gastrointestinal tract and / or blood of the animal.

[0010] In some embodiments, iv) the method according to the present invention for improving animal performance in stressed animals, in particular for improving average weight gain and / or reducing feed conversion ratio, relates to improving animal performance in stressed animals that are fed the oligosaccharide preparation compared to stressed animals that are not fed the oligosaccharide preparation. In some embodiments, v) the method according to the present invention for reducing cortisol levels in plasma, saliva and / or hair of stressed animals that are fed the oligosaccharide preparation relates to reducing cortisol in plasma, saliva and / or hair of stressed animals that are fed the oligosaccharide preparation compared to stressed animals that are not fed the oligosaccharide preparation.

[0011] In another aspect, the present invention provides a method for i) modulating animal behaviour, in particular reducing aggressive behaviour; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; and / or iv) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio in animals experiencing stress, comprising: 1) a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) 2) providing an oligosaccharide preparation by condensing at least a first sugar with at least a second sugar using a catalyst, the catalyst comprising methylphosphonic acid, thereby producing said oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; 2) feeding the oligosaccharide preparation to an animal; and 3) increasing the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, particularly in the gastrointestinal tract and / or blood of the animal.

[0012]

[0012] As used herein, the term "condense" or "condensation" should be interpreted as the step necessary to combine feed or starting sugars, e.g., simple sugars (monosaccharides), with one another to form larger sugars (disaccharides, oligosaccharides, or polysaccharides). For example, two monosaccharides can be combined with one another to form a disaccharide. Similarly, one monosaccharide can be condensed with a disaccharide to form a trisaccharide, etc. During such a condensation reaction, a water molecule is released. As used herein, condensation is initiated by applying heat to a mixture of feed sugars in the presence of a catalyst.

[0013] In some embodiments, the present invention provides a method for: i) modulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing cortisol levels in plasma, saliva and / or hair of animals experiencing stress, comprising: 1) using a catalyst to providing an oligosaccharide preparation produced or producible by condensing a first sugar with at least a second sugar, wherein the at least first sugar is N-acetylglucosamine, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; 2) feeding the oligosaccharide preparation to an animal; and 3) increasing the levels of gamma-aminobutyric acid, kynurenine, and / or serotonin in the animal, particularly in the gastrointestinal tract and / or blood of the animal.

[0014] In some embodiments, the present invention provides a method for: i) modulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing cortisol levels in the plasma, saliva and / or hair of animals experiencing stress, comprising: 1) using a catalyst the method comprising the steps of: 1) providing an oligosaccharide preparation produced or producible by condensing at least a first sugar with at least a second sugar using a catalyst that is methylphosphonic acid, wherein the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; 2) feeding the oligosaccharide preparation to an animal; and 3) increasing the levels of gamma-aminobutyric acid, kynurenine, and / or serotonin in the animal, particularly in the gastrointestinal tract and / or blood of the animal.

[0015] In some embodiments, the present invention provides a method for: i) modulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing cortisol levels in the plasma, saliva and / or hair of animals experiencing stress, comprising: 1) activating at least a first sugar using a catalyst; and (c) increasing the levels of gamma-aminobutyric acid, kynurenine, and / or serotonin in the animal's body, particularly in the gastrointestinal tract and / or blood of the animal.

[0016] In some embodiments, the at least second sugar in a) (using a catalyst to condense at least a first sugar with at least a second sugar, wherein the at least first sugar is N-acetylglucosamine) is also N-acetylglucosamine. In other embodiments, the at least second sugar in a) (using a catalyst to condense at least a first sugar with at least a second sugar, wherein the at least first sugar is N-acetylglucosamine) is selected from glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, and is preferably glucose.

[0017] In some embodiments, the at least first sugar and the at least second sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are not the same sugar as in a) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least a first sugar is N-acetylglucosamine). In some embodiments, the at least first sugar and the at least second sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are both glucose. In some embodiments, the at least first sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) is glucose, and the at least second sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably selected from arabinose, sucrose, xylose, mannose, lactose, and fructose.

[0018] In some embodiments, the at least first sugar and the at least second sugar of a) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose. In some embodiments, the at least first sugar and the at least second sugar of b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose. In some embodiments, methods according to the present invention comprise condensing at least a first sugar with at least a second sugar using a catalyst, preferably wherein the catalyst is methylphosphonic acid, the at least first sugar is N-acetylglucosamine, the at least second sugar is glucose, and the at least third sugar is selected from lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

[0019] In some embodiments, at least one of the sugars in a) is glucose, and the glucose is used in an amount of 50-99% (e.g., wt%) of all sugars used in the condensation step. In some embodiments, the at least second sugar is used in an amount of 1-50% (e.g., wt%) of all sugars used in the condensation step. In some embodiments, the at least third sugar is used in an amount of 1-50% (e.g., wt%) of all sugars used in the condensation step. In some embodiments, a) is carried out by condensing at least a first sugar with at least a second sugar and at least a third sugar using a catalyst, and the at least a first sugar, at least a second sugar, and at least a third sugar are used in a ratio of 50-99:1-50:1-50, preferably at least one of the at least a first sugar, at least a second sugar, and at least a third sugar is glucose.

[0020] In some embodiments of the method according to the present invention, at least one of the sugars in b) is glucose, and the glucose is used in an amount of 50-99% (e.g., wt%) of all sugars used in the condensation step. In some embodiments of the method according to the present invention, at least a second sugar is used in an amount of 1-50% (e.g., wt%) of all sugars used in the condensation step. In some embodiments of the method according to the present invention, at least a third sugar is used in an amount of 1-50% (e.g., wt%) of all sugars used in the condensation step. In some embodiments of the method according to the present invention, b) is carried out by condensing at least a first sugar with at least a second sugar and at least a third sugar using a catalyst, the catalyst being methylphosphonic acid, and the at least a first sugar, at least a second sugar, and at least a third sugar are used in a ratio of 50-99:1-50:1-50, preferably at least one of the at least a first sugar, at least a second sugar, and at least a third sugar is glucose.

[0021]

[0021] In another aspect, the present invention relates to a method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by a microbial population, the method comprising the steps of: 1) providing an oligosaccharide preparation produced or producible by: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid, wherein the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; and contacting a microbial population with the oligosaccharide preparation.

[0022]

[0022] In another aspect, the present invention relates to a method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by a microbial population, the method comprising the steps of: 1) providing an oligosaccharide preparation comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; and contacting a microbial population with the oligosaccharide preparation.

[0023]

[0023] This allows production of gamma-aminobutyric acid, kynurenine, and / or serotonin to be achieved independently of the animal host of the microbial population. In some instances, such methods can be used, for example, in screening tests for substances that can enhance or inhibit microbial production of gamma-aminobutyric acid, kynurenine, and / or serotonin.

[0024]

[0024] One skilled in the art will understand that contacting one or more cells (e.g., of a microbial community) with an oligosaccharide preparation will initiate production of gamma-aminobutyric acid, kynurenine and / or serotonin (e.g., by the microbial community), and the amount of gamma-aminobutyric acid, kynurenine and / or serotonin produced will increase with the ongoing incubation or fermentation time.

[0025] In some embodiments, the microorganisms are the gut microorganisms (i.e. those contained within or obtainable from a gut sample, for example from the cecum, ileum), preferably the gut microorganisms of an animal, for example the gut microorganisms of a pet animal, dog, cat, canary, guinea pig, hamster, rabbit, mouse, rat, deer, wild boar, zoo animal, horse, donkey, poultry, pig, ruminant, chicken, cow, sheep, goat, pig, piglet, turkey, aquaculture, fish, shrimp, prawn, crayfish, crab, oyster, mussel, bivalve, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chicken, pig, and ruminant such as cow.

[0026]

[0026] In another aspect, the present invention relates to a method for producing an oligosaccharide preparation for: i) modulating the behavior of an animal, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) increasing the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in an animal, in particular in the gastrointestinal tract and / or blood of the animal; and / or iv) reducing the cortisol concentration in the plasma, saliva and / or hair of an animal experiencing stress, the method comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid, thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0027]

[0027] In some embodiments, the present invention relates to a method for producing an oligosaccharide preparation for: i) regulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) increasing the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in an animal, in particular in the gastrointestinal tract and / or blood of the animal; and / or iv) reducing the cortisol concentration in the plasma, saliva and / or hair of an animal experiencing stress, the method comprising the step of condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine and the catalyst is methylphosphonic acid, thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0028]

[0028] In a further aspect, the present invention relates to an oligosaccharide preparation for: i) modulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress, and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio, in animals experiencing stress; and / or v) reducing cortisol levels in the plasma, saliva, and / or hair of animals experiencing stress, the oligosaccharide preparation being produced or producible by: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid; and the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0029]

[0029] In some embodiments, the present invention relates to an oligosaccharide preparation for: i) regulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing cortisol levels in the plasma, saliva and / or hair of animals experiencing stress, wherein the oligosaccharide preparation is produced or producible by condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine and the catalyst is methylphosphonic acid; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0030] In some embodiments, the at least second sugar in a) (using a catalyst to condense at least a first sugar with at least a second sugar, wherein the at least first sugar is N-acetylglucosamine) is also N-acetylglucosamine. In other embodiments, the at least second sugar in a) (using a catalyst to condense at least a first sugar with at least a second sugar, wherein the at least first sugar is N-acetylglucosamine) is selected from glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, and is preferably glucose.

[0031] In some embodiments of the oligosaccharide preparation, the at least first sugar and the at least second sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are not the same sugar as in a) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least a first sugar is N-acetylglucosamine). In some embodiments of the oligosaccharide preparation, the at least first sugar and the at least a second sugar in b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are both glucose. In some embodiments of the oligosaccharide preparation, the at least first sugar in b) (condensing the at least first sugar with the at least second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) is glucose, and the at least second sugar in b) (condensing the at least first sugar with the at least second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably selected from arabinose, sucrose, xylose, mannose, lactose, and fructose.

