WO2022218335A1 - 罗伊氏乳杆菌及其应用、组合物、药物和食品 - Google Patents

罗伊氏乳杆菌及其应用、组合物、药物和食品 Download PDF

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WO2022218335A1
WO2022218335A1 PCT/CN2022/086583 CN2022086583W WO2022218335A1 WO 2022218335 A1 WO2022218335 A1 WO 2022218335A1 CN 2022086583 W CN2022086583 W CN 2022086583W WO 2022218335 A1 WO2022218335 A1 WO 2022218335A1
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lactobacillus
lactobacillus reuteri
composition
reuteri
intestinal
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PCT/CN2022/086583
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English (en)
French (fr)
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段云峰
张烨
梁桦
崔雷
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 CA3215700A priority Critical patent/CA3215700A1/en
Priority to US18/287,028 priority patent/US20240197796A1/en
Priority to AU2022258164A priority patent/AU2022258164A1/en
Priority to GB2317588.8A priority patent/GB2622324A/en
Priority to JP2023563259A priority patent/JP2024516950A/ja
Publication of WO2022218335A1 publication Critical patent/WO2022218335A1/zh

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    • 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
    • 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
    • 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
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
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    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms
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    • 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
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    • 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
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/304—Foods, ingredients or supplements having a functional effect on health having a modulation effect on allergy and risk of allergy
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
    • 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
    • A23V2400/11—Lactobacillus
    • A23V2400/173—Reuteri
    • 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
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics
    • 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
    • C12R2001/225—Lactobacillus
    • 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 invention relates to the technical field of microorganisms, in particular to a Lactobacillus reuteri and its application, composition, medicine and food.
  • Allergic reaction refers to the tissue damage or functional disorder that occurs when the immune body is stimulated by the same antigen again. Globally, the number of people with allergies is increasing. In certain groups, the incidence of food allergy in children is as high as 10%.
  • Metabolites of the gut microbiota are important mediators between the microbiota and the host, by interacting with the immune system to inhibit its inflammatory response to allergens, or by reducing intestinal permeability to inhibit allergens from crossing the intestinal barrier , thereby promoting immune tolerance.
  • the disturbance of intestinal flora may cause allergy by increasing intestinal permeability.
  • the interior of the digestive tract is usually not immune-monitored, but if the permeability of the gut barrier is increased, food-derived proteins can leak out into the body, triggering an allergic reaction.
  • Some foods that cause children allergies, such as undigested proteins in peanuts and milk, are likely to pass through the damaged gut barrier intact and trigger an immune response.
  • mice were fed peanuts after treating their gut flora with antibiotics intact peanut protein was found in the blood of these mice.
  • 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 healthy women's milk (the deposit number is CGMCC No. 21577) can improve intestinal flora disturbance, reduce intestinal damage, improve intestinal permeability, and promote immune system. Restorative, has the effect of treating or preventing allergic reactions.
  • the present application provides a Lactobacillus reuteri, and the Lactobacillus reuteri has a deposit number of CGMCC No. 21577.
  • the present application also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating allergic reactions.
  • the present application also provides a composition comprising the above-mentioned Lactobacillus reuteri and/or the fermentation product of the above-mentioned Lactobacillus reuteri.
  • the present application also provides a medicine
  • the raw materials for preparing the medicine include the above-mentioned Lactobacillus reuteri or the above-mentioned composition, and pharmaceutically acceptable auxiliary materials.
  • the present application also provides a food, and the raw materials for preparing the food include the above-mentioned Lactobacillus reuteri or the above-mentioned composition.
  • the present application also provides a preparation method of the above-mentioned Lactobacillus reuteri, which adopts high-density fermentation to prepare the Lactobacillus reuteri, and the preparation method comprises the following steps:
  • the activated Lactobacillus reuteri was inoculated into a fermenter for fermentation.
  • the present application also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating and improving intestinal flora disturbance; or an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating intestinal leakage application.
  • the present application also provides a method for preventing or treating intestinal leakage and/or allergic reaction, the method comprises taking the above composition or taking the product prepared with the above composition as a raw material, wherein the Based on the number of probiotics in the composition, the dose is 3.0 ⁇ 10 6 CFU/kg body weight/day to 1.2 ⁇ 10 11 CFU/kg body weight/day.
  • Fig. 1 is a microscope observation diagram of Lactobacillus reuteri LR99 in Example 1;
  • Fig. 2 is the improvement situation of intestinal leakage after taking probiotics or placebo in Example 3;
  • Figure 3 is the improvement of CD4+ T cells after taking probiotics or placebo in Example 4.
  • Figure 4 is the composition of intestinal flora at the phylum level after taking probiotics or placebo in Example 4;
  • Figure 5 is a heat map of intestinal flora at the genus level after taking probiotics or placebo in Example 4;
  • Fig. 6 is the change of Shannon index of intestinal flora after taking probiotics or placebo in Example 4.
  • 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
  • the above-mentioned Lactobacillus reuteri can reprogram endothelial CD4+ T cells to immunoregulatory T cells, play an immunoregulatory role, promote the increase of Treg cells, and reduce the Th2 response, thereby facilitating intervention, alleviation or prevention of allergic reactions, and promoting immunity. system recovery.
  • the above-mentioned Lactobacillus reuteri also has the ability to metabolize tryptophan, and its metabolites of tryptophan can improve the intestinal barrier function, inhibit the increase of intestinal barrier permeability, reduce intestinal damage, prevent or alleviate Leaky gut can also further prevent or alleviate allergic reactions.
  • Lactobacillus reuteri can also improve the diversity of intestinal flora, increase the number of intestinal probiotics (such as Lactobacillus, Bifidobacterium, Akkermansia, etc.), and improve intestinal flora disturbance.
  • intestinal probiotics such as Lactobacillus, Bifidobacterium, Akkermansia, etc.
  • an embodiment of the present invention provides an application of the above Lactobacillus reuteri in preparing a product for preventing or treating allergic reactions.
  • the product is for the prevention or treatment of allergic diseases associated with neurodevelopmental disorders and/or mood disorders.
  • the developmental disorder of the nervous system is autism spectrum disorder (Autism Spectrum Disorder, ASD) or tic disorder; emotional disorders include ADHD, depression and the like.
  • Allergic diseases can occur at all ages, from newborns to the elderly, and often have obvious genetic predispositions.
  • immediate allergic reactions are more common, and the main types are skin allergic reactions, respiratory allergic reactions, digestive tract allergic reactions and anaphylactic shock.
  • an embodiment of the present invention also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating intestinal leakage.
  • an embodiment of the present invention also provides an application of the above-mentioned Lactobacillus reuteri in preparing a product for preventing or treating and improving intestinal flora disturbance.
  • An embodiment of the present invention 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 subjected to three times of activation and cultivation to improve the activity of the strain, and the temperature of the three times of 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 lyoprotectant 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 invention further provides a composition, the composition comprising the above-mentioned Lactobacillus reuteri and/or the fermentation product of the above-mentioned Lactobacillus reuteri.
