Nutritional composition comprising breast milk oligosaccharides and probiotics for improving depression and use thereof

Abstract

The invention belongs to the field of food, and particularly relates to a nutritional composition containing breast milk oligosaccharide and probiotics for improving depression and application of the nutritional composition. The invention provides application of a nutritional composition in preparation of food beneficial to improving constipation and anxiety state and / or depression state caused by constipation. Wherein the nutritional composition comprises the following components (i) and (ii): (i) a neutral fucosylated breast milk oligosaccharide, and (ii) a bifidobacterium probiotic; the neutral fucosylated breast milk oligosaccharide at least comprises 2 '-fucosylated lactose, and the bifidobacterium probiotics at least comprise bifidobacterium longum subsp. Longum. According to the present invention, the substance combination of the neutral fucosylated breast milk oligosaccharide and the bifidobacterium probiotic can improve the abnormal brain-intestine interaction, such that the constipation can be easily improved, and the anxiety and depression state caused by the constipation can be easily improved;

Description

Technical Field

[0001] The present invention belongs to the field of food, and in particular relates to a nutritional composition comprising human milk oligosaccharides and probiotics for improving depression and uses thereof. Background Art

[0002] The brain-gut axis (BGA) is a neuro-endocrine-immune network comprised of the central nervous system (CNS), autonomic nervous system (ANS), enteric nervous system (ENS), and hypothalamic-pituitary-adrenal axis (HPA). The brain and gastrointestinal tract engage in complex, bidirectional communication and interaction through the BGA. Through this BGA, the gastrointestinal tract is regulated by the CNS, ENS, and HPA at various levels. Internal and external stimuli, as well as emotional factors, can influence gastrointestinal function. Furthermore, gastrointestinal activity participates in the regulation of central sensory perception, emotion, and behavior.

[0003] Constipation is a common gastrointestinal complaint in infants, children, and adults. Constipation can be categorized as functional constipation (FC) or organic constipation, depending on whether there are organic changes. Functional constipation is characterized by hard stools, difficulty evacuating, infrequent bowel movements, or a feeling of incomplete defecation. Slow transit constipation (STC) is the most common type of functional constipation. Functional constipation accounts for over 90% of all childhood constipation. It is reported that the incidence of functional constipation in infants under one year old is 2.9%, rising to 10.1% in children aged one to two years and 14% in children. Long-term, recurrent constipation can lead to decreased appetite, mental disorders, and weakened immunity, impacting children's gastrointestinal function, memory, and intellectual development, and even causing enuresis and incontinence, seriously impacting their growth, development, and physical and mental health. If constipation in childhood is not effectively improved, about 25% of children will suffer from other digestive system diseases in adulthood, seriously affecting their quality of life.

[0004] The Rome IV criteria classify functional constipation as abnormal brain-gut interaction. Long-term constipation can not only cause intestinal and perianal lesions, but also cause mental and psychological disorders such as anxiety and depression, and even induce and aggravate neurological, cardiovascular and other systemic diseases, affecting physical and mental health and quality of life. Studies have found that children with long-term (≥2 years) slow-transmission constipation have significantly lower physical, psychological and social quality of life than healthy children. Therefore, slow-transmission constipation is considered to be a chronic debilitating state that affects children's physical and emotional functions. With the help of modern neuroimaging technology, it has been found that people with functional constipation have changes in the structure and functional connectivity of brain areas related to emotion regulation, somatic movement and sensory processing. The enteric nervous system, also known as the "gut brain", can directly regulate gastrointestinal motility through independent neurons and secreted neurotransmitters and hormones. Its functional abnormality is one of the important causes of functional constipation. Studies have found that people with functional constipation have abnormal morphological changes in the submucosal plexus and myenteric plexus.

[0005] The causes and mechanisms of functional constipation are complex, and the relevant people suffer from constipation for a long time, accompanied by psychological problems such as anxiety and depression. For constipation, many people use laxatives, prokinetic drugs, etc. to relieve it, which are effective in the short term, but the long-term effect is not good. For problems such as anxiety and depression, generally only patients who are diagnosed with anxiety or depression and whose condition is severe will choose to use drug intervention. Although these drugs play an important role in the treatment of anxiety or depression, they also have the disadvantages of large side effects and high treatment costs, and are not suitable for people with mild conditions. Therefore, from the perspective of brain-gut interaction, it is worth exploring to find non-toxic and economically priced nutrients that can not only relieve constipation but also adjust the anxiety and depression caused by constipation.

[0006] Reference 1 discloses a probiotic composition for relieving depression and its preparation method, aiming to address the technical problem that long-term use of existing antidepressants can damage intestinal flora, leading to intestinal dysbiosis and causing gastrointestinal reactions. The composition comprises probiotics, prebiotics, and nutrients. The probiotics include Bifidobacterium adolescentis, Bacillus coagulans, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus salivarius. The prebiotics include oligofructose, lactitol, L-arabinose, and inulin. The nutrients include vitamin B6, vitamin B9, and ginseng extract. The combined strains provided in this application have high activity, and the effective combination of probiotics and nutrients can significantly improve sleep, anxiety, and diarrhea in patients with depression.

[0007] Cited document 2 discloses a probiotic powder and its application that can relieve autism and regulate emotions. The probiotic powder is composed of Bifidobacterium bifidum BGN4, Bifidobacterium longum BORI, Bifidobacterium lactis AD011, prebiotics and multiple nutrients. It can regulate and balance the brain-gut axis microbial system, specifically improve the brain-gut microbial environment, can effectively relieve autism, regulate diarrhea, constipation, picky eating, allergies and other gastrointestinal discomfort problems associated with autism, and can effectively relieve emotions, improve sleep status and self-cognition.

[0008] Bifidobacterium longum subspecies longum BB536 is a probiotic of the genus Bifidobacterium that can provide benefits in multiple aspects, including immune regulation, regulation of intestinal microbiota, relief of allergic diseases, maintenance of intestinal health, and anti-infection.

[0009] Reference 3 discloses a study using Bifidobacterium longum subsp. longum BB536 to intervene in chronic constipation in the elderly. The results showed that Bifidobacterium longum subsp. longum BB536 supplementation is safe and partially effective in improving chronic constipation in the elderly.

[0010] 2'-fucosyllactose (2'-FL) is a neutral fucosylated human milk oligosaccharide that plays an important role in regulating intestinal flora, immune response, cognitive function, and reducing inflammation.

[0011] Reference 4 discloses a nutritional composition comprising at least one fucosylated oligosaccharide and at least one N-acetylated oligosaccharide, for preventing and / or treating constipation in infants or young children and / or for improving stool consistency and frequency in infants or young children. The fucosylated oligosaccharide may be 2'-FL.

[0012] Reference 5 discloses a synthetic composition for improving the symptoms of one or more comorbid psychiatric diseases in patients with irritable bowel syndrome, wherein the composition contains an effective amount of one or more neutral human milk oligosaccharides, which may be 2'-FL, etc. The comorbid psychiatric diseases are anxiety and / or depression.

[0013] Currently, there are few studies on the combination of Bifidobacterium probiotics and 2'-fucosyllactose, and the focus is mainly on the impact on intestinal health. For example, Reference 6 discloses the use of a combination for preparing a nutritional composition or preparation, wherein the nutritional composition is used to (a) reduce the production of intestinal gas in infants and young children; and (b) inhibit the excessive growth of Clostridium perfringens in the intestinal tract of infants and young children. The nutritional composition is composed of the following: (i) a human milk oligosaccharide (HMO), wherein the HMO is: 2'-fucosyllactose; and (ii) a probiotic microorganism, wherein the probiotic microorganism is a bifidobacterium, and the bifidobacterium is Bifidobacterium breve M-16V.

[0014] References:

[0015] Reference 1: CN117297098A;

[0016] Reference 2: CN114917255A;

[0017] Reference 3: Usefulness of Bifidobacterium longum BB536 in ElderlyIndividuals With Chronic Constipation: A Randomized Controlled Trial;

[0018] Reference 4: CN107846957A;

[0019] Reference 5: CN108348535B;

[0020] Reference 6: CN112075637B. Summary of the Invention

[0021] Problems to be solved by the invention

[0022] The causes and mechanisms of constipation are complex, and people with constipation suffer from it for a long time, accompanied by psychological problems such as anxiety and depression. Currently, medication is commonly used in clinical practice to treat constipation and the anxiety and depression it causes. However, medications for constipation are generally only effective in the short term and have poor long-term effects. Medications for anxiety and depression are generally only suitable for patients diagnosed with anxiety and depression with relatively severe symptoms, and are not suitable for people with milder conditions, making them not universally applicable.

