Lactobacillus reuteri, composition and application thereof

Abstract

The invention relates to the field of microorganisms, in particular to lactobacillus reuteri, a composition and application of the lactobacillus reuteri. The lactobacillus reuteri provided by the invention is preserved in the China General Microbiological Culture Collection Center on June 19, 2024, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.31007. The lactobacillus reuteri provided by the invention is obtained by screening feces of healthy puppies, has relatively strong acid resistance, bile salt resistance and intestinal tract adhesion capacity, has a good inhibition effect on common pathogenic bacteria, is sensitive to various antibiotics, has good probiotic potential, and can be used for preparing a feed additive. The invention provides a new candidate strain for pet food, health care and treatment products or probiotic preparations, and has wide application prospects in the aspects of preparation of pet food, health care products, medicines and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and specifically relates to a Lactobacillus reuteri, a composition and their applications. Background Art

[0002] Probiotics are a type of microbial preparation beneficial to the health of pets. They are safe and non-toxic, and are currently widely used in the pet food field, mainly focusing on treating and preventing diarrhea in dogs and cats, reducing intestinal inflammation, alleviating obesity, promoting growth, improving oral problems, etc. These bacteria can colonize, grow and reproduce in the pet intestine, and play many positive roles by regulating the intestinal microecological balance.

[0003] The mainstream consumption in the pet market in China is mainly pet dogs. At present, the number of pet dogs in China ranks first in the world. It is expected that by 2024, China will have 248 million pet dogs, far exceeding 172 million in the United States. With the increase in the number of pet dogs, more and more people begin to pay attention to the health and quality of life of pet dogs. Various pet dog foods, health products and nutritional products have also entered the families raising pet dogs, and the consumption scale of these products is also growing continuously. People's awareness of healthy pet raising has increased, and their consumption in pet medical care is also more diversified. Products that improve the intestinal flora structure of pets by adding probiotics, prebiotics, etc. to pet foods have received extensive attention, and the development of dog-specific probiotics has broad market prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a Lactobacillus reuteri, a composition and their applications. As a probiotic for regulating the gastrointestinal flora, Lactobacillus reuteri provides a new candidate strain for dog pet foods and health treatment products.

[0005] To achieve the above invention purposes, the present invention provides the following technical solutions: The present invention provides a strain of Lactobacillus reuteri, which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 31007 and the preservation date of June 19, 2024.

[0006] Wherein, Lactobacillus reuteri is the Latin name of Lactobacillus reuteri. Lactobacillus is the genus name, meaning the genus Lactobacillus, reuteri is the species name, Lactobacillus and reuteriCollectively, it specifically refers to Lactobacillus reuteri. The Lactobacillus reuteri of the present invention is deposited in the China General Microbiological Culture Collection Center, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China. In the specification of the present invention, the Lactobacillus reuteri claimed by the present invention has the number M60. In some embodiments, M60 is equivalent to the Lactobacillus reuteri claimed by the present invention.

[0007] In some embodiments, the Lactobacillus reuteri is isolated from the feces of puppies.

[0008] The advantage of extracting probiotics from puppy feces is that it itself originates from the canine intestine. Other microorganisms in the canine intestine have formed a specific ecosystem with it during its growth and development, enabling it to better adapt to physiological conditions such as pH and temperature at the action site. Moreover, compared with probiotics from other sources, the probiotics extracted from puppy feces have better compatibility with the existing microbial community in the canine intestine. They can better integrate into the existing microbial ecosystem, do not interfere with or destroy the original beneficial microbial community structure, but act synergistically with other bacterial groups to jointly maintain intestinal health. And the intestinal microbial composition of puppies is relatively simple compared with that of adult dogs, making it easy to screen and extract high-quality probiotics. Therefore, the Lactobacillus reuteri of the present invention is a strain with excellent probiotic properties selected after isolation, screening, and identification from puppy feces, and can be used to regulate gastrointestinal dysfunction caused by disordered intestinal flora structure.

