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 PDFInfo
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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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- A23V2200/304—Foods, ingredients or supplements having a functional effect on health having a modulation effect on allergy and risk of allergy
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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
- 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
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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
- 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
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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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
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- 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
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- 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
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- 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
Description
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 |
Claims (20)
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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。
- 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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CN113073066B (zh) | 2022-01-04 |
GB2622324A (en) | 2024-03-13 |
CA3215700A1 (en) | 2022-10-20 |
JP2024516950A (ja) | 2024-04-18 |
CN113073066A (zh) | 2021-07-06 |
US20240197796A1 (en) | 2024-06-20 |
AU2022258164A1 (en) | 2023-11-16 |
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