[0032] In some embodiments of the oligosaccharide preparation, the at least first and at least second sugars of a) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose. In some embodiments of the oligosaccharide preparation, b) (condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose. In some embodiments of the oligosaccharide preparation, the oligosaccharide preparation is produced or producible by condensing at least a first sugar with at least a second sugar using a catalyst, preferably wherein the catalyst is methylphosphonic acid, the at least first sugar is N-acetylglucosamine, the at least second sugar is glucose, and the at least third sugar is selected from lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

[0033]

[0033] In some aspects, the present invention relates to an oligosaccharide preparation for: i) regulating animal behavior, in particular reducing aggressive behavior; ii) improving animal welfare, in particular reducing anxiety, stress and / or fear disorders; iii) improving animal performance, in particular improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance in animals experiencing stress, in particular improving average weight gain and / or reducing feed conversion ratio; and / or v) reducing cortisol levels in the plasma, saliva and / or hair of animals experiencing stress, wherein the oligosaccharide preparation comprises at least two distinct monosaccharide units selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably selected from glucose and N-acetylglucosamine; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2. In some embodiments, the present invention relates to an oligosaccharide preparation for: i) modulating animal behavior, particularly reducing aggressive behavior; ii) improving animal welfare, particularly reducing anxiety, stress, and / or fear disorders; iii) improving animal performance, particularly improving average weight gain and / or reducing feed conversion ratio; iv) improving animal performance, particularly improving average weight gain and / or reducing feed conversion ratio, in animals experiencing stress; and / or v) reducing cortisol levels in plasma, saliva, and / or hair of animals experiencing stress, wherein the oligosaccharide preparation comprises at least two distinct monosaccharide units selected from glucose, N-acetylglucosamine, and at least a third distinct monosaccharide unit selected from lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0034]

[0034] In some embodiments, the oligosaccharide preparation according to the present invention comprises at least 10 wt% (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 wt%, etc.), preferably at least 20 wt%, of at least the first fraction.

[0035] In some embodiments, the oligosaccharide preparation according to the present invention is included in the nutritional composition at a content of at least 50 ppm (eg, at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

[0036]

[0036] In some embodiments, an oligosaccharide preparation according to the present invention comprises at least four fractions (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fractions, etc.), each having a distinct DP.

[0037] In some embodiments, an oligosaccharide preparation according to the invention comprises no more than three distinct monosaccharide units.

[0038] In some embodiments, an oligosaccharide preparation according to the invention comprises an anhydro-subunit-containing oligosaccharide in a relative abundance of less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%; and and / or each fraction of the oligosaccharide preparation comprises, by relative abundance, greater than 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydrosubunit-containing oligosaccharides.

[0039] In some embodiments, the oligosaccharide preparation according to the present invention has a saturation of 300-5000 g / mol (e.g., about 2000-2800 g / mol, 2100-2700 g / mol, 2200-2600 g / mol, 2300-2500 g / mol, or 2320-2420 g / mol), 500-5000 g / mol, 700-5000 g / mol, 500- 2000g / mol, 700~2000g / mol, 700~1500g / mol, 300~1500g / mol, 300~2000g / mol, 300~700g / mol , 400~1300g / mol, 400~1200g / mol, 400~1100g / mol, 500~1300g / mol, 500~1200g / mol, 500~110 and / or the oligosaccharide preparation has a weight-average molecular mass of 1000 to 2000 g / mol (e.g., 1100 to 1900 g / mol, 1200 to 1800 g / mol, 1300 to 1700 g / mol, 1400 to 1600 g / mol); , 300 to 1050 g / mol, 300 to 1020 g / mol, 300 to 1010 g / mol, 400 to 1050 g / mol, 400 to 1020 g / mol, 400 to 1010 g / mol, 500 to 1050 g / mol, 500 to 1020 g / mol, 500 to 1010 g / mol, or 1450 to 1550 g / mol).

[0040]

[0040] In further aspects, the present invention relates to the use of an oligosaccharide preparation according to the present invention and as referred to in the specification: i) for regulating animal behaviour, in particular for reducing aggressive behaviour; ii) for improving animal welfare, in particular for reducing anxiety, stress and / or fear disorders; iii) for improving animal performance, in particular for improving average weight gain and / or reducing feed conversion ratio; iv) for improving animal performance in animals facing stress, in particular for improving average weight gain and / or reducing feed conversion ratio; v) for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by the microbial population; and / or vi) for reducing cortisol levels in the plasma, saliva and / or hair of animals facing stress.

[0041]

[0041] In particular embodiments, the present invention relates to the use of an oligosaccharide preparation according to the present invention and as referred to in the specification: i) for regulating the behavior of healthy animals, in particular for reducing aggressive behavior; ii) for improving animal welfare in healthy animals, in particular for reducing anxiety, stress and / or fear disorders; iii) for improving animal performance in healthy animals, in particular for improving average weight gain and / or reducing feed conversion ratio; iv) for improving animal performance in animals experiencing stress, in particular for improving average weight gain and / or reducing feed conversion ratio in healthy animals experiencing stress; v) for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by the microbial population; and / or vi) for reducing cortisol levels in the plasma, saliva and / or hair of animals experiencing stress.

[0042]

[0042] In another aspect, the present invention relates to an oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid, kynurenine and / or cortisol, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation, and local inflammation, the oligosaccharide preparation being produced or producible by: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid; and the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0043]

[0043] In some embodiments, the present invention relates to an oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid, kynurenine and / or cortisol in an animal suffering from or at risk of suffering from one or more disorders associated with imbalanced levels of gamma-aminobutyric acid and / or kynurenine, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation, and local inflammation, and the oligosaccharide preparation is produced or producible by: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least the first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2. An animal suffering from one or more disorders associated with disproportionate levels of gamma-aminobutyric acid and / or kynurenine may be, for example, an animal (e.g., a pig) that is exposed to a situation that is likely to cause social anxiety in the animal, such as mixing of individuals between groups of animals. Similarly, for example, an animal about to be exposed to such a situation may be considered at risk for suffering from one or more disorders associated with disproportionate levels of gamma-aminobutyric acid and / or kynurenine.

[0044] In some embodiments, the oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis is an oligosaccharide preparation according to the present invention and described herein.

[0045]

[0045] In some embodiments, the present invention relates to an oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid, kynurenine and / or cortisol, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation, and local inflammation, and wherein the oligosaccharide preparation is included in a nutritional composition at a content of at least 50 ppm (e.g., at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

[0046] In some embodiments of the invention, the animals referred to herein are selected from the group consisting of pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, clams, trout, tilapia, salmon, carp, catfish, tuna, preferably the animals are selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows, preferably pigs.

[0047] It has been observed that the health, animal welfare and / or animal performance of the host animal are improved by the means, methods and uses of the present invention in four aspects. First, the welfare of the production animal herd is improved. In monogastric animals such as pigs, chickens or ducks kept in small spaces, it is a common problem for them to exhibit socially disruptive behaviors such as feather pecking or tail biting. Such disruptive behaviors cause poor welfare in the production animals and are therefore a persistent problem for animal breeders. The means, methods and uses according to the present invention help to improve animal welfare.

[0048]

[0048] Second, the health of the host animal can be improved by reducing the animal's systemic inflammation. Systemic inflammation is the result of the release of pro-inflammatory cytokines from immune-related cells and the chronic activation of the innate immune system. It contributes to the development of chronic disease states in animals. The method according to the present invention is useful for reducing the systemic inflammation in animals.

[0049]

[0049] Third, the health of a host animal can be improved by reducing local inflammation in the animal. Local inflammation occurs in the area affected by a harmful stimulus. Acute local inflammation develops within minutes or hours of the harmful stimulus, is short-lasting, and primarily involves the innate immune system. The method according to the present invention is useful for reducing local inflammation in an animal.

[0050] Fourth, the health of host animals can be improved by reducing the light regime / duration to the animal's daily circadian rhythm. Light-related circadian rhythms have a significant impact on the growth of production animals. In production animal breeding operations, one method for increasing growth rate and meat production is through extended lighting. In some extreme cases, poultry lighting is extended to 23 hours per day, with poultry being kept in the dark for only one hour per day. While such methods may increase productivity, they have adverse effects on animal health and welfare. It has been observed that the melatonin levels of chickens treated with 23 hours of light and 1 hour of darkness are reduced to less than half of the amount of melatonin in chickens treated with 16 hours of light and 8 hours of darkness. The means, methods, and uses according to the present invention increase the amount of melatonin and its precursor serotonin, and thus help restore melatonin levels in animals exposed to prolonged lighting. Since artificially prolonged light periods lead to abnormal behavior in poultry, such as aggressive interactions (tail biting, feather pecking, mobility / motility problems, etc.), restoring melatonin levels in such animals helps to improve their welfare. The inventors of the present application have discovered that by compensating melatonin production by applying the means, methods and uses according to the present invention, a stronger serotonergic flux goes into more melatonin, and therefore a reduction in the lighting regime and better animal welfare can be achieved.

[0051] The means, methods and uses of the present invention are particularly applicable to production animals in general and may be provided to any suitable animal. In some embodiments, the animal is monogastric. A monogastric animal is generally understood to have a single-chambered stomach. In other embodiments, the animal is a ruminant. A ruminant is generally understood to have a multi-chambered stomach. In some embodiments, the animal is a pre-ruminating ruminant. An example of such a pre-ruminating ruminant is a young cow.

