Human milk oligosaccharides for enhancing executive function

Abstract

Use of human milk oligosaccharides (HMOs) or nutritional compositions comprising human milk oligosaccharides for enhancing executive function in non-infants, preventing and / or reducing the risk of suboptimal executive function in non-infants, managing suboptimal executive function in non-infants, and / or improving myelination to mature the prefrontal cortex region of the brain in non-infants, and the use of synthetic compositions of HMOs and encapsulated products comprising at least 14 individual daily doses of an effective amount of at least one HMO in such applications.

Description

Technical Field

[0001] The present invention relates to the use of human milk oligosaccharides (HMO) or compositions comprising HMOs for enhancing executive function in non-infants. The present invention also relates to HMOs or compositions comprising HMOs for preventing or managing suboptimal executive function in non-infants. Background Art

[0002] Executive function is the ability to coordinate and integrate cognitive-perceptual processes related to time and space, determining the subject's ability to identify, evaluate, and make choices among various alternative options and strategies. It controls goal-directed behavior and plays a fundamental role in regulating higher-order cognitive processes such as problem solving, reasoning, flexible thinking, and decision-making. It is central to cognitive development and learning (especially new skills) and is closely associated with social and intellectual / academic success and / or achievement.

[0003] In humans, the abilities associated with executive functions do not develop all at once. Instead, they develop in a sequence, with one skill building on previous skills. All executive functions interact with each other and influence how humans regulate their behavior to create positive future outcomes. Typically, executive functions begin to develop at age two and are fully developed by age 30. Executive functions typically begin to decline after age 30, most commonly in relation to memory.

[0004] Executive function is believed to be mediated by the prefrontal cortex. Generally speaking, information is stored in the back of the brain, and humans use the prefrontal cortex to process stored information in order to be socially effective and successful in life. Executive function requires four main control circuits, as follows:

[0005] The task-oriented circuit, which controls working memory and allows execution of plans, specific steps required to complete a task, and achieve goals.

[0006] Time-oriented loops allow for the organization of the order in which tasks are completed and the scheduling of schedules.

[0007] The emotion-directed circuit, which controls emotions and mood.

[0008] The mind-directing circuit, which controls self-awareness of sensations and experiences.

[0009] Non-infant humans with insufficient executive function (or executive dysfunction - EFD) work hard to organize and regulate their behavior to help them achieve long-term goals. In general, non-infant humans with EFD are more motivated by short-term goals than long-term goals. The result of insufficient executive function is often less success in life.

[0010] Despite the very real impact of EFD, the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) does not recognize EFD as a specific mental health condition. Instead, problems with executive function are symptoms of other neurological, mental health, and behavioral disorders. Therefore, there is no diagnosis for EFD, only diagnoses of neurological, mental health, or behavioral disorders that include symptoms of executive function problems. Typical symptoms are difficulty controlling emotions or impulses, difficulty starting, organizing, planning, or completing tasks, difficulty listening or paying attention, problems with short-term memory, inability to multitask or balance tasks, socially inappropriate behavior, inability to learn from past consequences, difficulty problem solving, and difficulty learning or processing new information.

[0011] Once symptoms are evident, a variety of tests can be used to help assess a person's executive function. For example, tests such as the Stroop task, which assesses a person's mental control and selective attention, the trail-tracking test, the clock-drawing test, the verbal fluency test, and the card-sorting test. Otherwise, if a specific mental disorder is suspected, medical professionals may skip executive function testing and instead compare a person's symptoms to standard diagnostic criteria for that disorder or use an MRI scan to detect physical causes, such as a stroke injury or a brain tumor.

[0012] Diagnosable conditions that can cause executive function problems include attention deficit hyperactivity disorder (ADHD), depression and anxiety, bipolar disorder, schizophrenia, obsessive compulsive disorder (OCD), autism, Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, Tourette's syndrome, post-traumatic stress disorder (PTSD), and traumatic brain injury. However, executive function problems can also have temporary causes, such as exhaustion, severe pain, stress, distracting environments, drug abuse, alcohol abuse, and extreme boredom. In addition, people who are born prematurely or small for gestational age (SGA) appear to be at greater risk for executive function problems. When executive function problems or disorders are present, the type of treatment used depends on the diagnosed condition that is thought to be causing the problem or disorder. Treatment options may include stimulant medications, antidepressants, antipsychotics, psychoeducation, occupational or speech therapy, and cognitive behavioral therapy (CBT). First-line treatment is usually occupational or speech therapy, psychotherapy, reading tutoring, or CBT. CBT is highly effective in treating executive function problems in adults, including inhibition, emotion regulation, time management, and planning problems, when it is combined with medication to treat the diagnosed disorder. However, CBT is less effective in children. In addition, there are not always effective medications for the diagnosed disorder, or the medications have severe side effects, which can reduce patient compliance. Furthermore, there are extremely limited avenues for preventing executive function problems or disorders in at-risk non-infant humans.

[0013] Therefore, there remains a need for a safe, well-tolerated approach to prevent or manage executive function problems or disorders in non-infant humans. Summary of the invention

[0014] It has now been surprisingly discovered that human milk oligosaccharides may prevent executive function problems or disorders, may enhance executive function, and may better manage executive function problems or disorders in non-infant humans.

[0015] Therefore, in a first aspect, the present invention relates to a human milk oligosaccharide (HMO) or a synthetic composition comprising an HMO for use in

[0016] a) Enhance executive function in non-infants,

[0017] b) prevent and / or reduce the risk of suboptimal executive functioning in non-infants,

[0018] c) managing suboptimal executive functioning in non-infants, and / or

[0019] d) Improve myelination to mature the prefrontal cortex region of the brain in non-infants.

[0020] Preferably, the synthetic composition contains HMO in an amount of 0.5 g to 15 g; more preferably 1 g to 10 g. For example, the synthetic composition may contain 2 g to 7 g of HMO.

[0021] The synthetic composition may contain bifidobacteria; for example, Bifidobacterium longum and / or Bifidobacterium bifidum.

[0022] A second aspect of the present invention relates to a package for

[0023] a) Enhance executive function in non-infants,

[0024] b) prevent and / or reduce the risk of suboptimal executive functioning in non-infants,

[0025] c) managing suboptimal executive functioning in non-infants, and / or

[0026] d) improve myelination to mature the prefrontal cortex region of the brain in non-infants,

[0027] The package contains at least one human milk oligosaccharide (HMO) in an amount effective for at least 14 individual daily doses.

[0028] Preferably, each dose in the package contains about 0.5 g to 15 g of human milk oligosaccharide, more preferably 1 g to 10 g, even more preferably 2 g to 7 g.

[0029] The package preferably contains at least about 21 daily doses; for example, about 28 daily doses.

[0030] The encapsulate may contain bifidobacteria; for example, Bifidobacterium longum and / or Bifidobacterium spp.

[0031] A third aspect of the present invention provides a method for enhancing executive function in a non-infant, the method comprising administering to the non-infant an effective amount of at least one human milk oligosaccharide (HMO).

[0032] Preferably, the method is effective in enhancing the non-infant's ability to identify, evaluate and / or make choices among various alternative options and / or strategies.Preferably, the method further comprises providing cognitive behavioral therapy to the non-infant.

