Bifidobacterium composition for relieving emotional disorder caused by constipation and application of bifidobacterium composition

Abstract

The invention relates to a bifidobacterium composition for relieving mood disorder caused by constipation and application of the bifidobacterium composition, and belongs to the technical field of probiotics. According to the bifidobacterium composition containing the animal bifidobacterium subsp. Lactis Bb-12 and the bifidobacterium longum subsp. Longum BB536, through the synergistic effect of the Bb-12 and the BB536, the effect of relieving constipation can be achieved by improving the water content of excrement, improving the intestinal tract movement capacity and increasing the content of short-chain fatty acid in the intestinal tract. On the basis that constipation is relieved, emotional disorders caused by constipation can be further relieved, and particularly, the content of anxiety and depression related cranial nerve factors BDNF in hippocampus is increased, and the relative expression quantity of inflammatory factors TNF-alpha is reduced. A new thought is provided for the development of nutritional supplement food for relieving anxiety and depression caused by constipation.

Description

Technical Field

[0001] The present invention relates to a bifidobacterium composition for alleviating mood disorders caused by constipation and an application thereof, and more particularly to an application of the bifidobacterium composition comprising Bifidobacterium animalis subspecies lactis Bb-12 and Bifidobacterium longum subspecies longum BB536, belonging to the technical field of probiotics. Background Art

[0002] Constipation is a common gastrointestinal disease in infants, children, and adults. Constipation can be divided into functional constipation and organic constipation depending on the presence or absence of organic lesions. Functional constipation (FC) is a functional bowel disease with the main clinical features of hard stools, straining during defecation, reduced bowel movement, or a feeling of incomplete defecation, but without obvious organic lesions. Slow transit constipation (STC) is the most common clinical form of functional constipation. Infancy is an age group with a high incidence of FC, especially during dietary transitions (such as switching from breastfeeding to formula or adding complementary foods). Long-term and recurrent constipation in children can lead to decreased appetite, abdominal distension and pain, urinary tract problems, restricted growth and development, emotional problems, behavioral withdrawal, and even enuresis and incontinence, which seriously affect children's growth and development and physical and mental health. If childhood constipation is not effectively treated, it may develop into chronic constipation, making treatment more difficult. Some children will develop other digestive system diseases in adulthood, seriously affecting their quality of life.

[0003] According to the Rome IV diagnostic criteria published by the World Association of Gastroenterology in 2016, functional gastrointestinal disorders, including FC, are classified as abnormalities in brain-gut interaction. In other words, abnormalities in the brain-gut axis (BGA) are a key pathogenesis of disorders such as functional constipation (FC). The BGA is a complex network of bidirectional communication and regulation between the gut and the brain, involving multiple pathways, including the central nervous system (CNS), autonomic nervous system (ANS), enteric nervous system (ENS), and hypothalamic-pituitary-adrenal axis (HPA). Through the BGA, the gastrointestinal tract is regulated by the CNS, ENS, and HPA at various levels. Internal and external stimuli, as well as emotional and psychological factors, can influence gastrointestinal function. Furthermore, gastrointestinal activity is involved in the regulation of central sensory, emotional, and behavioral processes. The CNS is the highest neural center for gastrointestinal function regulation. It receives incoming gastrointestinal activity signals, processes and integrates them, and then feeds this regulatory information back to the ENS or gastrointestinal effector cells via the ANS and HPA, regulating intestinal motility, secretion, and permeability, and indirectly influencing the intestinal microbiota. Long-term constipation not only causes intestinal and perianal lesions, as well as psychological disorders such as anxiety and depression, but also can induce and aggravate systemic diseases such as neurological and cardiovascular diseases, impacting patients' physical and mental health and quality of life. International studies evaluating the physical, psychological, and social quality of life in children with long-term (≥2 years) STC have shown significant reductions in all three categories compared to healthy children, suggesting that STC is a chronic, debilitating condition that affects children's physical and emotional functioning.

[0004] Functional constipation has complex etiologies and pathogenesis, leading to chronic constipation and associated psychological disturbances such as anxiety and depression. Many patients resort to laxatives and prokinetics for treatment, which are effective in the short term but lack long-term efficacy. For conditions like anxiety and depression, antidepressants such as fluoxetine hydrochloride, paroxetine, and sertraline, which regulate serotonin reabsorption and play a crucial role in depression treatment, are typically reserved for patients with severe symptoms. However, existing studies have shown that over a third of patients do not respond to conventional treatments based on antidepressants and psychotherapy. These treatments also come with significant side effects and high costs, making them unsuitable for patients with milder symptoms. They are also inappropriate for preventing depression in sensitive individuals, particularly those with medication intolerances, as well as for pregnant women and children. In recent years, increasing research has revealed a close connection between gut health and emotional well-being. Therefore, the search for affordable, non-toxic nutrients suitable for a wide range of populations, capable of both maintaining gut health and potentially modulating mood, is a worthy area of ​​research.

[0005] Bb-12 is a strain of Bifidobacterium animalis subsp. lactis, belonging to the genus Bifidobacterium. It is a Gram-positive, strictly anaerobic bacterium. Most studies have focused on the benefits of Bb-12 for intestinal health, immune function, and nutrient absorption. It is commonly used in various probiotic products. For example, Reference 1 discloses a probiotic freeze-dried powder formulation containing Bb-12, which can improve intestinal microecological balance and enhance immunity. Reference 2 discloses a special medical purpose infant formula containing Bb-12 as an ingredient. This formula can reduce the intestinal digestive burden and improve the body's digestion and absorption rate.

[0006] BB536 is a strain of Bifidobacterium longum subsp. longum, isolated in 1969 from the intestinal flora of healthy breastfed infants. It belongs to the genus Bifidobacterium, subsp. longum. It is a Gram-positive, strictly anaerobic microorganism with primary benefits including improving gastrointestinal health, regulating immunity, and lowering cholesterol. For example, Reference 3 discloses that continuous administration of Bifidobacterium longum subsp. longum BB536 can improve bowel movement parameters and reduce ammonia levels in subjects with constipation, demonstrating its effectiveness in alleviating constipation.