[0052]

[0052] In some embodiments, the animal is poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cow, bovine, buffalo, bison, pig (e.g., young pig, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, or human.

[0053] In some embodiments, the animal is a livestock animal. In some embodiments, the animal is a companion animal. In some embodiments, the animal is poultry. Examples of poultry include chickens, ducks, turkeys, geese, quail, or Cornish game hens. In one variation, the animal is a chicken. In some embodiments, the poultry is a layer hen, a broiler chicken, or a turkey.

[0054] In other embodiments, the animal is a mammal, including, for example, a cow, pig, goat, sheep, deer, bison, rabbit, llama, mule, horse, reindeer, buffalo, yak, guinea pig, rat, mouse, alpaca, dog, or cat. In one variation, the animal is a cow. In another variation, the animal is a pig. In another variation, the animal is a sow.

[0055]

[0055] The present invention is further characterized by the following:

[0056]

[0056] Item 1: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of - a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0057]

[0057] Item 2: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0058]

[0058] Item 3: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0059]

[0059] Item 4: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of - providing an oligosaccharide preparation, a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0060]

[0060] Item 5: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of - providing an oligosaccharide preparation, a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0061]

[0061] Item 6: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) To improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio. The method of - providing an oligosaccharide preparation, b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - feeding the oligosaccharide preparation to the animal; - increasing the level of gamma-aminobutyric acid, kynurenine and / or serotonin in the animal, in particular in the gastrointestinal tract and / or blood of the animal; A method comprising:

[0062] Item 7: A method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g., gut microorganisms (i.e., contained in or obtainable from a gut sample, e.g., from the cecum, ileum), preferably the gut microorganisms of an animal, e.g., gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: - a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0063] Item 8: A method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g., gut microorganisms (i.e., those contained in or obtainable from a gut sample, for example from the cecum, ileum), preferably the gut microorganisms of an animal, for example gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0064] Item 9: A method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g., gut microorganisms (i.e., those contained in or obtainable from a gut sample, for example from the cecum, ileum), preferably the gut microorganisms of an animal, for example gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0065]

[0065] Item 10: Method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g. gut microorganisms (i.e. those contained in or obtainable from a gut sample, e.g. from the cecum, ileum), preferably the gut microorganisms of an animal, e.g. gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: - providing an oligosaccharide preparation, a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0066]

[0066] Item 11: A method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g. gut microorganisms (i.e. those contained in or obtainable from a gut sample, e.g. from the cecum, ileum), preferably the gut microorganisms of an animal, e.g. gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: - providing an oligosaccharide preparation, a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0067]

[0067] Item 12: Method for increasing the production of gamma-aminobutyric acid, kynurenine and / or serotonin by microorganisms (e.g. gut microorganisms (i.e. those contained in or obtainable from a gut sample, e.g. from the cecum, ileum), preferably the gut microorganisms of an animal, e.g. gut microorganisms from pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, tuna, preferably the gut microorganisms of an animal selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows), comprising: - providing an oligosaccharide preparation, b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation comprising: the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; - contacting the microbial population with an oligosaccharide preparation; A method comprising:

[0068]

[0068] Item 13: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. A method for producing an oligosaccharide preparation of a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0069]

[0069] Item 14: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. A method for producing an oligosaccharide preparation of a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0070]

[0070] Item 15: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal; 1. A method for producing an oligosaccharide preparation of b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0071]

[0071] Item 16: A method according to any of the preceding items, wherein the at least second sugar in step a) is selected from N-acetylglucosamine, glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably glucose.

[0072]

[0072] Item 17: A method according to any of the preceding items, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

[0073]

[0073] Item 18: A method according to any of the preceding items, wherein at least the first sugar in step a) and at least the second sugar in step a) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose.

[0074]

[0074] Item 19: A method according to any of the preceding items, wherein at least the first sugar in step b) and at least the second sugar in step b) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose.

[0075] Item 20: The method according to any of the preceding items, wherein the at least second sugar in step a) is glucose and the catalyst is selected from (+)-camphor-10-sulfonic acid and methylphosphonic acid.

[0076]

[0076] Item 21: A method according to any one of items 1 to 15, wherein at least the second sugar in step a) is glucose, and at least the third sugar in step a) is selected from lactose, mannose, arabinose, fructose, sucrose, and xylose, and optionally the catalyst is selected from (+)-camphor-10-sulfonic acid, methylphosphonic acid, and phosphoric acid.

[0077]

[0077] Item 22: A method according to any one of items 1 to 15, wherein at least the second sugar in step a) is glucose, at least the third sugar is lactose, and the catalyst is (+)-camphor-10-sulfonic acid.

[0078]

[0078] Item 23: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is lactose, and the catalyst is methylphosphonic acid.

[0079]

[0079] Item 24: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is mannose, and the catalyst is methylphosphonic acid.

[0080]

[0080] Item 25: A method according to any one of items 1 to 15, wherein at least the second sugar in step a) is glucose, at least the third sugar is arabinose, and the catalyst is (+)-camphor-10-sulfonic acid.

[0081]

[0081] Item 26: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is arabinose, and the catalyst is methylphosphonic acid.

[0082]

[0082] Item 27: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is arabinose, and the catalyst is phosphoric acid.

[0083]

[0083] Item 28: A method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is fructose, and the catalyst is (+)-camphor-10-sulfonic acid.

[0084]

[0084] Item 29: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is fructose, and the catalyst is methylphosphonic acid.

[0085]

[0085] Item 30: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is fructose, and the catalyst is methylphosphonic acid.

[0086]

[0086] Item 31: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is fructose, and the catalyst is phosphoric acid.

[0087]

[0087] Item 32: A method according to any one of items 1 to 15, wherein at least the second sugar in step a) is glucose, at least the third sugar is sucrose, and the catalyst is (+)-camphor-10-sulfonic acid.

[0088]

[0088] Item 33: A method according to any one of items 1 to 15, wherein at least the second sugar in step a) is glucose, at least the third sugar is sucrose, and the catalyst is (+)-camphor-10-sulfonic acid.

[0089]

[0089] Item 34: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is sucrose, and the catalyst is methylphosphonic acid.

[0090]

[0090] Item 35: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is sucrose, and the catalyst is methylphosphonic acid.

[0091]

[0091] Item 36: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is sucrose, and the catalyst is methylphosphonic acid.

[0092]

[0092] Item 37: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is sucrose, and the catalyst is phosphoric acid.

[0093]

[0093] Item 38: The method according to any one of items 1 to 15, wherein the at least second sugar in step a) is glucose, the at least third sugar is xylose, and the catalyst is methylphosphonic acid.

[0094]

[0094] Item 39: The method according to any one of Items 1 to 15, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is arabinose.

[0095]

[0095] Item 40: The method according to any one of Items 1 to 15, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is lactose.

[0096]

[0096] Item 41: The method according to any one of Items 1 to 15, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is N-acetylglucosamine.

[0097]

[0097] Item 42: The method according to any one of Items 1 to 15, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is xylose.

[0098]

[0098] Item 43: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is arabinose.

[0099]

[0099] Item 44: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is arabinose.

[0100]

[0100] Item 45: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is xylose, and at least the third sugar in step b) is arabinose.

[0101]

[0101] Item 46: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is arabinose, and at least the third sugar in step b) is lactose.

[0102]

[0102] Item 47: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is lactose.

[0103]

[0103] Item 48: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is N-acetylglucosamine, and at least the third sugar in step b) is lactose.

[0104]

[0104] Item 49: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is lactose.

[0105]

[0105] Item 50: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is xylose, and at least the third sugar in step b) is lactose.

[0106]

[0106] Item 51: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is mannose.

[0107]

[0107] Item 52: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is N-acetylglucosamine, and at least the third sugar in step b) is mannose.

[0108]

[0108] Item 53: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is mannose.

[0109]

[0109] Item 54: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is xylose, and at least the third sugar in step b) is mannose.

[0110]

[0110] Item 55: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is arabinose, and at least the third sugar in step b) is N-acetylglucosamine.

[0111]

[0111] Item 56: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is N-acetylglucosamine.

[0112]

[0112] Item 57: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is N-acetylglucosamine.

[0113]

[0113] Item 58: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is N-acetylglucosamine.

[0114]

[0114] Item 59: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is N-acetylglucosamine.

[0115]

[0115] Item 60: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is N-acetylglucosamine.

[0116]

[0116] Item 61: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is xylose, and at least the third sugar in step b) is N-acetylglucosamine.

[0117]

[0117] Item 62: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is fructose, and at least the third sugar in step b) is xylose.

[0118]

[0118] Item 63: A method according to any one of items 1 to 15, wherein at least the first sugar in step b) is glucose, at least the second sugar in step b) is sucrose, and at least the third sugar in step b) is xylose.

[0119]

[0119] Item 64: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. An oligosaccharide preparation comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. is generated or generateable by; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0120]

[0120] Item 65: An oligosaccharide preparation according to Item 64, wherein the at least second sugar in step a) is selected from glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably glucose.

[0121]

[0121] Item 66: An oligosaccharide preparation according to Item 64 or 65, wherein at least the first sugar in step b) is glucose and at least the second sugar in step b) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

[0122]

[0122] Item 67: An oligosaccharide preparation according to any one of Items 64 to 66, wherein at least the first sugar in either step a) or b) and at least the second sugar in either step a) or b) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose.

[0123]

[0123] Item 68: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. An oligosaccharide preparation comprising: An oligosaccharide preparation comprising at least two distinct monosaccharide units selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably glucose and N-acetylglucosamine; and the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0124]

[0124] Item 69: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. An oligosaccharide preparation comprising: An oligosaccharide preparation comprising at least two distinct monosaccharide units selected from glucose and N-acetylglucosamine and at least a third distinct monosaccharide unit selected from lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose; and the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0125]

[0125] Item 70: An oligosaccharide preparation according to any one of items 64 to 69, wherein the oligosaccharide composition comprises at least 10 wt% (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 wt%, etc.), preferably at least 20 wt%, of at least the first fraction.