[0033] A fourth aspect of the present invention provides a method for preventing and / or reducing the risk of suboptimal executive function in a non-infant, the method comprising administering to the non-infant an effective amount of at least one human milk oligosaccharide (HMO).

[0034] The HMO may be administered to a non-infant who is at risk for developing suboptimal executive functioning later in life. In this case, the method further comprises identifying a non-infant who is at risk for developing suboptimal executive functioning later in life. The non-infant may be an elderly person or a child older than 3 years who was born prematurely or small for gestational age. In addition, the HMO may be administered to a non-infant who previously suffered from suboptimal executive functioning to prevent and / or reduce the risk of relapse or the severity of relapse.

[0035] A fifth aspect of the present invention provides a method for managing suboptimal executive function in a non-infant, the method comprising administering to the non-infant an effective amount of at least one human milk oligosaccharide (HMO).

[0036] Preferably, the method is effective in managing a reduced ability of non-infants to identify, evaluate and / or make choices among various alternative options and / or strategies. The method is particularly effective in enhancing short-term and long-term memory in non-infants and through this, executive function.

[0037] Preferably, the method further comprises providing cognitive behavioral therapy to the non-infant.In one embodiment, the HMO is administered to the non-infant prior to adulthood (ie, prior to reaching 18 years of age).

[0038] Preferably, about 0.5 g to 15 g of human milk oligosaccharide per day is administered to the non-infant, more preferably 1 g to 10 g per day, even more preferably 2 g to 7 g per day.

[0039] The present invention also provides HMO or a composition comprising HMO for use in the preparation of a composition for preventing and / or treating suboptimal executive function in non-infants.

[0040] The non-infant may have one or more of attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, depression, anxiety, bipolar disorder, schizophrenia, obsessive compulsive disorder (OCD), autism, Alzheimer's disease, frontotemporal dementia, dementia with Lewy bodies, Tourette syndrome, post-traumatic stress disorder (PTSD), traumatic brain injury, or vascular dementia.

[0041] The HMO may be a fucosylated neutral HMO, a non-fucosylated neutral HMO and / or a sialylated HMO.

[0042] In one embodiment, the fucosylated neutral HMO may be 2'-fucosyllactose (2'-FL) and / or difucosyllactose (3,2'-difucosyllactose, DFL).

[0043] In one embodiment, the non-fucosylated neutral HMO may be LNT (lacto-N-tetraose), LNnT (lacto-N-neotetraose), or a combination thereof.

[0044] In one embodiment, the sialylated oligosaccharide may be 3'-sialyllactose (3'-SL) and / or 6'-sialyllactose (6'-SL).

[0045] In the context of the present invention, particularly effective combinations of HMOs may be:

[0046] -2'-FL, DFL, LNT and LNnT,

[0047] -3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), or

[0048] -2'-FL, DFL, LNT, LNnT, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0049] In a sixth aspect, the present invention provides a method for improving myelination to mature the prefrontal cortex region of the brain of a non-infant, the method comprising administering to the non-infant at least one sialylated human milk oligosaccharide (HMO). The sialylated HMO is 3'-SL, 6'-SL, or a combination thereof. Additional HMOs may be administered to the non-infant.

[0050] Preferably, the non-infant is administered an amount of 0.5 g to 15 g of HMO per day; more preferably 1 g to 10 g per day. For example, the non-infant may be administered 2 g to 7 g per day. Preferably, the non-infant is administered HMO for at least 1 week; more preferably at least 2 weeks.

[0051] A higher dose may be administered to a non-infant during the initial phase and a lower dose during the maintenance phase. Preferably, the HMO is administered to a non-infant for at least 1 week during the initial phase; more preferably for at least 2 weeks. During the maintenance phase, the HMO may be administered to a non-infant for at least 4 weeks; more preferably for at least 8 weeks. The dose administered during the initial phase is preferably about 3 g to about 10 g per day (e.g., about 4 g to about 7.5 g per day), and the dose administered during the maintenance phase is preferably about 2 g to about 7.5 g per day (e.g., about 2 g to about 5 g per day). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Performance scores of pigs on a pre-weaning diet supplemented with milk A, B, C, control milk or fed by sows, calculated to assess general working memory. Different letters indicate significant differences (p<0.05).

[0053] Figure 2 : Performance scores of pigs on a pre-weaning diet supplemented with milk A, B, C, control milk or fed by sows, calculated to assess reference memory. Different letters indicate significant differences (p<0.05).

[0054] Figure 3 : Performance scores of pigs on a pre-weaning diet supplemented with milk A, B, C, control milk or fed by sows, calculated to assess working memory. Different letters indicate significant differences (p<0.05).

[0055] Figure 4 : Expression of myelin genes in prefrontal and hippocampal brain samples from pups and adults that received milk without 6'-SL, normalized to expression levels in mice that received milk containing 6'-SL. Asterisks indicate significant differences from controls (p<0.05). DETAILED DESCRIPTION

[0056] It has now surprisingly been discovered that oral or enteral administration of one or more human milk oligosaccharides (HMOs) to a non-infant human a) enhances executive function in the non-infant, b) prevents and / or reduces the risk of suboptimal executive function in the non-infant, c) manages suboptimal executive function in the non-infant, and / or d) improves myelination to mature the prefrontal cortex region of the non-infant's brain.

[0057] As used herein, the following terms have the following meanings:

[0058] "Effective amount" means an amount of HMO sufficient to produce a desired result in a human body. An effective amount can be administered in one or more dosages to achieve the desired result.

[0059] "Enteral administration" means any conventional form of delivery of a composition to a patient that results in deposition of the composition in the gastrointestinal tract, including the stomach. Methods of enteral administration include administration via nasogastric or jejunal tube, oral, sublingual, and rectal administration.

[0060] “Executive function” refers to the ability to identify, evaluate, and choose among alternative options and strategies. The term encompasses goal-directed behavior, planning, and / or cognitive flexibility.

[0061] "Non-infant human" or "non-infant" means a human 3 years of age and older. A non-infant human may be a child, a teenager, an adult, or an elderly person (over 65 years of age).

[0062] "Human milk oligosaccharide" or "HMO" means a complex carbohydrate found in human milk (Urashima et al.: Milk Oligosaccharides. Nova Science Publisher (2011); Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). HMO has a core structure comprising a lactose unit at the reducing end, which may be extended by one or more β-N-acetyl-lactosamine units and / or one or more β-lacto-N-biose units, and the core structure may be partially substituted by αL-fucopyranosyl and / or α-N-acetyl-neuraminic acid (sialyl) groups. In this regard, non-acidic (or neutral) HMOs have no sialic acid residues, while acidic HMOs have at least one sialic acid residue in their structure. Non-acidic (or neutral) HMOs may be fucosylated or non-fucosylated. Examples of such neutral non-fucosylated HMOs include lactose-N-tetraose (LNT), lactose-N-neotetraose (LNnT), lactose-N-neohexose (LNnH), p-lactose-N-neohexose (pLNnH), p-lactose-N-hexaose (pLNH), and lactose-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2'-FL), lactose-N-fucopentaose I (LNFP-I), lactose-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3-FL), difucosyllactose (DFL), lactose-N-fucopentaose II (LNFP-II), lactose-N-fucopentaose III (LNFP-III), lactose-N-difucohexaose III (LNDFH-I). -III), fucosyl-lactose-N-hexaose II (FLNH-II), lactose-N-fucopentaose V (LNFP-V), lactose-N-fucopentaose VI (LNFP-VI), lactose-N-difucohexaose II (LNDFH-II), fucosyl-lactose-N-hexaose I (FLNH-I), fucosyl-p-lactose-N-hexaose I (FpLNH-I), fucosyl-p-lactose-N-neohexaose II (F-pLNnH II) and fucosyl-lactose-N-neohexaose (FLNnH).Examples of acidic HMOs include 3'-sialyllactose (3'-SL), 6'-sialyllactose (6'-SL), 3-fucosyl-3'-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b, fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH (SLNH), sialyl-lactose-N-hexaose (SLNH), sialyl-lactose-N-neohexose I (SLNH-I), sialyl-lactose-N-neohexose II (SLNH-II), and disialyl-lactose-N-tetraose (DSLNT).

