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

Abstract

The invention belongs to the field of food, and particularly relates to a nutritional composition containing probiotics and breast milk oligosaccharide for improving anxiety or 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 bifidobacterium probiotic, and (ii) a sialylated breast milk oligosaccharide; the bifidobacterium probiotics at least comprise animal bifidobacterium subsp. Lactis, the sialylated breast milk oligosaccharide at least comprises 6 '-sialylated lactose, and the animal bifidobacterium subsp. Lactis comprises a Bb-12 strain. The substance combination of the bifidobacterium probiotics and the sialylated breast milk oligosaccharide can synergistically improve abnormal brain-intestine interaction, and improve constipation and anxiety and / or depression state caused by constipation.

Description

Technical Field

[0001] The present invention belongs to the field of food, and particularly relates to a nutritional composition containing probiotics and human milk oligosaccharides for improving anxiety or depression and its uses. Background Art

[0002] The brain and the gastrointestinal tract achieve complex bidirectional communication through the brain-gut axis (BGA). The brain-gut axis is a neuro-endocrine-immune network jointly composed of the central nervous system (CNS), the autonomic nervous system (ANS), the enteric nervous system (ENS), and the hypothalamic-pituitary-adrenal axis (HPA), etc. Through the brain-gut axis, the gastrointestinal tract is coordinately regulated at various levels such as the central nervous system, the enteric nervous system, and the hypothalamic-pituitary-adrenal axis. Internal and external stimuli and mental and emotional factors can all affect gastrointestinal function, and at the same time, gastrointestinal activities are also involved in the regulation of central sensation, emotion, and behavior.

[0003] Constipation is a common gastrointestinal discomfort in infants, children, and adults. According to whether there is organic change, constipation can be divided into functional constipation and organic constipation. Functional constipation (FC) is mainly characterized by dry stools, difficult defecation, reduced defecation frequency, or a feeling of incomplete defecation. Slow transit constipation (STC) is the most common type of functional constipation. The incidence of pediatric functional constipation is over 90% of childhood constipation. It is reported that the incidence of functional constipation in infants within 1 year old is 2.9%, and it can rise to 10.1% in children aged 1 - 2 years old, and 14% in children. If constipation recurs for a long time, it can lead to decreased appetite, mental abnormalities, and reduced immunity, such as affecting the gastrointestinal function, memory, and intellectual development of children, and even can lead to enuresis, fecal and urinary incontinence, etc., seriously affecting the growth and development, physical and mental health of children. If constipation in childhood is not effectively improved, about 25% of these 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 an abnormality in brain-gut interaction. Chronic constipation can not only cause intestinal and perianal diseases, but also lead to mental and psychological disorders such as anxiety and depression. It can even induce and exacerbate diseases of the nervous, cardiovascular and other systems, affecting physical and mental health and quality of life. Some studies have found that the physical, psychological and social quality of life of children with slow transit constipation (≥2 years) is significantly lower than that of healthy children. Therefore, slow transit constipation is considered a chronic debilitating state that affects the physical and emotional functions of children. With the help of modern neuroimaging techniques, it has been found that the structure and functional connectivity of brain regions related to emotion regulation, somatic movement and sensory processing in people with functional constipation have changed. The enteric nervous system, also known as the "gut brain", can directly regulate gastrointestinal motility through independent neurons and secreted neurotransmitters, hormones, etc. Abnormalities in its function are one of the important causes of functional constipation. Some studies have found morphological abnormalities in the submucosal plexus and myenteric plexus in people with functional constipation.

[0005] The causes and mechanisms of functional constipation are complex. People with related problems have long suffered from constipation and are accompanied by psychological problems such as anxiety and depression. For constipation, many people use laxatives, prokinetic drugs, etc. to relieve it, which is effective in the short term but not in the long term. For problems such as anxiety and depression, generally only patients with diagnosed anxiety disorder or depression and severe conditions can use antidepressant drugs for intervention, such as drugs that regulate serotonin reabsorption like fluoxetine hydrochloride, paroxetine, sertraline, etc. Although these drugs play an important role in the treatment of depression, they also have the disadvantages of large side effects and high treatment costs. They are not suitable for people with mild conditions and cannot be used for the prevention of depression in sensitive populations. Therefore, from the perspective of brain-gut interaction, it is a field worthy of exploration to find non-toxic and side-effect-free and economically priced nutrients that can not only relieve constipation but also adjust the anxiety and depression states caused by constipation.

[0006] Citation 1 discloses a probiotic powder that can relieve autism and regulate emotions. The probiotic powder is composed of Bifidobacterium bifidum BGN4, Bifidobacterium longum BORI, Bifidobacterium lactis AD011, prebiotics and various nutrients. It can regulate and balance the brain-gut axis microbial system, specifically improve the brain-gut microbial environment, can well relieve autism, regulate gastrointestinal discomfort problems combined with autism such as diarrhea, constipation, picky eating, allergies, etc., and can effectively relieve emotions, improve sleep status and self-awareness.

[0007] Reference Document 2 discloses a probiotic composition for relieving depression and a preparation method thereof, aiming to solve the technical problems that long-term use of existing antidepressants can damage the intestinal flora, lead to intestinal ecological disorders, and cause gastrointestinal reactions. It includes 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 fructooligosaccharide, 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 the sleep, anxiety, and diarrhea of depressive patients.

[0008] Reference Document 3 discloses a probiotic composition and its application. The probiotic composition includes the following components in parts by weight: 0.2 - 2.5 parts of Bifidobacterium animalis subsp. lactis, 0.2 - 5 parts of Lactobacillus rhamnosus, 5 - 15 parts of stachyose, 10 - 40 parts of fructooligosaccharide, 1 - 5 parts of Bacillus coagulans, 10 - 25 parts of acetylated distarch adipate, and 10 - 40 parts of polydextrose. This probiotic composition regulates the balance of the intestinal flora and acts on the brain-gut axis to improve behavioral disorder problems such as autism and depression in pregnant mothers and offspring.

[0009] Bifidobacterium animalis subsp. lactis strain Bb-12 is a probiotic isolated from the feces of breastfed infants, which is part of the human intestinal indigenous flora and is the only Bifidobacterium strain with a complete gene sequence obtained. Since 1985, Bb-12 has been widely used in foods and supplements globally due to its high stability and safety. Bb-12 has health benefits for both infants and adults, including improving digestive system health, reducing diarrhea, enhancing immunity, and maintaining healthy cholesterol levels, etc. It has excellent acid and bile tolerance and intestinal adhesion ability, can inhibit pathogenic bacteria, and enhance immune interactions. Extensive clinical studies support that Bb-12 can survive in the gastrointestinal tract, maintain a healthy flora, improve intestinal function, prevent diarrhea, and reduce the side effects of antibiotic treatment.

[0010] The research in Reference Document 4 provides evidence of the positive effect of Bb-12 on defecation frequency and supports the potential benefits of Bb-12 as a probiotic in improving low defecation frequency and abdominal discomfort in healthy adults.

[0011] Reference Document 5 discloses the application of fermented milk containing active Bifidobacterium lactis in regulating the state of sub-health people. After using the fermented dairy product containing active Bifidobacterium lactis to intervene in people with intestinal sub-health, it has the effect of regulating the mental state of sub-health people, can relieve anxiety and depression, can be used to regulate the brain-gut axis pathway and mental emotions in sub-health state, and at the same time also shows the ability to reduce intestinal constipation or diarrhea, improve fecal characteristics and defecation frequency, and promote intestinal function.

[0012] Sialylated human milk oligosaccharides are an important type of human milk oligosaccharides, accounting for about 13% of the total human milk oligosaccharides. 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) are two of the simplest sialylated human milk oligosaccharides, with prebiotic effects, especially in promoting the proliferation of Bifidobacterium and shaping the gut microbiota, and at the same time showing a variety of health effects, including anti-adhesion and antibacterial abilities, antiviral activity, prevention of necrotizing enterocolitis, immunomodulatory activity, regulation of intestinal epithelial cell responses, promotion of brain development and cognitive improvement. 3'-SL and 6'-SL have been granted Generally Recognized as Safe approval in some countries or regions and are commercially added to infant formula.

[0013] The research in Reference Document 6 shows that when mice are fed 3'-SL or 6'-SL, it can reduce the changes in the structure of the colonic mucosa-associated microbiota caused by stress in mice and reduce the anxiety-like behaviors of mice in the light-dark preference and open field tests.

[0014] Reference Document 7 discloses an oligosaccharide composition for improving neurodevelopment and dysfunction. The oligosaccharide composition includes 6'-sialyllactose and lacto-N-triose. This oligosaccharide composition can effectively inhibit the accumulation of Aβ protein, thereby playing a role in protecting nerves and improving dysfunction. In addition, this oligosaccharide composition can also inhibit the activity of acetylcholinesterase, protect the normal development of cholinergic neurons and increase the content of neurotransmitters, which is beneficial for improving neurodevelopment, learning and memory ability, and improving the behavioral coordination of infants and young children.

[0015] Although the prior art has studied the effects of Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides on intestinal health, brain and nerve health, there is currently little research on their combination.

[0016] Reference Documents:

[0017] Reference Document 1: CN114917255A;

[0018] Reference Document 2: CN117297098A;

[0019] Reference Document 3: CN116850213B;

[0020] Citation Document 4: Effect of the probiotic strain Bifidobacterium animalissubsp.lactis, on defecation frequency in healthy subjects with lowdefecation frequency and abdominal discomfort:a randomised,double-blind,placebo-controlled,parallel-group trial.

