Bifidobacterium lactis and its use

Abstract

Bifidobacterium lactis is provided. The Bifidobacterium lactis is Bifidobacterium lactis BL-11, which has the accession number CGMCC No. 20847. The provided Bifidobacterium lactis can improve intestinal metabolic disorders and normalize the composition of intestinal microflora, thereby effectively improving height, preventing obesity, promoting the growth and development and intellectual development of children and adolescents, and preventing and treating mental disorders, including anxiety, depression, attention-deficit hyperactivity disorder, autism, isolationism, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette's syndrome, and Asperger's syndrome.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and specifically to Bifidobacterium lactis and its use.

Background Art

[0002] Studies have found that gut bacteria can affect the growth and development of the host, and some bacterial flora can mediate the host's growth and development process by affecting growth hormone (GH) / insulin-like growth factor-1 (IGF-1). Both germ-free mice and mice treated with antibiotics have slower growth, which is involved in the decrease in the levels of ghrelin, growth hormone, and IGF-1. Also, in mice and humans, the lack of microorganisms limits maturation and growth, leading to, for example, chronic malnutrition, anorexia nervosa, stunted growth, short stature, and further abnormal development of the nervous and immune systems. Insulin-like growth factor 1 (IGF-1) is a hormone with known effects on bone growth. With the increase in the colonization of gut microorganisms, the content of IGF-1 in serum increases significantly, and the contents of IGF-1 in the liver and adipose tissue also increase. After antibiotic treatment, the gut flora decreases significantly, which may reduce serum IGF-1 and suppress bone formation. Supplementing short-chain fatty acids (SCFAs), microbial metabolites, after antibiotic treatment can restore IGF-1 and bone mass to normal levels. Also, the level of IGF-1 in germ-free mice is low, but treatment with beneficial microorganisms can moderately increase bone mineral density, improve the level of IGF-1, and prevent bone loss. Insulin-like growth factor 1 (IGF-1) is a growth factor that affects the endocrine and paracrine / autocrine pathways of bone growth. Exogenous IGF-1 can promote the longitudinal growth of the femur, and it can be observed that after the deletion of the specific insulin-like growth factor I receptor (Igf1r) in cartilage, IGF-1 affects the maturation of the growth plate and the formation of secondary ossification centers. Studies have found that although the serum IGF-1 of liver-specific IGF-1-deficient mice decreased by 75%, they still showed relatively normal growth and development, indicating that some IGF-1 can also promote bone growth. Also, IGF-1 can promote osteoblasts (including affecting bone formation and bone resorption respectively). Therefore, the change in the gut microbiota can promote bone formation and resorption, thus causing net bone growth, and the microbiota can promote bone growth and remodeling by inducing IGF-1.Direct supplementation of short-chain fatty acids (SCFAs) produced after the fermentation of probiotics, prebiotics, or cellulose by microorganisms may induce an increase in IGF-1 and affect bone growth and health.

[0003] In addition, gut microbiota can control brain function and behavior through the gut-brain axis. The gut microbiota and the brain communicate bidirectionally through pathways and mediators such as the autonomic nervous system, enteric nervous system, immune system, olfactory system, gut endocrine signals, neurotransmitters, branched-chain amino acids, bile acids, short-chain fatty acids, spinal cord, hypothalamic-pituitary-adrenal axis, peptidoglycan, etc. The bidirectional communication between the gut microbiota and the central nervous system, i.e., the microbiota-gut-brain axis, affects the neural development and function of animals and their social behavior. Such bidirectional effects of the microbiota-gut-brain axis are influenced by internal factors such as gender and genetics, as well as external factors such as environment, diet, genetics, and stress. Therefore, the gut microbiota is involved in the development of multiple nervous system-related diseases, and at the same time, psychological and behavioral responses also affect the composition and function of the gut microbiota. According to existing evidence, intervention methods targeting the microbiota are expected to treat diseases such as anxiety, depression, schizophrenia, attention deficit hyperactivity disorder (ADHD), Tourette syndrome, Parkinson's disease, Alzheimer's disease, etc., related to the gut microbiota, as well as social disorders such as autism. Taking attention deficit hyperactivity disorder (ADHD) as an example, this is the most common neurodevelopmental disorder in children. The main symptoms are that it is easy for the affected children to be distracted inattentionally, have a narrow attention span, be overly active regardless of location, be prone to emotional impulsiveness, and generally be accompanied by cognitive impairment, conduct disorder, and learning difficulties. ADHD has a high incidence rate and clearly has an adverse impact on the academic performance, family, and social life of patients. However, the treatment methods for ADHD are still lacking.

[0004] Therefore, it is necessary to develop probiotics that can prevent obesity, promote the growth and development of children and adolescents, and prevent mental disorders, which is of great significance for children, adolescents, or patients with mental disorders and their families.

Summary of the Invention

[0005] This application provides Bifidobacterium lactis and its uses. The Bifidobacterium lactis can improve intestinal metabolic disorders and normalize the composition of the intestinal microbiota, effectively prevent obesity, promote the growth and development of children and adolescents, and promote their intellectual development. The Bifidobacterium lactis can also effectively improve intestinal permeability, reduce the levels of LPS and D-lactic acid in the blood, and play a role in preventing mental disorders. The mental disorders include anxiety, depression, attention deficit hyperactivity disorder, autism, asocial disorder, schizophrenia, hepatic encephalopathy, neurotic vomiting, Tourette syndrome, and Asperger syndrome.

[0006] To achieve the above object, the first aspect of this application provides Bifidobacterium lactis, which is Bifidobacterium lactis BL-11, and the deposit number of the Bifidobacterium lactis BL-11 is CGMCC No. 20847.

[0007] The second aspect of this application further provides a Bifidobacterium lactis preparation, which is a solid bacterial powder or a liquid beverage, and the Bifidobacterium lactis is the Bifidobacterium lactis described in the first aspect of this application.

[0008] The third aspect of this application further provides the use of the above Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for improving intestinal metabolic disorders and promoting the normalization of the composition of the intestinal microbiota.

[0009] The fourth aspect of this application further provides the use of the above Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for anti-obesity in children and adolescents.

[0010] The fifth aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the production of a food composition or a pharmaceutical composition for promoting the growth and development of children and adolescents.

[0011] The sixth aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the production of a food composition or a pharmaceutical composition for promoting the increase in height of children and adolescents.

[0012] The seventh aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the production of a food composition or a pharmaceutical composition for promoting the intellectual development of children and adolescents.

[0013] Optionally, the content of the Bifidobacterium lactis BL-11 is 1 to 25 parts by weight, preferably 1 to 15 parts by weight, based on 100 parts by weight of the food composition or the pharmaceutical composition. The Bifidobacterium lactis BL-11 is used in the form of viable cells of the Bifidobacterium lactis BL-11, inactivated cells of the Bifidobacterium lactis BL-11, or an extract of the Bifidobacterium lactis BL-11.

[0014] Optionally, the viable cell count of the Bifidobacterium lactis BL-11 is 1.0×10 6 ~1.5×10 12 CFU / g, preferably 3.0×10 10 ~5.0×10 11 CFU / g.

[0015] Optionally, the food composition is one or more of fermented milk, cheese, milk-based beverage, solid beverage, and powdered milk.

[0016] Optionally, based on the body weight of a human, the dosage of the Bifidobacterium lactis BL-11 is 2.0×10 6 CFU~1.5×10 11 CFU / kg / day, preferably 3.0×10 4 CFU~8.0×10 10It is CFU / kg / day.

[0017] The eighth aspect of the present application provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for preventing mental disorders.

[0018] Optionally, the mental disorders include anxiety, depression, attention deficit hyperactivity disorder, autism, isolation disorder, schizophrenia, hepatic encephalopathy, nervous anorexia, Tourette syndrome, Asperger syndrome.

[0019] Optionally, the food composition or the pharmaceutical composition further comprises one or more combinations of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malate, L-lactic acid, etc.

