Lactobacillus reuteri and its uses, compositions, pharmaceuticals and foods

Abstract

The present invention relates to Lactobacillus reuteri and its use, composition, medicine and food. The Lactobacillus reuteri has the accession number CGMCC No. 21577 and has the effects of improving the disorder of intestinal flora, reducing intestinal damage, improving intestinal permeability, promoting the recovery of the immune system, and treating or preventing nervous system-related diseases such as autism, hyperactivity, tic disorder and depression, as well as allergic reactions.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to Lactobacillus reuteri and its use, compositions, pharmaceuticals and foods.

Background Art

[0002] An allergic reaction refers to a reaction of tissue damage or dysfunction that occurs when a living body that already has immunity is stimulated by the same antigen again. People with allergies are constantly increasing all over the world. In a specific population, the incidence of pediatric food allergy is high, reaching 10%.

[0003] The idea that early contact with microorganisms has a positive impact on human immune health can be traced back at least to 1989, when the epidemiologist David Strachan at the London School of Hygiene and Tropical Medicine proposed the "hygiene hypothesis" that children growing up in smaller and cleaner homes are more likely to develop allergies. In the following decades, the hygiene hypothesis has already developed into a model centered on the microbiota, in which early contact with microorganisms from the family and environment plays an important role in reducing the risk of inflammatory diseases such as asthma, hay fever, eczema and food allergies. Currently, the "hygiene hypothesis" has evolved into the theory that "the early environment affects the microbiota and leads to immune dysfunction". When the intestinal flora is dysregulated in the early stage of life, for example, when the diversity is low and the ratio of Escherichia coli / Bacteroides is too high, it has a great impact on allergies. The metabolites of the intestinal flora are an important medium between the flora and the host, and by interacting with the immune system, they suppress the inflammatory response to allergens, or suppress the passage of allergens through the intestinal barrier by reducing the permeability of the intestinal tract, and promote immune tolerance.

[0004] Allergies caused by intestinal flora disorders may occur by increasing intestinal permeability. Research has found that infants with food allergies develop leaky gut and present as an increase in the migration of antigens and allergens from the intestinal tract into the blood. The inside of the digestive tract is not normally subject to immune surveillance, but when the permeability of the intestinal barrier increases, food-derived proteins seep into the body and trigger allergic reactions. Indigestible proteins in foods that cause pediatric food allergies, such as peanuts and milk, are likely to completely penetrate the damaged intestinal barrier without damage and trigger an immune response. Research has found that after treating the intestinal flora of mice with antibiotics and then administering peanuts, complete peanut proteins can be detected in the blood of these mice.

[0005] Lactobacillus reuteri is a lactic acid bacterium present in the intestinal tract, used as a probiotic, having a strong adhesion ability to the intestinal mucosa, excellent acid resistance, bile salt resistance, and broad-spectrum antibacterial properties, and can widely inhibit the growth of Gram-positive bacteria, Gram-negative bacteria, yeasts, fungi, and protozoa, etc.

Summary of the Invention

[0006] According to the research of the present application, Lactobacillus reuteri (deposit number CGMCC No. 21577) isolated from the breast milk of healthy women has been found to have the effects of improving intestinal flora disorders, reducing intestinal damage, improving intestinal permeability, promoting the recovery of the immune system, and treating or preventing allergic reactions.

[0007] The present application provides Lactobacillus reuteri, and the deposit number of the Lactobacillus reuteri is CGMCC No. 21577.

[0008] In addition, the present application provides the use of the above-mentioned Lactobacillus reuteri in the manufacture of products for preventing or treating allergic reactions.

[0009] The present application also provides a composition comprising the above Lactobacillus reuteri and / or a fermentation product of the above Lactobacillus reuteri.

[0010] The present application also provides a pharmaceutical product, and the raw materials for manufacturing the pharmaceutical product include the above Lactobacillus reuteri or the above composition and a pharmaceutically acceptable adjuvant.

[0011] The present application also provides a food product, and the raw materials for manufacturing the food product include the above Lactobacillus reuteri or the above composition.

[0012] The present application also provides a method for manufacturing the above Lactobacillus reuteri, which is fermented at a high density to produce the Lactobacillus reuteri. The manufacturing method includes: a step of activating the Lactobacillus reuteri; a step of inoculating the activated Lactobacillus reuteri into a fermenter and fermenting and culturing it.

[0013] The present application also provides the use of the above Lactobacillus reuteri in the manufacture of a product for preventing or treating intestinal flora disorder, or the use of the above Lactobacillus reuteri in the manufacture of a product for preventing or treating leaky gut.

[0014] The present application also provides a method for preventing or treating leaky gut and / or allergic reaction, the method including taking the above composition or a product manufactured using the above composition as a raw material, and based on the quantity 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.

[0015] Details of one or more embodiments of the present application are presented in the following drawings and description. Other features, objects, and advantages of the present application will become apparent from the specification, drawings, and claims.

[0016] To better explain the embodiments and / or examples of the invention disclosed in this application, one or more drawings can be referred to. The details or exemplifications of the drawings should not be construed as limitations on the scope of any of the disclosed invention, the currently described embodiments and / or examples, and the most preferred embodiments of these inventions that have been understood so far.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Modes for Carrying Out the Invention

[0018] The Lactobacillus reuteri related to the present application has a strain name of LR99, is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number being CGMCC No. 21577. The strain was received and registered at the deposit center on December 31, 2020, and was detected as a viable strain at the preservation center on December 31, 2020.

[0019] Hereinafter, for the purpose of facilitating the understanding of the present invention, the present invention will be described more comprehensively. However, the present invention may be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the disclosure content of the present invention more thorough.

