Combination of hmo mixtures with mfgm and uses thereof

Abstract

The present disclosure relates to compositions comprising milk fat globule membrane (MFGM) and an HMO mixture comprising at least one fucosylated HMO and at least one HMO selected from a neutral core HMO or a sialylated HMO. The compositions have potential health benefits in improving brain health, immunity, and recovery time after exercise by increasing the formation of glutamate.

Description

Technical Field

[0001] This disclosure relates to compositions comprising a mixture of a milk fat globule membrane (MFGM) and HMOs, the HMO mixture comprising at least one fucosylated HMO and at least one HMO selected from neutral core HMOs or sialylated HMOs. This composition has potential health benefits in improving brain health, immunity, and post-exercise recovery time by increasing glutamate formation. Background Technology

[0002] The human gut microbiome comprises bacteria, archaea, viruses, and eukaryotic microorganisms, primarily residing in the intestines. These microorganisms possess immense potential to influence human physiological functions in both healthy and disease states. The human gut microbiota is a complex and highly dynamic microbial ecosystem, believed to play many vital roles for its human host, including protecting it from pathogens, inducing immune regulation, and performing nutrient processing and metabolism—fundamental functions that directly or indirectly affect most of our physiological functions. Various gut bacteria, including certain Bifidobacteria, can ingest oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), and human milk oligosaccharides (HMOs), structures that are indigestible in humans. Because some oligosaccharides can be utilized by beneficial microorganisms and induce their growth or activity, these oligosaccharides are called prebiotics.

[0003] Human milk oligosaccharides (HMOs) are a heterogeneous mixture of soluble polysaccharides found in human milk. They are the third largest solid component of human milk after lactose and lipids, present at concentrations of 5–25 g / L. Certain HMOs are considered important for the development of the infant gut microbiota, particularly by enhancing the dominance of Bifidobacteria. HMOs have also shown beneficial effects in maintaining the adult gut microbiota, where they are referred to as prebiotics and are known to increase beneficial Bifidobacteria in the gut and support the establishment and maintenance of a balanced gut microbiota.

[0004] Human breast milk also contains lipids. Milk fat globule (MFG) is secreted by the mammary glands, and its core is mainly composed of triglycerides (98%), surrounded by a phospholipid trilayer structure called the milk fat globule membrane (MFGM). MFGM is composed of phospholipids such as sphingomyelin, phosphatidylcholine, gangliosides, and various proteins, including lactoferrin or mucin. There is evidence that MFGM may have beneficial effects on neurodevelopment and resistance to infection (see, for example, Hernell et al., 2016 J Pediatr173 Suppl:S60-5 doi: 10.1016 / j.jpeds.2016.02.077). MFGM also has benefits for adults, such as stimulating neuromuscular function and reducing stress and anxiety.

[0005] US 11,849,747 describes a combination of MFGM and 2'FL, along with at least one additional component selected from DHA, ARA, vitamin E, vitamin C, and sphingomyelin.

[0006] CN115887626 and CN116391759 describe a composition comprising MFGM, 2'FL and 6'SL or only 6'SL, as well as lactoferrin and osteopontin, for reducing bacterial infections such as Staphylococcus aureus. Staphylococcus golden ) and Escherichia coli ( E.coli )Infect.

[0007] WO2011 / 069987 describes a nutritional composition containing MFGM, probiotics, and prebiotics such as FOS and GOS for the prevention and / or treatment of infections and / or inflammatory conditions, and / or physiological and pathological intestinal discomfort.

[0008] WO2021 / 021746 describes the use of MFGM for the delivery of probiotics, which may be combined with prebiotics such as GOS or a single HMO, for the treatment of intestinal diseases. Summary of the Invention

[0009] This disclosure relates to a composition comprising a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises at least one fucosylated HMO and at least one HMO selected from neutral core HMOs and / or sialylated HMOs, wherein if 6'-sialyl lactose (6'-SL) is present in the mixture, the ratio (w / w) of the fucosylated HMO:6'-SL is greater than 4:1.

[0010] In this embodiment, the fucosylated oligosaccharide is selected from 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3FL), difucosylated lactose (DFL), lactose-N-fucopentose I (LNFP-I), lactose-N-difucohexasose I (LNDFH-I), or mixtures thereof. Preferably, the fucosylated oligosaccharide is fucosylated lactose, such as 2'FL, 3FL, or DFL, or mixtures thereof.

[0011] The sialylated HMO is preferably sialylated lactose, such as 3'-sialylated lactose (3'SL), 6'-sialylated lactose (6'SL), or a combination thereof. The neutral core HMO is preferably lactose-N-tetrasaccharide (LNT) or lactose-N-neotetrasaccharide (LNnT), or a combination thereof.

[0012] The preferred compositions disclosed herein are combinations of MFGM with one of the following HMO mixtures: a. 2'FL and LNT, or b. 2'FL and LNnT, or c. 2'FL, LNT, 6'SL, or d. 2'FL, LNnT and 6'SL, or e. 2'FL, LNnT, 6'SL and 3'SL, or f. 2'FL, DFL, LNnT, 6'SL, and 3'SL, or g. 2'FL, DFL, LNT, 6'SL and 3'SL, or h. 2'FL, 3'FL, LNT, 6'SL and 3'SL, or i. 2'FL, DFL, 3'SL and 6'SL.

[0013] In other aspects of this disclosure, the compositions described herein are used to support or improve brain health in subjects, such as improving cognition and / or immunity, such as reducing inflammation, including reduced symptoms of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) and / or post-exercise recovery time, including reduced muscle fatigue.

[0014] Furthermore, the compositions described herein can be used to increase the abundance of glutamate / glutamate in the gut of subjects compared to untreated subjects or the same subjects before administration of the composition.

[0015] In some implementations, the subject may be an infant or a non-infant, such as a child, or an older adult or elderly individual. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure and components of the milk fat globule membrane (MFGM).

[0017] Figure 2 Box plots showing the levels of the metabolite L-glutamate produced in different donors when using the test compositions in Table 1 are displayed. Sugar-free samples were fecal slurry and control samples without the test compositions. The baseline is represented by a dashed line, reflecting the glutamate level in the culture medium only sample. The Y-axis represents the relative change in glutamate compared to the culture medium only sample, which was set to 0. Detailed Implementation

[0018] This disclosure relates to a composition comprising a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises at least one fucosylated HMO and at least one HMO selected from neutral core HMOs and / or sialylated HMOs.

[0019] In this disclosure, the effects of the compositions described herein were tested in fecal samples undergoing anaerobic fermentation using the compositions of this disclosure and related control compositions. Fecal fermentation allows the microbiota in the sample to interact with the test compositions, indicating what will happen in the colon of a subject. The compositions described herein have been found to increase metabolites with relevant health benefits.

[0020] Specifically, as shown in Example 1, the fermented fecal sample showed an increase in glutamate or glutamate salts. Glutamate is a non-essential amino acid that can be produced by the body itself. Glutamate has been shown to have various health benefits, such as brain health, immunity, and post-exercise recovery.

