WO2022171203A1 - 一种乳双歧杆菌及其应用 - Google Patents

一种乳双歧杆菌及其应用 Download PDF

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WO2022171203A1
WO2022171203A1 PCT/CN2022/085752 CN2022085752W WO2022171203A1 WO 2022171203 A1 WO2022171203 A1 WO 2022171203A1 CN 2022085752 W CN2022085752 W CN 2022085752W WO 2022171203 A1 WO2022171203 A1 WO 2022171203A1
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bifidobacterium lactis
cfu
pharmaceutical composition
food composition
bifidobacterium
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PCT/CN2022/085752
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English (en)
French (fr)
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段云峰
张烨
梁桦
刘智
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中科微智（北京）生物科技有限公司
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Priority to EP22752363.6A priority Critical patent/EP4293103A1/en
Priority to JP2023549064A priority patent/JP2024507187A/ja
Priority to KR1020237030867A priority patent/KR20230170647A/ko
Priority to CA3208064A priority patent/CA3208064A1/en
Publication of WO2022171203A1 publication Critical patent/WO2022171203A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/515—Animalis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the technical field of microorganisms, and in particular, to a Bifidobacterium lactis and its application.
  • gut bacteria can affect the growth and development of the host, and some flora can mediate the growth and development of the host by affecting growth hormone (GH)/insulin-like growth factor-1 (IGF-1). Both germ-free and antibiotic-treated mice exhibited reduced growth and were associated with reduced levels of ghrelin, growth hormone and IGF-1. In addition, in mice and humans, lack of microbes can lead to maturation and growth restriction, such as chronic malnutrition, anorexia nervosa, growth retardation, short stature, and even neurological and immune system abnormalities. Insulin-like growth factor 1 (IGF-1), a hormone with a known effect on bone growth, was significantly increased in serum and liver and adipose tissue with increased gut microbial colonization.
  • IGF-1 Insulin-like growth factor 1
  • IGF-1 Insulin-like growth factor 1
  • Exogenous IGF-1 can promote longitudinal growth of the femur, and after cartilage-specific insulin-like growth factor I receptor (Igf1r) deletion, IGF-1 was observed to affect growth plate maturation and secondary ossification center formation.
  • IGF-1 can promote osteoblasts (including affecting bone formation and bone resorption, respectively). Therefore, alterations in the gut microbiota can promote bone formation and resorption, resulting in net bone growth, and the microbiota can promote bone growth and remodeling by inducing IGF-1.
  • Direct supplementation with probiotics, prebiotics, or short-chain fatty acids (SCFAs) produced after microbial fermentation of cellulose has the potential to induce an increase in IGF-1, which in turn affects bone growth and health.
  • SCFAs short-chain fatty acids
  • gut microbes can regulate brain function and behavior through the gut-brain axis.
  • gut microbiota and brain via autonomic, enteric, immune system, olfactory system, enteroendocrine signaling, neurotransmitters, branched-chain amino acids, bile acids, short-chain fatty acids, spinal cord, hypothalamic-pituitary-adrenal axis, peptides Glycans and other pathways and mediators communicate bidirectionally.
  • Two-way communication between the gut microbiota and the central nervous system, the microbiota-gut-brain axis can affect neurodevelopment and function in animals, thereby affecting social behavior.
  • This bidirectional influence of the microbiota-gut-brain axis is 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 pathogenesis of a variety of neurological-related diseases, and at the same time, psychological and behavioral responses will in turn affect the composition or function of the gut microbiota.
  • microbiota-targeted interventions hold promise for gut microbiota-related disorders such as anxiety, depression, schizophrenia, ADHD, tics, Parkinson’s, Alzheimer’s, and autism and other social barriers. Take, for example, attention-deficit/hyperactivity disorder (ADHD), the most common neurobehavioral developmental disorder in childhood.
  • ADHD attention-deficit/hyperactivity disorder
  • the main manifestations are easily distracted attention disproportionate to the age of the children, narrowed attention span, excessive activity regardless of the occasion, emotional impulsiveness and often accompanied by cognitive impairment, conduct disorder and learning difficulties.
  • the incidence of ADHD is high, and it has obvious adverse effects on patients' academic, family and social life. However, treatments for ADHD are still lacking.
  • the present disclosure provides a Bifidobacterium lactis and an application thereof.
  • the Bifidobacterium lactis can improve intestinal metabolic disorders and normalize the composition of the intestinal microbiome, thereby effectively resisting obesity, promoting the growth and development of children and adolescents, and promoting their intellectual development;
  • the Bifidobacterium lactis can also effectively improve intestinal permeability and reduce the levels of LPS and D-lactic acid in the blood, thereby preventing and treating mental disorders, including anxiety, depression, and attention deficit hyperactivity disorder. , autism, autism, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette's syndrome, Asperger's syndrome.
  • a first aspect of the present disclosure provides a Bifidobacterium lactis, the Bifidobacterium lactis is Bifidobacterium lactis BL-11, and the preservation number of the Bifidobacterium lactis BL-11 is CGMCC No.20847 .
  • the second aspect of the present disclosure also provides a Bifidobacterium lactis preparation, wherein the preparation is solid bacteria powder or liquid drinking liquid; the Bifidobacterium lactis is the Bifidobacterium lactis described in the first aspect of the disclosure.
  • the third aspect of the present disclosure also provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for improving intestinal metabolic disorder and promoting the normalization of intestinal microbiome composition.
  • the fourth aspect of the present disclosure also provides the application of the above-mentioned Bifidobacterium lactis in the preparation of an anti-obesity food composition or a pharmaceutical composition for children and adolescents.
  • the fifth aspect of the present disclosure also provides the application of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting the growth and development of children and adolescents.
  • the sixth aspect of the present disclosure also provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting height increase in children and adolescents.
  • the seventh aspect of the present disclosure also provides the application of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting the intellectual development of children and adolescents.
  • the content of Bifidobacterium lactis BL-11 is 1-25 parts by weight, preferably 1-15 parts by weight;
  • Bifidobacterium lactis BL-11 is used in the form of a living cell of the Bifidobacterium lactis BL-11, an inactivated cell of the Bifidobacterium lactis BL-11, or an extract of the 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, preferably 3.0 ⁇ 10 10 to 5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, beverages containing milk, solid beverages, and milk powder.
  • the dosage of 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.
  • the eighth aspect of the present disclosure provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for preventing and treating mental disorders.
  • the mental disorder includes anxiety, depression, attention deficit hyperactivity disorder, autism, autism, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette's syndrome, Asperger's syndrome.
  • the food composition or pharmaceutical composition also contains one or more of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, L-sodium ascorbate, L-malic acid, L-lactic acid, etc. kind of combination.
  • the food composition or pharmaceutical composition further contains flavoring agents, sweeteners, thickening agents, stabilizers, surfactants, lubricants, acid neutralizers, dispersing agents, buffers or buffers, Debitterizers, pH stabilizers, preservatives, sugar removers and/or colorants such as lactitol, sorbitol, maltitol, aspartame, stevia, monk fruit, sucralose, xylitol, vanilla , chocolate, fruit flavors, artificial flavors, or mixtures or combinations thereof.
  • flavoring agents such as lactitol, sorbitol, maltitol, aspartame, stevia, monk fruit, sucralose, xylitol, vanilla , chocolate, fruit flavors, artificial flavors, or mixtures or combinations thereof.
  • the food composition or pharmaceutical composition further contains vitamins, minerals and/or dietary supplements or prebiotic nutrients, at least one prebiotic, wherein optionally, the prebiotic comprises inulin, Artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharides, galactooligosaccharides, isomalt oligosaccharides, xylo-oligosaccharides, stachyose, mannose oligosaccharides, arabino-oligosaccharides, resistance Dextrin, resistant starch, or mixtures or combinations thereof.
  • the prebiotic comprises inulin, Artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharides, galactooligosaccharides, isomalt oligosaccharides, xylo-oligosaccharides, stachyose, mannose oligosaccharides, arabino-oligosaccharides, resistance Dextri
  • the food composition or pharmaceutical composition further contains ubiquinone (CoQ10), lycopene, beta-carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof.
  • ubiquinone CoQ10
  • lycopene beta-carotene
  • tryptophan vitamin B6, vitamin B12, or a mixture or combination thereof.
  • the food composition or pharmaceutical composition further contains probiotics, wherein optionally, the probiotics comprise cultured or fecal-extracted microorganisms or bacteria or bacterial components, and optionally, the Bacteria or bacterial components including or derived from Lactobacillus, Bifidobacterium, E coli, Prevotella, Faecalibacterium, Brautia (Blautia), Bacteroidetes (Bacteroidetes), Firmicutes (Firmicutes) and equivalents, or mixtures or combinations thereof.
  • the probiotics comprise cultured or fecal-extracted microorganisms or bacteria or bacterial components
  • Bacteria or bacterial components including or derived from Lactobacillus, Bifidobacterium, E coli, Prevotella, Faecalibacterium, Brautia (Blautia), Bacteroidetes (Bacteroidetes), Firmicutes (Firmicutes) and equivalents, or mixtures or combinations thereof.
  • the content of the Bifidobacterium lactis BL-11 is 0.5-20 parts by weight, preferably 1-15 parts by weight; the Bifidobacterium lactis BL-11 BL-11 is used in the form of the living cell of the Bifidobacterium lactis BL-11, the inactivated cell of the Bifidobacterium lactis BL-11 or the extract of the Bifidobacterium lactis BL-11;
  • the viable count of Bifidobacterium lactis BL-11 is 1.0 ⁇ 10 6 to 1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 to 5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, beverages containing milk, solid beverages, and milk powder.
  • 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.
  • the food composition or pharmaceutical composition can be in the form of powders, lozenges, liquids, chewing gums, gummies, tableted candies, yogurt, milk, cheese, ice cream, frozen foods, Health food, medicine or feed.
  • the Bifidobacterium lactis BL-11 of the present disclosure can improve intestinal permeability and reduce LPS and D-lactic acid levels in blood.
  • the Bifidobacterium lactis BL-11 of the present disclosure can be used to promote voluntary active activities, improve the memory function of one-time avoidance response, and improve anhedonia caused by stressful stimuli.
  • the probiotics provided by the present disclosure can effectively improve metabolic disorders and normalize the composition of intestinal microbiome, promote the growth and development of children and adolescents, improve intestinal permeability, reduce LPS in blood, D- lactate levels, thereby acting to prevent and treat mental disorders including anxiety, depression, attention deficit hyperactivity disorder, autism, autism, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette's , Asperger's syndrome.
  • Bifidobacterium lactis BL-11 classified as Bifidobacterium lactis (Bifidobacterium lactis)
  • Bifidobacterium lactis Bifidobacterium lactis
  • Fig. 1 is a microscopic view of Bifidobacterium lactis BL-11.
  • Figures 3 and 4 are the lengths of the femurs of mice treated with antibiotics and probiotics.
  • Figure 5 is the length of mouse leg bones after antibiotic and probiotic treatment.
  • Figure 6 shows the changes in the diversity of intestinal flora in mice treated with two probiotics.
  • Figure 7 is the ratio of bifidobacteria and lactobacilli in the intestinal tract of mice treated with two probiotics.
  • Figure 8 is a heat map analysis of the gut microbiota composition of mice treated with two probiotics.
  • Figure 9 is the change in human height from 0 to 12 weeks before and after taking probiotics or placebo.
  • Figure 10 is the change in body weight from 0 to 12 weeks before and after taking probiotics or placebo.
  • Figure 11 shows symptom improvement before and after taking probiotics or placebo.
  • Figure 12 shows the composition of gut microbiota at the genus level before and after taking probiotics or placebo.
  • Figure 13 is a comparison of the metabolic pathway levels of gut microbiota before and after taking probiotics or placebo.
  • Figure 14 shows the composition of gut microbiota at the phylum level before and after taking probiotics or placebo.
  • Figure 15 is a comparison of the composition of representative bacterial species in the intestinal flora before and after taking probiotics or placebo.
  • Figure 16 is a comparison of the gut microbiota at the functional gene level before and after taking probiotics or placebo.
  • Figure 17 is a comparison of gut microbiota levels in KO before and after taking probiotics or placebo.
  • Figure 18 is a comparison of the metabolic pathway levels of gut microbiota before and after taking probiotics or placebo.
  • Figure 19 is a graph of the results of the intestinal permeability assay.
  • Fig. 20 is a diagram showing the composition of the intestinal flora of each group at the genus level.
  • Figure 21 is the difference of Bifidobacterium between the two groups.
  • Figure 22 is the alpha diversity index results.
  • Figure 23 is the PCoA analysis result of the beta diversity index analysis.