  • the above-mentioned fermentation product of 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 above-mentioned Lactobacillus reuteri cultured culture solution or a lyophilized powder obtained by lyophilizing the culture solution is taken.
  • the above-mentioned composition comprises the above-mentioned Lactobacillus reuteri and auxiliary materials required for preparing a bacterial preparation.
  • the active ingredient of the above-mentioned composition includes the above-mentioned Lactobacillus reuteri.
  • the above-mentioned composition includes the above-mentioned Lactobacillus reuteri and a lyoprotectant. The lyoprotectant is as described above and will not be repeated here.
  • the above-described compositions further include other probiotics.
  • Probiotics are also used as active ingredients in the above-mentioned compositions. That is, the above-described composition includes active ingredients including the above-described Lactobacillus reuteri and other probiotics.
  • probiotics are selected from Lactobacillus (Lactobacillus), Bifidobacterium (Bifidobacterium), Streptococcus thermophilus (Streptococcus thermophilus), Lactococcus (Lactococcus), Propionibacterium (Propionibacterium), Leuconostoc (Leuconostoc) , Staphylococcus, Bacillus, Pediococcus, Escherichia.coli (eg Nissle1917), Prevotella, Faecalibacterium, Braut 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-described composition further includes a prebiotic.
  • the colonization and reproduction of the above-mentioned Lactobacillus reuteri are promoted by 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 composition further comprises a nutrient selected from the group consisting of GABA, tryptophan, lycopene, beta-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin At least one of , beta-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder and dietary fiber.
  • a nutrient selected from the group consisting of GABA, tryptophan, lycopene, beta-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin At least one of , beta-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder and dietary fiber.
  • the above-described composition 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 composition is a live bacteria. In other embodiments, the Lactobacillus reuteri in the above composition is an inactivated cell of Lactobacillus reuteri. In other embodiments, the Lactobacillus reuteri in the above composition is a mixture of live Lactobacillus reuteri and inactivated cells of Lactobacillus reuteri.
  • the dosage form of the above-mentioned composition is not particularly limited, for example, it can be powder, lozenge, tablet or capsule and the like.
  • the above-mentioned composition comprises 10-30 parts of Lactobacillus reuteri lyophilized powder, 15-25 parts of fructooligosaccharide, 40-65 parts of sorbitol or maltitol , and 5 to 10 parts of magnesium stearate, wherein, in the above composition, the content of Lactobacillus reuteri is 1.2 ⁇ 10 6 CFU/g to 1.5 ⁇ 10 10 CFU/g.
  • the above composition in parts by weight, comprises 0.5 to 30 parts of Lactobacillus reuteri LR99 freeze-dried bacteria powder, 1 to 20 parts of other probiotics, and 20 to 80 parts of probiotics Yuan, 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. Further, 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 substance 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-mentioned composition comprises the above-mentioned Lactobacillus reuteri and/or the fermentation product of the above-mentioned Lactobacillus reuteri, which can improve the diversity of intestinal flora and improve intestinal probiotics (such as Lactobacillus, Bifidobacterium and Ackermann). bacteria, etc.), improve intestinal flora disorder, reduce intestinal damage, prevent or alleviate intestinal leakage and allergic reactions, and promote the recovery of the immune system.
  • intestinal probiotics such as Lactobacillus, Bifidobacterium and Ackermann.
  • the above composition can be used as a raw material for preparing food, medicine, health product or animal feed.
  • it can be used as a raw material for preparing foods such as fermented milk, cheese, milk-containing beverages, solid beverages, milk powder, tablet candies, gelatin candies, fermented fruit and vegetable juices, and fermented soybean products.
  • the final product is made by direct mixing or fermentation according to the conventional process.
  • the above-mentioned composition can also be directly used as food or medicine.
  • 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.
  • An embodiment of the present invention also provides a food, and the raw materials for preparing the food include the above-mentioned Lactobacillus reuteri or the above-mentioned composition, and a food additive.
  • 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-mentioned food 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.
  • an embodiment of the present invention further provides a medicine, and the raw materials for preparing the medicine include the above-mentioned Lactobacillus reuteri or the above-mentioned composition, and pharmaceutically acceptable excipients .
  • an embodiment of the present invention also provides a method for preventing or treating intestinal leakage and/or allergic reaction, the method comprising taking the above composition or taking the above
  • the composition is a product prepared from raw materials, wherein, based on the number of probiotics in the composition, the dosage is 3.0 ⁇ 10 6 CFU/kg body weight/day to 1.2 ⁇ 10 11 CFU/kg body weight/day. Further, based on the number of microorganisms in the composition, 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 and named Lactobacillus reuteri LR99 (referred to as "LR99" or "LR-99”). 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.
  • Lactic acid has D type and L type.
  • the normal human body only has L-lactic acid.
  • D-lactic acid is a metabolite of bacterial fermentation. It can be produced by a variety of bacteria in the intestinal tract. Even if it is ingested from food, it is rarely absorbed into the blood under normal circumstances. , and mammals do not have an enzymatic system to rapidly degrade it.
  • 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, increasing the level of D-lactic acid in the blood.
  • Monitoring the level of D-lactic acid in blood can timely reflect the degree of intestinal mucosal damage and changes in permeability, and can be used for the auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory response, repeated fever, and vomiting.
  • LPS Lipopolysaccharide
  • TLR4 Lipopolysaccharide
  • 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 the intestinal epithelial cells is different, which can prevent the inflammatory response caused by LPS , and against pathogenic bacteria.
  • 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 content 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 intestinal infections. Auxiliary evaluation of endotoxemia, systemic inflammatory response, recurrent fever, vomiting, mental illness, stress response, etc.
  • Bacterial translocation refers to the passage of live intestinal bacteria from the gut through the epithelial mucosa into the body. Bacteria can enter the lymphatic system via the mesenteric lymph nodes and circulate throughout the body. Bacteria can enter the bloodstream causing bacteremia; they can also localize in tissues. Bacterial displacement can lead to small intestinal bacterial overgrowth, intestinal damage, and even shock.
  • the intestinal permeability of Lactobacillus reuteri LR99 was assessed by the content of D-lactic acid and LPS in mouse serum as follows:
  • mice Twelve 6-week-old C57BL/6J mice, 3 mice per cage, were raised with free food and water.
  • the growth environment of the mice was as follows: ambient temperature (23 ⁇ 2) °C, relative humidity (50 ⁇ 10 )%, light mode (12h dark/12h light).
  • the mice were randomly divided into a control group and a probiotic group, and the mice in the probiotic group were given a probiotic preparation (the probiotic preparation was prepared from Lactobacillus reuteri LR99 freeze-dried powder prepared in Example 2 and The composition of maltodextrin), the gavage dose was 10 billion CFU/mice/day; at the same time, the same amount of placebo (maltodextrin) was gavaged to the mice in the control group.