[0021] Glutamate is a major excitatory neurotransmitter in the brain, involved in many important brain functions, including cognitive functions such as learning and memory. In older adults, low prefrontal glutamate levels are associated with impaired working memory (see, for example, Rmus et al., eLife 2023;12:e85243. DOI: https: / / doi.org / 10.7554 / eLife.85243). In infants and young children, glutamate plays a crucial role in promoting neuronal activity and communication between brain cells. This activity is important for many aspects of brain development, including learning, memory, and the formation of new neural connections. Therefore, glutamate is beneficial for brain health, especially for developing infants and children.

[0022] As Jansson et al. (2014), Journal of Neural Transmission, Volume 121, pages 819-836, describe, glutamate also plays a role in the proliferation, migration, differentiation and survival of neural progenitor cells—and is therefore heavily involved in brain development, which is especially important for infants.

[0023] Glutamate is used by immune cells for energy and supports cell growth and division. It may also help reduce inflammation and improve gut health. Glutamate is important for maintaining the health of the intestinal lining and may help reduce symptoms of conditions such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). In healthy individuals, the concentration of glutamate in the gut is typically in the range of 0.1–1 mM.

[0024] Glutamate may help improve athletic performance by reducing muscle fatigue and improving post-exercise recovery time. Glutamate can be converted into glutamine, which is known to aid in post-exercise muscle recovery (see, for example, Córdova-Martínez et al. 2021 Nutrients 13(6): 2073).

[0025] The terms "microbiota," "microbial community," and "microbiome" are used interchangeably to refer to the living microbial community that typically inhabits an organ or part of the body in animals or humans. Specifically, in the gastrointestinal tract of animals or humans, the microbial community is referred to as the gastrointestinal tract or gut microbiome or microbiota. The most prominent members of the gastrointestinal microbiota in non-infant humans include the following microorganisms at the phylum level: Firmicutes (… Firmicutes Bacteroidetes ( Bacteroidetes ), Actinobacteria ( Actinobacteria Proteobacteria ( Proteobacteria Mutualistic bacteria ( Synergistetes ), Verrucous microbes ( Verrucomicrobia ), Fusobacteria ( Fusobacteria ) and Archaea ( Euryarchaeota ); at the genus level, Bacteroides ( Bacteroides ), Bacillus spp. Faecalibacterium Bifidobacterium spp. Bifidobacterium ), genus Roselle ( Rosemary ), genus *Alternaria* ( Common grebe Collins spp. Collinsella ), Broutella spp. Blueberry ), genus *Plasmodium* Coprococcus ), Rumenococcus ( Ruminococcus ), Eubacterium ( Eubacterium ) and Dorperella spp. Golden ); at the species level, monomorphobacteria ( Bacteroides uniform ), putrefactive bacteria ( Common grebe putrefaction ), fecal parabacterium ( Parabacteroids shit ), Ruminococcus bacillus ( Ruminococcus bromine ), Long-chain Dorperella ( Golden long-chain ), fecal bacterium ( Bacteroides poop ), Bacteroides multiforme ( Bacteroides thetaiotaomicron ), Hodgkin's Eubacterium ( Eubacterium garlic ), Ruminococcus twistens ( Ruminococcus torque ), Prevotella coli ( Faecalibacterium of Prausnitz ), Yogurt Ruminococcus ( Ruminococcus you are milking ), Collins gas-producing bacteria ( Collinsella aeronautical ), Formic acid-producing Dorper ( Golden ant-producing ), Bacteroides commonis ( Bacteroides popular ) and Rosbyrate ( Rosemary intestinal The gut microbiota includes mucosa-associated microbiota located on or attached to the mucus layer covering the gastrointestinal epithelium, and luminal-associated microbiota found in the gastrointestinal lumen.

[0026] The terms “intestine” or “gut” are used interchangeably in this article and refer to the gastrointestinal tract, which consists of the small intestine and the large intestine. The “large intestine” (intestinum crassum) is the lower part of the gastrointestinal tract and is also referred to as the “colon” ​​in this article.

[0027] As used herein, the term “milk fat globule membrane” (“MFGM”) includes lipid components such as phospholipids, cerebrosides, gangliosides, sphingoids or sphingolipids, and / or cholesterol. Furthermore, “milk fat globule membrane” as used herein may include milk fat globule membrane proteins such as mucin 1 (MUC1), mucin 15 (MUC15), lactolipoprotein, adipophilin, CD36, CD14, lactoglucosin (PAS6 / 7), xanthine oxidase, and fatty acid-binding proteins. MFGM proteins have been shown to have health benefits. Mucin 1 has a protective effect on epithelial cells and may help prevent infection and maintain gut health. Lactolipoprotein is involved in immune regulation and has potential anti-inflammatory properties. Adipophilin is involved in lipid metabolism and storage and may play a role in the management of metabolic health and obesity-related conditions. CD36 is involved in lipid metabolism and may have implications for cardiovascular health. Lactoglucosinolates have been shown to have antiviral properties, particularly against rotavirus, which causes severe diarrhea in infants. Xanthine oxidase has antioxidant properties, which can help reduce oxidative stress and may have effects on cardiovascular health. Figure 1A non-limiting structure of the milk fat globule membrane is shown. Phospholipids constitute 30% of the total lipid weight of the MFGM. Phospholipids include, for example, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin. Sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine each constitute 30% of the total phospholipid content in the MFGM. In embodiments, the MFGM used herein comprises or consists of lipids found in natural MFGM membranes, including protein fractions, particularly proteins such as mucin 1, mucin 15, lactopioids, adipocyte differentiation-associated proteins, CD36, lactoglucosin, and xanthine oxidase. In embodiments, the MFGM used herein comprises or consists of lipids found in natural MFGM membranes, without protein fractions. In embodiments, phospholipids constitute 30-60% of the lipid composition of the MFGM, cholesterol 10-30%, glycolipids 5% or less, such as between 0.5% and 3%, and other lipids such as sphingolipids and free fatty acids may also be present. In one embodiment, the MFGM comprises at least the following lipids: phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The MFGM may comprise, consist of, or substantially consist of one or more of the following components, in amounts per 100 g dry weight, (a) to (g) per 100 g dry weight: (a) phospholipids: 400-700 mg, wherein the phospholipids may be distributed as in (b) to (f): (b) phosphatidylcholine: 80-200 mg, (c) phosphatidylethanolamine: 80-200 mg, (d) phosphatidylinositol: 20-65 mg, (e) phosphatidylserine: 30-95 mg, (f) sphingomyelin: 80-200 mg, (g) gangliosides: 18-65 mg. It should be understood that the MFGM used in this disclosure differs from that found in breast milk because its structure is altered during the separation process. In one embodiment, MFGM is isolated from bovine, buffalo, horse, goat, and / or human milk. In another embodiment, MFGM can be provided by including enriched dairy products, such as enriched whey protein concentrate (eWPC), in the nutritional composition. Enriched dairy products generally refer to dairy products that have been enriched with certain milk fat globule membrane (MFGM) components, such as proteins and lipids found in MFGM. Enriched dairy products can be formed, for example, by grading non-human milk (such as cow's milk). The total protein level of the enriched dairy product can range from 20% to 90%, more preferably from 68% to 80%, of which 3% to 50% is MFGM protein; MFGM protein can account for 7% to 13% of the protein content of the enriched dairy product.Enriched dairy products also contain 0.5% to 5% (sometimes 1.2% to 2.8%) sialic acid, 2% to 25% (in some respects 4% to 10%) phospholipids, 0.4% to 3% sphingomyelin, 0.05% to 1.8% and in some respects 0.10% to 0.3% gangliosides, and 0.02% to about 1.2%, more preferably 0.2% to 0.9% cholesterol. MFGM is also available from commercial sources such as Lacprodan® MFGM-10 and Lacprodan PL-20 from Arla, NutriPRO™ milk fat globule membrane (MFGM) from Milk Specialities Global (MSG), MFGM from Univar Solutions, SureStart™ MFGM Lipid 70 / 100 from NZMP Fonterra Cooperative, and Vivinal® MFGM from Friesland Campina. These products may also contain some proteins and carbohydrates, which are not necessarily incorporated into the MFGM structure, but are merely residues from the milk used in production. (Not incorporated, e.g.) Figure 1 The components in the depicted MFGM membrane structure are not considered part of the MFGM.

