WO2022218336A1 - Application de lactobacillus reuteri dans la préparation d'un produit pour la prévention ou le traitement d'un trouble du développement - Google Patents

Application de lactobacillus reuteri dans la préparation d'un produit pour la prévention ou le traitement d'un trouble du développement Download PDF

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WO2022218336A1
WO2022218336A1 PCT/CN2022/086587 CN2022086587W WO2022218336A1 WO 2022218336 A1 WO2022218336 A1 WO 2022218336A1 CN 2022086587 W CN2022086587 W CN 2022086587W WO 2022218336 A1 WO2022218336 A1 WO 2022218336A1
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lactobacillus
lactobacillus reuteri
probiotics
application according
bifidobacterium
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PCT/CN2022/086587
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Chinese (zh)
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段云峰
张烨
梁桦
崔雷
刘智
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北京悦得微生物科技有限公司
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    • 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
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0321—Propionic acid bacteria
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0325—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using yeasts, alone or in combination with lactic acid bacteria or with fungi, without using other bacteria
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/061—Addition of, or treatment with, microorganisms
    • 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
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/09—Other cheese preparations; Mixtures of cheese with other foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/364—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
    • A23G3/366—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
    • A—HUMAN NECESSITIES
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    • A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
    • A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
    • A23G3/42—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds characterised by the carbohydrates used, e.g. polysaccharides
    • 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
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
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    • A23K20/10—Organic substances
    • A23K20/163—Sugars; Polysaccharides
    • 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
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    • A23L2/382—Other non-alcoholic beverages fermented
    • 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
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
    • 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
    • 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
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    • A23L33/14—Yeasts or derivatives thereof
    • 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
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • 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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
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    • 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
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
    • A23V2400/61—Propionibacterium

Definitions

  • the invention relates to the technical field of microorganisms, in particular to the application of Lactobacillus reuteri in the preparation of products for preventing or treating developmental disorders.
  • PWS Prader-Willi Syndrome
  • hypotonia-low intelligence-hypogonadism-obesity syndrome commonly known as chubby-Willi syndrome It is 1/12000 to 1/15000.
  • the cause is due to the gene defect of chromosome 15.
  • the patient has normal language ability, but the actual IQ is lower than that of ordinary people.
  • Prader-Willi Syndrome presents differently in different growth periods. Newborns and infants are manifested as poor muscle tone, difficulty in feeding, difficulty in gaining weight, poor limb mobility, and weak crying. Facial features include dark almond-shaped eyes, long straight eyelashes, thin, drooping lips, and a narrow forehead. Small hands and feet, short penis, undescended testicles, and poor scrotal development. Baby girl vulva dysplasia. Childhood manifestations: brain developmental delay with mild or moderate intellectual disability. Around the age of six, due to the dysfunction of the hypothalamus in the brain, the desire for food increases greatly and cannot be controlled, resulting in rapid weight gain. Adolescence manifests as: obesity, diabetes, scoliosis, lack of muscle coordination. Short stature, gonadal dysplasia, cryptorchidism, poor sexual development in girls, intellectual disability, and behavioral and emotional problems.
  • Lactobacillus reuteri is a lactic acid bacteria that exists in the intestinal tract. It is used as a probiotic and has strong adhesion to the intestinal mucosa, and also has excellent acid resistance, bile salt resistance and broad-spectrum antibacterial properties. Extensive inhibition of the growth of Gram-positive bacteria, Gram-negative bacteria, yeast, fungi and pathogens.
  • Lactobacillus reuteri isolated from the milk of healthy women can improve the intestinal flora of patients with developmental disorders (such as Prader-Willi syndrome). It can improve the composition of intestinal flora, prevent obesity or promote weight loss caused by diet, and can also improve the brain development of patients with developmental disorders, improve social communication disorders, communication disorders and improve fine motor skills. For the prevention or treatment of developmental disorders such as Prader-Willi syndrome and autism.