  • mice of each group were gavaged and fed for six weeks, and blood was collected from the tail vein terminal. The blood was centrifuged at 3000 g for 15 minutes.
  • the intestinal barrier function analysis system JY-DLT, Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd.
  • JY-DLT Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd.
  • SPSS SPSS statistical software
  • probiotics influence the differentiation of CD4+ T cells to regulate allergic diseases.
  • Probiotics can inhibit asthma, atopic dermatitis, allergic rhinitis, food, etc. by reducing Th2 cytokines (IL-4, IL-5, IL-13, etc.), reducing IgE, increasing IL-10, and promoting Treg differentiation. Allergies such as allergies and urticaria.
  • Studies have found that specific intestinal flora can metabolize tryptophan to indole-3-ethanol, indole-3-pyruvate or indole-3-acetaldehyde, which can improve the intestinal barrier function. In vivo, tryptophan metabolites can inhibit the increase in intestinal barrier permeability.
  • Lactobacillus reuteri LR99 can affect the differentiation of CD4+ T cells to regulate allergic diseases.
  • 18 6-week-old C57BL/6J mice were housed in groups of 3 mice per cage, with free food and water. After being adapted to the surrounding environment for one week, the mice were randomly divided into three groups, with 6 mice in each group, and were fed with placebo (control group), Lactobacillus reuteri LR99 freeze-dried powder (final viable count of 1 ⁇ 10 9 CFU/ only) (LR-99 group), and Lactobacillus reuteri LR99 lyophilized powder (the final viable count is 1 ⁇ 10 9 CFU/piece) + L-tryptophan (mass concentration 0.24%) (L-tryptophan Acid was obtained from Research Diets, LR-99 + tryptophan group).
  • Lactobacillus reuteri LR99 and Lactobacillus reuteri LR99 can significantly promote the differentiation of CD4+ T cells after binding to tryptophan (p ⁇ 0.05). Furthermore, L. reuteri LR99 alone and tryptophan did not significantly differ in increasing CD4+ T cell differentiation (p>0.05).
  • DNA of fecal flora was extracted from the cecal contents collected after each group of mice sacrificed in Example 4 using TIANmap Fecal DNA Kit (TIANGEN, catalog number DP328). The extracted DNA was quantitatively detected using a Qubit instrument. Use 1% agarose gel electrophoresis to detect: voltage 100V, 40min. The UVI gel imaging system photographed and recorded: DNA electrophoresis did not show hybrid bands and tailings, indicating that the DNA fragments were of good purity and had no obvious degradation. Take an appropriate amount of sample into a centrifuge tube and dilute the sample to 1ng/ ⁇ L with sterile water. DNA was stored in a -20°C refrigerator until use.
  • This example illustrates the improvement of allergic symptoms by Lactobacillus reuteri LR99 in children diagnosed with autism spectrum disorder (ASD).
  • ASD autism spectrum disorder

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Abstract

本发明涉及一种罗伊氏乳杆菌及其应用、组合物、药物和食品。该罗伊氏乳杆菌为保藏号为CGMCC No.21577，能够改善肠道菌群紊乱，减少肠道损伤，改善肠道通透性，促进免疫***恢复，具有治疗或预防自闭症、多动症、抽动症、抑郁症等神经***相关疾病过敏反应的效果。

Description

罗伊氏乳杆菌及其应用、组合物、药物和食品 技术领域
本发明涉及微生物技术领域，特别是涉及一种罗伊氏乳杆菌及其应用、组合物、药物和食品。
背景技术
过敏反应是指已产生免疫的机体在再次接受相同抗原刺激时所发生的组织损伤或功能紊乱的反应。在全球，过敏人群在不断增加。在特定群体中，儿童食物过敏的发病率高达10％。