[0063] "Oral administration" means any conventional form of delivering a composition to the human body through the oral cavity. Therefore, oral administration is a form of enteral administration.

[0064] "Synthetic composition" means an artificially prepared composition, preferably a composition containing at least one compound produced ex vivo chemically and / or biologically (e.g., by chemical reaction, enzymatic reaction, or recombination). In some embodiments, the synthetic composition may include one or more compounds or components other than the HMO. In addition, in some embodiments, the synthetic composition may include one or more nutritionally or pharmaceutically active components that do not adversely affect the efficacy of the HMO. Some non-limiting embodiments of the synthetic compositions of the present invention are also described below.

[0065] "Child older than 3 years born small for gestational age (SGA)" means a child born smaller than normal for gestational age, generally defined as weight less than the 10th percentile for gestational age. In some embodiments, SGA may be associated with intrauterine growth restriction (IUGR), which refers to a condition in which a fetus fails to reach its potential size.

[0066] "Low birth weight" means a birth weight of less than 2500g. It therefore covers:

[0067] - children who weigh between 1800 and 2500g at birth (often called 'low birth weight' or LBW),

[0068] - children born weighing between 1000 and 1800g (called 'very low birth weight' or VLBW),

[0069] - Children born weighing less than 1000g (called 'extremely low birth weight' or ELBW).

[0070] "Prophylactic treatment" or "prevention" means treatment given or actions taken to reduce the risk of disease onset or recurrence, or the severity of a disease if it occurs.

[0071] "Prophylactically reducing the severity and / or incidence of symptoms" means reducing the severity and / or incidence of symptoms at a later point in time.

[0072] "Secondary prevention" refers to preventing the onset of a condition in patients at high risk, or preventing the recurrence of symptoms in patients who already have the condition. A "high-risk" patient is an individual who is susceptible to the condition, such as someone with a family history of the condition.

[0073] "Management" means addressing a medical condition or disease with the goal of improving or stabilizing a non-infant's outcome or addressing an underlying nutritional need. Thus, management includes dietary or nutritional management of a medical condition or disease by addressing the nutritional needs of a non-infant. "Managing" and "management" have grammatical corresponding meanings. Management includes treatment of non-infants.

[0074] "Treatment" means treatment administered or action taken to reduce or eliminate the symptoms of a disease or pathological condition.

[0075] In a first aspect of the present invention, there is provided an HMO or a composition comprising the HMO for enhancing executive function in non-infants.

[0076] The HMO can be a fucosylated neutral HMO, a non-fucosylated neutral HMO, a sialylated HMO, or any combination of any of the foregoing.

[0077] In one embodiment, the HMO is a fucosylated neutral HMO.

[0078] In one embodiment, the HMO is a non-fucosylated neutral HMO.

[0079] In one embodiment, the HMO is a sialylated HMO.

[0080] In one embodiment, the HMO is a mixture comprising, consisting essentially of, or consisting of one or more fucosylated neutral HMOs and one or more non-fucosylated neutral HMOs.

[0081] In one embodiment, the HMO is a mixture comprising, consisting essentially of, or consisting of one or more fucosylated neutral HMOs, one or more non-fucosylated neutral HMOs, and one or more sialylated HMOs.

[0082] Specifically, one or more fucosylated neutral HMOs are selected from 2'-FL, 3-FL, DFL, LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LNDFH-I, LNDFH-II, LNDFH-III, FLNH-I, FLNH-II, FLNnH, FpLNH-I and F-pLNnH II, preferably 2'-FL, 3-FL and DFL. Particularly effective fucosylated oligosaccharides may be 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL).

[0083] Preferably, the one or more non-fucosylated neutral HMOs are selected from LNT, LNnT, LNH, LNnH, pLNH and pLNnH, preferably LNnT and LNT.

[0084] The one or more sialylated HMOs are preferably selected from 3'-SL, 6'-SL, LST a, LST b, LST c, DS-LNT, S-LNH and S-LNnH I, more preferably selected from 3'-SL and 6'-SL. HMOs containing fucosylated and sialylated moieties, such as FSL, can also be used.

[0085] In one embodiment, the HMO is selected from 2'-fucosyllactose (2'-FL), DFL, LNT, LNnT, sialyllactose, and any combination of the foregoing.

[0086] In one embodiment, the HMO is a combination of 2'-FL, DFL, LNT, and LNnT, comprising, consisting essentially of, or consisting of 2'-FL, DFL, LNT, and LNnT.

[0087] In one embodiment, the HMO is a combination of, comprises, consists essentially of, or consists of, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL).

[0088] In one embodiment, the HMO is a combination of 2'-FL, DFL, LNT, LNnT, 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL), which comprises, consists essentially of, or consists of 2'-FL, DFL, LNT, LNnT, 3'-sialyllactose (3'-SL), and 6'-sialyllactose (6'-SL).

[0089] HMO can be separated or enriched from the milk secreted by mammals by well-known methods, including but not limited to human, bovine, ovine, porcine or caprine species. HMO can also be produced by well-known methods using microbial fermentation, enzymatic methods, chemical synthesis or a combination of these techniques. As examples, LNnT can be prepared using chemical methods as described in WO 2011 / 100980 and WO 2013 / 044928, LNT can be synthesized as described in WO 2012 / 155916 and WO 2013 / 044928, a mixture of LNT and LNnT can be prepared as described in WO 2013 / 091660, 2'-FL can be prepared as described in WO 2010 / 115934 and WO 2010 / 115935, 3-FL can be prepared as described in WO 2013 / 139344, 6'-SL and its salts can be prepared as described in WO 2010 / 100979, sialylated oligosaccharides can be prepared as described in WO 2012 / 113404, and a mixture of human milk oligosaccharides can be prepared as described in WO 2012 / 113405. As examples of enzymatic production, sialylated oligosaccharides can be prepared as described in WO 2012 / 007588, fucosyl oligosaccharides can be prepared as described in WO 2012 / 127410, and advantageously, a diverse mixture of human milk oligosaccharides can be prepared as described in WO 2012 / 156897 and WO 2012 / 156898. In addition, WO 01 / 04341 and WO 2007 / 101862 describe how to use genetically modified E. coli to prepare core human milk oligosaccharides optionally substituted with fucose or sialic acid.

