Composition for improving gut health associated with a gut-brain-axis condition

Abstract

The present invention refers to a combination of prebiotic fibres with probiotic bacteria for use in preventing and treating gut brain axis-associated conditions, and compositions comprising the same. In particular, the combination comprises the prebiotic fibres GOS and FOS and probiotic strains of Lacticaseibacillus paracasei.

Description

[0001] COMPOSITION FOR IMPROVING GUT HEALTH ASSOCIATED WITH A GUT-BRAIN-AXIS

[0002] CONDITION

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to a combination of prebiotic fibres with probiotic bacteria for use in preventing and treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof, and compositions comprising the same. In particular, the combination comprises the prebiotic fibres GOS and FOS and probiotic species of Lacticaseibacillus.

[0005] BACKGROUND OF THE INVENTION

[0006] There is a bi-directional communication pathway between the gut and the brain called the microbiota-gut-brain axis. Brain function is influenced by the gut microbiota via neuroendocrine, neuroimmune and autonomic nervous systems (Mayer, E. A. Gut feelings: the emerging biology of gut-brain communication. Nat. Rev. Neurosci. 12, 453-466, 2011). An imbalance in the gut microbiota results in or affects the co-ordination of the microbiota-gut-brain axis in human health.

[0007] Altered gut microbiota in ASD children has been reported to include a diminished presence of Bacteroides, Lactobacillus, and Bifidobacterium (Kim, J. “Autism Spectrum Disorder and Eating Problems: The Imbalance of Gut Microbiota and the Gut-Brain Axis Hypothesis”. J Korean Acad Child Adolesc Psychiatry, 2024, 35(1):51-56). Moreover, it has been hypothesized that the degree of microbial alteration correlates with the severity of the disease since faecal microbiota and metabolomes alterations were higher in atypical children compared to neurotypical children (De Angelis et al. “Autism spectrum disorders and intestinal microbiota”. Gut Microbes, 6:3, 207-213; May / June 2015), and that ASD children present a much higher ratio of Enterobacteriaceae to Bifidobacteriaceae (Coretti et al. “Gut Microbiota Features in Young Children with Autism Spectrum Disorders” Frontiers in Microbiology, 2018, volume 9, article 3146). Veillonella is found to be more abundant in neurotypical children than in ASD children (Ye et al. Comparison of gut microbiota in autism spectrum disorders and neurotypical boys in China: A case-control study. Synth Syst Biotechnol. 2021 May 21 ;6(2):120-126; Strati et al. New evidences on the altered gut microbiota in autism spectrum disorders. Microbiome 5, 24 (2017)). Impaired protein digestion has been reported in patients suffering from AD. Sanctuary et al. (“Dietary Considerations in Autism Spectrum Disorders [ASD]: The Potential Role of Protein Digestion and Microbial Putrefaction in the Gut-Brain Axis”, Front. Nutr., vol. 5, 2018) reports that in children with ASD, reduced proteolytic capacity may cause gastrointestinal problems and also exacerbate ASD symptoms.

[0008] Digestion of protein starts in the gastric stage and continues in the duodenum and jejunum sections of the small intestine. Inadequate digestion at any stage leads to higher accumulation of protein in colon. Proteolytic fermentation is a process in which gut bacteria break down proteins in the colon and leads to the production of metabolites such as ammonia, phenols, and indoles, many of which are toxic to the gut lining and can promote inflammation. Several bacterial species such as Streptococcus, Propionibacterium, Clostridium, and Bacteroides are known to ferment protein (Korpela K. “Diet, Microbiota, and Metabolic Health: Trade-Off Between Saccharolytic and Proteolytic Fermentation” Annu Rev Food Sci Technol. 2018 Mar 25;9:65-84).

[0009] Dysbiosis is marked by a reduction in beneficial bacteria and an increase in potentially harmful, protein-fermenting species. This imbalance can compromise gut health and immune function. Dysbiosis and the toxic byproducts of proteolytic fermentation can disrupt this communication network and may increase gut permeability, trigger systemic inflammation, and alter the production of neurotransmitters. These disruptions have been linked to a range of neurological and psychiatric conditions, including depression, anxiety, autism spectrum disorders, and Parkinson’s disease, linking gut health to the brain conditions (Rodriguez-Romero JJ et al. “What we know about protein gut metabolites: Implications and insights for human health and diseases” Food ChemX. 2021 Dec 22;13:100195).

[0010] On the other hand, microbial metabolites produced from saccharolytic fermentation of dietary fibres are generally recognized as beneficial to human health. Saccharolytic fermentation results in the production of short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, which play essential roles in maintaining gut health. Butyrate, for example, is the primary energy source for colonocytes and supports the maintenance of the intestinal barrier by enhancing tight junction integrity. Acetate and propionate, on the other hand, are absorbed into the bloodstream and influence systemic metabolism and immune regulation. Collectively, SCFAs help lower the pH of the colon, creating an environment that inhibits the growth of pathogenic bacteria and supports microbial diversity. The overall impact of saccharolytic fermentation on the gut-brain axis is largely beneficial. By promoting a healthy gut environment, reducing inflammation, and supporting neurochemical balance, this process can contribute to improved mental health outcomes.

[0011] Supplementation with probiotics has suggested an amelioration of ASD’ symptoms (Sivamaruthi et al. The Role of Microbiome, Dietary Supplements, and Probiotics in Autism Spectrum Disorder Int. J. Environ. Res. Public Health 2020, 17(8), 2647). Other probiotic mixtures have also been suggested to improve depression and anxiety but not schizophrenia, thereby highlighting the specificity of the effects generated by probiotics in each microbiota state (Mbrkl et al. Probiotics and the Microbiota-Gut-Brain Axis: Focus on Psychiatry. Curr Nutr Rep 9, 171-182 (2020)).

[0012] Bifidobacterium has been proposed as a “psychobiotic” for its ability to produce neuromodulators and to influence gut-brain relationship through interaction with other commensal bacteria (Sarkar et al, 2016).

[0013] A synbiotic preparation comprising a mixture of probiotics strains in a total concentration of 1 x 1010CFU / day (5.0 x 109CFU of Lactobacillus paracasei HII01 and 5.0 x 109CFU of Bifidobacterium animalis subsp. lactis) and 10 g prebiotics (5 g galacto-oligosaccharides (GOS), and 5 g oligofructose (FOS)) has been proposed to attenuate cortisol levels in stressed population (as determined by ‘Thai Stress Test’), reduced negative feelings, and altered levels of IL-10, LPS and IgA (Lalitsuradej et al. “The Effects of Synbiotics Administration on Stress-Related Parameters in Thai Subjects — A Preliminary Study”, Foods, 2022, 11 , 759).