[0020] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. The terms used in this specification are for the purpose of explaining specific embodiments and are not intended to limit the present invention.

[0021] One embodiment of the present invention provides Lactobacillus reuteri LR99, which is isolated from the milk of healthy women. It was deposited on December 31, 2020, at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms (abbreviated as CGMCC, with the address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the postal code being 100101). It is classified and named as Lactobacillus reuteri, and the deposit number is CGMCC No. 21577.

[0022] According to verification, the above Lactobacillus reuteri can reprogram endothelial CD4+ T cells to reach immunoregulatory T cells, exert an immunomodulatory effect, promote the increase of Treg cells, and reduce the Th2 response. Therefore, it is advantageous for the intervention, alleviation or prevention of allergic reactions and can promote the recovery of the immune system. In addition, the above Lactobacillus reuteri also has the ability to metabolize tryptophan, and its metabolites of tryptophan can improve the intestinal barrier function, suppress the increase in intestinal barrier permeability, reduce intestinal damage, prevent or alleviate leaky gut, and further prevent or alleviate allergic reactions. In addition, the above Lactobacillus reuteri can improve the diversity of the intestinal flora, improve the quantity of intestinal probiotics (such as Lactobacillus, Bifidobacterium, Akkermansia muciniphila, etc.), and also improve the disorder of the intestinal flora.

[0023] Based on this, one embodiment of the present invention provides the use of the above Lactobacillus reuteri in the manufacture of a product for preventing or treating allergic reactions. Optionally, the product is used for the prevention or treatment of allergic diseases associated with neurodevelopmental disorders and / or mood disorders. Specifically, the neurodevelopmental disorder is Autism Spectrum Disorder (ASD) or tic disorder, and the mood disorder includes attention deficit hyperactivity disorder, depression, etc. Allergic diseases can occur in all age groups from newborns to the elderly and always have an obvious genetic tendency. In allergic diseases, immediate allergic reactions are common, and its main types include skin allergic reactions, respiratory allergic reactions, gastrointestinal allergic reactions, and anaphylactic shock, etc.

[0024] One embodiment of the present invention further provides the use of the above Lactobacillus reuteri in the manufacture of a product for preventing or treating leaky gut based on the above functions of the above Lactobacillus reuteri.

[0025] One embodiment of the present invention further provides the use of Lactobacillus reuteri in the manufacture of a product for preventing or treating intestinal flora disorder based on the above functions of Lactobacillus reuteri.

[0026] One embodiment of the present invention further provides a method for manufacturing Lactobacillus reuteri, which is fermented at a high density to produce Lactobacillus reuteri. Specifically, the manufacturing method includes steps S1 to S3.

[0027] In step S1, Lactobacillus reuteri is activated.

[0028] Specifically, in order to improve the activity of the strain, activation culture is performed on Lactobacillus reuteri three times. The temperature of the three activation cultures is 37 °C, and the time is 16 h to 18 h.

[0029] In step S2, the activated Lactobacillus reuteri is inoculated into a fermenter and fermented and cultured.

[0030] After the improved MRS medium is sterilized, it is cooled to 37 °C and inoculated into a fermenter at an inoculation amount of 2.5% - 3.5% (v / v) for fermentation. The pH and OD value of the fermentation broth are detected every hour. When the fermentation is carried out until the pH and OD value become relatively gentle, it indicates that the strain reaches the late logarithmic phase and enters the stable period, and the fermentation is completed. Then, the fermentation broth is cooled and centrifuged at a low temperature to collect the bacterial cells, which are washed with phosphate buffer solution (PBS) to produce the bacterial cells of Lactobacillus reuteri.

[0031] In step S3, the bacterial cells produced in step S2 are freeze-dried to produce freeze-dried powder.

[0032] Specifically, the bacterial cells obtained in step S2 are mixed with a cryoprotectant and then emulsified, and thereafter, freeze-dried under vacuum to produce a freeze-dried powder. Optionally, the cryoprotectant is at least one selected from skim milk powder, trehalose, fructooligosaccharide, lactose, glucose, sucrose, sodium L-ascorbate, L-malic acid, and L-lactic acid. Of course, in other embodiments, the cryoprotectant is not limited to the above, and may be other substances that can maintain the activity of the bacterial cells during the freeze-drying process. Optionally, the volume ratio of the bacterial cells to the cryoprotectant is 1:(2 to 10).

[0033] In addition, in some embodiments, step S3 can be omitted. At this time, what is produced is the bacterial cells of Lactobacillus reuteri.

[0034] The method for producing the above-mentioned Lactobacillus reuteri produces the above-mentioned Lactobacillus reuteri by high-density fermentation and has a high yield.

[0035] Further, an embodiment of the present invention further provides a composition containing the above-mentioned Lactobacillus reuteri and / or the fermentation product of the above-mentioned Lactobacillus reuteri based on the above-mentioned function of the above-mentioned Lactobacillus reuteri.

[0036] Specifically, the fermentation product of the above-mentioned Lactobacillus reuteri refers to the culture product of the above-mentioned Lactobacillus reuteri and includes at least one of the metabolites inside the bacterial cells and the metabolites secreted outside the bacterial cells. When in use, the culture solution after culturing the above-mentioned Lactobacillus reuteri or the freeze-dried powder obtained by performing freeze-drying treatment on the culture solution is used. Or, the solution obtained by dissolving after culturing the above-mentioned Lactobacillus reuteri or the freeze-dried powder obtained by performing purification and freeze-drying treatment on the solution is used.

[0037] In some embodiments, the composition includes the Lactobacillus reuteri and an auxiliary agent necessary for manufacturing the microbial agent. The active ingredient of the composition includes the Lactobacillus reuteri. In one selectable specific example, the composition includes the Lactobacillus reuteri and a cryoprotectant. The cryoprotectant is as described above and will not be described herein.

