A canine-derived Lactobacillus reuteri, bacterial agent and application thereof

Abstract

The present invention discloses a canine-derived Lactobacillus reuteri, a bacterial agent and an application thereof, and relates to the technical field of probiotics. The present invention separates and screens a canine-derived Lactobacillus reuteri, which has a high free radical scavenging ability and good antioxidant ability, is beneficial to animal anti-aging, and prolongs the life of pets. It has excellent acid resistance, good high temperature resistance, bile salt resistance, gastrointestinal fluid resistance, and is moderately hydrophobic. Therefore, the strain has good intestinal tolerance and adhesion, which is conducive to its colonization in the intestine and long-term use of its probiotic function. It can effectively inhibit the growth and proliferation of Escherichia coli, Staphylococcus aureus and Salmonella, and can be used to prevent or treat diarrhea. The bacterial agent provided by the present invention has a wide range of application prospects and can be used to prepare medicines and feeds.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a canine-derived Lactobacillus reuteri, a bacterial agent and applications thereof. Background Art

[0002] Lactobacillus reuteri ( Lactobacillus reuteri ) belongs to the genus Lactobacillus ( Lactobacillus ). Lactobacillus reuteri is an important heterofermentative probiotic found in the intestines of animals. Studies have shown that Lactobacillus reuteri can improve intestinal function, increase feed utilization, and improve animal growth performance; in addition, Lactobacillus reuteri adheres to intestinal epithelial cells and can communicate with other immune cells, has strain-specific immunomodulatory effects, and prevents and relieves inflammation; other studies have shown that Lactobacillus reuteri has antioxidant capacity and reduces animal stress damage; it can also regulate intestinal flora, inhibit the colonization of pathogenic microorganisms, prevent or alleviate diarrhea, maintain intestinal homeostasis, and enhance animal health.

[0003] Dogs and cats, as the main companion animals of humans, live together with humans and benefit each other. Now most pets feed on high carbohydrates, and pet health faces similar challenges as humans. It is well known that intestinal health is one of the main factors affecting animal health. With the introduction of the concept of scientific pet care, many functional pet foods that help improve intestinal health have emerged. At present, the commonly used probiotics in pet foods at home and abroad include Bifidobacterium, Lactobacillus acidophilus, Bacillus, Enterococcus, etc., all of which have the function of improving intestinal health. Although most probiotics have been "generally recognized as safe" (GRAS), it is still crucial to evaluate and verify the safety of each strain of bacteria. In addition, most of the probiotics currently used on the market are derived from humans rather than pet hosts. However, host-derived probiotics have co-evolved with the host for thousands of years, so they can better adapt to the gastrointestinal environment of the same animal, have better colonization and adhesion, and can better exert their probiotic properties. At present, there are few reports on canine-derived Lactobacillus reuteri, and there are even fewer corresponding products on the market.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a canine-derived Lactobacillus reuteri, a bacterial agent and application thereof to solve the above-mentioned technical problems.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a canine-derived Lactobacillus reuteri ( Lactobacillus reuteri ), its accession number is CCTCC NO: M 20232296.

[0008] The inventor isolated a bacterium from the rectal feces of adult healthy female beagles in Jimo Animal Husbandry Experimental Field, Qingdao, China. After morphological observation, microscopic examination, physiological and biochemical experiments, combined with 16S rRNA sequencing comparison, it was identified as Lactobacillus reuteri. The colony morphology is milky white, convex in the middle, neat edges, and smooth surface; facultative anaerobic, Gram staining is positive, and rod-shaped. Its 16S rRNA sequence is shown in SEQ ID NO.1. The strain has a high free radical scavenging ability and good antioxidant capacity, which is beneficial to animal anti-aging and prolongs the life of pets. It has excellent acid resistance, good high temperature resistance, bile salt resistance, gastrointestinal fluid resistance, and moderate hydrophobicity. Therefore, the strain has good intestinal tolerance and adhesion, which is conducive to its colonization in the intestine and long-term use of its probiotic function. It can effectively inhibit the growth and proliferation of Escherichia coli, Staphylococcus aureus and Salmonella, and can be used to prevent or treat diarrhea. It is highly sensitive to penicillin, piperacillin and imipenem and has high biosafety. This strain can increase the ratio of intestinal villus length to crypt depth (V / C value), improve intestinal morphology, and enhance the absorption capacity of nutrients. It has broad application prospects.

[0009] Lactobacillus reuteri of canine origin was deposited in the China Center for Type Culture Collection on November 22, 2023, with the deposit address being Wuhan University, Wuhan, China. The name of the deposited culture and the indicated identification characteristics are: Lactobacillus reuteri LRA7, its deposit number is CCTCC NO: M 20232296, and its identification result is alive.

[0010] In a second aspect, the present invention further provides a bacterial agent, which includes the above-mentioned canine Lactobacillus reuteri, a live fermentation product of canine Lactobacillus reuteri, or an inactivated fermentation product of canine Lactobacillus reuteri.