[0007] In addition, cited document 4 discloses a probiotic oligosaccharide composition comprising animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536, which can support the growth of symbiotic or beneficial intestinal flora while inhibiting the growth of harmful flora, thereby establishing a barrier function to protect the intestine.

[0008] Reference 5 discloses Bifidobacterium longum subsp. infantis CCFM687, fermented foods thereof, and their uses. Administration of the described Bifidobacterium longum subsp. infantis CCFM687 to a fluoxetine-treated mouse model of depression significantly improved the mice's depressive-like behavior and increased levels of serotonin, 5-hydroxytryptophan, and brain-derived neurotrophic factor in their brains. Furthermore, the mRNA level of tryptophan hydroxylase 1 in simulated enterochromaffin cells was increased, as was the secretion of 5-hydroxytryptophan by these cells, providing a precursor for the synthesis of serotonin in the brain.

[0009] Reference 6 discloses a probiotic for improving depression or anxiety and its use. The probiotic contains Bifidobacterium longum subsp. infantis BI03 and Lactobacius acidophilus LA05. This invention establishes a mouse model of depression and anxiety using the chronic unpredictable mild stimulation (CUMS) method. Applying the probiotic to mice with depression and anxiety significantly reduces anxiety and depressive-like behaviors, increases levels of short-chain fatty acids and neurotransmitters that regulate the brain-gut axis, and restores serum corticosterone and inflammatory cytokine levels.

[0010] It can be seen that although there have been some studies on the role of bifidobacteria in depression and anxiety, there have been no relevant reports on anxiety and depression caused by constipation.

[0011] Citations

[0012] Reference 1: CN118086131A

[0013] Reference 2: CN117378756A

[0014] Cited literature 3: Ogata T, Nakamura T, Anjitsu K, et al. Effect of Bifidobacterium longum BB536 Administration on the Intestinal Environment, Defecation Frequency and Fecal Characteristics of Human Volunteers[J]. Bioscience and Microflora, 1997, 16(2):53-58.

[0015] Reference 4: CN117678765A

[0016] Reference 5: CN109055269B

[0017] Reference 6: CN118406622B Summary of the Invention

[0018] Problems to be solved by the invention

[0019] Currently, the application of probiotics is largely limited to improving intestinal health. During their research, the present invention unexpectedly discovered that a composition containing a specific type of probiotic can also improve mood disorders caused by constipation, particularly functional constipation. Based on this, the present invention provides a novel use for a probiotic composition that can improve mood disorders by relieving constipation.

[0020] It should be noted that the mood disorder mentioned in the present invention refers to a non-disease state of poor health caused by constipation.

[0021] Solutions for solving problems

[0022] [1] Use of a bifidobacterium composition in preparing a food for alleviating mood disorders caused by constipation, wherein the bifidobacterium composition comprises Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536; or the present invention relates to use of a bifidobacterium composition in preparing a food having one or more of the following functions:

[0023] Alleviate depressive and / or anxiety-like behaviors, regulate the level of brain-derived neurotrophic factor in the hippocampus, and regulate the level of tumor necrosis factor α in the hippocampus.

[0024] The bifidobacterium composition comprises Bifidobacterium animalis subspecies lactis Bb-12 and Bifidobacterium longum subspecies longum BB536.

[0025] [2] The use according to [1], wherein the ratio of the amount of live bacteria of Bifidobacterium animalis subsp. lactis Bb-12 to that of Bifidobacterium longum subsp. longum BB536 in the composition is 1:15 to 15:1.

[0026] [3] The use according to [1] or [2], wherein the constipation is functional constipation; and the mood disorder caused by constipation includes depression and / or anxiety.

[0027] [4] The use according to any one of [1] to [3], wherein the relief of mood disorders caused by constipation comprises at least one of relieving depressive and / or anxiety-like behaviors, regulating the level of brain-derived neurotrophic factor in the hippocampus, and regulating the level of tumor necrosis factor α in the hippocampus.

[0028] [5] Use of a bifidobacterium composition in the preparation of a food for improving constipation, wherein the bifidobacterium composition comprises Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536;

[0029] The constipation includes functional constipation.

[0030] [6] The use according to [5], wherein the improvement of constipation includes at least one of increasing the water content of feces, improving intestinal motility, and increasing the content of short-chain fatty acids in the intestine.

[0031] [7] The use according to [6], wherein the ratio of the amount of live bacteria of Bifidobacterium animalis subsp. lactis Bb-12 to that of Bifidobacterium longum subsp. longum BB536 in the composition is 1:15 to 15:1.

[0032] [8] The use according to any one of [1] to [7], wherein the content of Bifidobacterium animalis subspecies lactis Bb-12 and Bifidobacterium longum subspecies longum BB536 in the food is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

[0033] [9] The use according to any one of [1] to [8], wherein the food is a beverage, milk and dairy products, baked goods or candy.

[0034]

[10] The use according to any one of [1] to [8], wherein the food is an oral preparation; the oral preparation comprises at least one of tablets, pills, granules, powders, teas, capsules and oral liquids.

[0035] Effects of the Invention

[0036] The bifidobacterium composition comprising animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies BB536 provided by the present invention, which acts synergistically through the bifidobacterium composition of animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies BB536, can improve constipation by increasing fecal water content, improving intestinal motility and increasing the content of short-chain fatty acids in the intestine. Moreover, among the many adverse health problems that may be caused by constipation, it has been further found that the above-mentioned specific combination can further alleviate the emotional disorders caused by constipation, specifically including increasing the content of BDNF, a brain nerve factor related to anxiety and depression, in the hippocampus and reducing the relative expression of inflammatory factor TNF-α. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Flowchart of an embodiment of the present invention.

[0038] Figure 2 The effect of the bifidobacterium composition on the defecation indexes of mice; wherein, A: change in fecal moisture content; B: time of first feces excretion; C: change in intestinal transit rate; different letters indicate significant differences between the examples (P<0.05).