[0038] In some embodiments, the composition further includes other probiotics. The probiotics also serve as the active ingredient of the composition. That is, the composition includes an active ingredient, and the active ingredient includes the Lactobacillus reuteri and other probiotics. Optionally, the other probiotics are at least one selected from Lactobacillus, Bifidobacterium, Streptococcus thermophilus, Lactococcus, Propionibacterium, Leuconostoc, Staphylococcus, Bacillus, Pediococcus, Escherichia coli (for example, Nissle 1917), Prevotella, Faecalibacterium, Blautia, Bacteroidetes, Firmicutes, and yeast.

[0039] In one selectable specific example, the Lactobacillus is at least one selected from Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus helveticus.

[0040] In one selectable specific example, the Bifidobacterium is at least one selected from Bifidobacterium adolescentic, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, and Bifidobacterium lactis.

[0041] In one selectable specific example, the Lactococcus is at least one selected from Lactococcus Lactis subsp. Lactis, Lactococcus Lactis subsp. Cremoris, and Lactococcus Lactis subsp. Diacetylactis.

[0042] In one selectable specific example, the Propionibacterium is at least one selected from Propionibacterium freudenreichii subsp. Shermanii and Propionibacterium acidipropionici. The Leuconostoc is Leuconostoc mesenteroides subsp. Mesenteroides. The Pediococcus is at least one selected from Pediococcus acidilactici and Pediococcus pentosaceus. The Staphylococcus is at least one selected from Staphylococcus vitulinus, Staphylococcus xylosus, and Staphylococcus carnosus. The Bacillus is Bacillus coagulans. The yeast is at least one selected from Kluyveromyces marxianus, Saccharomyces cerevisiae, Cadida atilis, Kluyveromyces lactis, and Saccharomyces carlsbergensis. Note that the other probiotics in this embodiment are not limited to those described above and may be other probiotics other than LR99.

[0043] In some embodiments, the composition further comprises prebiotics. According to the prebiotics, the colonization and proliferation of the above Lactobacillus reuteri are promoted, and the growth of other probiotics in the intestinal tract is also promoted. Optionally, the prebiotics are at least one selected from inulin, extract from artichoke, extract from chicory root, extract from Jerusalem artichoke root, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, xylooligosaccharide, stachyose, mannooligosaccharide, arabinooligosaccharide, indigestible dextrin, resistant starch. Of course, in other embodiments, the prebiotics in the composition are not limited to those described above, and may be other substances that can promote the growth and proliferation of probiotics.

[0044] In some embodiments, the composition further comprises nutrients, and the nutrients are at least one selected from GABA, tryptophan, lycopene, β-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, β-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder and dietary fiber.

[0045] In some embodiments, the composition further comprises antioxidants. The antioxidants are at least one selected from tocopherol, carotenoid, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenol, glutathione and superoxide dismutase.

[0046] In some embodiments, in the composition, the mass percentage of the above Lactobacillus reuteri is 1-30%. Further, the mass percentage of the above Lactobacillus reuteri is 1%-20%. Optionally, in the composition, the viable bacteria content of the above Lactobacillus reuteri is 1.2×10 6 CFU / g~1.6×10 12 CFU / g. Further, the viable bacteria content of the above Lactobacillus reuteri is 3.0×10 10CFU / g to 2.0×10 11 CFU / g.

[0047] In some embodiments, Lactobacillus reuteri in the above composition is viable bacteria. In some other embodiments, Lactobacillus reuteri in the above composition is inactivated cells of Lactobacillus reuteri. In some other embodiments, Lactobacillus reuteri in the above composition is a mixture of viable bacteria of Lactobacillus reuteri and inactivated cells of Lactobacillus reuteri.

[0048] The dosage form of the composition is not particularly limited and may be, for example, powder, tablet, tablet or capsule.

[0049] In some embodiments, based on parts by weight, the above composition contains 10 to 30 parts of freeze-dried powder of Lactobacillus reuteri, 15 to 25 parts of fructooligosaccharide, 40 to 65 parts of sorbitol or maltitol, and 5 to 10 parts of magnesium stearate. Here, in the above composition, the content of Lactobacillus reuteri is 1.2×10 6 CFU / g to 1.5×10 10 CFU / g.

[0050] In some embodiments, based on parts by weight, the above composition contains 0.5 to 30 parts of freeze-dried bacterial powder of Lactobacillus reuteri LR99, 1 to 20 parts of other probiotics, 20 to 80 parts of prebiotics, 2 to 10 parts of nutrients, and 0.1 to 10 parts of antioxidants. Other probiotics, prebiotics, nutrients and antioxidants are as described above and will not be described here. Further, the above composition contains 1 to 10 parts of freeze-dried bacterial powder of Lactobacillus reuteri LR99, 1 to 10 parts of other probiotics, 30 to 80 parts of prebiotics, 2 to 5 parts of nutrients, and 0.5 to 10 parts of antioxidants. Optionally, in the above composition, the content of Lactobacillus reuteri is 1.8×10 6 CFU / g to 6.5×1011 CFU / g, and the content of the single strain of other probiotics is 1×10 6 CFU / g to 6×10 9 CFU / g. Furthermore, the content of Lactobacillus reuteri is 2.5×10 7 CFU / g to 1×10 11 CFU / g.

[0051] The above composition contains the above Lactobacillus reuteri and / or the fermentation product of the above Lactobacillus reuteri, can improve the diversity of the intestinal flora, improve the quantity of probiotics in the intestinal tract (such as Lactobacillus, Bifidobacterium, Akkermansia, etc.), improve the disorder of the intestinal flora, reduce the damage of the intestinal tract, prevent or relieve leaky gut and allergic reactions, and promote the recovery of the immune system.