[0011] The fermentation live bacteria product includes but is not limited to the fermentation broth, the supernatant or bacterial sludge after centrifugation or static separation of the fermentation broth, or a mixture of cell lysate and bacteria after water extraction treatment.

[0012] The fermentation inactivated product includes but is not limited to the product obtained by inactivating the above-mentioned fermentation broth, fermentation supernatant, bacterial sludge or cell lysate and a mixture of bacteria. Inactivation includes but is not limited to heat inactivation, ultrasonic inactivation, etc.

[0013] It is easy for technicians in this field to think that postbiotics obtained after inactivation also have the same efficacy as live bacteria.

[0014] In a preferred embodiment of the present invention, the fermentation inactivated product is selected from at least one of a cell-free supernatant and an inactivated bacterial precipitate.

[0015] In an optional embodiment, the bacterial agent is liquid, solid or semisolid. Liquids include but are not limited to solutions, suspensions, emulsions, semi-emulsions, etc. Solids include but are not limited to: granules, powders, flakes, etc. Semisolid dosage forms include but are not limited to ointments, suppositories, pastes, etc.

[0016] In a third aspect, the present invention further provides the use of canine-derived Lactobacillus reuteri or the above-mentioned bacterial agent in the preparation of fermented food, wherein the fermented food is solid food, liquid food and semi-solid food; the food is pet food.

[0017] In a fourth aspect, the present invention further provides the use of canine-derived Lactobacillus reuteri or the above-mentioned bacterial agent in the preparation of a medicine or feed for any of the following purposes:

[0018] (1) Prevent or treat diarrhea;

[0019] (2) Enhance intestinal immunity;

[0020] (3) Anti-aging;

[0021] (4) Prevention and / or treatment of inflammatory diseases.

[0022] The feed is, for example, animal feed, including but not limited to pet feed, ruminant feed, poultry feed, fish feed, etc. For example, feed for dogs, cats, amphibian and reptile pets, pigs, sheep, cattle, horses, and fish.

[0023] In a preferred embodiment of the present invention, the prevention or treatment of diarrhea is achieved by inhibiting pathogenic microorganisms and / or improving the liver function of animals.

[0024] In a preferred embodiment of the present invention, improving intestinal immunity is achieved by enhancing intestinal barrier function or enhancing the body's antioxidant level.

[0025] The intestinal barrier is an important barrier that can prevent harmful substances such as pathogenic microorganisms and toxins from entering other tissues, organs and blood of the human body. The normal intestinal mucosal barrier is composed of mechanical barriers, chemical barriers, immune barriers and biological barriers. The use of the canine-derived Lactobacillus reuteri agent provided by the present invention to avoid or repair damage to the mechanical barrier, chemical barrier, immune barrier and / or biological barrier all falls within the scope of protection of the present invention.

[0026] In an optional embodiment, enhancing the intestinal barrier function comprises at least one of the following:

[0027] (1) maintain the integrity of the villus structure of the jejunum and / or ileum; (2) increase the ratio of villus length to crypt depth of the jejunum and / or ileum; (3) reduce or eliminate crypt abscesses or ruptures of the jejunum and / or ileum; and (4) reduce the crypt depth of the jejunum and / or ileum.

[0028] In an optional embodiment, enhancing the body's antioxidant level refers to enhancing the body's free radical scavenging ability;

[0029] In an optional embodiment, the free radical is selected from at least one of the following: a DPPH free radical, an ABTS free radical and a superoxide anion free radical.

[0030] In a preferred embodiment of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections or sprays.

[0031] In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as a solution, a suspension and a gel usually containing a liquid carrier, such as water and / or a pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include a pH regulator, an emulsifier or a dispersant, a buffer, a preservative, a wetting agent, a gelling agent (such as methylcellulose), a dye and / or a flavoring agent, such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be adjusted by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be adjusted by a pharmaceutically acceptable thickener such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

[0032] In an optional embodiment, the above-mentioned drug is a solid pharmaceutical preparation, such as freeze-dried bacterial powder, granular preparation, etc.

[0033] In an alternative embodiment, the medicament is formulated for oral administration, injection administration or intragastric administration.

[0034] In a fifth aspect, the present invention further provides a composition comprising the above-mentioned canine-derived Lactobacillus reuteri or the above-mentioned bacterial agent.

[0035] In a preferred embodiment of the present invention, the composition is a pharmaceutical composition, a vaccine composition or a feed composition.

[0036] The vaccine composition may also include an adjuvant, for example.

[0037] In a sixth aspect, the present invention further provides a method for preparing a bacterial agent, comprising: culturing canine-derived Lactobacillus reuteri at 20-40°C, preferably, at 37°C.