[0014] Wang et al 2019 (“Prebiotic supplementation of in vitro fecal fermentations inhibits proteolysis by gut bacteria, and host diet shapes gut bacterial metabolism and response to intervention. Appl Environ Microbiol 85:e02749-18) reports the impact of prebiotics on proteolysis within the gut. It was found that addition of fructan prebiotic Synergyl in faecal slurries increased the levels of bifidobacteria, and lowered the production of ammonia and branched-chain fatty acids (BCFA). BCFA are reliable markers of proteolytic fermentation as these are produced through the fermentation of branched-chain amino acids (Jardon et al., “Dietary macronutrients and the gut microbiome: a precision nutrition approach to improve cardiometabolic health”, Gut, 2022, 71 ; 1214 - 1226).

[0015] Wang et al 2020 (“Probiotics and fructo-oligosaccharide intervention modulate the microbiota-gut brain axis to improve autism spectrum reducing also the hyper-serotonergic state and the dopamine metabolism disorder”, Pharmacological Research, Elsevier, 2020, vol. 157) describes a composition administered to children with ASD, comprising a mixture of four probiotic strains, among these is Lactobacillus paracasei LPC-37) and further comprising FOS effectively improved symptoms in children with ASD based on questionnaires. In particular, it has been shown that SCFA levels increase upon administration of the probiotics + FOS and further suggests amelioration of leaky gut and gut dysbiosis.

[0016] There remains a need for improved compositions to prevent and treat gut brain axis-associated conditions by targeting key elements related to such conditions.

[0017] SUMMARY OF THE INVENTION

[0018] To test the effect of the combination according to the invention in dysbiosis induced by proteolytic fermentation, faecal samples from healthy children were used in an in vitro fermentation model to induce proteolysis thereby mimicking the dysbiotic pattern found in the gut microbiota of neurodivergent individuals. It has been unexpectedly found that the combination of the invention is able to modulate key bacterial species for gut health in an impaired gut microbiota environment such as that found in subjects suffering from ASD.

[0019] Accordingly, a first aspect of the invention relates to a combination of galacto-oligosaccharides and fructo-oligosaccharides with Lacticaseibacillus ssp. for use in preventing and / or treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof. Advantageously, the compositions of the invention are capable of substantially delay the increase in the ratio of relative abundance of Enterobacteria to Bifidobacteria as compared to a reference synbiotic composition in neurodivergent-like gut microbiota. In addition, it has been unexpectedly found that in the same model mimicking the dysbiotic pattern that the total amount of short chain fatty acids (SCFA) synergistically increased when the dysbiotic microbiota were supplemented with the synbiotic composition according to the invention. SCFA are a marker of saccharolytic fermentation and predominantly involves production of acetate, propionate and butyrate.

[0020] Furthermore, the inventors found that the amount of branched chain fatty acids (BCFA; a marker of proteolytic fermentation) decreased when the dysbiotic microbiota were supplemented with the synbiotic composition. It was found that the overall ratio of SCFA increased compared to BCFA, thereby indicating a shift to saccharolytic fermentation over time. Hence, a further advantage is that the compositions of the invention are capable of increasing the short chain fatty acid production in a dysbiotic-like gut microbiota thereby promoting the shift to saccharolytic fermentation instead of proteolytic fermentation found in neurodivergent-like gut microbiota. The present invention thus provides a safe and efficient therapeutic tool for the management of gut brain axis-associated conditions, particularly in individuals with impaired protein digestion.

[0021] A second aspect of the invention relates to a composition comprising the combination of prebiotics and probiotics as herein defined.

[0022] A third aspect of the invention relates to a non-therapeutic method for improving the gut microbiota in a subject having a first degree relative diagnosed as neurodivergent, said method comprising administering a combination of galacto-oligosaccharides and fructo-oligosaccharides with Lacticaseibacillus ssp to said subject, wherein the neurodivergent first degree relative has been diagnosed with one or more of ASD, ADHD, dyslexia, dyspraxia, dyscalculia, dysgraphia, Tourette Syndrome, Obsessive Compulsive disorder (OCD), bipolar disorder. Preferably the subject has a neurodivergent sibling or parent, preferably a neurodivergent sibling.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention concerns a combination of prebiotic fibre(s) selected from galactooligosaccharides and fructo-oligosaccharides with a probiotic strain(s) of Lacticaseibacillus ssp. for use in preventing and / or treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof.

[0025] In some jurisdictions the invention can also be worded as the use of prebiotic fibre(s) selected from galacto-oligosaccharides and fructo-oligosaccharides and of probiotic strain(s) of Lacticaseibacillus ssp. in the manufacture of a composition for use in preventing and / or treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof. In further jurisdictions, the invention can also be worded as a method for use in preventing and / or treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof by administering a combination of prebiotic fibre(s) selected from galacto-oligosaccharides and fructo-oligosaccharides with probiotic strain(s) of Lacticaseibacillus ssp. to the subject.

[0026] “Impaired gut health associated with a gut-brain-axis condition” refers to altered gut microbiota or gut function as prevalently found in subjects diagnosed with, or subjects likely to develop, gut brain axis conditions. It has been reported that impaired gut function in these subjects lead not only to worsening of Gl symptoms and but also linked to the onset and exacerbation of the neurodevelopmental condition (Sanctuary et al. 2018). In these subjects, microbial protein fermentation and inflammatory putrefactive metabolites are increased, whereas the levels of fiberfermenting bacteria and bacterial diversity are reduced. In other words, impaired gut health, preferably selected from gut dysbiosis, gut dysfunction, fragile gut and / or impaired protein digestion, associated with a gut-brain axis conditions, refers to altered gut microbiota or altered gut function occurring in subjects diagnosed with a gut-brain-axis condition, preferably the gut-brain-axis is a neurodevelopment / neurodivergent disorder. As described above, subjects with a gut-brain-axis condition have altered gut microbiota / gut function and it has been found that this specific target group particular have a shift towards a proteolytic gut ecosystem favouring proteolytic fermentation and have reduced fiber-fermenting bacteria and therewith reduced saccharolytic fermentation and reduced short-chain fatty acid production.

[0027] “Impaired gut health” can be routinely diagnosed by a medical professional. “Impaired gut health” is preferably selected from gut dysbiosis, gut dysfunction, fragile gut, and / or impaired protein digestion.