  • a first aspect of the present disclosure provides a Bifidobacterium lactis, the Bifidobacterium lactis is Bifidobacterium lactis BL-11, the preservation number of the Bifidobacterium lactis BL-11 is CGMCC No.20847.
  • the second aspect of the present disclosure also provides a Bifidobacterium lactis preparation, wherein the preparation is solid bacteria powder or liquid drinking liquid; the Bifidobacterium lactis is the Bifidobacterium lactis described in the first aspect of the disclosure.
  • the third aspect of the present disclosure also provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for improving intestinal metabolic disorder and promoting the normalization of intestinal microbiome composition.
  • the fourth aspect of the present disclosure also provides the use of the above-mentioned Bifidobacterium lactis in preparing an anti-obesity food composition or a pharmaceutical composition for children and adolescents.
  • the fifth aspect of the present disclosure also provides the application of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting the growth and development of children and adolescents.
  • the sixth aspect of the present disclosure also provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting height increase in children and adolescents.
  • the seventh aspect of the present disclosure also provides the application of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for promoting the intellectual development of children and adolescents.
  • the content of Bifidobacterium lactis BL-11 is 1-25 parts by weight, preferably 1-15 parts by weight;
  • Bifidobacterium lactis BL-11 is used in the form of a living cell of the Bifidobacterium lactis BL-11, an inactivated cell of the Bifidobacterium lactis BL-11, or an extract of the 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, preferably 3.0 ⁇ 10 10 to 5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, beverages containing milk, solid beverages, and milk powder.
  • the dosage of 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.
  • the eighth aspect of the present disclosure provides the use of the above-mentioned Bifidobacterium lactis in preparing a food composition or a pharmaceutical composition for preventing and treating mental disorders.
  • the mental disorder includes anxiety, depression, attention deficit hyperactivity disorder, autism, autism, schizophrenia, hepatic encephalopathy, anorexia nervosa, Tourette's syndrome, Asperger's syndrome.
  • the food composition or pharmaceutical composition also contains one or more of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, L-sodium ascorbate, L-malic acid, L-lactic acid, etc. kind of combination.
  • the food composition or pharmaceutical composition further contains flavoring agents, sweeteners, thickening agents, stabilizers, surfactants, lubricants, acid neutralizers, dispersing agents, buffers or buffers, Debitterizers, pH stabilizers, preservatives, sugar removers and/or colorants such as lactitol, sorbitol, maltitol, aspartame, stevia, monk fruit, sucralose, xylitol, vanilla , chocolate, fruit flavors, artificial flavors, or mixtures or combinations thereof.
  • flavoring agents such as lactitol, sorbitol, maltitol, aspartame, stevia, monk fruit, sucralose, xylitol, vanilla , chocolate, fruit flavors, artificial flavors, or mixtures or combinations thereof.
  • the food composition or pharmaceutical composition further contains vitamins, minerals and/or dietary supplements or prebiotic nutrients, at least one prebiotic, wherein optionally, the prebiotic comprises inulin, Artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharides, galactooligosaccharides, isomalt oligosaccharides, xylo-oligosaccharides, stachyose, mannose oligosaccharides, arabino-oligosaccharides, resistance Dextrin, resistant starch, or mixtures or combinations thereof.
  • the prebiotic comprises inulin, Artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharides, galactooligosaccharides, isomalt oligosaccharides, xylo-oligosaccharides, stachyose, mannose oligosaccharides, arabino-oligosaccharides, resistance Dextri
  • the food composition or pharmaceutical composition further contains ubiquinone (CoQ10), lycopene, beta-carotene, tryptophan, vitamin B6, vitamin B12, or a mixture or combination thereof.
  • ubiquinone CoQ10
  • lycopene beta-carotene
  • tryptophan vitamin B6, vitamin B12, or a mixture or combination thereof.
  • the food composition or pharmaceutical composition further contains probiotics, wherein optionally, the probiotics comprise cultured or fecal-extracted microorganisms or bacteria or bacterial components, and optionally, the Bacteria or bacterial components including or derived from Lactobacillus, Bifidobacterium, E coli, Prevotella, Faecalibacterium, Brautia (Blautia), Bacteroidetes (Bacteroidetes), Firmicutes (Firmicutes) and equivalents, or mixtures or combinations thereof.
  • the probiotics comprise cultured or fecal-extracted microorganisms or bacteria or bacterial components
  • Bacteria or bacterial components including or derived from Lactobacillus, Bifidobacterium, E coli, Prevotella, Faecalibacterium, Brautia (Blautia), Bacteroidetes (Bacteroidetes), Firmicutes (Firmicutes) and equivalents, or mixtures or combinations thereof.
  • the content of the Bifidobacterium lactis BL-11 is 0.5-20 parts by weight, preferably 1-15 parts by weight; the Bifidobacterium lactis BL-11 BL-11 is used in the form of the living cell of the Bifidobacterium lactis BL-11, the inactivated cell of the Bifidobacterium lactis BL-11 or the extract of the Bifidobacterium lactis BL-11;
  • the viable count of Bifidobacterium lactis BL-11 is 1.0 ⁇ 10 6 to 1.5 ⁇ 10 12 CFU/g, preferably 3.0 ⁇ 10 10 to 5.0 ⁇ 10 11 CFU/g.
  • the food composition is one or more of fermented milk, cheese, beverages containing milk, solid beverages, and milk powder.
  • 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.
  • the food composition or pharmaceutical composition can be in the form of powders, lozenges, liquids, chewing gums, gummies, tableted candies, yogurt, milk, cheese, ice cream, frozen foods, Health food, medicine or feed.
  • the Bifidobacterium lactis BL-11 of the present disclosure can improve intestinal permeability and reduce LPS and D-lactic acid levels in blood.
  • the Bifidobacterium lactis BL-11 of the present disclosure can be used to promote voluntary active activities, improve the memory function of one-time avoidance response, and improve anhedonia caused by stressful stimuli.
  • This example is used to illustrate Bifidobacterium lactis BL-11 and its performance characteristics.
  • Bifidobacterium lactis BL-11 was observed under a microscope, and the results are shown in Figure 1. The physical and chemical test results are shown in Tables 1 and 2.
  • + means able to metabolize; - means unable to metabolize.
  • Bifidobacterium is a genus of bacteria that are generally not acid-fast.
  • the artificial gastric juice and intestinal juice tolerance of Bifidobacterium lactis BL-11 of the present invention were tested, and at the same time, Bifidobacterium lactis Bb, which has excellent acid resistance and can survive through the gastrointestinal tract, is used in the laboratory. -XX for comparison.
  • the test results of the survival rate of the BL-11 strain in artificial gastric acid are shown in Table 3.
  • the survival rate of viable bacteria of Bb-XX was 44.7% when Bb-XX was treated in artificial gastric juice for 1h, and the survival rate of viable bacteria was 29.5% after treatment for 3h;
  • the survival rate of Bifidobacterium lactis BL-11 of the present invention was 86.2% when treated for 1 hour, and the survival rate of viable bacteria was 39.5% when treated for 3 hours. It is shown that the Bifidobacterium lactis BL-11 of the present invention has relatively good gastric acid resistance, and most of it can smoothly pass through the stomach and reach the intestinal tract to exert a probiotic effect.
  • the Bifidobacterium lactis BL-11 of the present invention is inoculated into the MRS liquid medium, anaerobic culture is carried out at 37° C. for 48 hours, and the number of viable bacteria of Bifidobacterium lactis BL-11 in the culture solution is counted to be 3.7 ⁇ 10 9 CFU/mL,
  • the culture stock solution was orally administered to the mice at a ratio of 20.0 mL/kg body weight for 3 consecutive days, and then observed for 7 days. Healthy male BALB/C mice, 6-8 weeks old, 16-18g, maintained at room temperature (25 ⁇ 2°C), relative humidity (55 ⁇ 2)%, 12h/12h light, free food and water. The results showed that compared with the control group, no toxic reaction or death was observed in the culture stock of Bifidobacterium lactis BL-11, and there was no statistical difference in the weight gain of the mice (p>0.05).
  • Bifidobacterium lactis BL-11 The antibiotic susceptibility of Bifidobacterium lactis BL-11 was evaluated by the method of SN/T 1944-2007 "Determination of Bacterial Resistance in Animals and Their Products". The evaluation results showed that Bifidobacterium lactis BL-11 was sensitive to Ampicillin, Penicillin G, Erythromycin, Chloramphenicol, Clindamycin, Vancomycin and Tetracycline. It meets the requirements of the European Food Safety Authority (European Food Safety Authority) for the evaluation of drug resistance of edible bacteria. Bifidobacterium lactis BL-11 does not contain exogenous antibiotic resistance genes and is safe to eat.
  • This example is used to illustrate the functional characteristics of Bifidobacterium lactis BL-11 in promoting growth and development.
  • Microbiota which promotes bone formation and resorption, resulting in net bone growth.
  • the microbiota induces the hormone-like insulin growth factor 1 (IGF-1), which promotes bone growth and remodeling.
  • IGF-1 hormone-like insulin growth factor 1
  • SCFAs Short-chain fatty acids
  • mice 2-month-old female BALB/c mice were treated with antibiotics and probiotics and housed under SPF conditions. Thirty mice were pooled and randomly assigned to treatment groups to minimize cage effects. Antibiotics were a mixture of 0.2 mg/mL gentamicin, 0.15 mg/mL ciprofloxacin, 2 mg/mL streptomycin, and 1 mg/mL bacitracin.
  • mice were treated with an antibiotic mixture added to their drinking water for 2 weeks to deplete the microorganisms, and then the mice were divided into three groups of 10 mice, one group served as a control group, fed normally, and the other two groups were given probiotics
  • Bacteria group: BL-11 or Bb-XX were added to the water, respectively, and fed continuously for 4 weeks.
  • 3% (g/100 mL) sucrose was added to all water to ensure palatability as specified by the animal facility.
  • the aqueous solution was freshly prepared and changed twice a week.
  • serum was prepared from blood samples collected by cardiac puncture using serum separator tubes after mice were sacrificed and using the murine IGF-1 standard ABTS ELISA development kit (PeproTech).
  • the lengths of the femurs of mice fed with normal feeding (CK), BL-11 and Bb-XX are shown in Figure 4, and the lengths of leg bones are shown in Figure 3.
  • the length of leg bones of mice fed BL-11 was significantly different from that of the control group (P ⁇ 0.05), while the length of leg bones of mice fed Bb-XX was not significantly different from that of the control group (P ⁇ 0.05).
  • Data are shown as mean ⁇ SD, and t-test was used to test whether the difference was significant. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • mice Both germ-free and antibiotic-treated mice exhibited reduced growth, reduced levels of ghrelin, growth hormone, and IGF, and gut microbiome disturbances, leading to growth restriction in mice. After the mice were treated with antibiotics, they were given probiotics to detect the serum IGF-1 level of the mice. The results are shown in Figure 5. After the antibiotic treatment, the mice that took probiotics increased the level of IGF-1 in the serum. , and taking BL-11 can significantly increase the level of IGF-1 in serum (P ⁇ 0.01), and the improvement effect is higher than that of Bb-XX.
  • This example is used to illustrate the preparation of BL-11 bacterial powder and its use in food production.
  • the Bifidobacterium lactis BL-11 provided by the present invention is anaerobic cultured in MRS broth liquid medium.
  • the fermentation broth after primary, secondary and expanded culture was cultured at 37°C for 24 hours and then centrifuged at 4°C and 3000 rpm for 10 min to collect the cells, washed with phosphate buffered saline (PBS), and then freeze-dried by adding skim milk. , Store below -20°C.
  • PBS phosphate buffered saline
  • the BL-11 bacteria powder prepared in this example can be used for food, medicine, health care product or animal feed.
  • the food can be common food or health food such as fermented milk, cheese, milk-containing beverage, solid beverage, and milk powder.
  • the recommended dose of Bifidobacterium lactis BL-11 for human use may be 1.0 ⁇ 10 3 CFU to 1.0 ⁇ 10 10 CFU/kg body weight/day, more preferably 1.0 ⁇ 10 4 CFU ⁇ 1.0 x 109 CFU/kg body weight/day.
  • This example is used to illustrate the functional characterization of growth and development in human clinical studies.
  • Subjects and Recruitment 65 patients with Prader-Willi syndrome aged 11 months to 16 years were recruited and randomly assigned to probiotics or placebo for a 12-week randomized, double-blind, placebo-controlled trial.
  • Inclusion criteria genetically proven PWS; no probiotics of any kind for four weeks; stable medication for at least four weeks; no planned pharmacological and psychological interventions during the trial; willingness to provide timely stool samples; willingness to participate in the study and interview process, and no other genetic disease, pregnancy or breastfeeding.