[0039] Figure 3The figure is a graph of the content of short-chain fatty acids in mouse feces; among them, A: acetic acid content; B: propionic acid content; C: butyric acid content; D: valeric acid content; different letters indicate significant differences between the examples (P<0.05).

[0040] Figure 4 The distribution and expression of BDNF in the DG region of the hippocampus and TNF-α protein in the CA1 region of the brain tissue of mice in different groups; A: representative immunohistochemical image of BDNF in the DG region of the hippocampus (10×); B: quantitative statistics of BDNF immunohistochemistry in the DG region of the hippocampus; C: representative immunohistochemical image of TNF-α in the CA1 region of the hippocampus (10×); D: quantitative statistics of TNF-α immunohistochemistry in the CA1 region of the hippocampus; different letters indicate significant differences among the treatments (P<0.05). DETAILED DESCRIPTION

[0041] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0042] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0043] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0044] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0045] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0046] As used herein, "optional" and "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0047] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0048] In addition, unless otherwise defined, other technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0049] The present invention is mainly completed based on the following insights:

[0050] Subspecies of Bifidobacterium animalis and Bifidobacterium longum have been shown to play important roles in improving intestinal health and regulating immunity. Furthermore, subspecies of Bifidobacterium longum have shown promising regulatory effects in mouse models of depression, including those induced by fluoxetine and those induced by chronic unpredictable mild stimulation. These effects include improving depressive-like behaviors and increasing brain levels of 5-hydroxytryptamine, 5-hydroxytryptophan, and brain-derived neurotrophic factor. However, studies examining anxiety and depression symptoms caused by constipation, particularly functional constipation, have been limited. The present invention uses loperamide to construct a constipation mouse model, and then gavages the constipated mice with animal Bifidobacterium lactis subsp. Bb-12, Bifidobacterium longum subsp. longum BB536, and a combination of Bifidobacterium animalis lactis subsp. Bb-12 and Bifidobacterium longum subsp. longum BB536, respectively. It is unexpectedly found that the constipation of the mice is significantly improved, and the depressive and anxiety-like behaviors of the mice are significantly improved. Further investigation shows that the content of short-chain fatty acids in the intestine of the mice is significantly increased, and the levels of brain-derived neurotrophic factor and inflammatory factors in the brain of the mice are significantly improved.

[0051] I. Bifidobacterium composition

[0052] The present invention provides a composition comprising animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536, wherein the composition of animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 can be dried by freeze-drying, and can contain an excipient used when drying a common bacterial strain or a composition derived from the bacterial strain. The excipient for freeze-drying can be selected from gluconic acid, alginic acid, sodium alginate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, methylcellulose, carbomer, hyaluronic acid, tragacanth gum, karaya gum, water-soluble starch, pectin, gelatin, polyvinyl alcohol, polyvinyl pyrrolidone, oligosaccharides, sugar alcohols, calcium gluconate, calcium lactate, polymethyl methacrylate, wheat protein, soy protein, and methylcellulose.

[0053] In some embodiments, the ratio of the viable bacteria of Bifidobacterium animalis subsp. lactis Bb-12 to Bifidobacterium longum subsp. longum BB536 in the composition is 1:15 to 15:1, preferably 1:10 to 10:1, more preferably 1:9 to 9:1, more preferably 1:9 to 2.3 and / or 2.3 to 9:1, for example, 1:1, 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 2:1, 2:2, 2:2.1, 2:2.2, 2:2.3, 2:2. 4, 2:2.5, 2:3, 2:4, 2:5, 2:6, 2:7, 2:8, 2:9, 2:10, 2:11, 2:12, 2:13, 2:14, 2:15, 3:1, 3:2, 3:2.1, 3:2.2, 3:2.3, 3:2.4, 3:2.5, 3:3, 3:4, 3:5, 3:6, 3:7, 3:8, 3:9, 3:10, 3:11, 3:12, 3:13, 3:14, 3:15, 4:1, 4:2, 4:2.1, 4:2.2, 4:2.3, 4:2.4, 4:2.5, 4:3, 4:5, 4:7, 4:9, 4:10, 4:11, 4:12, 4:13, 4:14, 4:15, 5:1, 5:2, 5:2.1, 5:2.2, 5:2.3, 5:2.4, 5:2.5, 5:3, 5:4, 5:6, 5:7, 5:8, 5:9, 5:11, 5:12, 5:13, 5:14, 6:1, 6:2.1, 6:2.2, 6:2.3, 6:2.4, 6:2.5, 6:5, 6:7, 6:11, 6:13, 7:1, 7:2, 7:2.1, 7:2.2, 7:2.3, 7:2.4, 7:2.5, 7:3, 7:4, 7:5, 7:6, 7:8, 7:9, 7:10, 7:11, 7:12, 7:13, 8:1, 8:2.1 , 8:2.2, 8:2.3, 8:2.4, 8:2.5, 8:5, 8:7, 8:9, 8:11, 8:13, 8:15, 9:1, 9:2, 9:2.1, 9:2.2, 9:2.3, 9:2.4, 9:2.5, 9:4, 9:5, 9:7, 9:8, 9:10, 9:11, 9:13, 9:14, 10:1, 10:2.1, 10:2.2, 10:2.3, 10:2.4, 10:2.5, 10:3, 10:7, 10:9, 10:11, 10:13, 11:1, 11:2, 11:2.1, 11:2.2, 11:2.3, 11:2.4, 11:2.5, 11:3, 11:4, 11:5, 11:6, 11:7, 11:8, 11:9, 11:10, 11:12, 11:13, 11:14, 11:15, 12:1, 12:2.1, 12:2.2, 12:2.3, 12:2.4, 12:2.5, 12:5, 12:7, 12:11, 12:13, 13:1, 13:2, 13:2.1, 13:2.2, 13:2.3, 13:2.4, 13:2.5, 13:3, 13:4, 13:5, 13:6, 1 3:7, 13:8, 13:9, 13:10, 13:11, 13:12, 13:14, 13:15, 14:1, 14:2.1, 14:2.2, 14:2.3, 14:2.4, 14:2.5, 14:3, 14:5, 14:9, 14:11, 14:13, 14:15, 15:1, 15:2, 15:2.1, 15:2.2, 15:2.3, 15:2.4, 15:2.5, 15:4, 15:7, 15:8, 15:11, 15:13, 15:14. .