[0023] While existing research, such as the aforementioned references, suggests using Bifidobacterium probiotics or human milk oligosaccharides to alleviate constipation or mental health issues, such studies are insufficient. Existing research typically focuses solely on improving constipation or anxiety and depression, without comprehensively examining constipation and the anxiety and depression it causes from the perspective of brain-gut interactions.

[0024] In this regard, the present invention has conducted extensive research from the perspective of brain-gut interaction and unexpectedly discovered that the combination of neutral fucosylated human milk oligosaccharides such as 2'-FL and Bifidobacterium probiotics such as Bifidobacterium longum subspecies longum can simultaneously help improve constipation and the anxiety and depression it causes. Based on this, the main purpose of the present invention is to provide a nutritional composition containing neutral fucosylated human milk oligosaccharides and Bifidobacterium probiotics for non-therapeutic purposes in helping to improve constipation and the anxiety and depression it causes.

[0025] Solutions for solving problems

[0026] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0027] [1] A nutritional composition for preparing a food for improving constipation and the anxiety and / or depression caused by constipation; wherein the nutritional composition comprises the following components (i) and (ii):

[0028] (i) neutral fucosylated human milk oligosaccharides,

[0029] (ii) Bifidobacterium probiotics;

[0030] in,

[0031] The neutral fucosylated human milk oligosaccharide comprises at least 2'-fucosyllactose,

[0032] The Bifidobacterium probiotics include at least Bifidobacterium longum subspecies longum.

[0033] [2] The use according to [1], wherein the neutral fucosylated human milk oligosaccharide further comprises any one or more of 3-fucosyllactose, lactose-N-fucopentaose I and difucosyllactose.

[0034] [3] The use according to [1] or [2], wherein the Bifidobacterium probiotic further comprises any one or more of Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum and Bifidobacterium breve.

[0035] [4] The use according to any one of [1] to [3], wherein the Bifidobacterium longum subspecies longum comprises the BB536 strain.

[0036] [5] The use according to any one of [1] to [4], wherein in the nutritional composition, the ratio of the mass of the (i) neutral fucosylated human milk oligosaccharide to the quantity of the (ii) Bifidobacterium probiotic is 1 mg:(2×10 5 -1×10 8 )CFU.

[0037] [6] The use according to any one of [1] to [5], wherein the method for improving constipation and the anxiety and / or depression caused by constipation includes the following improvements:

[0038] Increase the water content of feces.

[0039] [7] The use according to any one of [1] to [6], wherein the method for improving constipation and the anxiety and / or depression caused by constipation includes improving the following:

[0040] Improve gastrointestinal motility.

[0041] [8] The use according to any one of [1] to [7], wherein the method for improving constipation and the anxiety and / or depression caused by constipation includes the following improvements:

[0042] Improve environmental adaptability and action capabilities.

[0043] [9] The use according to any one of [1] to [8], wherein the method for improving constipation and the anxiety and / or depression caused by constipation comprises improving colon tissue damage;

[0044] The improvement of colon tissue damage includes any one or more of the following (b1)-(b4):

[0045] (b1) Improve crypt structure,

[0046] (b2) Reduce inflammatory cell infiltration,

[0047] (b3) Increase the number of goblet cells,

[0048] (b4) Increase the thickness of the colonic muscular layer.

[0049]

[10] The use according to any one of [1] to [9], wherein the method for improving constipation and the anxiety and / or depression caused by constipation includes improving brain tissue structural damage;

[0050] The improvement of brain tissue structural damage includes the following (c1) and / or (c2):

[0051] (c1) Promote regular arrangement of hippocampal neurons,

[0052] (c2) Increase the number of hippocampal neurons.

[0053]

[11] The use according to any one of [1] to

[10] , wherein the method for improving constipation and the anxiety and / or depression caused by constipation comprises improving the content of ghrelin in colon tissue and / or serum;

[0054] The improvement of the content of ghrelin in colon tissue and / or serum includes the following (d1) and / or (d2):

[0055] (d1) increasing the content of any one or more of 5-HT and SP in colon tissue and / or serum,

[0056] (d2) reducing the content of any one or more of NO and VIP in colon tissue and / or serum.

[0057]

[12] . The use according to any one of [1] to

[11] , wherein the food comprises any one or more of infant food, children's food, adolescent food, maternal and infant food, adult food and food for the middle-aged and elderly.

[0058]

[13] The use according to any one of [1] to

[12] , wherein the food is a candy, a beverage, a dairy product, a baked product, a dietary supplement or a food for special dietary uses.

[0059]

[14] The use according to any one of [1] to

[13] , wherein the content of the neutral fucosylated human milk oligosaccharide in the food is 0.01% to 4%, and the content of the Bifidobacterium probiotic is greater than 1×10 6 CFU / g.

[0060]

[15] Use of a nutritional composition in preparing a food having any one or more of the following effects (z1) to (z6):

[0061] (z1) Increase the water content of feces,

[0062] (z2) Improve gastrointestinal motility,

[0063] (z3) Improve environmental adaptability and action capabilities,

[0064] (z4) Improve colon tissue damage,

[0065] (z5) Improve brain tissue structural damage,

[0066] (z6) improving the content of brain-gut peptides in colon tissue and / or serum, wherein the brain-gut peptides include any one or more of 5-HT, SP, NO and VIP;

[0067] The nutritional composition is as defined in any one of [1]-[5].

[0068]

[16] . The use according to

[15] , wherein

[0069] The improvement of colon tissue damage includes any one or more of the following (b1)-(b4):

[0070] (b1) Improve crypt structure,

[0071] (b2) Reduce inflammatory cell infiltration,

[0072] (b3) Increase the number of goblet cells,

[0073] (b4) Increase the thickness of the colonic muscular layer.

[0074] The improvement of brain tissue structural damage includes the following (c1) and / or (c2):

[0075] (c1) Promote regular arrangement of hippocampal neurons,

[0076] (c2) Increase the number of hippocampal neurons.

[0077] The improvement of the content of ghrelin in colon tissue and / or serum includes the following (d1) and / or (d2):

[0078] (d1) increasing the content of any one or more of 5-HT and SP in colon tissue and / or serum,

[0079] (d2) reducing the content of any one or more of NO and VIP in colon tissue and / or serum.

[0080] Effects of the Invention

[0081] The present invention proposes that a nutritional composition comprising a combination of neutral fucosylated human milk oligosaccharides and a probiotic of the genus Bifidobacterium, particularly a nutritional composition comprising a combination of 2'-fucosyllactose and Bifidobacterium longum subspecies longum, can improve abnormal brain-gut interaction, not only helping to alleviate constipation but also helping to alleviate anxiety and depression caused by constipation. In other words, the nutritional composition comprising the above-mentioned substance combination proposed by the present invention can achieve bidirectional and simultaneous improvements in intestinal health and brain and nerve health through the brain-gut axis.

[0082] Experimental data show that consuming a combination of 2'-fucosyllactose and Bifidobacterium longum subsp. longum can improve gastrointestinal motility, increase stool volume and fecal water content, and improve colon tissue damage. Furthermore, this combination can improve the body's ability to adapt to the environment and mobility, and can also improve brain tissue structural damage. Therefore, intuitively speaking, consuming a combination of 2'-fucosyllactose and Bifidobacterium longum subsp. longum can effectively improve both constipation and the anxiety and / or depression it causes.

[0083] In this regard, the present invention has found through further in-depth research that the intake of a substance combination of 2'-fucosyllactose and Bifidobacterium longum subspecies longum can improve the content of brain-gut peptides (such as 5-HT, SP, NO and VIP, etc.) in the body, and these brain-gut peptides are widely present in the central nervous system and gastrointestinal tract, and can affect the interaction of various links in the brain-gut axis. Therefore, the nutritional composition comprising a substance combination of 2'-fucosyllactose and Bifidobacterium longum subspecies longum described in the present invention for the synchronous improvement of constipation and the anxiety and / or depression caused by it is based on the brain-gut axis, that is, the core of the improvement effect of the nutritional composition of the present invention on constipation and the anxiety and / or depression caused by it is the improvement of abnormal brain-gut interaction, and this synchronous improvement effect on the health of the intestines and brain nerves is a two-way regulation.

[0084] In addition, more importantly, the present invention has found that in terms of the above-mentioned effects, there is a synergistic effect between neutral fucosylated human milk oligosaccharides such as 2'-fucosyllactose and Bifidobacterium probiotics such as Bifidobacterium longum subspecies longum, and when the dosage ratio of neutral fucosylated human milk oligosaccharides and Bifidobacterium probiotics reaches a certain range, this synergistic effect is more obvious. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 : Intestinal transit diagram of each group of mice in the embodiment.

[0086] Figure 2 : Pathological sections of the colon tissues of mice in each group in the example.

[0087] Figure 3 : Pathological sections of the hippocampus of mice in each group of the embodiment.