[0052] The above composition can be used as a raw material for manufacturing foods, pharmaceuticals, dietary supplements or animal feeds. For example, it can be used as a raw material for manufacturing foods such as fermented milk, cheese, milk-based beverages, solid beverages, powdered milk, tablets, gel soft candies, fermented vegetable juices, fermented soy products, etc. When in use, it can be directly mixed in the normal process or the final product can be manufactured after fermentation. In some embodiments, the above composition can be directly used as a food or a pharmaceutical. Optionally, in food, the recommended dosage of the above 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. Furthermore, the recommended dosage of the above 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.

[0053] One embodiment of the present invention further provides a food, and the raw materials for manufacturing the food include the above-mentioned Lactobacillus reuteri or the above composition, and a food additive. Optionally, the food additive is at least one selected from seasonings, sweeteners, thickeners, stabilizers, surfactants, lubricants, acid neutralizers, dispersants, buffers or buffering agents, debittering agents, pH stabilizers, preservatives, desugaring agents, and colorants. In one specific example, the food additive is at least one selected from lactitol, sorbitol, maltitol, aspartame, stevia, lakanka, sucralose, xylitol, vanilla, chocolate, fruit flavors, and artificial essences.

[0054] In one example, the above food is fermented milk. The manufacturing method of the fermented milk includes the following steps.

[0055] In step a, the milk source is mixed with water and homogenized, and then sterilized to obtain a premix.

[0056] Specifically, the milk source includes at least one of raw milk, skim milk powder, and whole milk powder. The sterilization conditions are carried out at 120°C to 122°C for 250 s to 350 s. Of course, it is necessary to cool the premix to a temperature of 40°C to 45°C after sterilization.

[0057] In step b, an activated strain of Lactobacillus reuteri is added to the premix and fermented to produce a product of the fermentation process.

[0058] Specifically, the fermentation temperature is 40°C to 45°C, and the fermentation time is 8 h to 12 h.

[0059] In step c, the product of the fermentation process in step b is cooled and mixed with Lactobacillus reuteri again to obtain fermented milk with a high content of Lactobacillus reuteri.

[0060] In the fermented milk obtained by the method for producing the fermented milk described above, the content and activity of the above Lactobacillus reuteri are high, the nutritional value is high, and it can be further used for the treatment or prevention of disease symptoms.

[0061] One embodiment of the present invention further provides a pharmaceutical based on the function of the above Lactobacillus reuteri or the above composition, and the raw materials for producing the pharmaceutical include the above Lactobacillus reuteri or the above composition and a pharmaceutically acceptable adjuvant.

[0062] One embodiment of the present invention provides a method for preventing or treating leaky gut and / or allergic reactions based on the function of the above Lactobacillus reuteri or the above composition. The method includes taking the above composition or a product manufactured using the above composition as a raw material. Based on the quantity 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 quantity of probiotics in the composition, the dosage is 1.0×10 6 CFU / kg body weight / day to 6.0×10 10 CFU / kg body weight / day.

[0063] <Specific Examples> Hereinafter, it will be described in detail with reference to specific examples. The following examples do not contain other components except unavoidable impurities, unless otherwise specified. In the examples, the reagents and equipment are the usual selections in this field, unless otherwise specified. The experimental methods for which specific conditions are not specified in the examples are realized according to normal conditions, for example, the conditions described in the literature, books, or the methods recommended by the manufacturer.

[0064] Example 1 One strain of bacteria was isolated from the breast milk of healthy women using anaerobic culture, and after being tested through full-length 16S rRNA sequencing and mass spectrometry, the strain was found to be a new strain belonging to Lactobacillus reuteri, and was named Lactobacillus reuteri LR99 (abbreviated as "LR99" or "LR-99"). The strain was entrusted to the China General Microbiological Culture Collection Center (abbreviated as CGMCC, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing 100101) on December 31, 2020. The classification is named Lactobacillus reuteri, and the accession number is CGMCC No. 21577.

[0065] 1. Taxonomic characteristics of Lactobacillus reuteri LR99: (1) Lactobacillus reuteri LR99 was observed under a microscope, and the results are shown in Figure 1.

[0066] (2) The results of physicochemical tests are shown in Tables 1 and 2.

[0067]

Table 1

[0068] As can be seen from Table 1, Lactobacillus reuteri LR99 is a Gram-positive bacterium, does not form spores, is non-motile, is negative for catalase, is negative for oxidase, is anaerobic, and the appropriate temperature for culture is 37°C.

[0069]

Table 2

[0070] In Table 2, "+" indicates that metabolism is possible, and "-" indicates that metabolism is impossible.

[0071] As can be seen from Table 2, Lactobacillus reuteri LR99 can metabolize ribose, xylose, maltose, lactose, raffinose, inulin, starch, mannose, melibiose, galactose, sucrose, L - arabinose and salicin, but cannot metabolize trehalose, melezitose, fructose, cellobiose, sodium gluconate, mannitol and sorbitol.

[0072] 2. Resistance of Lactobacillus reuteri LR99 to artificial gastric juice and intestinal juice: The resistance of Lactobacillus reuteri LR99 to artificial gastric juice and intestinal juice was tested, and compared with Lactobacillus reuteri DSM17938, which has excellent acid resistance and can survive in the gastrointestinal tract, and was isolated from commercially available probiotic products stored in the current laboratory.

[0073] The detection results of the survival rates of Lactobacillus reuteri DSM17938 and Lactobacillus reuteri LR99 in artificial gastric acid (pH = 3) and artificial intestinal juice (pH = 8) are shown in Table 3.