[0090] In any aspect of the invention, one or more HMOs may be used as is, or they are (pure) without any carrier and / or diluent. In other embodiments, one or more HMOs are used in a synthetic composition together with one or more inert carriers / diluents acceptable in nutritional or pharmaceutical compositions, such as solvents (e.g., water, water / ethanol, oil, water / oil), dispersants, coatings, absorption promoters, controlled release agents, inert excipients (e.g., starch, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders, and disintegrants). These compositions do not contain prebiotics and / or probiotics. In other embodiments, one or more HMOs are used in a synthetic pharmaceutical or nutritional composition that may contain prebiotics and / or probiotics.

[0091] The non-infant may be a healthy non-infant not suffering from suboptimal executive function.

[0092] Executive function can be measured by methods well known to those skilled in the art, such as by an assessment, possibly a holistic assessment, of different cognitive skills known to constitute executive function, such as inhibition, working memory, cognitive flexibility, pattern recognition tests, etc. For children, this method can be, for example, a dimensional change card sorting (DCCS) task, and for adults, it can be, for example, a Wisconsin card sorting task. Participants are shown a target card and are asked to sort a series of bivalent test cards according to one dimension (e.g., color). During the post-conversion phase, they are told to sort the same type of test cards according to other dimensions (e.g., shape).

[0093] Non-infants who do not suffer from suboptimal executive functioning would have test scores within a range that is considered normal (non-pathological), eg, for the type and age of the non-infant.

[0094] It will be apparent to one skilled in the art that the HMOs or compositions comprising the HMOs as disclosed herein for enhancing executive function may also be used to prevent and / or treat suboptimal executive function in non-infants.

[0095] Therefore, in another aspect of the present invention there is provided a HMO or a composition comprising a HMO as disclosed herein for use in preventing and / or treating suboptimal functioning.

[0096] In one embodiment, the non-infant may be a non-infant who suffers from suboptimal executive functioning and therefore is in need of enhancing executive functioning.

[0097] A person with suboptimal executive function may be a non-infant whose test scores (on a standard test used to assess executive function) are not within a range that is considered normal (non-pathological), e.g., for the type and age of the non-infant. Determining when a non-infant has suboptimal executive function is well within the capabilities of one skilled in the art.

[0098] Suboptimal executive function may be associated with a variety of cognitive conditions, including attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, and vascular dementia. It is also thought to be more prevalent in non-infants who are born preterm or small for gestational age (SGA), and it is known that executive function declines with age. Therefore, there may be a particular need to treat and / or prevent suboptimal executive function in these patient populations.

[0099] Thus, in a more specific embodiment, the non-infant in need of enhanced executive function is a non-infant with ADHD, Alzheimer's disease, or vascular dementia, an elderly person, or a child born prematurely or small for gestational age (SGA).

[0100] In another aspect of the present invention, a method for preventing and / or treating suboptimal executive function in non-infants is provided, the method comprising the step of administering to the non-infant an HMO as disclosed herein or a composition comprising an HMO. The method may also optionally comprise the step of identifying a non-infant suffering from suboptimal executive function.

[0101] In another aspect of the present invention, a method of enhancing executive function in non-infants is provided, the method comprising the step of administering to the non-infant an HMO or a composition comprising an HMO as disclosed herein. The method may also optionally comprise the step of identifying a non-infant suffering from suboptimal executive function.

[0102] In any aspect of the present invention disclosed above, the synthetic composition can be a pharmaceutical composition. The pharmaceutical composition can contain a pharmaceutically acceptable carrier such as phosphate buffered saline, a mixture of ethanol in water, water and emulsions (such as oil / water or water / oil emulsions) and various wetting agents or excipients. The pharmaceutical composition can also contain other substances that will not produce adverse, allergic or other unwanted reactions when applied to non-infants. The carrier and other substances may include solvents, dispersants, coatings, absorption promoters, controlled release agents and one or more inert excipients, such as starch, polyols, granulating agents, microcrystalline cellulose, diluents, lubricants, binders and disintegrants. If desired, the tablet dosage of the composition can be coated by standard aqueous or non-aqueous techniques.

[0103] The pharmaceutical composition can be administered orally, for example as a tablet, capsule or pill containing a predetermined amount, or as a powder or granules containing a predetermined concentration, or as a gel, paste, solution, suspension, emulsion, syrup, bolus, ointment or slurry in an aqueous or non-aqueous liquid containing a predetermined concentration. Orally administered compositions may include adhesives, lubricants, inert diluents, flavoring agents and humectants. Orally administered compositions (such as tablets) may be optionally coated and may be formulated to provide a sustained, delayed or controlled release of the mixture therein.

[0104] The pharmaceutical compositions may also be administered by rectal suppository, aerosol tube, nasogastric tube, or direct infusion into the gastrointestinal tract.

[0105] The pharmaceutical composition may also include therapeutic agents, such as antivirals, antibiotics, probiotics, analgesics, and anti-inflammatory agents. Suitable dosages of these compositions for non-infant humans can be determined in a conventional manner based on factors such as immune status, weight, and age. In some cases, the concentration of the dosage will be similar to the concentration of HMO in human milk. The required amount is generally in the range of about 1 g to about 20 g per day, and in some embodiments, about 2 g to about 15 g per day, for example, about 3 g to about 7.5 g per day. Suitable dosage regimens can be determined by conventional methods.

[0106] In any aspect of the invention disclosed above, the composite composition may also be a nutritional composition. It may contain a source of protein, lipids and / or digestible carbohydrates and may be in powder or liquid form. The composition may be designed as a sole source of nutrition or a nutritional supplement.

[0107] Suitable protein sources include milk protein, soy protein, rice protein, pea protein and oat protein or mixtures thereof. The milk protein may be in the form of milk protein concentrate, milk protein isolate, whey protein or casein or a mixture of the two. The protein may be intact protein or hydrolyzed protein, partially hydrolyzed or extensively hydrolyzed. Hydrolyzed protein has the advantage of being easily digestible, which is very important for non-infants with gastrointestinal inflammation.

[0108] The protein may also be provided in the form of free amino acids.The protein may comprise from about 5% to about 30%, typically from about 10% to 20%, of the energy of the nutritional composition.

[0109] The protein source can be a source of glutamine, threonine, cysteine, serine, proline or a combination of these amino acids. The glutamine source can be glutamine dipeptide and / or a glutamine-rich protein. Glutamine can be included because enterocytes use glutamine as an energy source. Threonine, serine and proline are important amino acids for the production of mucin. Mucin lines the gastrointestinal tract and can improve mucosal healing. Cysteine ​​is the main precursor of glutathione, which is key to the body's antioxidant defenses.

[0110] Suitable digestible carbohydrates include maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, high fructose corn syrup, carbohydrates derived from rice, carbohydrates derived from peas, carbohydrates derived from potatoes, tapioca, sucrose, glucose, fructose, sucrose, lactose, honey, sugar alcohols (e.g. maltitol, erythritol, sorbitol) or mixtures thereof. Preferably, the composition does not contain lactose. Typically, digestible carbohydrates provide about 35% to about 55% of the energy of the nutritional composition. Preferably, the nutritional composition does not contain lactose. Particularly suitable digestible carbohydrates are low glucose equivalent (DE) maltodextrins.