[0088] Figure 4 : Schematic diagram of the overall experimental process and experimental results of the embodiment. DETAILED DESCRIPTION

[0089] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the invention. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0090] In the present invention, the terms "comprising," "having," "including," or "containing" may be inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising," "having," "including," or "containing" may also be closed-ended, excluding additional, unrecited elements or method steps.

[0091] In the present invention, the use of "may" includes both the meanings of performing a certain process and not performing a certain process.

[0092] In the present invention, "optional" or "optionally" means that certain substances, components, execution steps, application conditions and other factors are used or not used.

[0093] In the present invention, numerical ranges expressed using “numerical value A to numerical value B,” “numerical value A - numerical value B,” or “numerical value A or more / or less” refer to ranges including the endpoints A and B.

[0094] Throughout the present invention, the term "about" is used to define numerical ranges and parameters of the present invention. Numerical values are approximate, and the relevant numerical values in the specific embodiments are presented as accurately as possible. Unless otherwise expressly stated, all ranges, quantities, values, and percentages used herein are to be understood as modified by the word "about." As used herein, "about" generally refers to the actual value being within ±5%, ±3%, ±1%, or ±0.5% of a particular value or range. Furthermore, all numerical values and numerical ranges presented herein should be understood to include the inevitable systematic errors that occur in industrial production.

[0095] In the present invention, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0096] In the present invention, the unit names used are all international standard unit names, and unless otherwise stated, the "%" used represents weight or mass percentage.

[0097] In the present invention, "infants and young children" refers to a human group under 3 years old, including infants aged 0-6 months, older infants aged 6-12 months, and young children aged 12-36 months.

[0098] In the present invention, "children" refers to a human group aged 3-6 years.

[0099] In the present invention, "adolescents" refers to the human group aged 7-18 years old.

[0100] In the present invention, "pregnant women" include pregnant women and lactating women.

[0101] In the present invention, "middle-aged and elderly" refers to a human group over 41 years old.

[0102] Unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0103] I. Nutritional Composition

[0104] The nutritional composition of the present invention comprises the following components (i) and (ii): (i) neutral fucosylated human milk oligosaccharides, and (ii) Bifidobacterium probiotics; wherein the neutral fucosylated human milk oligosaccharides include at least 2'-fucosyllactose, and the Bifidobacterium probiotics include at least Bifidobacterium longum subspecies longum.

[0105] In some embodiments, in the nutritional composition described in the present invention, the (i) neutral fucosylated human milk oligosaccharides and (ii) Bifidobacterium probiotics are the main active ingredients, that is, the nutritional composition described in the present invention mainly relies on the (i) neutral fucosylated human milk oligosaccharides and (ii) Bifidobacterium probiotics contained therein to exert specific physiological active functions, such as helping to improve constipation and the anxiety and / or depression caused by it.

[0106] In some embodiments, in the nutritional composition described in the present invention, the (i) 2'-fucosyllactose in neutral fucosylated human milk oligosaccharides and (ii) Bifidobacterium longum subspecies longum in Bifidobacterium probiotics are the main active ingredients, that is, the nutritional composition described in the present invention mainly relies on the 2'-fucosyllactose and Bifidobacterium longum subspecies longum contained therein to exert specific physiological active functions, such as helping to improve constipation and the anxiety and / or depression caused by it.

[0107] The present invention has found that compared with single neutral fucosylated human milk oligosaccharides such as 2'-fucosyllactose and Bifidobacterium probiotics such as Bifidobacterium longum subsp. longum, the nutritional composition provided by the present invention can more effectively improve constipation and the anxiety and / or depression caused by it, that is, there is a synergistic effect between the two.

[0108] In some embodiments, in the nutritional composition of the present invention, the neutral fucosylated human milk oligosaccharide further comprises any one or more of 3-fucosyllactose, lactose-N-fucopentaose I and difucosyllactose.

[0109] In some embodiments, in the nutritional composition of the present invention, the neutral fucosylated human milk oligosaccharide is 2'-fucosyllactose.

[0110] The present invention does not particularly limit the source of the specific neutral fucosylated human milk oligosaccharides. Typically, they can be synthesized by common chemical synthesis methods in the art, such as by glycosylation reaction between a lactose acceptor and a fucosyl donor; they can also be obtained by microbial fermentation methods, for example, using exogenously added lactose as a substrate and 5'-diphosphate guanosine-fucose disodium salt formed by the microorganism's own metabolic pathway as a precursor to synthesize 2'-fucosyllactose under the action of fucosyltransferase.

[0111] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotics further include any one or more of Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum and Bifidobacterium breve.

[0112] The present invention does not particularly limit the specific strains of the above-mentioned bacterial species. For example, the animal Bifidobacterium lactis subspecies includes Bb-12 strain, HN109 strain and Bi-07 strain, etc., Bifidobacterium bifidum includes R0071 strain, etc., the Bifidobacterium longum subspecies infantis includes R0033 strain, etc., Bifidobacterium longum subspecies longus includes BB536 strain, etc., and the Bifidobacterium breve includes M-16V strain, etc.

[0113] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic is Bifidobacterium longum subsp. longum.

[0114] In some embodiments, in the nutritional composition of the present invention, the probiotic Bifidobacterium comprises Bifidobacterium longum subsp. longum BB536 strain.

[0115] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic is Bifidobacterium longum subsp. longum BB536 strain.

[0116] In order to obtain a better synergistic effect of improving constipation and the anxiety and / or depression caused by it, in some specific embodiments, in the nutritional composition, the ratio of the mass of the (i) neutral fucosylated human milk oligosaccharide to the quantity of the (ii) Bifidobacterium probiotic is 1 mg:(1×10 5 -5×10 8 ) CFU, for example, 1 mg: 1×10 5 CFU, 1mg: 2×10 5 CFU, 1mg:3×10 5 CFU, 1mg:4×10 5 CFU, 1mg: 5×10 5 CFU, 1mg: 6×105 CFU, 1mg:7×10 5 CFU, 1mg:8×10 5 CFU, 1mg:9×10 5 CFU, 1mg: 1×10 6 CFU, 1mg: 2×10 6 CFU, 1mg:3×10 6 CFU, 1mg:4×10 6 CFU, 1mg: 5×10 6 CFU, 1mg: 6×10 6 CFU, 1mg:7×10 6 CFU, 1mg:8×10 6 CFU, 1mg:9×10 6 CFU, 1mg: 1×10 7 CFU, 1mg: 2×10 7 CFU, 1mg:3×10 7 CFU, 1mg:4×10 7 CFU, 1mg: 5×10 7 CFU, 1mg: 6×10 7 CFU, 1mg:7×10 7 CFU, 1mg:8×10 7 CFU, 1mg:9×10 7 CFU, 1mg: 1×10 8 CFU, 1mg: 2×10 8 CFU, 1mg:3×10 8 CFU, 1mg:4×10 8 CFU or 1mg: 5×10 8 CFU, etc.; preferably, the ratio of the two can be 1mg:(2×10 5 -5×10 8 ) CFU; more preferably, the ratio of the two can be 1 mg:(2×10 5 -1×10 8 )CFU.

[0117] In some embodiments, the above ratio is the ratio of the content of 2'-fucosyllactose to that of Bifidobacterium longum subsp. longum BB536 in the nutritional composition of the present invention.

[0118] The present invention does not particularly limit the form of the nutritional composition, and typically, it can be liquid or solid, etc. From the perspective of production, transportation, storage and ease of use, the nutritional composition of the present invention is preferably a powdered solid.

[0119] II. Uses of the nutritional composition

[0120] The present invention proposes that compounding neutral fucosylated human milk oligosaccharides and Bifidobacterium probiotics, in particular compounding 2'-fucosyllactose and Bifidobacterium longum subspecies longum, especially compounding in a specific ratio, can help improve constipation and help improve anxiety and / or depression caused by constipation. At the same time, there is a synergistic effect between neutral fucosylated human milk oligosaccharides and Bifidobacterium probiotics, which is better than the single neutral fucosylated human milk oligosaccharides such as 2'-fucosyllactose and Bifidobacterium probiotics such as Bifidobacterium longum subspecies longum.

[0121] The method of the present invention for improving constipation and the anxiety and / or depression caused by it is not intended to treat or prevent diseases, and the constipation and the anxiety and / or depression caused by constipation described in the present invention have not reached the level that can be identified as a disease.

[0122] Furthermore, the present invention provides the use of the above nutritional composition in preparing a food that helps improve constipation and the anxiety and / or depression caused by it. Still further, the present invention provides the use of the above nutritional composition in preparing a food that helps improve constipation and the anxiety and / or depression caused by it in patients with non-anxiety disorders or depression.

[0123] In some embodiments, the present invention improves constipation by improving any one or more of the following conditions: hard stools, difficulty defecating, decreased bowel movement frequency, and a feeling of incomplete defecation. In some embodiments, the present invention improves anxiety and / or depression caused by constipation by primarily improving environmental adaptability and mobility.