[0074]

Table 3

[0075] As can be seen from Table 3, when Lactobacillus reuteri DSM17938 was treated with artificial gastric juice for 1 h, the survival rate of viable bacteria was 71.2%, and when treated for 1.5 h, the survival rate of viable bacteria was 33.1%. However, for Lactobacillus reuteri LR99, when treated for 1 h, the survival rate of viable bacteria was 93.1%, and when treated for 1.5 h, the survival rate of viable bacteria was 69.7%. This indicates that Lactobacillus reuteri LR99 has better gastric acid resistance and most of it reached the intestinal tract through the stomach and exerted its effect.

[0076] As can be seen from Table 3, when Lactobacillus reuteri DSM17938 was treated with artificial intestinal juice (pH = 8) for 1 hour, the viable cell survival rate was 25.8%, and the survival rate after 2-hour treatment was 18.5%. However, for Lactobacillus reuteri LR99, when treated with artificial intestinal juice for 1 hour, the viable cell survival rate was 48.8%, and the survival rate after 2-hour treatment was 37.8%.

[0077] As is clear from the above results, after digestion with artificial gastric juice and intestinal juice, Lactobacillus reuteri LR99 still has good survival ability. Lactobacillus reuteri LR99 has good resistance to digestive juices against commercial strains and can survive and grow smoothly in the intestinal tract.

[0078] 3. Toxicity test and safety detection of Lactobacillus reuteri LR99: (1) Lactobacillus reuteri LR99 was inoculated into MRS liquid medium and anaerobically cultured at 37°C for 48 hours. After counting, the viable cell count of Lactobacillus reuteri LR99 in the culture solution was 3.2×10 9 CFU / mL. Then, the undiluted culture was orally administered to mice (healthy male BALB / C mice, 6 - 8 weeks old, weighing 16 - 18 g, maintained at room temperature (25±2°C), relative humidity (55±2)%, 12h / 12h light irradiation, and allowed free access to food and water) at a rate of 20.0 mL / kg body weight continuously for 3 days, and then observed for 7 days. At the same time, MRS liquid medium was orally administered to mice in an amount of 20.0 mL / kg body weight as a control group.

[0079] As can be seen from the results, no toxic reactions or deaths were observed in the two groups of test mice for the undiluted culture of Lactobacillus reuteri LR99 compared with the control group, and there was no statistical difference in the weight gain of the mice (p>0.05).

[0080] (2) According to the method of SN / T 1944 - 2007 "Measurement of Bacterial Drug Resistance in Animals and Their Products", the antibiotic susceptibility of Lactobacillus reuteri LR99 was evaluated.

[0081] As can be seen from the evaluation results, Lactobacillus reuteri LR99 is sensitive to Ampicillin, Pencillin G, Erythromycin, Chloramphenicol, Clindamycin, Vancomycin, Tetracycine, etc. It meets the requirements in the evaluation criteria for the drug resistance of edible bacteria of the European Food Safety Authority. Lactobacillus reuteri LR99 does not contain foreign antibiotic resistance genes and can be safely consumed.

[0082] Example 2 This example is for explaining the high-density fermentation of Lactobacillus reuteri LR99 and the manufacturing process of freeze-dried bacterial powder.

[0083] (1) Lactobacillus reuteri LR99 was anaerobically cultured in the improved MRS medium to obtain the bacterial strain for fermentation.

[0084] (2) In order to improve the activity of the bacterial strain, activation culture was performed three times on the Lactobacillus reuteri LR99 bacterial strain. The three activation cultures were at a temperature of 37°C and a time of 16h - 18h. After the activation of the bacterial strain was completed, the detection results of the growth curve of the Lactobacillus reuteri LR99 bacterial strain are shown in Table 4.

[0085]

Table 4

[0086] As can be seen from the detection results of the growth curve of Lactobacillus reuteri LR99, Lactobacillus reuteri LR99 reached the logarithmic end stage, that is, the fermentation harvest point. The seeds of Lactobacillus reuteri LR99 were cultured until the logarithmic end stage and harvested. The harvested seeds were refrigerated in a refrigerator at 4°C.

[0087] (3) Upper tank of the fermenter: After sterilizing the improved MRS medium, quickly cool it to 37°C, inoculate the fermenter at an inoculation amount of 3% and ferment. Detect the pH and OD values of the fermentation broth every hour. When the fermentation is carried out until the pH and OD values become relatively gentle, it indicates that the strain reaches the end of the logarithmic phase and enters the stable period. At this time, complete the fermentation, immediately cool down, centrifuge the fermentation broth at low temperature, collect the bacterial cells, wash them with phosphate buffer (PBS), then add a lyophilization protectant (skim milk powder) and emulsify. After completion, perform vacuum freeze-drying to obtain bacterial powder. Store the produced freeze-dried bacterial powder at -20°C or below. Here, according to detection, the viable count of bacteria in the fermentation broth is 2.75×10 9 CFU / mL, the viable count of bacteria in the emulsion is 4.7×10 10 CFU / mL, and the viable count of bacteria in the freeze-dried powder is 1.55×10 11 CFU / g.

[0088] Example 3 This example is for explaining the effect of Lactobacillus reuteri LR99 on the improvement of leaky gut.

[0089] Lactic acid has D-type and L-type. The normal human body only has L-lactic acid. D-lactic acid is a metabolite fermented by bacteria and can be produced by various bacteria in the intestine. After being ingested from food, it is not normally absorbed into the blood, and mammals do not have an enzyme system to rapidly decompose it. When the permeability of the intestinal mucosa increases, a large amount of D-lactic acid produced by bacteria in the intestinal tract is taken into the blood by the damaged mucosa, increasing the level of D-lactic acid in the blood. By monitoring the level of D-lactic acid in the blood, the degree of damage to the intestinal mucosa and changes in permeability can be timely reflected. It can be used for auxiliary evaluation of intestinal infection, endotoxemia, systemic inflammatory response, repeated fever, vomiting, etc.