[0028] “Gut-brain-axis conditions” according to the invention can be clinically diagnosed as neurodevelopmental / neurodivergent disorders. Preferably, the gut brain axis conditions are selected from autism spectrum disorders (ASDs) or attention deficit hyperactivity disorder (ADHD). The ASDs are selected from autism disorder (AD), Asperger’s syndrome and pervasive developmental disorder-not otherwise specified (PDD-NOS). In a preferred embodiment, the gut brain axis condition is autism disorder (AD).

[0029] The subject is preferably a neurodivergent patient or a subject that is more likely to develop a neurodevelopmental disorder, such as a subject having a first degree relative already diagnosed as neurodivergent. Accordingly, the subject is preferably selected from a neurodivergent subject or a subject which has a neurodivergent sibling, more preferably the subject is a neurodivergent subject. In a further preferred embodiment, a neurodivergent subject is a subject diagnosed with one or more of ASD, ADHD, dyslexia, dyspraxia, dyscalculia, dysgraphia, Tourette Syndrome, Obsessive Compulsive disorder (OCD), bipolar disorder, more preferably diagnosed with ASD and / or, ADHD, most preferably diagnosed with ASD. The subject is an infant (0-12 months of age), toddler (12-36 months of age), child (3-12 years of age), teenager (12-18 years of age) or an adult (more than 18 years of age). Preferably, the subject is a toddler, child, teenager or an adult, more preferably, a child or teenager. In an alternative preferred embodiment, the subject is an infant of 0-12 months old.

[0030] “Gut dysbiosis” refers to a medical condition characterized by the imbalance of the gut microbial profile, i.e., the increase of pathogenic bacteria and / or decrease of beneficial bacteria. Gut dysbiosis may be transitory or permanent. For instance, transitory gut dysbiosis may occur over the course and / or following antibiotic therapy, but gut microbiota balance may be re-established on its own or as a response to dedicated therapy. Permanent gut dysbiosis may be identified in patients cosuffering from e.g. ASDs, inflammatory bowel syndrome, etc. In a preferred embodiment, the subject further suffers from permanent gut dysbiosis.

[0031] “Gut dysfunction” associated with a gut-brain-axis condition includes impaired gut permeability, reduction in effectiveness of digestion, and impaired nutrients absorption and uptake.

[0032] “Fragile gut” is defined as a gastrointestinal tract that is basically “healthy” (i.e., lack of defined gastrointestinal disease), but is not robust to stressors, challenges, or microbial insults such as indigestible or toxic components or infectious agents.

[0033] “Impaired protein digestion” is defined as a shift towards a proteolytic gut ecosystem favouring thus proteolytic fermentation, and is commonly identified in individuals suffering from gut brain axis- associated conditions. Proteolytic fermentation is the process in which proteins and / or peptides are anaerobically broken down by microorganisms in the gastro-intestinal tract of mammals. An increased level of proteobacteria, i.e., proteolytic bacteria, in the gastro-intestinal tract is considered an indicator of increased proteolytic fermentation, or the other way around a reduced level of proteobacteria, i.e., proteolytic bacteria, in the gastro-intestinal tract is considered an indicator of reduced proteolytic fermentation. There is mounting evidence that an increase in proteobacteria is linked to the appearance and development of ASDs and, conversely, reducing the relative abundance thereof prevents and ameliorates the symptoms of gut brain axis-associated conditions, such as ASDs.

[0034] According to one embodiment, the subject is diagnosed to suffer from at least one atypical eating behaviour. Atypical eating behaviours are herein defined include one or more of clinically identified food refusal patterns, food dislike patterns, food selectivity, limited food preference / repertoire, obsessive eating pattern, pica, pocketed food, anorexia, etc. Atypical eating behaviour and disorders can be clinically diagnosed and are well documented, such as in the “Clinical Handbook of Complex and Atypical Eating Disorders” (edited by Leslie K. Anderson, Stuart B. Murray, Walter H. Kaye, Oxford University Press, 2018). In one embodiment, preventing and / or treating impaired gut health associated with a gut-brain-axis condition comprises improving the gut microbiota of the subject. More preferably, improving gut microbiota comprises at least one of: a. increasing the relative abundance of saccharolytic bacterial taxa, preferably selected from Bifidobacteriaceae, Lactobacillaceae, Lachnospiraceae and Veillonellaceae; b. decreasing the relative abundance of proteolytic bacterial taxa, preferably selected from Enterobacterales, Peptostreptococcaceae, Peptostreptococcales-tissirierallales, Oscillospiraceae and Eggerthellaceae; c. decreasing the ratio of Enterobacterales to Bifidobacteriaceae; or d. increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales.

[0035] Worded differently, preventing and / or treating impaired gut health associated with a gut brain axis condition is at least one of: a. increasing the relative abundance of saccharolytic bacterial taxa, preferably selected from Bifidobacteriaceae, Lactobacillaceae, Lachnospiraceae and Veillonellaceae; b. decreasing the relative abundance of proteolytic bacterial taxa, preferably selected from Enterobacterales, Peptostreptococcaceae, Peptostreptococcales-tissirierallales, Oscillospiraceae and Eggerthellaceae; c. decreasing the ratio of Enterobacterales to Bifidobacteriaceae; or d. increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales.

[0036] Preferably, preventing and / or treating impaired gut health associated with a gut-brain-axis condition is decreasing the ratio of Enterobacterales to Bifidobacteriaceae and / or increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales, more preferably preventing and / or treating impaired gut health associated with a gut-brain-axis condition is decreasing the ratio of Enterobacterales to Bifidobacteriaceae.

[0037] Also, in further different wording, preventing and / or treating impaired gut health is at least one of: a. increasing the relative abundance of saccharolytic bacterial taxa, preferably selected from Bifidobacteriaceae, Lactobacillaceae, Lachnospiraceae and Veillonellaceae; b. decreasing the relative abundance of proteolytic bacterial taxa, preferably selected from Enterobacterales, Peptostreptococcaceae, Peptostreptococcales-tissirierallales, Oscillospiraceae and Eggerthellaceae; c. decreasing the ratio of Enterobacterales to Bifidobacteriaceae; or d. increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales.

[0038] Preferably, wherein preventing and / or treating impaired gut health is decreasing the ratio of Enterobacterales to Bifidobacteriaceae and / or increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales, more preferably preventing and / or treating impaired gut health is decreasing the ratio of Enterobacterales to Bifidobacteriaceae.