[0054] II. Food

[0055] The food of the present invention contains or uses the above-mentioned bifidobacterium composition, thereby containing Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536.

[0056] In the present invention, there is no particular limitation on the absolute content of Bifidobacterium animalis lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 in food, as long as it complies with the requirements of relevant laws and regulations. For example, the content of Bifidobacterium animalis lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL, for example, can be 1×10 6 CFU / g, 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g, 1×10 10 CFU / g, 1×10 6 CFU / mL, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL, etc.

[0057] The present invention is not particularly limited to the specific form of the food. The edible nutrient is solid, semi-solid or liquid, and can illustratively include a drinkable composition, a powdered or granular composition, a gel, a frozen or partially frozen composition. The food can optionally form part of a capsule filling, or can optionally form part of a beverage, dairy product, non-dairy cream, sauce or baked product.

[0058] In some specific embodiments, the food can be powdered reconstituted food (solid beverages, instant coffee, cereal powder, nut powder or lotus root powder, etc.), baked food (bread, cake or biscuit baked food, etc.), beverages (carbonated beverages, fruit and vegetable juice beverages, functional beverages, tea beverages, milk beverages or alcoholic beverages, etc.), candy (jelly candy, hard candy, compressed candy, etc.), milk and dairy products (fresh milk from fresh cow (sheep) milk, milk powder, whey powder, fermented milk, cheese or condensed milk, etc.), etc.

[0059] In some other specific embodiments, the food of the present invention may also be a health food, such as various types of oral preparations, including but not limited to tablets, pills, granules, powders, teas, capsules or oral liquids.

[0060] In addition to Bifidobacterium animalis lactis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536 in the bifidobacterium composition, depending on the type of food and the final needs of the applicable object, in some embodiments, the food of the present invention further contains any one or more of the following ingredients: plant product ingredients, animal dairy product ingredients, animal meat product ingredients, functional additives and any acceptable excipients.

[0061] For plant product ingredients, examples include fruits such as figs, pomegranates, kiwis, tangerines, oranges, pineapples, strawberries, apples, bananas, grapes, pears, cherries, blueberries, blackberries, blackcurrants, cranberries, raspberries, melons, emblica and bilberry or their extracts; vegetables such as onions, cucumbers, tomatoes, cauliflower, carrots, spinach, kale, Brussels sprouts, garlic, basil, oregano or their extracts; rice (indica rice) , japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, millet, yellow rice, buckwheat, soybeans, broad beans, peas, mung beans, red beans, kidney beans and other grains or their extracts; walnuts, pistachios, cashews, hazelnuts, almonds, apricots, pine nuts, peanuts, melon seeds, chestnuts, macadamia nuts, ginkgo nuts and other nuts or their extracts; coffee or its extract; and some Chinese medicinal plants with edible properties or their extracts.

[0062] Examples of animal dairy ingredients include fresh milk from mammals such as cows, sheep, and camels, as well as reprocessed dairy products such as whole milk powder, skim milk powder, concentrated whey protein powder, desalted whey powder, whey protein powder, and hydrolyzed whey protein powder.

[0063] Examples of animal meat product ingredients include meat product ingredients from pigs, cattle, sheep, aquatic products, poultry, and the like.

[0064] Examples of functional added ingredients include vitamin supplements, mineral supplements, nucleotide supplements, dietary fiber, functional polyunsaturated fatty acid supplements, and the like.

[0065] As for any acceptable excipients, examples include solvents, antioxidants, antibacterial agents, thickeners, diluents, cosolvents, stabilizers, emulsifiers, fillers, disintegrants, lubricants, coating materials, anti-caking agents, flavoring agents, sweeteners, edible flavors, edible pigments, etc.

[0066] III. Uses for improving constipation

[0067] The present invention has discovered that combining Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536 can effectively improve constipation, especially functional constipation. When the two strains are combined, there is a synergistic effect between the two, which can further enhance the effect of improving constipation. The constipation improvement includes increasing fecal moisture content, improving intestinal motility, and increasing the content of short-chain fatty acids in the intestine. For example, the intestinal circulation rate is increased, the time to first black stool is shortened, and the content of acetic acid, propionic acid, butyric acid, and valeric acid in the intestine is increased.

[0068] In some embodiments, improving constipation includes improving intestinal health for non-therapeutic purposes.

[0069] IV. Uses to help relieve mood disorders caused by constipation

[0070] The present invention unexpectedly discovered that when the animal Bifidobacterium lactis subspecies Bb-12 and / or Bifidobacterium longum subspecies longum BB536 were administered to constipated mice, the constipated mice not only had a significant alleviation of the constipation condition, but also had a corresponding significant alleviation of their mood disorders. Thus, the bifidobacterium composition comprising animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 has the effect of alleviating the mood disorders caused by constipation. Moreover, when animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 are combined, there is a synergistic effect between the two, which can further enhance the efficacy of alleviating the mood disorders caused by constipation.

[0071] Thus, the present invention provides use of a bifidobacterium composition comprising Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536 in preparing a food for alleviating mood disorders caused by constipation.

[0072] In some embodiments, the constipation is functional constipation.Also, the mood disorder comprises depression and / or anxiety.

[0073] In some embodiments, the alleviating mood disorders caused by constipation comprises at least one of alleviating depression-like and / or anxiety-like behaviors, regulating the level of brain-derived neurotrophic factor in the hippocampus, and regulating the level of inflammatory factors in the hippocampus.

[0074] In some specific embodiments, the relief of depression-like and / or anxiety-like behaviors includes a significant reduction in the immobility time, and a significant increase in the movement distance and the number of times crossing the platform of depressed and anxious mice in the open field test, tail suspension test, and forced swimming test.