[0124] In some embodiments, the improvement of constipation and the anxiety and / or depression caused by constipation includes any one or more of the following improvements (a1)-(a6): (a1) increasing stool water content, (a2) improving gastrointestinal motility, (a3) improving environmental adaptability and mobility, (a4) improving colon tissue damage, (a5) improving brain tissue structural damage, and (a6) improving the content of brain gut peptide in colon tissue and / or serum.

[0125] In some embodiments, the improvement of constipation and the anxiety and / or depression caused by constipation also includes the improvements described in (a1) to (a6).

[0126] The brain-gut peptide (BGP) described in the present invention is a small molecule polypeptide that regulates gastrointestinal function and participates in regulating gastrointestinal physiological activities in the brain-gut axis. It is dually distributed in the gastrointestinal tract and the nervous system. A variety of small molecule polypeptides with dual functions of neurotransmitters and hormones affect the interaction of various links in the brain-gut axis. There are more than 60 types of BGPs that have been discovered. BGP mediates intercellular information transmission in the brain-gut axis, exerts hormone and local transmitter effects, plays a role in building a connecting bridge and regulating functions, and can also directly act on the corresponding receptors of gastrointestinal sensory nerve endings or smooth muscle cells to regulate gastrointestinal function. BGP is mainly divided into three categories: gastrointestinal peptide hormones, gastrointestinal neuropeptides, and neuropeptides. Among them, gastrointestinal peptide hormones can be divided into two categories: excitatory and inhibitory types. These transmitters interact and influence each other. They play a regulatory role together.

[0127] In some specific embodiments, the improvement of colon tissue damage includes any one or more of the following (b1)-(b4): (b1) improving crypt structure, (b2) reducing inflammatory cell infiltration, (b3) increasing the number of goblet cells, and (b4) increasing the thickness of the colon muscle layer.

[0128] In some specific embodiments, the improving colon tissue damage includes the above-mentioned (b1)-(b4) at the same time.

[0129] In some specific embodiments, the improving brain tissue structural damage includes the following (c1) and / or (c2): (c1) promoting regular arrangement of hippocampal neurons, (c2) increasing the number of hippocampal neurons.

[0130] In some specific embodiments, the improvement of brain tissue structural damage includes both (c1) and (c2) above.

[0131] In some specific embodiments, the improvement of the content of brain gut peptide in colon tissue and / or serum includes the following (d1) and / or (d2): (d1) increasing the content of any one or more of 5-HT and SP in colon tissue and / or serum, (d2) reducing the content of any one or more of NO and VIP in colon tissue and / or serum.

[0132] In some specific embodiments, the improving the content of ghrelin in colon tissue and / or serum includes both (d1) and (d2) above.

[0133] 5-hydroxytryptamine (5-HT) is a brain-gut peptide with multiple biological effects. It primarily exists in the central nervous system and enteric nervous system. It promotes gastrointestinal motility, regulates the duodenogastric reflex, and modulates the secretion of pepsin and gastric acid. It also participates in regulating physiological activities such as emotion, cognition, mood, and sleep. 5-HT is a key neurotransmitter in the brain-gut axis, connecting the central nervous system and the enteric nervous system.

[0134] Substance P (SP) is the first identified, multiply distributed brain-gut peptide, mainly distributed in the central nervous system, dorsal roots of the spinal cord and enteric nervous system (most secreted in the proximal small intestine and colon). It has the functions of contracting gastrointestinal smooth muscle fibers to promote gastrointestinal motility, protecting the gastrointestinal epithelium to repair and heal damaged gastrointestinal mucosa, producing gastrointestinal mechanical hypersensitivity, and causing intestinal pain.

[0135] Vasoactive intestinal peptide (VIP) is a type of neuropeptide that inhibits gastrointestinal motility and has dual effects as a neurotransmitter and neuromodulator. It is expressed in large quantities in the central nervous system and gastrointestinal tract, and has the effects of inhibiting gastrointestinal motility and gastric emptying, slowing small intestinal motility, reducing the tension of the digestive sphincter, and dilating blood vessels.

[0136] Nitric oxide (NO) is an inhibitory neurotransmitter in brain-gut peptides. It is primarily distributed in the enteric nervous system and intestines of the antral muscularis and is produced in the human intestine in enzymatic, non-enzymatic, and bacterial forms. NO can induce smooth muscle relaxation, weaken antral contractions, and thus delay gastric emptying. It can also bidirectionally regulate the intensity of the fundus receptive reflex arc and the intestinal peristaltic reflex arc.

[0137] In some embodiments, the present invention also provides a use of the above nutritional composition in preparing a food having any one or more of the following effects (z1) to (z6):

[0138] (z1) Increase the water content of feces,

[0139] (z2) Improve gastrointestinal motility,

[0140] (z3) Improve environmental adaptability and action capabilities,

[0141] (z4) Improve colon tissue damage,

[0142] (z5) Improve brain tissue structural damage,

[0143] (z6) improving the content of brain-gut peptides in colon tissue and / or serum, wherein the brain-gut peptides include any one or more of 5-HT, SP, NO and VIP.

[0144] The present invention does not particularly limit the specific food containing the above nutritional composition or the food that can be prepared using the above nutritional composition.

[0145] In some embodiments, under normal temperature conditions, the food of the present invention is in the form of liquid or solid.

[0146] In some embodiments, the food of the present invention is candy, such as hard candy, jelly candy, crisp candy, compressed candy, and aerated candy.

[0147] In some embodiments, the food of the present invention is a beverage, such as a carbonated beverage, a tea beverage, a coffee beverage, a fruit and vegetable juice beverage, and a lactic acid bacteria beverage.

[0148] In some embodiments, the food of the present invention is a dairy product, such as milk powder, cheese, yogurt, liquid milk, etc.

[0149] In some embodiments, the food of the present invention is a baked food, such as bread, cakes, and biscuits.

[0150] In some embodiments, the food of the present invention is a dietary supplement, such as a hard capsule, a soft capsule, a tablet, an oral solution, a pill, a granule, a powder, and the like.

[0151] In some embodiments, the food of the present invention is a food for special dietary uses, such as infant formula, infant complementary food, complementary nutritional supplement, and special medical purpose formula.

[0152] In some embodiments, the food of the present invention includes any one or more of infant food, adolescent food, pregnant and lying-in women food, and middle-aged and elderly food.

[0153] For example, the food of the present invention may be infant formula milk powder, maternal and infant nutrition package, middle-aged and elderly milk powder, etc.

[0154] In some embodiments, in the food of the present invention, the neutral fucosylated human milk oligosaccharides and the Bifidobacterium probiotics are all derived from the nutritional composition. In other embodiments, in the food of the present invention, the neutral fucosylated human milk oligosaccharides and the Bifidobacterium probiotics are derived from other food raw materials, such as animal milk, in addition to the nutritional composition.

[0155] In some embodiments, in the food of the present invention, the ratio of the mass of neutral fucosylated human milk oligosaccharides to the number of Bifidobacterium probiotics can be 1 mg:(1×10 5 -5×10 8 ) CFU; preferably, the ratio of the two can be 1 mg:(2×10 5 -5×10 8 ) CFU; more preferably, the ratio of the two can be 1 mg:(2×10 5 -1×10 8 In some embodiments, the above ratio is the ratio of the content of 2'-fucosyllactose to the content of Bifidobacterium longum subsp. longum BB536 in the food of the present invention.

[0156] The present invention does not particularly limit the specific absolute content of the neutral fucosylated human milk oligosaccharides and the Bifidobacterium probiotics in food, as long as the requirements of local food-related laws and regulations are met.

[0157] In some embodiments, in the food, the content of the neutral fucosylated human milk oligosaccharide is 0.01% to 4%, and the content of the Bifidobacterium probiotic is greater than 1×10 6 CFU / g.

[0158] In addition to the components described above for the nutritional composition, the food may also contain other ingredients, such as proteins / amino acids, carbohydrates, fats, vitamins, minerals and other common food ingredients.

[0159] In addition, depending on the type of food and the final needs of the applicable object, in some embodiments, the food also contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional added ingredients and any acceptable excipients.

[0160] For plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, black currants, cranberries, raspberries, melons, emblica chinensis and bilberries or their extracts; fruits and vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; grains such as rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, millet, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans or their extracts; nuts such as walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts or their extracts; coffee or its extracts; and some botanical Chinese medicinal materials or their extracts that are both medicinal and edible.

[0161] Examples of animal dairy ingredients include fresh milk from cows, sheep, etc., as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, hydrolyzed whey protein powder, and casein powder.

[0162] Examples of animal meat product ingredients include meat product ingredients from pigs, cattle, sheep, aquatic products or poultry.