[0111] Suitable lipids include medium chain triglycerides (MCT) and long chain triglycerides (LCT). Preferably, the lipid is a mixture of MCT and LCT. For example, MCT may contain about 30% to about 70% (by weight), more specifically about 50% to about 60% (by weight) of lipid. MCT has the advantage of being easy to digest, which is very important for non-infants with gastrointestinal inflammation. Typically, lipids provide about 35% to about 50% of the energy of the nutritional composition. Lipids may contain essential fatty acids (ω-3 and ω-6 fatty acids). Preferably, the energy provided by these polyunsaturated fatty acids is less than about 30% of the total energy of the lipid source. It is particularly preferred that the composition contains a source of ω-3 polyunsaturated fatty acids; for example, fish oil, docosahexaenoic acid, α-linolenic acid and / or eicosapentaenoic acid. The polyunsaturated fatty acid may also be dihigh-dietary γ-linolenic acid (DGLA).

[0112] Suitable sources of long-chain triglycerides are rapeseed oil, sunflower oil, palm oil, soybean oil, butterfat, corn oil, high oleic oil and soy lecithin. Fractionated coconut oil is a suitable source of medium-chain triglycerides. The lipid profile of the nutritional composition is preferably designed to have a polyunsaturated fatty acid ω-6 (n-6) to ω-3 (n-3) ratio of about 4:1 to about 10:1. For example, the ratio of n-6 to n-3 fatty acids can be about 6:1 to about 9:1.

[0113] The nutritional composition preferably also includes vitamins and minerals. If the nutritional composition is intended to be the only source of nutrition, it preferably includes a complete vitamin and mineral spectrum. Examples of vitamins include vitamin A, B-complex (such as B1, B2, B6 and B12), C, D, E and K, niacin and acidic vitamins, such as pantothenic acid, folic acid and biotin. Examples of minerals include calcium, iron, zinc, magnesium, iodine, copper, phosphorus, manganese, potassium, chromium, molybdenum, selenium, nickel, tin, silicon, vanadium and boron. The source of vitamin D is particularly preferred.

[0114] The nutritional composition may also include carotenoids such as lutein, lycopene, zeaxanthin and beta-carotene. The total amount of the included carotenoids may vary within a range of about 0.001 μg / ml to about 10 μg / ml. Lutein may be included in an amount of about 0.001 μg / ml to about 10 μg / ml, preferably about 0.044 μg / ml to about 5 g / ml of lutein. Lycopene may be included in an amount of about 0.001 μg / ml to about 10 μg / ml, preferably about 0.0185 mg / ml to about 5 g / ml of lycopene. Beta-carotene may include about 0.001 μg / ml to about 10 mg / ml, for example, about 0.034 μg / ml to about 5 μg / ml of beta-carotene.

[0115] The nutritional composition preferably also contains sodium at a reduced concentration; for example, from about 300 mg / l to about 400 mg / l. The remaining electrolytes may be present at concentrations set to meet needs without causing undue renal solute burden on renal function. For example, potassium is preferably present in a range of about 1180 to about 1300 mg / l; and chloride is preferably present in a range of about 680 to about 800 mg / l.

[0116] The nutritional composition may also contain various other conventional ingredients, such as preservatives, emulsifiers, thickeners, buffers, fibers and prebiotics (e.g., fructooligosaccharides, galacto-oligosaccharides), probiotics (e.g., Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium lactis HN019, Bifidobacterium lactis Bi07, Bifidobacterium infantis ATCC 15697, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus HNOOI, Lactobacillus acidophilus LA-5, Lactobacillus acidophilus NCFM, Lactobacillus fermentum CECT5716, Bifidobacterium longum BB536, Bifidobacterium longum AH1205, Bifidobacterium longum AH1206, Bifidobacterium breve M-16V, Lactobacillus reuteri ATCC 55730, Lactobacillus reuteri ATCC PTA-6485, Lactobacillus reuteri DSM 17938), antioxidant / anti-inflammatory compounds including tocopherols, carotenoids, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenols, glutathione and superoxide dismutase (melon), other bioactive factors (e.g., growth hormones, cytokines, TFG-β), colorants, flavorings and stabilizers, lubricants, etc.

[0117] The nutritional composition may be in the form of a soluble powder, a liquid concentrate or a ready-to-use formulation. The composition may be fed to non-infants via a nasogastric tube or orally. Various flavorings, fibers and other additives may also be present.

[0118] The nutritional composition can be prepared by any common manufacturing technology for preparing the nutritional composition in solid or liquid form. For example, the composition can be prepared by merging various feed solutions. The lipid source can be heated and mixed, and then an emulsifier (such as lecithin), a fat-soluble vitamin and at least a portion of a protein source are added while heating and stirring to prepare a fat protein (protein-in-fat) feed solution. The carbohydrate feed solution is then prepared by adding minerals, trace and ultra-trace minerals, thickeners or suspending agents to water while heating and stirring. Before adding carbohydrates (such as HMO and digestible carbohydrate sources), the resulting solution is kept under continuous heating and stirring for 10 minutes. The resulting feed solution is then blended together when heated and stirred, and the pH is adjusted to 6.6-7.0, and the composition is then subjected to high temperature short-time processing, during which the composition is heat treated, emulsified and homogenized, and then cooled. Water-soluble vitamins and ascorbic acid are added, and if necessary, the pH is adjusted to the desired range, flavorings are added, and water is added to reach the desired total solid level.

[0119] For liquid products, the resulting solution can then be aseptically packaged to form an aseptically packaged nutritional composition. In this form, the nutritional composition can be in a ready-to-eat or concentrated liquid form. Alternatively, the composition can be spray dried and processed and packaged as a reconstitutable powder.

[0120] When the nutritional product is a ready-to-feed nutritional liquid, the total concentration of HMO in the liquid (by weight of the liquid) is from about 0.0001% to about 2.0%, including from about 0.001% to about 1.5%, including from about 0.01% to about 1.0%. When the nutritional product is a concentrated nutritional liquid, the total concentration of HMO in the liquid (by weight of the liquid) is from about 0.0002% to about 4.0%, including from about 0.002% to about 3.0%, including from about 0.02% to about 2.0%.

[0121] The composite composition, preferably the nutritional composition, may also be in a unit dosage form, such as a capsule, tablet, or sachet / stick pack. For example, the composite composition, preferably the nutritional composition, may be in tablet form or powder form, comprising the HMO and one or more additional components that aid in formulation and administration, such as diluents, excipients, antioxidants, lubricants, colorants, binders, disintegrants, etc.

[0122] Suitable diluents, excipients, lubricants, colorants, binders and disintegrants include polyethylene, polyvinyl chloride, ethylcellulose, acrylate polymers and copolymers thereof, hydroxyethylcellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO) or polyacrylamide (PA), carrageenan, sodium alginate, polycarbophil, polyacrylic acid, tragacanth, methylcellulose, pectin, natural gum, xanthan gum, guar gum, karaya gum, hypromellose, magnesium stearate, microcrystalline cellulose and colloidal silicon dioxide. Suitable antioxidants are vitamin A, carotenoids, vitamin C, vitamin E, selenium, flavonoids, polyphenols, lycopene, lutein, lignans, coenzyme Q10 ("CoQIO") and glutathione.

[0123] The unit dosage form, particularly in the form of a sachet or stick pack, may also include various nutrients, including macronutrients. Particularly preferred nutrients are sources of vitamin D, omega-3 polyunsaturated fatty acids and probiotics as described above.