[0028] In the context of this invention, the term "probiotic" refers to microbial cells or cell preparations, such as bacteria, that, when ingested in adequate amounts, provide benefits to a host (human or animal) by supplementing or otherwise complementing the natural gut microbiota, by eliminating unwanted bacteria in the gastrointestinal tract (GI), by carrying out beneficial metabolic activities along the gastrointestinal tract, or by stimulating the immune system. In some embodiments, the compositions disclosed herein do not contain probiotics because the compositions naturally increase beneficial bacteria, such as Bifidobacteria, in the gut microbiome.

[0029] In the context of this disclosure, bacteria belonging to the genus Bifidobacterium are selected from, but are not limited to, the following Bifidobacterium species: Bifidobacterium adolescentis (… B . of a young man ), Bifidobacterium angularis ( B . angular Bifidobacterium animalis ( B . animal Bifidobacterium animalis subspecies ( B . animal subsp. . animal Bifidobacterium animalis subsp. lactis ( B . animal subsp. . milk ), Bifidobacterium asteroides ( B . asteroidsBifidobacterium lactis ( B . biavatii Bifidobacterium bifidum ( B . bifid ), Bifidobacterium breve ( B . short ), Bifidobacterium chain ( B . chained Bacillus coagulans ( B . coagulating Bifidobacterium longum ( B . long ), Bifidobacterium infantis ( B . child Bifidobacterium longum infantis subspecies ( B . long subsp. . child Bifidobacterium longum subsp. ( B . long subsp. . long ), Bifidobacterium thermophilum ( B . great ), Bifidobacterium tumefaciens ( B . Coryneform Bifidobacterium densiflorum ( B . teeth ), Bifidobacterium galbana ( B . French ) or Bifidobacterium fibrosum ( B . subtle In a preferred embodiment, the genus Bifidobacterium is selected from Bifidobacterium difformis or Bifidobacterium longum, such as Bifidobacterium longum subsp. infantis.

[0030] Any use described herein that results in an increased benefit should be understood as relative to the same subject prior to administration of the composition described herein. Alternatively, the comparison parameter may also consist of two cohorts of individuals, one receiving the composition described herein, and one or more additional cohorts receiving a placebo, such as maltodextrin or lactose, or the same HMO mixture without MFGM. The term cohort is understood as a grouping of individuals with common characteristics, such as age, social, and health factors. The size of the cohort required for a comparative study depends on the statistical variability observed within the cohort.

[0031] "Prebiotic" is a term used to describe compounds in food that induce the growth or activity of beneficial microorganisms, such as bacteria and fungi in the gut of animals or humans. In the gastrointestinal tract, prebiotics can alter the composition of organisms in the gut microbiome. Dietary prebiotics are typically indigestible fibrous compounds that pass undigested through the upper gastrointestinal tract and stimulate the growth or activity of beneficial bacteria in the colon by serving as substrates for these bacteria. Common prebiotics used in food manufacturing include beta-glucan in oats and inulin in chicory roots. Oligosaccharides that are indigestible in humans and animals, such as fructooligosaccharides (FOS) and galactooligosaccharides (GOS), as well as polydextrose (PDX) and human milk oligosaccharides (HMOs), can act as prebiotics for certain bacterial species in the gut. In the context of this disclosure, the composition preferably contains only a mixture of HMOs as prebiotics. Preferably, the composition is free of GOS or PDX.

[0032] In the context of this invention, the term "oligosaccharide" refers to a sugar polymer containing a plurality of monosaccharide units. In some embodiments, preferred oligosaccharides are sugar polymers composed of three to nine monosaccharide units, and preferred oligosaccharides are trisaccharides, tetrasaccharides, pentasaccharides, or hexasaccharides. For the purposes of this invention, the oligosaccharide is a fucoidylated oligosaccharide, preferably a neutral fucoidylated human milk oligosaccharide.

[0033] In this context, the term "human milk oligosaccharide" or "HMO" refers to a complex carbohydrate found in human breast milk. HMOs have a core structure comprising a lactose unit at the reducing end, which can be elongated by one or more β-N-acetyl-lactosamine and / or one or more β-lactose-N-disaccharide units, and this core structure can be partially substituted by α-L-fucopyranosyl and / or α-N-acetyl-neuraminic acid (sialic acid) units. For example, the structure of an HMO was disclosed by Xi Chen in Advances in Carbohydrate Chemistry and Biochemistry 2015, Volume 72, Chapter 4.

[0034] In the context of this disclosure, disaccharides lactose, N-acetyllactosamine, or lactose-N-disaccharides are not considered HMO species.

[0035] HMOs can be non-acidic (or neutral) or acidic. Neutral HMOs lack sialic acid residues, while acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs can be fucosylated or non-fucosylated.

[0036] Examples of such neutral non-fucosylated (neutral core) HMOs include lactose-N-trisaccharide II (LNT-II), lactose-N-tetrasaccharide (LNT), lactose-N-neotetrasaccharide (LNnT), lactose-N-neohexasaccharide (LNnH), p-lactose-N-neohexasaccharide (pLNnH), p-lactose-N-hexasaccharide (pLNH), and lactose-N-hexasaccharide (LNH).