[0126]

[0126] Item 72: An oligosaccharide preparation according to any one of Items 64 to 71, wherein the oligosaccharide preparation is contained in the nutritional composition at a content of at least 50 ppm (e.g., at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

[0127]

[0127] Item 73: i) To regulate animal behavior, especially to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; and / or iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of an animal, in particular in the gastrointestinal tract and / or blood of the animal. 1. An oligosaccharide preparation comprising: 64. An oligosaccharide preparation produced or producible by the method according to any one of items 13 to 63.

[0128]

[0128] Item 74: The oligosaccharide preparation according to any one of Items 64 to 73, wherein the animal is selected from the group consisting of pet animals, dogs, cats, canaries, guinea pigs, hamsters, rabbits, mice, rats, deer, wild boars, zoo animals, horses, donkeys, poultry, pigs, ruminants, chickens, cows, sheep, goats, pigs, piglets, turkeys, aquaculture, fish, shrimp, prawns, crayfish, crabs, oysters, mussels, bivalves, trout, tilapia, salmon, carp, catfish, and tuna, preferably, the animal is selected from the group consisting of poultry such as chickens, pigs, and ruminants such as cows.

[0129]

[0129] Item 75: An oligosaccharide preparation according to any one of items 64 to 74, wherein the oligosaccharide preparation comprises at least four fractions (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fractions, etc.) each having a distinct DP.

[0130]

[0130] Item 76: An oligosaccharide preparation according to any one of items 64 to 75, wherein the oligosaccharide preparation comprises no more than three distinct monosaccharide units.

[0131] Item 77: At least one fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of anhydro-subunit-containing oligosaccharides by relative abundance; and / or each fraction of the oligosaccharide preparation 77. An oligosaccharide preparation according to any one of items 64 to 76, comprising greater than 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunit-containing oligosaccharides by relative abundance.

[0132] Item 78: The oligosaccharide preparation has a saturation of 300 to 5000 g / mol (e.g., about 2000 to 2800 g / mol, 2100 to 2700 g / mol, 2200 to 2600 g / mol, 2300 to 2500 g / mol, or 2320 to 2420 g / mol), 500 to 5000 g / mol, 700 to 5000 g / mol, 500 to 2000 g / mol, 700 to 2000 g / mol, or 800 to 8000 g / mol). 00g / mol, 700~1500g / mol, 300~1500g / mol, 300~2000g / mol, 300~700g / mol, 400~1300g / mol, 40 0~1200g / mol, 400~1100g / mol, 500~1300g / mol, 500~1200g / mol, 500~1100g / mol, 600~1300g / mol and / or the oligosaccharide preparation has a weight-average molecular mass of 1000 to 2000 g / mol (e.g., 1100 to 1900 g / mol, 1200 to 1800 g / mol, 1300 to 1700 g / mol, 1400 to 1600 g / mol, 300 to 1050 g / mol, 300 to 102 ... 78. The oligosaccharide preparation according to any one of items 64 to 77, having a number average molecular mass of 0 g / mol, 300 to 1010 g / mol, 400 to 1050 g / mol, 400 to 1020 g / mol, 400 to 1010 g / mol, 500 to 1050 g / mol, 500 to 1020 g / mol, 500 to 1010 g / mol, or 1450 to 1550 g / mol).

[0133]

[0133] Item 79: i) To regulate the behavior of animals (e.g., healthy animals), in particular to reduce aggressive behavior; ii) to improve animal welfare (e.g. in healthy animals), in particular to reduce anxiety, stress and / or fear disorders; iii) To improve animal performance (e.g. in healthy animals), in particular to improve average weight gain and / or reduce feed conversion ratio; iv) to improve animal performance in animals (e.g. healthy animals) facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or v) To increase the production of gamma-aminobutyric acid, kynurenine and / or serotonin by the microbial community 79. Use of an oligosaccharide preparation according to any one of items 64 to 78.

[0134] Item 80: An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid and / or kynurenine, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation, and local inflammation, and wherein the oligosaccharide preparation comprises: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. is generated or generateable by; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0135] Item 81: An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid and / or kynurenine in an animal suffering from or at risk of suffering from one or more disorders associated with imbalanced levels of gamma-aminobutyric acid and / or kynurenine, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation, and local inflammation, and the oligosaccharide preparation a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. is generated or generateable by; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

[0136]

[0136] Item 82: An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis according to Item 80 or 81, wherein the oligosaccharide preparation is an oligosaccharide preparation according to any one of Items 64 to 78.

[0137]

[0137] Item 83: An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis according to any one of items 80 to 82, wherein the oligosaccharide preparation is present in the nutritional composition at a content of at least 50 ppm (e.g., at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

[0138] It is also contemplated that the oligosaccharide preparation may be provided in the form of a powdered formulation comprising at least 20% (w / w) of the oligosaccharide preparation referred to herein, at least 25% (wt / wt) of a silica-based adsorbent (e.g., diatomaceous earth, amorphous precipitated silica) having an average particle size D of 3000 μm or less (e.g., 100-500, 200-500, 200-300 μm), and optionally 0-25% (wt / wt) of water and / or auxiliary substances (% based on the total weight of the powdered formulation). For example, such a powder formulation may contain 30-70% (wt / wt) of an oligosaccharide preparation as described herein, 30-70% (wt / wt) of a silica-based adsorbent (e.g., having an average particle size of at least 50 μm), and 0-21% (wt / wt) of water (% based on the total weight of the powder formulation). In some embodiments, the oligosaccharide preparation is formulated similarly to the protocol described in any one of Examples 22-26 and 33 of WO 2020 / 097458.

[0139] As used herein, an "oligosaccharide preparation" may include one or more monosaccharides or sugars, and / or anhydro-monosaccharides, and / or one or more compound molecules, e.g., disaccharides, trisaccharides, tetrasaccharides, or higher polymerized saccharides, containing two or more monosaccharide subunits linked by glycosidic bonds to form an oligosaccharide. "Oligosaccharide" may also refer to a compound molecule containing two or more monosaccharide subunits, in which at least one monosaccharide unit is replaced with an anhydro-subunit. "Oligosaccharides" may optionally be functionalized. As used herein, the term "oligosaccharide" encompasses all species of oligosaccharides, in which each of the monosaccharide subunits in an oligosaccharide is independently and optionally functionalized and / or replaced by its corresponding anhydro-monosaccharide subunit.

[0140] An "anhydro subunit" can be a monosaccharide or a product of reversible thermal dehydration of a monosaccharide subunit, or a sugar caramelization product. For example, an "anhydro subunit" can be an anhydro-monosaccharide, such as anhydro-glucose. As another example, an "anhydro subunit" can be linked to one or more regular monosaccharide subunits and / or one or more anhydro-monosaccharide subunits via glycosidic bonds.

[0141] Oligosaccharides can be characterized as containing two or more monosaccharide subunits linked by glycosidic bonds. In this regard, "gluco-oligosaccharide" can refer to a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds. "Gluco-oligosaccharide" can also refer to a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds, where at least one monosaccharide subunit is replaced by an anhydro-glucose subunit. Similarly, "galacto-oligosaccharide" can refer to a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. "Galacto-oligosaccharide" can also refer to a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, where at least one monosaccharide subunit is replaced by an anhydro-galactose subunit. Similarly, gluco-galactose-oligosaccharides can be compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. Gluco-galactose oligosaccharides can also refer to compounds in which at least one of the monosaccharide subunits is replaced with its respective anhydro-monosaccharide subunit. Gluco-galactose-xylo-oligosaccharides can refer to compounds produced by the condensation reaction of glucose, galactose, and xylose. Oligosaccharide preparations containing gluco-galactose-xylo-oligosaccharides can include gluco-galactose-oligosaccharides, gluco-xylo-oligosaccharides, galacto-xylo-oligosaccharides, gluco-galactose-xylo-oligosaccharides, and compounds containing one or more glucose monosaccharide subunits, one or more xylose monosaccharide subunits, and one or more galactose monosaccharide subunits linked by glycosidic bonds with these anhydro-monosaccharide subunits.

[0142] As used herein, the terms "monosaccharide unit" and "monosaccharide subunit" may be used interchangeably unless otherwise indicated. A "monosaccharide subunit" may refer to a monosaccharide monomer of an oligosaccharide. For oligosaccharides having a degree of polymerization (DP) of 1, the oligosaccharide may be referred to as a monosaccharide subunit or a monosaccharide. For oligosaccharides having a degree of polymerization greater than 1, the monosaccharide subunits are linked via glycosidic bonds.

[0143] As used herein, the term "normal monosaccharide" can refer to a monosaccharide that does not contain an anhydro subunit. The term "normal disaccharide" can refer to a disaccharide that does not contain an anhydro subunit. Thus, the term "normal subunit" can refer to a subunit that is not an anhydro subunit.

[0144]

[0144] As used herein, the terms "relative abundance" or "abundance" can refer to the abundance of a species in terms of how common or rare the species is. For example, a DP1 fraction containing 10% anhydro-subunit-containing oligosaccharides in relative abundance (i.e., a fraction with a DP of 1) can refer to a plurality of DP1 oligosaccharides, where 10% (by number) of the DP1 oligosaccharides are anhydro-monosaccharides.