早期接触微生物能对人体免疫健康产生积极影响的想法至少可以追溯到1989年，当时伦敦卫生与热带医学学院的流行病学家大卫·斯特拉昌(David Strachan)提出了“卫生假说”，认为更小、更清洁的家庭中长大的儿童更容易患过敏症。在随后的几十年中，卫生假说已经演变成以微生物群为中心的模型，在这个模型中，早期接触来自家庭和环境的微生物在降低哮喘、花粉热和湿疹等炎症性疾病以及食物过敏的风险方面发挥着关键作用。目前，“卫生假说”逐步演化为“早期环境影响微菌群从而导致免疫功能失调”的理论。在生命早期，肠道菌群失调如多样性低、肠杆菌/拟杆菌比高，对过敏会产生巨大影响。肠道菌群的代谢产物是菌群与宿主间的重要介质，通过与免疫***互作以抑制其对过敏原的炎症应答，或通过降低肠道通透性以抑制过敏原穿过肠道屏障，从而促进免疫耐受。
肠道菌群的紊乱引起过敏可能是通过增加肠道通透性发挥作用的。有研究 发现，食物过敏的婴儿会出现肠漏，表现为血液中抗原和过敏原从肠道转移的增加。消化道的内部通常不受免疫监测，但如果肠道屏障的通透性增加，食物衍生的蛋白质会渗出到达体内，引发过敏反应。一些引起儿童过敏的食物，例如花生和牛奶中未能消化的蛋白质，这些蛋白质很可能完好无损地穿过受损的肠道屏障，并引发免疫反应。有研究发现，当用抗生素处理小鼠的肠道菌群后，再给它们喂食花生，这些小鼠的血液中可以发现完整花生蛋白。
罗伊氏乳杆菌(Lactobacillus reuteri)是存在于肠道内的乳酸菌，作为益生菌使用，对肠黏膜具有很强的黏附能力，并且还具有优异的耐酸、耐胆盐和广谱抑菌特性，能广泛抑制革兰氏阳性菌、革兰氏阴性菌、酵母、真菌和病原虫等的生长。
发明内容
本申请经研究发现，从健康女性乳汁中分离的罗伊氏乳杆菌(保藏号为CGMCC No.21577)能够改善肠道菌群紊乱，减少肠道损伤，改善肠道通透性，促进免疫***恢复，具有治疗或预防过敏反应的效果。
因此，本申请提供了一种罗伊氏乳杆菌(Lactobacillus reuteri)，所述罗伊氏乳杆菌为保藏号为CGMCC No.21577。
此外，本申请还提供了一种上述罗伊氏乳杆菌在制备预防或治疗过敏反应的产品中的应用。
另外，本申请还提供了一种组合物，包括上述罗伊氏乳杆菌和/或上述罗伊氏乳杆菌的发酵产物。
另外，本申请还提供了一种药物，制备所述药物的原料包括上述罗伊氏乳杆菌或上述组合物，和药学上可接受的辅料。
另外，本申请还提供了一种食品，制备所述食品的原料包括权上述罗伊氏乳杆菌或上述组合物。
另外，本申请还提供了一种上述罗伊氏乳杆菌的制备方法，采用高密度发酵制备所述罗伊氏乳杆菌，所述制备方法包括以下步骤：
活化所述罗伊氏乳杆菌；及
将活化后的上所述罗伊氏乳杆菌接种到发酵罐中发酵培养。
另外，本申请还提供了一种上述罗伊氏乳杆菌在制备预防或治疗改善肠道菌群紊乱的产品中的应用；或上述罗伊氏乳杆菌在制备预防或治疗肠漏的产品中的应用。
另外，本申请还提供了一种用于预防或治疗肠漏和/或过敏反应的方法，所述方法包括服用上述的组合物或服用以上述的组合物为原料制备的产品，其中，以所述组合物中的益生菌的数量计，剂量为3.0×10 6CFU/kg体重/天～1.2×10 11CFU/kg体重/天。
本申请的一个或多个实施例的细节在下面的附图和描述中提出，本申请的其它特征、目的和优点将从说明书、附图及权利要求书变得明显。
附图说明
为了更好地描述和说明这里公开的那些发明的实施例和/或示例，可以参考一副或多副附图。用于描述附图的附加细节或示例不应当被认为是对所公开的发明、目前描述的实施例和/或示例以及目前理解的这些发明的最佳模式中的任何一者的范围的限制。
图1是实施例1中罗伊氏乳杆菌LR99显微镜观察图；
图2是实施例3中服用益生菌或安慰剂后肠漏改善情况；
图3是实施例4中服用益生菌或安慰剂后CD4+T细胞的改善情况；
图4是实施例4中服用益生菌或安慰剂后肠道菌群在门水平的组成；
图5是实施例4中服用益生菌或安慰剂后肠道菌群在属水平的热图；
图6是实施例4中服用益生菌或安慰剂后肠道菌群Shannon指数的改变；
图7～图9是实施例4中服用益生菌或安慰剂后肠道菌群在代表性有益菌种组成的对比。
本申请提供的罗伊氏乳杆菌(Lactobacillus reuteri)，菌株名为LR99，保藏于中国微生物菌种保藏管理委员会普通微生物中心，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，保藏编号为CGMCC No.21577，该菌株于2020年12月31日由保藏中心收到并登记入册，且经保藏中心于2020年12月31日检测为存活菌株。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使本发明公开内容更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本发明一实施方式提供了一种罗伊氏乳杆菌，该罗伊氏乳杆菌的编号为LR99，从健康女性乳汁中分离得到。已于2020年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：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，邮编100101)，分类命名为罗伊氏 乳杆菌(Lactobacillus reuteri)，保藏号为CGMCC No.21577。
经验证，上述罗伊氏乳杆菌可以重新编程内皮CD4+T细胞到免疫调节T细胞，发挥免疫调节作用，促进Treg细胞增加，使得Th2反应降低，从而利于干预、缓解或预防过敏反应，促进免疫***恢复。并且，上述罗伊氏乳杆菌也具有代谢色氨酸的能力，其色氨酸的代谢产物可以改善肠道的屏障功能，抑制肠道屏障通透性的增加，减少肠道损伤，预防或缓解肠漏，也能进一步预防或缓解过敏反应。此外，上述罗伊氏乳杆菌还能够提高肠道菌群的多样性，提高肠道益生菌(例如乳酸杆菌、双歧杆菌和阿克曼氏菌等)的数量，改善肠道菌群紊乱。
基于此，本发明一实施方式提供一种上述罗伊氏乳杆菌在制备预防或治疗过敏性反应的产品中的应用。可选地，产品是用于预防或治疗神经***发育障碍和/或情绪障碍伴随的过敏性疾病。具体地，神经***发育障碍为自闭症谱系障碍(Autism Spectrum Disorder，ASD)或抽动症；情绪障碍包括多动症、抑郁症等。过敏性疾病从新生儿到老年人的各个年龄阶段都可能发生，往往具有明显的遗传倾向。在过敏性疾病中，以速发型过敏反应比较常见，其主要类型有皮肤过敏反应、呼吸道过敏反应、消化道过敏反应及过敏性休克等。
基于上述罗伊氏乳杆菌的上述功能，本发明一实施方式还提供了一种上述罗伊氏乳杆菌在制备预防或治疗肠漏的产品中的应用。
基于上述罗伊氏乳杆菌的上述功能，本发明一实施方式还提供了一种上述罗伊氏乳杆菌在制备预防或治疗改善肠道菌群紊乱的产品中的应用。
本发明一实施方式还提供了一种上述罗伊氏乳杆菌的制备方法，该制备方法采用高密度发酵制备上述罗伊氏乳杆菌。具体地，该制备方法包括步骤S1～S3。
步骤S1：活化上述罗伊氏乳杆菌。
具体地，对罗伊氏乳杆菌菌行三次活化培养用于提高菌株活力，三次活化培养的温度为37℃，时间为16h～18h。
步骤S2：将活化后的上述罗伊氏乳杆菌接种到发酵罐中发酵培养。
将改良的MRS培养基进行灭菌处理后降温到37℃，以2.5％～3.5％(v/v)的接种量接种到发酵罐进行发酵，每小时检测发酵液的pH和OD值，待发酵到pH和OD值相对平缓则说明菌株到达对数末期即将进入稳定期，发酵完成。然后对发酵液降温、低温离心、收集菌体及磷酸盐缓冲液(PBS)洗涤，制备罗伊氏乳杆菌菌体。