[0037] Examples of neutral fucoidylated HMOs include 2'-fucosyllactose (2'FL), 3-fucosyllactose (3FL), difucosyllactose (DFL), lactose-N-fucopentose I (LNFP-I), lactose-N-difucohexasose I (LNDFH-I), lactose-N-fucopentose II (LNFP-II), lactose-N-fucopentose III (LNFP-III), and lactose-N-difucohexasose. LNDFH-III, Fucosyl-Lactose-N-Hexasaccharide II (FLNH-II), Lactose-N-Fucopentasaccharide V (LNFP-V), Lactose-N-Difucohexasaccharide II (LNDFH-II), Fucosyl-Lactose-N-Hexasaccharide I (FLNH-I), Fucosyl-p-Lactose-N-Hexasaccharide I (FpLNH-I), Fucosyl-p-Lactose-N-Neohexasaccharide II (F-pLNnH II), and Fucosyl-Lactose-N-Neohexasaccharide (FLNnH).

[0038] Examples of acidic HMOs or sialylated HMOs include 3'-sialyl lactose (3'SL), 6'-sialyl lactose (6'SL), 3-fucosyl-3'-sialyl lactose (FSL), 3'-O-sialyl lactose-N-tetrasaccharide a (LST a), fucosyl-LSTa (FLST a), 6'-O-sialyl lactose-N-tetrasaccharide b (LST b), fucosyl-LST b (FLST b), 6'-O-sialyl lactose-N-neotetrasaccharide (LST c), fucosyl-LST c (FLST c), 3'-O-sialyl lactose-N-neotetrasaccharide (LST d), and fucosyl-LST d (FLST d). d) Sialoyl-lactose-N-hexasaccharide (SLNH), sialyl-lactose-N-neohexose I (SLNH-I), sialyl-lactose-N-neohexose II (SLNH-II), and disialoyl-lactose-N-tetrasaccharide (DSLNT).

[0039] The production of various fucosylated oligosaccharides and certain specific HMOs is well known. For example, see the review on HMO production by Bych et al., Current Opinion in Biotechnology, 2019, 56:130-137. In preferred embodiments, fucosylated oligosaccharides (such as the fucosylated HMOs of the present invention) can be produced synthetically, meaning that it is produced in vitro by chemical and / or biological means, such as by chemical reactions, enzymatic reactions, or recombinant cell culture. For example, various fucosylated oligosaccharides can be prepared as described in WO2012 / 127410, WO 2010 / 115934, WO 2010 / 115935, WO2013 / 139344, or PCT / EP2021 / 086932.

[0040] The compositions described herein comprise a mixture of human milk oligosaccharides (HMOs) and milk fat globule membranes (MFGMs). The HMO mixture in the compositions comprises: a) at least one fucosylated HMO, and b) at least one additional HMO selected from neutral core HMOs and / or sialylated HMOs. Preferably, if 6'-sialyl lactose (6'-SL) is present in the HMO mixture, the ratio (w / w) of fucosylated HMO:6'-SL is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1. Even more preferably, at least one fucosylated HMO is 2'FL, and if 6'-sialyl lactose (6'-SL) is present in the mixture, the ratio (w / w) of fucosylated HMO:6'-SL is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1.

[0041] In an alternative embodiment, the HMO mixture in the composition described herein comprises: a) at least one fucosylated HMO, and b) at least one additional HMO selected from neutral core HMOs and / or sialylated HMOs, provided that if the mixture contains 2'FL and no neutral core HMOs are present, the ratio (w / w) of 2'FL:6'-SL (any 6'-SL present in the HMO mixture) will be greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1.

[0042] In one or more preferred embodiments, at least one fucosylated HMO is selected from 2'FL, 3FL, DFL, FSL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II and LNDFH-III or mixtures thereof.

[0043] In selected embodiments, the fucosylated HMO is selected from 2'FL, 3FL, DFL, LNFP-I, and LNDFH-I, or mixtures thereof. More preferably, the fucosylated HMO is fucosylated lactose, such as 2'FL, 3FL, DFL, or mixtures thereof. Most preferably, the fucosylated HMO comprises or consists of 2'FL and DFL.

[0044] In embodiments, the HMO mixture comprises or consists of fucosylated HMO and at least one neutral core HMO. The neutral core HMO is preferably selected from LNT-II, LNT and / or LNnT, and more preferably LNT and / or LNnT.

[0045] In one embodiment, the HMO mixture comprises or consists of 2'FL and DFL, and at least one additional HMO selected from neutral core HMOs.

[0046] In embodiments, the HMO mixture comprises or consists of fucosylated HMO and at least one sialylated HMO. The sialylated HMO is preferably selected from 3'SL, 6'SL, LST a, LST b, LST c, and FSL, or mixtures thereof. More preferably, the sialylated HMO is selected from 3'SL or 3'SL and 6'SL.

[0047] In one embodiment, the HMO mixture comprises or consists of 2'FL and DFL and at least one additional HMO selected from 3'SL or 3'SL and 6'SL or 6'SL, wherein the ratio (w / w) of 2'FL:6'-SL is greater than 4:1, for example greater than 5:1, for example between 4 and 10:1, for example between 4.5 and 8:1, for example between 5.5 and 7:1.

[0048] In one embodiment, the HMO mixture is fucoidan lactose such as 2'FL, 3FL, DFL, or a mixture thereof with LNT.

[0049] In another embodiment, the HMO mixture is fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with LNnT.

[0050] In another embodiment, the HMO mixture is fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with LNT and LNnT.

[0051] In another embodiment, the HMO mixture is either 2'FL alone or a combination of 2'FL and DFL with LNnT.

[0052] In another embodiment, the HMO mixture is either 2'FL alone or a combination of 2'FL and DFL with LNT.

[0053] In another embodiment, the HMO mixture is either 2'FL alone or a combination of 2'FL and DFL with LNT and LNnT.

[0054] In one embodiment, the HMO mixture is fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with 3'SL.

[0055] In another embodiment, the HMO mixture is fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with 6'SL.

[0056] In one embodiment, the HMO mixture is fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with 3'SL and 6'SL.

[0057] In another embodiment, the HMO mixture is either a single 2'FL or a combination of 2'FL and DFL with 3'SL.

[0058] In another embodiment, the HMO mixture is either a single 2'FL or a combination of 2'FL and DFL with 6'SL.

[0059] In another embodiment, the HMO mixture is either a single 2'FL or a combination of 2'FL and DFL with 3'SL and 6'SL.

[0060] In one embodiment, the HMO mixture is a combination of fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with 6'SL and LNT.

[0061] In one embodiment, the HMO mixture is a combination of fucoidosyl lactose such as 2'FL, 3FL, DFL, or a mixture thereof with 6'SL and LNnT.

[0062] In one embodiment, the HMO mixture is a combination of 2'FL with 6'SL and LNT or LNnT.

[0063] In one embodiment, the HMO mixture is a combination of fucoidosyl lactose such as 2'FL, 3FL, DFL or a mixture thereof with 3'SL and 6'SL and LNT or LNnT.

[0064] In another embodiment, the HMO mixture is either 2'FL alone or a combination of 2'FL and DFL with 3'SL and 6'SL and LNnT.

[0065] In another embodiment, the HMO mixture is either a single 2'FL or a combination of 2'FL and DFL with 3'SL and 6'SL and LNT.