[0145] Degree of polymerization (DP) distribution: The distribution of the degree of polymerization of an oligosaccharide preparation can be determined by any suitable analytical method and instrument, including, but not limited to, end-group analysis, osmotic pressure (osmometry), ultracentrifugation, viscometry, light scattering, size exclusion chromatography (SEC), SEC-MALLS, field-flow fractionation (FFF), asymmetric flow field-flow fractionation (A4F), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). For example, the distribution of the degree of polymerization can be determined and / or detected by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. For another example, the distribution of the degree of polymerization can be determined and / or detected by SEC, such as gel permeation chromatography (GPC). As yet another example, the distribution of the degree of polymerization can be determined and / or detected by HPLC, FFF, or A4F. In another example, the degree of polymerization of an oligosaccharide preparation can be determined based on its molecular weight and molecular weight distribution (see WO 2020 / 097458 for a more detailed description).

[0146] Anhydro Subunit Levels: An oligosaccharide preparation can comprise n distinct fractions, each fraction having a distinct DP, where n is any integer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. In some embodiments, each of the n fractions of oligosaccharides of an oligosaccharide preparation described herein independently comprises an anhydro subunit level. For example, in some embodiments, the DP1 fraction comprises 10% anhydro subunit-containing oligosaccharides in relative abundance, and the DP2 fraction comprises 15% anhydro subunit-containing oligosaccharides in relative abundance. For another example, in some embodiments, the DP1, DP2, and DP3 fractions each comprise 5%, 10%, and 2% anhydro subunit-containing oligosaccharides in relative abundance, respectively. In other embodiments, two or more fractions of oligosaccharides may comprise similar levels of anhydro subunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each contain about 5% relative abundance of anhydro-subunit-containing oligosaccharides.

[0147] The level of anhydro subunits can be determined by any suitable analytical method, such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, HPLC, FFF, A4F, or any combination thereof. In some embodiments, the level of anhydro subunits is determined, at least in part, by mass spectrometry, such as MALDI-MS. In some embodiments, the level of anhydro subunits can be determined, at least in part, by NMR. In some embodiments, the level of anhydro subunits can be determined, at least in part, by HPLC. For example, in some embodiments, the level of anhydro subunits can be determined by MALDI-MS, as described in more detail in WO 2020 / 097458.

[0148] Glycosidic linkages: In some embodiments, the oligosaccharide preparations described herein comprise various glycosidic linkages. The type and distribution of glycosidic linkages can depend on the source and production method of the oligosaccharide preparation. In some embodiments, the type and distribution of various glycosidic linkages can be determined and / or detected by any suitable method known in the art, such as NMR. For example, in some embodiments, glycosidic linkages are determined and / or detected by proton NMR, carbon NMR, 2D NMR, such as 2D JRES, HSQC, HMBC, DOSY, COSY, ECOSY, TOCSY, NOESY, or ROESY, or any combination thereof. In some embodiments, glycosidic linkages are determined and / or detected, at least in part, by proton NMR. In some embodiments, glycosidic linkages are determined and / or detected, at least in part, by carbon NMR. In some embodiments, glycosidic linkages are determined and / or detected, at least in part, by 2D HSQC NMR.

[0149] In some embodiments, the oligosaccharide preparation comprises one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-β glycosidic linkages, or any combination thereof. may include:

[0150] In some embodiments, the oligosaccharide preparation has an α-(1,6) glycosidic bond content of about 0 to 60 mol%, 5 to 55 mol%, 5 to 50 mol%, 5 to 45 mol%, 5 to 40 mol%, 5 to 35 mol%, 5 to 30 mol%, 5 to 25 mol%, 10 to 60 mol%, 10 to 55 mol%, 10 to 50 mol%, 10 to 45 mol%, 10 to 40 mol%, 10 to 35 mol%, 15 to 60 mol%, 15 to 55 mol%, mol%, 15 to 50 mol%, 15 to 45 mol%, 15 to 40 mol%, 15 to 35 mol%, 20 to 60 mol%, 20 to 55 mol%, 20 to 50 mol%, 20 to 45 mol%, 20 to 40 mol%, 20 to 35 mol%, 25 to 60 mol%, 25 to 55 mol%, 25 to 50 mol%, 25 to 45 mol%, 25 to 40 mol%, or 25 to 35 mol%.

[0151] Molecular Weight: The molecular weight and molecular weight distribution of the oligosaccharide preparations can be determined by any suitable analytical means and instrumentation, such as end-group methods, osmotic pressure (osmometry), ultracentrifugation, viscometry, light scattering, SEC, SEC-MALLS, FFF, A4F, HPLC, and mass spectrometry. In some embodiments, the molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, the molecular weight and molecular weight distribution are determined by size exclusion chromatography (SEC), such as gel permeation chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined by HPLC. In some embodiments, the molecular weight and molecular weight distribution are determined by MALDI-MS.

[0152] In some embodiments, the oligosaccharide preparation has a saturation of 300-5000 g / mol (e.g., about 2000-2800 g / mol, 2100-2700 g / mol, 2200-2600 g / mol, 2300-2500 g / mol, or 2320-2420 g / mol), 500-5000 g / mol, 700-5000 g / mol, 500-2000 g / mol, or 500-2000 g / mol. g / mol, 700~2000g / mol, 700~1500g / mol, 300~1500g / mol, 300~2000g / mol, 300~700g / mol, 40 0~1300g / mol, 400~1200g / mol, 400~1100g / mol, 500~1300g / mol, 500~1200g / mol, 500~1100g / mol mol, 600-1300 g / mol, 600-1200 g / mol, or 600-1100 g / mol); and / or the oligosaccharide preparation has a weight-average molecular mass of 1000-2000 g / mol (e.g., 1100-1900 g / mol, 1200-1800 g / mol, 1300-1700 g / mol, 1400-1600 g / mol, 300-3100 g / mol, 3100-3200 g / mol, 3200-3300 g / mol, 3300-3400 g / mol, 3400-3500 g / mol, 3500-3600 g / mol, 3600-3700 g / mol, 3700-3800 g / mol, 3800-3900 g / mol, 3900-4000 g / mol, 400-4100 g / mol, 4100-4200 g / mol, 4200-4300 g / mol, 4300-4400 g / mol, 4400-4500 g / mol, 4500-4600 g / mol, 4600-4700 g / mol, 4700-4800 g / mol, 4800-4900 g / mol, 4900-5000 g / mol, 5000-5100 g / mol, 5100-5200 g / mol, 5200-5300 g / mol, 5300-5400 g / mol, 5400-5500 g / mol, 5500-5600 g / mol, 5600-5700 g / mol, The number average molecular mass of the copolymer is 00 to 1050 g / mol, 300 to 1020 g / mol, 300 to 1010 g / mol, 400 to 1050 g / mol, 400 to 1020 g / mol, 400 to 1010 g / mol, 500 to 1050 g / mol, 500 to 1020 g / mol, 500 to 1010 g / mol, or 1450 to 1550 g / mol).

[0153] Oligosaccharide species: In some embodiments, the species of oligosaccharides present in the oligosaccharide preparations referred to herein may depend on the type of feed sugar. For example, in some embodiments, if the feed sugar comprises glucose, the oligosaccharide preparation comprises gluco-oligosaccharides. For example, in some embodiments, if the feed sugar comprises fructose, the oligosaccharide preparation comprises fructose-oligosaccharides. For another example, in some embodiments, if the feed sugar comprises fructose and glucose, the oligosaccharide preparation comprises gluco-fructo-oligosaccharides.

[0154] In some embodiments, the oligosaccharide preparation comprises one or more species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation may comprise oligosaccharides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different species of monosaccharide subunits.

[0155] Methods for Producing Oligosaccharide Preparations: Oligosaccharide preparations can be produced by heating an aqueous composition comprising one or more feed sugars and a catalyst at a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinesulfonic acid; (β)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methyl Phosphonic acid;Phenylphosphinic acid;Phenylphosphonic acid;tert-Butylphosphonic acid;(SS)-VAPOL hydrogen phosphate;6-Quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate;2-(2-Pyridinyl)ethanesulfonic acid;3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate;1,1'-Binaphthyl-2,2'-diyl hydrogen phosphate;Bis(4-methoxyphenyl)phosphinic acid;Phenyl(3,5-xylyl)phosphinic acid; L-Cysteic acid monohydrate;Poly(styrenesulfonic acid-co-divinylbenzene);Lysine;Ethanedisulfonic acid;Ethanesulfonic acid;Isethionic acid;Homocysteic acid;HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid));HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid;Methanesulfonic acid;Methaniazid;Naphthalene-1-sulfonic acid;Naphthalene The amino acids selected from the group consisting of 2-chloro-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; triflic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; and tryptophan.

[0156] In some embodiments, polymerization of the feed sugar is achieved by step-growth polymerization. In some embodiments, polymerization of the feed sugar is achieved by polycondensation.

[0157] Feed sugars: The one or more feed sugars used in the methods for producing an oligosaccharide preparation described herein can comprise one or more types of sugars. In some embodiments, the one or more feed sugars comprise a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, or any mixture thereof. The feed sugar can be, for example, one or more selected from glucose, lactose, galactose, mannose, glucosamine, N-acetylglucosamine, arabinose, xylose, sucrose, and fructose.

[0158] As used herein, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "agent" includes a plurality of such agents, and reference to an "oligosaccharide" includes one or more oligosaccharides (or oligosaccharides) and equivalents thereof known to those skilled in the art.

[0159]

[0159] When ranges are used herein for physical properties such as molecular weight or chemical properties such as chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is approximate within experimental variation (or within statistical experimental error), and thus, in some cases, the numerical value or numerical range may vary by 1% to 15% of the stated numerical value or numerical range.

[0160]

[0160] In the following, the present invention will be further illustrated by non-limiting examples.

[0161] [Example]

[0161] The present invention as disclosed herein is not limited to the specific embodiments, diagrams, methodologies, examples, protocols, etc. described herein, but is defined only by the claims.