步骤S3：将步骤S2制得的菌体冻干，制成冻干粉。
具体地，将步骤S2制得的菌体与冻干保护剂混合后乳化，然后真空冷冻干燥，制备冻干粉。可选地，冻干保护剂选自脱脂奶粉、海藻糖、低聚果糖、乳糖、葡萄糖、蔗糖、L-抗坏血酸钠、L-苹果酸及L-乳酸中的至少一种。当然，在其他实施例中，冻干保护剂不限于上述，还可以是其他能够在冻干过程中保持菌体活性的物质。可选地，菌体与冻干保护剂的体积之比为1：(2～10)。
可以理解的是，在一些实施例中，步骤S3可以省略。此时，制备的是罗伊氏乳杆菌的菌体。
上述罗伊氏乳杆菌的制备方法采用高密度发酵制备上述罗伊氏乳杆菌，产量高。
此外，基于上述罗伊氏乳杆菌的上述功能，本发明一实施方式还提供了一种组合物，该组合物包括上述罗伊氏乳杆菌和/或上述罗伊氏乳杆菌的发酵产物。
具体地，上述罗伊氏乳杆菌的发酵产物是指上述罗伊氏乳杆菌的培养产物，包括位于菌体内的代谢产物和分泌到菌体外的代谢产物中的至少一种。在使用时，取上述罗伊氏乳杆菌培养之后的培养液或将该培养液的进行冻干处理而得 到的冻干粉末。或者，取上述罗伊氏乳杆菌培养之后裂解的裂解液或将该裂解液的进行纯化、冻干处理而得到的冻干粉末。
在一些实施例中，上述组合物包括上述罗伊氏乳杆菌和制成菌剂所需的辅料。上述组合物的活性成分包括上述罗伊氏乳杆菌。在一个可选地具体示例中，上述组合物包括上述罗伊氏乳杆菌和冻干保护剂。冻干保护剂如上文所述，此处不再赘述。
在一些实施例中，上述组合物还包括其他益生菌。益生菌也作为上述组合物的活性成分。也即是，上述组合物包括活性成分，该活性成分包括上述罗伊氏乳杆菌和其他益生菌。可选地，其他益生菌选自乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)、嗜热链球菌(Streptococcus thermophilus)、乳球菌(Lactococcus)、丙酸杆菌(Propionibacterium)、明串球菌(Leuconostoc)、葡萄球菌(Staphylococcus)、芽孢杆菌(Bacillus)、片球菌(Pediococcus)、大肠杆菌(Escherichia.coli)(例如Nissle1917)、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)和酵母菌中的至少一种。
在一个可选地具体示例中，乳酸杆菌选自植物乳杆菌(Lactobacillus plantarum)、发酵乳杆菌(Lactobacillus fermentum)、嗜酸乳杆菌(Lactobacillus acidophilus)、干酪乳杆菌(Lactobacillus casei)、卷曲乳杆菌(Lactobacillus crispatus)、保加利亚乳杆菌(Lactobacillus bulgaricus)、德氏乳杆菌乳亚种(Lactobacillus delbrueckii subsp.Lactis)、格氏乳杆菌(Lactobacillus gasseri)、约氏乳杆菌(Lactobacillus johnsonii)、副干酪乳杆菌(Lactobacillus paracasei)、罗伊氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、唾液乳杆菌(Lactobacillus salivarius)、清酒乳杆菌(Lactobacillus sakei)和瑞士 乳杆菌(Lactobacillus helveticus)中的至少一种。
在一个可选地具体示例中，双歧杆菌选自青春双歧杆菌(Bifidobacterium adolescentic)、短双歧杆菌(Bifidobacterium breve)、长双歧杆菌(Bifidobacterium longum)、婴儿双歧杆菌(Bifidobacterium infantis)、两歧双歧杆菌(Bifidobacterium bifidum)、动物双歧杆菌(Bifidobaterium animalis)和乳双歧杆菌(Bifidobaterium lactis)中的至少一种。
在一个可选的具体示例中，乳球菌选自乳酸乳球菌乳酸亚种(Lactococcus Lactis subsp.Lactis)、乳酸乳球菌乳脂亚种(Lactococcus Lactis subsp.Cremoris)和乳酸乳球菌双乙酰亚种(Lactococcus Lactis subsp.Diacetylactis)中的至少一种。
在一个可选的具体示例中，丙酸杆菌选自费氏丙酸杆菌谢氏亚种(Propionibacterium freudenreichii subsp.Shermanii)和产丙酸丙酸杆菌(Propionibacterium acidipropionici)中的至少一种。明串球菌为肠膜明串珠菌肠膜亚种(Leuconostoc mesenteroides subsp.Mesenteroides)。片球菌选自乳酸片球菌(Pediococcus acidilactici)和戊糖片球菌(Pediococcus pentosaceus)中的至少一种。葡萄球菌选自小牛葡萄球菌(Staphylococcus vitulinus)、肉葡萄球菌(Staphylococcus xylosus)和木糖葡萄球菌(Staphylococcus carnosus)中的至少一种。芽孢杆菌为凝结芽孢杆菌(Bacillus coagulans)。酵母菌选自马克斯克鲁维酵母(Kluyveromyces marxianus)、酿酒酵母(Saccharomyces cerevisiae)、产朊假丝酵母(Cadida atilis)、乳酸克鲁维酵母(Kluyveromyces lactis)和卡氏酵母(Saccharomyces carlsbergensis)中的至少一种。可以理解的是，本实施方式中的其他益生菌不限于上述，还可以是除其他LR99外的益生菌。
在一些是实施例中，上述组合物还包括益生元。通过益生元促进上述罗伊 氏乳杆菌的定植及繁殖，同时也促进肠道中的其他益生菌的生长。可选地，益生元选自菊粉、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***糖、抗性糊精和抗性淀粉中的至少一种。当然，在其他实施例中，上述组合物中的益生元不限于上述，还可以是其他可以促进益生菌生长和繁殖的物质。
在一些实施例中，上述组合物还包括营养物质，所述营养物质选自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、柑橘纤维、苹果纤维、小球藻、苜蓿粉、青汁粉和膳食纤维中的至少一种。
在一些实施例中，上述组合物还包括抗氧化剂。具体地，抗氧化剂选自生育酚、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中的至少一种。
在一些实施例中，在上述组合物中，上述罗伊氏乳杆菌的质量百分含量为1％～30％。进一步地，上述罗伊氏乳杆菌的质量百分含量为1％～20％。可选地，在上述组合物中，上述罗伊氏乳杆菌的活菌含量为1.2×10 6CFU/g～1.6×10 12CFU/g。进一步地，上述罗伊氏乳杆菌的活菌含量为3.0×10 10CFU/g～2.0×10 11CFU/g。
在一些实施例中，上述组合物中的罗伊氏乳杆菌为活菌。在另一些实施例中，上述组合物中的罗伊氏乳杆菌为罗伊氏乳杆菌的灭活菌体。在另一些实施例中，上述组合物中的罗伊氏乳杆菌为罗伊氏乳杆菌活菌和罗伊氏乳杆菌的灭活菌体的混合物。
上述组合物的剂型没有特别限制，例如可以是粉剂、锭剂、片剂或胶囊剂等。