[0039] Relative abundance of gut bacteria in the gut microbiota, as herein referred, can be suitably determined in sample stools of a subject using microbiomics techniques known in the art, such as amplification kits commercially available and sequencing using 16S rRNA gene sequencing protocols. Such techniques are increasingly used in medical practice to confirm clinical diagnostic of gut dysbiosis, impaired gut microbiota, and associated conditions.

[0040] In the context of the present invention, increasing or reducing relative abundance is compared to a control which the reference value from a subject that has not been administered the combination according to the invention. Increase or decrease of relative abundance refers to a difference of at least 10% compared to control, more preferably at least 20% compared to control, more preferably at least 40% compared to control, more preferably at least 60% compared to control.

[0041] In a further embodiment, preventing and / or treating impaired gut health associated with a gut brain axis condition is increasing saccharolytic fermentation by gut microbiota and / or decreasing proteolytic fermentation by gut microbiota, preferably both. More preferably, preventing and / or treating impaired gut health associated with a gut brain axis condition at least one of: increasing the relative amount of short chain fatty acid (SCFA) in the gut; decreasing the relative amount of branched chain fatty acids (BCFA) in the gut; increasing the ratio of SCFA to BCFA in the gut.

[0042] Most preferably, preventing and / or treating impaired gut health associated with a gut-brain axis condition is all of the above.

[0043] Worded differently, preventing and / or treating impaired gut health is increasing saccharolytic fermentation by gut microbiota and / or decreasing proteolytic fermentation by gut microbiota, preferably both. More preferably, preventing and / or treating impaired gut health is at least one of: increasing the relative amount of short chain fatty acid (SCFA) in the gut; decreasing the relative amount of branched chain fatty acids (BCFA) in the gut; increasing the ratio of SCFA to BCFA in the gut.

[0044] Most preferably, preventing and / or treating impaired gut health is all of the above.

[0045] The combination according to the invention is also referred to as a synbiotic. As used herein, “synbiotics” refer to a mixture comprising microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host, e.g., a combination of probiotics and prebiotic fibres.

[0046] Prebiotics

[0047] The combination according to the invention comprises galacto-oligosaccharides (GOS) and fructooligosaccharides (FOS). GOS are preferably selected from the group consisting of betagalactooligosaccharides, alphagalacto-oligosaccharides, and galactan. According to a preferred embodiment GOS are betagalacto-oligosaccharides. Preferably the GOS comprise galactooligosaccharides with beta(1 ,4), beta(1 ,3) and / or beta(1 ,6) glycosidic bonds and a terminal glucose. Transgalacto-oligosaccharides is for example available under the trade name VivinalOGOS (Domo FrieslandCampina Ingredients), Bi2muno (Clasado), Cup-oligo (Nissin Sugar), Oligomate55 (Yakult), Promovita (Dairy Crest), Bioligo (Ingredion). Fructo-oligosaccharides may in other context have names like fructopolysaccharides, oligofructose, polyfructose, polyfructan, inulin, levan and fructan and may refer to oligosaccharides comprising beta-linked fructose units, which are preferably linked by beta(2,1) and / or beta(2,6) glycosidic linkages, and a preferable DP between 2 and 200. Preferably, the fructo-oligosaccharides contain a terminal beta(2,1) glycosidic linked glucose. Preferably, the fructo-oligosaccharides contain at least 7 beta-linked fructose units. Inulin is a type of fructo-oligosaccharides wherein at least 75% of the glycosidic linkages are beta(2,1) linkages. Typically, inulin has an average chain length between 8 and 60 monosaccharide units. Preferred fructo-oligosaccharides for use in the combination of the present invention is commercially available under the trade names RaftilineOHP (Orafti), Fibrulose and Fibruline (Cosucra) and Frutafit and Frutalose (Sensus).

[0048] The present combination comprises a mixture of GOS and FOS. Preferably the mixture of GOS and FOS is present in a weight ratio of from 1 / 99 to 99 / 1 , more preferably from 1 / 19 to 19 / 1 , more preferably from 1 / 1 to 19 / 1 , more preferably from 2 / 1 to 15 / 1 , more preferably from 5 / 1 to 12 / 1 , even more preferably from 8 / 1 to 10 / 1 , even more preferably in a ratio of about 9 / 1 . This weight ratio is particularly advantageous when the GOS have a low average DP and FOS have a relatively high DP.

[0049] Preferably the GOS are short-chain galacto-oligosaccharides (scGOS) and the FOS are long-chain fructo-oligosaccharides (IcFOS). Most preferred is a mixture of GOS with an average DP below 10, preferably below 6, and FOS with an average DP above 7, preferably above 11 , even more preferably above 20. Preferably the GOS have an average DP in the range from 3-7 and the FOS have an average DP in the range from 20-40. Preferably, GOS and FOS are present in a weight ratio in the range from 5 :1 to 12: 1 , preferably in a weight ratio of from 8 : 1 to 10 : 1 .

[0050] Lacticaseibacillus ssp.

[0051] The combination according to the invention comprises Lacticaseibacillus ssp.. In a preferred embodiment, the Lacticaseibacillus ssp. is Lacticaseibacillus paracasei. Commercially available Lacticaseibacillus paracasei include e.g. Lacticaseibacillus ssp. LPC-37 (ATCC 334).

[0052] The subject according to the invention is an infant, a toddler, a child, a teenager, an adult or an elderly subject. Preferably, the subject is 12 months to 12 years old, 12-18 years old, 18-45 years old. In an alternative embodiment, the subject is an infant of 0-12 months old.

[0053] The combination for use according to the invention may be used on its own or may be comprised in a composition, such as a nutritional composition. The combination for use according to the present invention is preferably comprised in a nutritional composition. Hereafter the combination comprised in a nutritional composition is also referred to as the nutritional composition for use according to the invention or the nutritional composition according to the invention or the present nutritional composition. In one embodiment, the nutritional composition is directed to infant or clinical nutrition. Preferably, the nutritional composition is an infant formula, follow on formula, growing up milk, fortified milk, milk supplement, and the like. Preferably, the nutritional composition is an infant formula or follow on formula. The nutritional composition can be advantageously applied as a complete nutrition for infants. Preferably the present nutritional composition is an infant formula. An infant formula is defined as a formula for use in infants and can for example be a starter formula, intended for infants of 0 to 6 or 0 to 4 months of age. A follow on formula is intended for infants of 4 or 6 months to 12 months of age. At this age infants start weaning on other food. A toddler or growing up milk or formula is intended for children of 12 to 36 months of age. The present nutritional composition preferably comprises a lipid component, protein component and carbohydrate component and is preferably administered in liquid form. The present nutritional composition may also be in the form of a dry food, preferably in the form of a powder which is accompanied with instructions as to mix said dry food, preferably powder, with a suitable liquid, preferably water. The present nutritional composition preferably comprises other fractions, such as vitamins, minerals, trace elements and other micronutrients in order to make it a complete nutritional composition. Preferably infant formulas comprise vitamins, minerals, trace elements and other micronutrients according to international directives.