[0038] The present invention has the following beneficial effects:

[0039] The present invention separates and screens a canine-derived Lactobacillus reuteri strain, which has a high free radical scavenging ability and good antioxidant capacity, is beneficial to animal anti-aging and prolongs the life of pets. It has excellent acid resistance, good high temperature resistance, bile salt resistance, gastrointestinal fluid resistance, and is moderately hydrophobic. Therefore, the strain has good intestinal tolerance and adhesion, is conducive to its colonization in the intestine, and exerts its probiotic function for a long time. It can effectively inhibit the growth and proliferation of Escherichia coli, Staphylococcus aureus and Salmonella, and can be used to prevent or treat diarrhea.

[0040] In addition, it is highly sensitive to penicillin, piperacillin and imipenem, and has high biosafety. The strain can increase the ratio of intestinal villus length to crypt depth (V / C value) of animals, improve intestinal morphology, and enhance the absorption capacity of nutrients. It has broad application prospects and can be used to prepare drugs and feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0042] Figure 1 Figure 2 shows the colony morphology (A) and cell morphology (B) of Lactobacillus reuteri LRA7;

[0043] Figure 2 is a phylogenetic tree diagram;

[0044] Figure 3 This is the growth curve of Lactobacillus reuteri LRA7 under different pH conditions;

[0045] Figure 4 It is the growth curve and acid production curve of Lactobacillus reuteri LRA7;

[0046] Figure 5 This is a graph showing the high temperature resistance characteristics of Lactobacillus reuteri LRA7;

[0047] Figure 6 The antibacterial images of Lactobacillus reuteri LRA7 (Escherichia coli (A), Staphylococcus aureus (B) and Salmonella (C));

[0048] Figure 7 This is the picture of hemolysis of Lactobacillus reuteri LRA7 (Staphylococcus aureus (A), LRA7 (B));

[0049] Figure 8 This is a statistical diagram of the body weight and food intake of mice fed with LRA7;

[0050] Fig. 9 HE staining results of mouse jejunum and ileum ((AD) shows the HE staining picture of mouse jejunum, (EH) shows the HE staining picture of ileum);

[0051] Fig.10 is the genome circos map;

[0052] Fig.11 This is the classification diagram of COG results;

[0053] Fig.12 This is the classification diagram of KEGG pathway results;

[0054] Fig.13 This is a classification diagram of GO annotation results. DETAILED DESCRIPTION

[0055] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0056] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0057] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0058] Example 1

[0059] This example is to isolate and identify canine-derived Lactobacillus reuteri.

[0060] 1. Preparation of separation medium:

[0061] MRS culture medium, the specific components are shown in Table 1:

[0062] Table 1 MRS medium composition

[0063]

[0064] pH 6.5±0.2, autoclave at 121℃ for 15 min.

[0065] 2. Separation method

[0066] (1) Sample collection and microbial isolation: The samples were collected from three healthy adult female beagles at Jimo Animal Husbandry Experimental Farm in Qingdao, China. Rectal feces were collected by sterile swabs and quickly placed in a 50 mL sterile test tube containing physiological saline. They were transported to the Special Economic Animal Nutrition Laboratory of Qingdao Agricultural University in cold storage and immediately diluted in gradients, with three plates per gradient, separated and cultured by pouring method, and inverted at 37°C.

[0067] (2) Isolation and purification: Take the above plates with various bacteria, select single lactic acid bacteria colonies with different colony morphologies, and use the continuous streaking method to separate and purify them. Repeat the purification steps 3 to 5 times until a pure strain is obtained.

[0068] (3) Observe the characteristics of the colonies, perform Gram staining and microscopic examination, and observe the cell morphology of the strain.

[0069] (4) Bacterial DNA was extracted according to the instructions of the Bacterial Genome Extraction Kit (D1600) of Beijing Solebow Technology Co., Ltd., and the conserved sequences of the obtained strains were amplified and sequenced using the bacterial universal primers 27F and 1492R. Primer synthesis and sequencing were completed by Beijing Qingke Biotechnology Co., Ltd. Then the 16S rRNA sequence was compared for homology. The amplification system of 16SrDNA was as follows. The total volume of the amplification system was 50 μl, including 19 μL of ddH2O, 2 μL of DNA plate, 2 μL of Primer F, 2 μL of Primer R, and 25 μL of 2×Es Taq MasterMix (containing dye). PCR reaction program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 45 s, 56℃ annealing for 30 s, 72℃ extension for 45 s, a total of 30 cycles, 72℃ extension for 10 min, and storage at 4˚C.

[0070] 3. Isolation and identification of Lactobacillus reuteri

[0071] Through morphological observation, microscopic examination, physiological and biochemical experiments, combined with 16S rRNA sequencing, three strains of Lactobacillus reuteri were identified and named LR67, LRA1 and LRA7. The 16S rRNA gene sequence of LRA7 is shown below. The colony morphology of Lactobacillus reuteri LRA7 is milky white, convex in the middle, with neat edges and smooth surface; it is facultative anaerobic, Gram-positive, and rod-shaped. The colony morphology of LRA7 is shown below. Figure 1 As shown in A, the microscopic morphology is Figure 1 As shown in B.