[0021] Citation Document 5: CN118716406A;

[0022] Citation Document 6: The prebiotics 3'Sialyllactose and 6'Sialyllactose diminishstressor-induced anxiety-like behavior and colonic microbiota alterations:Evidence for effects on the gut-brain axis.

[0023] Citation Document 7: CN118104826A。 SUMMARY OF THE INVENTION

[0024] Problems to be Solved by the Invention

[0025] The causes and mechanisms of constipation are relatively complex. Related people have long suffered from constipation and are accompanied by psychological problems such as anxiety and depression. Currently, for constipation and the resulting anxiety and depression, drugs are usually used for intervention clinically. However, drugs for treating constipation are usually only effective in the short term, but have poor long-term effects. Drugs for treating anxiety and depression are usually only suitable for patients diagnosed with anxiety disorder and depression and with relatively severe symptoms, and are not suitable for people with mild conditions, lacking universality.

[0026] Although prior art such as the cited references above have proposed using probiotics of the genus Bifidobacterium or sialylated human milk oligosaccharides to improve constipation or mental problems, such research cannot be said to be sufficient. Prior art usually only studies the improvement of constipation or the improvement of anxiety and depression separately, rather than conducting an overall study on constipation and the resulting anxiety and depressive states from the perspective of brain-gut interaction. At the same time, prior art mainly focuses on the individual effects of Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides, and there is less research on their combination.

[0027] In view of this, the present invention has conducted extensive research from the perspective of brain-gut interaction and unexpectedly found that after the combination of probiotics of the genus Bifidobacterium such as Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides such as 6'-SL, it can simultaneously contribute to the improvement of constipation and the resulting anxiety and depressive states. Based on this, the main object of the present invention is to provide a non-therapeutic application of a nutritional composition containing probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides in contributing to the improvement of constipation and the resulting anxiety and depressive states.

[0028] Solutions for Solving the Problems

[0029] To solve the above technical problems, the present invention provides the following technical solutions:

[0030] [1]. Use of a nutritional composition in the preparation of a food that helps to improve constipation and the resulting anxiety state and / or depressive state; wherein, the nutritional composition comprises the components shown in the following (i) and (ii):

[0031] (i) Probiotics of the genus Bifidobacterium,

[0032] (ii) Sialylated human milk oligosaccharides;

[0033] The probiotics of the genus Bifidobacterium include at least Bifidobacterium animalis subsp. lactis, the sialylated human milk oligosaccharides include at least 6'-sialyllactose, and the Bifidobacterium animalis subsp. lactis includes the Bb-12 strain.

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

[0035] [3]. The use according to [1] or [2], wherein the sialylated human milk oligosaccharides further include sialylated human milk oligosaccharides different from 6'-sialyllactose.

[0036] [4]. Use according to any one of [1]-[3], wherein, in the nutritional composition, the ratio of the number of probiotics of the genus Bifidobacterium in (i) to the mass of sialylated human milk oligosaccharides in (ii) is (1×10 6 - 8×10 9 ) CFU: 1 mg.

[0037] [5]. Use according to any one of [1]-[4], wherein, the improvement that helps to improve constipation and the anxiety state and / or depressive state caused thereby includes the following improvements:

[0038] Increase the water content of feces.

[0039] [6]. Use according to any one of [1]-[5], wherein, the improvement that helps to improve constipation and the anxiety state and / or depressive state caused thereby includes the following improvements:

[0040] Increase the gastrointestinal motility ability.

[0041] [7]. Use according to any one of [1]-[6], wherein, the improvement that helps to improve constipation and the anxiety state and / or depressive state caused thereby includes the following improvements:

[0042] Increase the environmental adaptability and locomotor ability.

[0043] [8]. Use according to any one of [1]-[7], wherein, the improvement that helps to improve constipation and the anxiety state and / or depressive state caused thereby includes improving the content of brain-gut peptides in the colon tissue and / or serum;

[0044] The improvement of the content of brain-gut peptides in the colon tissue and / or serum includes the following (b1) and / or (b2):

[0045] (b1) Increase the content of any one or more of 5-HT and SP in the colon tissue and / or serum,

[0046] (b2) Decrease the content of any one or more of NO and VIP in the colon tissue and / or serum.

[0047] [9]. Use according to any one of [1]-[8], wherein, the improvement that helps to improve constipation and the anxiety state and / or depressive state caused thereby includes improving the colon tissue damage;

[0048] The improvement of the colon tissue damage includes any one or more of the following (c1)-(c4):

[0049] (c1) Improve the crypt structure,

[0050] (c2) Reduce the phenomenon of inflammatory cell infiltration,

[0051] (c3) Increase the number of goblet cells,

[0052] (c4) Increase the thickness of the muscular layer of the colon.

[0053]

[10] . The use according to any one of [1]-[9], wherein the assistance in improving constipation and the anxiety state and / or depressive state caused thereby includes improving the damage to the brain tissue structure;

[0054] The improvement of the damage to the brain tissue structure includes the following (d1) and / or (d2):

[0055] (d1) Promote the regular arrangement of hippocampal neurons,

[0056] (d2) Increase the number of hippocampal neurons.

[0057]

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

[10] , wherein the food includes any one or more of infant food, children's food, adolescent food, pregnant and lying-in women's food, adult food, and middle-aged and elderly food.

[0058]

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

[11] , wherein the food is candy, beverage, dairy product, baked food, dietary supplement, or food for special dietary use.

[0059]

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

[12] , wherein, in the food, the content of the probiotic of the genus Bifidobacterium is greater than 1×10 6 CFU / g, and the content of the sialylated human milk oligosaccharide is 0.01% - 0.5%.

[0060]

[14] . The use of a nutritional composition in the preparation of a food for assisting in improving constipation and the anxiety state and depressive state caused thereby; wherein the nutritional composition comprises the components shown in the following (i) and (ii):

[0061] (i) Probiotic of the genus Bifidobacterium,

[0062] (ii) Sialylated human milk oligosaccharide;

[0063] The probiotic of the genus Bifidobacterium includes at least Bifidobacterium animalis subsp. lactis, the sialylated human milk oligosaccharide includes at least 6'-sialyllactose, and the Bifidobacterium animalis subsp. lactis includes the Bb-12 strain.

[0064]

[15] . Use according to

[14] , wherein the probiotic of the genus Bifidobacterium further comprises any one or more of Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium breve.

[0065]

[16] . Use according to

[13] or

[14] , wherein the sialylated human milk oligosaccharide further comprises sialylated human milk oligosaccharides different from 6'-sialyllactose.

[0066]

[17] . Use according to any one of

[13] -

[16] , wherein in the nutritional composition, the ratio of the number of the (i) probiotic of the genus Bifidobacterium to the mass of the (ii) sialylated human milk oligosaccharide is (1×10 6 -8×10 9 ) CFU: 1 mg.

[0067]

[18] . Use according to any one of

[13] -

[17] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes the following improvements:

[0068] Increase in fecal water content.

[0069]

[19] . Use according to any one of

[13] -

[18] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes the following improvements:

[0070] Increase in gastrointestinal motility.

[0071]

[20] . Use according to any one of

[13] -

[19] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes the following improvements:

[0072] Increase in environmental adaptability and locomotor ability.

[0073]

[21] . Use according to any one of

[13] -

[20] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes improving the content of brain-gut peptides in the colon tissue and / or serum;

[0074] The improvement of the content of brain-gut peptides in the colon tissue and / or serum includes the following (b1) and / or (b2):

[0075] (b1) Increase in the content of any one or more of 5-HT and SP in the colon tissue and / or serum,

[0076] (b2) Decrease in the content of any one or more of NO and VIP in the colon tissue and / or serum.

[0077]

[22] . Use according to any one of

[13] -

[21] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes improvement of colon tissue damage;

[0078] The improvement of colon tissue damage includes any one or more of the following (c1)-(c4):

[0079] (c1) Improvement of crypt structure,

[0080] (c2) Reduction of inflammatory cell infiltration,

[0081] (c3) Increase in the number of goblet cells,

[0082] (c4) Increase in the thickness of the muscular layer of the colon.

[0083]

[23] . Use according to any one of

[13] -

[22] , wherein the improvement of constipation and the anxiety state and depressive state caused thereby includes improvement of brain tissue structure damage;

[0084] The improvement of brain tissue structure damage includes the following (d1) and / or (d2):

[0085] (d1) Promotion of regular arrangement of hippocampal neurons,

[0086] (d2) Increase in the number of hippocampal neurons.

[0087]

[24] . Use according to any one of

[13] -

[23] , wherein the food includes any one or more of infant food, children's food, adolescent food, pregnant and lying-in women's food, adult food, and middle-aged and elderly food.

[0088]

[25] . Use according to any one of

[13] -

[24] , wherein the food is candy, beverage, dairy product, baked food, dietary supplement, or food for special dietary use.

[0089]

[26] . Use according to any one of

[13] -

[26] , wherein, in the food, the content of probiotics of the genus Bifidobacterium is greater than 1×10 6 CFU / g, and the content of sialylated human milk oligosaccharide is 0.01% - 0.5%.