[0162] [Example 1] A library of synthetic oligosaccharide preparations was prepared using different combinations of feed sugars and catalysts. The feed sugars were selected from glucose, lactose, galactose, mannose, glucosamine, N-acetylglucosamine, arabinose, xylose, sucrose, and fructose. The catalysts were sulfuric acid, acetic acid, propionic acid, butanoic acid, L-glutamic acid, L-lysine, phosphoric acid, (+)-camphor-10-sulfonic acid, methylphosphonic acid, L-cysteic acid, hydrochloric acid, citric acid, lactic acid, 3-(1-pyridinio)-1-propanesulfonate, 2-pyridinesulfonic acid, 3-pyridinesulfonic acid, α-hydroxy-2-pyridinemethanesulfonic acid, (β)-camphor-10-sulfonic acid, butylphosphonic acid, and diphenylphosphine. The acid was selected from hexylphosphonic acid, phenylphosphinic acid, phenylphosphonic acid, tert-butylphosphonic acid, 2-(2-pyridinyl)ethanesulfonic acid, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate, 1,1'-binaphthyl-2,2'-diyl-hydrogen phosphate, bis(4-methoxyphenyl)phosphinic acid, and phenyl(3,5-xylyl)phosphinic acid.

[0163]

[0163] In principle, 10 g scale reactions were carried out by placing respective masses of powdered sugar (dry solids basis) into 20 mL scintillation vials, melting the contents by heating to 135°C with stirring, adding catalyst, and allowing oligomerization to occur by continuing heating to release water from the reaction vessel until adequate conversion is achieved, as indicated by an increase in viscosity, brown or amber color, for up to several hours. Table 1 below shows exemplary combinations of feed sugars and respective catalysts used to synthesize oligosaccharide preparations. The OPs listed in Table 1 below, in order, The sugars fed to IDs 1 to 61 were 98:2, 75:5:20, 85:5:10, 93:5:2, 97:3, 90:5:5, 90:5:5, 68:2:30, 90:5:5, 90:10, 65:5:30, 95:5, 90:5:5, 67:3:30, 95:5, 94:5:1, 95:5, 92:5:3, 90:5:5, 94:5:1, 90:10, 90:5:5, 90:5:5, 95:5, 95:5, 95:5, 85:5:10, 94:5:1, 85:5:10, 94:5:1, 85:5:10, 94:5:1, 94:5:10 ... :1:5, 92:5:3, 98:2, 85:5:10, 90:5:5, 90:5:5, 97:3, 95:5, 90:5:5, 75:5:20, 90:5:5, 98:2, 99:1, 94:5:1, 93:5:2, 85:5:10, 92:5:3, 90:5:5, 92:5:3, 92:5:3, 98:2, 99:1, 90:5:5, 88:2:10, 94:5:1, 92:5:3, 92:5:3, 90:5:5, 93:2:5, 85:5:10, and 94:5:1 ratios were used.

[0164] [Table 1]

[0165] [Table 2]

[0166] [Example 2] The amount of catalyst present in the resulting oligosaccharide preparation was quantified by anion chromatography. Analysis was performed on a Thermo-Fisher ICS-3000 ion chromatography system equipped with an EG-3000 eluent generator, an AS autosampler, and a CD-3000 conductivity detector. Separation was achieved on an AS19 column (4.0 mm × 250 mm) protected by an AG19 precolumn (4.0 mm × 50 mm). The mobile phase was a KOH gradient (0–55 mM) generated by an eluent generator (EGC II EluGen® cartridge). Chromatographic parameters were a flow rate of 1.0 mL / min, an injection volume of 25 μL, and a column temperature of 30°C. Calculations were based on peak areas. Quantitative results were obtained by comparing peak areas with a calibration curve of external standards. All applied catalysts could be quantified and were less than 0.1%, i.e., less than 1000 ppm, after the condensation reaction.

[0167] Samples of the oligosaccharide preparation prepared in Example 1 were further analyzed by gel permeation chromatography on an Agilent 1260 Infinity system containing a degasser, binary pump, and thermostatted column compartment equipped with an Agilent 1200 refractive index detector. The columns used were a PL aquagel-OH guard column, 7.5 × 50 mm, 5 μM (Agilent part number: PL1149-1530), and a PL aquagel-OH, 7.5 × 300 mm, 5 μM (Agilent part number: PL1120-6520). The solvent used was 200 mM aqueous sodium nitrate at a flow rate of 0.9 mL / min and a column temperature of 40°C. Both glucose (for quantification of total sugar content) and pullulan standards (for size distribution) were used as standards. The oligosaccharide preparations of OP IDs 50, 43, 57, and 54 were found to have DP1 contents (DP: degree of polymerization; DP1:1 degree of polymerization) of 29.5 wt%, 27.3 wt%, 11.6 wt%, and 50.5 wt%, respectively, and DP2+ contents (oligosaccharides with a degree of polymerization of 2 or more) of 42.2 wt%, 39.2 wt%, 45.0 wt%, and 22.1 wt%, respectively. Further analysis of OP ID 54 by liquid chromatography with an evaporative light scattering detector coupled to high-resolution mass spectrometry (LC-ELSD-HRMS) confirmed a DP1 content of 69.22 area%, a DP2 content of 24.64 area%, and a DP3+ content of 6.14 area%. Furthermore, the number-average molecular mass (Mn) and weight-average molecular mass (Mw) were determined. OP IDs 50, 43, 57, and 54 had Mn values ​​of 477, 574, 710, and 690 g / mol, respectively, and Mw values ​​of 578, 779, 1059, and 1008 g / mol, respectively. The higher Mn and Mw values ​​of OP ID 57 compared to the other OD IDs tested were consistent with the high DP2+ content of this oligosaccharide preparation. The total dry glycan contents were 71.7, 66.5, 56.6, and 72.6 wt%, respectively.

[0168]

[0166] Additionally, the refractive indices were determined at 78.9, 79.6, 77.0 and 73.6 Brix for the oligosaccharide preparations of OP IDs 50, 43, 57 and 54, respectively.

[0169] [Example 3] To test for increased GABA production by the porcine intestinal microbiome, the following assay was performed under anaerobic conditions. For this purpose, a minimal medium solution, a glycan solution (containing the tested oligosaccharide preparation), a feed extract solution, and a porcine cecal slurry (containing the intestinal microbiota) were prepared.

[0170] The minimal medium solution contained 900 mg / L sodium chloride, 26 mg / L calcium chloride dihydrate, 20 mg / L magnesium chloride hexahydrate, 40 mg / L ammonium sulfate, 300 mg / L potassium phosphate dibasic, 1500 mg / L sodium phosphate dibasic, 5000 mg / L sodium bicarbonate, 150 mg / L histidine, 75 mg / L glycine, 150 mg / L tryptophan, 150 mg / L arginine, 150 mg / L methionine, 150 mg / L triphosphate. It was prepared to contain leonine, 225 mg / L valine, 225 mg / L isoleucine, 300 mg / L leucine, 400 mg / L cysteine, 450 mg / L proline, 75 mg / L phenylalanine, 75 mg / L tyrosine, 75 mg / L glutamic acid, 10 mg / L manganese chloride solution, 4 mg / L ferrous sulfate solution, 1 mg / L cobalt chloride solution, 1 mg / L pyridoxine solution, 1 mg / L pantothenate solution, and 0.125 mg / L biotin solution.

[0171]

[0169] Glycan solutions were prepared by diluting each of the individual oligosaccharide preparations to be tested with distilled water to a concentration of 6 Brix. For this purpose, oligosaccharide preparations OP ID1-61 prepared in Example 1 were used.

[0172] A feed extract solution was prepared by mixing ground swine feed (detailed in Table 2) with 20 mL of distilled water, followed by incubation at 125° C. for 15 minutes. The mixture was then pelleted by centrifugation, and the supernatant was incubated at 121° C. for 15 minutes. Sterile distilled water was then added to a final volume of 20 mL.

[0173] [Table 3]

[0174]

[0171] Pig cecal slurry was prepared by mixing 3.8 g of cecal sample in 25 mL of PBS (phosphate buffered saline: 137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassium dihydrogen phosphate, pH 7.4). After large particles settled in the mixture, the unsettled supernatant was decanted into a new container. This supernatant was then centrifuged (500 rpm, 5 min). After centrifugation, the supernatant was collected and mixed with 50% glycerol to a final glycerol concentration of 15% (v / v), thus forming the pig cecal slurry.

[0175] To mimic the actual diversity of the pig cecal microbiota, three cecal samples containing different microbiome compositions were tested. As determined by metagenomic analysis, the cecal samples differed in diversity and microbial composition and ratios. Specifically, in terms of phylum, cecal sample 1 contained 48.0%, 33.3%, 15.4%, and 3.3% of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, respectively. Cecal sample 2 contained 54.9%, 27.2%, 15.3%, and 2.7% of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, respectively. Cecal sample 3 contained 67.0%, 20.4%, 10.4%, and 2.3% of Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteriae, respectively. In terms of genera, cecal sample 1 contained 15.1%, 8.5%, 14.6%, 5.2%, 3.8%, 3.6%, 4.2%, 23.4%, 6.2%, 11.1%, and 4.3% of Bacteroides, Blautia, Clostridium, Escherichia, Faecalibacterium, Lachnoclostridium, Phocaeicola, Prevotella, Roseburia, Streptococcus, and Turicibacter, respectively.Cecal sample 2 contained 10.8%, 5.5%, 3.7%, 12.3%, 6.0%, 10.3%, 4.9%, 17.9%, 4.4%, 9.9%, and 14.3% of Bacteroides, Blautia, Campylobacter, Clostridium, Escherichia, Lactobacillus, Limosilactobacillus, Prevotella, Roseburia, Sarcina, and Streptococcus, respectively. Cecal sample 3 contained 6.6%, 5.9%, 4.4%, 1.8%, 2.7%, 41.9%, 18.1%, 2.0%, 10.5%, 4.4%, and 1.8% of Bacteroides, Blautia, Clostridium, Faecalibacterium, Lachnoclostridium, Lactobacillus, Limosilactobacillus, Phocaeicola, Prevotella, Roseburia, and Ruminococcus, respectively.