[0072] Phylogenetic analysis of the 16S rRNA gene of LRA7 confirmed that it was closely related to Lactobacillus reuteri. Figure 2 shown.

[0073] The sequence of Lactobacillus reuteri LRA7 16S rRNA gene is shown in SEQ ID NO.1.

[0074]

[0075] 4. Physiological and biochemical identification

[0076] Take the isolated bacterial liquid after 18 hours of activation culture, and identify the physiological and biochemical characteristics of lactic acid bacteria with reference to the "Bergey's Bacterial Identification Manual", "Common Bacterial System Identification Manual" and "Lactic Acid Bacteria Classification and Identification and Experimental Methods", and identify the biochemical characteristics such as sugar fermentation test, VP test, methyl red (MR) test, gelatin test, etc.

[0077] The physiological and biochemical characteristics of Lactobacillus reuteri LRA7 are shown in Table 2. Lactobacillus reuteri LRA7 can utilize monosaccharides such as galactose, glucose, arabinose, and xylose, disaccharides such as sucrose, maltose, and lactose, and oligosaccharides such as raffinose.

[0078] Table 2 Physiological and biochemical characteristics of Lactobacillus reuteri LRA7

[0079]

[0080] +: positive reaction; -: negative reaction

[0081] Example 2

[0082] The strains LR67, LRA1 and LRA7 were screened for their antioxidant and acid resistance abilities.

[0083] (1) DPPH free radical scavenging ability

[0084] The DPPH scavenging ability of the isolated strains was determined using the Solebow DPPH detection kit. The operation was carried out according to the instructions. In short, 100 μL of the isolated bacterial culture after 18 h of culture was added to 900 μL of the extract, vortexed and mixed, centrifuged at 10000 rpm at room temperature for 10 min, 25 μL of the supernatant was added to 975 μL of the DPPH working solution, vortexed and mixed, and allowed to stand at room temperature in the dark for 30 min. The absorbance was recorded at 515 nm using an ELISA reader. The calculation formula for DPPH free radical scavenging rate is as follows:

[0085] (1)

[0086] in: is the absorbance of the sample to be tested; The absorbance of the supernatant and the mixture of anhydrous ethanol was used to isolate the strain; is the absorbance of the mixture of the extract and the working solution.

[0087] (2) ABTS free radical scavenging ability

[0088] The ABTS scavenging ability of the isolated strains was determined using the Solebow ABTS detection kit. The operation was carried out according to the instructions. In short, 100 μL of the isolated bacterial culture after 18 h of culture was added to 900 μL of the extract, vortexed and mixed, centrifuged at 10000rpm for 10 min at room temperature, and the supernatant was taken and placed on ice for testing. 50 μL of the supernatant was added to 850 μL of ABTS working solution, and then 100 μL of reagent four working solution was added, mixed thoroughly, and allowed to stand at room temperature in the dark for 6 min. The absorbance was measured at 405 nm using an enzyme reader, and blank and control were also performed. The calculation formula for ABTS free radical scavenging rate is as follows:

[0089] (2)

[0090] in: is the absorbance of the sample to be tested; is the absorbance of the control tube; The absorbance of the blank tube.

[0091] (3) Superoxide anion free radical scavenging test

[0092] The superoxide anion scavenging ability of the isolated strains was determined using the Solebow Superoxide Anion Free Radical Scavenging Detection Kit according to the instructions. The absorbance of the blank tube and the assay tube was measured at 530 nm and recorded as A blank and A assay, respectively. The calculation formula is as follows:

[0093] (3).

[0094] The scavenging rates of strains LR67, LRA1 and LRA7 for ABTS, DPPH and superoxide anion free radicals are shown in Table 3. The scavenging rates of strains LRA1 and LRA7 for ABTS were significantly higher than those of strain LR67; the scavenging rate of LRA7 for DPPH was significantly higher than that of LR67, but there was no significant difference with LRA1; there was no significant difference in the scavenging rate of superoxide anions among the three strains.

[0095] Table 3 Free radical scavenging rate of Lactobacillus reuteri LR67, LRA1 and LRA7%

[0096]

[0097] Note: Data in the same row with different lowercase letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05), the same as in the following table.

[0098] (3) Acid resistance

[0099] Take the isolated bacterial suspension after activation culture for 18 hours, centrifuge at 4℃, 10000 rpm for 10 min, discard the supernatant, wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in sterile PBS buffer (pH 2.5). After culturing at 37℃ for 0 h and 3 h, spread 10× serial dilutions of the culture on MRS agar plates, and then incubate at 37℃ for 24 h. The acid tolerance of the isolated strains was evaluated by the number of viable colonies. The test was repeated 3 times.