[0020] Optionally, the food composition or the pharmaceutical composition further comprises a flavoring agent, a sweetening agent, a thickening agent, a stabilizer, a surfactant, a lubricant, an acid neutralizer, a dispersant, a buffer solution or a buffering agent, a debittering agent, a pH stabilizer, a preservative, a de-sugaring agent and / or a coloring agent, for example, lactitol, sorbitol, maltitol, aspartame, stevia, lakanka, sucralose, xylitol, vanilla, chocolate, fruit flavor, artificial essence, or a mixture or combination thereof.

[0021] Optionally, the food composition or the pharmaceutical composition further comprises vitamins, minerals and / or supplements or prebiotic nutrients, at least one prebiotic, and optionally, the prebiotic comprises inulin, artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, mannooligosaccharide, arabinooligosaccharide, resistant dextrin, resistant starch or a mixture or combination thereof.

[0022] Optionally, the food composition or pharmaceutical composition further comprises ubiquinone (CoQ10), lycopene, β-carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof.

[0023] Optionally, the food composition or pharmaceutical composition further comprises probiotics, and optionally, the probiotics comprise microorganisms or bacteria or bacterial components cultured or extracted from feces, and optionally, the bacteria or bacterial components comprise Lactobacillus, Bifidobacterium, Escherichia coli, Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes and equivalents, or a mixture or combination thereof.

[0024] Optionally, the content of Bifidobacterium lactis BL-11 is 0.5 to 20 parts by weight, preferably 1 to 15 parts by weight, per 100 parts by weight of the food composition or pharmaceutical composition. The Bifidobacterium lactis BL-11 is used in the form of viable cells of Bifidobacterium lactis BL-11, inactivated cells of Bifidobacterium lactis BL-11, or an extract of Bifidobacterium lactis BL-11. The viable count of Bifidobacterium lactis BL-11 is 1.0×10 6 ~1.5×10 12 CFU / g, preferably 3.0×10 10 ~5.0×10 11 CFU / g.

[0025] Optionally, the food composition is one or more of fermented milk, cheese, milk-based beverages, solid beverages, and powdered milk.

[0026] Optionally, based on the body weight of a human, the dosage of Bifidobacterium lactis BL-11 is 2.0×10 9CFU~1.5×10 11 CFU / kg / day, preferably 3.0×10 4 CFU~8.0×10 10 CFU / kg / day.

[0027] Optionally, different delivery forms and carriers are used, and the food composition or pharmaceutical composition may be a powder, tablet, liquid, gum, soft candy, lozenge, yogurt, milk, cheese, ice cream, frozen food, health supplement, pharmaceutical, or feed.

[0028] Bifidobacterium lactis BL-11 of the present application can improve intestinal permeability and reduce the levels of LPS and D-lactic acid in the blood.

[0029] Bifidobacterium lactis BL-11 of the present application can promote autonomous activity, improve the memory function of one trial avoidance response, and be used to improve anhedonia caused by stress stimuli.

[0030] According to the above technical solution, the probiotics according to the present application can effectively improve metabolic disorders, normalize the composition of the intestinal microbiota, promote the growth of children and adolescents, improve intestinal permeability, reduce the levels of LPS and D-lactic acid in the blood, and play a role in preventing mental disorders. The mental disorders include anxiety, depression, attention deficit hyperactivity disorder, autism, asocial disorder, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette syndrome, and Asperger syndrome.

[0031] Other features and advantages of the present application will be described in detail in the following specific embodiment section.

[0032] <Biomass Entrustment Information> Bifidobacterium lactis BL-11 is named Bifidobacterium lactis, preserved in the China General Microbiological Culture Collection Center, with the deposit address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being October 10, 2020, and the deposit number being CGMCC No. 20847.

Brief Description of the Drawings

[0033] The drawings are provided for a better understanding of the present application, form a part of the specification, and are used together with the following specific embodiments to explain the present application, but do not limit the present application. The drawings are as follows.

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Mode for Carrying Out the Invention

[0034] Hereinafter, specific embodiments of the present application will be described in detail with reference to the drawings. It should be understood that the specific embodiments described herein are for the purpose of explaining and interpreting the present application and are not intended to limit the present application.

[0035] The first aspect of the present application provides Bifidobacterium lactis, and the Bifidobacterium lactis is Bifidobacterium lactis BL-11, and the Bifidobacterium lactis BL-11 has an accession number of CGMCC No. 20847.

[0036] The second aspect of the present application further provides a Bifidobacterium lactis preparation, the preparation being a solid bacterial powder or a liquid beverage, and the Bifidobacterium lactis being the Bifidobacterium lactis described in the first aspect of the present application.

[0037] The third aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for improving intestinal metabolic disorders and promoting normalization of the composition of the intestinal microbiota.

[0038] The fourth aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of an anti-obesity food composition or a pharmaceutical composition for children and adolescents.

[0039] The fifth aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for promoting the growth and development of children and adolescents.

[0040] The sixth aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for promoting an increase in height of children and adolescents.

[0041] The seventh aspect of the present application further provides the use of the above-mentioned Bifidobacterium lactis in the manufacture of a food composition or a pharmaceutical composition for promoting the intellectual development of children and adolescents.

[0042] Optionally, the content of the Bifidobacterium lactis BL-11 is 1 to 25 parts by weight, preferably 1 to 15 parts by weight, based on 100 parts by weight of the food composition or pharmaceutical composition. The Bifidobacterium lactis BL-11 is used in the form of viable cells of the Bifidobacterium lactis BL-11, inactivated cells of the Bifidobacterium lactis BL-11, or an extract of the Bifidobacterium lactis BL-11.

[0043] Optionally, the viable count of the Bifidobacterium lactis BL-11 is 1.0×10 6 ~1.5×10 12 CFU / g, preferably 3.0×10 10 ~5.0×10 11 CFU / g.

[0044] Optionally, the food composition is one or more of fermented milk, cheese, milk-based beverage, solid beverage, and powdered milk.

[0045] Optionally, based on the body weight of a human, the dosage of the Bifidobacterium lactis BL-11 is 2.0×10 6 CFU~1.5×10 11 CFU / kg / day, preferably 3.0×10 4 CFU~8.0×10 10 CFU / kg / day.

[0046] The eighth aspect of the present application provides the use of the above Bifidobacterium lactis in the manufacture of a food composition or pharmaceutical composition for preventing mental disorders.

[0047] Optionally, the mental disorders include anxiety, depression, attention deficit hyperactivity disorder, autism, isolation disorder, schizophrenia, hepatic encephalopathy, nervous anorexia, Tourette syndrome, and Asperger syndrome.

[0048] Optionally, the food composition or pharmaceutical composition further comprises one or more combinations of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malic acid, L-lactic acid, and the like.

[0049] Optionally, the food composition or pharmaceutical composition further comprises a seasoning, a sweetener, a thickener, a stabilizer, a surfactant, a lubricant, an acid neutralizer, a dispersant, a buffer solution or buffer, a debittering agent, a pH stabilizer, a preservative, a de-sugaring agent and / or a colorant, for example, lactitol, sorbitol, maltitol, aspartame, stevia, lakanka, sucralose, xylitol, vanilla, chocolate, fruit flavor, artificial essence, or a mixture or combination thereof.

[0050] Optionally, the food composition or pharmaceutical composition further comprises vitamins, minerals and / or supplements or prebiotic nutrients, at least one prebiotic, and optionally, the prebiotic comprises inulin, artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, mannooligosaccharide, arabinooligosaccharide, indigestible dextrin, resistant starch or a mixture or combination thereof.

[0051] Optionally, the food composition or pharmaceutical composition further comprises coenzyme Q10 (CoQ10), lycopene, β-carotene, tryptophan, vitamin B6, vitamin B12 or a mixture or combination thereof.

[0052] Optionally, the food composition or pharmaceutical composition further comprises probiotics, and optionally, the probiotics comprise cultured or fecal-extracted microorganisms or bacteria or bacterial components, and optionally, the bacteria or bacterial components comprise Lactobacillus, Bifidobacterium, Escherichia coli, Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes and equivalents, or mixtures or combinations thereof.