[0054] In some embodiments, the combination is suitable to be comprised in composition for children from 36 months to 18 years of age. The combination according to the invention can be included in powdered compositions to be dissolved in water, milk or juice, capsules or pills, saches, liquid drinks, fortified drinks, supplements, nutraceutical gums and jellies, weaning foods, semi-solid or solid foods, and the like.

[0055] In some embodiments the combination is comprised in a supplement or therapeutic compositions directed to adults, and optionally comprising further pharmaceutically or nutritionally accepted adjuvants.

[0056] The nutritional composition according to the invention preferably comprises 0.13 - 2.7 g GOS per 100 ml, more preferably 0.20 - 2.0 g, even more preferably 0.40 - 1 .3 g GOS per 100 ml as a drink or as a powder in reconstituted ready-to-drink form. Based on dry weight, the nutritional composition according to the invention preferably comprises 1 .0 - 20 g GOS per 100 g, more preferably 1 .5 - 15 g, even more preferably 3.0 - 10 g GOS per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.2 - 4.0 g beta galactooligosaccharides per 100 kcal, more preferably 0.3 - 3.0 g, even more preferably 0.6 - 2.0 g GOS per 100 kcal. Preferably, GOS is beta-galactooligosaccharides.

[0057] Furthermore, the nutritional composition according to the invention preferably comprises 0.02- 1 .5 g FOS per 100 ml, more preferably 0.04- 1 .0 g, even more preferably 0. 08- 0.5 g FOS per 100 ml. Based on dry weight, the nutritional composition according to the invention preferably comprises 0.1 - 2.0 g FOS per 100 g, more preferably 0.2 -1.5 g, even more preferably 0.4- 1.0 g FOS per 100 g. Based on calories, the nutritional composition according to the invention preferably comprises 0.02 - 0.4 g FOS per 100 kcal, more preferably 0.04- 0.6 g, even more preferably 0.06 - 0.3 g FOS per 100 kcal.

[0058] The present nutritional composition comprises preferably 0.5 to 20 wt% of the combination of GOS, FOS, more preferably 1 .5 to 15 wt%, even more preferably 2.5 to 12 wt%, most preferably 5.0 to 10.0 wt%, based on dry weight of the nutritional composition. Based on 100 ml the present nutritional composition preferably comprises 0.35 to 2.5 wt% combination of GOS, FOS, more preferably 0.35 to 2.0 wt%, even more preferably 0.4 to 1 .5 wt%, based on 100 ml of the nutritional composition. A lower amount of the combination will be less effective in preventing or treating the conditions of the invention, whereas a too high amount will result in side-effects of bloating and abdominal discomfort.

[0059] Preferably, the present nutritional composition comprises 2 x103to 3 x 1012colony forming units (cfu) Lacticaseibacillus ssp. per gram dry weight of the nutritional composition, more preferably 2 x 104to 3 x 1011colony forming units (cfu) Lacticaseibacillus ssp. per gram dry weight, even more preferably 2 x 105to 3 x 109colony forming units (cfu) Lacticaseibacillus ssp. per gram dry weight, most preferably 3 x 105to 3 x 108colony forming units (cfu) Lacticaseibacillus ssp. per gram dry weight of nutritional composition.

[0060] In one embodiment, the nutritional composition further comprises non digestible oligosaccharides other than GOS and FOS. Such other NDOs are preferably selected from the group consisting of human milk oligosaccharides (HMO), such as 2’-FL, 3’-SL, 6SL, and the like.

[0061] The present nutritional composition preferably comprises lipid, protein and digestible carbohydrate wherein the lipid provides 5 to 50% of the total calories, the protein provides 5 to 50% of the total calories, and the digestible carbohydrate provides 15 to 90% of the total calories. Preferably, in the present nutritional composition the lipid provides 35 to 50% of the total calories, the protein provides 7.0 to 12.5% of the total calories, and the digestible carbohydrate provides 40 to 55% of the total calories. For calculation of the % of total calories for the protein, the total of energy provided by proteins, peptides and amino acids needs to be taken into account. Preferably the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 4 g per 100 kcal, preferably 1 .7 to 2.5 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition. Preferably the present nutritional composition comprises lipid providing 4 to 6 g per 100 kcal, protein providing 1 .6 to 2.0 g per 100 kcal, more preferably 1 .7 to 1 .9 g per 100 kcal and digestible carbohydrate providing 8 to 15 g per 100 kcal of the nutritional composition. In one embodiment, the lipid provides 3 to 7 g lipid per 100 kcal, preferably 4 to 6 g per 100 kcal, the protein provides 1.6 to 2.1 g per 100 kcal, preferably 1 .6 to 2.0 g per 100 kcal and the digestible carbohydrate provides 5 to 20 g per 100 kcal, preferably 8 to 15 g per 100 kcal of the nutritional composition and wherein preferably the digestible carbohydrate component comprises at least 60 wt.% lactose based on total digestible carbohydrate, more preferably at least 75 wt.%, even more preferably at least 90 wt.% lactose based on total digestible carbohydrate. The amount of total calories is determined by the sum of calories derived from protein, lipids, digestible carbohydrates and non-digestible oligosaccharide.

[0062] The present nutritional composition preferably comprises a digestible carbohydrate component. Preferred digestible carbohydrate components are lactose, glucose, sucrose, fructose, galactose, maltose, starch and maltodextrin. Lactose is the main digestible carbohydrate present in human milk. The present nutritional composition preferably comprises lactose. Preferably the present nutritional composition does not comprise high amounts of carbohydrates other than lactose. Compared to digestible carbohydrates such as maltodextrin, sucrose, glucose, maltose and other digestible carbohydrates with a high glycaemic index, lactose has a lower glycaemic index and is therefore preferred. The present nutritional composition preferably comprises digestible carbohydrate, wherein at least 35 wt.%, more preferably at least 50 wt.%, more preferably at least 60 wt.%, more preferably at least 75 wt.%, even more preferably at least 90 wt.% , most preferably at least 95 wt.% of the digestible carbohydrate is lactose. Based on dry weight the present nutritional composition preferably comprises at least 25 wt.% lactose, preferably at least 40 wt.%, more preferably at least 50 wt.% lactose.