  • Informed consent was obtained from the subjects' parents or legal guardians as required by the IRB for the research protocol and was conducted in accordance with the Declaration of Helsinki.
  • RESULTS Randomized and blinded testing: A randomized double-blind placebo-controlled design was used, with random assignment concealment of subjects by a statistician not part of the team, allowing each unidentified subject to generate a random sample number.
  • Beijing Huayuan Institute of Biotechnology provided identical-looking coded probiotics and placebos to ensure allocation concealment and blind spots maintained.
  • the patients were randomly assigned to receive daily Bifidobacterium lactis BL- 11 probiotic (6 x 1010 CFU) or placebo bar packs.
  • the weight, height, ASQ-3, ABC, SRS-2 and CGI-1 of the two groups were compared at 6 and 12 weeks of treatment.
  • CGI consists of two single measures assessing the following: (a) psychopathology severity (CGI-S) from 1 to 7 and (b) change in symptoms before and after treatment on a seven-point scale from start to finish (CGI-S) -I).
  • the probiotic group is Bifidobacterium lactis BL-11 in the form of sticks and packs.
  • Each bag of Bifidobacterium lactis supplement contains 3 x 10 10 Colony Forming Units (CFU); the placebo is maltodextrin in the same package, similar in color, taste and flavor to Bifidobacterium lactis stick packs.
  • Subjects received a sachet of Bifidobacterium lactis or a placebo orally with water twice daily for 12 weeks. Maltodextrin as a supplement had minimal side effects and as a placebo maltodextrin also had minimal adverse effects.
  • Weight and height were measured by parents using standard scales and collected by the researchers, using the age-increasing reference provided by WHO as a reference to convert weight, height, and BMI to z-scores.
  • ASQ-3 Age and Stage Questionnaire, Third Edition
  • the ASQ-3 is one of the most widely used developmental screening tools for children and adolescents and has five dimensions: communication, gross motivation, fine motivation, problem solving, and personal socialization. The subject's total score was calculated according to five dimensions to evaluate the trial effect.
  • ABS Abnormal Behavior Inventory
  • SRS Social Responsiveness Scale
  • Fecal samples were collected in DNA/RNA shielded fecal collection tubes (Zymo, Cat #R1101) containing 1 mL of preservation solution and transported to the laboratory via ice packs and then frozen at -80°C.
  • DNA was extracted using the TIANmap Fecal DNA Kit (TIANGEN, cat. no. DP328) according to the manufacturer's instructions, and DNA samples were carefully quantified using a Nanodrop spectrophotometer. The ratio of A260/A280 was measured to confirm the yield of high-purity DNA. DNA samples were frozen at -20°C until use.
  • the PCR product was purified with 1xKAPA AMPure (KAPA, cat. no. KK8002) by (95°C for 2 min, then at 95°C for 30s, 55°C for 30s, and 72°C for 30s for 25 cycles) followed by a final extension at 72°C for 5min. , the product was subjected to a second PCR reaction step (95°C for 2 min, followed by eight cycles of 95°C for 30s, 55°C for 30s and 72°C for 30s, and a final extension at 72°C for 5min).
  • the PCR product was purified by 1 ⁇ KAPA AMPure , and quantitatively analyzed by real-time PCR using the Bioanalyzer DNA kit.
  • CGI Clinical Global Impression
  • Gastrointestinal symptoms were assessed based on the total number of gastrointestinal symptoms present at baseline, including constipation, diarrhea, abdominal pain, flatulence, blood in the stool, nausea, dysphagia, loss of appetite, dyspepsia, and acid regurgitation.
  • FDRs false discovery rates
  • Quality control filtered sequencing reads using QIIME2 (v2019.10).
  • the default parameters were denoised using Deblur and the abundance table of the samples was obtained by amplicon sequence variants (ASVs).
  • Alpha diversity was calculated using QIIME2.
  • Bray-Curtis distance was used to characterize microbiome beta diversity.
  • ASVs were assigned using a sklearn based classifier trained on sequences with 99% similarity to Greengenes v13.8. Significant differences in relative abundances of microbial phyla, genera and alpha diversity between the placebo and probiotic groups were confirmed by the Kruskal–Wallis test. False discovery rate (FDR) based on Benjamini-Hochberg (BH) adjustment was used for multiple comparisons.
  • FDR False discovery rate
  • BH Benjamini-Hochberg
  • PICSRUSt2 was used to infer the functional content of microorganisms based on the abundance table of ASVs, and then generated the Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs (KO), enzyme taxonomy and pathway abundance tables. The ratios between the probiotic and placebo groups were analyzed for differences using permutation-based nonparametric tests, and the most significant differences were plotted with Calour. All raw data from 16s rRNA Illumina amplicon sequencing has been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA, PRJNA643297).
  • NCBI National Center for Biotechnology Information
  • Table 4 summarizes the demographic characteristics and comorbid gastrointestinal (GI) symptoms of the 65 participants, and no between-group differences were observed (P>0.05). 47.5% of the subjects showed one or more gastrointestinal symptoms in the study population. Although the proportion of gastrointestinal symptoms in the probiotic group was 37.4% lower than that in the placebo group, the difference did not reach significant (P>0.05). ).
  • Bifidobacterium lactis BL-11 subjects experienced a significant increase in height and a slight change in weight.
  • the present disclosure provides new evidence for Bifidobacterium lactis BL-11 as an early intervention for PWS patients.
  • the present disclosure found that there was a significant difference in gut microbiome beta diversity between the treated probiotic group and the placebo group, with baseline beta diversity directly correlated with long-term weight loss when adhering to a controlled diet. Therefore, supplementation with Bifidobacterium lactis BL-11 may improve gut microbiota composition, prevent obesity or promote diet-induced weight loss. Supplementation with Bifidobacterium lactis BL-11 may also improve the growth and development of children and adolescents.
  • This example is used to illustrate the preparation method of BL-11 freeze-dried bacterial powder.
  • the BL-11 preserved bacterial solution was recovered in a 37°C water bath until the liquid in the cryopreservation tube was completely thawed; the MRS medium was zoned and streaked, and anaerobic culture at 37°C for 12-24 hours; single colonies were picked and inoculated Put it into an anaerobic culture tube containing a liquid medium, seal it, and cultivate it anaerobically at 37 °C for 6-12 hours. Monitor the OD600 value of the bacterial liquid to stop growing, and stop the fermentation; carry out bacterial fermentation production, and culture at 40 °C with constant temperature stirring for 6 ⁇ 12 hours.
  • Add freeze-drying protective agent stir and mix evenly, put it into freeze-drying machine for freeze-drying of bacterial powder; collect freeze-dried powder, pulverize and pack according to quality requirements.
  • This example is used to illustrate that BL-11 freeze-dried bacterial powder is used in the production of food.
  • the freeze-dried bacteria powder prepared by the Bifidobacterium lactis BL-11 provided by the invention can be used for common food or health food such as yogurt, fermented milk, cheese and the like.
  • the dosage of Bifidobacterium lactis BL-11 for making yogurt is 1.0 ⁇ 10 6 CFU ⁇ 1.0 ⁇ 10 8 CFU/kg, more preferably 1.2 ⁇ 10 7 CFU ⁇ 1.5 ⁇ 10 8 CFU/kg.
  • the production method is direct investment or post-addition. It is used as a fermentation strain during direct injection. After the raw milk is sterilized, it will be added in proportion when the temperature is reduced to an appropriate temperature, and then fermented at 40-43 °C for 10-48 hours. After mixing and mixing, it is dispensed into cups or bags as finished products.
  • adding in the post-adding method after the fermentation of the yogurt is completed, add it according to a certain proportion and stir it evenly.
  • This example is used to illustrate that BL-11 freeze-dried bacterial powder is used for the production of dietary nutritional supplements and probiotics.
  • the freeze-dried bacteria powder prepared by the Bifidobacterium lactis BL-11 provided by the invention can be used for the production of probiotic bacteria.
  • Probiotic lyophilized powder contains 0.5-30 parts of BL-11 lyophilized powder, 5-20 parts of Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus reuteri, Lactobacillus plantarum, Bifidobacterium adolescentis, Bifidobacterium breve Bacillus, Bifidobacterium longum and other strains, 20-70 parts of galacto-oligosaccharides, fructooligosaccharides, inulin and other prebiotics, 5-10 parts of GABA, tryptophan, lycopene, ⁇ -carotene, Nutrients such as vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, beta-glucan, fucose, carrageenan, guar
  • the total dosage of the viable count of probiotics is as follows: the dosage of BL-11 is 2.0 ⁇ 10 6 CFU to 2.0 ⁇ 10 10 CFU/g, more preferably 3 ⁇ 10 7 CFU to 3 ⁇ 10 10 CFU/g.
  • the dosage of single bacteria for other strains is 1 ⁇ 10 6 CFU to 3 ⁇ 10 9 CFU/g.
  • the preparation process includes: weighing raw materials in proportion, mixing, sub-packaging and quality inspection.
  • This example is used to illustrate the effect of BL-11 freeze-dried bacterial powder on improving intestinal permeability and behavior.
  • Gut microbes and the gut-brain axis play a two-way communication in the regulation of stress responses.
  • Microbes communicate with the gut-brain axis through different mechanisms. Directly interacts with mucosal cells, affects brain development and behavior through immune cells and through contact with nerve endings. Stress in the brain can also have an effect on the gut microbiome through GBA, which is responsible for gastrointestinal dysfunction and dysregulation.
  • the stress response also affects the synthesis of microbial by-products and precursors that enter the brain through the blood and hindbrain regions, release cytokines through mucosal immune cells, and release gut hormones such as serotonin through enteroendocrine cells ( 5-HT), or through afferent neural pathways, including the enteric nervous system.
  • 5-HT enteroendocrine cells
  • probiotic preparations for intervention were freeze-dried powder containing BL-11, ⁇ -carotene, vitamin B6, vitamin B12, coenzyme Q10 and maltodextrin, and the control group was maltodextrin.
  • the dose is 10 billion CFU/only/day.
  • mice Twenty 6-week-old C57BL/6J mice were housed in groups of 5 mice per cage, with free food and water. Food and water intake were recorded twice a week. The mice were divided into two groups, the control group and the probiotic group, with 10 mice in each group. After one week of acclimatization, the experimental mice were randomly divided into the model group and the probiotic intervention group. The growth environment conditions of the experimental mice were: ambient temperature (23 ⁇ 2)° C., relative humidity (50 ⁇ 10)%, and light mode (12h dark/12h light).
  • a chronic stress model was used, and the specific stimulation methods included: (1) fasting for 24 hours; (2) continuous light for 24 hours; (3) iron clips on the tail, 1 cm from the tip of the tail, for 3 min/hour times; (4) restraint stress, circular sleeve restraint action 2h/day. 1-2 different stress stimuli were used every day, and the time of each stimulation was not fixed, and the modeling time lasted for 4 weeks.
  • Platform jumping device experimental box (DTT-2 mouse jumping platform, Institute of Materia Medica, Chinese Academy of Medical Sciences).
  • the platform box is about 120cm long, 12cm wide and 30cm high, made of plexiglass. It is divided into 6 chambers, each chamber is 12cm long, 12cm wide and 30cm high.
  • the experimental box can allow 6 mice to conduct experiments at the same time.
  • the bottom of the experimental box is covered with a copper grid, which is connected to the power supply through wires, and the current and voltage are set to 36V.
  • An insulating table (a pentagon-shaped wooden block with a long diameter of 5.7 cm, a short diameter of 4.5 cm, and a height of 4.8 cm) was placed on the copper grid in the experimental box near the corner of the box as a safe area for animals to avoid electric shock.
  • the test device is connected with a computer automatic recording system.
  • the mice were put into the experiment box of the platform jumping apparatus and adapted to the environment for 5 minutes. After that, the mice were gently placed on the platform and the copper grid was energized. When the mice jumped off the platform and touched the copper grid, they would receive an electric shock.
  • the normal avoidance response is to jump on the platform to return to the safe area, avoid the electric shock, and study for 5 minutes.
  • the memory ability test was carried out, and the mice were placed on the platform, and the time from staying on the platform to the first jump to receive an electric shock, i.e. the latency period and the number of errors within 5 minutes (the mice’s limbs simultaneously contacted the copper grid for electric shock) were recorded. The number of times) was used as an evaluation index of memory function.
  • mice in the probiotic group could improve the impairment of one-time avoidance response memory function (see Table 5).