[0075] In the present invention, the "hippocampus" is an important component of the limbic system of the brain, located in the medial temporal lobe and named because of its shape similar to the seahorse. It plays a core role in memory formation (especially episodic and spatial memory), learning, emotional regulation, and spatial navigation. The hippocampus includes: (1) hippocampus; (2) dentate gyrus (DG); (3) subiculum; (4) entorhinal cortex; (5) hippocampal appendages surrounding the corpus callosum. The hippocampus is often divided into four regions: CA1, CA2, CA3, and CA4.

[0076] In some specific embodiments, regulating the level of brain-derived neurotrophic factor in the hippocampus includes increasing the expression of brain-derived neurotrophic factor (BDNF) in the DG region of the hippocampus. Regulating the level of inflammatory factors in the hippocampus includes decreasing the expression of tumor necrosis factor α (TNF-α) in the CA1 region of the hippocampus. This can alleviate the levels of depression and anxiety caused by constipation, particularly functional constipation.

[0077] Therefore, the bifidobacterium composition containing Bifidobacterium animalis subsp. lactis Bb-12 and Bifidobacterium longum subsp. longum BB536, as well as the food containing the bifidobacterium composition, described in the present invention, can help alleviate mood disorders caused by constipation, especially functional constipation. The present invention's relief of mood disorders is not intended to prevent or treat diseases.

[0078] Example

[0079] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0080] 1 Materials and Methods

[0081] 1.1 Raw materials:

[0082] Bifidobacterium animalis subsp. lactis Bb-12: Chr. Hansen

[0083] Bifidobacterium longum subsp. longum BB536: Morinaga

[0084] 1.2 Animal Experiment Design

[0085] Ninety healthy male BALB / c mice (6 weeks old, 20 g) were housed under a 12 h light / dark cycle, 25 ± 2 ° C temperature, and 55-65% humidity. Sterile water and normal feed were freely available throughout the experiment. After one week of adaptive feeding, they were randomly divided into 9 groups of 10 mice each, including a blank group (Comparative Example 1), a constipation anxiety model group (Comparative Example 2), a positive control group (Comparative Example 3), a Bb-12 group (Experimental Example 1), a BB536 group (Experimental Example 2), and a Bb-12 combined with BB536 group (Experimental Example 3, Experimental Example 4, Experimental Example 5, and Experimental Example 6).

[0086] Except for the blank group, mice in other groups were gavaged with 0.2 mL of loperamide (10 mg / kg BW) once daily at 9:00 am. The blank group was gavaged with normal saline for 7 days. Starting from the 8th day, the blank group was gavaged with normal saline daily, while the other groups were gavaged with 0.2 mL of loperamide (10 mg / kg BW). Two hours after gavage with loperamide, the positive group was gavaged with 2.5 mg / kg / d of mosapride. The Bb-12 and BB536 groups were gavaged with 5×10 10 and 5×10 10 The CFU / kg·bw of Bb-12 and BB536, and the dosage of the composition are shown in Table 1. On day 29, the mice were fasted for 12 h (with free access to drinking water) for subsequent index detection.

[0087] Table 1 Experimental groups

[0088]

[0089] 1.3 Health Index

[0090] During the mouse breeding period, the mice were observed daily for defecation, activity, and death. In addition, the mice's weight, water intake, and food intake were recorded at the same time every day.

[0091] 1.4 Determination of constipation-related indicators

[0092] 1.4.1 Water content of feces

[0093] Feces were collected on days 14 and 28 of feeding and dried to constant weight to obtain the dry weight and moisture content of the feces. Meanwhile, fecal samples collected on day 28 were stored at -80°C for analysis of short-chain fatty acids and intestinal microbiota. Fresh mouse feces were weighed and recorded as wet weight. The feces were then dried at 105°C for 5 hours to constant weight, then weighed and recorded as dry weight:

[0094] Fecal moisture content = (wet weight - dry weight) / dry weight × 100%

[0095] 1.4.2 Determination of defecation time

[0096] On day 28, all mice were fasted overnight for 12 hours and then gavaged with activated carbon solution (10% activated carbon, 0.5% carboxymethylcellulose suspension) before being placed in metabolic cages. The time between the mice's ingestion of activated carbon and their defecation of dark feces was recorded.

[0097] 1.4.3 Gastrointestinal transit rate

[0098] On day 29, the animals were gavaged with activated carbon solution. Thirty minutes later, all animals were euthanized by cervical dislocation, and the small intestine was collected by dissection. The distance from the pylorus to the border of the activated carbon was considered the migration distance. After measuring this length, the gastrointestinal transit rate was calculated using the following formula:

[0099] Gastrointestinal transit rate = activated carbon migration distance / total length of small intestine × 100%

[0100] 1.5 Mouse behavioral experiments

[0101] 1.5.1 Open field test (OFT)

[0102] The open field test is a classic emotion-related behavioral test. It is used to detect the behavior of animals in unfamiliar environments, including spontaneous activity and exploratory behavior. It is a method to evaluate the autonomous behavior, exploratory behavior and tension of animals in a new environment. Each mouse was randomly selected and placed in an apparatus (50×50cm 2The test was conducted individually in a chamber (40 cm and 40 cm high). The bottom of the apparatus was divided into 16 squares. Each subject was placed in a corner of the apparatus for 1 minute to adapt. All spontaneous activities were recorded (15 minutes) and calculated using an automatic movement activity recording tracker. The apparatus was cleaned with 75% ethanol solution before and after each test. The total distance traveled, average speed, total distance moved in the center area, and number of times the mice entered the center area were calculated for each group.