  • the present invention provides an application of Lactobacillus reuteri in the preparation of products for preventing or treating developmental disorders.
  • Disorders are Prader-Willi syndrome or autism.
  • Fig. 1 is a microscope observation diagram of Lactobacillus reuteri LR99 in Example 1;
  • Fig. 2 is the overall composition of intestinal flora at the genus level before and after taking probiotics or placebo in Example 3;
  • Fig. 3 is a comparison chart of the alpha diversity index of intestinal flora after taking probiotics or placebo for six weeks in Example 3;
  • Fig. 4 is a comparison chart of the ⁇ diversity index of intestinal flora after taking probiotics or placebo in Example 3;
  • Figure 5 is a comparison diagram of the changes in the intestinal flora before and after taking probiotics or placebo in Example 3 before and after the intervention of representative strains.
  • the Lactobacillus reuteri provided by this application, the strain name is LR99, is preserved in the General Microbiology Center of the China Microorganism Culture Collection and Management Committee, and the address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, the deposit number is CGMCC No. 21577, the strain was received and registered by the collection center on December 31, 2020, and was detected as a viable strain by the collection center on December 31, 2020.
  • Lactobacillus reuteri the number of which is LR99, is isolated from the milk of healthy women. On December 31, 2020, it was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101), classified It is named Lactobacillus reuteri, and the deposit number is CGMCC No.21577.
  • CGMCC General Microbiology Center of the China Microorganism Culture Collection Management Committee
  • Lactobacillus reuteri has been verified to improve the composition of the intestinal flora in patients with Prader-Willi syndrome, improve intestinal flora disturbance, prevent obesity or promote diet-induced weight loss, and also improve the general Brain development of patients with Rad-Willi syndrome, improve social interaction, communication and fine motor skills, can be used to prevent or treat developmental disorders such as Prader-Willi syndrome and autism .
  • An embodiment of the present application also provides a preparation method of the above-mentioned Lactobacillus reuteri, and the preparation method adopts high-density fermentation to prepare the above-mentioned Lactobacillus reuteri.
  • the preparation method includes steps S1-S3.
  • Step S1 Activating the above-mentioned Lactobacillus reuteri.
  • the Lactobacillus reuteri is activated and cultivated for three times to improve the activity of the strain, and the temperature of the three activation and cultivation is 37° C. and the time is 16 h to 18 h.
  • Step S2 Inoculate the activated Lactobacillus reuteri into a fermenter for fermentation culture.
  • the improved MRS medium was sterilized and then cooled to 37°C, inoculated into the fermenter with an inoculation amount of 2.5% to 3.5% (v/v) for fermentation, and the pH and OD values of the fermentation broth were detected every hour.
  • the pH and OD values are relatively flat, it means that the strain reaches the end of logarithmic phase and is about to enter the stable phase, and the fermentation is completed.
  • the fermentation broth was cooled, centrifuged at low temperature, the cells were collected and washed with phosphate buffered saline (PBS) to prepare Lactobacillus reuteri cells.
  • PBS phosphate buffered saline
  • Step S3 freeze-dry the thalli obtained in step S2 to prepare freeze-dried powder.
  • the thalline obtained in step S2 is mixed with a freeze-drying protective agent, emulsified, and then freeze-dried in a vacuum to prepare a freeze-dried powder.
  • the lyoprotectant is selected from at least one of skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malic acid and L-lactic acid.
  • the freeze-drying protective agent is not limited to the above, and can also be other substances that can maintain the activity of the cells during the freeze-drying process.
  • the volume ratio of the bacterial cells to the lyophilized protective agent is 1:2-10.
  • step S3 may be omitted.
  • cells of Lactobacillus reuteri were prepared.
  • the above-mentioned preparation method of Lactobacillus reuteri adopts high-density fermentation to prepare the above-mentioned Lactobacillus reuteri, and the yield is high.
  • an embodiment of the present application provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating Prader-Willi syndrome.