[0090]

[27] . Use of a nutritional composition in the preparation of a food having any one or more of the following effects (z1)-(z6):

[0091] (z1) Increase in fecal water content,

[0092] (z2) Increase in gastrointestinal motility,

[0093] (z3) Improve environmental adaptability and mobility,

[0094] (z4) Improve the content of brain-gut peptides in colon tissue and / or serum, where the brain-gut peptides include any one or more of 5-HT, SP, NO, and VIP,

[0095] (z5) Improve colon tissue damage,

[0096] (z6) Improve brain tissue structure damage;

[0097] The nutritional composition is defined as in any one of [1]-[4].

[0098]

[28] . According to the use described in

[27] , where,

[0099] The improvement of the content of brain-gut peptides in colon tissue and / or serum includes the following (b1) and / or (b2):

[0100] (b1) Increase the content of any one or more of 5-HT and SP in colon tissue and / or serum,

[0101] (b2) Decrease the content of any one or more of NO and VIP in colon tissue and / or serum;

[0102] The improvement of colon tissue damage includes any one or more of the following (c1)-(c4):

[0103] (c1) Improve the crypt structure,

[0104] (c2) Reduce the phenomenon of inflammatory cell infiltration,

[0105] (c3) Increase the number of goblet cells,

[0106] (c4) Increase the thickness of the colon muscular layer;

[0107] The improvement of brain tissue structure damage includes the following (d1) and / or (d2):

[0108] (d1) Promote the regular arrangement of hippocampal neurons,

[0109] (d2) Increase the number of hippocampal neurons.

[0110] Effects of the Invention

[0111] The present invention provides a nutritional composition comprising a combination of substances of probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides, in particular a nutritional composition comprising a combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose, which can improve abnormal brain-gut interaction. Through the brain-gut axis, it can not only improve constipation, but also improve the anxiety state and / or depressive state caused by constipation. That is to say, the above-mentioned combination of substances proposed by the present invention can achieve a two-way synchronous improvement of intestinal health and brain and nerve health through the brain-gut axis.

[0112] Experimental data show that the intake of the combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose can improve the gastrointestinal motility ability of the body, increase the defecation volume and fecal water content of the body, and can improve colon tissue damage. At the same time, the intake of the combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose can also improve the environmental adaptability and locomotor ability of the body, and can improve brain tissue damage. Therefore, from the intuitive performance, the intake of the combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose can effectively improve constipation and the anxiety state and / or depressive state caused by it at the same time.

[0113] In this regard, through further in-depth research, the present invention finds that the intake of the combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose 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 of the brain-gut axis. Therefore, the nutritional composition comprising the combination of substances of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose described in the present invention for the synchronous improvement of constipation and the anxiety state and / or depressive state caused by it is completed based on the brain-gut axis. That is to say, the core of the improvement effect of the nutritional composition described in the present invention on constipation and the anxiety state and / or depressive state caused by it is the improvement of abnormal brain-gut interaction, and this synchronous improvement effect on intestinal and brain nerve health is bidirectional regulation.

[0114] In addition, more importantly, the present invention finds that in the above-mentioned effects, there is a synergistic effect between probiotics of the genus Bifidobacterium such as Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides such as 6'-sialyllactose, and when the dosage ratio of probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides reaches a certain range, this synergistic effect is more obvious. Description of the Drawings

[0115] Figure 1 : Intestinal transit map of mice in each group of the examples.

[0116] Figure 2 : Pathological section map of colon tissue of mice in each group of the examples.

[0117] Figure 3 : Hippocampal pathological section images of mice in each group of the examples.

[0118] Figure 4 : Schematic diagram of the overall experimental process and experimental results of the examples. Detailed implementation manners

[0119] The following describes the implementation manners of the present invention, but the present invention is not limited thereto. The present invention is not limited to the respective components described below. Various changes can be made within the scope claimed in the invention, and the implementation manners and examples obtained by appropriately combining the technical means respectively disclosed in different implementation manners and examples are also included in the technical scope of the present invention.

[0120] In the present invention, the terms "comprise", "have", "include" or "contain" can mean inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprise", "have", "include" or "contain" can also mean closed-ended, excluding additional, unrecited elements or method steps.

[0121] In the present invention, the meaning expressed by "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0122] In the present invention, "optional" or "optionally" means that certain substances, components, execution steps, applied conditions and other factors are used or not used.

[0123] In the present invention, the numerical range expressed by "numerical value A to numerical value B", "numerical value A - numerical value B", "numerical value A or more / less" means a range including the endpoint numerical values A and B.

[0124] In the present invention, the term "about" is used to define that the numerical ranges and parameters of the present invention are approximate numerical values, and the relevant numerical values in the specific embodiments have been presented as precisely as possible here. Unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values and percentages used in the present invention are modified by "about". Here, "about" generally means that the actual numerical value is within ±5%, ±3%, ±1% or ±0.5% of a certain specific numerical value or range. And the numerical values and numerical ranges appearing in the present invention should all be understood to include the systematic errors inevitable in industrial production.

[0125] In the present invention, the "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc. mentioned refer to the specific elements (e.g., features, structures, properties, and / or characteristics) related to the embodiments, which are included in at least one of the embodiments described herein, and may or may not exist in other embodiments. Additionally, it should be understood that the elements can be combined in various embodiments in any suitable manner.

[0126] In the present invention, the unit names used are all international standard unit names, and if not otherwise specified, the "%" used represents weight or mass percentage.

[0127] In the present invention, "infants and young children" refers to the human group under 3 years old, including infants aged 0 - 6 months, older infants aged 6 - 12 months, and toddlers aged 12 - 36 months.

[0128] In the present invention, "children" refers to the human group aged 3 - 6 years old.

[0129] In the present invention, "adolescents" refers to the human group aged 7 - 18 years old.

[0130] In the present invention, "pregnant and lactating women" includes pregnant women and lactating women.

[0131] In the present invention, "middle - aged and elderly" refers to the human group over 41 years old.

[0132] Unless otherwise defined, other technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0133] I. Nutritional composition

[0134] The nutritional composition provided by the present invention comprises the components shown in the following (i) and (ii): (i) probiotics of the genus Bifidobacterium, (ii) sialylated human milk oligosaccharides; the probiotics of the genus Bifidobacterium include at least Bifidobacterium animalis subsp. lactis, and the sialylated human milk oligosaccharides include at least 6'-sialyllactose.

[0135] In some embodiments, in the nutritional composition described in the present invention, the (i) probiotics of the genus Bifidobacterium and (ii) sialylated human milk oligosaccharides are the main active ingredients, that is, the nutritional composition described in the present invention mainly relies on the (i) probiotics of the genus Bifidobacterium and (ii) sialylated human milk oligosaccharides it contains to exert specific physiological and active functions, such as the function of helping to improve constipation and the anxiety state and / or depressive state caused by it.

[0136] In some specific embodiments, in the nutritional composition of the present invention, the (i) Bifidobacterium probiotic of the subspecies Bifidobacterium animalis subsp. lactis and (ii) 6'-sialyllactose in sialylated human milk oligosaccharides are the main active ingredients, that is, the nutritional composition of the present invention mainly relies on the Bifidobacterium animalis subsp. lactis and 6'-sialyllactose it contains to exert specific physiological and active functions, such as helping to improve constipation and the anxiety state and / or depressive state caused by it.

[0137] The present invention discovers that, compared with the Bifidobacterium probiotic alone such as Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides such as 6'-sialyllactose, the nutritional composition provided by the present invention can more effectively improve constipation and the anxiety state and / or depressive state caused by it, that is, there is a synergistic effect between the two.

[0138] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic further includes any one or more of Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium breve.

[0139] The present invention does not make special limitations on the specific strains of the above various strains. Exemplarily, the Bifidobacterium animalis subsp. lactis includes strains such as Bb-12, HN109, and Bi-07, etc., Bifidobacterium bifidum includes strain R0071, etc., Bifidobacterium longum subsp. infantis includes strain R0033, etc., Bifidobacterium longum subsp. longum includes strain BB536, etc., and Bifidobacterium breve includes strain M-16V, etc.

[0140] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic includes the Bb-12 strain of Bifidobacterium animalis subsp. lactis.

[0141] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic is Bifidobacterium animalis subsp. lactis.

[0142] In some embodiments, in the nutritional composition of the present invention, the Bifidobacterium probiotic is the Bb-12 strain of Bifidobacterium animalis subsp. lactis.

[0143] In some embodiments, in the nutritional composition of the present invention, the sialylated human milk oligosaccharides further include sialylated human milk oligosaccharides different from 6'-sialyllactose, such as 3'-sialyllactose.

[0144] In some embodiments, in the nutritional composition of the present invention, the sialylated human milk oligosaccharides are 6'-sialyllactose.

[0145] The present invention does not make a special limitation on the specific source of sialylated human milk oligosaccharides. Typically, they can be prepared by chemical synthesis, microbial fermentation, and enzymatic catalysis. The chemical method synthesizes lactosylsialic acid through automatic solid-phase synthesis. The biological fermentation method mainly constructs the synthesis pathway through genetic engineering modification. The enzymatic catalysis method mainly relies on two strategies. One strategy is to use sialidase, including exo-α-sialidase and endo-α-sialidase. Exo-α-sialidase has the ability of trans-sialylation and uses lactose as the acceptor and sialoside as the donor to synthesize 6'-SL. The other strategy is to use sialyltransferase, which can use cytidine 5'-monophosphate-activated sialic acid as the donor substrate to combine with lactose to achieve the natural transfer of sialic acid. In addition, sialylated human milk oligosaccharides can also be derived from animal milk such as cow milk, goat milk, etc.