  • the opening experiment analysis system is to observe and study the neuropsychiatric changes of experimental animals and various behaviors after entering the open environment. For example, animals are afraid of the new open environment, but they mainly move in the peripheral area, and less in the central area, but the animal's exploratory characteristics In turn, the motivation to act in the central area can also be observed, and the resulting anxiety can also be observed. Used to assess the voluntary active activity and anxiety levels of animals.
  • mice 60 minutes before the experiment, the mice were moved to the test room of the opening experiment to adapt to the environment in advance. During the experiment, the mice were taken out of the cage and placed in the behavioral experimental station in the open-field experimental device (box length ⁇ width ⁇ height: 100 cm ⁇ 100 cm ⁇ 40 cm, the inner and bottom surfaces were blue, and a camera was placed just above the central area. After the start of the experiment, put the mouse in a fixed position in the central area, place the head fixed to one side each time, and quickly pull up the hood. After recording the mouse number, date, and status in the operating software, open the recording system, and select Jiugongge Mode, the central area ratio is 0.5, through the camera above the opening equipment and the monitor connected to it.
  • Each mouse is measured for 5 minutes, and the activity of the mice is recorded.
  • the measurement indicators include exercise time, total distance, and percentage of residence time in the central area (central area).
  • Stay time LPMM s), percentage of horizontal movement in central area (horizontal movement distance in central area L horizontal movement distance), percentage of horizontal movement in four-side area (horizontal movement distance in four-side area L horizontal movement distance) and percentage of horizontal movement in four corner areas (horizontal movement in four corner areas) Exercise L horizontal movement distance). Record the number of times of standing and grooming, then clean the environment in the box with 75% alcohol, wait for the alcohol to evaporate and tasteless, and then proceed to the next one. The results are shown in Table 6.
  • D-lactic acid is a metabolite of bacterial fermentation, which can be produced by a variety of bacteria in the gut. Even after being ingested from food, it is rarely absorbed into the blood under normal circumstances, and mammals do not have an enzyme system to rapidly degrade it. Therefore, when the permeability of the intestinal mucosa increases, a large amount of D-lactic acid produced by bacteria in the intestinal tract enters the blood through the damaged mucosa, which increases the level of D-lactic acid in the blood.
  • D-type and L-type lactic acid There are D-type and L-type lactic acid.
  • the normal human body only has L-lactic acid, and microorganisms such as bacteria can produce D-lactic acid.
  • Monitoring the level of D-lactic acid in blood can timely reflect the degree of intestinal mucosal damage and changes in permeability. It can be used for the auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory response, repeated fever, vomiting, etc.
  • LPS Lipopolysaccharide
  • lipid A lipid A
  • core oligosaccharide lipid A
  • hypervariable polysaccharide domain O antigen.
  • the structure of LPS determines its agonist/antagonist action on TLR4.
  • LPS binds to the TLR4/MD-2 receptor complex and activates different signaling pathways through Myd88-dependent or TRIF-dependent pathways; the expression of TLR in different parts of intestinal epithelial cells is different, which can prevent the inflammatory response caused by LPS , and against pathogenic bacteria.
  • Bacterial translocation is the movement of live intestinal bacteria from the gut across the epithelial mucosa into the body. Bacteria can enter the lymphatic system via the mesenteric lymph nodes and circulate throughout the body. Bacteria can also enter the blood circulation, causing bacteremia, and can also localize in tissues. Bacterial displacement can lead to bacterial overgrowth in the small intestine, intestinal damage and shock. Any stress response, both psychological and physiological, that leads to gut permeability could potentially lead to bacterial displacement.
  • LPS is associated with the pathogenesis of various diseases, such as intestinal diseases such as IBD and enterocolitis, and even Parkinson's and Alzheimer's diseases. LPS can not only enter the blood, but also enter and remain in the brain for a lifetime, possibly causing Alzheimer's disease.
  • the level of LPS in the blood can reflect the permeability of the intestinal tract.
  • the normal intestinal barrier does not allow the entry of LPS.
  • a higher level of LPS in the blood indicates that the intestinal bacteria or LPS has migrated to the blood, indicating that the intestinal tract is permeable.
  • the amount of LPS in the blood can also indicate the inflammatory response and stress state. Too much LPS can cause abnormality of the human immune system, chronic or acute inflammatory response, and acute inflammation such as fever and pain. It can be used for the auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory response, repeated fever, vomiting, mental illness, stress response, etc.
  • the intestinal barrier function analysis system JY-DLT, Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd. was used to detect the content of D-lactic acid and LPS in serum according to the operating instructions.
  • This example is used to illustrate the effect of BL-11 freeze-dried bacterial powder on intestinal microbial composition.
  • Bacterial 16S rRNA gene amplification using the diluted genomic DNA as the template, according to the selection of the sequencing region, using specific primers with Barcode, using bacterial universal primers 341F (CCTAYGGGRBGCASCAG SEQ ID NO.3) and 806R (GGACTACNNGGGTATCTAAT SEQ ID NO. 4) Amplify the V3-V4 region of the bacterial 16S rRNA gene. 100 ng of extracted DNA was subjected to PCR for strand renaturation at 56°C, starting with 4 minutes of denaturation at 94°C, followed by 30 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute.
  • Amplicon gene sequencing The library construction kit of Illumina's TruSeq DNA PCR-Free Library Preparation Kit was used to construct the library. The constructed library was quantified and tested by Qubit. After passing the test, the Illumina HiSeq2500PE250 sequencing platform was used for bacterial population sequencing. .