[0163] Examples of functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0164] As for any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0165] Example

[0166] The embodiments of the present invention will be described in detail below with reference to the examples. However, it will be understood by those skilled in the art that the following examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used were commercially available conventional products.

[0167] Nutrients used in the embodiments of the present invention and their sources:

[0168] 2'-fucosyllactose (2'-FL): DSM;

[0169] Bifidobacterium longum subsp. longum BB536: Morinaga.

[0170] Animal experiment design in the embodiment of the present invention:

[0171] One hundred and forty healthy male BALB / c mice (6 weeks old, 20 g) were housed under 12 h light / dark cycle, 25 ± 2 °C temperature, and 55%-65% humidity with free access to sterile water and normal chow throughout the experiment.

[0172] After one week of adaptive feeding, the rats were randomly divided into 14 groups, each with 10 rats, including a blank group (K), a constipation anxiety model group (M), a positive control group (Y), a high-dose 2'-FL group (H2), a medium-dose 2'-FL group (M2), a low-dose 2'-FL group (L2), a high-dose BB536 group (HB), a low-dose BB536 group (LB), a high-dose 2'-FL combined with a high-dose BB536 group (HH), a medium-dose 2'-FL combined with a high-dose BB536 group (MH), a low-dose 2'-FL combined with a high-dose BB536 group (LH), a high-dose 2'-FL combined with a low-dose BB536 group (HL), a medium-dose 2'-FL combined with a low-dose BB536 group (ML), and a low-dose 2'-FL combined with a low-dose BB536 group (LL). Specific experimental group information is shown in Table 1.

[0173] Except for the blank group, mice in other groups were gavaged with 0.2 mL (10 mg / kg.bw) of loperamide once a day at 9:00 am. The blank group was gavaged with normal saline for 7 days. On the 8th day, the blank group was gavaged with normal saline every day, while the other groups were gavaged with 0.2 mL (10 mg / kg.bw) of loperamide. Two hours after gavage with loperamide, the positive group was gavaged with 2.5 mg / kg / d of mosapride. The high-dose 2'-FL, medium-dose 2'-FL, and low-dose 2'-FL groups were gavaged with 2500 mg / kg.bw, 1500 mg / kg.bw, and 500 mg / kg.bw of 2'-FL, respectively. The high-dose BB536 and low-dose BB536 groups were gavaged with 5×10 10 CFU / kg.bw and 5×10 8 CFU / kg.bw of BB536, the high-dose 2'-FL + high-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (2500 mg / kg.bw and 5×10 10 CFU / kg.bw), the medium-dose 2'-FL + high-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (1500 mg / kg.bw and 5×10 10CFU / kg.bw), the low-dose 2'-FL + high-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (500 mg / kg.bw and 5×10 10 CFU / kg.bw), the high-dose 2'-FL + low-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (2500 mg / kg.bw and 5×10 8 CFU / kg.bw), the medium-dose 2'-FL + low-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (1500 mg / kg.bw and 5×10 8 CFU / kg.bw), the low-dose 2'-FL + low-dose BB536 group was orally gavaged with a mixed solution of 2'-FL and BB536 (500 mg / kg.bw and 5×10 8 CFU / kg.bw).

[0174] On day 29, mice were fasted for 12 hours (with free access to water), and orbital blood was collected from each mouse and allowed to rest for 1 hour. The collected blood was centrifuged at 4000×g for 15 minutes to obtain serum for biochemical assays. The whole brain was quickly dissected and weighed. The colon tissue was straightened and the length from the anus to the end of the cecum was measured with a ruler and recorded. A 1-cm segment of the colon was cut and immersed in a 4% paraformaldehyde solution for subsequent histopathological sectioning. All remaining animal tissues were frozen in a -80°C ultra-low temperature freezer until use.

[0175] Table 1 Experimental groups

[0176]

[0177] Example 1: Effects of nutrients on physiological parameters of mice

[0178] 1 Experimental Methods

[0179] 1.1 Mouse health index

[0180] During the mouse rearing period, observe the mice's growth status, mental state, hair changes, defecation, activity status, and other clinical manifestations, as well as whether they died, every day. Observe and record the mice's weight, water intake, and food intake at the same time every day.

[0181] 1.2 Statistical analysis

[0182] The experimental data are expressed as mean ± standard deviation. P < 0.05 indicates a significant difference between the data, and P > 0.05 indicates an insignificant difference between the data.

[0183] 2 Experimental results

[0184] After oral administration, the body weight, food consumption, and water intake of mice in each group are shown in Table 2. The body weights of mice in groups K, M, and Y were 26.65±1.32g, 24.75±0.90g, and 25.71±1.00g, respectively. Compared with groups K and Y, group M had a relatively lower body weight, but there were no significant differences among the three groups (P>0.05). Furthermore, there were no significant differences in food consumption and water intake among the groups (P>0.05).

[0185] Table 2 Body weight, food intake and water intake of mice

[0186]

[0187] Example 2: Effects of nutrients on defecation indicators in mice

[0188] 1 Experimental Methods

[0189] 1.1 Fecal water content

[0190] Feces were collected on days 14 and 28 of feeding and dried to constant weight to calculate fecal dry weight and moisture content. Fecal samples collected on day 28 were stored at -80°C for analysis of short-chain fatty acids and intestinal microbiota. Fresh mouse feces were weighed and recorded as wet weight. Feces were then dried at 105°C for 5 hours to constant weight and weighed and recorded as dry weight.

[0191] Fecal moisture content = (wet weight - dry weight) / dry weight × 100%.

[0192] 1.2 Determination of defecation time

[0193] On day 28, all mice were fasted overnight for 12 hours and then gavaged with activated carbon solution (10% activated carbon, 0.5% carboxymethylcellulose suspension) before being placed in metabolic cages. The time between the mice's ingestion of activated carbon and their defecation of dark feces was recorded.

[0194] 1.3 Gastrointestinal transit rate

[0195] On day 29, the animals were gavaged with activated carbon solution. Thirty minutes later, all animals were euthanized by cervical dislocation, and the small intestine was collected by dissection. The distance from the pylorus to the border of the activated carbon was considered the migration distance. After measuring this length, the gastrointestinal transit rate was calculated using the following formula:

[0196] Gastrointestinal transit rate = activated carbon migration distance / total length of small intestine × 100%.

[0197] 1.4 Statistical analysis

[0198] The experimental data are expressed as mean ± standard deviation. P < 0.05 indicates a significant difference between the data, and P > 0.05 indicates an insignificant difference between the data.

[0199] 2 Experimental results

[0200] Fecal water content is one of the important indicators reflecting the degree of constipation in mice. The effects of 2'-FL and BB536 on the fecal water content of mice are shown in Table 3. Compared with the K group, the fecal water content of mice in the M group was significantly reduced (P<0.05), indicating that the administration of loperamide caused the feces to become dry and hard. Compared with the M group, the fecal water content of mice in the Y group was significantly increased (P<0.05), indicating that mosapride can effectively improve fecal water content. After intervention with high and medium doses of 2'-FL or high and low doses of BB536 alone, the fecal water content of Experimental Examples 1, 2, 4, and 5 was significantly higher than that of the M group (P<0.05). Although the fecal water content of Experimental Example 3 was higher than that of the M group, there was no significant difference (P>0.05). In addition, the fecal water content of all 2'-FL and BB536 combined treatment groups was significantly higher than that of the M group (P<0.05). Moreover, compared with the increase in fecal water content of the 2'-FL or BB536 single treatment groups relative to the M group, the 2'-FL and BB536 combined treatment groups had more advantages. Taking Experimental Examples 2, 5, and 10 as examples, the increase in fecal water content of the ML group relative to the M group (30.05%) was greater than the sum of the increase in fecal water content of the M2 group relative to the M group (5.67%) and the increase in fecal water content of the LB group relative to the M group (10.63%).

[0201] The time to first black stool reflects the motility of the entire gastrointestinal tract. The longer the time to first black stool, the weaker the motility of the mouse's gastrointestinal tract. As shown in Table 3, the time to first black stool in Group M was 144.64±5.89 minutes, significantly higher than the 78.23±4.87 minutes in Group K. Compared with Group M, the time to first black stool in Group Y was significantly reduced (P<0.05), indicating that mosapride can effectively improve gastrointestinal motility. Compared with Group M, the time to first black stool was significantly reduced after intervention with different doses of 2'-FL and BB536 (P<0.05). Among them, compared with the reduction in the time to first black stool in the 2'-FL or BB536 single treatment group relative to the M group, the 2'-FL and BB536 combined treatment group has more advantages. Taking experimental examples 1, 5, and 9 as examples, the reduction value of the HL group relative to the M group (60.75 min) is greater than the sum of the reduction value of the H2 group relative to the M group (31.59 min) and the reduction value of the LB group relative to the M group (25.68 min).