[0100] (4)

[0101] in, and The numbers of surviving bacteria (log CFU / mL) before (0 h) and after (3 h) incubation are shown.

[0102] As shown in Table 4, the survival rate of Lactobacillus reuteri LRA7 was 79.98% after 3 h of inoculation in PBS buffer at pH = 2.5. The acid tolerance of LRA7 was significantly higher than that of LR67 and LRA1. Figure 3 It can be seen that the growth of Lactobacillus reuteri LRA7 can be completely inhibited when pH < 3, the strain can grow when pH ≥ 4, and pH = 6 is the optimal growth pH.

[0103] Table 4 Acid resistance results of Lactobacillus reuteri LRA7%

[0104]

[0105] Example 3

[0106] Based on the free radical scavenging ability and acid resistance test results, Lactobacillus reuteri LRA7 was selected for probiotic property evaluation and safety evaluation.

[0107] 1. Growth performance and acid production performance determination

[0108] The isolated bacterial culture liquid after 18 hours of activation culture was inoculated into MRS medium at a 2.0% inoculum and cultured at 37°C for 24 hours. The control group was a liquid culture medium without bacterial culture liquid. The culture liquid OD was measured at 0, 1, 2, 3, 6, 9, 12, 15, 18, 24, 30, 36, and 48 hours. 600 The absorbance value was measured by taking samples at 0, 1, 2, 3, 6, 9, 12, 15, 18, 24, 30, 36, and 48 h, respectively. 600 The growth curve and acid production curve are drawn with the nm absorbance value and the pH of the culture medium as the ordinate.

[0109] like Figure 4As shown in the figure, Lactobacillus reuteri LRA7 grew slowly from 0 to 3 h and was in the growth retardation phase. 600 The absorbance value of nm increased rapidly, and it was in the logarithmic growth phase. After 12 hours, the absorbance of the bacterial solution tended to be stable, and it entered the stable growth phase. The pH curve of the bacterial solution acid production was consistent with the growth curve. The pH dropped slowly within 3 hours; after 3 hours, the pH drop rate accelerated, and after 12 hours, the pH change of the bacterial solution tended to be gentle and stabilized at about 4.4.

[0110] 2. High temperature resistance test

[0111] Take the isolated bacterial culture after 18 hours of activation culture, treat it in a water bath at 37℃, 50℃, 60℃, 70℃, 80℃ and 90℃ for 5 minutes, and put it in an ice box immediately after treatment. Take 100 μL of culture solution for gradient dilution and spread it on the plate, and calculate the number of viable bacteria by plate count method, with lg CFU / mL as the counting unit.

[0112] Depend on Figure 5 It can be seen that compared with the normal culture condition of 37℃, the survival number of Lactobacillus reuteri LRA7 after high temperature treatment at 70℃ is still more than 6 lg (CFU / mL), and its survival rate is more than 50% after high temperature treatment at 80℃. Therefore, Lactobacillus reuteri has good high temperature resistance.

[0113] 3. Gastrointestinal colonization ability

[0114] (1) Bile salt tolerance test

[0115] The isolated bacterial suspension after 18 h of activation culture was centrifuged at 4°C, 10,000 rpm for 10 min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), and resuspended in PBS buffer containing 0.1%, 0.3%, and 0.5% (w / v) porcine bile salts (Solarbio, China). After incubation at 37°C for 0 h and 4 h, 10× serial dilutions of the culture were spread on MRS agar plates and incubated in a 37°C incubator for 24 h. The tolerance of LRA7 to bile salts was evaluated by counting viable colonies. The experiment was repeated 3 times.

[0116] (5)

[0117] in, and The numbers of surviving bacteria (log CFU / mL) before (0 h) and after (4 h) incubation are shown.

[0118] As shown in Table 5, the survival rates of Lactobacillus reuteri LRA7 in 0.1%, 0.3% and 0.5% bile salt were 98.77%, 92.95% and 91.25%, respectively, and the viable counts were 1.5×10 8 CFU / mL, 1.2×10 6 CFU / mL and 2.0×10 5 CFU / mL.

[0119] Table 5 Survival rate of bile salt tolerance of Lactobacillus reuteri LRA7%

[0120]

[0121] (2) Artificial gastrointestinal fluid tolerance test

[0122] The preparation of artificial gastric juice and artificial intestinal juice refers to the 2010 edition of the "Chinese Pharmacopoeia".

[0123] The isolated bacterial suspension after 18 hours of activation culture was centrifuged at 4°C, 10,000 rpm for 10 min, the supernatant was discarded, washed twice with sterile neutral PBS buffer (pH 7.0), resuspended in artificial gastric juice, and incubated at 37°C, 200 rpm for 1.5 h. After incubation, the bacterial precipitate was washed twice with sterile neutral PBS buffer (pH 7.0), centrifuged at 4°C, 10,000 rpm for 10 min, then resuspended in artificial intestinal juice and incubated at 37°C, 200 rpm for 3 h. Before and after incubation in artificial gastric juice and intestinal juice, the bacterial precipitate activity was calculated by the plate count dilution method, and the experiment was repeated 3 times.