[0053] Optionally, the content of Bifidobacterium lactis BL-11 is 0.5 to 20 parts by weight, preferably 1 to 15 parts by weight, based on 100 parts by weight of the food composition or pharmaceutical composition. The Bifidobacterium lactis BL-11 is used in the form of viable cells of Bifidobacterium lactis BL-11, inactivated cells of Bifidobacterium lactis BL-11, or an extract of Bifidobacterium lactis BL-11. The viable count of Bifidobacterium lactis BL-11 is 1.0×10 6 ~1.5×10 12 CFU / g, preferably 3.0×10 10 ~5.0×10 11 CFU / g.

[0054] Optionally, the food composition is one or more of fermented milk, cheese, milk-based beverages, solid beverages, and powdered milk.

[0055] Optionally, based on the body weight of a human, the dosage of Bifidobacterium lactis BL-11 is 2.0×10 9 CFU~1.5×10 11 CFU / kg / day, preferably 3.0×10 4 CFU~8.0×10 10 CFU / kg / day.

[0056] Optionally, different delivery forms and carriers are used, and the food composition or pharmaceutical composition may be a powder, tablet, liquid, gum, soft candy, lozenge, yogurt, milk, cheese, ice cream, frozen food, health supplement, pharmaceutical product or feed.

[0057] Bifidobacterium lactis BL-11 of the present application can improve intestinal permeability and reduce the levels of LPS and D-lactic acid in the blood.

[0058] Bifidobacterium lactis BL-11 of the present application can be used to promote autonomous activity, improve the memory function of one-trial avoidance response, and improve anhedonia caused by stress stimulation.

[0059] Hereinafter, the present application will be further described by way of examples, but the present application is not limited thereto.

[0060] (Example 1) This example is for explaining Bifidobacterium lactis BL-11 and its performance characteristics.

[0061] <1. Taxonomic characteristics of Bifidobacterium lactis BL-11> The results of observing Bifidobacterium lactis BL-11 under a microscope are shown in FIG. 1. The results of physicochemical experiments are shown in Tables 1 and 2.

[0062]

Table 1

[0063]

Table 2

[0064] <2. Tolerance of Bifidobacterium lactis BL-11 to artificial gastric juice and intestinal juice> Bifidobacterium lactis is usually a genus of bacteria that is sensitive to acids. In this example, while testing the resistance of Bifidobacterium lactis BL-11 of the present application to artificial gastric juice and intestinal juice, Bifidobacterium lactis Bb-XX, which has extremely excellent acid resistance and can survive through the gastrointestinal tract and is currently stored in the laboratory, was used as a control.

[0065] The detection results of the survival rate of the BL-11 strain in artificial gastric acid (pH = 3) are as shown in Table 3. For Bb-XX, the viable bacteria survival rate when treated with artificial gastric juice for 1 hour is 44.7%, and the viable bacteria survival rate when treated for 3 hours is 29.5%. However, for Bifidobacterium lactis BL-11 of the present application, the viable bacteria survival rate when treated for 1 hour is 86.2%, and the viable bacteria survival rate when treated for 3 hours is 39.5%. This indicates that Bifidobacterium lactis BL-11 of the present application has relatively good gastric acid resistance, and most of it can smoothly pass through the stomach and reach the intestine to exert beneficial effects.

[0066] The detection results of the survival rate of the BL-11 strain in artificial small intestinal juice (pH = 8) are shown in Table 3. As can be seen from the data, for Bb-XX, the viable bacteria survival rate when treated with artificial intestinal juice (pH = 8) for 1 hour is 66.1%. However, for Bifidobacterium lactis BL-11 of the present application, the viable bacteria survival rate when treated with artificial intestinal juice for 1 hour is 67.5%. After 3 hours of treatment, the survival rates of the two strains are 49.4% and 32.1% respectively.

[0067] As can be seen from the above results, Bifidobacterium lactis BL-11 can still survive well after digestion with artificial gastric juice and intestinal juice (Figure 2). Bifidobacterium lactis BL-11 of the present application has relatively good digestive juice resistance compared to the reference bacteria and can survive and grow smoothly in the intestine.

[0068]

Table 3

[0069] <3. Toxicity Test and Safety Detection of Bifidobacterium lactis BL-11> The Bifidobacterium lactis BL-11 of the present application was inoculated into MRS liquid medium and anaerobically cultured at 37 °C for 48 hours. The viable count of Bifidobacterium lactis BL-11 in the culture solution was counted to be 3.7×10 9 CFU / mL. The culture stock solution was orally administered to mice continuously for 3 days at a rate of 20.0 mL / kg body weight, and then observed for 7 days. Healthy BALB / C male mice aged 6 - 8 weeks and weighing 16 - 18 g were selected, and maintained at room temperature (25±2 °C), relative humidity (55±2)%, and 12h / 12h light irradiation, and allowed to freely ingest food and water. As can be seen from the results, no toxic reactions or deaths were observed in the two groups of test mice for the culture stock solution of Bifidobacterium lactis BL-11 compared with the control group, and there was no statistical difference in the weight gain of the mice (p>0.05).

[0070] The antibiotic susceptibility of Bifidobacterium lactis BL-11 was evaluated by the method of SN / T 1944-2007 "Measurement of Bacterial Drug Resistance in Animals and Their Products". As can be seen from the evaluation results, Bifidobacterium lactis BL-11 is sensitive to Ampicillin, PencillinG, Erythromycin, Chloramphenicol, Clindamycin, Vancomycin, Tetracycine, etc., and meets the requirements in the evaluation specifications of the antibiotic resistance of edible bacteria by the European Food Safety Authority. Bifidobacterium lactis BL-11 does not contain foreign antibiotic resistance genes and is safe for consumption.

[0071] (Example 2) This example is for explaining the functional characteristics of Bifidobacterium lactis BL-11 in promoting growth and development.

[0072] The microbiota causes net bone growth by promoting bone formation and resorption. The microbiota induces the hormone-like insulin growth factor 1 (IGF-1) and promotes bone growth and remodeling. Short-chain fatty acids (SCFAs) generated by the fermentation of fibers by the microbiota also induce IGF-1, presenting the mechanism by which the microbiota affects bone health.

[0073] Two-month-old BALB / c female mice were treated with antibiotics and probiotics and bred under SPF conditions. A total of 30 mice were randomly assigned to treatment groups to minimize cage effects. The antibiotic is a mixture of 0.2 mg / mL gentamicin, 0.15 mg / mL Ciprofloxacin, 2 mg / mL streptomycin, and 1 mg / mL Bacitracin.

[0074] First, the mixture of antibiotics was added to the drinking water of the mice and the mice were treated for 2 weeks to consume the microorganisms. Then, the mice were divided into 3 groups, with 10 mice in each group. One group was used as the control group and bred normally, and the other 2 groups were probiotic groups. BL-11 or Bb-XX was added to the water respectively, and the breeding continued for 4 weeks. According to the regulations of the animal facility, 3% (g / 100 mL) of sucrose was added to all the water to ensure palatability. The aqueous solution was freshly prepared and changed twice a week. After four weeks, after the mice were killed, serum was produced from the blood samples collected by cardiac puncture using serum separation tubes, and a kit (PeproTech) for the development of ABTS ELISA for mouse IGF-1 standard was used.

[0075] The femur lengths of mice bred with normal feed (CK), BL-11, and Bb-XX are shown in Figure 4, and the leg bone lengths are shown in Figure 3. The femur length of the mice bred with BL-11 was significantly different compared to the control group (P < 0.05), and the femur length of the mice bred with Bb-XX was not significantly different compared to the control group (P < 0.05). All data are shown as mean ± SD, and whether the difference is significant was verified by t. *P < 0.05, ** P < 0.01, *** P < 0.001.