[0202] The effects of 2'-FL, BB536, and 2'-FL combined with BB536 on the number of fecal particles in mice within 3 hours are shown in Table 3. The number of fecal particles excreted by mice within 3 hours was significantly reduced in the M group compared with the K group (P<0.05). Compared with the M group, the number of fecal particles in the mice increased significantly after intervention with positive drugs, different doses of 2'-FL, and BB536 (P<0.05). Among them, the number of fecal particles in the LB group was higher than that in the M group, but the data did not show significant differences (P>0.05). In addition, compared with the increase in the number of fecal particles in the 2'-FL or BB536 single treatment group relative to the M group, the 2'-FL and BB536 combined treatment group was more advantageous. Taking experimental examples 3, 4, and 8 as examples, the increase in the number of fecal particles in the LH group relative to the M group (8) was greater than the sum of the increase in the number of fecal particles in the L2 group relative to the M group (3.67) and the increase in the number of fecal particles in the HB group relative to the M group (3.34).

[0203] The intestinal transit rate can reflect the motility of the entire gastrointestinal tract and is an important indicator for evaluating constipation. The faster the intestinal transit rate, the more conducive it is to the excretion of feces. The effects of 2'-FL, BB536, and 2'-FL combined with BB536 on intestinal transit in mice are shown in Table 3. The intestinal transit rate of mice in group K reached 83.58±9.56%. Compared with group K, the intestinal transit rate of mice in group M was significantly reduced (P<0.05). In addition, the intestinal transit diagrams of groups K, M, and Y are shown in Figure 3. Figure 1 As shown, it is obvious that the distance of activated carbon transport in the M group is shorter, which indicates that the constipation model has been successfully established. Compared with the M group, after intervention with positive drugs, different doses of 2'-FL and BB536, the intestinal transit rate of mice increased significantly (P<0.05). Among them, although the intestinal transit rate of the L2 group was higher than that of the M group, the data were not significantly different, which was similar to the results of the water content of mouse feces. In addition, compared with the increase in the intestinal transit rate of the 2'-FL or BB536 single treatment group relative to the M group, the 2'-FL and BB536 combined treatment group was more advantageous. Taking Experimental Examples 3, 5, and 11 as examples, the increase in the LL group relative to the M group (29.66%) was greater than the sum of the increase in the L2 group relative to the M group (5.58%) and the increase in the LB group relative to the M group (14.93%).

[0204] The above results showed that 2'-FL, BB536 and the combination of 2'-FL and BB536 could significantly improve constipation in mice, but the combination group showed a synergistic effect.

[0205] Table 3 Effects of nutrients on defecation indicators in mice

[0206]

[0207] Example 3: Effects of nutrients on behavioral indicators in mice

[0208] 1 Experimental methods

[0209] 1.1 Elevated Plus Maze (EPM) experiment

[0210] The EPM experimental apparatus consists of a "+"-shaped maze, 50 cm above the ground, with two opposing open arms, two opposing closed arms, and a 5×5 cm central area. Each arm is 30 cm long and 5 cm wide. The closed arms have 15 cm high walls, while the open arms have 3 mm high rock shelves. Each mouse is placed in the center of the apparatus, facing an open arm. All spontaneous activities are recorded (5 min) and calculated using SMART3.0. The performance of mice in the elevated plus maze test can reflect their ability to adapt to different environments, and the time it takes to enter the open arm is used as an indicator of anxiety-like behavior. The apparatus was cleaned with 75% ethanol solution before and after each experiment.

[0211] 1.2 Open field test (OFT)

[0212] OFT is a classic method for evaluating the anxiety behavior of rodents to assess their locomotor behavior, exploratory behavior and tension. Each mouse was randomly selected and placed in an apparatus (50×50cm 2 The test was conducted individually in a chamber (40 cm and 40 cm high). The bottom of the apparatus was divided into 16 squares. Each subject was placed in a corner of the apparatus for 1 minute to adapt. All spontaneous activities were recorded (15 minutes) and calculated using an automatic movement activity recording tracker. The apparatus was cleaned with 75% ethanol solution before and after each experiment. The total distance traveled, average speed, total distance moved in the center area, and number of times the mice entered the center area were calculated for each group.

[0213] 1.3 Tail Suspension Test (TST)

[0214] The TST is widely used to study depressive states in mice. The mouse's tail is secured with tape and suspended in mid-air, with its head positioned 15 cm above a surface, for 6 minutes. Behavioral tracking software records video footage from the second to sixth minute, precisely capturing the duration of the mouse's immobility.

[0215] 1.4 Forced swimming test (FST)

[0216] The FST is used to assess depressive-like behaviors. Mice are placed in a clear glass tube 35 cm high and 15 cm in diameter. The water is maintained at a depth of 10 cm and a temperature of 25 ± 1°C. The mice are allowed to swim for 6 minutes, and their activity is recorded using a video capture system. A stopwatch is used to record the duration of immobility for 4 minutes after the end of the experiment.

[0217] 1.5 Statistical analysis

[0218] The experimental data are expressed as mean ± standard deviation. P < 0.05 indicates a significant difference between the data, and P > 0.05 indicates an insignificant difference between the data.

[0219] 2 Experimental Results

[0220] 2.1 Elevated plus maze test

[0221] The elevated plus maze test primarily assesses anxiety by comparing the time mice spend in the open arms. Table 4 shows the effects of different doses of 2'-FL, BB536, and 2'-FL combined with BB536 on the time mice spend in the open arms during the elevated plus maze test. Compared with group K, group M significantly decreased the time mice spent in the open arms (P < 0.05). Compared with group M, treatment with 2'-FL alone, high-dose BB536 alone, or both treatments significantly increased the time mice spent in the open arms (P < 0.05). Furthermore, compared with the increase in arm open time relative to group M observed in groups treated with either 2'-FL or BB536 alone, the combined treatment with 2'-FL and BB536 was superior. For example, in Experiments 3, 5, and 11, the increase in arm open time in group LL (32.67 seconds) relative to group M was greater than the sum of the increases in arm open time in group L2 (10 seconds) and group LB (8 seconds). Therefore, the BB536 and 2'-FL combination group showed good synergistic effect in alleviating anxiety in constipated mice.

[0222] Table 4 Effects of nutrients on the open arm activity time of mice in the elevated plus maze test

[0223]

[0224] 2.2 Open field experiment

[0225] The open field test is a commonly used behavioral assay for assessing anxiety in mice. When animals are placed in an unfamiliar open field (a strange environment), fear of the unfamiliar environment causes them to primarily move in the peripheral area. However, the novelty of the unfamiliar environment prompts them to explore the central area, creating a conflict. The open field test measures anxiety levels by observing spontaneous movements in a novel environment. The effects of different doses of 2'-FL, BB536, and 2'-FL combined with BB536 on the locomotor activity of mice are shown in Table 5. Compared with group K, mice in group M showed significant decreases in total distance traveled in the open field test, distance traveled in the central area, number of platform crossings, and average speed (P < 0.05). These results indicate that a mouse anxiety model has been successfully established. Compared with group M, after positive drug intervention, mice in group Y showed significant increases in total distance traveled in the open field test, distance traveled in the central area, number of platform crossings, and average speed (P < 0.05). In addition, after intervention with high or medium doses of 2'-FL or high dose of BB536 alone, the total movement distance, central area movement distance, number of platform crossings and average speed of experimental cases 1, 2 and 4 were significantly higher than those of group M (P<0.05). After intervention with low doses of 2'-FL and low dose of BB536 alone, the total movement distance, central area movement distance, number of platform crossings and average speed of experimental cases 3 and 5 were higher than those of group M, but there was no significant difference (P>0.05). In addition, the total movement distance, movement distance in the central area, number of platform crossings and average speed of all 2'-FL and BB536 combined treatment groups were significantly higher than those of the M group (P<0.05). At the same time, compared with the increase in total movement distance, movement distance in the central area, number of platform crossings and average speed of the 2'-FL or BB536 single treatment groups relative to the M group, the 2'-FL and BB536 combined treatment group had more advantages. Taking experimental cases 2, 5 and 10 as examples, in terms of total movement distance, the increase in the ML group relative to the M group (2549.33 cm) was greater than the increase in the M2 group relative to the M group (1421.60 cm) and the increase in the LB group relative to the M group (672.45 cm). cm); in terms of central area movement distance, the increase in the ML group relative to the M group (1409.18 cm) was greater than the sum of the increase in the M2 group relative to the M group (670.72 cm) and the increase in the LB group relative to the M group (445.44 cm); in terms of the number of platform crossings, the increase in the ML group relative to the M group (62.58 times) was greater than the sum of the increase in the M2 group relative to the M group (38.67 times) and the increase in the LB group relative to the M group (16 times); in terms of average speed, the increase in the ML group relative to the M group (2.77 cm / s) was greater than the sum of the increase in the M2 group relative to the M group (1.54 cm / s) and the increase in the LB group relative to the M group (0.73 cm / s). These results indicate that the combination of 2'-FL and BB536 exhibits a synergistic effect in relieving anxiety caused by constipation compared to either of them alone.