[0009] In some embodiments, the 16s rRNA gene sequence of the Lactobacillus reuteri ( Lactobacillus reuteri ) is as shown in SEQ ID NO: 01.

[0010] In some embodiments, the Lactobacillus reuteri inhibits intestinal pathogenic bacteria, and the intestinal pathogenic bacteria are selected from at least one of Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, and Methicillin-resistant Staphylococcus aureus; and / or The Lactobacillus reuteri is non-hemolytic.

[0011] The Lactobacillus reuteri of the present invention has a certain inhibitory effect on intestinal pathogenic bacteria and is non-hemolytic. Therefore, the Lactobacillus reuteri of the present invention can be used as a probiotic to prepare pet food, with the characteristic of high safety. Further, the puppy-derived Lactobacillus reuteri M60 of the present invention has an inhibition zone diameter of ≥20 mm against Escherichia coli, ≥18 mm against Streptococcus pneumoniae, ≥25 mm against Staphylococcus aureus, ≥21 mm against Salmonella, and ≥24 mm against Methicillin-resistant Staphylococcus aureus.

[0012] In some embodiments, the Lactobacillus reuteri is resistant to quinolone antibiotics, aminoglycoside antibiotics or penicillin; and / or the Lactobacillus reuteri is sensitive to at least one of macrolide antibiotics, cephalosporin antibiotics, chloramphenicol antibiotics, and ampicillin.

[0013] Among them, quinolone antibiotics include antibiotics such as norfloxacin and ciprofloxacin, aminoglycoside antibiotics are antibiotics including gentamicin, macrolide antibiotics are antibiotics including chloramphenicol, and cephalosporin antibiotics are antibiotics including cefazolin. The Lactobacillus reuteri of the present invention is sensitive to most antibiotics, which can avoid the problem of antibiotic resistance to a certain extent, prevent the imbalance of the intestinal flora, and has a certain safety.

[0014] In some embodiments, the Lactobacillus reuteri has acid tolerance; and / or the Lactobacillus reuteri has bile salt tolerance; and / or the Lactobacillus reuteri has intestinal adhesion ability.

[0015] The Lactobacillus reuteri of the present invention is acid-tolerant, bile salt-tolerant and has intestinal adhesion ability, can stably survive and colonize in the host, and can play a probiotic role in the intestine to regulate gastrointestinal dysfunction caused by the disorder of the intestinal flora structure.

[0016] Furthermore, the survival rate of M60 is ≥101% after surviving for 3 h at pH 2, ≥94% after surviving for 3 h at pH 1.5, and ≥103% after surviving for 3 h at pH 2.5. The survival rate of M60 is ≥87% after surviving for 3 h under 0.3% bile salt condition, ≥27% after surviving for 3 h under 0.1% bile salt condition, and ≥84% after surviving for 3 h under 0.2% bile salt condition. M60 has extremely strong cell adhesion ability, and the adhesion rate reaches 67%, with good probiotic potential. Adhesion and colonization in the intestine are important prerequisites for lactic acid bacteria to exert their ecological effects and physiological functions, and are one of the important bases for evaluating their probiotic potential. The adhesion of Lactobacillus reuteri to epithelial cells is the main basis for its probiotic function. The adhered Lactobacillus reuteri can exist in the intestine and can resist the invasion of pathogenic bacteria, and produce metabolites such as antibacterial peptides to help effectively kill pathogenic bacteria, achieving functions such as preventing the intestine from being infected by pathogenic bacteria and enhancing the immune ability of the body.

[0017] In some embodiments, the Lactobacillus reuteri has antioxidant properties.

[0018] The Lactobacillus reuteri of the present invention has certain antioxidant properties, among which the DPPH free radical scavenging rate is greater than 48%, and the ABTS free radical scavenging rate is greater than 96%.