  • products for preventing or treating Prader-Willi syndrome include the above-mentioned Lactobacillus reuteri and/or the fermentation products of the above-mentioned Lactobacillus reuteri.
  • the fermentation product of the above-mentioned Lactobacillus reuteri refers to the culture product of the above-mentioned Lactobacillus reuteri, including at least one of metabolites located in the bacteria and metabolites secreted outside the bacteria.
  • the culture solution after the above-mentioned Lactobacillus reuteri culture or the freeze-dried powder obtained by lyophilizing the culture solution is taken.
  • the above-mentioned product includes the above-mentioned Lactobacillus reuteri and the auxiliary materials required for preparing a bacterial preparation.
  • the active ingredient of the above product includes the above-mentioned Lactobacillus reuteri.
  • the above-mentioned product includes the above-mentioned Lactobacillus reuteri and a lyoprotectant. The lyoprotectant is as described above and will not be repeated here.
  • the products described above also include other probiotics.
  • Probiotics are also used as active ingredients in the above-mentioned compositions. That is, the aforementioned product includes an active ingredient including the aforementioned Lactobacillus reuteri and other probiotics.
  • probiotics are selected from Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), Streptococcus thermophilus (Streptococcus thermophilus), Lactococcus (Lactococcus), Propionibacterium (Propionibacterium), Leuconostoc ), Staphylococcus, Bacillus, Pediococcus, Escherichia.coli (eg Nissle1917), Prevotella, Faecalibacterium, Blau At least one of Blautia, Bacteroidetes, Firmicutes and yeast.
  • the lactobacillus is selected from Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus ( Lactobacillus crispatus), Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei ( At least one of Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei and Lactobacillus helveticus A sort of.
  • the Bifidobacterium is selected from Bifidobacterium adolescentic, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, At least one of Bifidobacterium bifidum, Bifidobaterium animalis, and Bifidobaterium lactis.
  • the Lactococcus is selected from Lactococcus Lactis subsp. Lactis, Lactococcus Lactis subsp. Cremoris and Lactococcus Lactis subsp. At least one of Lactis subsp. Diacetylactis).
  • the Propionibacterium is selected from at least one of Propionibacterium freudenreichii subsp. Shermanii and Propionibacterium acidipropionici. Leuconostoc mesenteroides subsp. Mesenteroides.
  • the Pediococcus is selected from at least one of Pediococcus acidilactici and Pediococcus pentosaceus.
  • the staphylococcus is at least one selected from Staphylococcus vitulinus, Staphylococcus xylosus and Staphylococcus carnosus.
  • Bacillus is Bacillus coagulans.
  • the yeast is selected from the group consisting of Kluyveromyces marxianus, Saccharomyces cerevisiae, Cadida atilis, Kluyveromyces lactis and Saccharomyces carlsbergensis. at least one. It can be understood that other probiotics in this embodiment are not limited to the above, and may also be probiotics other than LR99.
  • the above product further includes prebiotics.
  • the colonization and reproduction of the above-mentioned Lactobacillus reuteri are promoted through prebiotics, and the growth of other probiotics in the intestinal tract is also promoted.
  • the prebiotic is selected from inulin, artichoke extract, chicory root extract, Jerusalem artichoke root extract, fructooligosaccharides, galactooligosaccharides, isomalt oligosaccharides, xylo-oligosaccharides, stachyose, low At least one of polymannose, arabino-oligosaccharide, resistant dextrin and resistant starch.
  • the prebiotics in the above composition are not limited to the above, and may also be other substances that can promote the growth and reproduction of probiotics.
  • the above-mentioned product further comprises nutrients selected from the group consisting of 4-aminobutyric acid (GABA), tryptophan, lycopene, beta-carotene, vitamin B6, vitamin B12, coenzyme Q10, At least one of taurine, pectin, beta-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder, and dietary fiber.