[0124] (6)

[0125] in, and The numbers of surviving bacteria (log CFU / mL) before and after incubation in artificial gastric or intestinal fluid, respectively.

[0126] As shown in Table 6, the survival rates of Lactobacillus reuteri LRA7 in artificial gastric juice, intestinal juice and gastrointestinal juice were 85.41%, 84.57% and 91.12%, respectively, and the viable counts were 2.06×10 6 CFU / mL, 1.82×10 8 CFU / mL and 7.06×10 7 CFU / mL.

[0127] Table 6 Tolerance of Lactobacillus reuteri LRA7 to artificial gastrointestinal fluid

[0128]

[0129] (3) Surface hydrophobicity test

[0130] The bacterial adhesion to hydrocarbons (BATH) method was used to reflect the hydrophobicity of the strain surface through the affinity of lactic acid bacteria to hydrocarbons. The isolated bacterial culture after 18 h of activation culture was centrifuged at 4°C, 10,000 rpm for 10 min, washed three times with sterile neutral PBS buffer (pH 7.0), and the OD of the bacterial culture was adjusted. 630 The absorbance value of nm was 0.25±0.05 (A1) and it was set aside. Then, xylene and chloroform were added in a ratio of 1:1, vortexed for 3 min, and incubated at 37°C for 4 h and 8 h. Finally, the aqueous phase was carefully aspirated and the OD was measured. 630 The absorbance value (A2) was measured at 100 nm, and the experiment was repeated 3 times. The hydrophobicity calculation formula is as follows:

[0131] Hydrophobicity CSH (%) = (1-A2 / A1) × 100% (7)

[0132] A1 and A2 are the OD values ​​before and after incubation, respectively. 630 nm absorbance value.

[0133] As shown in Table 7, there was no significant difference in the surface hydrophobicity of Lactobacillus reuteri LRA7 in xylene, chloroform and xylene-chloroform 1:1 for 4 h and 8 h, which was moderately hydrophobic.

[0134] Table 7 Surface hydrophobicity of Lactobacillus reuteri LRA7%

[0135]

[0136] (4) Self-cohesion test

[0137] Take the isolated bacterial solution cultured overnight for 18 h, centrifuge at 10,000 rpm for 10 min at 4°C, wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in an equal volume of sterile neutral PBS buffer (pH 7.0) to measure the OD 630 The sample was then vortexed for 10 s and incubated at 37 °C for 4 h and 8 h, avoiding stirring. The upper portion was carefully absorbed and the OD was measured. 630 The absorbance value was measured by the nm method (A4), and the experiment was repeated 3 times. The formula for calculating the self-aggregation rate is as follows:

[0138] Autoaggregation ability(%)=(1-A4 / A3)×100%(8)

[0139] Among them, A3 and A4 are the OD before and after incubation, respectively. 630nm absorbance value.

[0140] As shown in Table 8, the self-aggregation rates of Lactobacillus reuteri LRA7 at 4 h and 8 h were 21.85% and 44.62%, respectively.

[0141] Table 8 Self-aggregation rate of Lactobacillus reuteri LRA7%

[0142]

[0143] (5) Antibacterial activity

[0144] The antibacterial properties of the strains were determined by the Oxford cup agar diffusion method. The test strains were Escherichia coli ATCC25922, Salmonella ATCC14028, and Staphylococcus aureus ATCC25923. The concentration was 1×10 7 100 μL of pathogenic bacteria with CFU / mL was spread on TSA agar plates. After the bacterial solution was completely absorbed, 200 μL of bacterial fermentation broth, cell-free supernatant and bacterial protein were inoculated into each well. Then the TSA plates were placed upright in a 37°C constant temperature incubator for incubation. After 24 hours, the formation of inhibition zones was observed and the size of the inhibition zones was accurately measured.

[0145] The cell-free supernatant was prepared by the following method: Lactobacillus reuteri LRA7 was cultured at 37°C for 18 h, and then centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was aspirated in an ultra-clean workbench and filtered with a 0.22 μm sterile filter to obtain the cell-free supernatant.

[0146] Depend on Figure 6 It can be seen that the bacterial suspension (b) and cell-free supernatant (a) of Lactobacillus reuteri LRA7 can effectively inhibit the growth and proliferation of Escherichia coli (A), Staphylococcus aureus (B) and Salmonella (C), and the bacterial precipitate (c) has no antibacterial effect. As shown in Table 9, the antibacterial effect of LRA7 cell-free supernatant (a) is Salmonella>Staphylococcus aureus>Escherichia coli, and there is no significant difference in the antibacterial effect of the bacterial suspension on the three pathogens.