[0019] In summary, the Lactobacillus reuteri provided by the present invention has the ability to produce acid and adhere, can regulate the gastrointestinal flora, and maintain the balance of the intestinal microecology. It has an antagonistic effect on pathogenic microorganisms in animals, can competitively inhibit pathogenic microorganisms, enhance the immune function of the animal body, produce beneficial metabolites, activate the activity of acid protease, participate in the metabolism of the body, and prevent the generation of harmful substances. At the same time, the Lactobacillus reuteri of the present invention is derived from the feces of puppies and has various physiological functions such as regulating the gastrointestinal flora, enhancing the immune function, and anti-inflammatory effect. Isolating and screening excellent Lactobacillus reuteri from puppies has important scientific significance and practical application value, can meet the market demand, promote the development of the probiotic industry, facilitate scientific research, improve the health level of animals, and promote the development of related application fields.

[0020] Based on the above technical solutions, the present invention also provides a composition, which comprises the above-mentioned Lactobacillus reuteri ( Lactobacillus reuteri ) or its metabolites.

[0021] In some embodiments, the composition is a probiotic preparation, a functional food, a health product or a drug.

[0022] Among them, the functional foods include but are not limited to: yogurt, milk powder, cheese, snacks and / or freeze-dried staple foods or pet dry foods, etc.

[0023] In addition, the present invention also provides the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri ) or the above composition for use in the preparation of a drug or food for treating, preventing or alleviating gastrointestinal dysfunction.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a new strain of Lactobacillus reuteri M60 from puppies, which has strong adhesion ability and good probiotic ability.

[0025] (2) The Lactobacillus reuteri M60 from puppies provided by the present invention has no hemolytic property and high biosafety.

[0026] (3) The Lactobacillus reuteri M60 from puppies provided by the present invention has good acid tolerance and bile salt tolerance.

[0027] (4) The Lactobacillus reuteri M60 from puppies provided by the present invention is a strain with excellent probiotic properties selected after isolation, screening and identification from the feces of puppies. It has a good inhibitory effect on common pathogenic bacteria and is sensitive to a variety of antibiotics, providing a new candidate strain for pet foods, health care products or probiotic preparations, and having broad application prospects in the preparation of pet foods, health products, drugs, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0029] Figure 1 It is the acid resistance test result at pH = 2 in Example 1.

[0030] Figure 2 It is the bile salt resistance test result at a bile salt concentration of 0.3% in Example 1.

[0031] Figure 3 It is the antibacterial ability result of Lactobacillus reuteri M60 from puppies in Example 1.

[0032] Figure 4 It is the BLAST database annotation result in Example 2.

[0033] Figure 5 It is the colony morphology of Lactobacillus reuteri M60 from puppies in Example 2.

[0034] Figure 6 It is the growth curve of Lactobacillus reuteri M60 from puppies in Example 3.

[0035] Figure 7 It is the in vitro acid resistance ability result of Lactobacillus reuteri M60 from puppies in Example 3.

[0036] Figure 8 It is the in vitro bile salt resistance ability result of Lactobacillus reuteri M60 from puppies in Example 3.

[0037] Figure 9 It is the hemolytic ability result of Lactobacillus reuteri M60 from puppies in Example 3.

[0038] Figure 10 It is the adhesion experiment result of Lactobacillus reuteri M60 from puppies in Example 3.

[0039] Figure 11 It is the DPPH free radical scavenging test result of Lactobacillus reuteri M60 from puppies in Example 3.

[0040] Figure 12 It is the ABTS free radical scavenging test result of Lactobacillus reuteri M60 from puppies in Example 3. Specific embodiments

[0041] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0042] Example 1: Screening and Identification of Lactobacillus reuteri M60 from Puppies 1. Cultivation and Isolation of Strains Select fresh puppy fecal samples, perform gradient dilution with sterilized PBS buffer (purchased from Wuhan Sevier Biotechnology Co., Ltd.), and coat them on MRS solid medium containing 1% CaCO3. Culture in an anaerobic incubator at 37°C for 48 h. Pick single colonies that are white or milky white with obvious transparent circles for Gram staining and microscopic examination. Select rod-shaped and Gram-positive bacteria strains. Select strains M21, M74, and M60 with better growth from numerous strains, and perform enrichment culture in an anaerobic incubator at 37°C; mix the bacterial liquid with sterilized 30% glycerol in a ratio of 1:1 and store it in a -80°C refrigerator for later use.