在一些实施例中，以重量份数计，上述组合物包括罗伊氏乳杆菌冻干粉10份～30份、低聚果糖15份～25份、山梨糖醇或麦芽糖醇40份～65份、和硬脂酸镁5份～10份，其中，在上述组合物中，罗伊氏乳杆菌的含量为1.2×10 6CFU/g～1.5×10 10CFU/g。
在一些实施例中，以重量份数计，上述组合物包括0.5份～30份的罗伊氏乳杆菌LR99冻干菌粉、1份～20份的其他益生菌、20份～80份的益生元、2份～10份的营养物质和0.1份～10份的抗氧化剂。其他益生菌、益生元、营养物质和抗氧化剂如上述，此处不再赘述。进一步地，上述组合物包括1份～10份的罗伊氏乳杆菌LR99冻干菌粉、1份～10份的其他益生菌、30份～80份的益生元、2份～5份的营养物质和0.5份～10份的的抗氧化剂。可选地，在上述组合物中，罗伊氏乳杆菌的含量为1.8×10 6CFU/g～6.5×10 11CFU/g，其它益生菌的单菌的含量为1×10 6CFU/g～6×10 9CFU/g。进一步地，罗伊氏乳杆菌的含量为2.5×10 7CFU/g～1×10 11CFU/g。
上述组合物包括上述罗伊氏乳杆菌和/或上述罗伊氏乳杆菌的发酵产物，能够提高肠道菌群的多样性，提高肠道益生菌(例如乳酸杆菌、双歧杆菌及阿克曼氏菌等)的数量，改善肠道菌群紊乱，减少肠道损伤，能够预防或缓解肠漏和过敏反应，促进免疫***恢复。
上述组合物可以作为制备用于食品、药品、保健品或动物饲料的原料。例如可以作为制备发酵乳、乳酪、含乳饮料、固体饮料、乳粉、压片糖果、凝胶软糖、发酵果蔬汁、发酵豆制品等食品的原料。使用时，按常规工艺，直接混合或经过发酵之后做出终产品。可以理解的是，在一些实施方式中，上述组合物也直接可以作为食品或药品。可选地，在食品中，上述罗伊氏乳杆菌供人类使用的建议的用量为1.0×10 3CFU/kg体重/天～1.0×10 10CFU/kg体重/天。进一步 地，上述罗伊氏乳杆菌供人类使用的建议用量为1.0×10 4CFU/kg体重/天～1.0×10 9CFU/kg体重/天。
本发明一实施方式还提供了一种食品，制备该食品的原料包括上述罗伊氏乳杆菌或上述组合物、和食品添加剂。可选地，食品添加剂选自调味剂、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、脱糖剂和着色剂中的至少一种。在一个具体的示例中，食品添加剂选自乳糖醇、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、水果香料和人造香精中的至少一种。
在其中一个实施例中，上述食品为发酵乳。该发酵乳的制备方法包括以下步骤：
步骤a：将奶源与水混合并均质后灭菌，得到预混料。
具体地，奶源包括鲜奶、脱脂乳粉和全脂乳粉中的至少一种。灭菌的条件为：120℃～122℃的条件下进行250s～350s，当然，在灭菌之后需要冷却，以使预混料的温度为40℃～45℃。
步骤b：向预混料中加入活化好的罗伊氏乳杆菌菌种进行发酵，制备发酵处理产物。
具体地，发酵温度40℃～45℃；发酵时间8h～12h。
步骤c：将步骤b的发酵处理产物进行冷却，再次与罗伊氏乳杆菌进行混合，以便获得高含量罗伊氏乳杆菌的发酵乳。
上述发酵乳的制备方法获得的发酵乳中上述罗伊氏乳杆菌的含量和活性高，营养价值高，可以进一步用于疾病症状的治疗或预防。
基于上述罗伊氏乳杆菌或上述组合物的功能，本发明一实施方式还提供了 一种药品，制备该药品的原料包括上述罗伊氏乳杆菌或上述组合物、和药学上可接受的辅料。
基于上述罗伊氏乳杆菌或上述组合物的功能，本发明一实施方式还提供了一种用于预防或治疗肠漏和/或过敏反应的方法，该方法包括服用上述组合物或服用以上述组合物为原料制备的产品，其中，以组合物中的益生菌的数量计，剂量为3.0×10 6CFU/kg体重/天～1.2×10 11CFU/kg体重/天。进一步地，以组合物中的微生物的数量计，剂量为1.0×10 6CFU/kg体重/天～6.0×10 10CFU/kg体重/天。
具体实施例
以下结合具体实施例进行详细说明。以下实施例如未特殊说明，则不包括除不可避免的杂质外的其他组分。实施例中采用试剂和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，按照常规条件，例如文献、书本中所述的条件或者生产厂家推荐的方法实现。
实施例1
从健康女性的乳汁中采用厌氧培养分离一株菌株，经16S rRNA全长测序鉴定和质谱鉴定，该菌株是新菌株，属于罗伊氏乳杆菌(Lactobacillus reuteri)，命名为罗伊氏乳杆菌LR99(简称“LR99”或“LR-99”)。该菌株已于2020年12月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：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，邮编100101)，分类命名为罗伊氏乳杆菌(Lactobacillus reuteri)，保藏号为CGMCC No.21577。
1.罗伊氏乳杆菌LR99的分类学特征：
(1)显微镜下观察罗伊氏乳杆菌LR99，结果如图1所示。
(2)理化试验结果如表1和2所示。
表1
Figure PCTCN2022086583-appb-000001
由表1可知，罗伊氏乳杆菌LR99为革兰氏阳性菌，不形成芽孢，无运动性，接触酶阴性，氧化酶阴性，厌氧，适宜培养温度为37℃。
表2
Figure PCTCN2022086583-appb-000002
表2中“+”表示能够代谢；“-”表示无法代谢。
由表2可知，罗伊氏乳杆菌LR99能够代谢核糖、木糖、麦芽糖、乳糖、棉子糖、菊粉、淀粉、甘露糖、蜜二糖、半乳糖、蔗糖、L-***糖和水杨苷，但不能代谢海藻糖、松三糖、果糖、纤维二糖、葡萄糖酸钠、甘露醇和山梨醇。
2.罗伊氏乳杆菌LR99的人工胃液和肠液耐受性：
测试罗伊氏乳杆菌LR99的人工胃液和肠液耐受性，同时以目前实验室保存的，从市售益生菌产品中分离的耐酸性能极好、可以通过胃肠道存活的罗伊氏乳杆菌DSM17938作为对比。
罗伊氏乳杆菌DSM17938和罗伊氏乳杆菌LR99在人工胃酸(pH＝3)和人工肠液(pH＝8)中的存活率检测结果如表3所示。
表3
Figure PCTCN2022086583-appb-000003
由表3可知，罗伊氏乳杆菌DSM17938在人工胃液中处理1h时活菌存活率71.2％，处理1.5h活菌存活率33.1％，而罗伊氏乳杆菌LR99处理1h时活菌存活率93.1％，处理1.5h活菌存活率69.7％，这表明罗伊氏乳杆菌LR99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作用。
由表3可知，罗伊氏乳杆菌DSM17938在人工肠液(pH＝8)中处理1小时活菌存活率25.8％，处理2小时后的存活率为18.5％；而罗伊氏乳杆菌LR99在人工肠液中处理1小时活菌存活率48.8％，处理2h后的存活率为37.8％。
上述结果表明，经过人工胃液和肠液消化后罗伊氏乳杆菌LR99仍能较好的存活，罗伊氏乳杆菌LR99相对商业化菌株具有较好耐消化液能力，可以在肠道内顺利存活并定殖。
3.罗伊氏乳杆菌LR99毒力实验及安全性检测：
(1)将罗伊氏乳杆菌LR99接种于MRS液体培养基中，37℃厌氧培养48小时，计数培养液中罗伊氏乳杆菌LR99活菌数为3.2×10 9CFU/mL，然后将培养液原液经口以20.0mL/kg体重的比例连续给小鼠(健康雄性BALB/C小鼠，6～8周龄，体重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)灌胃3天，之后再观察7天。同时将MRS液体培养基以 20.0mL/kg体重的量灌胃小鼠作为对照组。