[0176] GABA production by the microbiota was initiated by mixing 50 μL of porcine cecal slurry with 80 μL of feed extract solution, 70 μL of glycan solution, and 800 μL of minimal medium solution in a single well of a 96-deep-well plate. The glycan solution was varied by testing several different oligosaccharide preparations prepared in Example 1. Controls included a blank control in which the glycan solution was replaced with minimal medium solution, as well as a glucose control in which the glycan solution contained 1% (v / v) glucose instead of the oligosaccharide preparations mentioned herein. The glucose control was included to obtain a basal GABA response from the microbiome as a reference for calculating fold changes. Because glucose is largely absorbed in the small intestine before actually reaching the cecum, the 1% glucose control adequately mimicked the in vivo situation. The 96-deep-well plate was sealed with porous, breathable adhesive film and incubated anaerobically at 37°C for 24 hours. The 96 deep-well plates were then centrifuged to pellet the microbiota and undissolved material, and the supernatant was used for further analysis, particularly for determination of GABA and / or KYN production.

[0177] [Example 4] GABA was measured by liquid chromatography with tandem mass spectrometry (HPLC-MS / MS). The supernatants obtained from the in vitro fermentation in Example 3 were centrifuged. 200 μL of the supernatants were then transferred to a 96-well plate. The components contained in these supernatants were separated using an Acquity UPLC H-class system equipped with a TQDetector (Waters), a degasser, a quaternary pump, a sample manager, and a thermostatted column compartment. The columns used were a UPLC BEH C18 guard column, 2.1 × 50 mm, 1.7 μM (Waters Reference Number: 1866003975), and an Acquity UPLC BEH C18, 2.1 × 150 mm, 1.7 μM (Waters Reference Number: 176001048). The eluents were A 99.8% HO + 0.2% formic acid and B 99.8% ACN (LC-MS grade) + 0.2% formic acid. The flow rate was 0.3 mL / min and the column temperature was 40 °C. The autosampler was set at 15 °C and the run time was 7.5 min. The gradient profile is shown in Table 3.

[0178] [Table 4]

[0179] GABA powder was used as a standard with a calibration range of 3 to 0.0003 μg / mL. The chromatographic injection volume was 2 μL, and the syringe was washed after each injection. The MS method used was multiple reaction monitoring (MRM) with positive electrospray ionization. For GABA, the parent fragment m / z was 103.72 and the daughter fragment m / z was 85.85. The dwell time was 0.025 s, the cone voltage was 10 V, and the collision voltage was 13 V.

[0180] Kynurenine (KYN) quantification was performed using an ELISA kit (FI-EM1862; Euromedex) according to the supplier's recommendations. Briefly, 50 μL of standard, blank, or sample was added to 50 μL of biotin-labeled antibody standard solution and incubated at 37°C for 45 min. The plate was washed three times with wash buffer, and 100 μL of HRP-streptavidin conjugate was added to each well. The plate was incubated at 37°C for 30 min. The plate was then washed five times with wash buffer. 90 μL of TMB substrate was added to each well, followed by incubation at 37°C in the dark for 10–20 min. Then, 50 μL of stop solution was added to each well, and absorbance was measured at 450 nm and correlated with a standard curve of known KYN concentrations.

[0181] [Example 5] The synthetic oligosaccharide preparations produced in Example 1 were individually exposed to porcine microbiota during in vitro fermentation as described in Example 3 and analyzed as outlined in Example 4. The effect of contacting the microbiota with any one of the synthetic oligosaccharide preparations on GABA production was calculated as the log2 fold change in GABA produced compared to the GABA concentration obtained from the microbiota contacted with the glucose control (see Table 4). While some oligosaccharide preparations had no or only a slight effect on GABA production compared to the glucose control (e.g., OP IDs 1, 6, 14, 17, 21, 23, 24, 38, 41, 46, and 59), other oligosaccharide preparations were found to be able to induce microbial production of GABA significantly greater than the glucose control. In particular, oligosaccharide preparations containing N-acetylglucosamine were found to increase GABA production. Similarly, oligosaccharide preparations produced using methylphosphonic acid as a catalyst were found to increase microbial GABA production. In particular, oligosaccharide preparations produced from the same sugar feed but using catalysts other than methylphosphonic acid were found to have no effect on GABA or to have significantly less effect than their corresponding oligosaccharide preparations using methylphosphonic acid as the catalyst (e.g., compare OP IDs 30 and 58, 48 and 23, 10 and 21, 9 and 39, 15 and 25, and 26).

[0182] [Table 5]

[0183] [Example 6] To demonstrate the translatability of the in vitro results from the preceding examples to in vivo effects, a 14-day feeding study was conducted in pigs. To this end, five different treatments were tested, with 12 animals per treatment. The basal pig diet was used as a negative control. As a positive control, the same basal pig diet was supplemented with 30 g of GABA per ton of feed. Such 30 g / MT GABA supplementation has been reported to improve growth performance, improve stress-related parameters, and reduce the incidence of biting (Li et al. 2015. Can. J. Anim. Sci. 95:165-171; Bi et al. 2020. J Anim Physiol Anim Nutr. 104:590-596). Three test treatments were prepared by spraying one of the oligosaccharide preparations (OP IDs 50, 43, and 57) onto the basal pig diet as mash. The oligosaccharide preparation was supplied in syrup form at a concentration of 70 Brix and added to the diet at an inclusion level of 1000 mg / kg. For the negative and positive controls, the same volume of water was sprayed onto the basal pig diet as mash instead of the syrup of any of the test treatments.

[0184] The composition of the basal pig diet is shown in Table 5 below. The diet was given ad libitum as mash throughout the experimental period. 100 mg / kg feed of phytase (Ronozme HiPhos) was included in the basal diet in premix form using ground corn as a carrier.

[0185] [Table 6]

[0186] Zootechnical parameters (mortality, weight gain, final weight, FCR - feed conversion ratio) were monitored. GABA and KYN were determined in plasma and fecal samples, and cortisol (COR), adrenocorticotropic hormone (ACTH), serum neuropeptide Y (NPY), and lactate were determined in plasma samples.

[0187] Statistical analysis of performance: In this study, the experimental unit was the pen (n=4 pigs / pen). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Treatment effects were considered significant at p<0.05. JMP software (version 16.0) was used for the analysis. Statistical biological analysis: In this study, for GABA and KYN, and the stress-related markers COR, ACTH, NPY, glucose / lactose, the experimental unit was the pig (n=12 pigs / treatment). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Treatment effects were considered significant at p<0.05. JMP software (version 16.0) was used for the analysis.

[0188] On day 12, the fold changes of plasma GABA compared to day 0 were 1.07 and 1.09 in the cases of treatment with the positive control and OP ID57, respectively, slightly increased to 1.12 in the case of treatment with OP ID50, and significantly increased to 1.73 in the case of treatment with OP ID43. The fold changes of plasma COR (compared to day 0) as an index of stress resistance were found to be significantly decreased in the cases of treatment with OP ID50 and 43 (0.27 and 0.38, respectively) compared to the fold changes in the cases of treatment with the negative control, positive control, and OP ID57 (0.66, 0.70, and 0.77, respectively). Consistent with these findings, the fold change in plasma NPY (compared to day 0), as an index of stress tolerance, was found to be significantly increased with treatment with OP ID50 and 43 (0.93 and 1.60, respectively) compared with the fold changes with the negative control, positive control, and OP ID57 (0.57, 0.51, and 0.50, respectively). Thus, the in vitro results were confirmed to be extendable to in vivo effects. Further consequences of the significant differences in GABA and stress-related markers were observed: a reduction in the incidence of aggressive behaviors, such as tail biting, along with reduced stress, fear, and anxiety, and improved animal performance in terms of weight gain and FCR.

[0189] [Example 7] In another feeding study, 288 pigs were used in a 44-day study, including a 28-day growing phase and a 16-day fattening phase. Six treatments were tested, with 48 animals per treatment. A basal diet was provided as a negative control. The basal diet is shown in Table 6 below. The GABA control was prepared by supplementing the basal diet with 30 mg of GABA per kg of feed. In the four test treatments, the oligosaccharide preparation of OP ID54 was added to the basal diet at a content of 50, 150, 250, or 500 mg of oligosaccharide preparation per kg of feed. Silica was added to compensate for the mass difference between the treatment diet containing 500 mg of oligosaccharide preparation per kg of feed and the other treatment diets. Specifically, 0.5 kg SiO2 per 1000 kg of feed was added to the negative control treatment diet; 0.47 kg SiO2 per 1000 kg of feed was added to the GABA control treatment diet in addition to 0.03 kg GABA per 1000 kg of feed; and 0.45 kg, 0.35 kg, and 0.25 kg SiO2 per 1000 kg of feed were added to the treatment diets containing 50, 150, and 250 mg of the oligosaccharide preparation per 1000 kg of feed, respectively.

[0190] As in the previous examples, the diet was given ad libitum as mash throughout the experimental period. 100 mg / kg of Ronozme HiPhos phytase was included in the basal diet in premix form using ground corn as a carrier (2 x 150 g). The feed base was ground in a hammer mill using a 4 mm sieve and mixed with other ingredients (minerals, vitamins, and amino acids) in a 1500 L mixer. The glycan product was provided in the form of a syrup sprayed onto the mash diet. The negative control diet was sprayed with the same volume of water.