[0146] To obtain a better synergistic effect that helps to improve constipation and the anxiety state and / or depressive state caused thereby, in some specific embodiments, in the nutritional composition, the ratio of the number of the (i) probiotics of the genus Bifidobacterium to the mass of the (ii) sialylated human milk oligosaccharides is (1×10 6 -8×10 9 ) CFU: 1 mg. For example, it can be 1×10 6 : 1 mg, 2×10 6 : 1 mg, 3×10 6 : 1 mg, 4×10 6 : 1 mg, 5×10 6 : 1 mg, 6×10 6 : 1 mg, 7×10 6 : 1 mg, 8×10 6 : 1 mg, 9×10 6 : 1 mg, 1×10 7 : 1 mg, 2×10 7 : 1 mg, 3×10 7 : 1 mg, 4×10 7 : 1 mg, 5×10 7 : 1 mg, 6×10 7 : 1 mg, 7×10 7 : 1 mg, 8×10 7 : 1 mg, 9×10 7 : 1 mg, 1×10 8 : 1 mg, 2×10 8 : 1 mg, 3×10 8 : 1 mg, 4×10 8 : 1 mg, 5×10 8 : 1 mg, 6×10 8 : 1 mg, 7×108 : 1 mg, 8 × 10 8 : 1 mg, 9 × 10 8 : 1 mg, 1 × 10 9 : 1 mg, 2 × 10 9 CFU: 1 mg, 3 × 10 9 CFU: 1 mg, 4 × 10 9 CFU: 1 mg, 5 × 10 9 CFU: 1 mg, 6 × 10 9 CFU: 1 mg, 7 × 10 9 CFU: 1 mg or 8 × 10 9 CFU: 1 mg etc.; preferably, the ratio of the two can be (2 × 10 6 - 4 × 10 9 ) CFU: 1 mg; more preferably, the ratio of the two can be (4 × 10 6 - 2 × 10 9 ) CFU: 1 mg. In some embodiments, the above ratio is the content ratio of Bifidobacterium animalis subsp. lactis strain Bb - 12 and 6'-sialyllactose in the nutritional composition described in the present invention.

[0147] The present invention does not make any particular limitation on the form of the nutritional composition. Typically, it can be liquid, solid, etc. Considering the convenience of production, transportation, storage and use, the nutritional composition described in the present invention is preferably a powdery solid.

[0148] II. Use of the nutritional composition

[0149] The present invention proposes that the combination of probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides, especially the combination of Bifidobacterium animalis subsp. lactis and 6'-sialyllactose, especially when combined in a specific ratio, helps to improve constipation, and helps to improve the anxiety state and / or depressive state caused by constipation. At the same time, there is a synergistic effect between probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides. Compared with single probiotics of the genus Bifidobacterium such as Bifidobacterium animalis subsp. lactis and sialylated human milk oligosaccharides such as 6'-sialyllactose, the above effects are better.

[0150] The improvement of constipation and the anxiety state and / or depressive state caused thereby described in the present invention are not for the purpose of treating or preventing diseases, and the constipation and the anxiety state and / or depressive state caused by constipation described in the present invention do not reach the level that can be recognized as diseases.

[0151] Furthermore, the present invention provides the use of the above-mentioned nutritional composition in the preparation of a food that helps to improve constipation and the anxiety state and / or depressive state caused thereby. Further still, the present invention provides the use of the above-mentioned nutritional composition in the preparation of a food that helps to improve constipation and the anxiety state and / or depressive state caused thereby in patients without anxiety disorder or depressive disorder.

[0152] In some embodiments, the improvement of constipation in the present invention is achieved by alleviating any one or more of the following conditions: dry stools, difficult defecation, reduced frequency of defecation, and a feeling of incomplete defecation. In some embodiments, the improvement of the anxiety state and / or depressive state caused by constipation in the present invention mainly includes improving the environmental adaptability and locomotor ability.

[0153] In some embodiments, the help in improving constipation and the anxiety state and / or depressive state caused thereby includes any one or more of the following improvements (a1)-(a6): (a1) increasing the water content of feces, (a2) increasing the gastrointestinal motility, (a3) improving the environmental adaptability and locomotor ability, (a4) improving the content of brain-gut peptides in the colon tissue and / or serum, (a5) improving the damage of colon tissue, and (a6) improving the damage of brain tissue structure.

[0154] In some embodiments, the help in improving constipation and the anxiety state and / or depressive state caused thereby simultaneously includes the above improvements (a1)-(a6).

[0155] In some embodiments, the improvement of the content of brain-gut peptides in the colon tissue and / or serum includes the following (b1) and / or (b2): (b1) increasing the content of any one or more of 5-HT and SP in the colon tissue and / or serum; (b2) decreasing the content of any one or more of NO and VIP in the colon tissue and / or serum.

[0156] In some specific embodiments, the improvement of the content of brain-gut peptides in the colon tissue and / or serum simultaneously includes the above (b1) and (b2).

[0157] The brain-gut peptide (BGP) described in the present invention is a small molecule polypeptide that regulates gastrointestinal function and participates in the regulation of gastrointestinal physiological activities in the brain-gut axis. It is distributed in both the gastrointestinal tract and the nervous system, and the interaction of various small molecule polypeptides with dual functions of neurotransmitters and hormones affects all aspects of the brain-gut axis. More than 60 kinds of BGP have been discovered so far. BGP mediates intercellular information transmission in the brain-gut axis, exerts the effects of hormones and local transmitters, plays the role of 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 functions. BGP is mainly divided into three categories: gastrointestinal peptide hormones, gastrointestinal neuropeptides, and neuropeptides. Among them, gastrointestinal peptide hormones can be divided into excitatory and inhibitory types, and these transmitters interact with and influence each other to jointly play a regulatory role.

[0158] 5-Hydroxy tryptamine (5-HT) is a brain-gut peptide with multiple biological effects. It is mainly present in the central nervous system and the enteric nervous system, and has the effects of promoting gastrointestinal motility, regulating the duodenogastric reflex, regulating the secretion of pepsin and gastric acid, and participating in the regulation of physiological activities such as emotion, cognition, mood, and sleep. 5-HT is a key neurotransmitter connecting the central nervous system and the enteric nervous system on the brain-gut axis.

[0159] Substance P (SP) is the first identified and multiply distributed brain-gut peptide. It is mainly distributed in the central nervous system, the dorsal root of the spinal cord, and the enteric nervous system (the most secreted in the proximal small intestine and colon), and has the effects of contracting gastrointestinal smooth muscle fibers to promote gastrointestinal peristalsis, protecting the gastrointestinal epithelium to repair and heal damaged gastrointestinal mucosa, producing gastrointestinal mechanical hypersensitivity, and causing intestinal pain.

[0160] Vasoactive intestinal peptide (VIP) is a neuropeptide that inhibits gastrointestinal motility and has dual functions of neurotransmitter and neuromodulator. It is highly expressed in the central nervous system and the gastrointestinal tract, and has the effects of inhibiting gastrointestinal motility and gastric emptying, slowing down small intestine motility, reducing the tone of digestive sphincters, and dilating blood vessels.

[0161] Nitric oxide (NO) is an inhibitory neurotransmitter in brain-gut peptides. It is mainly distributed in the enteric nervous system of the gastric antral muscular layer and the intestine, and exists in the human intestine in the forms of enzymes, non-enzymes, and bacteria. NO can cause smooth muscle relaxation, weaken gastric antral contraction to delay gastric emptying, and can also bidirectionally regulate the intensity of the receptive relaxation reflex arc and peristaltic reflex arc of the gastric fundus.

[0162] In some embodiments, the improvement of colon tissue injury includes any one or more of the following (c1)-(c4): (c1) improving the crypt structure, (c2) reducing the phenomenon of inflammatory cell infiltration, (c3) increasing the number of goblet cells, (c4) increasing the thickness of the muscularis propria of the colon.

[0163] In some embodiments, the improvement of colon tissue injury simultaneously includes the above (c1)-(c4).

[0164] In some embodiments, the improvement of brain tissue structure injury includes the following (d1) and / or (d2): (d1) promoting the regular arrangement of hippocampal neurons, (d2) increasing the number of hippocampal neurons.

[0165] In some embodiments, the improvement of brain tissue structure injury simultaneously includes the above (d1) and (d2).

[0166] In some embodiments, the present invention also provides the use of the above nutritional composition in the preparation of foods having any one or more of the following effects (z1)-(z6):

[0167] (z1) increasing the water content of feces,

[0168] (z2) increasing the gastrointestinal motility ability,

[0169] (z3) increasing the environmental adaptability and locomotor ability,

[0170] (z4) improving the content of brain-gut peptides in colon tissue and / or serum, the brain-gut peptides including any one or more of 5-HT, SP, NO, and VIP,

[0171] (z5) improving colon tissue injury,

[0172] (z6) improving brain tissue structure injury.

[0173] For specific foods containing the above nutritional composition or prepared with the above nutritional composition, the present invention is not particularly limited.

[0174] In some embodiments, at room temperature, the food of the present invention is in the form of a liquid or a solid.

[0175] In some embodiments, the food of the present invention is a candy, such as hard candy, gel candy, crispy candy, tablet candy, and aerated candy, etc.

[0176] In some embodiments, the food of the present invention is a beverage, such as carbonated beverage, tea beverage, coffee beverage, fruit and vegetable juice beverage, and lactic acid bacteria beverage, etc.