[0226] Table 5 Effects of nutrients on the results of open field test in mice

[0227]

[0228] 2.3 Tail suspension test

[0229] The tail suspension test is a model of despair, and the length of time a mouse stops struggling and stops moving is used as an indicator to reflect its state of despair. The tail suspension test is usually used as one of the common behavioral tests to assess depression in mice. The effects of oral gavage of 2'-FL and BB536 on the depressive behavior of mice are shown in Table 6. The immobility time of mice in the blank K group in the tail suspension test was 59.33±6.43s. Compared with the blank K group, the immobility time of mice in the M group in the tail suspension test was significantly increased to 141.33±8.62s (P<0.05), indicating that mice gavaged with loperamide showed depressive behavior. Compared with the M group, after intervention with high or medium doses of 2'-FL or high dose of BB536 alone, the immobility time of the mice in Experimental Examples 1, 2, and 4 in the tail suspension test was significantly reduced (P<0.05). After intervention with low doses of 2'-FL and low doses of BB536 alone, the immobility time of the mice in Experimental Examples 3 and 5 in the tail suspension test was lower than that of the M group, but there was no significant difference (P>0.05). At the same time, the immobility time of mice treated with different doses of 2'-FL and BB536 was significantly lower than that of the M group in the tail suspension test (P<0.05), and compared with the reduction in the tail suspension immobility time of the 2'-FL or BB536 alone treatment group relative to the M group, the 2'-FL and BB536 combined treatment group had an advantage. Taking experimental examples 3, 5, and 11 as an example, the reduction value of the LL group relative to the M group (50.91s) was greater than the sum of the reduction value of the L2 group relative to the M group (22s) and the reduction value of the LB group relative to the M group (17s), indicating that the combination of 2'-FL and BB536 can synergistically relieve depression caused by constipation.

[0230] Table 6 Effects of nutrients on tail suspension immobility time in mice

[0231]

[0232] 2.4 Forced swimming test

[0233] Forced swimming is a behavioral despair test and a commonly used behavioral test for assessing depression in mice. The effects of different doses of 2'-FL, BB536, and 2'-FL combined with BB536 on the immobility time of mice in the forced swim test are shown in Table 7. The results of the forced swim test showed that oral administration of loperamide induced an increase in the immobility time of mice, reaching 127.67±4.93 seconds. Compared with the mice in the M group, treatment with the positive drug, different doses of 2'-FL, BB536, and 2'-FL combined with BB536 all reduced the immobility time of the mice in the water. Compared with the M group, treatment with high or medium doses of 2'-FL or high dose of BB536 alone significantly reduced the immobility time of mice in Experiments 1, 2, and 4 (P < 0.05). Treatment with low doses of 2'-FL or low dose of BB536 alone resulted in lower immobility time in the swim test in Experiments 3 and 5, but the difference was not significant (P > 0.05). At the same time, the immobility time of mice treated with different doses of 2'-FL and BB536 was significantly lower than that of the M group in the swimming experiment (P<0.05), and compared with the reduction in swimming immobility time of the 2'-FL or BB536 single treatment group relative to the M group, the 2'-FL and BB536 combination treatment group had more advantages. Taking Experimental Examples 3, 4, and 8 as an example, the reduction value of the LH group relative to the M group (46.67s) was greater than the sum of the reduction value of the L2 group relative to the M group (17.67s) and the reduction value of the HB group relative to the M group (22s), indicating that the combination of 2'-FL and BB536 can synergistically relieve depression caused by constipation.

[0234] Table 7 Effects of nutrients on immobility time in mice forced swimming test

[0235]

[0236] Example 4: Effects of nutrients on indicators related to the brain-gut axis

[0237] 1 Experimental Methods

[0238] 1.1 Determination of biochemical markers of the brain-gut axis

[0239] A portion of frozen colon tissue was homogenized and pre-treated according to the test kit instructions. Experiments were performed strictly according to the protocol to measure 5-hydroxytryptamine (5-HT), substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide (NO). Serum samples from all groups were thawed at 4°C, and serum 5-HT, SP, VIP, and NO levels were measured according to the kit instructions.

[0240] 1.2 Statistical analysis

[0241] The experimental data are expressed as mean ± standard deviation. SPSS 26.0 software was used for data analysis, and one-way analysis of variance was performed. Turkey test was used to compare whether there were significant differences between the treatment groups. P < 0.05 indicated a significant difference between the data, and P > 0.05 indicated an insignificant difference between the data.

[0242] 2 Experimental results

[0243] Brain-gut peptides can connect and regulate all aspects of the brain-gut axis interaction. They are widely distributed in the gastrointestinal tract and brain tissue, and have the dual effects of hormones and neurotransmitters. The effects of various brain-gut peptides are different, and they are interconnected and jointly participate in the regulation of gastrointestinal motility, sensation and secretion, as well as central emotions and behaviors. In people with functional constipation and abnormal brain-gut interaction, their brain-gut peptide secretion disorders gradually worsen, and their anxiety and depression tendencies also increase. These negative emotions can affect the distribution of some brain-gut peptides in the gastrointestinal tract and serum, suggesting that brain-gut peptides interact with anxiety and depression. SP and 5-HT are the main excitatory brain-gut peptides, which can promote gastrointestinal motility, while VIP and NO are inhibitory brain-gut peptides, which have an inhibitory effect on gastrointestinal motility. The regulation of brain-gut peptide levels is closely related to constipation, anxiety, and brain-gut interaction.

[0244] The effects of nutrients on colonic ghrelin levels in mice are shown in Table 8. After oral administration of loperamide, colonic 5-HT and SP levels in mice in group M were significantly decreased (P < 0.05), while colonic NO and VIP levels were significantly increased (P < 0.05) compared with group K. Compared with group M, colonic 5-HT and SP levels were significantly increased (P < 0.05), while colonic NO and VIP levels were significantly decreased (P < 0.05) after treatment with the positive drug mosapride. Compared with group M, colonic 5-HT levels were significantly increased (P < 0.05), while VIP levels were significantly decreased (P < 0.05) after treatment with high-dose 2'-FL or high-dose BB536. Colonic SP levels were significantly increased (P < 0.05), while NO levels were significantly decreased (P < 0.05) after treatment with high- and medium-dose 2'-FL or high-dose BB536. There was no significant difference in the colon 5-HT, SP, VIP and NO contents of mice in the low-dose 2'-FL group of Experimental Example 3 and the low-dose BB536 group of Experimental Example 5 compared with those in the M group. In addition, the colon 5-HT and SP contents of mice treated with different doses of 2'-FL and BB536 were significantly increased (P<0.05), and the colon NO and VIP contents were significantly decreased (P<0.05). Among them, compared with the increased 5-HT and SP values and decreased VIP and NO values of the 2'-FL or BB536 single treatment groups relative to the M group, the 2'-FL and BB536 combined treatment group had more advantages. Taking Experimental Examples 1, 5, and 9 as examples, in terms of 5-HT content, the increased value of the HL group relative to the M group (90.04) was greater than the sum of the increased value of the H2 group relative to the M group (52.69) and the increased value of the LB group relative to the M group (16.21); in terms of SP content, the increased value of the HL group relative to the M group (51.7 6) was greater than the sum of the increased value of the H2 group relative to the M group (27.81) and the increased value of the LB group relative to the M group (18.68); in terms of VIP content, the decreased value of the HL group relative to the M group (79.36) was greater than the sum of the decreased value of the H2 group relative to the M group (53.1) and the decreased value of the LB group relative to the M group (25.51); in terms of NO content, the decreased value of the HL group relative to the M group (1.87) was greater than the sum of the decreased value of the H2 group relative to the M group (0.96) and the decreased value of the LB group relative to the M group (0.14).