[0103] 1.5.2 Tail Suspension Test (TST)

[0104] The TST is widely used to study depressive states in mice. The mouse is fixed to its tail with tape and suspended in mid-air, with its head positioned 15 cm above a surface, for 6 minutes. Behavioral tracking software records video footage from the 2nd to 6th minute, accurately capturing the duration of the mouse's immobility. 1.5.3 Forced Swimming Test (FST)

[0105] The FST is used to assess depressive-like behaviors. Mice are placed in a clear glass tube 35 cm high and 15 cm in diameter. The water is maintained at a depth of 10 cm and a temperature of 25 ± 1°C. The mice are allowed to swim for 6 minutes, and their activity is recorded using a video capture system. A stopwatch is used to record the duration of immobility for 4 minutes after the end of the experiment.

[0106] 1.6 Determination of fecal short-chain fatty acids (SCFAs) content

[0107] Levels of acetic acid, propionic acid, butyric acid, and valeric acid in mouse feces were determined using gas chromatography-mass spectrometry (GC-MS). Briefly, 100 mg of fecal sample was homogenized with 1 mL of 0.5% phosphoric acid and centrifuged at 12,000 × g for 10 min. The collected supernatant was mixed with 500 μL of ethyl acetate and then analyzed by passing through a 0.22 μm PVDF membrane. SCFAs were separated using a GC-MS system equipped with an Agilent DB-WAX capillary column (30 m × 0.25 mm ID × 0.25 μm, 0.25 μm film thickness, 5% phenylmethylsiloxane). Helium (99.999%) was used as the carrier gas at a rate of 1.00 mL / min. The column temperature was initially set to 90°C, gradually increased to 150°C at 10°C / min, then to 230°C at 20°C / min, and finally held for 3 min. The ion source temperature, forward sample orifice temperature, and interface temperature were set at 230, 250, and 280° C. The detector was operated in electron impact ionization mode (electron energy 70 eV) with a scan range of 30–250 m / z.

[0108] 1.7 Brain Tissue Immunohistochemistry (IHC)

[0109] Paraffin-embedded slides of brain tissue were treated with methanol containing 3% hydrogen peroxide and then incubated with 10% goat serum. After washing three times, brain tissue sections (TNF-α, BDNF) were incubated overnight with the primary antibody ionized calcium-binding adapter molecule 1 (Iba-1) and then with horseradish peroxidase (HRP)-conjugated immunoglobulin G (IgG) (1:1000, ab6721). The sections were then stained with 3,3′-diaminobenzidine (DAB) and observed under a fluorescence inverted microscope.

[0110] 1.8 Statistical analysis:

[0111] The experimental data are expressed as mean ± standard deviation. P < 0.05 indicates a significant difference between the data, and P > 0.05 indicates an insignificant difference between the data.

[0112] 2 Experimental results

[0113] 2.1 Effects of Bifidobacterium Composition on Defecation Indices of Mice

[0114] Fecal moisture content is one of the important indicators reflecting the degree of constipation in mice. Figure 2 As shown in Figure A, compared with the blank group, the fecal moisture content of mice in the model group was significantly reduced (P < 0.05), indicating that loperamide administration caused the feces to become dry and hard. Compared with the model group, the fecal moisture content of mice in the positive group was significantly increased (P < 0.05), indicating that mosapride can effectively improve fecal moisture content. After intervention with Bb-12 and BB536, the fecal moisture content of all treatment groups was significantly higher than that of the model group (P < 0.05). The fecal moisture content of the combined Bb-12 and BB536 treatment group was significantly higher than that of the individual treatment groups (P < 0.05), approaching that of the blank group (P > 0.05). In particular, the increase in fecal moisture content in the combined Bb-12 and BB536 treatment group compared with the model group was greater than the combined increase in fecal moisture in the Bb-12 and BB536 treatment groups alone.

[0115] The time to the first black stool can reflect the motility of the entire gastrointestinal tract. The longer the time to the first black stool, the weaker the motility of the gastrointestinal tract of the mouse. Figure 2As shown in Figure B, the time to first black stool in the model group was significantly longer than that in the blank group. Compared with the model group, the time to first black stool in the positive group was significantly reduced (P < 0.05), indicating that mosapride can effectively improve gastrointestinal motility. Compared with the model group, the time to first black stool was significantly reduced after both Bb-12 and BB536 intervention (P < 0.05). The time to first black stool in the combined Bb-12 and BB536 treatment group was significantly shorter than that in the individual Bb-12 and BB536 treatment groups (P < 0.05), approaching that in the blank and positive groups (P > 0.05). In particular, the reduction in time to first black stool in the combined Bb-12 and BB536 treatment group compared with the model group was greater than the combined reduction in time in the Bb-12 and BB536 treatment groups alone.

[0116] Intestinal transit rate can also reflect the motility of the entire gastrointestinal tract and is an important indicator for evaluating constipation. The faster the intestinal transit rate, the more conducive it is to the excretion of feces. Figure 2 As shown in C. Compared with the blank group, the intestinal transit rate of mice in the model group was significantly reduced (P<0.05). Compared with the model group, the intestinal transit rate of mice was significantly increased after intervention with positive drugs, Bb-12 and BB536 (P<0.05). Among them, the intestinal transit rate of each group of the combination of Bb-12 and BB536 was significantly higher than that of the single group (P<0.05), and was close to that of the positive group and the blank group (P>0.05); in particular, the intestinal transit rate of the combined treatment group of Bb-12 and BB536 compared with the model group was greater than the sum of the intestinal transit rates of the Bb-12 single treatment group and the BB536 single treatment group.

[0117] The above results indicate that the combination of Bb-12 and BB536 can synergistically improve constipation in mice.

[0118] 2.2 Effects of nutrients on behavioral indicators of mice

[0119] 2.2.1 Open field experiment

[0120] The open field test is a classic method for evaluating the anxiety behavior of experimental rodents. When the animals are placed in an unfamiliar open field (strange environment), they will mainly move in the peripheral area due to their fear of the unfamiliar environment. However, the novelty of the unfamiliar environment will prompt them to explore the central area, thus creating a conflict "contradiction". The open field test measures the anxiety level of mice by observing spontaneous movements in a new environment. The effects of the bifidobacterium composition on the autonomous activity ability of mice are shown in Table 2. Compared with the blank group, the total distance moved, the average speed, the distance moved in the central area, and the number of times the platform was crossed in the open field test of the model group mice were significantly reduced (P<0.05). These results indicate that the anxiety model of mice has been successfully established.