[0177] In some embodiments, the food of the present invention is a dairy product, such as milk powder, cheese, yogurt, liquid milk, etc.

[0178] In some embodiments, the food of the present invention is a baked food, such as bread, cake, biscuits, etc.

[0179] In some embodiments, the food of the present invention is a dietary supplement, such as hard capsules, soft capsules, tablets, oral liquids, pills, granules, powders, etc.

[0180] In some embodiments, the food of the present invention is a food for special dietary uses, such as infant formula foods, complementary foods for infants, supplementary nutritional products, and formula foods for special medical purposes, etc.

[0181] In some embodiments, the food of the present invention includes any one or more of infant foods, adolescent foods, maternal and child foods, and elderly foods.

[0182] Exemplarily, the food of the present invention can be infant formula milk powder, maternal and child nutrition sets, elderly milk powder, etc.

[0183] In some embodiments, in the food of the present invention, the probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides all originate from the nutritional composition. In other embodiments, in the food of the present invention, the probiotics of the genus Bifidobacterium and sialylated human milk oligosaccharides, in addition to originating from the nutritional composition, can also originate from other food raw materials, such as animal milk, etc.

[0184] In some embodiments, in the food of the present invention, the ratio of the number of the probiotics of the genus Bifidobacterium to the mass of the sialylated human milk oligosaccharides can be (1×10 6 -8×10 9 ) CFU: 1 mg; preferably, the ratio of the two can be (2×10 6 -4×10 9 ) CFU: 1 mg; more preferably, the ratio of the two can be (4×10 6 -2×10 9 ) CFU: 1 mg. In some embodiments, the above ratio is the content ratio of Bifidobacterium animalis subsp. lactis strain Bb-12 and 6'-sialyllactose in the food of the present invention.

[0185] The present invention does not particularly limit the specific absolute content of the probiotics of the genus Bifidobacterium, such as Bifidobacterium animalis subsp. lactis, and the sialylated human milk oligosaccharides, such as 6'-sialyllactose, in the food, as long as the requirements of local food-related laws and regulations are met.

[0186] In some embodiments, in the food, the content of probiotic Bifidobacterium in the food is greater than 1×10 6 CFU / g, and the content of sialylated human milk oligosaccharide is 0.01% - 0.5%.

[0187] In addition to the components described above for the nutritional composition, the food may further contain other ingredients, such as common food ingredients like proteins / amino acids, carbohydrates, fats, vitamins, minerals, etc.

[0188] Furthermore, according to the type of food and the ultimate needs of the target users, in some embodiments, the food further contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additive ingredients, and any acceptable excipients.

[0189] For plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, oranges, tangerines, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, phyllanthus emblica, and cornelian cherries, or their extracts; fruit and vegetable substances such as onions, cucumbers, tomatoes, broccoli, carrots, spinach, Chinese kale, Brussels sprouts, garlic, basil, oregano, or their extracts; grains such as indica rice, japonica rice, glutinous rice, wheat, barley, oats, rye, corn, sorghum, millet, panicled millet, buckwheat, soybeans, broad beans, peas, mung beans, adzuki beans, kidney beans, or their extracts; nut substances such as walnuts, pistachios, cashews, hazelnuts, almonds, pine nuts, peanuts, sunflower seeds, chestnuts, macadamia nuts, ginkgo nuts, or their extracts; coffee or its extract; and some plant-based Chinese medicinal materials with both medicinal and edible properties or their extracts.

[0190] For animal dairy product ingredients, examples include fresh milk from cows, sheep, etc., and reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, demineralized whey powder, whey protein powder, hydrolyzed whey protein powder, casein powder, etc.

[0191] For animal meat product ingredients, examples include meat product ingredients of pigs, cows, sheep, aquatic products, or poultry.

[0192] For functional additive ingredients, examples include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, etc.

[0193] For any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, solubilizers, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anticaking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0194] Examples

[0195] The embodiments of the present invention will be described in detail below in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used are conventional products that can be obtained commercially.

[0196] Nutrients and their sources used in the embodiments of the present invention:

[0197] 6'-Sialyllactose (6'-SL): DSM;

[0198] Bifidobacterium animalis subsp. lactis Bb-12: Chr. Hansen.

[0199] Animal experiment design in the embodiments of the present invention:

[0200] 140 healthy male BALB / c mice (6 weeks old, 20 g) were housed under a 12 h light / dark cycle, at a temperature of 25 ± 2 °C, and a humidity of 55% - 65%. During the entire experiment, sterile water and normal feed were freely available.

[0201] After one week of adaptive feeding, they were randomly divided into 14 groups of 10 mice each, specifically including a blank group (K), a constipation anxiety model group (M), a positive control group (Y), a high-dose 6'-SL group (H6), a medium-dose 6'-SL group (M6), a low-dose 6'-SL group (L6), a high-dose Bb-12 group (HB), a low-dose Bb-12 group (LB), a high-dose 6'-SL combined with high-dose Bb-12 group (HH), a medium-dose 6'-SL combined with high-dose Bb-12 group (MH), a low-dose 6'-SL combined with high-dose Bb-12 group (LH), a high-dose 6'-SL combined with low-dose Bb-12 group (HL), a medium-dose 6'-SL combined with low-dose Bb-12 group (ML), and a low-dose 6'-SL combined with low-dose Bb-12 group (LL).

[0202] Except for the blank group, mice in other groups were gavaged with loperamide at a dose of 10 mg / kg.bw every morning at 9:00, once a day for 7 days. The blank group was gavaged with an equal volume of normal saline. On the 8th day, the K group was gavaged with normal saline every day, while the other groups were gavaged with loperamide at a dose of 10 mg / kg.bw. 2 hours after gavaging with loperamide, the K group was gavaged with normal saline, the Y group was gavaged with mosapride at a dose of 2.5 mg / kg.bw, the H6 group, M6 group and L6 group were respectively gavaged with 6'-SL at doses of 500 mg / kg.bw, 300 mg / kg.bw and 100 mg / kg.bw, and the HB group and LB group were respectively gavaged with Bb-12 at doses of 2×10 11 CFU / kg.bw and 2×10 9 CFU / kg.bw of Bb-12. The HH group was gavaged with a mixed solution of 6'-SL and Bb-12 (500 mg / kg.bw and 2×10 11 CFU / kg.bw), the MH group was gavaged with a mixed solution of 6'-SL and Bb-12 (300 mg / kg.bw and 2×10 11 CFU / kg.bw), the LH group was gavaged with a mixed solution of 6'-SL and Bb-12 (100 mg / kg.bw and 2×10 11 CFU / kg.bw), the HL group was gavaged with a mixed solution of 6'-SL and Bb-12 (500 mg / kg.bw and 2×10 9 CFU / kg.bw), the ML group was gavaged with a mixed solution of 6'-SL and Bb-12 (300 mg / kg.bw and 2×10 9 CFU / kg.bw), and the LL group was gavaged with a mixed solution of 6'-SL and Bb-12 (100 mg / kg.bw and 2×10 9 CFU / kg.bw). The specific experimental groups are shown in Table 1.

[0203] On the 29th day, the mice were fasted (with free access to water) for 12 hours, and orbital blood was collected from each mouse and allowed to stand 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. After straightening the colon tissue, the length from the anus to the end of the cecum was measured with a ruler and recorded. A 1-cm colon segment was cut and immersed in 4% paraformaldehyde solution for subsequent histological sectioning. The remaining animal tissues were stored in a -80°C ultra-low temperature freezer for later use.

[0204] Table 1 Experimental groups

[0205]

[0206] Example 1: Effects of nutrients on physiological indices of mice

[0207] 1 Experimental method

[0208] 1.1 Mouse health index

[0209] During the mouse feeding period, observe the clinical manifestation status of the mice every day, such as growth status, mental state, hair changes, defecation, activity status, etc., as well as whether they die. Observe and record the body weight, water intake, and food intake of the mice at the same time every day.

[0210] 1.2 Statistical analysis

[0211] The experimental data are expressed as mean ± standard deviation. P<0.05 is considered to be a significant difference between the data, and P>0.05 is considered to be no significant difference between the data.

[0212] 2 Experimental results

[0213] After the experiment, as shown in Table 2, there were no significant differences in food consumption and water intake among the mice in each group (P>0.05). The body weights of the mice in the K group, M group, and Y group were 26.58±2.15 g, 25.19±1.37 g, and 26.79±1.06 g, respectively. Compared with the K group and Y group, the body weight of the M group was relatively lower, but there was no significant difference among the three groups (P>0.05). In addition, there were no significant differences in the body weights of the mice in the HH, MH, LH, HL, ML, and LL groups of the 6'-SL and Bb-12 combined treatment group compared with those in the K group and Y group (P>0.05).

[0214] Table 2 Mouse body weight, food intake, and water intake

[0215]

[0216] Example 2: Effects of nutrients on mouse defecation indexes

[0217] 1 Experimental method

[0218] 1.1 Fecal water content

[0219] Collect feces at 14 d and 28 d of feeding. After the feces are dried until the weight remains unchanged, obtain the dry weight and moisture content of the feces. At the same time, store the fecal samples collected at 28 d at -80 °C for analysis of short-chain fatty acids and gut microbiota. Weigh the obtained fresh mouse feces and record it as the wet weight. Then, dry the feces at 105 °C for 5 h until constant weight, and then weigh and record it as the dry weight:

[0220] Fecal water content = (wet weight - dry weight) / dry weight × 100%.