  • GABA 4-aminobutyric acid
  • tryptophan tryptophan
  • lycopene beta-carotene
  • vitamin B6, vitamin B12 vitamin B12
  • coenzyme Q10 coenzyme Q10
  • taurine At least one of taurine, pectin, beta-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder, and dietary fiber.
  • the above-described product further includes an antioxidant.
  • the antioxidant is selected from at least one of tocopherols, carotenoids, ascorbic acid/vitamin C, ascorbyl palmitate, polyphenols, glutathione, and superoxide dismutase.
  • the mass percentage content of the above Lactobacillus reuteri is 1% to 30%. Further, the mass percentage content of the above Lactobacillus reuteri is 1% to 20%.
  • the viable bacteria content of the above Lactobacillus reuteri is 1.2 ⁇ 10 6 CFU/g to 1.6 ⁇ 10 12 CFU/g. Further, the viable bacteria content of the above Lactobacillus reuteri is 3.0 ⁇ 10 10 CFU/g to 2.0 ⁇ 10 11 CFU/g.
  • the Lactobacillus reuteri in the above product is a live bacteria. In other embodiments, the Lactobacillus reuteri in the above product is an inactivated cell of Lactobacillus reuteri. In other embodiments, the Lactobacillus reuteri in the above product is a mixture of live Lactobacillus reuteri and inactivated cells of Lactobacillus reuteri.
  • the dosage form of the above product is not particularly limited, for example, it can be powder, lozenge, tablet or capsule and the like.
  • the above product comprises 10 to 30 parts of Lactobacillus reuteri lyophilized powder, 15 to 25 parts of fructooligosaccharide, 40 to 65 parts of sorbitol or maltitol, and 5 to 10 parts of magnesium stearate, wherein, in the above product, the content of Lactobacillus reuteri is 1.2 ⁇ 10 6 CFU/g to 1.5 ⁇ 10 10 CFU/g.
  • the product in parts by weight, includes 0.5 to 30 parts of Lactobacillus reuteri LR99 lyophilized powder, 1 to 20 parts of other probiotics, and 20 to 80 parts of prebiotics , 2 to 10 parts of nutrients and 0.1 to 10 parts of antioxidants.
  • Other probiotics, prebiotics, nutrients and antioxidants are described above and will not be repeated here. It can be understood that the nutrients can be omitted, and can be added separately during use.
  • the above composition comprises 1 to 10 parts of Lactobacillus reuteri LR99 freeze-dried bacteria powder, 1 to 10 parts of other probiotics, 30 to 80 parts of prebiotics, and 2 to 5 parts of nutrition substances and 0.5 to 10 parts of antioxidants.
  • the content of Lactobacillus reuteri is 1.8 ⁇ 10 6 CFU/g ⁇ 6.5 ⁇ 10 11 CFU/g, and the content of single bacteria of other probiotics is 1 ⁇ 10 6 CFU/g ⁇ 6 ⁇ 10 9 CFU/g. Further, the content of Lactobacillus reuteri is 2.5 ⁇ 10 7 CFU/g to 1 ⁇ 10 11 CFU/g.
  • the above product includes the above-mentioned Lactobacillus reuteri and/or the fermented product of the above-mentioned Lactobacillus reuteri, improves intestinal flora disturbance, prevents obesity or promotes diet-induced weight loss, and also improves Prader-Willi
  • the brain development of patients with the syndrome can improve the social interaction disorder, communication disorder and fine motor ability of patients, and can be used to prevent or treat developmental disorders such as Prader-Willi syndrome and autism.
  • the above products are food, medicine, health care products or animal feed.
  • the final product is made by direct mixing or fermentation according to the conventional process.
  • the recommended dosage of the above-mentioned Lactobacillus reuteri for human use is 1.0 ⁇ 10 3 CFU/kg body weight/day to 1.0 ⁇ 10 10 CFU/kg body weight/day.