[0090] Lipopolysaccharide (LPS), also known as bacterial endotoxin, is a component on the cell wall of Gram-negative bacteria, and LPS is a toxic substance to animals. The structure of LPS can be divided into three parts such as glycolipid domain - lipid A, short chain of sugar residues - core oligosaccharide, and highly variable polysaccharide domain - O antigen. The structure of LPS determines its agonist / antagonist effect on TLR4. In the body, LPS binds to the complex of TLR4 / MD-2 receptor and activates different signaling pathways through Myd88-dependent or TRIF-dependent pathways. The expression levels of TLRs in intestinal epithelial cells at different sites are different, and LPS can prevent the inflammatory reaction and fight against pathogenic bacteria.

[0091] LPS is involved in the onset of various diseases, such as intestinal diseases like IBD and enterocolitis, and further Parkinson's and Alzheimer's diseases. LPS can not only enter the blood but also enter the brain and stay there for life, which may cause Alzheimer's disease.

[0092] The LPS level in the blood can reflect intestinal permeability. A normal intestinal barrier does not allow the entry of LPS. A relatively high level of LPS in the blood indicates that intestinal bacteria or LPS have moved into the blood, which means an increase in intestinal permeability and an increased probability of the appearance of leaky gut symptoms. The content level of LPS in the blood can also indicate the inflammatory reaction and stress state. Excessive LPS may cause abnormalities in the human immune system, trigger chronic or acute inflammatory reactions, and may cause acute inflammations such as fever and pain. It can be used for the auxiliary evaluation of intestinal infections, endotoxemia, systemic inflammatory reactions, repeated fevers, vomiting, mental diseases, stress reactions, etc.

[0093] Any stress response that results in increased intestinal permeability includes psychological and physiological effects and may potentially cause bacterial translocation. Bacterial translocation refers to the entry of intestinal endogenous bacteria into the body through the epithelial mucosa from the intestine. Bacteria can enter the lymphatic system through the mesenteric lymph nodes and circulate throughout the body. Bacteria can enter the bloodstream and cause bacteremia, or they may be located in tissues. Bacterial translocation can lead to overgrowth of small intestinal bacteria, intestinal damage, and ultimately shock.

[0094] The permeability of Lactobacillus reuteri LR99 to the intestine was evaluated based on the contents of D-lactic acid and LPS in the serum of mice.

[0095] Twelve 6-week-old C57BL / 6J mice were assigned to three mice per cage and raised, allowing them to eat and drink freely. The conditions of the mice's growth environment were environmental temperature (23 ± 2) °C, relative humidity (50 ± 10)%, and light irradiation mode (12 h dark / 12 h light irradiation). After the mice had adapted to the environment for one week, they were randomly assigned to a control group and a probiotics group. The mice in the probiotics group were administered a probiotics preparation (the probiotics preparation consisted of the freeze-dried powder of Lactobacillus reuteri LR99 produced in Example 2 and maltodextrin) by intragastric administration, and the intragastric administration dose was 10 billion CFU / mouse / day. The mice in the control group were administered an equal amount of placebo (maltodextrin) by intragastric administration.

[0096] From the second week, while administering placebo or probiotics, a combined stress stimulus of noise and nocturnal lighting was applied to each group.

[0097] After the mice in each group were orally administered and raised for 6 weeks, blood was collected from the peripheral vein of the tail. The blood was centrifuged at 3000 g for 15 minutes. Using an intestinal barrier function analysis system (JY-DLT, Beijing Zhongsheng Jinyu Diagnostic Technology Co., Ltd.), according to the instruction manual, the contents of D-lactic acid and LPS in the serum were detected. Next, the data obtained by processing the contents of D-lactic acid and LPS in the serum of the mice in each group with SPSS statistical software were expressed as mean ± standard deviation, and the comparison between groups was performed using the independent sample t-test. P<0.05 was considered to have statistical significance, and the results are shown in Figure 2.

[0098] As can be seen from Figure 2, the probiotics group had significantly lower levels of LPS and D-lactic acid compared to the control group (P<0.05). This indicates that stress stimulation increases intestinal permeability, and probiotics can reduce intestinal permeability and the risks of endotoxemia, systemic inflammatory response, etc.

[0099] Example 4 This example is for explaining the promoting effect of Lactobacillus reuteri LR99 on the immune system.

[0100] In animal model and human studies, probiotics affect the differentiation of CD4+ T cells and regulate allergic diseases. Probiotics can suppress allergic diseases such as asthma, atopic dermatitis, allergic rhinitis, food allergy, and urticaria by means of reducing Th2 cytokines (IL-4, IL-5, IL-13, etc.), reducing IgE, increasing IL-10, and promoting Treg differentiation. Studies have found that specific intestinal flora can metabolize tryptophan into indole-3-ethanol, indole-3-pyruvic acid, or indole-3-acetaldehyde, and can improve the barrier function of the intestinal tract. In the body, the metabolites of tryptophan can suppress the increase in the permeability of the intestinal barrier.