[0063] The present nutritional composition preferably comprises at least one lipid selected from the group consisting of animal lipid (excluding human lipids) and vegetable lipids. Preferably the present nutritional composition comprises a combination of vegetable lipids and at least one oil selected from the group consisting of fish oil, animal oil (including animal fat, for instance milk fat), algae oil, fungal oil, and bacterial oil. Herein, the terms oil and fat are interchangeably used. The lipid of the present nutritional composition preferably provides 3 to 7 g per 100 kcal of the nutritional composition, preferably the lipid provides 4 to 6 g per 100 kcal. When in liquid form, e.g. as a ready- to-feed liquid, the nutritional composition preferably comprises 2.1 to 6.5 g lipid per 100 ml, more preferably 3.0 to 4.0 g per 100 ml. Based on dry weight the present nutritional composition preferably comprises 12.5 to 40 wt.% lipid, more preferably 19 to 30 wt.%. Preferably the lipid comprises the essential fatty acids alpha-linolenic acid (ALA), linoleic acid (LA) and / or long chain polyunsaturated fatty acids (LC-PUFA). The LC-PUFA, LA and / or ALA may be provided as free fatty acids, in triglyceride form, in diglyceride form, in monoglyceride form, in phospholipid form, or as a mixture of one of more of the above. Preferably the present nutritional composition comprises at least one, preferably at least two lipid sources selected from the group consisting of rape seed oil (such as colza oil, low erucic acid rape seed oil and canola oil), high oleic sunflower oil, high oleic safflower oil, olive oil, marine oils, microbial oils, coconut oil, palm kernel oil. The present nutritional composition is not human milk. The present nutritional composition preferably comprises protein. The protein used in the nutritional composition is preferably selected from the group consisting of non-human animal proteins, preferably milk proteins, vegetable proteins, such as preferably soy protein and / or rice protein, and mixtures thereof. The present nutritional composition preferably contains casein, and / or whey protein, more preferably bovine whey proteins and / or bovine casein. Thus, in one embodiment the protein in the present nutritional composition comprises protein selected from the group consisting of whey protein and casein, preferably whey protein and casein, preferably the whey protein and / or casein is from cow’s milk. Preferably the protein comprises less than 5 wt.% based on total protein of free amino acids, dipeptides, tripeptides or hydrolyzed protein. The present nutritional composition preferably comprises casein and whey proteins in a weight ratio casein: whey protein of 10 : 90 to 90 : 10, more preferably 20 : 80 to 80 : 20, even more preferably 35 : 65 to 55 : 45.

[0064] The wt.% protein based on dry weight of the present nutritional composition is calculated according to the Kjeldahl-method by measuring total nitrogen and using a conversion factor of 6.38 in case of casein, or a conversion factor of 6.25 for other proteins than casein. The term ‘protein’ or ‘protein component’ as used in the present invention refers to the sum of proteins, peptides and free amino acids.

[0065] The nutritional composition preferably comprises 0.8 - 2.7 g protein per 100 ml, more preferably 0.9 - 2.1 g per 100 ml, more preferably 1 .1- 1 .6 g per 100 ml nutritional composition. Based on dry weight, the nutritional composition preferably comprises 6 - 20 g protein per 100 g, more preferably 7 - 16 g per 100 g, more preferably 8 - 12 g protein per 100 g dry weight of the nutritional composition. Based on calories, the nutritional composition preferably comprises 1.2 - 4.0 g protein per 100 kcal, more preferably 1.4- 3.2 g per 100 kcal, more preferably 1.6- 2.4 g protein per 100 kcal of the nutritional composition. The protein preferably provides 5 - 15%, more preferably 6 - 13% even more preferably 7 - 10% based on total calories of the composition. Protein is to be taken as the sum of proteins, peptides, and free amino acids. The amount of protein can be calculated according to the amount of nitrogen multiplied by 6.25. The present nutritional composition preferably comprises casein and / or whey proteins. Preferably the weight ratio caseimwhey protein is 0:100 to 90:10, more preferably 20:80 to 90:10, more preferably 40:60 to 80:20.

[0066] In order to meet the caloric requirements of an infant or toddler, when the composition is of the infant formulae type, for example when the composition is an infant formula or follow-on formula, the nutritional composition preferably comprises 45 to 200 kcal / 100 ml liquid. For infants the nutritional composition has more preferably 60 to 90 kcal / 100 ml liquid, even more preferably 65 to 75 kcal / 100 ml liquid. This caloric density ensures an optimal ratio between water and calorie consumption. For toddlers, human subjects with an age between 12 and 36 months, the nutritional composition more preferably has a caloric density between 45 and 65, even more preferably between 50 and 60 kcal / 100 ml. The osmolarity of the present composition is preferably between 150 and 420 mOsmol / l, more preferably 260 to 320 mOsmol / l. The low osmolarity aims to further reduce the gastro-intestinal stress.

[0067] When the nutritional composition is in a ready to feed, liquid form, the preferred volume administered on a daily basis is in the range of about 80 to 2500 ml, more preferably about 200 to 1200 ml per day. Preferably, the number of feedings per day is between 1 and 10, preferably between 3 and 8. In one embodiment the nutritional composition is administered daily for a period of at least 2 days, preferably for a period of at least 4 weeks, preferably for a period of at least 8 weeks, more preferably for a period of at 25 least 12 weeks, in a liquid form wherein the total volume administered daily is between 200 ml and 1200 ml and wherein the number of feedings per day is between 1 and 10.

[0068] In a second aspect, the present invention concerns a composition comprising, based on dry weight, 6 - 20 g protein per 100g of the composition, 2 x 103to 3 x 1012colony forming units (cfu) Lacticaseibacillus ssp. per gram of the composition, and 1-20 g of a combination of GOS and FOS per 100g of the composition. In a preferred embodiment, the composition is an infant formula or follow-on formula.

[0069] Preferably, the composition further comprises a protein selected from casein, whey or a plant-based protein.

[0070] In a preferred embodiment, the composition comprises, on dry weight, 0.1 - 2.0 g FOS per 100 g of the composition and / or 1 .0 - 20 g GOS per 100 g of the composition.

[0071] The combination^) and composition(s) according to the invention are preferably enterally administered, more preferably orally.