[0066] In one embodiment, the HMO mixture is a combination of 2'FL and 3FL with 3'SL and 6'SL and LNT or LNnT.

[0067] In one embodiment, the HMO mixture is a combination of 2'FL and 3FL with 3'SL and 6'SL and LNT.

[0068] In one embodiment, the HMO mixture is a combination of 2'FL and 3FL with 3'SL and 6'SL and LNnT.

[0069] In a preferred embodiment, the fucoidylated HMO comprises at least 35 wt%, such as at least 40 wt%, such as at least 45 wt%, such as at least 50 wt%, of the HMO mixture. Preferably, 2'FL comprises at least 35 wt%, such as at least 40 wt%, such as at least 45 wt%, such as at least 50 wt%, of the HMO mixture.

[0070] In some embodiments, the HMO mixture accounts for at least 90 wt%, such as at least 95 wt%, such as at least 98 wt%, of the total HMO in the composition, and the HMO mixture is selected from mixtures comprising or consisting of the following: a) 2'-FL and 3'-SL, with a 2'-FL:3'-SL ratio between 9.5 and 18:1, such as between 11 and 16:1, or b) 2'-FL and 6'-SL, with a 2'-FL:6'-SL ratio between 4-10:1, such as between 4.5-8:1, or between 5.5-7:1, or c) 2'-FL and LNnT, with a 2'-FL:LNnT ratio between 2 and 8:1, such as between 4 and 6.5:1, or d) 2'-FL and LNT, with a 2'-FL:LNT ratio between 1.5 and 6:1, such as between 2 and 4:1, or e) 2'-FL, LNT, and LNnT, with the ratio of 2'-FL:LNT:LNnT between 4-8:1-3:1, such as between 5-7:1.5-2.5:1, or f) 2'-FL, LNnT, and DFL, with the ratio of 2'-FL:LNnT:DFL between 6-11:0.5-3:1, such as between 7-9:1-2:1, or g) 2'-FL, LNT, and DFL, with the ratio of 2'-FL:LNT:DFL between 6-11:0.5-4:1, such as between 7-9:1.5-3:1, or h) 2'-FL, LNnT, and 3'-SL, with the ratio of 2'-FL:LNnT:3'-SL between 10-18:1-5:1, such as between 12-16:2-4:1, or i) 2'-FL, 6'-SL, and LNnT, with the ratio of 2'-FL:6'-SL:LNnT between 3-9:0.5-2:1, such as between 4-8:1-1.5:1, or j) 2'-FL, LNT, and 3'-SL, with the ratio of 2'-FL:LNT:3'-SL between 8-16:2-6:1, such as between 10-14:3-5:1, or k) 2'-FL, LNT, and 6'-SL, with the ratio of 2'-FL:LNT:6'-SL between 4-8:1 and 4:1, or between 5-7:1 and 3:1, or l) 2'-FL, DFL, and 3'-SL, with the ratio of 2'-FL:DFL:3'-SL between 6-10:0.5-3:1, such as between 7-9:1-2:1, or m) 2'-FL, DFL, and 6'-SL, with the ratio of 2'-FL:DFL:6'-SL between 5-10:0.5-3:1, such as between 7-9:1-2:1, or n) 2'-FL, DFL, 6'-SL, and 3'SL, with the ratio of 2'-FL:DFL:6'-SL:3'SL between 8-16:0.5-3:1-4:1, such as between 9-14:1-2:1.5-3:1, or o) 2'-FL, LNnT, 6'-SL, and 3'SL, with the ratio of 2'-FL:LNnT:6'-SL:3'SL between 8-16:2-6:1-4:1, such as between 9:14:3-5:1-3:1, or p) 2'-FL, LNT, 6'-SL and 3'SL, 2'-FL:LNT:6'-SL:3'SL between 8-16:1-4:1-4:1, such as between 9:14:1-3:1-3:1, or q) 2'-FL, 3'-FL, LNT, 6'-SL, and 3'-SL, with the ratio of 2'-FL:3'-FL:LNT:6'-SL:3'-SL between 8-16:2-6:2-6:0.5-4:1, and between 10-14:3-5:3-5:1-3:1. r) 2'-FL, DFL, LNnT, 6'-SL and 3'-SL, the ratio of 2'-FL:DFL:LNnT:6'-SL:3'-SL is between 8-16:1-3:0.5-4:0.5-4:1, such as between 10-14:1-2:3-5:3-5:1.

[0071] In embodiments of this disclosure, the composition comprises a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture is selected from one of the following mixtures: a. 2'FL and LNT, or b. 2'FL and LNnT, or c. 2'FL, LNT, 6'SL, or d. 2'FL, LNnT and 6'SL, or e. 2'FL, LNnT, 6'SL and 3'SL, or f. 2'FL, DFL, LNnT, 6'SL, and 3'SL, or g. 2'FL, DFL, LNT, 6'SL and 3'SL, or h. 2'FL, 3'FL, LNT, 6'SL and 3'SL, or i. 2'FL, DFL, 3'SL and 6'SL, and The selected HMO mixture accounts for at least 90 wt% of the total HMO in the composition, such as at least 95 wt% or at least 98 wt%.

[0072] In a preferred embodiment of this disclosure, the composition comprises a milk fat globule membrane (MFGM) and a mixture of human milk oligosaccharides (HMOs) comprising 2'FL, DFL, 3'SL, and 6'SL, wherein the HMO mixture comprises at least 90 wt%, such as at least 95 wt%, such as at least 98 wt%, of the total HMOs in the composition.

[0073] In a preferred embodiment of this disclosure, the composition comprises a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture is substantially composed of the following: a. 2'FL and LNT, or b. 2'FL and LNnT, or c. 2'FL, LNT, 6'SL, or d. 2'FL, LNnT and 6'SL, or e. 2'FL, LNnT, 6'SL and 3'SL, or f. 2'FL, DFL, LNnT, 6'SL, and 3'SL, or g. 2'FL, DFL, LNT, 6'SL and 3'SL, or h. 2'FL, 3'FL, LNT, 6'SL and 3'SL, or i. 2'FL, DFL, 3'SL and 6'SL.

[0074] In a preferred embodiment of this disclosure, the composition comprises a milk fat globule membrane (MFGM) and a mixture of human milk oligosaccharides (HMOs) consisting essentially of 2'FL, DFL, 3'SL, and 6'SL.