[0076] Analysis of growth regulatory activity: Both germ-free mice and mice treated with antibiotics showed slow growth, reduced levels of ghrelin, growth hormone, and IGF, and intestinal microbiota disorders, resulting in restricted mouse growth. After treating the mice with antibiotics, they were given probiotics, and the serum IGF-1 level of the mice was detected. As shown in Figure 5, the mice that were given probiotics after treatment with antibiotics had increased serum IGF-1 levels, and taking BL-11 could significantly improve the serum IGF-1 level (P < 0.01), and its improvement effect was higher than that of Bb-XX. Intestinal flora diversity: As can be seen from the analysis of the α-diversity index, there was no significant difference in the diversity index between BL-11 and Bb-XX. BL-11 was slightly higher than Bb-XX in the Shannon index, but the significant difference could not be achieved (P > 0.05). The results are as shown in Figure 6. As can be seen from the analysis of the two types of probiotics in the intestines of the two groups of mice, using BL-11 could significantly increase the ratio of Bifidobacterium and Lactobacillus in the intestine, and the results are shown in Figure 7.

[0077] According to the heatmap analysis (Figure 8), at the genus level, Faecalibacterium, Lachnospira, Lachnospiraceae_UCG_004, and Sutterella increased in the BL-11 group, and the increase of these bacteria may be involved in the increase of IGF-11 in the serum.

[0078] (Example 3) This example is for explaining the use of BL-11 bacterial powder in the production of food.

[0079] Bifidobacterium lactis BL-11 according to the present application is anaerobically cultured in MRS Broth liquid medium. The fermented liquid after primary and secondary culturing is expanded, cultured at 37 °C for 24 hours, then centrifuged at 4 °C and 3000 rpm for 10 min to collect the bacterial cells, washed with phosphate buffer (PBS), added with skim milk and freeze-dried, and stored at -20 °C or lower.

[0080] The BL-11 bacterial powder produced in this example can be used for food, pharmaceuticals, health supplements or animal feed.

[0081] The food may be ordinary food or health supplements such as fermented milk, cheese, milk-based beverages, solid beverages, powdered milk, etc. Preferably, in the food, the proposed dosage of Bifidobacterium lactis BL-11 for human use is 1.0×10 3 CFU~1.0×10 10 CFU / kg body weight / day, and more preferably 1.0×10 4 CFU~1.0×10 9 CFU / kg body weight / day.

[0082] (Example 4) This example is for explaining the functional characteristics of promoting growth and development in human clinical research.

[0083] <Subjects and Recruitment> 65 Prader-Willi syndrome patients aged from 11 months to 16 years were publicly recruited and randomly assigned to the probiotics group or the placebo group for a 12-week randomized, double-blind, placebo-controlled trial.

[0084] <Selection Criteria> Genetically confirmed to have PWS, not taking any probiotics within 4 weeks, taking stable medications for at least 4 weeks, no planned drug and psychological interventions during the test period, able to provide fecal samples in a timely manner, able to participate in the research and consultation process, and no other genetic diseases, pregnancy or lactation status. In accordance with IRB requirements, the research protocol obtains the informed consent of the subject's parent or legal guardian and is conducted based on the Declaration of Helsinki.

[0085] <Method> Randomized and blinded trials: Adopt a randomized, double-blind, placebo-controlled design. A statistician not belonging to the team conducts random concealed allocation for the subjects and generates a random sample number for each subject with unknown identity. Beijing Huayuan Biotechnology Research Institute provides probiotics and placebo with the same appearance codes, ensures allocation concealment, and maintains the blind spot. These patients are randomly assigned to receive either the probiotic of Bifidobacterium lactis BL-11 (6×10 10 CFU) or a placebo packet daily. When treating for 6 weeks and 12 weeks, compare the weight, height, ASQ-3, ABC, SRS-2, and CGI-1 of the two groups. CGI includes two single measurements and evaluates the following: (a) a mental symptom rating scale from 1 to 7 (CGI-S), and (b) an evaluation of the symptom change (CGI-I) before and after treatment from start to end using a 7-point scale.

[0086] <Materials> The probiotic group is the packet-form powdered Bifidobacterium lactis BL-11. Each supplement of Bifidobacterium lactis is 3×10 10It contains colony - forming units (CFU), and the placebo is maltodextrin packaged in the same way, with color, taste and flavor similar to those of the Bifidobacterium lactis packet. The subjects were orally administered one bag of Bifidobacterium lactis or placebo twice a day with water for 12 consecutive weeks. Maltodextrin has minimal side effects as a supplement and also minimal adverse reactions as a placebo.

[0087] <Measurement of primary results> 1. Parents measured the weight and height using a standard scale, which was collected by the researchers and used with reference to the aging provided by the WHO, and the weight, height and BMI were converted to z - scores.

[0088] 2. Psychological tests (1) "Ages and Stages Questionnaire", Third Edition (ASQ - 3). ASQ - 3 is one of the most widely used developmental screening tools for children and adolescents, and has five domains: communication, gross motor, fine motor, problem - solving, and personal - social. The total score of the subjects was calculated from the five domains to evaluate the test effect.

[0089] (2) Aberrant Behavior Checklist (ABC). ABC is a 58 - item behavior evaluation scale that evaluates behavior problems in five sub - scales: anger, lethargy / social withdrawal, stereotyped behavior, hyperactivity / non - compliant behavior, and inappropriate speech. The total score of the subjects was calculated from the above - mentioned behavior problems to evaluate the test effect.

[0090] (3) Social Responsiveness Scale (SRS). SRS contains 65 items and quantitatively evaluates the severity of social behavior. The total score of the subjects was calculated from the above - mentioned social behavior to evaluate the test effect.

[0091] (4) Restricted and Repetitive Behaviors (RRB) based on the 4 - point scale (0 - 3) adopted by the Gilliam Autism Rating Scale (Third Edition) (GARS - 3). The total score of the subjects was calculated from the above - mentioned behaviors to evaluate the test effect.

[0092] <Measurement of secondary results> 1. Fecal microbiota (1). Sample processing and collection Fecal samples were collected using DNA / RNA Shield fecal collection tubes (Zymo, Cat#R1101) containing 1 mL of preservation solution, transported to the laboratory with ice packs, and then frozen at -80°C. Based on the manufacturer's instructions, DNA (TIANGEN, catalog number DP328) was extracted using the TIANmap fecal DNA kit, and the DNA samples were carefully quantified using a Nanodrop spectrophotometer. The ratio of A260 / A280 was measured to confirm the yield of high-purity DNA. The DNA samples were frozen at -20°C until use.

[0093] (2) Amplicon sequencing of the 16S rRNA gene. A 16S rRNA V3-V4 library was constructed using the following primers by two rounds of PCR.

[0094] 341F: 5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG-3’ (SEQ ID NO.1) 805R: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC-3’ (SEQ ID NO.2)

[0095] After performing 25 cycles in the reaction process of 95°C for 2 min, and then 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, it was finally extended at 72°C for 5 min. The PCR products were purified with 1xKAPA AMPure (KAPA, catalog number KK8002). Then, the products were subjected to the second round of PCR reaction process (95°C for 2 min, and then 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 8 cycles, and finally extended at 72°C for 5 min). The PCR products were purified with 1×KAPA AMPure, and quantitative analysis was performed by real-time PCR using a Bioanalyzer DNA kit.

[0096] 2. The Clinical Global Impression (CGI) was developed for use in clinical trials and aims to simply and independently evaluate, by clinicians, how the overall function of patients appears before and after the start of using the investigational drug. The CGI includes two single measurements and evaluates the following: (a) the Psychiatric Symptom Rating Scale (CGI-S) from 1 to 7, and (b) the evaluation of changes after the start of treatment (CGI-I) using a similar 7-point scale.

[0097] 3. Gastrointestinal symptoms are evaluated based on the total number of gastrointestinal symptoms present at baseline, including constipation, diarrhea, abdominal pain, gastrointestinal ballooning, bloody stools, nausea, dysphagia, loss of appetite, indigestion, and acid reflux.

[0098] <Analysis of Data> All primary data are recorded and processed in Microsoft Excel 2007 and R. The data presentation is for reporting the results of a randomized, double-blind, clinical placebo-controlled trial in accordance with the CONSORT recommendations. Statistical processing was performed using α = 0.05 as the significance level. This application explored the changes at 0 - 6 weeks and 0 - 12 weeks in the z-scores of weight and height, the total scores and subscores of ASQ-3, ABC, and SRS, and each item at baseline using the Wilcoxon rank sum test. The linear mixed model was also used to analyze repeated measurements.