  • the raw data of bacterial population sequencing was imported into QIIME (2019.4), denoised with DADA2 to obtain representative amplicon variants (ASVs) and constructed a phylogenetic tree. After quality control, the filtered ASVs were The bayes classifier (NBC) method was used for alignment and species annotation with gene sequences in the Greengenes (V_13.5) database. In performing Alpha and Beta diversity analyses, the resampling depth was 10,000 sequences per sample to ensure sufficient sequences. In order to reduce the influence of too many species on the results, the statistical results were corrected by calculating the false discovery rate (FDR).
  • FDR false discovery rate
  • This example is used to illustrate the results of the intervention of probiotics consisting of BL-11 bacteria on children diagnosed with autism spectrum disorder (ASD) at the age of 3 years and 6 months.
  • ASD autism spectrum disorder
  • the taking cycle is 90 days.
  • the child's defecation habits have changed.
  • the frequency of defecation has been changed from once every 3-5 days to once every 1-2 days.
  • Parents reported that the child's vocabulary expression ability, socialization and eye contact frequency increased, and the words spoken Increase the volume by 3-5 words.
  • the frequency of active feedback on their own feelings has increased, and the number of active requests has increased. There were fewer repetitive movements than before the intervention. The parents chose to continue taking it, and the continuous improvement is still being observed and recorded.
  • This example is used to illustrate the improvement of attention hyperactivity deficit disorder (ADHD) by probiotics composed of BL-11 bacteria.
  • ADHD attention hyperactivity deficit disorder
  • the treatment method is oral probiotic freeze-dried powder treatment, dosage: 2 times a day, 60 billion CFU each time. Take it with warm water in the morning and after dinner, respectively. Continuous treatment for 12 weeks. Conners parent questionnaire was used for assessment, including behavior, somatic, anxiety, learning, hyperactivity, and other indicators. The results showed that the children had lower scores on impulsivity, anxiety, and hyperactivity, improved bowel function, from once every 3-4 days to 1-2 times a day, sometimes once every 2-3 days, increased stool volume, and The bloating and pain gradually disappeared. The overall mental health improved markedly. Parents are satisfied with the results.

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Abstract

提供一种乳双歧杆菌，该乳双歧杆菌为乳双歧杆菌BL-11，所述乳双歧杆菌BL-11的保藏编号为CGMCC No.20847。提供的乳双歧杆菌可以改善肠道代谢紊乱并使肠道微生物组的组成正常化从而有效改善身高、抑制肥胖、促进儿童青少年生长发育和智力发展、防治精神障碍的作用，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。

Description

一种乳双歧杆菌及其应用 技术领域
本公开涉及微生物技术领域，具体地，涉及一种乳双歧杆菌及其应用。
背景技术
有研究发现，肠道细菌可以影响宿主的生长发育，部分菌群可以通过影响生长激素(GH)/胰岛素样生长因子-1(IGF-1)介导宿主生长发育过程。无菌小鼠和经抗生素治疗的小鼠均表现出生长减慢，并与生长素释放肽，生长激素和IGF-1水平降低相关。此外，在小鼠和人类中，缺乏微生物会导致成熟与生长受限，比如会导致慢性营养不良、神经性厌食症、生长发育迟缓、身材矮小甚至神经和免疫系统发育异常等。胰岛素样生长因子1(IGF-1)是一种对骨骼生长有已知作用的激素，随着肠道微生物定植的增加，血清中IGF-1的含量显著增加，并且肝脏和脂肪组织IGF-1的含量也会增加。而抗生素治疗后，肠道菌群大幅减少，可降低血清IGF-1并抑制骨形成。抗生素治疗后补充微生物代谢产物短链脂肪酸(SCFA)可将IGF-1和骨量恢复至正常水平。此外，无菌小鼠的IGF-1水平较低，然而，采用有益微生物治疗可以适度增加骨矿物质密度，提高IGF-1水平并防止骨损失。胰岛素样生长因子1(IGF-1)是影响骨骼生长的内分泌和旁分泌/自分泌途径的生长因子。外源性IGF-1可以促进股骨的纵向生长，并且软骨特异性胰岛素样生长因子I受体(Igf1r)缺失后，可以观察到IGF-1影响生长板成熟和次级骨化中心的形成。有研究发现，肝脏特异性IGF-1缺陷型小鼠的血清IGF-1降低了75％，但仍表现出相对正常的生长发育，这表明局部IGF-1也可以促进骨骼生长。此外，IGF-1可以促进成骨细胞(包括分别影响骨形成和骨吸收)。因此，肠道微生物群的改变，可以促进骨骼的形成以及再吸收，从而导致骨骼净生长，微生物群可以通过诱导IGF-1促进骨骼生长和重塑。通过直接补充益生菌、益生元或微生物发酵纤维素后产生的短链脂肪酸(SCFA)有可能诱导IGF-1的增加，进而影响骨骼生长和健康。
另外，肠道微生物可以通过肠-脑轴调控脑功能和行为。而且肠道菌群与脑通过自主神经、肠神经、免疫系统、嗅觉系统、肠内分泌信号、神经递质、支链氨基酸、胆汁酸、短链脂肪酸、脊髓、下丘脑-垂体-肾上腺轴、肽聚糖等途径和介质进行双向交流。肠道菌群和中枢神经系统之间的双向交流，即菌群-肠-脑轴，可影响动物的神经发育和功能，从而影响社会行为。这种菌群-肠-脑轴的双向影响受到性别和遗传等内部因素，以及环境、饮食、遗传、压力等外部因素影响。因此，肠道菌群参与了多种神经系统相关疾病的发病，与此同时，心理和行为反应也会反过来影响肠道菌群的组成或功能等。已有证据证明，靶向菌群的干预方法有望治疗与肠道菌群相关的焦虑、抑郁、精神分裂、多动症、抽动症、帕金森氏症、阿尔兹海默症等疾病，以及自闭症等社交障碍。以注意缺陷多动障碍(attention-deficit/hyperactivity disorder，ADHD)为例，这是一种儿童时期最常见的神经行为发育障碍性疾病。主要表现为与患儿年龄不相称的注意力易分散、注意广度缩小、不分场合的过度活动、情绪冲动并常伴有认知障碍、品行障碍和学习困难等。ADHD的发病率较高，对患者的学业、家庭和社会生活等方面产生了明显不良的影响。但是针对ADHD的治疗方法仍比较欠缺。
因此，亟需开发出一种抗肥胖、促进儿童青少年生长发育，又能防治精神障碍的益生菌，对于儿童青少年或者精神障碍患者及其家属来说具有重要意义。
发明内容
本公开提供了一种乳双歧杆菌及其应用，该乳双歧杆菌可以改善肠道代谢紊乱并使肠道微生物组组成正常化从而有效抗肥胖、促进儿童青少年生长发育，促进其智力发展；该乳双歧杆菌还能有效可以改善肠道通透性，降低血液中的LPS，D-乳酸水平，从而起到防治精神障碍的作用，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。
为了实现上述目的，本公开第一方面提供了一种乳双歧杆菌，该乳双歧杆菌为乳双歧杆菌BL-11，所述乳双歧杆菌BL-11的保藏编号为CGMCC No.20847。
本公开第二方面还提供了一种乳双歧杆菌制剂，其中，所述制剂为固态菌粉或者液态饮液；所述乳双歧杆菌为本公开第一方面所述的乳双歧杆菌。
本公开第三方面还提供了上述乳双歧杆菌在制备改善肠道代谢紊乱促进肠道微生物组组成正常化的食品组合物或药物组合物中的应用。
本公开第四方面还提供了上述乳双歧杆菌在制备儿童青少年抗肥胖的食品组合物或药物组合 物中的应用。
本公开第五方面还提供了上述乳双歧杆菌在制备促进儿童青少年生长发育的食品组合物或药物组合物中的应用。
本公开第六方面还提供了上述乳双歧杆菌在制备促进儿童青少年身高增加的食品组合物或药物组合物中的应用。
本公开第七方面还提供了上述乳双歧杆菌在制备促进儿童青少年智力发展的食品组合物或药物组合物中的应用。
可选地，其中，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含量为1-25重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用。
可选地，其中，所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
可选地，其中，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
可选地，其中，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 6CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
本公开第八方面提供了上述乳双歧杆菌在制备防治精神障碍的食品组合物或药物组合物中的应用。
可选地，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。
可选地，所述食品组合物或药物组合物还含有脱脂奶粉、海藻糖、低聚果糖、乳糖、葡萄糖、蔗糖、L-抗坏血酸钠、L-苹果酸、L-乳酸等的一种或几种组合。
可选地，所述食品组合物或药物组合物还含有调味剂、甜味剂、增稠剂、稳定剂、表面活性剂、润滑剂、酸性中和剂、分散剂、缓冲液或缓冲剂、脱苦剂、pH稳定剂、防腐剂、脱糖剂和/或着色剂，如乳糖醇、山梨糖醇、麦芽糖醇、阿斯巴甜、甜叶菊、罗汉果、三氯蔗糖、木糖醇、香草、巧克力、水果香料、人造香精、或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有维生素、矿物质和/或膳食补充剂或益生元营养物质、至少一种益生元，其中任选地，所述益生元包括菊粉、洋蓟提取物、菊苣根提取物、菊芋根提取物、低聚果糖、低聚半乳糖、低聚异麦芽、低聚木糖、水苏糖、低聚甘露糖、低聚阿拉伯糖、抗性糊精、抗性淀粉或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有泛醌(CoQ10)、番茄红素、β-胡萝卜素、色氨酸、维生素B6、维生素B12或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有益生菌，其中任选地，所述益生菌包括培养的或粪便中提取的微生物或细菌或细菌组分，并且任选地，所述细菌或细菌组分包括或者来源于乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)、大肠杆菌(E coli)、普雷沃氏杆菌(Prevotella)、粪栖杆菌(Faecalibacterium)、布劳特氏菌(Blautia)、拟杆菌(Bacteroidetes)、厚壁菌(Firmicutes)以及等同物，或者它们的混合物或组合。
可选地，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含量为0.5-20重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用；所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
可选地，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
可选地，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 9CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
可选地，采用不同的递送形式和载体，所述食品组合物或药物组合物可以为粉剂、锭剂、液体、口香糖、软糖、压片糖果、酸奶、牛奶、奶酪、冰淇淋、冰冻食品、保健食品、药品或饲料。
本公开的乳双歧杆菌BL-11可以改善肠道通透性，降低血液中的LPS，D-乳酸水平。
本公开的乳双歧杆菌BL-11可以用于促进自主主动活动、改善一次性回避反应记忆功能、改善应激刺激导致的快感缺失。