[0043] 2. Screening of Strains Under sterile conditions, inoculate the strains of M21, M74, and M60 preserved with glycerol into MRS broth medium, culture anaerobically at 37°C for 24 h, and activate for three generations continuously; after centrifuging the bacterial liquid, wash the medium with sterilized PBS 2-3 times. Perform acid tolerance, bile salt tolerance, and antibacterial tests on strains M21, M74, and M60. The test steps for acid tolerance, bile salt tolerance, and adhesion are as follows:

[0044] (1) Acid Tolerance Test Resuspend the washed medium in MRS broth medium with a pH of 2, culture anaerobically at 37°C, take samples for gradient dilution at 0 h and 3 h respectively, coat them on MRS solid medium plates, and count after anaerobic culture at 37°C for 48 h.

[0045] Survival rate of strain (%) = lg (N1 / N0) * 100%; Where: N0 is the viable count at 0 h (cfu / mL); N1 is the viable count of the strain after 3 h of acid tolerance (cfu / mL).

[0046] The experimental results are as Figure 1 shown. The survival rate of strain M60 is the highest, up to 101.97%.

[0047] (2) Bile Salt Tolerance Test The washed culture medium was resuspended in MRS broth medium containing 0.3% bile salt and cultured under anaerobic conditions at 37°C. Samples were taken at 0 h and 3 h for gradient dilution, spread on MRS solid medium plates, and cultured under anaerobic conditions at 37°C for 48 h before counting.

[0048] Survival rate of the strain (%) = lg (N1 / N0) * 100%; Where: N0 is the number of viable bacteria at 0 h (cfu / mL); N1 is the number of viable bacteria after 3 h of bile salt tolerance of the strain (cfu / mL).

[0049] The experimental results are as Figure 2 shown, and the survival rate of strain M60 is 87.31%.

[0050] (3)Determination of the antibacterial ability of Lactobacillus reuteri M60 from puppies Pet food contains a large amount of nutrients. In daily life, food spoilage and deterioration not only harm the health of pets but also cause economic losses. Microbial contamination and growth are the main reasons for food spoilage and deterioration. Therefore, the antibacterial performance of M60 is one of the safety indicators for its application in food.

[0051] Using three common foodborne pathogenic bacteria, Escherichia coli, Staphylococcus aureus, and Salmonella, as indicator bacteria, after activating the indicator bacteria for three generations, the Oxford cup method was used for the preliminary determination of antibacterial activity.

[0052] Using the Oxford cup method, the specific method is as follows: Take the activated strain and inoculate it into MRS broth medium at an inoculation amount of 2% (V / V), and culture it under anaerobic conditions at 37°C for 24 h. Dilute the activated Escherichia coli, Staphylococcus aureus, and Salmonella bacterial suspensions by 10-fold gradients, and select the bacterial suspension with a dilution of 10 -2 for use. Add 5 mL of LB agar medium to the culture dish for bottom sealing. After cooling and solidifying, place Oxford cups evenly. Take 100 μL of each gradient of indicator bacteria suspension and add it to 15 mL of LB agar at 50 - 60°C, mix well and slowly pour it into the plate with Oxford cups. After condensation, use forceps to pull out the Oxford cups to form round holes with a size of 8×6×10. Add 200 μL of the strain fermentation supernatant to the round holes, diffuse it in a 4°C refrigerator for 12 h, and then culture it under anaerobic conditions at 37°C for 9 - 12 h. Observe whether an antibacterial zone is formed, and use a vernier caliper to measure the diameter of the antibacterial zone. Each group of experiments is repeated three times in parallel, and the results are expressed as x±s.