[0099] This application adjusted the multiple comparison results using the False Discovery Rate (FDR). Secondary outcomes were analyzed in the same way as the primary outcomes. Also, linear regression was performed to examine the correlation between clinical indicators and the composition of the microbiota.

[0100] <Data Processing and Analysis of Microbiota> As quality control, sequence numbers were filtered using QIIME2 (v2019.10). Deblur was used to remove noise for the default parameters, and an abundance table of samples was obtained by amplicon sequence variants (ASVs). Alpha diversity was calculated using QIIME2. Bray-Curtis distance was used to represent the β-diversity of the microbiota. ASVs were assigned using the classification method of a classifier based on sklearn, and the classifier was trained with sequences that had 99% similarity to Greengenes v13.8. The Kruskal-Wallis test confirmed that there were significant differences between the relative abundances of the phyla, genera, and α-diversity of the microbiota in the placebo group and the probiotics group. The false discovery rate (FDR) by Benjamini-Hochberg (BH) adjustment was used for multiple comparisons.

[0101] PICSRUSt2 is used to estimate the functional content of the microbiota based on the abundance table of ASVs and generate Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO), enzyme classification numbers, and pathway abundance tables. A ratio difference analysis between the probiotics and the placebo group was performed using a nonparametric test based on sequences, and the most significant differential features were plotted with Calour. All primary data derived from 16s rRNA Illumina amplicon sequences are stored in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, PRJNA643297).

[0102] <Results> 1. Demographic characteristics of PWS subjects Table 4 summarizes the demographic characteristics of 65 participants and the gastrointestinal (GI) symptoms of comorbidities. No differences between groups were observed (P>0.05). 47.5% of the subjects showed one or more GI symptoms in the study group, and the proportion of GI symptoms in the probiotics group decreased by 37.4% compared to the placebo group, but the difference did not reach significance (P>0.05).

[0103]

Table 4

[0104] No major defects were found. This is because there was no significant difference between the two groups in all observed defect events and the main causes of school dropout (P > 0.05).

[0105] 2. Effects of probiotics on body weight, height, psychological measurements, and CGI-I Throughout the treatment process, anthropometric data were collected and analyzed. The probiotics group had significantly higher height from 6 weeks to 12 weeks than the placebo group (the probiotics intervention group was significantly higher than the placebo group, with an average of 2.58 cm higher, P < 0.05, Figure 9). The weight of the probiotics group decreased over time compared to the placebo group, but the difference between the two groups was not significant (Figure 10). The scores of psychological measurements (including ASQ-3, ABC, SRS, and RRB) were observed by linear mixed-effects model analysis. The probiotics group tended to improve more than the placebo group, but the difference was not significant (P > 0.05). The overall improvement of symptoms during the treatment process was measured using the CGI-1 scale. Compared with the placebo group, the overall improvement of symptoms in the probiotics group was more significant (Figure 11, P < 0.05).

[0106] 3. Changes in the composition and function of the microbiota in probiotic intervention After sequencing, during the intervention process, the differences in the composition of the gut microbiota of PWS individuals in the two groups were abundant. The overall specific bacterial flora had relative abundance ratios as shown in Figure 12. After 6 weeks, compared with the placebo group, the α-diversity in the probiotics group increased slightly, but the difference was not significant. By arranging the β-diversity display of multivariate analysis of variance (PERMANOVA) analysis, the separation of the two groups by probiotic treatment can be achieved (F statistic = 2.2526, R 2 = 0.035613, P < 0.05, NMDS stress = 0.19048, Figure 13).

[0107] To represent the possibility of changes in the abundance of bacteria with clinical significance throughout the intervention process, this application introduced several changes in the multiples of selected bacterial genera and families, as shown in Figure 14. In the probiotics group, at week 6 and week 12, the relative abundances of Lachnospiraceae ND3007, Ruminococcaceae UCG-003, Streptococcus mutans, Comamonadaceae (genus Comamonas), Alistipes, and Rothia were lower than the baseline. In the probiotics group, Bifidobacterium, Lactobacillus, and Prevotella 9 increased significantly compared to the baseline at 12 weeks (Figure 15).

[0108] As revealed by the predictive analysis of functional genes, after 12 weeks of treatment, some genes in the probiotics group had different abundances. Genes encoding biosynthesized ubiquinone protein (ubiB, k03688), removing saturated enzyme (EC: 1.3.99.29) in parallel, removing saturated enzyme (lycopene formation) (EC: 1.3.99.31) in parallel, upregulating the gene of all-trans-ζ-carotene desaturase (EC: 1.3.99.26), and downregulating genes encoding dimethylarginase (k01482) and acid phosphatase (phoN, k09474, EC: 3.1.3.2) (Figure 16). These findings did not meet the false discovery criteria for multiple comparisons significance. The analysis results of the predicted KEGG pathways and predicted KOs shown in Figures 17 and 18 further compared the gene expressions of the probiotics and placebo groups. 4. Correlation between the abundance of gut microbiota and clinical indicators

[0109] Clinical indicators are involved in the abundance ratio of bacterial genera. Two correlations were found in the probiotics group, but no correlation was observed in the placebo group. At week 6, there was a positive correlation between the RRB score of the probiotics group and Rothia (R = 0.97, p < 0.005).

[0110] In the 12-week randomized, double-blind, and placebo-controlled experiment of 65 PWS patients in this application, the height of the Bifidobacterium lactis BL-11 subjects increased significantly, and the weight changed slightly. This application provides new evidence for the early intervention of Bifidobacterium lactis BL-11 in PWS patients. In addition, this application found that there was a significant difference in the beta diversity of the gut microbiota between the probiotics group and the placebo group after treatment, and when diet was strictly controlled, it was found that the baseline beta diversity was directly correlated with long-term weight loss. Therefore, the supplementation of Bifidobacterium lactis BL-11 can improve the composition of the gut flora, prevent obesity, or promote weight loss by diet. The supplementation of Bifidobacterium lactis BL-11 can also improve the growth and development status of children and adolescents.

[0111] In addition, since this application found that the improvement of the overall symptoms between the probiotics group and the placebo group after treatment was obvious and the indicators of psychological measurement were improved, the supplementation of Bifidobacterium lactis BL-11 may have the effect of improving the intellectual development, behavior, or emotional state of children and adolescents.

[0112] (Example 5) This example is for explaining the manufacturing method of the BL-11 freeze-dried bacterial powder. The BL-11 preserved bacterial solution was resuscitated in a 37°C water bath until all the liquid in the cryopreservation tube had melted. It was partitioned on MRS medium and anaerobically cultured at 37°C for 12 - 24 hours. Then it was inoculated into an anaerobic culture tube containing liquid medium, sealed, and anaerobically cultured at 37°C for 6 - 12 hours. The fermentation was stopped by monitoring that the growth of the bacterial solution OD600 value had ceased. Fermentation production of the bacterial strain was carried out, stirred at a constant temperature of 40°C for 6 - 12 hours of culture, maintaining the constant pH of the bacterial solution at 5.4 ± 0.5, and the fermentation was immediately stopped until it was monitored that the growth of the bacterial solution OD600 value had ceased. The bacterial sludge was collected by centrifugation, and a cryoprotectant was added at a volume ratio of bacterial sludge:cryoprotectant = 1:1 - 10, stirred, and uniformly mixed. Then it was put into a freeze dryer for freeze-drying of the bacterial powder. The freeze-dried powder was collected, pulverized according to the quality requirements, and packaged.

[0113] (Example 6) This example is for explaining the use of the Bifidobacterium lactis BL-11 freeze-dried bacterial powder in food production.

[0114] The freeze-dried bacterial powder produced by Bifidobacterium lactis BL-11 according to the present application can be used in ordinary foods such as yogurt, fermented milk, cheese, or health supplements. Preferably, in the said food, the addition amount of Bifidobacterium lactis BL-11 in the production of yogurt is 1.0×10 6 CFU~1.0×10 8 CFU / kg, and more preferably, 1.2×10 7 CFU~1.5×10 8 CFU / kg. The production method is direct administration or post-addition. In the case of direct administration, as a fermentation bacterial strain, after the sterilization of raw milk is completed, it is added at a certain ratio when the temperature is lowered to an appropriate temperature, and then incubated at 40 - 43°C for 10 - 48 hours for fermentation. After stirring and blending, it is dispensed into cups or bags as a finished product. When added in the post-addition method, after the fermentation of yogurt is completed, it is added at a certain ratio and then uniformly stirred, and after the blending is completed, it is dispensed as a finished product.