[0121] Compared with the model group, the total distance traveled, average speed, distance traveled in the central area, and number of platform crossings in the open field test were significantly increased after treatment with the positive drug (P < 0.05), indicating that the positive drug has a certain alleviating effect on the anxiety of constipated mice. Furthermore, compared with the model group, the total distance traveled, average speed, distance traveled in the central area, and number of platform crossings in mice treated with Bb-12 and BB536 were significantly increased (P < 0.05). In the open field test, the total distance traveled, distance traveled in the central area, number of platform crossings, and average speed in mice treated with the combination of Bb-12 and BB536 were significantly higher than those in the Bb-12 or BB536 groups alone (P < 0.05). In particular, the total distance traveled in the combined Bb-12 and BB536 group, compared with the model group, was greater than the total distance traveled in the Bb-12 and BB536 groups alone combined. The combination groups demonstrated a synergistic effect in alleviating constipation-induced anxiety.

[0122] Table 2 Open field test

[0123]

[0124] Note: Different letters indicate significant differences among the examples (P<0.05).

[0125] 2.2.2 Tail suspension test

[0126] The tail suspension test is a model of despair. The length of time a mouse gives up struggling and stops moving in the tail suspension test is used as an indicator to reflect its state of despair. This behavioral despair model is similar to depression. The tail suspension test is usually used as one of the commonly used behavioral experiments to assess depression in mice. The effects of Bb-12 and BB536 intervention on the depressive behavior of mice are shown in Table 3. Compared with the blank group, the immobility time of mice in the model group in the tail suspension test was significantly increased (P<0.05), indicating that mice gavage with loperamide showed depressive behavior. Compared with the model group, the immobility time of mice after Bb-12 and BB536 intervention in the tail suspension test was significantly reduced (P<0.05). Furthermore, mice treated with the combination of Bb-12 and BB536 showed significantly lower immobility time in the tail suspension test compared to mice treated with either Bb-12 or BB536 alone (P<0.05). In particular, the reduction in immobility time in the combined Bb-12 and BB536 group compared to the model group was greater than the combined reduction in immobility time in the Bb-12 and BB536 groups alone, indicating that the Bb-12 and BB536 combination can synergistically alleviate depression caused by constipation.

[0127] Table 3 Tail suspension immobility time of mice

[0128] Example Dose group Bb-12: BB536 Immobility time (s) Comparative Example 1 K <![CDATA[57.42±5.28 e ]]> Comparative Example 2 M <![CDATA[145.92±6.45 a ]]> Comparative Example 3 Y <![CDATA[116.67±4.77 bc ]]> Experimental Example 1 Bb-12 <![CDATA[112.5±5.74 bc ]]> Experimental Example 2 BB536 <![CDATA[130.25±5.11 ab ]]> Experimental Example 3 Experimental Example 3 2.3：1 <![CDATA[86.08±5.42 d ]]> Experimental Example 4 Experimental Example 4 9：1 <![CDATA[91.58±3.46 d ]]> Experimental Example 5 Experimental Example 5 1：2.3 <![CDATA[85.83±5.06 d ]]> Experimental Example 6 Experimental Example 6 1：9 <![CDATA[93.5±4 d ]]>

[0129] Note: Different letters indicate significant differences among the examples (P<0.05).

[0130] 2.2.3 Forced swimming

[0131] Forced swimming is a behavioral despair test method and one of the commonly used behavioral experiments to evaluate depression by showing a typical "immobility state" in animals. The effects of Bb-12 and BB536 intervention on the immobility time of mice in the forced swimming test are shown in Table 4. The results showed that oral administration of loperamide in the model group induced an increase in the immobility time of mice. Compared with the model group mice, the intervention of positive drugs, Bb-12, BB536 and the combination of Bb-12 and BB536 significantly reduced the immobility time of mice in the water (P<0.05). Among them, the immobility time of mice in the Bb-12 and BB536 combination group was significantly lower than that of the Bb-12 and BB536 groups alone, indicating that the bifidobacterium combination can synergistically alleviate depression caused by constipation.

[0132] Table 4 Mouse forced swimming test

[0133] Example Dose group Bb-12: BB536 Immobility time (s) Comparative Example 1 Blank group <![CDATA[60.33±4.06 e ]]> Comparative Example 2 Model Group <![CDATA[130.83±3.94 a ]]> Comparative Example 3 Positive group <![CDATA[102.75±5.62 b ]]> Experimental Example 1 Bb-12 group <![CDATA[102.5±3.95 b ]]> Experimental Example 2 BB536 group <![CDATA[102.67±4.7 b ]]> Experimental Example 3 Bb-12 combination BB536 2.3：1 <![CDATA[74.58±4.88 d ]]> Experimental Example 4 Bb-12 combination BB536 9：1 <![CDATA[79.42±5.82 cd ]]> Experimental Example 5 Bb-12 combination BB536 1：2.3 <![CDATA[80±2.41 cd ]]> Experimental Example 6 Bb-12 combination BB536 1：9 <![CDATA[86.29±4.08 cd ]]>

[0134] Note: Different letters indicate significant differences among the examples (P<0.05).