结果显示，与对照组相比，罗伊氏乳杆菌LR99的培养物原液，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)。
(2)采用SN/T 1944-2007《动物及其制品中细菌耐药性的测定》方法，评估罗伊氏乳杆菌LR99的抗生素敏感性能。
结果显示，罗伊氏乳杆菌LR99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；并且罗伊氏乳杆菌LR99不含外源抗生素耐药基因，食用安全。
实施例2
本实施例用于说明罗伊氏乳杆菌LR99高密度发酵及冻干菌粉的制备过程。
(1)将罗伊氏乳杆菌LR99在采用改良MRS培养基中进行厌氧培养，获得发酵用菌种。
(2)对罗伊氏乳杆菌LR99菌株进行三次活化培养用于提高菌株活力，三次活化培养的温度为37℃，时间为16h～18h之间，菌株活化完成后，对罗伊氏乳杆菌LR99株菌进行生长曲线检测，结果如表4所示。
表4
时间 pH OD
5h 4.48 4.5
6h 4.31 4.71
7h 4.17 6.05
8h 4.06 6.97
9h 3.73 7.26
10h 3.67 7.78
由罗伊氏乳杆菌LR99的生长曲线检测结果可知，罗伊氏乳杆菌LR99到达生长对数末期的时间点，即发酵收获点。对罗伊氏乳杆菌LR99种子进行培养达到对数末期时进行收获。收获的种子冷藏在4℃冰箱。
(3)发酵罐上罐：将改良的MRS培养基进行灭菌处理后迅速降温到37℃，以3％接种量接种到发酵罐进行发酵，每小时检测发酵液的pH和OD值，待发酵到pH和OD值相对平缓则说明菌株到达对数末期即将进入稳定期，此时发酵完成，立即进行降温，发酵液进行低温离心、收集菌体，磷酸盐缓冲液(PBS)洗涤后，加冻干保护剂(脱脂奶粉)后进行乳化，完成后进行真空冷冻干燥，得到菌粉，将制备的冻干菌粉放置于-20℃以下保存。其中，经检测，发酵液中的活菌数为2.75×10 9CFU/mL，乳化液中的活菌数为4.7×10 10CFU/mL，冻干粉中的活菌数为1.55×10 11CFU/g。
实施例3
本实施例用于说明罗伊氏乳杆菌LR99对改善肠漏的影响
乳酸有D型和L型，正常人体只有L-乳酸，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***。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。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-核心寡糖和高变的多糖结构域-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。
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***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。
任何导致肠道通透性增加的应激反应，包括心理的和生理的，都可能潜在地导致细菌位移。细菌移位是指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***进入淋巴***在全身循环。细菌能进入血液循环导致菌血症；还可以位于组织中。细菌位移可使小肠细菌过度生长、肠道损伤，甚至休克。
通过小鼠血清中D-乳酸和LPS含量来评估罗伊氏乳杆菌LR99对肠道通透性，具体如下：
将6周龄的C57BL/6J小鼠12只，每笼3只小鼠进行饲养，自由进食和饮水，小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)。待小鼠适应环境一周后，随机分为对照组和益生菌组， 并对益生菌组小鼠灌胃益生菌制剂(益生菌制剂由实施例2制备的罗伊氏乳杆菌LR99冻干粉和麦芽糊精组成)，灌胃剂量为100亿CFU/只/天；同时对对照组小鼠灌胃等量的安慰剂(麦芽糊精)。
从第2周开始，饲喂安慰剂或益生菌，同时给各组进行噪声和夜间光照的复合应激刺激。
将各组小鼠灌胃并饲养六周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***(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-乳酸和LPS含量。然后将各组小鼠血清中D-乳酸和LPS含量采用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，结果如图2所示。
由图2可知，与对照组相比，益生菌组的LPS和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，全身性炎症反应等风险。
实施例4
本实施例用于说明罗伊氏乳杆菌LR99对免疫***的促进作用
在动物模型及人体研究中，益生菌影响CD4+T细胞分化以调控过敏性疾病。益生菌可通过减少Th2细胞因子(IL-4、IL-5、IL-13等)、减少IgE、增加IL-10、促进Treg分化等方式，抑制哮喘、特应性皮炎、过敏性鼻炎、食物过敏及荨麻疹等过敏性疾病。研究发现，特定肠道菌群可以将色氨酸代谢为吲哚-3-乙醇、吲哚-3-丙酮酸或吲哚-3-乙醛，可改善肠道的屏障功能。在体内，色氨酸代谢产物可以抑制肠道屏障通透性的增加。
为了检验罗伊氏乳杆菌LR99是否能影响CD4+T细胞分化以调控过敏性疾病，将6周龄的C57BL/6J小鼠18只，每笼3只小鼠分组饲养，自由进食和饮水。适应周围环境一周之后，随机分为三组，每组小鼠6只，分别每天喂养安慰剂(对照组)、罗伊氏乳杆菌LR99冻干粉(终活菌数为1×10 9CFU/只)(LR-99组)、和罗伊氏乳杆菌LR99冻干粉(终活菌数为1×10 9CFU/只)+L-色氨酸(质量浓度0.24％)(L-色氨酸从Research Diets获得，LR-99+色氨酸组)。喂养4周后，处死小鼠后将肠道和脾脏取出。通过机械破坏法制备脾脏的单细胞悬液以分离T细胞。脾脏用胶原酶D 100ng/mL(Invitrogen)消化以进行DC分离。对于流式细胞术，使用以下荧光团标记的单克隆抗体：来自BD Biosciences的抗CD8(53-6.7)，抗CD4(GK1.5)对于幼稚T细胞分离，用“幼稚的CD4+T细胞分离试剂盒”(Miltenyi Biotec)富集单细胞悬液，然后分选CD4+细胞。使用FACSCantoII(BD Biosciences)处理样品，使用FACSAria II(BD Biosciences)进行荧光激活的细胞分选，用FlowJo软件(TreeStar)分析数据，结果如图3所示。
由图3可知，罗伊氏乳杆菌LR99和罗伊氏乳杆菌LR99结合色氨酸后均可显著促进CD4+T细胞分化(p<0.05)。此外，单独罗伊氏乳杆菌LR99与结合色氨酸之后对增加CD4+T细胞分化没有显著差异(p>0.05)。
实施例5
本实施例用于说明LR99对肠道微生物组成的影响
(1)提取DNA：将实施例4处死的各组小鼠后收集的盲肠内容物采用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***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。