[0124] The appropriate dosage of an HMO or synthetic composition can be determined in a conventional manner based on factors such as the severity of the symptoms, immune status, body weight and age. In some cases, the concentration of the dosage is similar to the concentration of HMO in human breast milk. The required amount will generally be in the range of about 1 g to about 20 g per day, in certain embodiments about 1 g to about 15 g per day, for example about 2 g to about 10 g per day, and in certain embodiments about 3 g to about 7.5 g per day. A suitable dosage regimen can be determined by conventional methods. Ideally, a dosage of about 2 g to about 10 g is administered daily, with the exact dosage depending on whether the symptoms are being treated (usually higher doses) or secondary prevention (lower doses) is intended.

[0125] The HMO or synthetic composition may be in the form of a package containing an effective amount of human milk oligosaccharides for at least 14 individual daily doses. The daily dose is preferably in the form of a sachet / stick package, but may be in any suitable form. Each dose preferably contains from about 1 g to about 20 g of human milk oligosaccharides, more preferably from about 2 g to about 15 g, for example from about 2 g to about 10 g. Preferably, the package contains at least 21 daily doses, more preferably at least 28 daily doses. Most suitable packages contain enough for 4 weeks or a full month. The package may include instructions for use.

[0126] The appropriate dosage can be determined based on several factors, for example, including, for example, weight and / or condition, severity of symptoms, incidence and / or severity of side effects, and mode of administration. The effective amount can be any amount that enhances non-infant executive function. The enhancement of executive function can be measured by the well-known tests detailed above. The enhancement of executive function may only be detected after more than 6 months, 1 year, for example, more than 5 years, more than 10 years, more than 20 years. It is entirely within the capabilities of those skilled in the art to determine the effective dosage based on the information herein and the knowledge in the art.

[0127] Suitable dosage ranges can be determined by methods known to those skilled in the art. During the initial treatment phase, the dosage may be higher (e.g., 3 g to 15 g per day, preferably 4 g to 10 g per day, more preferably 4 g to 7.5 g per day). During the maintenance phase, the dosage may be reduced (e.g., 1 to 10 g per day, preferably 2 mg to 5 g per day, more preferably 2 g to 5 g per day). If the composition comprising HMO comprises 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), then 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) may be particularly beneficial if they are included in the composition in a weight ratio between 10:1 and 1:10 (such as between 10:1 and 2:1, between 8:1 and 3:1, between 6:1 and 3:1, between 5:1 and 3:1, between 5:1 and 4:1, or between 4.7:1 and 4.1:1).

[0128] The duration of treatment can be determined based on several factors, including, for example, weight and / or condition, severity of symptoms, incidence and / or severity of side effects. Preferably, the duration is at least 14 days, more preferably at least 3 weeks; even more preferably more than 4 weeks. The synthetic composition or HMO can also be taken chronically.

[0129] A certain aspect of the invention is the use of the following for dietary management of a non-infant human being in need of enhanced executive function:

[0130] - one or more human milk oligosaccharides (HMO),

[0131] - a synthetic composition comprising one or more human milk oligosaccharides (HMO), or

[0132] - An encapsulation comprising an effective amount of one or more human milk oligosaccharides for at least 14 individual daily doses.

[0133] The HMOs, synthetic compositions and encapsulations are as disclosed above.

[0134] It should be understood that all features of the present invention disclosed herein are freely combinable and can be changed and modified without departing from the scope of the present invention defined in the claims. In addition, where there are known equivalents for specific features, such equivalents are incorporated into this specification as if specifically mentioned in this specification.

[0135] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an ingredient" or "the ingredient" includes two or more ingredients. The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." As used herein, the term "example," particularly when followed by a list of terms, is exemplary and illustrative only and should not be considered exclusive or comprehensive.

[0136] As used herein, "about" is understood to refer to a number within a numerical range, such as a range of -10% to +10% of the mentioned number, preferably within a range of -5% to +5% of the mentioned number, more preferably within a range of -1% to +1% of the mentioned number, and most preferably within a range of -0.1% to +0.1% of the mentioned number. The range "between" two values ​​includes both values. In addition, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions within the range. In addition, these numerical ranges should be interpreted as supporting claims for any number or subset of numbers within the range. For example, a disclosure from 1 to 10 should be interpreted as supporting ranges from 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0137] Unless otherwise indicated, all percentages expressed herein are by weight based on the total weight of the composition. When pH values ​​are mentioned, the values ​​correspond to the pH values ​​measured at 25°C using standard equipment.

[0138] The relative terms "enhance" and "reduce" refer to the effects of an HMO or a composition comprising an HMO as disclosed herein on executive function in a non-infant (e.g., effects on different cognitive skills known to constitute executive function, such as attention or impulsivity, working memory, cognitive flexibility. This may need to be considered holistically) compared to a non-infant not administered the HMO or composition comprising the HMO. It is well within the capabilities of the skilled artisan to assess the improvement, increase, or enhancement. An enhancement of executive function (a reduction in different cognitive skills known to constitute executive function, such as cognitive flexibility, working memory, attention, and / or impulsivity) may only be detectable after more than 1 year, e.g., more than 5 years, more than 10 years, more than 20 years.

[0139] The compositions disclosed herein may lack any element not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments consisting of "essentially" as well as "consisting of" the components identified. Similarly, the methods disclosed herein may lack any steps not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments consisting of "essentially" as well as "consisting of" the steps identified. Furthermore, describing some steps as "optional" does not mean that other steps that are not explicitly described as optional are required. When a composition is simply mentioned herein, this may be a nutritional composition.

[0140] The following is a series of non-limiting examples used to illustrate the present invention.

[0141] Example

[0142] Example 1

[0143] Methods - Behavioral Research

[0144] 53 female piglets (Miniature piglets from Ellegaard Gottingen, Denmark) were subjected to the orifice test after weaning. At 1 week of age, the piglets fed with formula milk powder (N=45) were separated from the sows and housed in mixed groups of 2 piglets in a pen (2.5m×1m) rich in chopped straw as bedding and equipped with a squeeze ball and a dog bed. In addition, 8 piglets were cross-fed and kept together with 3 sows until 10 weeks of age (naturally suckled piglets). After weaning from breast milk, at 10 weeks of age, the naturally suckled piglets were housed under the same conditions as the formula-fed piglets. Two metal chains were also added to the pen as additional enrichment materials.

[0145] Treatment. The duration of milk intervention ranged from 1 week to 10 weeks of age. At 1 week of age, formula-fed piglets were randomly assigned to 1 of 4 milk formulas enriched with different prebiotic mixtures (milk A: 3'-SL and 6'-SL, milk B: 2'-FL+DFL+LNT+LNnT, milk C: 2'-FL+DFL+LNT+LNnT+3'-SL+6'-SL) or without prebiotics (control milk). Naturally lactating piglets were fed with breast milk throughout the nutritional intervention period. After weaning, at 10 weeks of age, piglets were fed a high-energy fattening diet. Five treatment groups were set up in this experiment: milk A (N=12), milk B (N=12), milk C (N=10), control milk (N=11), and natural lactation (N=8).