[0191] [Table 7]

[0192] A mixing task was conducted to study the effects of providing animals with the oligosaccharide preparation of the present invention, particularly with regard to animal welfare. Agonistic behavior after mixing individual pigs between groups is known to be an animal welfare concern, resulting in skin lesions on the body, reduced growth performance, and increased aggressive behavior. At the start of the study, pigs were grouped by weight. On day 28, all pigs were weighed before mixing, and then one pig per pen was selected and replaced with a pig of similar weight from the same treatment group but another pen. Performance data (weight, weight gain, FCR), social behavior, and skin lesions were monitored. Metagenomic analysis was performed on fecal samples. GABA, serotonin (5-HT), Trp (tryptophan), and KYN were analyzed from plasma and fecal samples. COR, ACTH, NPY, chromogranin A (CgA), and haptoglobin (HPT) were analyzed from plasma samples. Statistics were performed as in the previous examples.

[0193] Consistent with the previous examples, when the oligosaccharide preparation of the present invention was provided to animals with diet, an increased fold change in plasma GABA was observed compared to the negative control and the GABA control. Also, when the oligosaccharide preparation of the present invention was provided to animals with diet, a significant decrease in plasma cortisol fold change and a significant increase in plasma 5-HT fold change were observed compared to the negative control. Between days 0 and 28 of the study, i.e., before the mixing task, average daily weight gain (ADG) was 3.0% higher in the test treatment group compared to the negative control group, at 862 g / d compared to 837 g / d, respectively. Mixing had a negative effect on ADG, as reflected by ADGs from days 28 to 42 of 769 g / d and 810 g / d for the negative control and test treatment groups, respectively. However, despite the stress imposed on the animals during mixing, the test treatment group given the oligosaccharide preparation had a 5.3% higher ADG than the negative control. When comparing ADG from days 0 to 42, the test treatment group (845 g / d) was significantly (P=0.04) higher than the negative control group (814 g / d). Thus, feeding the oligosaccharide preparation according to the present invention was found to improve the animals' performance in terms of ADG in general, and especially when the animals faced stress. Average daily feed intake (ADFI) was found not to be significantly affected in the negative control and test treatment groups, with 2004 g / day and 1991 g / day from days 0 to 28; 2577 g / day and 2605 g / day from days 28 to 42; and 2195 g / day and 2196 g / day from days 0 to 42, respectively. Thus, feed conversion ratios from days 0 to 28 were reduced by 3.3% in the test treatment group (2.32) compared to the negative control (2.40); and from days 28 to 42, they were reduced by 1.1% in the test treatment group (3.32) compared to the negative control (3.36); and from days 0 to 42, they were significantly (P=0.07) reduced by 3.5% in the test treatment group (2.60) compared to the negative control (2.70). Therefore, it was concluded that energy from the diet consumed during stress is not converted into animal growth by animals in the negative control group, while animals fed the oligosaccharide preparation were less sensitive to stress and more successful in converting energy from the diet into growth.

[0194] [Example 8] Further animal studies were conducted using 144 un-tailed, [Large White x Landrace] x Pietran breeding pigs (72 females and 72 intact males). Two treatments were tested: the basal diet shown in Table 7 was given as a negative control. The test treatment was the basal diet supplemented with 250 g of dry OP ID54 per MT of feed. Feed was provided ad libitum in pellet form.

[0195] The study was conducted for a total of 105 days, with dietary treatment commencing on day 0 after an initial 7-day adaptation period (day -7 to day 0). On days 55 and 56, animals underwent a mixed task similar to that described in Example 7. The study was terminated on day 98, with final body weights of approximately 100-105 kg.

[0196]

[0189] Feeding the oligosaccharide composition of the present invention was found to improve animal performance (e.g., in terms of daily weight gain, feed intake, and feed-to-gain ratio) throughout the study, especially during the stress period established by the mixed challenge. In particular, during the mixed challenge week (days 51-59), animals fed the oligosaccharide composition of the present invention showed significant improvements in growth performance (final weight, weight gain, FCR). Details are shown in Tables 8-11 below.

[0197] Saliva samples were collected on days -7 (baseline), 58 (post-mixed challenge), and 97 (end of study). Hair samples were collected on days -7 and 98 (to detect potential cortisol accumulation during the treatment period from day -7 to day 98). Saliva and hair samples were tested for cortisol content as a non-invasive method for measuring stress response. Notably, cortisol concentrations in saliva samples were significantly reduced by 24.9% and 23.9%, respectively, in animals in the test treatment group compared with control animals. Similarly, cortisol concentrations in hair samples on day 98 were reduced by 17.5% in animals in the test treatment group compared with control animals. Thus, animals fed the oligosaccharide preparation of the present invention showed a significantly reduced stress response after the mixed challenge, as seen by a lower accumulation of cortisol, a stress biomarker, in hair samples, indicating that the mixed challenge causes less chronic stress. Consistent with these findings, the amount of severe or moderate-to-severe lesions on the ears, body and / or tail was found to be reduced in animals fed the oligosaccharide preparation of the present invention one and two days after the mixed challenge, thus indicating a reduction in aggressive behavior in these animals compared to animals fed the control diet.

[0198] [Table 8]

[0199] [Table 9]

[0200] [Table 10]

[0201] [Table 11]

[0202] Table 12

Claims

1. i) to regulate animal behavior, in particular to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; iv) to improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio; and / or v) to reduce cortisol concentrations in the plasma, saliva and / or hair of animals facing stress The method of a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; feeding the oligosaccharide preparation to the animal; increasing the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of said animal, particularly in the gastrointestinal tract and / or blood of said animal; A method comprising:

2. 1. A method for increasing gamma-aminobutyric acid, kynurenine and / or serotonin production by a microbial population, comprising: The method comprises: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. providing an oligosaccharide preparation produced or producible by the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2; contacting the microbial population with the oligosaccharide preparation; A method comprising:

3. i) to regulate animal behavior, in particular to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of said animal, in particular in the gastrointestinal tract and / or blood of said animal; and / or iv) to reduce cortisol concentrations in the plasma, saliva and / or hair of animals experiencing stress 1. A method for producing an oligosaccharide preparation of a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. thereby producing an oligosaccharide preparation, the oligosaccharide preparation comprising at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

4. 4. The method according to any one of claims 1 to 3, wherein the at least second sugar in step a) is selected from glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably glucose.

5. 5. The method of claim 1, wherein the at least a first sugar in step b) is glucose and the at least a second sugar in step b) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

6. 6. The method of any one of claims 1 to 5, wherein the at least a first sugar in either step a) or b); and the at least a second sugar in either step a) or b) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose.

7. i) to regulate animal behavior, in particular to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to increase the levels of gamma-aminobutyric acid, kynurenine and / or serotonin in the body of said animal, in particular in the gastrointestinal tract and / or blood of said animal; and / or iv) to reduce cortisol concentrations in the plasma, saliva and / or hair of animals experiencing stress 1. An oligosaccharide preparation comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. is generated or producible by; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

8. 8. The oligosaccharide preparation of claim 7, wherein the at least second sugar in step a) is selected from glucose, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably glucose.

9. 9. The oligosaccharide preparation of claim 7 or 8, wherein the at least a first sugar in step b) is glucose and the at least a second sugar in step b) is selected from N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose.

10. 10. The oligosaccharide preparation according to any one of claims 7 to 9, wherein the at least first sugar in either step a) or b) and the at least second sugar in either step a) or b) are condensed with at least a third sugar selected from glucose, N-acetylglucosamine, lactose, mannose, glucosamine, fructose, sucrose, xylose, and arabinose, preferably lactose.

11. 11. The oligosaccharide preparation according to any one of claims 7 to 10, wherein the oligosaccharide composition comprises at least 10 wt% (such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 wt%), preferably at least 20 wt% of the at least first fraction.

12. The oligosaccharide preparation according to any one of claims 7 to 11, wherein the oligosaccharide preparation is present in a nutritional composition at a content of at least 50 ppm (e.g., at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

13. i) to regulate animal behavior, in particular to reduce aggressive behavior; ii) to improve animal welfare, in particular to reduce anxiety, stress and / or fear disorders; iii) to improve animal performance, in particular to improve average weight gain and / or reduce feed conversion ratio; iv) To improve animal performance in animals facing stress, in particular to improve average weight gain and / or reduce feed conversion ratio; v) to increase the production of gamma-aminobutyric acid, kynurenine and / or serotonin by the microbial population; and / or vi) to reduce cortisol concentrations in the plasma, saliva and / or hair of animals experiencing stress Use of the oligosaccharide preparation according to any one of claims 7 to 12.

14. 1. An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid, kynurenine and / or cortisol, wherein said one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation and local inflammation, said oligosaccharide preparation comprising: a) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the at least first sugar is N-acetylglucosamine; or b) condensing at least a first sugar with at least a second sugar using a catalyst, wherein the catalyst is methylphosphonic acid. is generated or producible by; and the oligosaccharide preparation comprises at least a first fraction having a degree of polymerization (DP) of 1, at least a second fraction having a DP of 2, and at least a third fraction having a DP greater than 2.

15. 15. An oligosaccharide preparation for use in the treatment, amelioration, prevention and / or prophylaxis of one or more disorders associated with imbalanced levels of gamma-aminobutyric acid, kynurenine and / or cortisol, wherein the one or more disorders are selected from the group consisting of anxiety, stress, fear disorders, systemic inflammation and local inflammation according to claim 14, wherein the oligosaccharide preparation is comprised in a nutritional composition at a content of at least 50 ppm (e.g., at least 50, 70, 100, 150, 200, 300, 400, 500 ppm).

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