[0147] Table 9 Antibacterial test results of Lactobacillus reuteri LRA7

[0148]

[0149] Note: Data in the same column without letters or with the same letters indicate no significant difference (P>0.05), and data with different lowercase letters indicate significant difference (P<0.05).

[0150] (6) Antibiotic sensitivity test

[0151] The antibiotic sensitivity of the strains was determined by disk diffusion method. According to the recommendations for evaluating the safety of probiotics and combined with the common antibiotics on the market, 30 antibiotics were selected for testing in the form of 6 mm disks (BIO-KONT, China): penicillin, oxacillin, ampicillin, piperacillin, cephalexin, cefazolin, cefuroxime sodium, ceftazidime, ceftriaxone, cefoperazone, lincomycin, vancomycin, polymyxin B, cotrimoxazole, chloramphenicol, amikacin, gentamicin, kanamycin, streptomycin, tetracycline, minocycline, erythromycin, azithromycin, norfloxacin, ciprofloxacin, clindamycin, levofloxacin, imipenem, doxycycline, and florfenicol.

[0152] Take the isolated bacterial suspension after 18 hours of activation culture, centrifuge at 4℃, 10000 rpm for 10 min, wash twice with sterile neutral PBS buffer (pH 7.0), and then resuspend in sterile neutral PBS buffer (pH 7.0) to obtain a bacterial suspension in the logarithmic growth phase. Take 100 uL of the suspension of the isolated strain and spread it on the MRS agar plate. Finally, stick the antibiotic paper on the surface of the MRS agar plate within 15 minutes, and incubate the plate at 37℃ for 24 hours to observe whether there is an inhibition zone formed, and measure the diameter of the inhibition zone with a vernier caliper.

[0153] The diameter of the drug sensitivity sheet is 6 mm, so the diameter of the inhibition zone greater than 7 mm is considered to be antibacterial, and less than or equal to 7 mm is considered to be non-inhibitory. As shown in Table 10, strain LRA7 has different sensitivities to various common antibiotics. It is highly sensitive to penicillin, piperacillin and imipenem, with an inhibition zone diameter greater than 23 mm, moderately sensitive to ampicillin, chloramphenicol and tetracycline, with an inhibition zone diameter between 15 and 22 mm, and has an inhibition zone diameter between 7 and 14 mm for cephalexin, lincomycin and ciprofloxacin. It is insensitive to oxacillin, vancomycin and streptomycin, with no inhibition zone.

[0154] Table 10 Antibiotic susceptibility of Lactobacillus reuteri LRA7

[0155]

[0156] Note: Diameter of inhibition zone (mm): +++: 23-30 mm; ++: 15-22 mm; +: 7-14 mm; −: no inhibition zone.

[0157] Example 4

[0158] This example performs a safety evaluation.

[0159] (1) Hemolysis test

[0160] The isolates were streaked on tryptic soy agar (TSA) containing 5.0% (w / v) sheep blood (Oxoid, Germany) after 18 h of activation culture. The plates were incubated at 37°C for 48 h and the blood agar plates were examined for signs of β-hemolysis (clear areas around colonies), α-hemolysis (green areas around colonies), or γ-hemolysis (no areas around colonies). Staphylococcus aureus (ATCC25923) strain was used as a positive control.

[0161] Staphylococcus aureus was used as a positive control ( Figure 7 a), obvious β-hemolysis was observed (clear areas around the colonies), but LRA7 ( Figure 7 b) It is γ-hemolytic (no hemolytic ring around the colonies), which can be preliminarily judged that strain LRA7 is safe and non-pathogenic.

[0162] (2) Safety test in mice

[0163] For in vivo safety evaluation, 80 healthy Kunming mice with similar body weight were randomly divided into 4 groups, with 20 animals in each group. The mice in the control group (CON) were gavaged with 0.2 mL of sterile saline, while the mice in the other three groups were gavaged with 0.2 mL of 1×10 9 (LR), 1×10 10 (MR) and 1×10 11 (HR) CFU / mL. Body weight and food intake were measured every 3 days, and the health status of the mice was recorded. After 28 days, the mice were fasted for 12 hours and then anesthetized with 0.3% sodium pentobarbital (0.15 mL / 10g). Blood was collected from the abdominal aorta, and serum was collected by centrifugation (4000 rpm, 4°C, 10min) for further analysis. After the mice were killed, the visceral poisoning of the mice in each group was observed by dissection, and organs such as liver, kidney, spleen and thymus were collected and weighed. The organ coefficient was calculated as organ weight / body weight × 100. About 1 cm of the middle jejunum and middle ileum of the mice were collected, and the intestinal contents were gently rinsed with saline, and the samples were quickly immersed in 4% paraformaldehyde for tissue fixation for 24 hours. Then, HE staining of the tissue was performed, the sections were observed using an optical microscope, and the length of the villi (length villous) and the depth of the crypt (intestinal crypt) were measured using Image J, and the V / C value was calculated.