[0053] The experimental results are as Figure 3 shown, and strain M60 has strong inhibitory effects on both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli, Salmonella), and the diameter of the antibacterial zone is above 20 mm.

[0054] ByFigure 1 , Figure 2 and Figure 3 The results show that Lactobacillus reuteri M60 from puppies has extremely strong antibacterial ability, reaching more than 20 mm, far higher than other Lactobacillus reuteri from puppies; and after surviving for 3 h under the acid tolerance condition of pH = 2 and the bile salt tolerance condition of 0.3% bile salt concentration, the survival rates reach 101% and 87% respectively, indicating good acid tolerance and bile salt tolerance.

[0055] Example 2: Identification of Lactobacillus reuteri M60 from puppies The sequencing results were used for homology search in the gene bank by applying BLAST on the NCBI website. The results showed that the highest similarity was 99.93% with Lactobacillus reuteri, and the results were as Figure 4 shown. 99.93% of the genes conformed to the genus Lactobacillus reuteri.

[0056] The strain M60 was diluted and spread on the plate, and the colony status was observed. As Figure 5 shown, combining with the morphological characteristics of the colonies of M60, the strain M60 was identified as Lactobacillus reuteri. In addition, M21 and M74 belong to Lactobacillus reuteri.

[0057] Example 3: Determination of the probiotic properties of Lactobacillus reuteri M60 from puppies 1. Determination of the growth curve of Lactobacillus reuteri M60 from puppies The cultured M60 bacterial solution was activated for three generations and inoculated into MRS broth medium, placed in an incubator at 37°C, and anaerobically cultured. The viable counts of each bacterium were measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h respectively; The growth curve of Lactobacillus reuteri M60 from puppies was plotted, and the results are shown in Figure 6 which, with the OD value on the vertical axis.

[0058] 2. Determination of the in vitro acid and bile salt tolerance of Lactobacillus reuteri M60 from puppies The cultured M60 bacterial solution was activated for three generations and inoculated into MRS broth media with pH values of 1.5, 2.5 and bile salt concentrations of 0.1%, 0.2% respectively, placed in an incubator at 37°C, and anaerobically cultured. Samples were taken for plate counting at 0 h and 3 h respectively; The experimental results are shown in Figure 7 and Figure 8 respectively. As can be seen from Figure 7 , when the bacteria were cultured in the environment of pH 1.5 and pH 2.5 for 0 - 3 h, the growth of the bacteria was stable, and the viable counts were all above 10 8 cfu / mL. The survival rate of the bacteria was 88.89% after being cultured in the environment of pH 1.5 for 3 h, indicating strong acid tolerance. As can be seen from Figure 8 , the survival rate of the bacteria was 84.10% after being cultured in the medium with 0.2% bile salt for 3 h, indicating relatively strong bile salt tolerance.

[0059] 3. Determination of the antibiotic sensitivity of Lactobacillus reuteri M60 from puppies The excessive use of antibiotics in the pharmaceutical industry can lead to an increase in the drug resistance of pathogenic strains, and the flora in the human intestine is prone to disorder and imbalance. Therefore, obtaining lactic acid bacteria that are sensitive to antibiotics has become one of the important indicators for their safety in food applications.

[0060] Adjust the concentrations of the fermentation broths of each strain cultured for 24 h to 10 8 CFU / mL. Add 100 μL of the bacterial solution to the MRS solid plate, spread it evenly, and let it dry. Place 5 - 6 antibiotic susceptibility discs in each plate on average. After placing them in a 4°C refrigerator for diffusion for 12 h, culture them anaerobically at 37°C for 9 - 12 h. The sensitivity of the strain to the drug was judged with reference to the instructions for using the antibiotic susceptibility discs.

[0061] The results are shown in Table 1. As can be seen from Table 1, Lactobacillus reuteri M60 is tolerant to norfloxacin, ciprofloxacin, gentamicin, and penicillin, and sensitive to ampicillin, chloramphenicol, and cefazolin.