[0221] 1.2 Determination of defecation time

[0222] After all mice were fasted overnight for 12 h on the 28th day, they were gavaged with activated carbon solution (10% activated carbon, 0.5% carboxymethyl cellulose suspension), and then placed in metabolic cages separately. The time between the intake of activated carbon by the mice and the defecation of dark feces was recorded.

[0223] 1.3 Gastrointestinal transit rate

[0224] On the 29th day, the activated carbon solution was gavaged. After 30 min, all mice were euthanized by cervical dislocation, and the small intestine was dissected and collected. The distance from the pylorus to the activated carbon boundary was regarded as the migration distance. After measuring the length, the gastrointestinal transit rate was calculated using the following formula:

[0225] Gastrointestinal transit rate = activated carbon migration distance / total length of small intestine × 100%.

[0226] 1.4 Statistical analysis

[0227] The experimental data were expressed as mean ± standard deviation. P < 0.05 was considered to indicate a significant difference between the data, and P > 0.05 was considered to indicate no significant difference between the data.

[0228] 2 Experimental results

[0229] Fecal water content is one of the important indicators reflecting the degree of constipation in mice. The effects of 6'-SL and Bb-12 on fecal water content in 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 dry and hard state of feces. Compared with the M group, the fecal water content of mice in the Y group was significantly increased (P < 0.05), indicating that mosapride could effectively improve fecal water content. After separate intervention with different doses of 6'-SL or different doses of Bb-12, the fecal water content was significantly higher than that in the M group (P < 0.05). In addition, the fecal water content of all 6'-SL and Bb-12 combination treatment groups was significantly higher than that in the M group (P < 0.05). Moreover, compared with the increase value of fecal water content in the M group of the 6'-SL or Bb-12 single treatment groups, the 6'-SL and Bb-12 combination treatment groups had more advantages. Taking Experimental Examples 2, 5, and 10 as examples, the increase value of fecal water content in the ML group relative to the M group (19.74%) was greater than the sum of the increase values of fecal water content in the M6 group relative to the M group (10.16%) and the LB group relative to the M group (7.89%).

[0230] The time of the first black stool is the total time it takes for the chyme to pass through the intestinal tract after leaving the stomach and finally be excreted as feces, which can reflect the peristaltic ability of the entire gastrointestinal tract. As shown in Table 3, the time of the first black stool in the M group was significantly higher than that in the K group. Compared with the M group, the time of the first black stool in the Y group of mice was significantly reduced (P<0.05), indicating that mosapride can effectively improve the peristaltic ability of the gastrointestinal tract. Compared with the M group, after intervention with high, medium doses of 6'-SL or high dose of Bb-12, the time of the first black stool was significantly reduced (P<0.05). Among them, compared with the reduction values of the time of the first black stool in the single treatment groups of 6'-SL or Bb-12 relative to the M group, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 1, 5, and 9 as examples, the reduction value of the time of the first black stool in the HL group relative to the M group (70.6 min) was greater than the sum of the reduction values of the time of the first black stool in the H6 group relative to the M group (37.93 min) and the reduction value of the time of the first black stool in the LB group relative to the M group (20.05 min).

[0231] The effects of 6'-SL, Bb-12, and the combination of 6'-SL and Bb-12 on the number of fecal particles in mice within 3 h are shown in Table 3. The number of fecal particles excreted by mice within 3 h was significantly reduced in the M group compared with the K group (P<0.05). Compared with the M group, after intervention with the positive drug, high, medium doses of 6'-SL, and high dose of Bb-12, the number of fecal particles in mice increased significantly (P<0.05). In addition, compared with the increase values of the number of fecal particles in the single treatment groups of 6'-SL or Bb-12 relative to the M group, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 3, 4, and 8 as examples, the increase value of the number of fecal particles in the LH group relative to the M group (17.67) was greater than the sum of the increase values of the number of fecal particles in the L6 group relative to the M group (5) and the increase value of the number of fecal particles in the HB group relative to the M group (8).

[0232] The distance of activated carbon transported in the intestine is one of the important indicators for evaluating constipation. The effects of 6'-SL, Bb-12, and the combination of 6'-SL and Bb-12 on intestinal transit in mice are shown in Table 3. The intestinal transit rate of M mice was significantly reduced compared with the K group (P<0.05). In addition, the intestinal transit diagrams of the K, M, and Y groups are as follows Figure 1As shown, it is obvious that the transport distance of activated carbon in group M is shorter, indicating that the constipation model has been successfully established. Compared with group M, after intervention with positive drugs, different doses of 6'-SL, and Bb-12, the intestinal transport rate of mice increased significantly (P<0.05). In addition, compared with the increase in intestinal transport rate of the single-treatment groups of 6'-SL or Bb-12 relative to group M, the combined treatment group of 6'-SL and Bb-12 has more advantages. Taking experimental examples 3, 5, and 11 as examples, the increase in intestinal transport rate of the LL group relative to group M (46.66%) is greater than the sum of the increase in intestinal transport rate of the L6 group (21.22%) and the LB group relative to group M (17.8%).

[0233] The above results indicate that 6'-SL, Bb-12, and the combination of 6'-SL and Bb-12 can all significantly improve constipation in mice, but the combined group shows a synergistic effect.

[0234] Table 3 Effects of nutrients on defecation indexes of mice

[0235]

[0236] Example 3: Effects of nutrients on behavioral indexes of mice

[0237] 1 Experimental method

[0238] 1.1 Elevated Plus Maze (EPM) experiment

[0239] The EPM experimental device consists of a "+"-shaped maze, 50 cm above the ground, two opposite open arms, two opposite closed arms, and a central area of 5×5 cm. Each arm is 30 cm long and 5 cm wide. The closed arms have walls 15 cm high, while the open arms have ledges 3 mm high. Each mouse is placed in the center of the apparatus, facing an open arm. All spontaneous activities are recorded (5 min) and calculated by SMART 3.0. Generally, mice tend to stay in the closed arms because it provides visual shelter and makes them feel safe. On the contrary, the open arms are exposed, which is likely to cause anxiety in mice. Therefore, by observing the behavioral performance of mice in the open arms and closed arms, their anxiety levels can be preliminarily evaluated. The instrument is cleaned with 75% ethanol solution before and after each trial.

[0240] 1.2 Open field test (OFT)

[0241] The OFT is a method for evaluating the spontaneous locomotor behavior, exploratory behavior, and tension of rodents. The OFT design uses an open environment model to assess the anxiety level of mice. Each mouse is randomly selected and tested individually in an apparatus (50×50 cm 2 and 40 cm high). The bottom of the apparatus is evenly divided into 16 squares. The mouse is placed in a corner of the apparatus to acclimate for 1 min. After the experiment starts, a video device monitors the movement path of the mouse in the apparatus for 5 min. After the experiment ends, the mouse is returned to the cage, and the activity traces on the open ground are cleaned with ethanol for subsequent experiments. Calculate the total travel distance, average speed, total distance moved in the central area, and the number of entries into the central area of each group of mice.

[0242] 1.3 Tail Suspension Test (TST)

[0243] The TST is widely used to study depressive-like states in mice. The mice are acclimated to the test environment for 30 min. The mouse's tail is fixed with tape and suspended in mid-air, with the head 15 cm above the surface, and this configuration is maintained for 6 min. Behavioral tracking software records the video clip from the 2nd minute to the 6th minute, precisely capturing the duration of the mouse's immobility.

[0244] 1.4 Forced swimming test (FST)

[0245] The FST is used to test depressive-like behaviors. Before the experiment, the mice are acclimated to the test environment for 30 min. Subsequently, the mice are placed in a transparent glass tube with a height of 35 cm and a diameter of 15 cm, the water depth is controlled at 10 cm, and the water temperature is controlled at 25±1°C. A video detection device is used to clearly capture the behavior of the mice during the 6 min. The duration of the mouse's immobility is recorded starting from the 2nd minute of the test.

[0246] 1.5 Statistical analysis

[0247] The experimental data are expressed as mean ± standard deviation. P<0.05 is considered to indicate a significant difference between the data, and P>0.05 is considered to indicate no significant difference between the data.

[0248] 2 Experimental results

[0249] 2.1 Elevated plus maze experiment

[0250] The elevated plus-maze test was used to evaluate the anxiety behavior of mice. The experimental results are shown in Table 4. Compared with the K group, the open-arm activity time of the M group mice was significantly reduced (P<0.05). Compared with the M group, after intervention with different concentrations of 6'-SL or Bb-12 or their combination, the open-arm activity time of the mice was significantly higher than that of the M group (P<0.05). In addition, compared with the increase in the open-arm activity time of the M group in the individual treatment groups of 6'-SL or Bb-12, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 2, 4, and 7 as examples, the increase in the open-arm activity time of the MH group relative to the M group (44.68 s) was greater than the sum of the increase in the open-arm activity time of the M6 group relative to the M group (18.38 s) and the increase in the open-arm activity time of the HB group relative to the M group (21.79 s). Therefore, the combined group of 6'-SL and Bb-12 showed better synergistic effects in relieving the anxiety of constipated mice.