[0072] EXAMPLES

[0073] Example 1

[0074] To test the effect of the combination according to the invention in dysbiosis induced by proteolytic fermentation, faecal samples from healthy children (18-24 months old) were used in an in vitro fermentation model to induce proteolysis thereby mimicking the dysbiotic pattern found in the gut microbiota of patients with protein digestion problems. Proteolysis was induced by adding tryptone (20 g / L concentration) for 24 hours.

[0075] A combination according to the invention (“A”) comprising 0.5% w / w scGOS / lcFOS (ratio 9:1) and 107-108cfu / ml of Lacticaseibacillus paracasei was tested and compared with a reference composition (“Ref’) comprising 0.5% w / w Synergyl (commercially available prebiotic fiber comprising oligofructose-enriched inulin) and 107-108cfu / ml L. paracasei. The scGOS was VivinalGOS® the IcFOS was RaftilineHP®.

[0076] Test started after 24 tryptone fermentation (tO) by adding the respective test compositions. The total test time was 56 hours. To reach a steady state, mixtures were added every 4 hours during the day and once in the end of the day for overnight. The 4h intervals allow all fibers to be utilized by the microbial community and reach a steady state (data not shown). Samples were collected in the beginning (tO) and at the end of test (t56h) for microbiota profiling analysis (16S rRNA sequencing).

[0077] Under anaerobic conditions, the faecal samples were thawed and approximately a 10% (w / v) suspension of the fecal samples were made in age adapted Colonic Microbiota medium containing 25 mM acetate and 12 mM lactate, 25 mg / L bile acids (Sigma), 15 mmol / L ammonium sulphate without carbon source adjusted to toddler stool pH (6.5) in order to mimic uncompromised intestinal conditions. The diluted faecal samples were homogenized, allowed to sediment for 5 minutes, and subsequently filtered over a Millex 100 pm vacuum filter. The filtered fecal samples were pooled together in 1 :1 :1 ratio. A Biolector Pro plate (BOH2 round well, M2P-labs) with pH optodes was used. One feeding row of the plate was filled with sterile 3M NaOH for the pH adjustment. Thirty- two wells of the plate were filled with 800 pL of the pooled faecal solution (10% w / v) and 800 pL of Tryptone (40 g / L) were added to the wells with the faecal samples. After this, the plates were sealed with ventilated silicone foil with slits. The plate was incubated (85% moisture, 37 degrees, 600 rpm, anaerobic) in BioLector Pro for 24 hours. At the end of the protein fermentation, the experiment was paused, the faecal slurry of each well was harvested and shortly centrifuged, under a-septical anaerobic conditions. The supernatant was frozen for further analyses, three faecal pellets were frozen for 16S rRNA sequencing analysis while the rest of the faecal pellets was resuspended into 800 pL of fresh colonic microbiota medium and pipetted back in the wells of a new BOH2 plate.

[0078] At the beginning of the fermentation, 800 pL of sterile water solution was added to control wells, while sterile GOS / FOS (1 %) and Synergyl tested solutions were added to the respective test wells. To the wells with GOS / FOS and Synergyl , the cells of active culture of L paracasei were added in a concentration of 2x10E7 CFUs / mL. The experiment was started with pH at setpoint 6.5 for toddler samples with continuous pH control. After 4 hours the experiment was paused, the faecal slurry of each well was harvested and shortly centrifuged, the supernatant was frozen for further analyses while the faecal pellet was resuspended in fresh medium with carbohydrates and L paracasei cells and pipetted back in the original well of BOH2 plate. The procedure was repeated for 3 days, with 3 times manual feeding every 4 hours per day. Subsequently, the faecal pellets from the last time point (t56) were stored for 16S rRNA sequencing analysis.

[0079] The results of the combination according to the invention versus the comparative combination is shown in Figure 1. The results show that the composition according to the invention is advantageously capable of modulating the gut microbiota in dysbiotic state induced by proteolytic fermentation. In particularly, in Figure 1 , it is shown that a synbiotic composition according to the invention substantially reduced the ratio of Enterobacterales to Bifidobacteriaceae when compared to the synbiotic composition comprising reference composition (Synergyl and L. paracasei), thus bringing the ratio closer to that of healthy controls (HC), ie, prior to the disturbance caused by the proteolytic fermentation.

[0080] It was further observed that the ratio of Veillonellaceae + Bifidobacteriaceae over Enterobacterales is closer to the healthy controls after synbiotics treatment, when compared to the reference combination (Fig. 2).

[0081] Example 2

[0082] To mimic the dysbiosis induced by proteolytic fermentation, the in vitro fermentation protocol of Example 1 was followed. In this experiment, the combination of prebiotics (0.5% w / v) and probiotic comprising L paracasei L411 (between 107and 108CFUs / mL) was studied for 56 hours after the induction of proteolytic dysbiosis to observe any effects on microbial fermentation in this state. The study arms included:

[0083] - prebiotics GOS / FOS (0.5% w / v)

[0084] - probiotic L paracasei L411 (between 107and 108CFUs / mL)

[0085] - synbiotic GOS / FOS (0.5% w / v) and L paracasei 411 (between 107and 108CFUs / mL.

[0086] - control: water only, referred to as blank.

[0087] In addition, the expected sum of individual components has been taken into account for comparison of effect to the synbiotic composition.

[0088] During the study, the supplements were given every 4 hours during the day and once at the end of the day for the overnight fermentation. Samples were collected in the beginning (TO), during (T8 and T32) and at the end (T56) of the in vitro fermentation protocol to collect metabolites and study the metabolite profile (SCFAs and BCFAs). Table 1 indicates the delta change of total SCFAs over the time period T32-T8 and T56-T8. Table 2 indicates the delta change of total BCFAs over the time period T32-T8 and T56-T8. The time point reference of T8 instead of TO was chosen to allow the tryptone fermentation inducing proteolytic dysbiosis to settle.

[0089] Table 1. Total change in SCFA production over time in fecal dysbiotic samples.

[0090] Blank reflects the effect of water on SCFA production and the concentration of SCFA is very low or even negative mimicking the dysbiotic state in which SCFA production is hardly present. The total amount of SCFA increased over time in the fecal dysbiotic samples and peaked around T32, indicating a shift to saccharolytic fermentation (fiber-driven, beneficial). The synbiotic composition synergistically increased the SCFA production in the dysbiotic samples more than was expected based on the additive effect of GOS / FOS and L paracasei L411 .

[0091] Table 2. Total change in BCFA production over time in fecal dysbiotic samples.