[0245] The effects of nutrients on serum ghrelin levels in mice are shown in Table 9. After oral administration of loperamide, compared with group K, serum 5-HT and SP levels in group M were significantly decreased (P < 0.05), while serum NO and VIP levels were significantly increased (P < 0.05). Compared with group M, treatment with the positive drug mosapride significantly increased serum 5-HT and SP levels (P < 0.05), while serum NO and VIP levels were significantly decreased (P < 0.05). Compared with group M, treatment with high and medium doses of 2'-FL or high dose of BB536 significantly increased serum 5-HT and SP levels (P < 0.05), while treatment with high doses of 2'-FL or high dose of BB536 significantly decreased serum VIP levels (P < 0.05). Treatment with high, medium, and low doses of 2'-FL or high and low doses of BB536 significantly decreased serum NO levels (P < 0.05). There was no significant difference in the serum 5-HT, SP and VIP levels between the low-dose 2'-FL group of Experimental Example 3 and the low-dose BB536 group of Experimental Example 5 and the M group. In addition, the serum 5-HT and SP levels of mice treated with different doses of 2'-FL and BB536 were significantly increased (P<0.05), and the serum NO and VIP levels were significantly decreased (P<0.05). Among them, compared with the increased 5-HT and SP values and decreased VIP and NO values of the 2'-FL or BB536 single treatment groups relative to the M group, the 2'-FL and BB536 combined treatment group had more advantages. Taking Experimental Examples 3, 4, and 8 as examples, in terms of 5-HT content, the increased value of the LH group relative to the M group (32.54) was greater than the sum of the increased value of the L2 group relative to the M group (11.25) and the increased value of the HB group relative to the M group (16.49); in terms of SP content, the increased value of the LH group relative to the M group (23.0 6) was greater than the sum of the increased value of the L2 group relative to the M group (7.47) and the increased value of the HB group relative to the M group (10.51); in terms of VIP content, the decreased value of the LH group relative to the M group (55.84) was greater than the sum of the decreased value of the L2 group relative to the M group (10.46) and the decreased value of the HB group relative to the M group (31.58); in terms of NO content, the decreased value of the LH group relative to the M group (5.73) was greater than the sum of the decreased value of the L2 group relative to the M group (2.56) and the decreased value of the HB group relative to the M group (2.89).

[0246] The above results indicate that the combination of 2'-FL and BB536 can synergistically regulate the levels of brain-gut peptides, which are closely related to constipation, anxiety, and brain-gut interaction.

[0247] Table 8 Effects of nutrients on 5-HT, SP, VIP and NO levels in the colon

[0248]

[0249] Table 9 Effects of nutrients on serum 5-HT, SP, VIP and NO levels

[0250]

[0251] Example 5: Effects of nutrients on colon and brain tissue in mice

[0252] 1 Experimental Methods

[0253] 1.1 Histopathology

[0254] Colon and brain tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. The sections were fixed, dewaxed with xylene, and dehydrated with graded ethanol. The sections were then stained with hematoxylin and eosin, destained again with alcohol and xylene, and mounted. Histological morphology was observed and images were collected under a light microscope.

[0255] 2 Experimental results

[0256] The results of colon tissue pathology of mice in each group are as follows Figure 2 As shown. It can be seen that the colon tissue structure of the blank K group mice is intact, the texture is clear, the mucosal crypt structure is normal, the depth is uniform, a large number of goblet cells can be seen, and the muscle layer thickness is normal. No adverse phenomena such as abnormal crypt structure and abnormal infiltration of inflammatory cells occurred. However, after gavage with loperamide model, the colon tissue of the M group mice showed severe abnormal infiltration of inflammatory cells, some crypts disappeared, goblet cells were lost, the submucosal layer was edematous, and the thickness of the colon muscle layer was reduced. After intervention with positive drugs, high, medium and low doses of 2'-FL, high and low doses of BB536, and different doses of 2'-FL combined with BB536, the colon tissue damage of mice in each group was recovered to varying degrees. After intervention with 2'-FL alone (H2, M2 and L2), the colon tissue still had obvious abnormal morphology, the abnormal infiltration of inflammatory cells was restored to some extent, but there was still obvious abnormal infiltration of inflammatory cells, the crypt structure was slightly improved, but the crypt structure was still distorted and atrophied. After intervention with BB536 (HB and LB) alone, goblet cells increased and abnormal inflammatory cell infiltration improved, but significant abnormal morphology of the colon tissue still existed. After combined intervention with 2'-FL and BB536, abnormal inflammatory cell infiltration improved and goblet cells increased. Among them, the improvement effect was more obvious after combined intervention with high-dose 2'-FL and high-dose BB536, as evidenced by clear and complete colon tissue morphology, distinct U-shaped crypts, abundant goblet cells, and a colon muscle layer thickness close to that of the blank K group. In summary, 2'-FL combined with BB536 can effectively improve colon tissue damage in mice with constipation, anxiety, and depression.

[0257] According to the H&E staining experiment, it is possible to observe whether there are any abnormalities in the structure, nerve cell arrangement, and nuclear structure of the mouse hippocampus, thereby judging the pathological changes in anxiety and depression-like behaviors induced by constipation in mice. Figure 3 As shown, the neurons in the CA1 and DG regions of mice in the blank group K group were regularly arranged, with clear structures and good staining. Compared with the blank group K group, the neurons in the loperamide-induced constipation mice (M group) changed in morphology, with a decrease in the number of neurons, shrinkage of the nuclei in the CA1 and DG regions, and disordered neuronal arrangement. Compared with the model (M group), after intervention with different doses of 2'-FL and BB536, the neuronal damage in the brain tissue of each group of mice was alleviated, and the number of neurons increased to varying degrees. In addition, the brain tissue recovery effect of the combined intervention of 2'-FL and BB536 was more obvious than that of either alone. In summary, 2'-FL combined with BB536 can effectively improve brain tissue damage in mice with constipation, anxiety, and depression.

[0258] In general, the experimental process and experimental results of the present invention are as follows Figure 4 shown.

Claims

1. Use of a nutritional composition in preparing a food that helps improve constipation and the anxiety and / or depression caused by it; wherein: The nutritional composition comprises the following components (i) and (ii): (i) neutral fucosylated human milk oligosaccharides, (ii) Bifidobacterium probiotics; in, The neutral fucosylated human milk oligosaccharide comprises at least 2'-fucosyllactose, The Bifidobacterium probiotics include at least Bifidobacterium longum subspecies longum.

2. The use according to claim 1, characterized in that The neutral fucosylated human milk oligosaccharide further comprises any one or more of 3-fucosyllactose, lactose-N-fucopentaose I and difucosyllactose.

3. The use according to claim 1 or 2, characterized in that The Bifidobacterium probiotics further include any one or more of Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium animalis subsp. animalis, Bifidobacterium animalis subsp. lactis, Bifidobacterium bifidum and Bifidobacterium breve.

4. The use according to any one of claims 1 to 3, characterized in that The Bifidobacterium longum subspecies longum includes the BB536 strain.

5. The use according to any one of claims 1 to 4, characterized in that The ratio of the mass of the (i) neutral fucosylated human milk oligosaccharide to the quantity of the (ii) Bifidobacterium probiotics is 1 mg:(1×10 5 -5×10 8 )CFU.

6. The use according to any one of claims 1 to 5, characterized in that The benefits described for improving constipation and the anxiety and / or depression it causes include improvements in: Increase the water content of feces.

7. The use according to any one of claims 1 to 6, characterized in that The benefits described for improving constipation and the anxiety and / or depression it causes include improvements in: Improve gastrointestinal motility.

8. The use according to any one of claims 1 to 7, characterized in that The benefits described for improving constipation and the anxiety and / or depression it causes include improvements in: Improve environmental adaptability and action capabilities.

9. The use according to any one of claims 1 to 8, characterized in that The said helping to improve constipation and the anxiety and / or depression caused by it includes improving colon tissue damage; The improvement of colon tissue damage includes any one or more of the following (b1)-(b4): (b1) Improve crypt structure, (b2) Reduce inflammatory cell infiltration, (b3) Increase the number of goblet cells, (b4) Increase the thickness of the colonic muscular layer.

10. The use according to any one of claims 1 to 9, characterized in that The said helping to improve constipation and the anxiety and / or depression caused by it includes improving brain tissue structural damage; The improvement of brain tissue structural damage includes the following (c1) and / or (c2): (c1) Promote regular arrangement of hippocampal neurons, (c2) Increase the number of hippocampal neurons.

11. The use according to any one of claims 1 to 10, characterized in that The method of helping to improve constipation and the anxiety and / or depression caused by it includes improving the content of brain gut peptide in colon tissue and / or serum; The improvement of the content of ghrelin in colon tissue and / or serum includes the following (d1) and / or (d2): (d1) increasing the content of any one or more of 5-HT and SP in colon tissue and / or serum, (d2) reducing the content of any one or more of NO and VIP in colon tissue and / or serum.

12. The use according to any one of claims 1 to 11, characterized in that The food includes any one or more of infant food, children's food, adolescent food, pregnant and lying-in women food, adult food and middle-aged and elderly food.

13. The use according to any one of claims 1 to 12, characterized in that The food is candy, beverage, dairy product, baked food, dietary supplement or food for special dietary uses.

14. The use according to any one of claims 1 to 13, characterized in that In the food, the content of the neutral fucosylated human milk oligosaccharide is 0.01% to 4%, and the content of the Bifidobacterium probiotic is greater than 1×10 6 CFU / g.

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