[0115] (Example 7) This example is for explaining the use of BL-11 freeze-dried bacterial powder in the production of nutritional supplements and probiotics.

[0116] The freeze-dried bacterial powder produced by Bifidobacterium lactis BL-11 according to the present application can be used in the production of probiotics. The freeze-dried powder of probiotics contains 0.5 to 30 parts of BL-11 freeze-dried bacterial powder, 5 to 20 parts of fermented Lactobacillus, Lactobacillus swiss, Lactobacillus reuteri, Lactobacillus plantarum, Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium longum and other bacterial species, 20 to 70 parts of prebiotics such as galactooligosaccharide, fructooligosaccharide, inulin, 5 to 10 parts of nutrients such as GABA, tryptophan, lycopene, β-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, β-glucan, fucose, carrageenan, guar gum, dietary fiber, 0.1 to 5 parts of antioxidants / anti-inflammatory substances such as tocopherol, carotenoid, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenol, glutathione and superoxide dismutase. The total added amount of viable bacteria in the probiotics is such that the added amount of BL-11 is 2.0×10 6 CFU~2.0×10 10 CFU / g, and more preferably 3×10 7 CFU~3×10 10 CFU / g. The added amount of a single bacterium of other bacterial species is 1×10 6 CFU~3×10 9 CFU / g. The manufacturing process includes weighing, mixing, dispensing and quality inspection of raw materials in a certain ratio.

[0117] (Example 8) This example is for explaining that the BL-11 freeze-dried bacterial powder improves intestinal permeability and behavioral effects.

[0118] The gut microbiota and the gut-brain axis (GBA) play a two-way communication role in the control of the stress response. Microorganisms communicate with the gut-brain axis through different mechanisms. They interact directly with mucosal cells and affect brain development and behavior through contact with immune cells and peripheral nerves. Brain stress can also affect gut microbiota through the GBA. The gut microbiota is a cause of gastrointestinal dysfunction and disorders. The stress response also affects the synthesis of microbial by-products and precursors. These by-products and precursors enter the brain through the blood and the hindbrain, release cytokines through mucosal immune cells, release gut hormones such as serotonin (5-hydroxy tryptamine, 5-HT) through enteroendocrine cells, or are released through neural pathways including the enteric nervous system.

[0119] Manufacture of an intervention probiotic preparation: The probiotic preparation contains freeze-dried powder of BL-11, β-carotene, vitamin B6, vitamin B12, coenzyme Q10 and maltodextrin. The control group is maltodextrin, and the intragastric administration dose for experimental mice is 10 billion CFU / mouse / day.

[0120] Twenty 6-week-old C57BL / 6J mice were assigned to cages with 5 mice per cage and raised, allowing them to eat and drink freely. The food and water intake was recorded twice a week. The mice were assigned to two groups, a control group and a probiotic group, with 10 mice in each group. After the experimental mice adapted to the environment for one week, they were randomly assigned to the model group and the probiotic intervention group. The conditions of the growth environment for the experimental mice were environmental temperature (23±2) °C, relative humidity (50±10)%, and light irradiation mode (12h dark / 12h light irradiation).

[0121] A chronic stress model induced by physical stimuli was adopted. As specific stimulation methods, (1) fasting and water deprivation for 24 h, (2) continuous light irradiation for 24 h, (3) clamping a 1-cm portion of the tail tip with an iron clamp and lasting for 3 min each time, and (4) restraint stress, restraining the behavior with a circular sleeve for 2 h per day were included. One or two different stress stimuli were adopted daily, the stimulation time each time was not constant, and the modeling time was continued for 4 weeks.

[0122] From the 6th week, the stress stimulation was stopped, and experiments on sucrose preference, step-down test, and open field test were conducted. After the behavioral experiments were completed, physicochemical indexes of the mice in each group were measured. The data analysis adopted the data obtained by processing with SPSS statistical software and was expressed as mean ± standard deviation. Independent sample t-tests were used to compare between groups, and P < 0.05 was considered to have statistical significance.

[0123] 1) Experiment on sucrose preference Before the start of the experiment, two identical water bottles were simultaneously placed in the cage rack. One bottle was filled with pure water, and the other bottle was filled with 1% sucrose solution. First, the experimental mice were adapted to the sucrose solution for 24 h. To avoid inhibiting the drinking habits of the experimental mice, the positions of the water bottles were adjusted every 1 h. After the adaptation was completed, the experimental mice were fasted and water-deprived for 24 h. Before the start of the experiment, one bottle of pure water and one bottle of 1% sucrose solution were placed in the breeding cage, and the consumption of sucrose solution and pure water was recorded once every 3 h.

[0124] Sucrose preference = consumption of sucrose solution / (consumption of sucrose solution + consumption of pure water) × 100%.

[0125] As can be seen from the experimental results, the control group reduced the preference for the sucrose solution (49.63% ± 15.79), and after supplementing with probiotics, the preference for the sucrose solution was improved (68.79% ± 12.34), indicating that probiotics can improve the anhedonia caused by stress stimulation.

[0126] 2) Step-down test Platform experimental box (platform for DTT-2 type mice, Institute of Materia Medica, Chinese Academy of Medical Sciences). The platform box is about 120 cm in length, 12 cm in width, and 30 cm in height, and is made of organic glass material. It has a total of 6 rooms, each room being 12 cm in length, 12 cm in width, and 30 cm in height. The experimental box was used to conduct experiments on 6 mice simultaneously. A copper grid was laid on the bottom surface of the experimental box, and the copper grid was connected to the power supply via an electric wire, with the voltage of the current set at 36V. An insulating platform (a pentagonal wooden piece with a major diameter of 5.7 cm, a minor diameter of 4.5 cm, and a height of 4.8 cm) was placed at the corner inside the box on the copper grid in the experimental box as a safe area for animals to avoid electric shock. The test device is connected to the automatic recording system of a computer. During the experiment, the mice were placed into the platform experimental box, allowed to adapt to the environment for 5 min, and then gently placed on the platform. The copper grid was electrified, and when the mice jumped from the platform and their limbs contacted the copper grid, they received an electric shock. The normal avoidance reaction is to jump onto the platform and return to the safe area to avoid the electric shock. In this way, they learned for 5 min, and the number of electric shocks (error times) within this 5 min was recorded as the learning performance. After 24 h, a memory ability test was conducted. The mice were placed on the platform, and the latency, which is the time from when the mice stayed on the platform until they jumped and received the first electric shock, and the number of error times (the number of times the mice's limbs simultaneously contacted the copper grid) within 5 min were recorded as the evaluation indicators of the memory function.

[0127] Compared with the normal control group, in the probiotics group, the latency in the step-down test was significantly shortened (P<0.05), and there was no significant difference in the number of error times in the step-down test, but the probiotics group showed a tendency of decreasing the number of error times. The mice in the probiotics group can improve the impairment of the one-trial avoidance reaction memory function (see Table 5).

[0128]

Table 5

[0129] 3) Open-field experiment The open-field analysis system observes the neuropsychiatric changes of the experimental animals studied and various behaviors after they enter a large environment. For example, animals mainly move in the peripheral area due to fear of the new open environment and have little activity in the central area. However, the animals' obsessive characteristics generate the motivation to move in the central area, and the resulting anxiety can also be observed. The autonomous activity and anxiety level of the animals are evaluated.