[0075] In embodiments of this disclosure, the composition comprises a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises or is substantially composed of the following: a. 60 wt% to 80 wt% 2'FL and 20 wt% to 40 wt% LNT, or b. 65 wt% to 90 wt% 2'FL and 10 wt% to 35 wt% LNnT, or c. 60 wt% to 75 wt% 2'FL, 15 wt% to 25 wt% LNT, 5 wt% to 15 wt% 6'SL, or d. 65 wt% to 85 wt% 2'FL, 5 wt% to 20 wt% LNnT and 8 wt% to 20 wt% 6'SL, or e. 60 wt% to 75 wt% 2'FL, LNnT, 6'SL and 3'SL, or f. 60 wt% to 75 wt% 2'FL, 5 wt% to 12 wt% DFL, 5 wt% to 15 wt% LNnT, 5 wt% to 15 wt% 6'SL and 2 wt% to 10 wt% 3'SL, or g. 50 wt% to 70 wt% 2'FL, 3 wt% to 12 wt% DFL, 12 wt% to 25 wt% LNT, 5 wt% to 15 wt% 6'SL and 2 wt% to 10 wt% 3'SL, or h. 45 wt% to 65 wt% 2'FL, 10 wt% to 20 wt% 3FL, 10 wt% to 25 wt% LNT, 5 wt% to 15 wt% 6'SL and 2 wt% to 8 wt% 3'SL, or i. 45 wt% to 70 wt% 2'FL, 2 wt% to 10 wt% DFL, 5 wt% to 15 wt% 6'SL and 5 wt% to 15 wt% 3'SL, and The selected HMO mixture accounts for at least 95 wt% of the total HMO in the composition, such as at least 97 wt%, at least 98 wt%, at least 99 wt%, or at least 100 wt%. In a preferred embodiment of this disclosure, the composition comprises a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises or substantially comprises 45 wt% to 70 wt% 2'FL, 2 wt% to 10 wt% DFL, 5 wt% to 15 wt% 6'SL and 5 wt% to 15 wt% 3'SL, and wherein the selected HMO mixture accounts for at least 95 wt%, such as at least 97 wt%, such as at least 98 wt%, such as at least 99 wt%, such as 100 wt% of the total HMOs in the composition.

[0076] The compositions disclosed herein may contain or contain a mixture of about 25 wt% to 50 wt% MFGM and 50 wt% to 75 wt% HMO.

[0077] In one embodiment, the MFGM is primarily composed of lipids, such as 70% to 100% lipids. In another embodiment, phospholipids comprise 20%-50% of the MFGM, and cholesterol comprises 3%-10%. Furthermore, glycolipids may comprise 0.5% to 5% of the MFGM, such as 1% to 3%, and other lipids such as sphingolipids and fatty acids may constitute the remaining lipid component of the MFGM. In embodiments, the MFGM comprises, or is composed of, phospholipids, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Preferably, the MFGM phospholipids comprise 20%-30% phosphatidylcholine and 20%-30% phosphatidylethanolamine, 5%-15% phosphatidylserine, and 15%-30% sphingomyelin.

[0078] The compositions disclosed herein may contain additional ingredients, such as nutrients, fillers, or carriers. In such compositions, the ratio between the MFGM and HMO mixture is between 1 and 3, such as between 1 and 2.5, such as between 1 and 2, such as between 1 and 1.5, such as 1:1.

[0079] In a preferred embodiment, the compositions of this disclosure may contain non-HMO oligosaccharides (cellulose), such as FOS, GOS, and / or PDX. Preferably, the non-HMO oligosaccharides constitute less than 10 wt%, such as less than 5 wt%, such as less than 2 wt%, of the compositions of this disclosure. Preferably, the compositions of this disclosure are substantially free of FOS, GOS, and PDX.

[0080] In some implementations, the MFGM component and the HMO mixture are formulated together. Alternatively, the MFGM component and the HMO mixture may be formulated as two separate compounds (e.g., in a kit of parts), which are then mixed in the final product, such as a nutritional or pharmaceutical product.

[0081] The compositions disclosed herein may be in the form of pharmaceutical compositions or medical foods.

[0082] The compositions disclosed herein are preferably nutritional compositions, such as dietary supplements, infant formula, growing milk, or food supplements. It should be understood that a nutritional composition refers to a formulated mixture of individual ingredients designed to provide essential dietary elements. A synthetic nutritional composition is a composition in which at least one ingredient is obtained through a biological process (such as enzymatic or fermentation) or a chemical process. In the context of this disclosure, breast milk is not considered a nutritional composition because it is not formulated from individual ingredients. In a preferred embodiment, the nutritional composition is a synthetic nutritional composition.

[0083] In the nutritional compositions disclosed herein, MFGM is preferably added in an amount of 0.2 to 10 mg / ml, such as 0.5 to 5 mg / ml, such as 1 to 10 mg / ml, such as 1.5 mg / ml to about 7.5 mg / ml, and HMO mixture is preferably added in an amount of 0.2 to 20 mg / ml, such as 0.5 to 10 mg / ml, such as 1 to 15 mg / ml.

[0084] The nutritional composition may additionally contain one or more ingredients selected from the following: i. Carbohydrates such as lactose and / or inositol; ii. Protein, preferably plant-based protein such as algal protein, pea protein or rapeseed protein, and some proteins may be hydrolyzed protein; iii. Long-chain polyunsaturated fatty acids (LCPUFA), such as docosahexaenoic acid (DHA) and / or arachidonic acid (ARA). iv. Vitamins, such as one or more of vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6, vitamin B12, folic acid (vitamin B9), pantothenic acid (vitamin B5) and / or choline; v. Salts, such as one or more of calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, selenium, sodium, potassium and / or chlorides; vi. One or more enzymes or functional proteins, such as biotin, coenzyme Q10, and / or lactoferrin; and vii. Antioxidants, such as one or more of resveratrol, α-carotene, β-carotene, β-cryptoxanthin, lycopene, lutein, zeaxanthin, retinol and / or α-tocopherol.

[0085] Lactoferrin is a multifunctional protein belonging to the transferrin family. It is a globular glycoprotein with a molecular weight of approximately 80 kDa. Lactoferrin is one of the transferrins that transports iron into cells and controls the level of free iron in the blood and exocrine glands. Lactoferrin has a very high isoelectric point (~p1 9), and its cationic properties play a major role in its ability to resist bacterial, viral, and fungal pathogens. The lactoferrin used in this disclosure can be isolated, for example, from the milk of non-human animals or produced by genetically modified organisms. The oral electrolyte solutions described herein may contain non-human lactoferrin, non-human lactoferrin produced by genetically modified organisms, and / or human lactoferrin produced by genetically modified organisms.

[0086] In one embodiment, the composition containing MFGM and HMO disclosed herein does not contain lactoferrin.

[0087] Osteopontin is a highly glycosylated and phosphorylated protein found in high concentrations (140 mg / L) in breast milk. Known as milk protein, it is believed to mediate the cognitive, gut, and immune system development of newborns.

[0088] In one embodiment, the composition containing MFGM and HMO disclosed herein does not contain osteopontin.