  • the recommended dosage of the above-mentioned Lactobacillus reuteri for human use is 1.0 ⁇ 10 4 CFU/kg body weight/day to 1.0 ⁇ 10 9 CFU/kg body weight/day.
  • the above-mentioned product is a food
  • the raw materials for preparing the food include the above-mentioned Lactobacillus reuteri and/or the fermentation product of the above-mentioned Lactobacillus reuteri, and food additives.
  • the food additive is selected from flavoring agents, sweeteners, thickeners, stabilizers, surfactants, lubricants, acid neutralizers, dispersants, buffers or buffers, debittering agents, pH stabilizers , at least one of preservatives, desugaring agents and coloring agents.
  • the food additive is selected from lactitol, sorbitol, maltitol, aspartame, stevia, monk fruit, sucralose, xylitol, vanilla, chocolate, fruit flavors and artificial flavors at least one.
  • the above product is fermented milk.
  • the preparation method of this fermented milk comprises the following steps:
  • Step a The milk source is mixed with water, homogenized and then sterilized to obtain a premix.
  • the milk source includes at least one of fresh milk, skimmed milk powder and whole milk powder.
  • the sterilization conditions are: 120°C to 122°C for 250s to 350s.
  • cooling is required after sterilization so that the temperature of the premix is 40°C to 45°C.
  • Step b adding the activated Lactobacillus reuteri strain to the premix for fermentation to prepare a fermented product.
  • the fermentation temperature is 40°C to 45°C; the fermentation time is 8h to 12h.
  • Step c cooling the fermented product of step b, and mixing with Lactobacillus reuteri again, so as to obtain fermented milk with a high content of Lactobacillus reuteri.
  • the fermented milk obtained by the above-mentioned fermented milk preparation method has high content and activity of the above-mentioned Lactobacillus reuteri and high nutritional value, and can be further used for the treatment or prevention of disease symptoms.
  • the above-mentioned product is a medicine
  • the raw materials for preparing the medicine include the above-mentioned Lactobacillus reuteri and/or the fermentation product of the above-mentioned Lactobacillus reuteri and pharmaceutically acceptable adjuvants.
  • an embodiment of the present application also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating intestinal flora disturbance.
  • the specific composition of the product for preventing or treating intestinal flora disturbance can be referred to the product for preventing or treating Prader-Willi syndrome, which will not be repeated here.
  • an embodiment of the present application also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating autism.
  • the specific composition of the product for preventing or treating autism developmental disorder refers to the product for preventing or treating Prader-Willi syndrome, which will not be repeated here.
  • an embodiment of the present application also provides a method for preventing or treating Prader-Willi syndrome, intestinal disorders and/or autism A method, the method comprising taking the above product, wherein the dose is 3.0 ⁇ 10 6 CFU/kg body weight/day to 1.2 ⁇ 10 11 CFU/kg body weight/day based on the quantity of probiotic bacteria in the product. Further, in terms of the number of microorganisms in the product, the dose is 1.0 ⁇ 10 6 CFU/kg body weight/day to 6.0 ⁇ 10 10 CFU/kg body weight/day.
  • a strain was isolated from the milk of healthy women by anaerobic culture. After 16S rRNA full-length sequencing and mass spectrometry identification, the strain was a new strain, belonging to Lactobacillus reuteri, named Reuteri Lactobacillus LR99 (or "LR99"). This strain has been deposited in the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) on December 31, 2020 , the classification name is Lactobacillus reuteri, and the deposit number is CGMCC No.21577.
  • CGMCC General Microbiology Center of China Microorganism Culture Collection Management Committee
  • Lactobacillus reuteri LR99 was observed under a microscope, and the results were shown in FIG. 1 .
  • Lactobacillus reuteri LR99 is a gram-positive bacterium, does not form spores, has no motility, is contact enzyme negative, oxidase negative, anaerobic, and the suitable culture temperature is 37°C.