[0130] Before the experiment, the mice were transferred to the open-field laboratory in 60 minutes to adapt to the environment earlier. During the experiment, the mice were taken out of the cage and placed in the open-field experimental device (the length × width × height of the box: 100 cm × 100 cm × 40 cm, both the inner surface and the bottom surface are blue, and a camera is placed directly above the central area) placed on the behavioral experiment station. After the experiment started, the mice were placed at a fixed position in the central area, placed on the side where the head was fixed each time, and the shade curtain was quickly pulled. After recording the mouse number, date, and status in the operation software, the recording system was executed, the nine-square grid mode was selected, the ratio of the central area was 0.5, and it was measured by the camera above the open-field equipment and the monitor connected to it. Each mouse was measured for 5 minutes, and the activity status of the mouse was recorded. The measurement indicators include movement time, total distance, residence time fraction in the central area (residence time in the central area LPMM = s), horizontal movement percentage in the central area (horizontal movement distance in the central area / horizontal movement distance), horizontal movement percentage in the four-side area (horizontal movement distance in the four-side area / horizontal movement distance), and horizontal movement percentage in the four-corner area (horizontal movement in the four-corner area / horizontal movement distance). The number of times of standing upright and the number of grooming times were recorded. Subsequently, the inside of the box was cleaned with 75% alcohol, and after the alcohol had completely evaporated, the next mouse was tested. The results are shown in Table 6.

[0131] Compared with the control group, the number of times the mice in the probiotics group entered the central area and the residence time in the central area were significantly higher than those in the control group (P < 0.05). The number of times of standing upright and grooming increased significantly (P < 0.05), and the difference in the number of times of entering the central area was not large. No obvious differences were found in the remaining open-field results.

[0132]

Table 6

[0133] 4) Detection of intestinal permeability To evaluate the intestinal permeability in the body, the D-lactic acid and LPS contents in serum were measured to evaluate the intestinal permeability.

[0134] D-lactic acid is a metabolite fermented by bacteria and can be produced by multiple bacteria in the intestinal tract. Even if it is ingested from food, it is rarely absorbed by the blood under normal circumstances. In addition, mammals do not have an enzyme system that rapidly degrades it. Therefore, when the permeability of the intestinal mucosa increases, a large amount of D-lactic acid produced by bacteria in the intestinal tract is taken into the blood by the damaged mucosa, increasing the level of D-lactic acid in the blood.

[0135] Lactic acid has D-type and L-type. In a normal human body, there is only L-lactic acid, and D-lactic acid can be produced by microorganisms such as bacteria. By monitoring the level of D-lactic acid in the blood, the degree of intestinal mucosal damage and changes in permeability can be timely reflected. It can be used for auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory response, repeated fever, vomiting, etc.

[0136] Lipopolysaccharide (LPS) is also called bacterial endotoxin and is a component on the cell wall of Gram-negative bacteria. LPS is a toxic substance for animals. The structure of LPS can be divided into three parts: glycolipid domain - lipid A, short-chain sugar residue - core oligosaccharide, and highly variable polysaccharide domain - O antigen. The structure of LPS determines its agonist / antagonist action on TLR4. In the body, LPS binds to the complex of TLR4 / MD-2 receptor and activates different signal pathways through the Myd88-dependent or TRIF-dependent pathway. The expression levels of TLRs in intestinal epithelial cells at different sites are different, and LPS can prevent the inflammatory reaction and fight against pathogenic bacteria.

[0137] Bacterial translocation refers to the entry of intestinal active bacteria into the body through the epithelial mucosa from the intestinal tract. Bacteria can enter the lymphatic system through the mesenteric lymph nodes and circulate throughout the body. Bacteria can enter the blood circulation, cause bacteremia, and can also be located in tissues. Bacterial translocation can cause overgrowth of bacteria in the small intestine, intestinal injury, and shock. Any stress response that leads to intestinal permeability includes psychological and physiological effects and may potentially cause bacterial translocation.

[0138] LPS is involved in the onset of various diseases such as intestinal diseases like IBD and enterocolitis, as well as Parkinson's and Alzheimer's diseases. LPS can not only enter the blood but also enter the brain and stay there for a lifetime, potentially causing Alzheimer's disease.

[0139] The LPS level in the blood can reflect intestinal permeability. A normal intestinal barrier does not allow the entry of LPS, and a high level of LPS in the blood indicates the movement of intestinal bacteria or LPS into the blood, meaning an increase in intestinal permeability and an increased probability of the appearance of leaky gut symptoms. The content level of LPS in the blood can also indicate an inflammatory response and a stress state. Excessive LPS can cause immune system abnormalities in the human body, trigger chronic or acute inflammatory responses, and present acute inflammations such as fever and pain. It can be used for the auxiliary evaluation of intestinal infections, endotoxemia, systemic inflammatory responses, repeated fevers, vomiting, mental diseases, stress responses, etc.

[0140] After the experiment, blood was collected from the peripheral vein of the tail. The blood was centrifuged at 3000g for 15 minutes. Using an intestinal barrier function analysis system (JY-DLT, Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd.), the D-lactic acid and LPS contents in the serum were detected according to the instruction manual.

[0141] As can be seen from the results, the LPS and D-lactic acid levels of the probiotics group were significantly decreased compared with the control group (P<0.05). It was demonstrated that stress stimulation increased intestinal permeability, while probiotics decreased intestinal permeability, reducing the risks of endotoxemia, systemic inflammatory response, etc.

[0142] (Example 9) This example is for explaining the effect of BL-11 freeze-dried bacterial powder on the composition of intestinal microorganisms.

[0143] After the above behavioral experiment was completed, the collected cecal contents were stored at -80°C. Feces of the two groups of mice were collected, and DNA of fecal flora was extracted using the TIANmap fecal DNA kit (TIANGEN, catalog number DP328). The extracted DNA was quantitatively detected using a Qubit meter. Detection was performed using 1% agarose gel electrophoresis (voltage 100V, 40 min). The UVI gel imaging system took pictures for recording. For DNA electrophoresis, no non-specific bands or smears occurred, indicating good purity of DNA fragments and no obvious degradation. An appropriate amount of sample was placed in a centrifuge tube and diluted to 1 ng / μL with sterile water. The DNA was stored in a -20°C refrigerator for use.

[0144] Amplification of bacterial 16S rRNA gene: Using the diluted genomic DNA as a template, according to the selection of the sequencing region, specific primers with barcodes were used, and the V3-V4 region of the bacterial 16S rRNA gene was amplified using the bacterial universal primers 341F (CCTAYGGGRBGCASCAG SEQ ID NO.3) and 806R (GGACTACNNGGGTATCTAAT SEQ ID NO.4). 100 ng of the extracted DNA was used for PCR at 56°C for strand regeneration. First, it was denatured at 94°C for 4 minutes, then lowered to 94°C for 30 seconds, lowered to 56°C for 30 seconds, and lowered to 72°C for 1 minute, and 30 cycles were performed.

[0145] Amplicon gene sequencing: Libraries were constructed using the library kit of the Illumina TruSeq DNA PCR-Free Library Preparation Kit. After the constructed libraries passed Qubit quantification and library detection, the microbiota was sequenced using the Illumina HiSeq2500 PE250 sequencing platform.

[0146] Processing and analysis of sequencing data: The primary data of the microbiota sequencing was imported into QIIME (2019.4), and noise reduction was performed with DADA2 to obtain representative amplicon sequence variants (ASVs), which were used to construct a phylogenetic tree. After quality control, the filtered ASVs were compared with gene sequences in the Naive bayes classifier (NBC) method and the Greengenes (V_13.5) database for species annotation. In Alpha and Beta diversity analyses, the resampling depth was 10,000 sequences per sample to ensure sufficient sequences. The aggregation results were corrected by calculating the false discovery rate (FDR) to reduce the impact of too many species on the results.

[0147] The results are shown in Figures 20 - 23 and Table 7. As can be seen from the results, at the genus level, there are significant differences in the intestinal microbiota between the control group and the probiotics. The content of the genus Bifidobacterium in the control group is significantly lower than that in the probiotics group. Also, the alpha diversity index of the probiotics group is higher than that of the control group, and the index difference is not significant (Shannon index p = 0.9118 (Mann-Whitney statistic); Shannon index P = 0.35268 (Mann-Whitney statistic)). In the β diversity analysis, there are differences in the overall composition of the intestinal microbiota between the control group and the probiotics group. In the PCoA analysis results, the two groups can be clearly distinguished (F-value = 2.4268, R-square = 0.1188, p = 0.009 (PERMANOVA)).