[0092] BCFA are indicators of proteolytic fermentation (protein-driven, detrimental) and it was found that proteolytic fermentation decreases over time and its decrease peaked around T32 in the synbiotic composition emphasizing the shift to saccharolytic fermentation that was observed at the same time period. The synbiotic composition synergistically decreased the BCFA production in the dysbiotic samples more than was expected based on the additive effect of GOS / FOS and L paracasei L411 but this was not observed at the time period between T8 and T56.

[0093] In addition, Figure 1 shows the ratio of ASCFA / ABCFA over time. ASCFAs / ABCFAs is a dynamic marker of microbial fermentation balance: Positive values are indicators of dominant proteolytic fermentation (protein-driven, potentially harmful) while negative values are indicators of dominant saccharolytic fermentation (fibre-driven, beneficial). The synbiotic combination (GOS / FOS and L. paracasei strain L411) increased the ratio of the delta change of the Total SCFAs over the delta change of the Total BCFAs (ASCFA / ABCFA) more than compared to GOS / FOS and L411 individually as well as compared to their theoretical effect (sum of their individual values). This figure indicates the ability of the synbiotic product to induce a shift towards saccharolytic fermentation over proteolytic fermentation by producing more SCFAs and at the same time decreasing the concentration of BCFAs and therewith improving gut health in a dysbiotic condition seen in neurodivergent / neurodevelopmental disorders.

[0094] Example 3

[0095] A powdered nutritional composition for young children, which after reconstitution with water (1 scoop of 14.8g powder) provides a ready to feed liquid composition comprising per 100 ml:

[0096] - 67 kcal

[0097] - 1.3 g protein (milk protein)

[0098] - 3.4 g lipids (mainly vegetable lipids)

[0099] - 7.3 g digestible carbohydrate (mainly lactose)

[0100] - 1.15 g of scGOS (source VivinalGOS) and IcFOS (source RaftilinHP) in a 9:1 weight ratio.

[0101] - about 109cfu Lacticaseibacillus paracasei strain 37

[0102] - Vitamins and minerals as known in the art. Example 4

[0103] Supplement to be added to milk, yoghurt, porridge and the like. Sachet comprising per pack:

[0104] 4 g fibres,

[0105] 2.1 g Galacto-oligosaccharides (VivinalGOS powder), - 0.5 g FOS (Raftiline HP), about 109cfu of L. paracasei LPC37.

Claims

Claims1 . A combination of galacto-oligosaccharides and fructo-oligosaccharides with Lacticaseibacillus ssp. for use in preventing and / or treating impaired gut health associated with a gut-brain-axis condition in a subject in need thereof, wherein impaired gut health is selected from gut dysbiosis, gut dysfunction, fragile gut, and / or impaired protein digestion, wherein the gut-brain- axis condition is a neurodivergent / neurodevelopmental disorder and wherein the combination is comprised in an infant formula or follow on formula.

2. The combination for use according to claim 1 or 2, wherein the gut brain axis condition is selected from autism spectrum disorders (ASDs), wherein ASDs are preferably selected from autism disorder (AD), Asperger’s syndrome and pervasive developmental disorder-not otherwise specified (PDD-NOS).

3. The combination for use according to any of the preceding claims, wherein the subject is a neurodivergent subject or has a neurodivergent sibling, preferably is a neurodivergent subject.

4. The combination for use according to any of the preceding claims, wherein the subject suffers from at least one atypical eating behaviour.

5. The combination for use according to any of the preceding claims, wherein the subject is suffering from a gut-brain axis condition selected from autism spectrum disorders (ASDs) or attention deficit hyperactivity disorder (ADHD).

6. The combination for use according to any one of the preceding claims, wherein preventing and / or treating impaired gut health is at least one of: a. increasing the relative abundance of saccharolytic bacterial taxa, preferably selected from Bifidobacteriaceae, Lactobacillaceae, Lachnospiraceae and Veillonellaceae; b. decreasing the relative abundance of proteolytic bacterial taxa, preferably selected from Enterobacterales, Peptostreptococcaceae, Peptostreptococcales- tissirierallales, Oscillospiraceae and Eggerthellaceae; c. decreasing the ratio of Enterobacterales to Bifidobacteriaceae; or d. increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales.

7. The combination for use according to any of the preceding claims, wherein preventing and / or treating impaired gut health is decreasing the ratio of Enterobacterales to Bifidobacteriaceae and / or increasing the ratio of Veillonellaceae plus Bifidobacteriaceae to Enterobacterales.

8. The combination for use according to any of the preceding claims, wherein preventing and / or treating impaired gut health is decreasing the ratio of Enterobacterales to Bifidobacteriaceae.

9. The combination for use according to any of the preceding claims, wherein Lacticaseibacillus ssp. is Lacticaseibacillus paracasei.

10. The combination for use according to any of the preceding claims, wherein the galactooligosaccharides have an average degree of polymerisation in the range from 3-7 and the fructo-oligosaccharides have an average degree of polymerisation in the range from 20-40.11 . The combination for use according to any of the preceding claims, wherein the weight ratio of galacto-oligosaccharides to fructo-oligosaccharides is in the range from 5 : 1 to 12 : 1 , preferably in the range from 8 : 1 to 10 : 1 .

12. The combination for use according to any of the preceding claims, wherein preventing and / or treating impaired gut health is increasing saccharolytic fermentation by gut microbiota and / or decreasing proteolytic fermentation by gut microbiota, preferably both.

13. The combination for use any of the preceding claims, wherein preventing and / or treating impaired gut health is at least one of: increasing the relative amount of short chain fatty acid (SCFA) in the gut; decreasing the relative amount of branched chain fatty acids (BCFA) in the gut; increasing the ratio of SCFA to BCFA in the gut; preferably preventing and / or treating impaired gut health is all of the above.

14. The combination for use according to any of the preceding claims, wherein the combination is comprised in a nutritional composition in an amount of 0.5 to 20 wt%, preferably 1 .5 to 15 wt%, more preferably 2.5 to 12 wt%, even more preferably 5.0 to 10.0 wt%, based on dry weight of the nutritional composition.

15. An infant formula or follow-on formula composition comprising, based on dry weight, 6 - 20 g protein per 100g of the composition, 2 x 103to 3 x 1012colony forming units (cfu) Lacticaseibacillus ssp. per gram of the composition, and 1-20 g of a combination of GOS and FOS per 100g of the composition, preferably wherein the composition comprises, on dry weight, 0.1 - 2.0 g FOS per 100 g of the composition and / or 1 .0 - 20 g GOS per 100 g of the composition.

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