[0089] Use of the composition This disclosure relates to one or more methods for providing health benefits. These health benefits may be non-medical, but may also involve the treatment of a condition, disease, or disorder. A method for providing health benefits is achieved by administering to a subject a composition comprising a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises a) at least one fucosylated HMO, and b) at least one HMO selected from neutral core HMOs and / or sialylated HMOs. Preferably, if 6'-sialyl lactose (6'-SL) is present in the HMO mixture, the ratio (w / w) of fucosylated HMO:6'-SL is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1. Even more preferably, at least one fucosylated HMO is 2'FL, and if 6'-sialyl lactose (6'-SL) is present in the mixture, the fucosylated HMO:6'-SL ratio (w / w) is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1. The amount of the composition administered is preferably in the range of 1 to 15 g MFGM per day and 1 to 20 g HMO mixture per day. Preferably, the compositions disclosed herein are administered in an amount (effective amount) that effectively achieves the desired health benefits. Efficacy can be assessed by comparison with pre-treatment subjects or control subjects who have not received the treatment.

[0090] Furthermore, this disclosure provides for non-medical uses of the compositions described herein. In particular, such non-medical uses may be for use in dietary supplements or infant formula or growing milk or food supplements.

[0091] One aspect of this disclosure is a composition as described herein, such as a composition comprising a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises a) at least one fucosylated HMO, and b) at least one HMO selected from neutral core HMOs and / or sialylated HMOs, for use in a subject to support or improve one or more of the following: a. Brain health, and / or b. Immunity, and / or c. Recovery time after exercise.

[0092] Preferably, if 6'-sialyl lactose (6'-SL) is present in the HMO mixture, the ratio (w / w) of fucosylated HMO:6'-SL is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1. Even more preferably, at least one fucosylated HMO is 2'FL, and if 6'-sialyl lactose (6'-SL) is present in the mixture, the ratio (w / w) of fucosylated HMO:6'-SL is greater than 4:1, such as greater than 5:1, such as between 4 and 10:1, such as between 4.5 and 8:1, such as between 5.5 and 7:1.

[0093] One embodiment is the use of the compositions of this disclosure in supporting or improving brain health in a subject (non-medical), particularly relating to the use of improving cognitive, learning, and memory abilities. Preferably, the subject is an infant or an elderly person. In infants, the use of the compositions of this disclosure promotes brain development by supporting the proliferation, migration, differentiation, and survival of neural progenitor cells, as well as promoting neuronal activity and communication between brain cells and the formation of new neural connections.

[0094] One embodiment is the use of the compositions disclosed herein for the purpose of supporting gut health and a healthy immune system by maintaining the health of the intestinal lining, particularly for non-medical purposes.

[0095] The compositions disclosed herein can also be used to reduce inflammation or its symptoms, particularly in subjects who require this. This, in turn, supports a healthy immune system.

[0096] The compositions disclosed herein can also be used to prevent or alleviate symptoms associated with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Preferably, the compositions disclosed herein are administered to subjects in need at an effective dose.

[0097] One embodiment is the use (non-medical) of the compositions disclosed herein for improving athletic performance by reducing muscle fatigue and improving post-exercise recovery time. The compositions disclosed herein may be particularly useful for athletes with high levels of physical activity. The compositions may be sports nutrition products.

[0098] In embodiments, the use (non-medical or therapeutic) of the compositions of this disclosure is to increase the abundance / concentration / formation of glutamate in the gut of a subject. This increase is preferably assessed relative to the level of glutamate in the gut prior to administration of the compositions of this disclosure. Alternatively, the ability of a subject to produce glutamate after administration of the compositions of this disclosure can be assessed by fermenting a fecal sample of the subject according to the method of Example 1.

[0099] In some embodiments, the “subject” can be a human or mammal, or other livestock with a gut microbiome, such as pets (cats, dogs, rodents, rabbits, birds, reptiles, etc.), livestock and domesticated animals (pigs, poultry, goats, sheep, and dairy cows), and working animals (horses, cattle, camels, donkeys, and elephants). Preferably, the subject is a human, such as an infant or a non-infant human. The term “infant human” or “infant” refers to a person under 3 years of age, and an infant is a child aged 1 to 3 years. An infant can be a premature infant, meaning delivered before 37 weeks of gestation. An infant can be delivered by cesarean section, meaning that the infant is not exposed to the natural microbiome in the woman’s vagina. The term “non-infant human” or “non-infant” refers to a human aged 3 years and older. A non-infant human can be a child (3 to 12 years old), an adolescent (13 to 19 years old), an adult, or an elderly person. In the human context, the term “elderly person” refers to someone who has been at least 60 years old since birth, preferably over 65 years old, and more preferably over 70 years old. In human context, the term "older adult" refers to someone who has been at least 40 years old since birth, preferably over 50 years old, more preferably over 55 years old, including elderly individuals.

[0100] As used herein, the term "treat" (or "treatment" or "treating") refers to treating an existing disease (such as a disease, symptom, or disorder as referred to herein) or preventing disease, i.e., prevention. In the context of this invention, maintaining and / or promoting the health of an individual who is not suffering from a disease but may be susceptible to developing unhealthy conditions is considered non-medical treatment.

[0101] The “effective amount” of the compositions disclosed herein refers to the amount that produces the desired health benefit / therapeutic outcome in a subject to which it is administered. The effective amount may be administered in one or more doses to achieve the desired therapeutic effect.

[0102] Preferably, the effect of the use of the composition disclosed herein is evaluated after daily administration of one dose of the composition disclosed herein for at least one week, such as at least two weeks, such as at least three weeks.

[0103] "Enterovenous administration" refers to any conventional method of delivering a composition to a subject, resulting in the deposition of the composition in the gastrointestinal tract, including the stomach. Methods of enteral administration include feeding via nasogastric tube or jejunal tube, oral administration, direct delivery to the intestine, sublingual administration, and rectal administration.

[0104] "Oral administration" refers to any conventional form of delivery of the composition to a non-infant via oral administration. Therefore, oral administration is a form of enteral administration.

[0105] The appropriate dosage of the combinations disclosed herein can be determined at least in part based on factors such as immune status, weight, and age. In some cases, the dosage of the HMO mixture will be similar to the amount of a specific HMO component (e.g., 2'FL, DFL, 3FL, LNT, LNnT, 3'SL, 6'SL) found in human breast milk. The recommended amount of the HMO mixture is generally in the range of about 1 g to about 20 g per day, in some embodiments about 2 g to about 15 g per day, about 3 g to about 10 g per day, and in some embodiments about 1 g to about 10 g per day. The appropriate dosage regimen can be determined based on the present disclosure and / or factors known to those skilled in the art. The dosage of the MFGM component in the compositions disclosed herein is generally in the range of about 1 g to about 15 g per day, in some embodiments about 1.5 g to about 10 g per day, about 2 g to about 5 g per day, and in some embodiments about 1 g to about 7 g per day.

[0106] Example Material Prepare the mixtures listed in Table 1 using the following products. 2'FL: GlyCare 2FL 9000 2'FL / DFL: GlyCare 2FL / DFL 8001 3FL: dsm-firmenich development LNnT: GlyCare LNnT 9000 LNT: GlyCare LNT 8001 3'SL: GlyCare 3SL 9001 6'SL: GlyCare 6SL 9001 MFGM: SureStart™ MFGM LIPID 70 GOS: Available, for example, from BOC Sciences (catalog number: B1999-003025), Biosynth (OG32134), or NZMP's SureStart GOS. PDX: Sigma 68424-04-4 Example 1 - Glutamic acid In this embodiment, the effect of MFGM on HMO mixtures was tested.