[0148] At the genus level, there are many specific differential bacteria, among which Coprobacillus is significantly increased (FDR < 0.001) after using probiotics.

[0149]

Table 7

[0150] (Example 10) This example is to explain the results of the intervention of probiotics consisting of BL-11 bacteria on a 3-year-old and 6-month-old child suffering from autism spectrum disorder (ASD). The lyophilized powder was taken orally, 50 billion CFU each time, twice a day, and the administration period was 90 days. After taking it, the child's defecation habit changed, and the number of defecations changed from once every 3 - 5 days to once every 1 - 2 days. According to the feedback from the parents on the child's vocabulary expression ability, socialization and the frequency of eye contact increased, and the amount of spoken vocabulary increased by 3 - 5. The frequency of spontaneously providing feedback on one's own physical sensations increased, and the number of times of spontaneously submitting requests also increased. Repetitive movements were less than before the intervention. The parents chose to continue taking it and continued to observe and record further improvement situations.

[0151] (Example 11) This example is to explain the improvement of attention deficit hyperactivity disorder (ADHD) by probiotics consisting of BL-11 bacteria.

[0152] The case is a person from Hebei Children's Hospital, diagnosed by a doctor according to the ADHD criteria in the Diagnostic and Statistical Manual of Mental Disorders in the United States, aged 6.5 years, with the hyperactive-impulsive type, and schizophrenia, mood disorder, epilepsy and other organic diseases were excluded. Also, there are chronic constipation and abdominal discomfort.

[0153] The treatment method uses oral administration of the freeze-dried powder of probiotics for treatment, with a dosage of 60 billion CFU twice a day. It was taken with warm water after breakfast and dinner respectively. The treatment was carried out continuously for 12 weeks. Evaluation using the Conners Parent Scale includes indicators such as behavior, body, anxiety, learning, hyperactivity, etc. As can be seen from the results, the scores of impulse, anxiety and hyperactivity of the children decreased, the defecation function improved, from once every 3 - 4 days to 1 - 2 times a day, and sometimes defecated once every 2 - 3 days. The fecal volume increased, and the abdominal distension and pain gradually disappeared. The overall mental improvement was obvious. The parents were satisfied with this result.

[0154]

Table 8

Claims

1. Bifidobacterium lactis which is Bifidobacterium lactis BL-11, wherein the Bifidobacterium lactis BL-11 has a deposit number of CGMCC No. 20847, characterized Bifidobacterium lactis.

2. A Bifidobacterium lactis preparation, wherein the preparation is a solid bacterium powder or a liquid beverage, and the Bifidobacterium lactis is the Bifidobacterium lactis according to Claim 1, Bifidobacterium lactis preparation.

3. Use of the Bifidobacterium lactis according to Claim 1 in the manufacture of a food composition or a pharmaceutical composition for increasing the abundance ratio of intestinal probiotics.

4. Use of the Bifidobacterium lactis according to Claim 1 in the manufacture of a food composition or a pharmaceutical composition for promoting bone growth in children and adolescents.

5. Use of the Bifidobacterium lactis according to Claim 1 in the manufacture of a food composition or a pharmaceutical composition for promoting height increase in children and adolescents.

6. Use of the Bifidobacterium lactis according to Claim 1 in the manufacture of a food composition or a pharmaceutical composition for promoting memory ability in children and adolescents.

7. The content of the Bifidobacterium lactis BL-11 is 1 to 25 parts by weight based on 100 parts by weight of the food composition or the pharmaceutical composition, and the Bifidobacterium lactis BL-11 is used in the form of viable cells of the Bifidobacterium lactis BL-11, inactivated cells of the Bifidobacterium lactis BL-11, or an extract of the Bifidobacterium lactis BL-11, The viable cell count of the Bifidobacterium lactis BL-11 was 1.0 × 10 ６ ~1.5 x 10 １２ CFU / g, Use according to any one of Claims 3 to 6.

8. The content of the Bifidobacterium lactis BL-11 is 1 to 15 parts by weight based on 100 parts by weight of the food composition or the pharmaceutical composition, and the viable count of the Bifidobacterium lactis BL-11 is 3.0×10¹⁰ to 5.0×10¹¹ CFU / g, Use according to Claim 7.

9. The food composition is one or more of fermented milk, cheese, milk-based beverage, solid beverage, and powdered milk, Use according to any one of Claims 3 to 6.

10. Based on the body weight of a human, the dosage of said Bifidobacterium lactis BL-11 is 2.0×10 ９ CFU to 1.5×10 １１ CFU / kg / day, Use according to any one of Claims 3 to 6. **Claim 11**: Based on the body weight of a human, the dosage of said Bifidobacterium lactis BL-11 is 3.0×10⁴ CFU to 8.0×10¹⁰ CFU / kg / day. The use according to claim 10. **Claim 12** The use of the Bifidobacterium lactis according to claim 1 in the manufacture of a food composition or a pharmaceutical composition for preventing mental disorders, wherein the mental disorders include anxiety, depression, autism, and attention deficit hyperactivity disorder. **Claim 13** The food composition or the pharmaceutical composition further comprises one or more combinations of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malic acid, and L-lactic acid. The use according to claim 12. **Claim 14** The food composition or the pharmaceutical composition further comprises a flavoring agent, a sweetening agent, a thickening agent, a stabilizer, a surfactant, a lubricant, an acid neutralizer, a dispersant, a buffer solution or a buffering agent, a debittering agent, a pH stabilizer, a preservative, a de-sugaring agent, and / or a coloring agent, and optionally further comprises lactitol, sorbitol, maltitol, aspartame, stevia, lakanka, sucralose, xylitol, vanilla, chocolate, fruit flavor, artificial essence, or a mixture or combination thereof. The use according to claim 12. **Claim 15** The food composition or the pharmaceutical composition further comprises vitamins, minerals and / or supplements or prebiotic nutrients, and at least one prebiotic, and optionally, the prebiotic comprises inulin, artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, mannooligosaccharide, arabinooligosaccharide, resistant dextrin, resistant starch, or a mixture or combination thereof. The use according to claim 12. **Claim 16** The food composition or the pharmaceutical composition further comprises ubiquinone (CoQ10), lycopene, β-carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof. The use according to claim 12. **Claim 17** The food composition or pharmaceutical composition further contains probiotics, and optionally, the probiotics contain microorganisms or bacteria or bacterial components cultured or extracted from feces, and optionally, the bacteria or bacterial components include Lactobacillus, Bifidobacterium, Escherichia coli, Prevotella, Faecalibacterium, Blautia, Bacteroides, Firmicutes and equivalents, or mixtures or combinations thereof. The use according to claim 12.

18. The content of Bifidobacterium lactis BL-11 is 0.5 to 20 parts by weight based on 100 parts by weight of the food composition or pharmaceutical composition. The Bifidobacterium lactis BL-11 is used in the form of viable cells of Bifidobacterium lactis BL-11, inactivated cells of Bifidobacterium lactis BL-11, or an extract of Bifidobacterium lactis BL-11. The viable count of the Bifidobacterium lactis BL-11 is 1.0×10 ６ to 1.5×10 １２ CFU / g, The use according to any one of claims 12 to 17.

19. The content of Bifidobacterium lactis BL-11 is 1 to 15 parts by weight based on 100 parts by weight of the food composition or pharmaceutical composition. The viable count of Bifidobacterium lactis BL-11 is 3.0×10^10 to 5.0×10^11 CFU / g. The use according to claim 18.

20. The food composition is one or more of fermented milk, cheese, milk-based beverages, solid beverages, and powdered milk. The use according to any one of claims 12 to 17.

21. Based on the body weight of a human, the dosage of the Bifidobacterium lactis BL-11 is 2.0×10 ９ CFU to 1.5×10 １１ CFU / kg / day, The use according to any one of claims 11 to 17.

22. Based on the body weight of a human, the dosage of Bifidobacterium lactis BL-11 is 3.0×10^4 CFU to 8.0×10^10 CFU / kg / day. The use according to claim 21.

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