[0135] 2.3 Short-chain fatty acids

[0136] Short-chain fatty acids are the main metabolites produced by intestinal microorganisms through anaerobic fermentation of indigestible polysaccharides such as dietary fiber and resistant starch, mainly including acetic acid, propionic acid, butyric acid and valeric acid. These fatty acids are present in large quantities in the intestine and play an important role in the gut-brain axis. They have the ability to penetrate the intestinal mucosal barrier, enter the blood circulation, and eventually reach the brain. Short-chain fatty acids can relieve anxiety and depression by directly stimulating neural pathways or through indirect central effects of neuroendocrine and immune activation. The effects of Bb-12 and BB536 on the levels of short-chain fatty acids including acetic acid, propionic acid, butyric acid and valeric acid in the intestines of mice are as follows: Figure 3As shown. Compared with the blank group, the levels of acetic acid, propionic acid, butyric acid and valeric acid in the intestinal contents of the model group mice were significantly reduced (P<0.05). Compared with the model group mice, the intervention of Bb-12 and BB536 significantly increased the content of short-chain fatty acids in the intestinal tract of mice (P<0.05). Among them, compared with Bb-12 or BB536 alone, the combination group significantly increased the content of short-chain fatty acids in the intestine of mice (P<0.05); in particular, the increased levels of acetic acid, propionic acid, butyric acid and valeric acid in the combined treatment group of Bb-12 and BB536 compared with the model group were greater than the sum of the increased levels of acetic acid, propionic acid, butyric acid or valeric acid in the Bb-12 alone treatment group and the BB536 alone treatment group. The results show that the bifidobacterium composition has the effect of synergistically restoring the content of short-chain fatty acids in the intestine of mice, thereby indirectly affecting mood improvement.

[0137] 2.4 Distribution and expression of BDNF in the DG region of the hippocampus and TNF-α protein in the CA1 region of the brain

[0138] Brain-derived neurotrophic factor (BDNF) is an important brain neurotrophic factor that can mediate a variety of brain functions and plays a key role in synaptic regulation. Many studies have shown that the expression level of BDNF is closely related to depression. Tumor necrosis factor α (TNF-α) is a pleiotropic proinflammatory cytokine that plays an important role in the hippocampus through multiple mechanisms, including inducing neuroinflammation, promoting neuronal apoptosis, inhibiting neurogenesis and affecting cognitive function. These effects play a key role in a variety of neurodegenerative diseases and mood disorders. IHC was used to further study the changes in hippocampal BDNF and TNF-α in the hippocampus of anxious and depressed mice induced by constipation. Figure 4 A and Figure 4 As shown in Figure C, the distribution of BDNF in the DG region of the hippocampus of depressed mice (model group) was significantly lower than that of the control group, while the distribution of TNF-α in the CA1 region of the hippocampus was significantly higher than that of the control group. Compared with the model group, after Bb-12 and BB536 treatment, the number of BDNF-expressing cells and the staining depth in the DG region of the hippocampus of mice increased to varying degrees, while the number of TNF-α-expressing cells and the staining depth were significantly reduced.

[0139] ImageJ was used to analyze the immunohistochemistry results. Figure 4 B and Figure 4As shown in Figure D, the mean optical density of BDNF in the model group was significantly lower than that in the blank control group (P < 0.05), while the mean optical density of TNF-α was significantly higher than that in the blank control group (P < 0.05). Positive drug intervention reversed these changes. Compared with the model group, the mean optical density of BDNF and TNF-α in mice treated with Bb-12, BB536, and the combined treatment of Bb-12 and BB536 all showed varying degrees of recovery. It is worth noting that the average optical density of BDNF in the DG region of the hippocampus of mice treated with the combined treatment of Bb-12 and BB536 was significantly higher than that in the Bb-12 group alone and the BB536 group alone (P<0.05), while the average optical density of TNF-α was significantly lower than that in the Bb-12 group alone and the BB536 group alone (P<0.05); in particular, the increased average optical density of BDNF in the DG region of the hippocampus in the combined treatment of Bb-12 and BB536 group compared with the model group was greater than the sum of the increased average optical density of BDNF in the DG region of the hippocampus in the Bb-12 and BB536 group alone; the decreased average optical density of TNF-α in the combined treatment of Bb-12 and BB536 group compared with the model group was greater than the sum of the decreased average optical density of TNF-α in the Bb-12 group alone and the BB536 group alone. In summary, the IHC staining results showed that Bb-12 and BB536 intervention can synergistically improve the anxiety and depression of mice caused by constipation by regulating the expression of BDNF and TNF-α.

Claims

1. Use of a bifidobacterium composition in preparing a food for alleviating mood disorders caused by constipation, characterized in that: The bifidobacterium composition comprises Bifidobacterium animalis subspecies lactis Bb-12 and Bifidobacterium longum subspecies longum BB536.

2. The use according to claim 1, characterized in that The ratio of the live bacteria amount of Bifidobacterium animalis subspecies lactis Bb-12 to Bifidobacterium longum subspecies longum BB536 in the composition is 1:15 to 15:

1.

3. The use according to claim 1 or 2, characterized in that The constipation is functional constipation; and the mood disorders caused by constipation include depression and / or anxiety.

4. The use according to any one of claims 1 to 3, characterized in that The relief of mood disorders caused by constipation comprises at least one of relieving depression-like and / or anxiety-like behaviors, regulating the level of brain-derived neurotrophic factor in the hippocampus, and regulating the level of tumor necrosis factor α in the hippocampus.

5. Use of a bifidobacterium composition in preparing a food for improving constipation, characterized in that: The bifidobacterium composition comprises Bifidobacterium animalis subspecies lactis Bb-12 and Bifidobacterium longum subspecies longum BB536; The constipation includes functional constipation.

6. The use according to claim 5, characterized in that The improvement of constipation includes at least one of increasing the water content of feces, improving intestinal motility and increasing the content of short-chain fatty acids in the intestine.

7. The use according to claim 6, characterized in that The ratio of the live bacteria amount of Bifidobacterium animalis subspecies lactis Bb-12 to Bifidobacterium longum subspecies longum BB536 in the composition is 1:15 to 15:

1.

8. The use according to any one of claims 1 to 7, characterized in that The content of animal Bifidobacterium lactis subspecies Bb-12 and Bifidobacterium longum subspecies longum BB536 in the food is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

9. The use according to any one of claims 1 to 8, characterized in that The food is beverage, milk and dairy products, baked food or candy.

10. The use according to any one of claims 1 to 8, characterized in that The food is an oral preparation; the oral preparation includes at least one of tablets, pills, granules, powders, teas, capsules and oral liquids.

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