[0062]

[0063] S: susceptible, sensitive; I: intermediate, moderately sensitive; R: resistance, tolerant 4. Determination of the hemolytic ability of Lactobacillus reuteri M60 from puppies Dip a inoculation loop into a small amount of bacterial solution and streak it on a Columbia blood agar plate. Culture it anaerobically at 37°C for 24 - 36 h, and observe whether there is a hemolytic zone on the plate. According to the hemolytic phenomenon, it can be divided into α-hemolysis (greenish hemolysis), β-hemolysis (completely transparent hemolytic ring), and γ-hemolysis (non-hemolysis).

[0064] The hemolysis experiment can detect the safety of the strain and determine whether it has hemolytic activity. The results are as Figure 9 shown. No hemolysis occurred in this strain on the blood plate, which was γ-hemolysis. Staphylococcus aureus was selected as the positive control, and a completely transparent hemolytic ring was found around Staphylococcus aureus, which was β-hemolysis. The above results indicate that this Lactobacillus reuteri strain has no hemolytic activity and is a safe and non-toxic strain.

[0065] 5. Adhesion test After resuscitating Caco-2 cells (human colon cancer cells), inoculate them into a cell culture flask, add fresh DMEM complete medium (DMEM high-glucose medium containing double antibiotics was purchased from Beijing Solarbio Science & Technology Co., Ltd., 20% fetal bovine serum), and culture them in an incubator at 37°C and 5% CO2. Change the cell culture medium every two days. When the cell confluence reaches about 70% - 80%, passage them with 0.25% trypsin-EDTA (purchased from Beijing Solarbio Science & Technology Co., Ltd.) mixed digestive solution. The last medium used was DMEM incomplete medium.

[0066] Preparation of FITC solution: FITC (fluorescein isothiocyanate) labeling solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.): Before preparation, place the FITC stored at 4°C at room temperature for 30 min. Accurately weigh 0.5 g and dissolve it in 1 mL of dimethyl sulfoxide (DMSO) to prepare a 500 mg / mL solution. Dilute it 1000-fold (take 10 μL and add it to a 15 mL brown EP tube containing 9.99 mL of DMSO), and the working concentration of the labeling solution is 500 μg / mL, which is stored at 4°C.

[0067] Fluorescent labeling: Take 1 mL of freshly cultured probiotic suspension in a 1.5 mL brown EP tube and centrifuge it (4°C, 4000 rpm, 5 min). Wash it continuously 3 times with sterile PBS. After the last wash, resuspend the cells with 1 mL of FITC solution that has been placed at room temperature for 30 min, and incubate it with shaking in the dark at 37°C for 2 h. After the dark incubation, centrifuge it (4°C, 4000 rpm, 5 min) and wash the strain 3 times with sterile PBS to wash away the unbound FITC labeling solution, and resuspend it with 1 mL of DMEM cell culture medium. Take 200 μL and add it to a black 96-well plate to measure the relative fluorescence intensity value (RFU) of the strain at a wavelength of 485 nm (absorption wavelength) and a wavelength of 530 nm (emission wavelength), which is recorded as the relative fluorescence intensity value R0 of the strain before adhesion.

[0068] Adhesion test: Passage Caco-2 cells more than 3 generations continuously and prepare for the experiment.

[0069] Before the experiment, adjust the cell concentration to 5×10 5 cell / mL with cell culture medium. Spread 1 mL of cell suspension per well on a 24-well cell culture plate and continue culturing. After observing that the cells in the 24-well plate have grown to a monolayer (about 24 h of culture), discard the cell culture medium, wash it 3 times with sterile PBS, and then add 0.6 mL of the labeled probiotic suspension. Set three replicate wells for each strain and incubate it in the dark at a constant temperature of 37°C and 5% CO2 for 2 h. After taking it out, discard the culture medium and wash it 3 times with sterile PBS to remove the strains that have not adhered to the cells; add 0.3 mL of trypsin to each well to digest the cells for 5 min, and terminate the digestion with DMEM cell culture medium. Measure the relative fluorescence intensity value (RFU) of the cell suspension under the same wavelength conditions, which is recorded as the relative fluorescence intensity value R of the strain after adhesion.