  • Lactobacillus reuteri LR99 can metabolize ribose, xylose, maltose, lactose, raffinose, inulin, starch, mannose, melibiose, galactose, sucrose, L-arabinose and salicylic glycosides, but cannot metabolize trehalose, melanose, fructose, cellobiose, sodium gluconate, mannitol and sorbitol.
  • Lactobacillus reuteri LR99 To test the artificial gastric and intestinal juice tolerance of Lactobacillus reuteri LR99, and at the same time use the Lactobacillus reuteri isolated from commercial probiotic products, which has excellent acid resistance and can survive through the gastrointestinal tract. DSM17938 as a comparison.
  • Lactobacillus reuteri LR99 can still survive well after being digested by artificial gastric juice and intestinal juice. Compared with commercial strains, Lactobacillus reuteri LR99 has better resistance to digestive juice, and can survive and stabilize in the intestinal tract. Colonize.
  • Lactobacillus reuteri LR99 was resistant to Ampicillin, Penicillin G, Erythromycin, Chloramphenicol, Clindamycin, and Vancomycin. and tetracycline (Tetracycline) sensitive. It meets the requirements of the European Food Safety Authority (European Food Safety Authority) for the evaluation of drug resistance of edible bacteria; and Lactobacillus reuteri LR99 does not contain exogenous antibiotic resistance genes and is safe to eat.
  • This example is used to illustrate the preparation process of Lactobacillus reuteri LR99 high-density fermentation and freeze-dried bacterial powder.
  • Lactobacillus reuteri LR99 was anaerobic cultured in a modified MRS medium to obtain strains for fermentation.
  • Lactobacillus reuteri LR99 From the detection results of the growth curve of Lactobacillus reuteri LR99, it can be known that the time point when Lactobacillus reuteri LR99 reaches the end of the growth logarithm is the fermentation harvest point. Lactobacillus reuteri LR99 seeds were harvested when they reached late log phase. Harvested seeds were refrigerated at 4°C.
  • Fermentation tank upper tank the improved MRS medium is sterilized and then rapidly cooled to 37°C, inoculated into the fermentation tank with 3% inoculum for fermentation, the pH and OD values of the fermentation broth are detected every hour, and the fermentation
  • the pH and OD values are relatively flat, it means that the strain reaches the logarithmic end phase and is about to enter the stable phase.
  • the fermentation is completed, and the temperature is immediately lowered.
  • the fermentation broth is centrifuged at low temperature to collect the bacterial cells. After drying the protective agent (skimmed milk powder), emulsification is carried out, and after completion, vacuum freeze-drying is carried out to obtain bacterial powder, and the prepared freeze-dried bacterial powder is placed under -20° C. for storage.
  • the number of viable bacteria in the fermentation broth was 2.75 ⁇ 10 9 CFU/mL
  • the number of viable bacteria in the emulsion was 4.7 ⁇ 10 10 CFU/mL
  • the number of viable bacteria in the freeze-dried powder was 1.55 ⁇ 10 11 CFU/g.
  • Lactobacillus reuteri LR99 can improve the composition of intestinal flora in patients with Prader-Willi syndrome, reduce body weight, reduce obesity, and at the same time can improve brain development and improve the growth and development of patients. , improve autism symptoms. Specifically as follows:
  • Subjects and Recruitment Open enrollment of 71 patients with Prader-Willi syndrome aged 0.5 to 22 years who were randomly assigned to probiotics or placebo for a 12-week randomized, double-blind, placebo-controlled study test.
  • 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.
  • the probiotic group was Lactobacillus reuteri LR99 in powdered form in strips. Each bag of L. reuteri LR99 contains 3 x 10 10 colony forming units (CFU); the placebo is maltodextrin in the same package, similar in color, taste and flavor to the L. reuteri LR99 stick pack. Subjects received one sachet of Lactobacillus reuteri or 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.
  • CFU colony forming units
  • 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 Child Development Screening Scale
  • 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.
  • 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.