通过上述技术方案，本公开提供的益生菌可以有效改善代谢紊乱并使肠道微生物组的组成正常化，促进儿童青少年生长发育，而且可以改善肠道通透性，降低血液中的LPS，D-乳酸水平，从而起到防治精神障碍的作用，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤 独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
生物材料保藏信息
乳双歧杆菌BL-11，分类命名为乳双歧杆菌(Bifidobacterium lactis)，保藏于中国普通微生物菌种保藏管理中心，保藏地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期为2020年10月10日，保藏编号为CGMCC No.20847。
附图说明
附图是用来提供对本公开的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本公开，但并不构成对本公开的限制。在附图中：
图1是乳双歧杆菌BL-11显微镜观察图。
图2是乳双歧杆菌BL-11在人工胃液(pH＝3)和人工肠液(pH＝8)中的存活率检测结果。
图3、4是抗生素和益生菌处理后的小鼠股骨长度。
图5是抗生素和益生菌处理后的小鼠腿骨长度。
图6是两种益生菌处理后的小鼠肠道菌群多样性变化。
图7是两种益生菌处理后的小鼠肠道中双歧杆菌和乳酸杆菌的比例。
图8是两种益生菌处理后的小鼠肠道菌群组成的热图分析。
图9是服用益生菌或安慰剂前后0-12周人体身高变化。
图10是服用益生菌或安慰剂前后0-12周人体体重变化。
图11是服用益生菌或安慰剂前后症状改善情况。
图12是服用益生菌或安慰剂前后肠道菌群在属水平的组成。
图13是服用益生菌或安慰剂前后肠道菌群在代谢通路水平的对比。
图14是服用益生菌或安慰剂前后肠道菌群在门水平的组成。
图15是服用益生菌或安慰剂前后肠道菌群在代表性菌种组成的对比。
图16是服用益生菌或安慰剂前后肠道菌群在功能基因水平的对比。
图17是服用益生菌或安慰剂前后肠道菌群在KO水平的对比。
图18是服用益生菌或安慰剂前后肠道菌群在代谢通路水平的对比。
图19是肠道通透性检测的结果图。
图20是在属水平各组肠道菌群的构成图。
图21是双歧杆菌属在两组之间的差异。
图22是α多样性指数结果。
图23是β多样性指数分析的PCoA分析结果。
具体实施方式
以下结合附图对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开第一方面提供了一种乳双歧杆菌，该乳双歧杆菌为乳双歧杆菌BL-11，所述乳双歧杆菌BL-11的保藏编号为CGMCC No.20847。
本公开第二方面还提供了一种乳双歧杆菌制剂，其中，所述制剂为固态菌粉或者液态饮液；所述乳双歧杆菌为本公开第一方面所述的乳双歧杆菌。
本公开第三方面还提供了上述乳双歧杆菌在制备改善肠道代谢紊乱促进肠道微生物组组成正常化的食品组合物或药物组合物中的应用。
本公开第四方面还提供了上述乳双歧杆菌在制备儿童青少年抗肥胖的食品组合物或药物组合物中的应用。
本公开第五方面还提供了上述乳双歧杆菌在制备促进儿童青少年生长发育的食品组合物或药物组合物中的应用。
本公开第六方面还提供了上述乳双歧杆菌在制备促进儿童青少年身高增加的食品组合物或药物组合物中的应用。
本公开第七方面还提供了上述乳双歧杆菌在制备促进儿童青少年智力发展的食品组合物或药物组合物中的应用。
可选地，其中，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含 量为1-25重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用。
可选地，其中，所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
可选地，其中，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
可选地，其中，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 6CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
本公开第八方面提供了上述乳双歧杆菌在制备防治精神障碍的食品组合物或药物组合物中的应用。
可选地，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。
可选地，所述食品组合物或药物组合物还含有脱脂奶粉、海藻糖、低聚果糖、乳糖、葡萄糖、蔗糖、L-抗坏血酸钠、L-苹果酸、L-乳酸等的一种或几种组合。
可选地，所述食品组合物或药物组合物还含有调味剂、甜味剂、增稠剂、稳定剂、表面活性剂、润滑剂、酸性中和剂、分散剂、缓冲液或缓冲剂、脱苦剂、pH稳定剂、防腐剂、脱糖剂和/或着色剂，如乳糖醇、山梨糖醇、麦芽糖醇、阿斯巴甜、甜叶菊、罗汉果、三氯蔗糖、木糖醇、香草、巧克力、水果香料、人造香精、或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有维生素、矿物质和/或膳食补充剂或益生元营养物质、至少一种益生元，其中任选地，所述益生元包括菊粉、洋蓟提取物、菊苣根提取物、菊芋根提取物、低聚果糖、低聚半乳糖、低聚异麦芽、低聚木糖、水苏糖、低聚甘露糖、低聚阿拉伯糖、抗性糊精、抗性淀粉或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有泛醌(CoQ10)、番茄红素、β-胡萝卜素、色氨酸、维生素B6、维生素B12或者它们的混合物或组合。
可选地，所述食品组合物或药物组合物还含有益生菌，其中任选地，所述益生菌包括培养的或粪便中提取的微生物或细菌或细菌组分，并且任选地，所述细菌或细菌组分包括或者来源于乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)、大肠杆菌(E coli)、普雷沃氏杆菌(Prevotella)、粪栖杆菌(Faecalibacterium)、布劳特氏菌(Blautia)、拟杆菌(Bacteroidetes)、厚壁菌(Firmicutes)以及等同物，或者它们的混合物或组合。
可选地，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含量为0.5-20重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用；所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
可选地，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
可选地，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 9CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
可选地，采用不同的递送形式和载体，所述食品组合物或药物组合物可以为粉剂、锭剂、液体、口香糖、软糖、压片糖果、酸奶、牛奶、奶酪、冰淇淋、冰冻食品、保健食品、药品或饲料。
本公开的乳双歧杆菌BL-11可以改善肠道通透性，降低血液中的LPS，D-乳酸水平。
本公开的乳双歧杆菌BL-11可以用于促进自主主动活动、改善一次性回避反应记忆功能、改善应激刺激导致的快感缺失。
下面通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。
实施例1
本实施例用于说明乳双歧杆菌BL-11及其性能特征。
1.乳双歧杆菌BL-11的分类学特征：
显微镜下观察乳双歧杆菌BL-11，结果如图1所示。理化试验结果如表1和2所示。
表1
Figure PCTCN2022085752-appb-000001
表2
Figure PCTCN2022085752-appb-000002
+表示能够代谢；-表示无法代谢。
2、乳双歧杆菌BL-11的人工胃液、肠液耐受性：
双歧杆菌为通常不抗酸的菌属。本实施例中，测试了本发明的乳双歧杆菌BL-11的人工胃液、肠液耐受性，同时以目前实验室保存的耐酸性能极好、可以通过胃肠道存活的乳双歧杆菌Bb-XX作为对比。
BL-11菌株在人工胃酸(pH＝3)中的存活率检测结果如表3所示，Bb-XX在人工胃液中处理1h时活菌存活率44.7％，处理3h活菌存活率29.5％；而本发明的乳双歧杆菌BL-11处理1h时活菌存活率86.2％，处理3h活菌存活率39.5％。表明本发明的乳双歧杆菌BL-11具有相对较好的耐胃酸能力，可以大部分顺利通过胃到达肠道发挥益生作用。
BL-11菌株在人工小肠液(pH＝8)中的存活率检测结果参见表3。数据显示，Bb-XX在人工肠液(pH＝8)中处理1小时活菌存活率66.1％；而本发明的乳双歧杆菌BL-11在人工肠液中处理1小时活菌存活率67.5％，处理3h后则两株菌的存活率分别为49.4％和32.1％。
上述结果表明，经过人工胃液和肠液消化后乳双歧杆菌BL-11仍能较好的存活(图2)。本发明的乳双歧杆菌BL-11相对参照菌具有较好耐消化液能力，可以在肠道内顺利存活并定殖。
表3
Figure PCTCN2022085752-appb-000003
3、乳双歧杆菌BL-11毒力实验及安全性检测：
将本发明的乳双歧杆菌BL-11接种于MRS液体培养基中，37℃厌氧培养48小时，计数培养液中乳双歧杆菌BL-11活菌数为3.7×10 9CFU/mL，将培养物原液经口以20.0mL/kg体重的比例连续给小鼠灌胃3天，之后再观察7天。健康雄性BALB/C小鼠，6-8周龄，16-18g，维持室温(25±2℃)，相对湿度(55±2)％，12h/12h光照，自由进食和饮水。结果表明：乳双歧杆菌BL-11的培养物原液与对照组相比，未观察到两组受试小鼠有毒性反应或死亡，小鼠体重增长也无统计学差异(p＞0.05)。
采用SN/T 1944-2007《动物及其制品中细菌耐药性的测定》方法，评估乳双歧杆菌BL-11的抗生素敏感性能。评价结果显示，乳双歧杆菌BL-11对氨苄西林Ampicillin、青霉素G PenicillinG、红霉素Erythromycin、氯霉素Chloramphenicol、克林霉素Clindamycin、万古霉素Vancomycin和四环素Tetracycline等敏感。符合欧洲食品安全委员会(EuropeanFood Safety Authority)对食用细菌耐药性评价规范中的要求。乳双歧杆菌BL-11不含外源抗生素耐药基因，食用安全。
实施例2
本实施例用于说明乳双歧杆菌BL-11促进生长发育的功能特征。
微生物群，促进骨骼的形成以及再吸收，从而导致骨骼的净生长。微生物群诱导激素样胰岛 素生长因子1(IGF-1)，它促进骨骼生长和重塑。微生物菌群发酵纤维产生的短链脂肪酸(SCFA)也诱导IGF-1，提示微生物菌群影响骨骼健康的机制。
使用抗生素和益生菌处理2个月大的雌性BALB/c小鼠，并在SPF条件下饲养。合并30只小鼠并随机分配至治疗组以使笼效应最小化。抗生素为0.2mg/mL庆大霉素，0.15mg/mL环丙沙星，2mg/mL链霉素和1mg/mL杆菌肽的混合物。
首先，用抗生素混合物加入小鼠饮用水中处理小鼠2周以消耗微生物，然后再将小鼠分为三组，每组10只，一组作为对照组，正常饲喂，另外两组为益生菌组：分别将BL-11或Bb-XX添加到水中，持续饲喂4周。按照动物设施的规定，将3％(g/100mL)的蔗糖添加到所有水中以确保适口性。水溶液是新鲜制备的，每周更换两次。四周之后，处死小鼠后使用血清分离管从通过心脏穿刺收集的血液样本中制备血清，并使用鼠IGF-1标准ABTS ELISA开发试剂盒(PeproTech)。
正常饲喂(CK)、BL-11和Bb-XX饲喂的小鼠股骨长度如图4所示，腿骨长度如图3所示。BL-11饲喂的小鼠腿骨长度与对照组有显著差异(P<0.05)，Bb-XX饲喂的小鼠腿骨长度与对照组无明显差异(P<0.05)。数据均显示为平均值±SD，使用t检验差异是否显著。*P<0.05；**P<0.01；***P<0.001。
生长调节活性分析：无菌小鼠和经抗生素治疗的小鼠均表现出生长减慢，生长素释放肽、生长激素和IGF水平降低，肠道微生物紊乱，导致小鼠生长受限。将小鼠进行抗生素处理后，再另其服用益生菌，检测小鼠血清IGF-1水平，结果如图5所示，抗生素处理之后再服用益生菌的小鼠，血清中的IGF-1水平增加，并且服用BL-11可以明显提高血清中IGF-1的水平(P<0.01)，提高效果高于Bb-XX。肠道菌群多样性：α多样性指数分析显示，使用BL-11与Bb-XX之间的多样性指数没有显著差异，BL-11在Shannon指数上比Bb-XX稍高，但未能达到显著差异(P>0.05)，结果如图6所示，对两组小鼠肠道中两种有益菌的分析显示，使用BL-11可明显增加肠道中双歧杆菌和乳酸杆菌的比例，结果如图7所示。
通过热图分析(图8)，在属水平上，粪杆菌属Faecalibacterium，毛螺菌属Lachnospira，Lachnospiraceae_UCG_004，萨特氏菌属Sutterella在BL-11组中升高，这些菌的升高可能与IGF-11在血清中的增加有关。
实施例3
本实施例用于说明BL-11菌粉的制备及其用于食品的生产。
本发明提供的乳双歧杆菌BL-11在MRS肉汤液体培养基中进行厌氧培养。经一级、二级并扩大培养后的发酵液，在37℃下培养24h后于4℃、3000rpm下离心10min，收集菌体，磷酸盐缓冲液(PBS)洗涤后，加脱脂乳进行冷冻干燥，-20℃以下保存。
本实施例所制得的BL-11菌粉，可以用于食品、药品、保健品或动物饲料用途。
所述食品可以是发酵乳、乳酪、含乳饮料、固体饮料、乳粉等普通食品或保健食品。优选地，在所述食品中，乳双歧杆菌BL-11供人类使用的建议的剂量可为1.0×10 3CFU～1.0×10 10CFU/kg体重/天，更优选为1.0×10 4CFU～1.0×10 9CFU/kg体重/天。
实施例4
本实施例用于说明在人体临床研究中促进生长发育的功能特征。
受试者和招募：公开招募65名11个月至16岁的Prader-Willi综合征患者随机分配为益生菌组或安慰剂组，进行为期12周的随机、双盲、安慰剂对照试验。
入选标准：经遗传学证实患有PWS；四周内没有服用过任何形式的益生菌；服用稳定药物至少四周；在试验期间没有计划的药物和心理干预措施；愿意及时提供粪便样本；愿意参与研究和访谈过程，并且没有其他遗传疾病、怀孕或哺乳情况。根据IRB要求，研究方案获得了受试者父母或法定监护人的知情同意书，是根据赫尔辛基宣言进行的。
方法：随机法和盲法测试：采用随机双盲安慰剂对照设计，由不属于团队的统计学家对受试者进行随机分配隐藏，使每个身份不明的受试者生成随机抽样数。北京华元生物技术研究院提供了外观相同的编码益生菌和安慰剂，以确保分配隐藏，保持盲点。这些患者被随机分配为每日接受乳双歧杆菌BL-11种益生菌(6×10 10CFU)或安慰剂条包。治疗第6和12周时比较两组的体重、身高、ASQ-3、ABC、SRS-2和CGI-1。CGI包括两个单项测量，评估以下内容：(a)从1到7的精神病理学严重程度(CGI-S)和(b)从开始到结束，以七分制方式评估治疗前后的症状变化(CGI-I)。