(2)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V3-V4通用引物341F(CCTACGGGNBGCASCAG，SEQ ID No.1)和805R(:GACTACNVGGGTATCTAATCC，SEQ ID No.2)扩增细菌16S rRNA基因的V3-V4区域。100ng的样本中提取的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。
(3)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 HiSeq2500 PE250测序平台进行菌群测序。
(4)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***发育树。经质控后，将过滤后的ASVs用
Figure PCTCN2022086583-appb-000004
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。
结果表明，在门水平，对照组、益生菌组(LR99)和益生菌结合色氨酸组(LR99+TRP)的肠道菌群存在明显差异。对照组的拟杆菌门低于益生菌组(图4)。益生菌组的alpha多样性指数也高于对照组，Shannon指数差异不显著 (p＝0.45064(Mann-Whitney statistic))(图5)。热图分析显示，在属水平，三组菌群组成存在差异(图6)。在属水平，具体的差异菌较多，其中使用LR99后，乳酸杆菌(Lactobacillus)、双歧杆菌(Bifidobacterium)和阿克曼氏菌(Akkermansia)明显增多，尤其是对于乳酸杆菌，色氨酸的加入似乎可以更加促进其丰度增多(图7～图9)。
实施例6
本实施例用于说明由罗伊氏乳杆菌LR99菌对被诊断患有自闭症谱系障碍(ASD)的儿童过敏症状的改善。
儿童年龄7岁，男孩。以口服罗伊氏乳杆菌LR99冻干粉(组成为罗伊氏乳杆菌LR99和冻干粉保护剂)的形式，每天3次，每次600亿CFU，服用周期30天。
在服用前后，记录孩子的肠道，皮肤症状。采集血液检测IgE和慢性食物过敏。30天后，该儿童腹泻、腹胀症状有所改变，大便呈粘液状改善，出现数次成型便，排便次数由1天2次～3次，改为1天～2天一次；皮肤炎症状改善，抓挠减少。同时，家长反馈孩子烦躁的次数减少，词汇量有少量增加。血液检测结果显示IgE由服用前的572.2IU/mL，降低到368.7IU/mL。慢性食物过敏检测显示，鸡蛋和牛奶指标稍有降低。家长选择了继续服用，仍在观察和记录持续改善情况。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的 普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (20)

  1. 一种罗伊氏乳杆菌(Lactobacillus reuteri)，所述罗伊氏乳杆菌为保藏号为CGMCC No.21577。
  2. 权利要求1所述的罗伊氏乳杆菌在制备预防或治疗过敏反应的产品中的应用。
  3. 根据权利要求2所述的应用，其特征在于，所述产品是用于预防或治疗神经***发育障碍和/或情绪障碍伴随的过敏性疾病。
  4. 根据权利要求3所述的应用，其特征在于，所述神经***发育障碍为自闭症谱系障碍或抽动症。
  5. 根据权利要求3所述的应用，其特征在于，所述情绪障碍包括多动症。
  6. 一种组合物，包括权利要求1所述的罗伊氏乳杆菌和/或所述罗伊氏乳杆菌的发酵产物。
  7. 根据权利要求6所述的组合物，其特征在于，还包括其他益生菌。
  8. 根据权利要求7所述的组合物，其特征在于，所述其他益生菌选自乳酸杆菌、双歧杆菌、嗜热链球菌、乳球菌、丙酸杆菌、明串球菌、葡萄球菌、芽孢杆菌、片球菌、大肠杆菌、普雷沃氏杆菌、粪栖杆菌、布劳特氏菌、拟杆菌、厚壁菌和酵母菌中的至少一种。
  9. 根据权利要求8所述的组合物，其特征在于，所述乳酸杆菌选自植物乳杆菌、发酵乳杆菌、嗜酸乳杆菌、干酪乳杆菌、卷曲乳杆菌、保加利亚乳杆菌、德氏乳杆菌乳亚种、格氏乳杆菌、约氏乳杆菌、副干酪乳杆菌、罗伊氏乳杆菌、鼠李糖乳杆菌、唾液乳杆菌、清酒乳杆菌和瑞士乳杆菌中的至少一种；
    和/或，所述双歧杆菌选自青春双歧杆菌、短双歧杆菌、长双歧杆菌、婴儿双歧杆菌、两歧双歧杆菌、动物双歧杆菌和乳双歧杆菌中的至少一种；
    和/或，所述乳球菌选自乳酸乳球菌乳酸亚种、乳酸乳球菌乳脂亚种和乳酸乳球菌双乙酰亚种中的至少一种；
    和/或，所述丙酸杆菌选自费氏丙酸杆菌谢氏亚种和产丙酸丙酸杆菌中的至少一种；
    和/或，所述明串球菌为肠膜明串珠菌肠膜亚种；
    和/或，所述片球菌选自乳酸片球菌和戊糖片球菌中的至少一种；
    和/或，所述葡萄球菌选自小牛葡萄球菌、肉葡萄球菌和木糖葡萄球菌中的至少一种；
    和/或，所述芽孢杆菌为凝结芽孢杆菌；
    和/或，所述酵母菌选自马克斯克鲁维酵母、酿酒酵母、产朊假丝酵母、乳酸克鲁维酵母和卡氏酵母中的至少一种。
  10. 根据权利要求6所述的组合物，其特征在于，所述罗伊氏乳杆菌为活菌和/或灭活菌体。
  11. 根据权利要求6所述的组合物，其特征在于，所述组合物还包括益生元。
  12. 根据权利要求11所述的组合物，其特征在于，所述益生元选自菊粉、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***糖、抗性糊精和抗性淀粉中的至少一种。
  13. 根据权利要求6所述的组合物，其特征在于，所述组合物还包括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、柑橘纤维、苹果纤维、小球藻、苜蓿粉、青汁粉和膳食纤维中的至少一种。
  14. 根据权利要求6所述的组合物，其特征在于，所述组合物还包括抗氧化 剂；所述抗氧化剂选自生育酚、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中的至少一种。
  15. 一种药物，制备所述药物的原料包括权利要求1所述的罗伊氏乳杆菌或权利要求6～14任一项所述的组合物，和药学上可接受的辅料。
  16. 一种食品，制备所述食品的原料包括权利要求1所述的罗伊氏乳杆菌或权利要求6～14任一项所述的组合物。
  17. 根据权利要求16所述的食品，其特征在于，所述食品为发酵乳，所述发酵乳的制备方法包括以下步骤：
    将奶源与水混合并均质后灭菌，得到预混料；
    向预混料中加入活化好的所述罗伊氏乳杆菌进行发酵，制备发酵处理产物；及
    将所述发酵处理产物冷却，再次与所述罗伊氏乳杆菌进行混合，获得含罗所述伊氏乳杆菌的发酵乳。
  18. 权利要求1所述的罗伊氏乳杆菌的制备方法，采用高密度发酵制备所述罗伊氏乳杆菌，所述制备方法包括以下步骤：
    活化所述罗伊氏乳杆菌；及
    将活化后的上所述罗伊氏乳杆菌接种到发酵罐中发酵培养。
  19. 权利要求1所述的罗伊氏乳杆菌在制备预防或治疗改善肠道菌群紊乱的产品中的应用；
    或权利要求1所述的罗伊氏乳杆菌在制备预防或治疗肠漏的产品中的应用。
  20. 一种用于预防或治疗肠漏和/或过敏反应的方法，所述方法包括服用权利要求6～14任一项所述的组合物或服用以所述组合物为原料制备的产品，其中，以所述组合物中的益生菌的数量计，剂量为3.0×10 6CFU/kg体重/天～1.2× 10 11CFU/kg体重/天。
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