[0101] To verify whether Lactobacillus reuteri LR99 can affect the differentiation of CD4+ T cells and regulate allergic diseases, 18 six-week-old C57BL / 6J mice were housed with three mice assigned to each cage and allowed free access to food and water. After one week of adaptation to the surrounding environment, they were randomly assigned to three groups, with six mice in each group. Each group was fed daily with placebo (control group), freeze-dried powder of Lactobacillus reuteri LR99 (the viable cell count was 1×10 9 CFU / mouse) (LR-99 group), and freeze-dried powder of Lactobacillus reuteri LR99 (the viable cell count was 1×10 9 CFU / mouse) + L-tryptophan (mass concentration 0.24%) (L-tryptophan was obtained from Research Diets, designated as the LR-99 + tryptophan group).

[0102] After four weeks of breeding, the mice were sacrificed and the intestinal tract and spleen were removed. A single-cell suspension of the spleen was prepared by mechanical disruption to isolate T cells. The spleen was digested with 100 ng / mL of collagenase D (Invitrogen) for DC isolation. For flow cytometry, fluorescently labeled monoclonal antibodies such as anti-CD8 (53-6.7) and anti-CD4 (GK1.5) from BD Biosciences were used. For the isolation of naive T cells, after concentrating the single-cell suspension using the "Naive CD4+ T Cell Isolation Kit" (Miltenyi Biotec), CD4+ cells were sorted. Samples were processed using FACSantoII (BD Biosciences), fluorescently activated cells were sorted using FACSAria II (BD Biosciences), and the data were analyzed with FlowJo software (TreeStar), and the results are shown in Figure 3.

[0103] As can be seen from Figure 3, after being combined with tryptophan, both Lactobacillus reuteri LR99 and Lactobacillus reuteri LR99 can significantly promote the differentiation of CD4+ T cells (p<0.05). In addition, there was no significant difference in the differentiation of CD4+ T cells after Lactobacillus reuteri LR99 alone was combined with tryptophan (p>0.05).

[0104] Example 5 This example is for explaining the influence of LR99 on the composition of intestinal microbiota.

[0105] (1) DNA extraction: The cecal contents collected after sacrificing the mice in each group in Example 4 were used to extract the DNA of the fecal flora using the TIANmap fecal DNA kit (TIANGEN, catalog number DP328). The extracted DNA was quantitatively detected using a Qubit meter. Detection was performed using 1% agarose gel electrophoresis (voltage 100V, 40 min). The UVI gel imaging system took pictures and recorded them. For DNA electrophoresis, no non-specific bands or smears occurred, and the purity of the DNA fragments was good, indicating that there was no obvious degradation. An appropriate amount of the sample was placed in a centrifuge tube and diluted to 1 ng / μL using sterile water. The DNA was stored in a -20°C refrigerator for standby.

[0106] (2) Amplification of bacterial 16S rRNA gene: Using the diluted genomic DNA as a template, according to the selection of the sequencing region, specific primers with barcodes were used, and the V3-V4 region of the bacterial 16S rRNA gene was amplified using the V3-V4 universal primers 341F (CCTACGGGNBGCASCAG, SEQ ID No.1) and 805R (GACTACNVGGGTATCTAATCC, SEQ ID No.2). The DNA extracted from 100 ng of the sample was used for PCR at 56°C for strand regeneration. First, it was denatured at 94°C for 4 minutes, and then 30 cycles were performed at 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 1 minute.

[0107] (3) Amplicon gene sequencing: The library was constructed using the library kit of Illumina's TruSeq DNA PCR-Free Library Preparation Kit. After passing Qubit quantification and library detection, the constructed library was sequenced for the bacterial flora using the Illumina HiSeq2500 PE250 sequencing platform.

[0108] (4) Processing and analysis of sequencing data: The primary data of the bacterial flora sequencing was introduced into QIIME (2019.4), and noise was reduced with DADA2 to obtain representative amplicon sequence variants (ASVs), which were then constructed into a phylogenetic tree. After quality control, the filtered ASVs were compared with gene sequences in the Naive bayes classifier (NBC) method and the Greengenes (V_13.5) database for species annotation. In Alpha and Beta diversity analyses, the resampling depth was 10,000 sequences per sample to ensure sufficient sequences. The statistical results were corrected by calculating the false discovery rate (FDR) to reduce the influence of too many types of results on the results.

[0109] As can be seen from the results, at the phylum level, there are significant differences in the gut microbiota of the control group, the probiotics group (LR99), and the probiotics combined with tryptophan group (LR99+TRP). The Bacteroidetes phylum in the control group was lower than that in the probiotics group (Figure 4). The alpha diversity index of the probiotics group was also higher than that of the control group, and the difference in the Shannon index was not significant (p = 0.45064 (Mann-Whitney statistic)) (Figure 5). The heatmap analysis shows that there are differences in the composition of the microbiota of the three groups at the genus level (Figure 6). At the genus level, there are many specific differential bacteria. After using LR99, Lactobacillus, Bifidobacterium, and Akkermansia increased significantly. In particular, for Lactobacillus, the addition of tryptophan is considered to be able to further promote the increase in its abundance (Figures 7-9).

[0110] Example 6 This example is to explain the improvement of allergic symptoms in children diagnosed with autism spectrum disorder (ASD) by Lactobacillus reuteri LR99.

[0111] The child is a 7-year-old boy. The lyophilized powder of Lactobacillus reuteri LR99 (the composition is Lactobacillus reuteri LR99 and a lyophilized powder protectant) was orally administered 3 times a day, 60 billion CFU each time, and the administration period was 30 days.

[0112] Before and after administration, the intestinal and skin symptoms of the child were recorded. Blood was collected to detect IgE and chronic food allergies. After 30 days, the diarrhea and peritoneal symptoms of the child improved, the mucous stools improved, and several formed stools appeared. The frequency of bowel movements changed from 2 to 3 times a day to once every 1 to 2 days, the skin inflammation improved, and the scratching decreased. At the same time, feedback from the parents indicated that the frequency of the child's irritability decreased and the vocabulary increased slightly. The blood test results showed that IgE decreased from 572.2 IU / mL before administration to 368.7 IU / mL. The detection of chronic food allergies showed that the egg and milk indicators decreased slightly. The parents chose to continue the administration and are still observing and recording the continuous improvement.