材料：益生菌组为条包形式粉状的乳双歧杆菌BL-11。每袋乳双歧杆菌补充剂包含3×10 10集落形成单位(CFU)；安慰剂为同样包装的麦芽糊精，颜色、味道和风味与乳双歧杆菌条包相似。 受试者每天两次用水口服一袋乳双歧杆菌或安慰剂，持续12周。麦芽糊精作为补充剂，具有最小的副作用，作为安慰剂麦芽糊精的不良反应也最小。
初级结果测量：
1.由父母使用标准秤测量体重和身高，并由研究人员收集，使用WHO提供的年龄增长作为参考，将体重、身高和BMI转换为z评分。
2.心理测试：
(1)《年龄和阶段问卷》，第三版(ASQ-3)。ASQ-3是最广泛使用的针对儿童青少年的发育筛查工具之一，具有五个维度：交流，总动力，精细动力，解决问题和个人社交。根据五个维度计算受试者总分数以评估试验效果。
(2)异常行为清单(ABC)，ABC是一个58项行为评估量表，用于衡量五个子量表的行为问题：易怒，嗜睡/社交退缩，刻板印象行为，多动/不遵从行为和不适当的言语。根据以上行为问题计算受试者总分数以评估试验效果。
(3)社会反应量表(SRS)，SRS包含65个项目，用于定量评估社会行为的严重性。根据以上社会行为计算受试者总分数以评估试验效果。
(4)基于Gilliam自闭症评分量表(第三版)(GARS-3)所采用的4分制(0-3)的限制性和重复性行为(RRB)。根据以上行为计算受试者总分数以评估试验效果。
二级结果测量：
1、粪便微生物组
(1)样品处理和收集
用含有1mL保存溶液的DNA/RNA屏蔽粪便收集管(Zymo,Cat#R1101)收集粪便样品，并通过冰袋将其运送至实验室，然后在-80℃下冷冻。根据制造商的说明，使用TIANmap粪便DNA试剂盒提取DNA(TIANGEN，目录号DP328)，并使用Nanodrop分光光度计对DNA样品进行仔细定量。测量A260/A280的比例以确认高纯度DNA的产量。将DNA样品在-20℃冷冻直至使用。
(2)16S rRNA基因扩增子测序
通过两轮PCR，使用以下引物构建16S rRNA V3-V4文库：
341F:5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAGCCTACGGGNBGCASCAG-3’(SEQ ID NO.1)
805R:5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGACTACNVGGGTATCTAATCC-3’(SEQ ID NO.2)
通过(95℃2min，然后在95℃30s，55℃30s，和72℃30s反应过程进行25个循环，然后在72℃最终延伸5min。用1xKAPA AMPure(KAPA，目录号KK8002)纯化PCR产物。然后，使产物经历第二个PCR反应步骤(95℃2min，然后在95℃30s，55℃30s和72℃30s进行八个循环，最后在72℃下放置延伸5min)。1×KAPA AMPure纯化PCR产物，并使用Bioanalyzer DNA试剂盒通过实时PCR进行定量分析。
2、临床整体印象(CGI)是为在临床试验中使用而开发的，目的是在开始使用研究药物之前和之后，将临床医生对患者整体功能的看法进行简短的独立评估。CGI包括两个伴随的单项测量，评估以下内容：(a)从1到7的精神病理学严重程度(CGI-S)和(b)以类似的七点量表(CGI-I)从治疗开始后的变化。
3.胃肠道症状是根据基线时存在的胃肠道症状的总数进行评估的，包括便秘、腹泻、腹痛、肠胃气胀、便血、恶心、吞咽困难、食欲不振、消化不良和反酸。
数据分析
所有原始数据均在Microsoft Excel 2007和R中记录和处理。数据显示遵循CONSORT建议，用于报告双盲随机临床安慰剂对照试验的结果。使用α＝0.05作为显着性水平进行统计学程序。本公开应用Wilcoxon秩和检验来探索基线上体重、身高的z得分，ASQ-3、ABC、SRS的总分和子得分，每个项目在0～6周和0～12周的变化。线性混合模型也用于分析重复测量。
本公开使用错误发现率(FDR)来调整多个比较结果。使用与主要结果相似的方法分析了次要结果。另外，进行线性回归以检查临床指标和微生物组组成之间的相关性。
微生物组数据处理和分析：
质控使用QIIME2(v2019.10)过滤测序读数。使用Deblur对默认参数进行去噪，并通过扩增子序列变体(ASVs)获得样品的丰度表。使用QIIME2计算Alpha多样性。Bray-Curtis距离用于表征微生物组β多样性。使用基于sklearn的分类器分类法分配ASV，该分类器在与Greengenes v13.8相似度为99％的序列上进行训练。通过Kruskal–Wallis测试确认安慰剂组和益生菌组的微生物门、 属和α多样性的相对丰度之间存在显着差异。基于Benjamini-Hochberg(BH)调整的错误发现率(FDR)用于多重比较。
PICSRUSt2用于根据ASV的丰富表来推断微生物的功能含量，然后生成《京都基因与基因组百科全书》(KEGG)直系同源物(KO)，酶分类号和途径丰度表。使用基于排列的非参数测试对益生菌和安慰剂组之间的比率进行差异分析，并用Calour绘制最显著的差异特征。来自16s rRNA Illumina扩增子测序的所有原始数据已保存在美国国家生物技术信息中心(NCBI)序列读取档案(SRA，PRJNA643297)中。
结果
1.PWS受试者的人口统计学特征
表4概述了65名参与者的人口统计学特征和合并症的胃肠道(GI)症状，没有观察到组间差异(P>0.05)。47.5％的受试者在研究人群中显示一种或多种胃肠道症状，益生菌组的胃肠道症状比例虽然比安慰剂组降低了37.4％，但差异未能达到显著(P>0.05)。
表4
Figure PCTCN2022085752-appb-000004
没有观察到严重的不良事件。所有观察到的不良事件和辍学的主要原因，两组之间没有显着差异(P>0.05)。
2.益生菌对体重、身高、心理测量和CGI-I的影响
在整个治疗过程中，人体测量数据被收集和分析。益生菌组从6周到12周的身高显着高于安慰剂组(益生菌干预组比安慰剂组增高明显，平均增高2.58cm，P<0.05，图9)。益生菌组比安慰剂组的体重随时间下降更多，但两组差异未达到显著(图10)。心理测量(包括ASQ-3、ABC、SRS和RRB)得分，经线性混合效应模型分析观察到，益生菌组比安慰剂组有改善趋势，但差异未达显著(P>0.05)。使用CGI-1量表测量治疗过程中症状的总体改善，与安慰剂组相比，益生菌组的症状整体改善更明显(图11，P<0.05)。
3.益生菌干预下微生物组组成和功能的变化
测序后，在干预过程中，两组中PWS个体的肠道微生物组组成差异丰富。特定细菌类群的总体相对丰度级别如图12所示。6周后，与安慰剂组相比，益生菌组中的α多样性略有增加，差异不显著。通过排列多元方差分析(PERMANOVA)分析的β多样性显示，用益生菌治疗可实现两组的分离(F统计＝2.2526，R 2＝0.035613，P<0.05，NMDS压力＝0.19048，图13)。
为了表征整个干预过程中可能具有临床意义的细菌的丰度变化，本公开介绍了几个选定细菌属和科的倍数变化，如图14所示。益生菌组在第6周和第12周时，相对于基线，Lachnospiraceae ND3007，Ruminococcaceae UCG-003，Streptococcus mutans，Comamonadaceae，Alistipes和Rothia的相对丰度比基线降低。在益生菌组中，有益菌属，如双歧杆菌，乳杆菌和普雷沃氏菌9在12周时相比基线明显增加(图15)。
功能基因预测分析表明，在12周的治疗期后，益生菌组中的几个基因具有不同的丰度。编码泛醌生物合成蛋白(ubiB，k03688)，并列去饱和酶(EC：1.3.99.29)，并列去饱和酶(番茄红素形成)(EC：1.3.99.31)和全反式-ζ-胡萝卜素去饱和酶的基因(EC：1.3.99.26)均被上调，而编码二甲基精氨酸酶(k01482)和酸性磷酸酶(phoN，k09474，EC：3.1.3.2)的基因被下调(图16)。这些发现不符合多重比较显著性的错误发现标准。图17、18中显示的预测的KEGG途径和预测的KO的分析结果进一步比较了益生菌和安慰剂组的基因表达。
4.肠道菌群丰度与临床指标之间的相关性
临床指标与细菌属的丰度相关。益生菌组中发现了两种相关性，而安慰剂组中没有发现相关性。在第6周，益生菌组的RRB评分与Rothia之间存在正相关(R＝0.97，p<0.005)。
本公开的65名PWS患者在为期12周的随机、双盲和安慰剂对照实验中，乳双歧杆菌BL-11受试者的身高明显增加而体重稍有改变。本公开为乳双歧杆菌BL-11作为PWS患者的早期干预提供了新的证据。并且，本公开发现治疗后的益生菌组和安慰剂组之间的肠道微生物组β多样性存在明显差异，坚持控制饮食时，基线β多样性与长期体重减轻直接相关。因此，补充乳双歧杆菌BL-11可能具有改善肠道菌群构成，预防肥胖作用或可以促进饮食引起的体重减轻。补充乳双歧杆菌BL-11还可能改善儿童青少年的生长发育状况。
此外，本公开发现治疗后的益生菌组和安慰剂组之间的的整体症状改善明显，心理测量指标有改善，因此，补充乳双歧杆菌BL-11可能具有改善儿童青少年智力发育，行为或情绪状态的作用。
实施例5
本实施例用于说明BL-11冻干菌粉的制备方法。
BL-11保存菌液在37℃水浴锅内进行菌种复苏，至冻存管内液体全部融化；在MRS培养基上分区划线，37℃厌氧培养12～24小时；挑取单菌落，接种至装有液体培养基的厌氧培养管中，密封，37℃厌氧培养6-12小时，监测到菌液OD600值停止增长，停止发酵；进行菌种发酵生产，40℃恒温搅动培养6～12小时，维持菌液的恒pH为5.4±0.5，直至监测到菌液OD600值停止增长，立即停止发酵；离心收集菌泥，按照菌泥：冻干保护剂＝1:1～10的体积比添加冻干保护剂，搅拌、混合均匀后放入冷冻干燥机内进行菌粉的冻干；收集冻干粉，按质量要求进行粉碎后包装。
实施例6
本实施例用于说明BL-11冻干菌粉用于食品的生产。
本发明提供的乳双歧杆菌BL-11制作的冻干菌粉，可以用于酸奶、发酵乳、乳酪等普通食品或保健食品。优选地，在所述食品中，乳双歧杆菌BL-11供制作酸奶的添加剂量为1.0×10 6CFU～1.0×10 8CFU/kg，更优选为1.2×10 7CFU～1.5×10 8CFU/kg。生产方式为直投或后添加。直投时作为发酵菌种，在原料乳杀菌结束后，降低到合适温度时按比例添加，之后40-43℃保温发酵10-48小时。经过搅拌复配后分装到杯子或袋子中作为成品。后添加方式添加时，在酸奶发酵结束后，按照的一定的比例添加后搅拌均匀，复配结束后分装作为成品。
实施例7
本实施例用于说明BL-11冻干菌粉用于膳食营养补充剂、益生菌的生产。
本发明提供的乳双歧杆菌BL-11制作的冻干菌粉，可以用于益生菌的生产。益生菌冻干粉包含0.5-30份的BL-11冻干菌粉，5-20份的发酵乳杆菌、瑞士乳杆菌、罗伊氏乳杆菌、植物乳杆菌、青春双歧杆菌、短双歧杆菌、长双歧杆菌等菌种，20-70份的低聚半乳糖、低聚果糖、菊粉等益生元，5-10份的GABA、色氨酸、番茄红素、β-胡萝卜素、维生素B6、维生素B12、辅酶Q10、牛磺酸、果胶、β-葡聚糖、岩藻糖、卡拉胶、瓜尔豆胶、膳食纤维等营养物质，还包括0.1-5份的抗氧化剂/抗炎物质，如生育酚、类胡萝卜素、抗坏血酸/维生素C、抗坏血酸棕榈酸酯、多酚、谷胱甘肽和超氧化物歧化酶等。益生菌的活菌数添加总剂量为：BL-11的添加量为2.0×10 6CFU～2.0×10 10CFU/g，更优选为3×10 7CFU～3×10 10CFU/g。其它菌种的单菌添加剂量为1×10 6CFU～3×10 9CFU/g。制备流程包括：按比例称量原料、混合、分装和质检等。
实施例8
本实施例用于说明BL-11冻干菌粉改善肠道通透性和行为作用。
肠道微生物与肠-脑轴(GBA)在应激反应调控中发挥双向通讯。微生物通过不同的机制与肠-脑轴进行沟通。与粘膜细胞直接相互作用，通过免疫细胞以及通过接触神经末梢影响大脑发育和行为。大脑的应激也可以通过GBA对肠道微生物产生影响，肠道微生物群是导致胃肠功能紊乱和失调的原因。应激反应也会影响微生物的副产品和前体的合成，这些副产品和前体通过血液和后脑区进入大脑，通过黏膜免疫细胞释放细胞因子，通过肠内分泌细胞释放肠道激素，如5-羟色胺(5-HT)，或通过传入神经通路，包括肠道神经系统。
干预用益生菌制剂制备：益生菌制剂为含有BL-11的冻干粉、β-胡萝卜素、维生素B6、维 生素B12、辅酶Q10和麦芽糊精，对照组为麦芽糊精，实验鼠的灌胃剂量为100亿CFU/只/天。
将6周龄的C57BL/6J小鼠20只，每笼5只小鼠分组饲养，自由进食和饮水。食物和水摄入量每周记录两次。将小鼠分为对照组和益生菌组2组，每组10只。实验鼠适应环境一周后，随机分组：模型组，益生菌干预组。实验鼠的生长环境条件为：环境温度(23±2)℃、相对湿度(50±10)％、光照模式(12h黑暗/12h光照)。
采用认为物理刺激造成慢性应激模型，具体刺激方式包括：(1)禁食、禁水24h；(2)持续光照24h；(3)铁夹夹尾，夹住尾尖1cm处，持续3min/次；(4)束缚应激，圆形套筒约束行动2h/天。每天采用1-2种不同应激刺激，每次刺激时间不固定，持续造模时间4周。
从第六周开始，停止应激刺激，开展糖水偏好实验、跳台实验和开场实验，行为实验结束后对各组小鼠进行理化指标测定。数据分析采用SPSS统计软件处理所得数据，以均值±标准差表示，采用独立样本检验进行组间比较，P<0.05认为差异有统计学意义。
1)糖水偏好实验
在实验开始前，在笼架上同时放置两个相同的水瓶，其中一个瓶盛装纯水，一个瓶盛装1％浓度的蔗糖水，先让实验鼠进行24h的糖水适应。为了避免实验鼠饮水习惯带来的干扰，每隔1h，对调一次水瓶的位置。适应结束后，实验鼠禁食、禁水24h。实验开始前在每个饲养笼上放置一瓶纯水和一瓶1％浓度的蔗糖水，每隔3h记录一次糖水和纯水的消耗量。
糖水偏好程度＝糖水消耗量/(糖水消耗量+纯水消耗量)×100％。
实验结果显示，对照组对糖水的偏好程度降低(49.63％±15.79)，而在补充益生菌之后，对糖水偏好的程度得到提升(68.79％±12.34)，表明益生菌能改善应激刺激导致的快感缺失。
2)跳台实验
跳台仪实验箱(DTT-2型小鼠跳台，中国医学科学院药物研究所)。跳台箱长约120cm，宽12cm，高30cm，有机玻璃材质。共分为6个室，每个室长12cm，宽12cm，高30cm。实验箱可允许6只小鼠同时进行实验。实验箱底面铺有铜栅，铜栅通过电线与电源相连，电流电压设置为36V。实验箱内铜栅上靠箱内一角置绝缘台(长径5.7cm，短径4.5cm，高4.8cm的五边形木块)作为动物回避电击的安全区。试验装置与计算机自动记录系统相连。实验时将小鼠放入跳台仪实验箱内，适应环境5min，之后将小鼠轻放于跳台上，并将铜栅通电，当小鼠从跳台上跳下四肢接触铜栅时会受到电击，正常回避反应是跳上跳台返回安全区，逃避电击，如此学习5min，并记录此5min内的触电次数(错误次数)，以此为学习成绩。24h后进行记忆能力测试，将小鼠置于跳台上，记录小鼠从停留在跳台上至第1次跳下受电击的时间即潜伏期和5min内的错误次数(小鼠四肢同时接触铜栅触电的次数)，以此作为记忆功能的评价指标。
与正常对照组比较，益生菌组在跳台实验中的潜伏期均明显缩短(P<0.05)，跳台试验的错误次数无显著差异，但益生菌组呈现错误次数减少趋势。表明益生菌组小鼠可改善一次性回避反应记忆功能的障碍(见表5)。
表5益生菌对回避反应记忆功能的影响
  错误次数(次) 潜伏期(S)
对照组 1.45±0.82 268.32±50.63