[0251] Table 4 Effects of nutrients on the open-arm activity time of mice in the elevated plus-maze test

[0252]

[0253] 2.2 Open-field test

[0254] The open field test is one of the common behavioral tests for evaluating anxiety in mice. When an animal is placed in a strange open field (strange environment), due to the fear of the strange environment, it will mainly move in the peripheral area, but the novelty of the strange environment will also prompt it to explore the central area, thus generating a conflict "contradiction". The open field test measures its anxiety level by observing spontaneous movement in the new environment. The effects of different doses of 6'-SL, Bb-12, and 6'-SL combined with Bb-12 on the spontaneous activity ability of mice are shown in Table 5. Compared with the K group, the total movement distance, movement distance in the central area, number of times crossing the platform, and average speed of the mice in the M group were significantly reduced (P<0.05) in the open field test. These results indicate that the mouse anxiety model has been successfully established. Compared with the M group, after intervention with the positive drug, the total movement distance, movement distance in the central area, number of times crossing the platform, and average speed of the mice in the Y group were significantly increased (P<0.05) in the open field test. In addition, after intervention with different doses of 6'-SL alone, the total movement distance and average speed of the treatment groups in Experimental Examples 1, 2, and 3 were significantly higher than those in the M group (P<0.05), while although the total movement distance and average speed of the treatment groups with different doses of Bb-12 were higher than those in the M group, there was no significant difference (P>0.05). After intervention with high and medium doses of 6'-SL or high dose of Bb-12 alone, the movement distance in the central area and number of times crossing the platform of the treatment groups in Experimental Examples 1, 2, and 4 were significantly higher than those in the M group (P<0.05), and although the movement distance in the central area and number of times crossing the platform of the low dose 6'-SL and low dose Bb-12 treatment groups in Experimental Examples 3 and 5 were higher than those in the M group, there was no significant difference (P>0.05).In addition, the total movement distance, the movement distance in the central area, the number of times crossing the platform, and the average speed of all the combined treatment groups of 6'-SL and Bb-12 were significantly higher than those of the M group (P<0.05). At the same time, compared with the increased values of the total movement distance, the movement distance in the central area, the number of times crossing the platform, and the average speed of the individual treatment groups of 6'-SL or Bb-12 relative to the M group, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 2, 5, and 10 as examples, in terms of the total movement distance, the increased value of the ML group relative to the M group (4127.49 cm) was greater than the sum of the increased values of the M6 group relative to the M group (2312.34 cm) and the LB group relative to the M group (1631.96 cm); in terms of the movement distance in the central area, the increased value of the ML group relative to the M group (740.61 cm) was greater than the sum of the increased values of the M6 group relative to the M group (518.62 cm) and the LB group relative to the M group (159.87 cm); in terms of the number of times crossing the platform, the increased value of the ML group relative to the M group (59.33 times) was greater than the sum of the increased values of the M6 group relative to the M group (35 times) and the LB group relative to the M group (15.33 times); in terms of the average speed, the increased value of the ML group relative to the M group (4.52 cm / s) was greater than the sum of the increased values of the M6 group relative to the M group (2.55 cm / s) and the LB group relative to the M group (1.81 cm / s). The above results indicate that the combination of 6'-SL and Bb-12 shows a synergistic effect in relieving anxiety caused by constipation compared with the individual use.

[0255] Table 5 Effects of Nutrients on the Results of the Open Field Test in Mice

[0256]

[0257] 2.3 Tail Suspension Test

[0258] The tail suspension test is an animal model experiment used to study depression. This experiment evaluates the helplessness and emotional state of mice by measuring the time they stop moving in a suspended state. As shown in Table 6, compared with the blank K group, the immobility time of mice in the M group was significantly increased in the tail suspension test, indicating that mice treated with loperamide by gavage showed depressive behavior. Compared with the M group, after intervention with positive drugs, 6'-SL, Bb-12 alone or in combination, the immobility time of mice in the tail suspension test was significantly decreased (P<0.05). At the same time, compared with the decrease in the immobility time of the M group in the individual treatment groups of 6'-SL or Bb-12, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 3, 5, and 11 as examples, the decrease in the immobility time of the LL group relative to the M group (48.17 s) was greater than the sum of the decrease in the immobility time of the L6 group relative to the M group (20.17 s) and the decrease in the immobility time of the LB group relative to the M group (14.33 s), indicating that the combination of 6'-SL and Bb-12 could synergistically relieve depression caused by constipation.

[0259] Table 6 Effects of nutrients on the immobility time of mice in the tail suspension test

[0260]

[0261] 2.4 Forced swimming

[0262] Forced swimming is a behavioral despair experimental method and one of the commonly used behavioral experiments to evaluate depression by the typical "immobile state" shown by animals. The effects of different doses of 6'-SL and Bb-12 and the combination of 6'-SL and Bb-12 on the immobility time of mice in the FST are shown in Table 7. Compared with the blank K group, the immobility time of mice in the M group was significantly increased in the tail suspension test, indicating that mice treated with loperamide by gavage showed depressive behavior. Compared with the M group, after intervention with positive drugs, 6'-SL, Bb-12 alone or in combination, the immobility time of mice in the forced swimming test was significantly decreased (P<0.05). At the same time, compared with the decrease in the immobility time of the M group in the individual treatment groups of 6'-SL or Bb-12, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 3, 4, and 8 as examples, the decrease in the immobility time of the LH group relative to the M group (48 s) was greater than the sum of the decrease in the immobility time of the L6 group relative to the M group (13.75 s) and the decrease in the immobility time of the HB group relative to the M group (28.25 s), indicating that the combination of 6'-SL and Bb-12 could synergistically relieve depression caused by constipation.

[0263] Table 7 Effects of nutrients on the immobility time of mice in the forced swimming test

[0264]

[0265] Example 4: Effects of Nutrients on Brain-Gut Axis-Related Indicators

[0266] 1 Experimental Method

[0267] 1.1 Determination of Brain-Gut Axis Biochemical Indicators

[0268] Take a part of the frozen colon tissue to make a homogenate, complete the pretreatment according to the requirements in the detection kit instructions, and strictly follow the operation steps for the experiment to detect the levels of 5-hydroxytryptamine (5-HT), substance P (SP), vasoactive intestinal peptide (VIP), and nitric oxide (NO). The serum samples of all groups were thawed at 4°C, and the levels of 5-HT, SP, VIP, and NO in the serum of each group of mice were detected according to the kit instructions.

[0269] 1.2 Statistical Analysis

[0270] The experimental data are expressed as mean ± standard deviation. P<0.05 is considered to indicate a significant difference between the data, and P>0.05 is considered to indicate no significant difference between the data.

[0271] 2 Experimental Results

[0272] Brain-gut peptides can connect and regulate all aspects of the brain-gut axis interaction, are widely distributed in the gastrointestinal tract and brain tissue, and have the dual functions 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 abnormal brain-gut interaction in functional constipation, the secretion disorder of brain-gut peptides gradually worsens, and the tendency of anxiety and depression also increases. These negative emotions can also affect the distribution of some brain-gut peptides in the gastrointestinal tract and serum, suggesting that brain-gut peptides and anxiety and depression emotions interact with each other. SP and 5-HT are the main excitatory brain-gut peptides, which can promote gastrointestinal motility, and VIP and NO are inhibitory brain-gut peptides, which play an inhibitory role in gastrointestinal motility. The regulation of brain-gut peptide levels is closely related to constipation, anxiety, and brain-gut interaction.

[0273] The effects of nutrients on the levels of brain-gut peptides in the colon of mice are shown in Table 8. After intragastric administration of loperamide, compared with the K group, the contents of 5-HT and SP in the colon of mice in the M group were significantly decreased (P<0.05), while the contents of NO and VIP in the colon were significantly increased (P<0.05). Compared with the M group, after intervention with the positive drug mosapride, the contents of 5-HT and SP in the colon of mice were significantly increased (P<0.05), while the contents of NO and VIP in the colon were significantly decreased (P<0.05). Compared with the M group, after intervention with 6'-SL or Bb-12, the content of 5-HT in the colon of mice in each group was significantly increased (P<0.05), the content of VIP was significantly decreased (P<0.05), and the content of NO was significantly decreased (P<0.05). After intervention with high and medium doses of 6'-SL or high dose of Bb-12, the content of SP in the colon of mice was significantly increased (P<0.05). In addition, after intervention with different doses of the combined group of 6'-SL and Bb-12, the contents of SP and 5-HT in the colon of mice were significantly increased (P<0.05), while the contents of VIP and NO in the colon were significantly decreased (P<0.05). Among them, compared with the increased values of 5-HT and SP and the decreased values of VIP and NO in the M group in the single treatment groups of 6'-SL or Bb-12, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 1, 5, and 9 as examples, in terms of the content of SP, the increased value of the HL group relative to the M group (41.72) was greater than the sum of the increased values of the H6 group (22.68) relative to the M group and the increased value of the LB group relative to the M group (13.14); in terms of the content of 5-HT, the increased value of the HL group relative to the M group (90.73) was greater than the sum of the increased values of the H6 group relative to the M group (53.63) and the increased value of the LB group relative to the M group (33.95); in terms of the content of VIP, the decreased value of the HL group relative to the M group (83.71) was greater than the sum of the decreased values of the H6 group relative to the M group (49.42) and the decreased value of the LB group relative to the M group (23.53); in terms of the content of NO, the decreased value of the HL group relative to the M group (1.84) was greater than the sum of the decreased values of the H6 group relative to the M group (1.24) and the decreased value of the LB group relative to the M group (0.46).