[0070] Calculation formula for adhesion rate (%): R / R0×100% CFU (Colony Forming Unit): The total number of bacterial communities in a unit volume; when counting viable bacteria in culture, the colonies formed by single bacteria or multiple bacteria aggregated into clusters during growth and reproduction on solid medium are called colony forming units, and the number of viable bacteria is expressed by them.

[0071] The experimental results are shown in Figure 10 As shown, the adhesion rate of strain M60 reached 67.07%.

[0072] 6. Determination of antioxidant capacity (1)DPPH free radical scavenging assay Collect the bacterial liquid and adjust the concentration of the bacterial liquid to 10 -8 , centrifuge to obtain the bacterial cells and the supernatant of the strain, and use the DPPH free radical scavenging capacity kit (purchased from Beijing Solarbio Science & Technology Co., Ltd.) for the experiment. The specific operation is carried out according to the steps in the instruction manual.

[0073] DPPH free radical scavenging rate of the sample (%) = (1 - (A measurement - A control) / A blank) × 100% The experimental results are shown in Figure 11 , the DPPH free radical scavenging rate of strain M60 was 57.37%, and the DPPH free radical scavenging rate of the supernatant of strain M60 was 48.06%, showing certain antioxidant properties.

[0074] (2)ABTS free radical scavenging assay Collect the bacterial cells and adjust the concentration of the bacterial liquid to 10 -8 , use the ABTS free radical scavenging capacity reagent gold (Beijing Solarbio Science & Technology Co., Ltd.), and the specific operation is carried out according to the steps in the instruction manual.

[0075]

[0076] ABTS free radical scavenging rate (%) = [A blank - (A measurement - A control)] / A blank × 100%.

[0077] The experimental results are shown in Figure 12 , the ABTS free radical scavenging rate of strain M60 was 96.38%, showing certain antioxidant properties.

[0078] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Lactobacillus reuteri, characterized in that, The Lactobacillus reuteri is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC NO. 31007 and the deposit date of June 19, 2024.

2. The Lactobacillus reuteri according to claim 1, characterized in that, The Lactobacillus reuteri is isolated from the feces of puppies.

3. The Lactobacillus reuteri according to claim 1, characterized in that, The 16s rRNA gene sequence of the Lactobacillus reuteri is as shown in SEQ ID NO:

01.

4. The Lactobacillus reuteri according to claim 1, characterized in that, The Lactobacillus reuteri inhibits intestinal pathogenic bacteria, and the intestinal pathogenic bacteria are selected from at least one of Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus, Salmonella, and Methicillin-resistant Staphylococcus aureus; and / or The Lactobacillus reuteri is non-hemolytic.

5. The Lactobacillus reuteri according to claim 1, characterized in that, The Lactobacillus reuteri is resistant to quinolone antibiotics, aminoglycoside antibiotics or penicillin; and / or The Lactobacillus reuteri is sensitive to at least one of macrolide antibiotics, cephalosporin antibiotics, chloramphenicol antibiotics, and ampicillin.

6. The Lactobacillus reuteri according to claim 1, characterized in that, The Lactobacillus reuteri has acid tolerance ability; and / or The Lactobacillus reuteri has bile salt tolerance ability; and / or The Lactobacillus reuteri has intestinal adhesion ability.

7. The Lactobacillus reuteri according to claim 1, characterized in that, The Lactobacillus reuteri has antioxidant property.

8. A composition, characterized in that, The composition comprises the Lactobacillus reuteri as claimed in any one of claims 1 to 7 or its metabolites.

9. The composition according to claim 8, wherein The composition is a probiotic preparation, functional food, health product or medicine.

10. Use of the Lactobacillus reuteri as claimed in any one of claims 1 to 7 or the composition as claimed in claim 8 or 9 in the preparation of a drug or food for treating, preventing or alleviating gastrointestinal dysfunction.

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