益生菌组 1.07±0.87 248.05±82.33
P value 0.0348 0.0782
3)开场实验
开场实验分析系统是观察研究实验动物神经精神变化、进入开阔环境后的各种行为，例如动物对新开阔环境的恐惧，而主要在周边区域活动，在中央区域活动较少，但动物的探究特性又促使其产生在中央区域活动的动机，也可观察由此而产生的焦虑心理。用于评估动物的自主主动活动和焦虑水平。
实验前60min将小鼠移到开场实验测试房，提前适应环境。实验时将小鼠从笼中取出置于行为学实验站中的开场实验装置(箱长×宽×高为:100cm×100cm×40cm，内面及底面均为蓝色，中央区正上方安置摄像头。实验开始后，将小鼠放到中央区固定位置，每次放置头部固定朝向一侧，迅速拉上遮光罩帘。在操作软件中记录小鼠编号、日期、状态后打开记录系统，选取九宫格模式，中心区比例为0.5，通过开场设备上方的摄像机及与其相连的监视器。每只测量5min，记录小鼠的活动情况，测量指标包括运动时间、总距离、中央区停留时间百分率(中央区停留时间LPMM＝s)、中央区水平运动百分率(中央区水平运动距离L水平运动距离)、四边区域水平运动百分率(四边区域水平运动距离L水平运动距离)和四角区域水平运动百分率(四角区域水平运 动L水平运动距离)。记录直立次数及理毛次数，随后用75％酒精清洁箱内环境，等酒精挥发无味后，再进行下1只。结果见表6。
与对照组相比，益生菌组小鼠的中央区进入次数、中央区停留时间显著高于对照组(P<0.05)。直立次数及理毛次数显著增加(P<0.05)，而中央区进次数差异不大。其余开场行为学结果未见明显差异。
表6小鼠开场行为实验结果
Figure PCTCN2022085752-appb-000005
4)肠道通透性检测
为了评估体内肠道通透性，测量血清中的D-乳酸，LPS含量来评估肠道通透性。
D-乳酸是细菌发酵的代谢产物，肠道多种细菌均可产生，即使从食物中摄取后，正常情况下很少被吸收入血，并且哺乳动物不具备将其快速降解的酶系统。因此，当肠黏膜通透性增加时，肠道中的细菌产生的大量D-乳酸通过受损的黏膜入血，使血中D-乳酸的水平升高。
乳酸有D型和L型，正常人体只有L-乳酸，细菌等微生物可以产生D乳酸。监测血中D-乳酸的水平可及时反映肠黏膜损害程度和通透性变化。可用于肠道感染，内毒素血症，全身性炎症反应，反复发烧，呕吐等的辅助评价。
脂多糖(LPS)也称为细菌内毒素，是由革兰氏阴性菌细胞壁上的成分，LPS对动物来说是一种毒性物质。LPS的结构可分为3个部分：糖脂结构域—脂质A、糖残基短链—核心寡糖、高变的多糖结构域—O抗原。LPS的结构决定了其对TLR4的激动剂/拮抗剂作用。在体内，LPS与TLR4/MD-2受体复合物结合，通过Myd88依赖性或TRIF依赖性途径激活不同的信号通路；不同部位的肠道上皮细胞TLR表达量不同，能够防止LPS引起的炎症反应，并对抗致病菌。
细菌移位是肠道活细菌从肠道穿过上皮粘膜进入机体。细菌可以经由肠系膜淋巴结进入淋巴系统在全身循环。细菌还能进入血液循环，导致菌血症，还可位于组织中。细菌位移可在小肠细菌过度生长，肠道损伤和休克。任何导致肠道通透性的应激反应，包括心理的和生理的，都可能潜在地导致细菌位移。
LPS与多种疾病的发病有关，如IBD及小肠结肠炎等肠道疾病，甚至帕金森和阿尔兹海默症等。LPS不仅可以进入血液，还能够进入并可一生滞留在脑内，可能引起阿尔兹海默病。
血液中的LPS水平可反映肠道的通透性，正常的肠道屏障是不允许LPS进入的，血液中较高水平的LPS预示着肠道细菌或LPS移位到了血液，说明肠道通透性的增加，出现肠漏症状的几率增加。血液中LPS的含量多少还能指示炎症反应和应激状态，过多的LPS可引起人体免疫系统异常，出现慢性或急性炎症反应，可出现发热、疼痛等急性炎症等。可用于肠道感染，内毒素血症，全身性炎症反应，反复发烧，呕吐、精神疾病、应激反应等的辅助评价。
实验结束后，从尾静脉末梢收集血液。将血液3000g下离心15分钟。采用肠道屏障功能分析系统(JY-DLT，北京中生金域诊断技术股份有限公司)，按照操作说明书，检测血清中的D-乳酸，LPS含量。
结果显示，与对照组相比，益生菌组LPS，D-乳酸水平明显降低(P<0.05)。表明应激刺激导致肠道通透性的增加，益生菌可以降低肠道通透性，减少内毒素血症，全身性炎症反应等的风险。
实施例9
本实施例用于说明BL-11冻干菌粉对肠道微生物组成的影响。
在上述行为实验结束后，收集的盲肠内容物储存在-80℃下。采集两组小鼠粪便，采用TIANmap粪便DNA试剂盒(TIANGEN，目录号DP328)提取粪便菌群的DNA。使用Qubit仪对提取的DNA进行定量检测。使用1％琼脂糖凝胶电泳检测：电压100V，40min。UVI凝胶成像系统拍照记录：DNA电泳未出现杂带、拖尾，说明DNA片段纯度良好，无明显降解。取适量样品于离心管当中，使用无菌水稀释样品至1ng/μL。DNA于-20℃冰箱内储存备用。
细菌16S rRNA基因扩增：以稀释后的基因组DNA为模板，根据测序区域的选择，使用带Barcode的特异引物，采用细菌通用引物341F(CCTAYGGGRBGCASCAG SEQ ID NO.3)和806R(GGACTACNNGGGTATCTAAT SEQ ID NO.4)扩增细菌16S rRNA基因的V3-V4区域。 100ng的提取的DNA在56℃进行PCR用于链复性，开始在94℃下变性4分钟，然后以94℃下降30秒、56℃下降30秒、72℃下降1分钟进行30个循环。
扩增子基因测序：使用Illumina公司TruSeq DNA PCR-Free Library Preparation Kit的建库试剂盒进行文库的构建，构建好的文库经过Qubit定量和文库检测，合格后，使用Illumina HiSeq2500PE250测序平台进行菌群测序。
测序数据的处理和分析：将菌群测序的原始数据导入QIIME(2019.4)，用DADA2降噪以获得代表性扩增子变异体(ASVs)并将其构建系统发育树。经质控后，将过滤后的ASVs用
Figure PCTCN2022085752-appb-000006
bayes classifier(NBC)方法与Greengenes(V_13.5)数据库中的基因序列进行比对和物种注释。在进行Alpha和Beta多样性分析中，重抽样深度为每样本10000条序列以保证足够序列。为了减少物种数过多对结果的影响，统计结果计算错误发现率(false discovery rate，FDR)进行校正。
结果如图20-23和表7所示。结果表明，在属水平，对照组和益生菌肠道菌群存在明显差异。对照组的双歧杆菌属含量明显低于益生菌组。此外，益生菌组的alpha多样性指数也高于对照组，指数差异不显著(Shannon指数p＝0.9118(Mann-Whitney statistic)；Simpson指数P＝0.35268(Mann-Whitney statistic))。在β多样性分析上，对照组和益生菌组的肠道菌群整体构成存在差异，在PCoA分析结果上两组可明显区分开(F-value＝2.4268；R-squared＝0.1188；p＝0.009(PERMANOVA))。
在属水平，具体的差异菌较多，其中Coprobacillus在使用益生菌后明显增多(FDR<0.001)。
表7两组之间属水平肠道菌群的差异分析
Figure PCTCN2022085752-appb-000007
Figure PCTCN2022085752-appb-000008
实施例10
本实施例用于说明由BL-11菌组成的益生菌对对被诊断患有自闭症谱系障碍(ASD)的3岁6个月的儿童进行干预的结果。以口服冻干粉的形式，每天2次，每次500亿CFU，服用周期90天。在服用后，该儿童排便习惯有所改变，排便次数由3-5天一次，改为1-2天一次；家长反馈孩子的词汇表达能力，社会化和眼神交流频率升高，说出的词汇量又增加3-5个词。主动反馈自身感受频率增加，主动提出需求次数增加。重复性动作较干预前较少。家长选择了继续服用，仍在观察和记录持续改善情况。
实施例11
本实施例用于说明由BL-11菌组成的益生菌对注意力多动缺陷障碍(ADHD)的改善。
病例来自河北省儿童医院，医师按照美国精神障碍诊断及统计手册中的ADHD标准确诊，年龄6.5岁，为多动冲动型，排除精神分裂，情感障碍，癫痫和其他器质性疾病。也具有慢性便秘和腹部不适。
治疗方式采用口服益生菌冻干粉治疗，用量：每日2次，每次600亿CFU。分别在早、晚饭后温水送服。连续治疗12周。采用Conners父母问卷进行评估，包括行为、躯体、焦虑、学习、多动、等指标。结果表明，患儿的冲动、焦虑和多动得分降低，排便功能得到改善，从每3-4天一次变为每天1-2次，有时2-3天1次排便，粪便量又增加，并且腹胀和疼痛逐渐消失。整体的精神改善明显。家长对该结果满意。
表8治疗前后Conners父母问卷结果
  行为 学习 心身 冲动 焦虑 多动
治疗前 1.6 2.1 0.35 1.34 0.52 1.38
治疗后 1.2 1.8 0.29 1.32 0.35 1.23

Claims (20)

  1. 一种乳双歧杆菌，该乳双歧杆菌为乳双歧杆菌BL-11，其特征在于，所述乳双歧杆菌BL-11的保藏编号为CGMCC No.20847。
  2. 一种乳双歧杆菌制剂，其中，所述制剂为固态菌粉或者液态饮液；所述乳双歧杆菌为权利要求1所述的乳双歧杆菌。
  3. 权利要求1所述的乳双歧杆菌在制备改善肠道代谢紊乱促进肠道微生物组组成正常化的食品组合物或药物组合物中的应用。
  4. 权利要求1所述的乳双歧杆菌在制备儿童青少年抗肥胖的食品组合物或药物组合物中的应用。
  5. 权利要求1所述的乳双歧杆菌在制备促进儿童青少年生长发育的食品组合物或药物组合物中的应用。
  6. 权利要求1所述的乳双歧杆菌在制备促进儿童青少年身高增加的食品组合物或药物组合物中的应用。
  7. 权利要求1所述的乳双歧杆菌在制备促进儿童青少年智力发展的食品组合物或药物组合物中的应用。
  8. 根据权利要求3-7中任意一项所述的应用，其中，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含量为1-25重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用；所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
  9. 根据权利要求3-7中任意一项所述的应用，其中，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
  10. 根据权利要求3-7中任意一项所述的应用，其中，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 9CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
  11. 权利要求1所述的乳双歧杆菌在制备防治精神障碍的食品组合物或药物组合物中的应用。
  12. 根据权利要求11所述的应用，其中，所述精神障碍包括焦虑、抑郁、注意缺陷多动障碍、自闭症、孤独症、精神分裂症、肝性脑病、神经性厌食症、妥瑞症、艾斯伯格症候群。
  13. 根据权利要求11所述的应用，其中，所述食品组合物或药物组合物还含有脱脂奶粉、海藻糖、低聚果糖、乳糖、葡萄糖、蔗糖、L-抗坏血酸钠、L-苹果酸、L-乳酸等的一种或几种组合。
  14. 根据权利要求11所述的应用，其中，所述食品组合物或药物组合物还含有 调味剂、甜味剂、增稠剂、稳定剂、表面活性剂、润滑剂、酸性中和剂、分散剂、缓冲液或缓冲剂、脱苦剂、pH稳定剂、防腐剂、脱糖剂和/或着色剂，如乳糖醇、山梨糖醇、麦芽糖醇、阿斯巴甜、甜叶菊、罗汉果、三氯蔗糖、木糖醇、香草、巧克力、水果香料、人造香精、或者它们的混合物或组合。
  15. 根据权利要求11所述的应用，其中，所述食品组合物或药物组合物还含有维生素、矿物质和/或膳食补充剂或益生元营养物质、至少一种益生元，其中任选地，所述益生元包括菊粉、洋蓟提取物、菊苣根提取物、菊芋根提取物、低聚果糖、低聚半乳糖、低聚异麦芽、低聚木糖、水苏糖、低聚甘露糖、低聚阿拉伯糖、抗性糊精、抗性淀粉或者它们的混合物或组合。
  16. 根据权利要求11所述的应用，其中，所述食品组合物或药物组合物还含有泛醌(CoQ10)、番茄红素、β-胡萝卜素、色氨酸、维生素B6、维生素B12或者它们的混合物或组合。
  17. 根据权利要求11所述的应用，其中，所述食品组合物或药物组合物还含有益生菌，其中任选地，所述益生菌包括培养的或粪便中提取的微生物或细菌或细菌组分，并且任选地，所述细菌或细菌组分包括或者来源于乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)、大肠杆菌(E coli)、普雷沃氏杆菌(Prevotella)、粪栖杆菌(Faecalibacterium)、布劳特氏菌(Blautia)、拟杆菌(Bacteroidetes)、厚壁菌(Firmicutes)以及等同物，或者它们的混合物或组合。
  18. 根据权利要求11-17中任意一项所述的应用，其中，相对于100重量份的食品组合物或药物组合物，所述乳双歧杆菌BL-11的含量为0.5-20重量份，优选为1-15重量份；所述乳双歧杆菌BL-11以所述乳双歧杆菌BL-11的活菌体、所述乳双歧杆菌BL-11的灭活菌体或所述乳双歧杆菌BL-11的提取物的形式使用；所述乳双歧杆菌BL-11的活菌数为1.0×10 6～1.5×10 12CFU/g，优选为3.0×10 10～5.0×10 11CFU/g。
  19. 根据权利要求11-17中任意一项所述的应用，其中，所述食品组合物是发酵乳、乳酪、含乳饮料、固体饮料、乳粉中的一种或多种。
  20. 根据权利要求11-17中任意一项所述的应用，其中，以人体体重为基准，所述乳双歧杆菌BL-11的服用剂量为2.0×10 9CFU～1.5×10 11CFU/kg/天，优选为3.0×10 4CFU～8.0×10 10CFU/kg/天。
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CN116769654A (zh) * 2023-06-14 2023-09-19 内蒙古农业大学 一株动物双歧杆菌乳亚种及其应用
CN116746683A (zh) * 2023-08-01 2023-09-15 河北源民生物科技有限公司 防消化不良胀气、促吸收的动物双歧杆菌乳亚种bal-28及发酵产物、菌群cw和应用
CN117025489A (zh) * 2023-10-09 2023-11-10 杭州微致生物科技有限公司 一种动物双歧杆菌乳亚种vb315及其应用
CN117025489B (zh) * 2023-10-09 2024-03-19 杭州微致生物科技有限公司 一种动物双歧杆菌乳亚种vb315及其应用
CN117431190A (zh) * 2023-12-14 2024-01-23 深圳未知君生物科技有限公司 一株能够缓解自闭症谱系障碍的短双歧杆菌及其应用
CN117431190B (zh) * 2023-12-14 2024-03-12 深圳未知君生物科技有限公司 一株能够缓解自闭症谱系障碍的短双歧杆菌及其应用

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