  • V3-V4 universal primers 341F (CCTACGGGNBGCASCAG, SEQ ID No. 1) and 805R (GACTACNVGGGTATCTAATCC, SEQ ID No. 2)
  • the PCR product was purified with 1 x KAPA AMPure (KAPA, cat. no. KK8002) by (95°C for 2 min, then 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s), followed by a final extension at 72°C for 5 min. Then, 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). 1 ⁇ KAPA AMPure purification PCR products were quantified by real-time PCR using the Bioanalyzer DNA kit.
  • FDR false discovery rate
  • 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 were confirmed between the placebo and probiotic groups 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 5 summarizes the demographic characteristics of the participants. No between-group differences were observed (P>0.05). Of the 71 people, 15 fell off, and the actual number was 56.
  • probiotic treatment also altered the composition of the patient's microbiome, enhancing microbiota that favors weight loss and gut health.
  • the gut microbiota encodes genes that are linked to calcium signaling, The abundance of genes involved in anti-inflammatory responses, antioxidant production and carbohydrate metabolism was enhanced.
  • Lactobacillus reuteri LR99 subjects significantly reduced body weight, and some communication impairments and motor coordination were significantly improved.
  • This study provides new evidence for Lactobacillus reuteri LR99 as an early intervention in PWS patients.
  • the present study found significant differences in gut microbiome beta diversity between the probiotic and placebo groups after treatment, and baseline beta diversity was directly associated with long-term weight loss when a controlled diet was adhered to. Therefore, supplementation with Lactobacillus reuteri LR99 has the effect of improving gut microbiota composition, preventing obesity or promoting diet-induced weight loss.
  • supplementation with Lactobacillus reuteri LR99 can also improve the growth and development of children and adolescents, especially social communication, social interaction and fine motor function, the improvement is the most obvious.

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Abstract

L'invention concerne une application de Lactobacillus reuteri dans la préparation d'un produit pour la prévention ou le traitement d'un trouble du développement. Le lactobacillus reuteri, dont le numéro de dépôt est CGMCC No. 21577, peut améliorer la composition du microbiote intestinal chez une personne atteinte d'un trouble du développement tel que le syndrome de Prader-Willi ou l'autisme, améliorer une perturbation du microbiote intestinal, prévenir l'obésité ou favoriser la perte de poids par le régime alimentaire, et peut également améliorer le développement du cerveau chez une personne atteinte d'un trouble du développement tel que le syndrome de Prader-Willi ou l'autisme, atténuer un trouble social ou un trouble de la communication de ladite personne, et améliorer la motricité fine.
PCT/CN2022/086587 2021-04-16 2022-04-13 Application de lactobacillus reuteri dans la préparation d'un produit pour la prévention ou le traitement d'un trouble du développement WO2022218336A1 (fr)

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CN115838676A (zh) * 2022-12-29 2023-03-24 青岛康迈臣生物科技有限责任公司 利用新型植物乳植杆菌菌株中药发酵及应用
CN116042470A (zh) * 2022-12-29 2023-05-02 青岛康迈臣生物科技有限责任公司 利用新型鼠李糖乳酪杆菌株中药发酵及其应用
CN116904371A (zh) * 2023-08-22 2023-10-20 湖南湘农动物药业有限公司 一种促生长的丁酸梭菌与罗伊氏乳杆菌后生元复合物及其制备方法
CN116904371B (zh) * 2023-08-22 2024-05-24 湖南湘农动物药业有限公司 一种促生长的丁酸梭菌与罗伊氏乳杆菌后生元复合物及其制备方法
CN117866854A (zh) * 2024-03-08 2024-04-12 广州同康生物科技有限公司 修复胃肠黏膜损伤的罗伊氏粘液乳杆菌bn01及其后生元
CN117866854B (zh) * 2024-03-08 2024-06-07 广州同康生物科技有限公司 修复胃肠黏膜损伤的罗伊氏粘液乳杆菌bn01及其后生元

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