[0107] A series of HMO mixtures (test compositions) containing at least one fucosylated HMO and at least one neutral core HMO or sialylated HMO were compared with the same HMO mixtures without MFGM.

[0108] The mixtures are shown in Table 1.

[0109] Table 1: The amounts of each component in the mixtures used in this embodiment are expressed in g / L of the final measured volume. The experimental setup was as follows. Fecal samples were obtained from infant donors (2 to 5 months of age) (n=5). The samples were prepared into a 10% slurry using PBS and glycerol. The slurry was aliquoted into 48-well multi-well plates (MTP) and added to Prodigest minimal nutrient medium. Two control samples were added to each plate: one containing fecal slurry without the test mixture (sugar-free), and one containing only the culture medium. Fecal samples were added to the test mixture listed in Table 1 (n=2). Five donors were used to prepare a final volume of 0.6 mL, and the samples were incubated in a BioLector XT at 37 °C for 24 hours with shaking at 600 rpm under anaerobic conditions and a continuous nitrogen flow. The samples were then transferred to 96-well plates and centrifuged at 4000 rpm for at least 10 minutes at 4 °C. The supernatant was collected for analysis or stored at -80 °C until further analysis.

[0110] The sample supernatant was analyzed internally using liquid chromatography-mass spectrometry (LC-MS) metabolomics.

[0111] To enable comparison of metabolite profiles across analytical batches, all data were center-scaled. This normalization technique adjusts the data so that the mean is zero, mitigating any bias caused by variations in sample volume. Furthermore, for each Biolectrode run, the metabolite concentrations measured in the basal medium were subtracted from the concentrations measured in all experiments within that run. This step removes background signals from the medium itself, allowing direct comparison of cell-produced metabolites across different experiments.

[0112] result Data such as Figure 2 As shown.

[0113] The data show that MFGM alone (01) had no effect on glutamate formation compared to the sugar-free control. For samples containing HMO mixtures, these samples were tested in pairs, one with MFGM (even-numbered) and one without (odd-numbered). This indicates that all HMO mixtures without MFGM had a negative effect on glutamate formation compared to the sugar-free sample. Interestingly, all samples with MFGM and containing 2'FL and at least one neutral core HMO and / or sialylated HMO showed increased glutamate formation compared to the sugar-free control. This is highly surprising and clearly a synergistic effect, as the individual components of the tested mixtures (MFGM or HMO mixtures) showed no benefit, so combining them could not have been expected to increase glutamate levels. In particular, the effect on glutamate was significantly different for the mixture containing MFGM + 2'FL / DFL + 6'SL + 3'SL (06) compared to the HMO mixture without MFGM (07).

Claims

1. A composition comprising a mixture of milk fat globule membrane (MFGM) and human milk oligosaccharides (HMOs), wherein the HMO mixture comprises: a. at least one fucosylated HMO, and b. At least one HMO selected from neutral core HMOs and / or sialylated HMOs, in, If 6'-sialyl lactose (6'-SL) is present in the mixture, the ratio (w / w) of the fucosylated HMO:6'-SL is greater than 4:

1.

2. The composition according to claim 1, wherein the fucosylated HMO is selected from 2'-fucosylated lactose (2'FL), 3-fucosylated lactose (3FL), difucosylated lactose (DFL), lactose-N-fucopentose I (LNFP-I), and lactose-N-difucohexasose I (LNDFH-I) or mixtures thereof.

3. The composition according to claim 1 or 2, wherein the fucosylated HMO accounts for at least 35 wt% of the HMO mixture.

4. The composition according to any one of the preceding claims, wherein the neutral core HMO is selected from lactose-N-tetrasaccharide (LNT) and lactose-N-neotetrasaccharide (LNnT) or a mixture thereof.

5. The composition according to any one of the preceding claims, wherein the sialylated HMO is selected from 3'-sialyl lactose (3'SL) and 6'-sialyl lactose (6'SL) or a mixture thereof.

6. The composition according to any one of the preceding claims, wherein the composition comprises at least 2'FL, preferably both 2'-FL and DFL.

7. The composition according to any one of the preceding claims, wherein the composition further comprises 3'-SL or 6'SL or both.

8. The composition according to any one of the preceding claims, wherein the composition comprises LNT or LNnT or both.

9. The composition according to any one of the preceding claims, wherein the HMO mixture is selected from one of the following mixtures: a. 2'FL and LNT, or b. 2'FL and LNnT, or c. 2'FL, LNT, 6'SL, or d. 2'FL, LNnT and 6'SL, or e. 2'FL, LNnT, 6'SL and 3'SL, or f. 2'FL, DFL, LNnT, 6'SL, and 3'SL, or g. 2'FL, DFL, LNT, 6'SL and 3'SL, or h. 2'FL, 3'FL, LNT, 6'SL and 3'SL, or i. 2'FL, DFL, 3'SL and 6'SL, and The selected HMO mixture accounts for at least 95 wt% of the total HMO in the composition.

10. The composition according to claim 1 or 9, wherein the HMO mixture is substantially composed of: a. 2'FL and LNT, or b. 2'FL and LNnT, or c. 2'FL, LNT, 6'SL, or d. 2'FL, LNnT and 6'SL, or e. 2'FL, LNnT, 6'SL and 3'SL, or f. 2'FL, DFL, LNnT, 6'SL, and 3'SL, or g. 2'FL, DFL, LNT, 6'SL and 3'SL, or h. 2'FL, 3'FL, LNT, 6'SL and 3'SL, or i. 2'FL, DFL, 3'SL and 6'SL.

11. The composition according to claim 9 or 10, wherein the HMO mixture comprises or consists of 2'FL, DFL, 3'SL and 6'SL.

12. The composition according to any one of the preceding claims, wherein the composition is a nutritional composition, such as infant formula or growing milk.

13. The nutritional composition of claim 12, wherein the MFGM is added in an amount of 1 to 10 mg / ml, and the HMO mixture is added in an amount of 1 to 15 mg / ml.

14. The composition according to any one of claims 1 to 13 supports or improves the use of one or more of the following in a subject: a. Brain health, such as improved cognition, learning, and / or memory, and / or b. Immunity, such as reducing inflammation, including reducing symptoms of irritable bowel syndrome (IBS) and / or inflammatory bowel disease (IBD), and / or c. Post-exercise recovery time, including reducing muscle fatigue.

15. Use of the composition according to claim 14, wherein the improvement in brain health and / or immunity and / or recovery time is based on increasing the production (concentration) of glutamate in the gut.

16. Use of the composition according to claim 14 or 15, wherein the improvement of immunity encompasses the activation and regulation of immune cells, the promotion of the body's defense against pathogens, and the maintenance of immune homeostasis.

17. Use of the composition according to claims 14 to 16, wherein the subject is an infant, a child, an older adult, or an elderly individual.

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