[0146] Two pigs from each pen were tested individually in the spatial hole board task to evaluate their spatial cognition (memory and learning) performance. The hole board arena (3 m × 3 m) had black, wooden, 80 cm high walls and 4 gated entrances. In the arena, 16 gray metal buckets ( =The piglets were fed with food overnight during the entire orifice test period.

[0147] From about 16.5 to 19 weeks of age, in 12 consecutive working days, 2 batch tests (i.e., performed a few minutes apart) were performed on the piglets individually every day, i.e., 24 collection tests. Different entrances were used every day, wherein 2 different entrances were tested every day (i.e., 1 entrance per test). The test was started when the piglets' 4 legs were in the hole plate field, and the test was terminated when the piglets found all 4 rewards or after 180 seconds. Whenever the piglets first visited the bucket with bait, a click was produced to promote learning. If the piglets completed the task (i.e., found 4 rewards in less than 180 seconds), the exit (south) gate was opened, and the piglets received half of the white grapes. If the piglets did not complete the task within 180 seconds, a siren sound was produced; The piglets did not receive a reward. After 2 piglets were tested twice in each pen, all pig pen companions were guided back to their family circle, and their morning milk rations were distributed.

[0148] After the acquisition phase was completed, the piglets were given 16 reverse trials individually and 2 batches per day for 8 consecutive weekdays. The procedure was the same as in the acquisition phase, but the piglets were assigned to different configurations of baited buckets.

[0149] The following parameters were scored in real time using Observer XT 10 (Noldus Information Technology, Wageningen, The Netherlands): all visits and revisits to all buckets, waiting time for all bucket visits, trial duration, total number of defecation, urination and escape attempts during the trial. Based on the parameters recorded during the test, variables were calculated empirically according to Neurosci.Biobehav.Rev.36, 379 (2012) by van der Staay et al. (Table 1).

[0150] Table 1. Parameters calculated empirically in the hole plate task

[0151]

[0152] During the acquisition phase, all pigs showed improvements in executive function performance as evidenced by linear increases in WM, RM, and general WM scores over time, and a linear decrease in the number of WM, RM, and general WM errors over time.

[0153] During the reversal phase, all pigs showed an improvement in executive function performance as seen by a linear increase in WM, RM and general WM scores over time, and a linear decrease in the number of WM, RM and general WM errors over time. This improvement in RM, WM and general WM performance was reduced in the control milk group compared to naturally suckling piglets (significant for general WM and RM), and surprisingly, this deficiency was restored to levels similar to those observed in the naturally suckling group by the presence of HMO in milk (see Figure 1-3 ).

[0154] Example 2

[0155] Animals and housing conditions

[0156] Adult wild-type (WT) B6.129 and heterozygous (HZ) B6.129-St6gal1 tm2JxmBreeding pairs (four males and four females and three males and four females, respectively) were purchased from commercial breeding (The Jackson Laboratory). After arrival, same-sex mice were housed in 2-3 same-sex groups in type 1 polycarbonate cages (33.0 × 13.0 × 14.0 cm), equipped with sawdust mats, enrichment bags (Mucedola, Settimo Milanese, Italy), metal tops and arbitrary water and food pellets (Mucedola, Settimo Milanese, Italy). In an air-conditioned room (temperature 21 ± 1 ° C and relative humidity 60 ± 10%), mice were kept in an inverted 12-hour light-dark cycle (lights on at 7:00 p.m.). After arriving two weeks later, a three-mouse group (one male, two females) was formed. After mating for two weeks, male mice were removed, and female individuals were individually housed in standard type 1 cages. The time of delivery of females was checked daily, and the day of their delivery was designated as postpartum day (PND) 0. Except for weekly cage cleaning, mother mice and their offspring remain undisturbed until weaning (at PND 25). At weaning, male and female mice are separated and placed in the same feeding cage of the same sex; In addition, male mice are marked by ear clips, and the ear tissue removed by this procedure is used for genotyping. Then homozygous knockout (KO) and WT mice are used for experiments.

[0157] Feeding procedures and feeding

[0158] Fourteen wild-type (WT) and 14 St6Gal1 homozygous (hereinafter referred to as KO) female mice were mated with seven WT and seven KO male mice, respectively. In this batch, 10 WT and 10 KO female mice gave birth to live offspring. The birth day was designated as postnatal day (PND) 0. The nursing procedure was performed between 10:00 and 13:00 (for details, see Figure 1 ) requires the use of four mothers (two WT and two KO) at the same time. Therefore, in order to minimize the number of individuals abandoned due to lack of nursing mothers, the nursing procedure was performed between 24 and 60 hours after birth. On the nursing day, we first removed the mother from its cage, and then determined the sex of the offspring by toe tattoo acupuncture and marked it. After completing the sex determination and marking procedures, the mice were transferred to the cage containing the nursing mother and covered with sawdust. Each offspring was transferred to the nursing mother so that all experimental individuals were in the same conditions. Each mother raised mixed pups consisting of WT and KO male and female offspring (the ratio between all variables was as close as possible to 1:1). At the time of eye opening and adulthood, prefrontal cortex and hippocampus brain samples were collected for gene expression analysis.

[0159] The following experimental groups were formed:

[0160] -WT offspring raised by WT dams (WT to WT)

[0161] -WT offspring raised by KO mothers (WT vs. KO)

[0162] -KO offspring were raised by WT mothers (KO vs WT)

[0163] -KO offspring are raised by KO mothers (KO to KO)

[0164] Total RNA extraction and QC

[0165] Total RNA was extracted using the Agencourt RNAdvance Tissue Kit (Beckman Coulter): Lyse in 450 μL. Extract 400 μL of lysate. Elution volume 50 μL

[0166] Quantification was performed using Quant It Ribogreen assay reagent (Life Technologies)

[0167] QC assessment was performed using the Standard Sensitivity RNA Kit on a Fragment Analyzer 96 (Agilent).

[0168] Sample library preparation

[0169] Libraries were generated using the QuantSeq 3' mRNA-Seq Library Preparation Kit for Illumina (FWD) HT from Lexogen. It is designed to generate Illumina compatible sequences close to the 3' end of polyadenylated RNA.

[0170] The kit uses total RNA as input, so no prior poly(A) enrichment or rRNA depletion is required. Library generation begins with oligodT priming containing the Illumina-specific Read 2 adapter sequence. After first-strand synthesis, RNA is removed. Second-strand synthesis begins by random priming and DNA polymerase. Random primers contain the Illumina-specific Read 1 adapter sequence. No purification is required between first-strand synthesis and second-strand synthesis. Second-strand synthesis is followed by a bead-based purification step. Sequences required for cluster generation are introduced during the library amplification step for sequencing. Double-stranded cDNA is amplified by PCR. During this step, individual barcode indexes are introduced to facilitate multiplexing of samples. NGS reads are generated for the poly(A) tail and correspond directly to the mRNA. For more information, see Figure 1. Libraries were quantified using Quant it Picogreen (Life Technologies). Size patterns were controlled using a high sensitivity NGS fragment analysis kit on a fragment analyzer (Agilent). Libraries were combined in equimolar ratios (i.e., equal amounts of each sample library) and clustered on a single read (SR) sequencing flow cell (Illumina) at a concentration of 9 pM. Sequencing was performed on a HiSeq 2500 (Illumina) for 65 cycles using the HiSeq SR Cluster Kit v4 cBot, HiSeq SBS Kit V4 50 Cycle Kit (sequencing by synthesis). Raw data quality control was performed during the sequencing run to ensure optimal flow cell loading (cluster density) and to check quality metrics (QC30) for the sequencing run. The optimal value for cluster density detected by image analysis was between 850K / mm 2 and 1000K / mm 2 Performing a run at optimal cluster density involves finding a balance between under-clustering, which maintains good quality data but results in lower data output, and over-clustering, which can result in poor run performance. Percent ≥ Q30 refers to the percentage of bases with a quality Phred score of 30 or higher. It is a measure of the quality of the nucleic acid bases generated for identification. The Phred score is related to the logarithm of the error probability. A Phred score equal to 30 means a probability of 1 in 1000 of incorrect base assignment, so the base call accuracy is 99.9%. With respect to Illumina specifications, the score should be a minimum of 80%.