[0274] The effects of 6'-SL and Bb-12 on the levels of brain-gut peptides in mouse serum are shown in Table 9. After intragastric administration of loperamide, compared with the K group, the contents of 5-HT and SP in the serum of mice in the M group were significantly decreased (P<0.05), while the contents of NO and VIP in the serum were significantly increased (P<0.05). Compared with the M group, after intervention with the positive drug mosapride, the contents of 5-HT and SP in the serum of mice were significantly increased (P<0.05), while the contents of NO and VIP in the serum were significantly decreased (P<0.05). Compared with the M group, after intervention with 6'-SL or Bb-12, the content of 5-HT in the serum of each group of mice was significantly increased (P<0.05), the content of VIP was significantly decreased (P<0.05), and the content of NO was significantly decreased (P<0.05). After intervention with high, medium, and low doses of 6'-SL or high dose of Bb-12, the content of SP in the serum of mice was significantly increased (P<0.05). In addition, in the combined treatment groups of different doses of 6'-SL and Bb-12, the contents of SP and 5-HT in the serum of mice were significantly increased (P<0.05), while the contents of VIP and NO in the serum were significantly decreased (P<0.05). Among them, compared with the increase values of 5-HT and SP and the decrease values of VIP and NO in the M group in the single treatment groups of 6'-SL or Bb-12, the combined treatment group of 6'-SL and Bb-12 had more advantages. Taking Experimental Examples 3, 4, and 8 as examples, in terms of the content of SP, the increase value of the LH group relative to the M group (20.54) was greater than the sum of the increase values of the L6 group relative to the M group (7.69) and the HB group relative to the M group (10.65); in terms of the content of 5-HT, the increase value of the LH group relative to the M group (30.95) was greater than the sum of the increase values of the L6 group relative to the M group (13.26) and the HB group relative to the M group (13.49); in terms of the content of VIP, the decrease value of the LH group relative to the M group (86.81) was greater than the sum of the decrease values of the L6 group relative to the M group (31.79) and the HB group relative to the M group (51.23); in terms of the content of NO, the decrease value of the LH group relative to the M group (4.62) was greater than the sum of the decrease values of the L6 group relative to the M group (1.77) and the HB group relative to the M group (2.43).

[0275] The above results indicate that the combination of 6'-SL and Bb-12 can synergistically regulate the levels of brain-gut peptides that are closely related to constipation, anxiety, and brain-gut interaction.

[0276] Table 8 Effects of nutrients on the contents of SP, 5-HT, VIP, and NO in the colon

[0277]

[0278] Table 9 Effects of nutrients on the contents of SP, 5-HT, VIP, and NO in the serum

[0279]

[0280] Example 5: Effects of Nutrients on Mouse Colonic and Brain Tissues

[0281] 1 Experimental Methods

[0282] 1.1 Histopathology

[0283] Fresh colonic and brain tissues were fixed with 4% paraformaldehyde for 48 h, dehydrated with gradient ethanol for colonic and brain tissues respectively, made transparent with xylene, and then embedded in paraffin and cut into 4-μm thick sections. After staining with hematoxylin and eosin, the sections were sealed with neutral resin, and the tissue morphology was observed and images were collected under a fluorescence inverted microscope.

[0284] 2 Experimental Results

[0285] The pathological sections of the colonic tissues of mice in different groups are as Figure 2 shown. In the blank group of mice (Group K), the colonic tissue structure was intact, the texture was clear, the mucosal crypt structure was normal, there were a large number of goblet cells, and there were no adverse phenomena such as abnormal crypt structure and abnormal infiltration of inflammatory cells. However, in Group M treated with loperamide by gavage, submucosal edema was observed, some crypt cells disappeared, goblet cells were absent, and abnormal infiltration of inflammatory cells occurred. After intervention with different doses of 6'-SL (H6, M6, L6), Bb-12 (HB, LB) and 6'-SL combined with Bb-12 (HH, MH, LH, HL, ML, LL), the colonic tissue damage of mice was recovered to varying degrees. After intervention with different doses of 6'-SL, there were still obvious abnormal epithelial morphologies in the colonic tissues, and the abnormal infiltration of some inflammatory cells was restored but there was still obvious abnormal infiltration of inflammatory cells, and the crypt structure was slightly improved but the crypt structure was still distorted and atrophied. After intervention with low-dose Bb-12 (Group LB), there was still obvious abnormal infiltration of inflammatory cells in the mouse colon, some crypt cells disappeared, but the number of goblet cells increased. After intervention with high-dose Bb-12 (Group HB), the improvement effect was more obvious than that of Group LB. After combined intervention with 6'-SL and Bb-12, the recovery effect of the pathological damage of the mouse colonic tissue was better. Among them, after intervention with high-dose 6'-SL and high-dose Bb-12 combined (Group HH), the colonic tissue morphology of the mice was clear and intact, with obvious U-shaped crypts, abundant goblet cells, and the thickness of the colonic muscular layer was close to that of the blank Group K. In summary, 6'-SL combined with Bb-12 can effectively improve the colonic tissue damage in constipated, anxious and depressed mice.

[0286] To clarify the effects of 6'-SL and Bb-12 alone or in combination on the brain tissue pathology of anxious and depressive mice, we performed H&E staining on the hippocampus of the brain. According to the H&E staining experiment, it is possible to observe whether there are abnormalities in the structure of the hippocampus of the mouse brain, the arrangement of nerve cells, the structure of the cell nucleus, etc., so as to judge the pathological conditions of constipation-induced depressive-like behaviors in mice. The changes in the number and morphology of pyramidal neurons in the CA1 region and DG region of the hippocampus of depressive mice after 6'-SL and Bb-12 alone or in combination interventions are as Figure 3 shown. In group K mice, the neurons in the CA1 region and DG region were arranged regularly, with clear structure and good staining. Compared with the blank group K, the neuronal morphology of loperamide-induced constipated mice (group M) changed, the number of neurons decreased, nuclear shrinkage occurred in the CA1 region and DG region, and the neuronal arrangement was disordered. Compared with the model (group M) mice, after single or combined interventions with different doses of 6'-SL and Bb-12, the neuronal damage in the brain tissues of each group of mice was alleviated to varying degrees, and the number of neurons increased to varying degrees. Among them, the intervention effect of group HH on the brain tissue was more obvious than that of group H6 and group HB alone. To sum up, 6'-SL combined with Bb-12 can effectively improve the brain tissue damage of constipated, anxious and depressive mice.

[0287] Generally speaking, the experimental procedures and results of the present invention are as Figure 4 shown.

Claims

1. Use of a nutritional composition in the preparation of a foodstuff for helping to improve constipation and the anxiety state and / or depressive state caused thereby; wherein, The nutritional composition comprises the components shown in the following (i) and (ii): (i) Probiotics of the genus Bifidobacterium, (ii) Sialylated human milk oligosaccharides; The probiotics of the genus Bifidobacterium include at least Bifidobacterium animalis subsp. lactis, the sialylated human milk oligosaccharides include at least 6'-sialyllactose, and the Bifidobacterium animalis subsp. lactis includes the Bb-12 strain.

2. The use according to claim 1, wherein The probiotics of the genus Bifidobacterium further include any one or more of Bifidobacterium animalis subsp. animalis, Bifidobacterium longum subsp. longum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Bifidobacterium breve.

3. The use according to claim 1 or 2, characterized in that, The sialylated human milk oligosaccharides further include sialylated human milk oligosaccharides different from 6'-sialyllactose.

4. Use according to any one of claims 1 - 3, characterized in that, In the nutritional composition, the ratio of the amount of the (i) probiotic bacteria of the genus Bifidobacterium to the mass of the (ii) sialylated human milk oligosaccharides is (1×10 6 -8×10 9 )CFU:1mg.

5. The use according to any one of claims 1 to 4, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes the following improvements: Increasing the water content of feces.

6. The use according to any one of claims 1-5, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes the following improvements: Increasing the gastrointestinal motility.

7. The use according to any one of claims 1-6, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes the following improvements: Increasing the environmental adaptability and locomotor ability.

8. The use according to any one of claims 1-7, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes improving the content of brain-gut peptides in the colon tissue and / or serum; The improvement in the content of brain-gut peptides in the colon tissue and / or serum includes the following (b1) and / or (b2): (b1) Increasing the content of any one or more of 5-HT and SP in the colon tissue and / or serum, (b2) Decreasing the content of any one or more of NO and VIP in the colon tissue and / or serum.

9. The use according to any one of claims 1-8, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes improving the damage of colon tissue; The improvement in the damage of colon tissue includes any one or more of the following (c1)-(c4): (c1) Improving the crypt structure, (c2) Reducing the infiltration of inflammatory cells, (c3) Increasing the number of goblet cells, (c4) Increasing the thickness of the muscular layer of the colon.

10. The use according to any one of claims 1-9, characterized in that, The improvement in constipation and the anxiety state and / or depressive state caused thereby includes improving the damage of brain tissue structure; The improvement in the damage of brain tissue structure includes the following (d1) and / or (d2): (d1) Promoting the regular arrangement of hippocampal neurons, (d2) Increasing the number of hippocampal neurons.

11. The use according to any one of claims 1 to 10, characterized in that, The food includes any one or more of infant food, children's food, adolescent food, maternal food, adult food, and food for the middle-aged and elderly.

12. The use according to any one of claims 1-11, characterized in that, The food is candy, beverage, dairy product, baked food, dietary supplement, or food for special dietary use.

13. The use according to any one of claims 1-12, characterized in that, In the food, the content of probiotics of the genus Bifidobacterium is greater than 1×10 6 CFU / g, and the content of sialylated human milk oligosaccharides is 0.01% to 0.5%.

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