[0113] Each of the technical features of the foregoing embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, it should be understood that they are within the scope described in this specification.

[0114] The above embodiments merely illustrate some embodiments of the present invention. It should be understood that although the description is specific and detailed, it does not limit the scope of the claims. For those skilled in the art, without departing from the idea of the present invention, some modifications and improvements are possible, and all of these belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Lactobacillus reuteri, wherein the Lactobacillus reuteri has a deposit number of CGMC No. 21577 Lactobacillus reuteri.

2. Use of the Lactobacillus reuteri according to claim 1 in the manufacture of a product for preventing or treating allergic reactions.

3. The product is used for preventing or treating allergic diseases associated with neurodevelopmental disorders and / or mood disorders The use according to claim 2, characterized in that.

4. The neurodevelopmental disorder is an autism spectrum disorder or a tic disorder The use according to claim 3, characterized in that.

5. The mood disorder includes attention deficit hyperactivity disorder The use according to claim 3, characterized in that.

6. A composition comprising the Lactobacillus reuteri according to claim 1 and / or a fermentation product of the Lactobacillus reuteri.

7. Further comprising other probiotics The composition according to claim 6, characterized in that.

8. The other probiotics are at least one selected from Lactobacillus, Bifidobacterium, Streptococcus thermophilus, Lactococcus, Propionibacterium, Leuconostoc, Staphylococcus, Bacillus, Pediococcus, Escherichia coli, Prevotella, Faecalibacterium, Blautia, Bacteroides, Firmicutes and yeast The composition according to claim 7, characterized in that.

9. The Lactobacillus is at least one selected from Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus delbrueckii subspecies, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sakei, Lactobacillus helveticus, and / or The bifidobacterium is at least one selected from Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, and Bifidobacterium lactis, and / or, The lactococcus is at least one selected from Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. diacetylactis, and / or, The propionibacterium is at least one selected from Propionibacterium freudenreichii subsp. shermanii and Propionibacterium acidipropionici, and / or, The leuconostoc is Leuconostoc mesenteroides subsp. mesenteroides, and / or, The pediococcus is at least one selected from Pediococcus acidilactici and Pediococcus pentosaceus, and / or, The staphylococcus is at least one selected from Staphylococcus pulvereri, Staphylococcus carnosus, and Staphylococcus xylosus, and / or, The bacillus is Bacillus coagulans, and / or, The yeast is at least one selected from Kluyveromyces marxianus, Saccharomyces cerevisiae, Candida utilis, Kluyveromyces lactis, and Saccharomyces carlsbergensis. The composition according to claim 8, characterized in that.

10. The Lactobacillus reuteri is viable bacteria and / or inactivated bacterial cells. The composition according to claim 6, characterized in that.

11. The composition further contains prebiotics. The composition according to claim 6, characterized in that.

12. The prebiotics are at least one selected from inulin, extracts from artichokes, extracts from chicory roots, extracts from Jerusalem artichoke roots, fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, xylooligosaccharides, stachyose, mannooligosaccharides, arabinooligosaccharides, indigestible dextrin, and resistant starch. The composition according to claim 11, characterized in that.

13. The composition further contains at least one of GABA, tryptophan, lycopene, β-carotene, vitamin B6, vitamin B12, coenzyme Q10, taurine, pectin, β-glucan, fucose, carrageenan, guar gum, citrus fiber, apple fiber, chlorella, alfalfa powder, green juice powder, and dietary fiber. The composition according to claim 6, characterized in that.

14. The composition further contains an antioxidant, The antioxidant is at least one selected from tocopherol, carotenoid, ascorbic acid / vitamin C, ascorbyl palmitate, polyphenol, glutathione, and superoxide dismutase. The composition according to claim 6, characterized in that.

15. Lactobacillus reuteri according to claim 1, or the composition according to any one of claims 6 to 14, and a pharmaceutically acceptable adjuvant, A pharmaceutical product comprising.

16. A food product comprising Lactobacillus reuteri according to claim 1, or the composition according to any one of claims 6 to 14.

17. A method for producing fermented milk, comprising: the fermented milk contains Lactobacillus reuteri according to claim 1, or the composition according to any one of claims 6 to 14, The method for producing the fermented milk is as follows: A step of mixing a milk source with water, homogenizing it, and then sterilizing it to obtain a premix; A step of adding the activated strain of Lactobacillus reuteri to the premix and fermenting it to produce a product of the fermentation treatment; A step of cooling the product of the fermentation treatment and mixing it again with the Lactobacillus reuteri to obtain fermented milk containing the Lactobacillus reuteri. A method for producing fermented milk, comprising.

18. A method for producing Lactobacillus reuteri according to claim 1, comprising: Fermenting at high density to produce the Lactobacillus reuteri, The production method includes: A step of activating the Lactobacillus reuteri; A step of inoculating the activated Lactobacillus reuteri into a fermenter and fermenting and culturing it. A method for producing Lactobacillus reuteri.

19. Use of Lactobacillus reuteri according to claim 1 in the production of a product for preventing or treating intestinal flora disorder, or Use of Lactobacillus reuteri according to claim 1 in the production of a product for preventing or treating leaky gut. A method for preventing and / or treating leaky gut and / or allergic reactions in animals other than humans, comprising: administering the composition according to any one of claims 6 to 14, or a product manufactured using said composition as a raw material; Based on the quantity 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, A method for preventing and / or treating leaky gut and / or allergic reactions.

Images