[0171] Data analysis

[0172] Genes with very low counts are unlikely to be differentially expressed between groups. We filtered low expressed genes by selecting only genes with at least 5 reads in at least 8 samples. The threshold on the number of samples ensured that the gene would be expressed in the smallest group (which contained 8 samples). The filtering step was performed on the CPM value taking into account the library size. In our case, it corresponded to a threshold of 2.427 on the CPM value. We also discarded genes without annotations. We retained 11523 features with these filtering criteria. We used the TMM method for normalization.

[0173] result

[0174] The expression of multiple genes associated with myelination was reduced in mice receiving milk without 6'-SL during early life only in the prefrontal cortex, which was not observed in adulthood or in the hippocampus at either age. Figure 4It was shown that in mice that received milk without 6'-SL, the expression of myelin basic protein (MBP) and myelin associated glycoprotein (MAG), two key proteins of myelin, was reduced by 50% (gene expression quantified as fold change relative to control mice). When we studied the effects of the absence of 6'-SL on Kyoto Encyclopedia of Genes and Genomes pathways in early life, we identified that both myelination and myelination pathways were downregulated in mice that received milk without 6'-SL. This was also only seen in the prefrontal cortex and not in the hippocampus (in adulthood or early life age). These results clearly indicate that the presence of 6'-SL in breast milk is necessary to observe optimal myelination of the prefrontal cortex, especially in early life. Interestingly, the prefrontal cortex is known to be a key brain area that mediates executive function.

[0175] Example 3 -Nutritional composition

[0176] The ready-to-eat nutritional composition is prepared from the following substances: water, maltodextrin, corn syrup, sugar, milk protein concentrate, vegetable oils (canola, high oleic sunflower and corn), soy protein isolate, gum arabic, flavors, 2'-FL and DFL, potassium citrate, magnesium phosphate, cellulose gel and gum, calcium carbonate, sodium ascorbate, soy lecithin, choline bitartrate, calcium phosphate, alpha-tocopheryl acetate, ascorbic acid, carrageenan, ferric pyrophosphate, flavors, sweetener (Stevia), vitamin A palmitate, niacinamide, vitamin D3, calcium pantothenate, manganese sulfate, copper sulfate, pyridoxine hydrochloride, thiamine hydrochloride, beta-carotene, riboflavin, chromium chloride, folic acid, biotin, potassium iodide, phytonadione, sodium selenite, sodium molybdate, vitamin B12.

[0177] The composition provides a nutritional supplement that is a good source of protein, low fat, vitamins, minerals and antioxidants and meets the FODMAP standard. In addition, the composition contains 2'-FL and DFL, which can promote the growth of beneficial intestinal bacteria and improve intestinal barrier function.

[0178] Example 4 – Capsule composition

[0179] Capsules were prepared by filling about 1 g of 2'-FL and DFL into 000 gelatin capsules using a filling machine. The capsules were then closed. 2'-FL and DFL were in the form of free-flowing powders.

Claims

1. Use of human milk oligosaccharides (HMO) in the preparation of a nutritional composition or a pharmaceutical composition for use in non-infants - Enhance working memory, and / or - Improve myelination to allow maturation of the prefrontal cortex region of the brain, The human milk oligosaccharide is a combination of 2'-FL, DFL, LNT and LNnT.

2. Use of the synthetic composition in the preparation of a nutritional composition or a pharmaceutical composition for use in non-infants - Enhance working memory, and / or - Improve myelination to allow maturation of the prefrontal cortex region of the brain, The composition comprises at least one human milk oligosaccharide (HMO), The human milk oligosaccharide is a combination of 2'-FL, DFL, LNT and LNnT.

3. Use of the encapsulated material in the preparation of a nutritional preparation or a medicine for use in non-infants - Enhance working memory, and / or - Improve myelination to allow maturation of the prefrontal cortex region of the brain, The package comprises at least one human milk oligosaccharide (HMO) in an amount effective for at least 14 individual daily doses, The human milk oligosaccharide is a combination of 2'-FL, DFL, LNT and LNnT.

4. The use according to any one of claims 1 to 3, wherein the non-infant - have attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, depression, anxiety, bipolar disorder, schizophrenia, obsessive compulsive disorder (OCD), autism, frontotemporal dementia, dementia with Lewy bodies, Tourette syndrome, post-traumatic stress disorder (PTSD), traumatic brain injury, or vascular dementia, - are elderly, or - Are or have been born prematurely or small for gestational age.

5. The use according to any one of claims 1 to 3, wherein the HMO is contained in an amount of 0.5 g to 15 g.

6. The use according to any one of claims 1 to 3, wherein the HMO is contained in an amount of 1 g to 10 g.

7. Use of at least one human milk oligosaccharide (HMO) in the preparation of a nutritional composition or a pharmaceutical composition for enhancing working memory in a non-infant, wherein an effective amount of at least one human milk oligosaccharide (HMO) is administered to the non-infant, wherein the human milk oligosaccharide is a combination of 2'-FL, DFL, LNT and LNnT.

8. The use of claim 7, wherein the non-infant suffers from attention deficit hyperactivity disorder (ADHD), Alzheimer's disease, depression, anxiety, bipolar disorder, schizophrenia, obsessive compulsive disorder (OCD), autism, frontotemporal dementia, dementia with Lewy bodies, Tourette syndrome, post-traumatic stress disorder (PTSD), traumatic brain injury, or vascular dementia.

9. Use according to claim 7 or 8, wherein the non-infant is also provided with cognitive behavioral therapy.

10. Use of at least one human milk oligosaccharide (HMO) in the preparation of a nutritional composition or a pharmaceutical composition for improving myelination to mature the prefrontal cortex region of the brain of a non-infant, wherein at least one human milk oligosaccharide (HMO) is administered to the non-infant, wherein the human milk oligosaccharide is a combination of 2'-FL, DFL, LNT and LNnT.

11. Use according to any one of claims 7 to 8 and 10, wherein the HMO is administered to the non-infant in an amount of 0.5 g to 15 g per day.

12. Use according to any one of claims 7 to 8 and 10, wherein the HMO is administered to the non-infant in an amount of 1 g to 10 g per day.

13. The use according to any one of claims 7 to 8 and 10, wherein the HMO is administered to the non-infant for a period of at least 1 week.

14. The use according to any one of claims 7 to 8 and 10, wherein the HMO is administered to the non-infant for a period of at least 2 weeks.

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