[0164] Clinical observation showed that all mice were in good mental condition, could eat and drink normally, had normal feces, and no experimental mice died during the experiment. After the experiment, the necropsy observation of their internal organs showed no abnormal pathological histological changes and bacterial translocation, indicating that the strain had no toxic side effects on the clinical application of mice.

[0165] Body weight, food intake, organ index and serum biochemical index are commonly used indicators for evaluating the health status of animals, and we use them to evaluate the safety of isolated strains. First, this example observed the effect of LRA7 supplementation on the body weight and food intake of mice. Figure 8 It can be seen that there were no significant differences in body weight, feed intake, average daily weight gain (ADG) and average daily feed intake (ADFI) between the mice supplemented with different doses of LRA7 and the CON group. The above results show that the addition of LRA7 has no adverse effects on the body weight and feed intake of mice.

[0166] This example also studies the effect of LRA7 on mouse organ indexes. As shown in Table 11, there was no significant difference in the organ indexes of the heart, liver, spleen, kidney, and thymus of mice in the CON group and the experimental group. This shows that strain LRA7 has no adverse effects on animal growth and development, is safe, and has no toxic side effects.

[0167] Table 11 Effect of feeding LRA7 on organ indexes of mice %

[0168]

[0169] Biochemical analysis of mouse serum showed that, as shown in Table 12, the AST level of mice in the experimental group was significantly lower than that in the CON group, and the ALT levels of mice in the MR and HR groups were significantly reduced; in terms of antioxidant activity, the SOD level of mice in the MR group was significantly increased, and the MDA level of mice in the HR group was significantly decreased, indicating that supplementation with LRA7 may improve the liver function of animals and enhance the body's antioxidant level.

[0170] Table 12 Effects of feeding LRA7 on serum biochemical parameters of mice

[0171]

[0172] This example also provides the effect of feeding LRA7 on the intestinal morphology of mice. Fig. 9 (AD) shows the HE staining images of mouse jejunum. Fig. 9 (EH) are HE staining images of ileum. Fig. 9 As shown in Table 13, the length of jejunal villi in the experimental group was significantly greater than that in the control group, the depth of jejunal crypts in the MR and HR groups were significantly smaller than those in the CON and LR groups, and the jejunal V / C values ​​in the MR and HR groups were significantly greater than those in the CON and LR groups; the length of ileal villi and V / C values ​​in the MR and HR groups were significantly greater than those in the CON and LR groups.

[0173] Table 13 Villus length, crypt depth and ratio of ileum and jejunum in mice fed LRA7

[0174]

[0175] Example 5

[0176] In this example, whole genome sequencing of the strain was performed.

[0177] Illumina high-throughput sequencing platform NovaSeq 6000 was used for joint sequencing, and Unicycler (version: 0.5.0, https: / / github.com / rrwick / Unicycler) was used for genome assembly. The assembled genome was predicted by Prokka (version: 1.14.6, https: / / github.com / tseemann / prokka) software for coding gene prediction. MinCED (version: 0.4.2) was used to predict clustered regularly interspaced short palindromic repeats (CRISPR) in the genome. The predicted gene sequences were compared with these functional databases using GO, COG, and KEGG to obtain gene function annotation results. The software Hmmer (version: 3.3.2) was used for functional annotation based on the Pfam and TIGERFAMs databases, and the genome circle map was drawn using the R package.

[0178] To understand the properties of probiotics and explore the prebiotic potential of LRA7, whole genome sequencing was performed. Fig.10 The results of the complete circular genome map of LRA7 are shown in Figure 1. The complete genome of LRA7 consists of a 2.021M circular chromosome and a circular plasmid, with (G+C) contents of 38.86% and 34.33%, respectively. It is a medium-sized genome, and these bacteria usually have strong metabolism, tolerance and adaptability.

[0179] The COG annotation results of strain LRA7 genome are as follows Fig.11 , LRA7 was mainly enriched in ribosome structure and biogenesis, major function prediction, amino acid transport metabolism, mobilization group, phage progenitor, transposon, and transcription.

[0180] The KEGG annotation results of strain LRA7 genome are as follows Fig.12 The top five annotated signaling pathways are: Global and overview maps (327), Carbohydrate metabolism (92), Translation (80), Amino acid metabolism (75), and Metabolism of cofactors and vitamins (74).

[0181] The GO annotation results of strain LRA7 genome are as follows Fig.13The annotation results showed that the top three enriched biological processes were translation, DNA integration, transcriptional regulation, and DNA templating; the top three enriched cellular components were membrane components, cytoplasm, and cytoplasmic membrane; and the top three enriched molecular functions were ATP binding, DNA binding, and metal ion binding.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A canine-derived Lactobacillus reuteri ( Lactobacillus reuteri ), characterized in that, Its accession number is CCTCCNO: M 20232296.

2. A bacterial agent, characterized in that The invention comprises the canine-derived Lactobacillus reuteri or a fermented live bacteria product of canine-derived Lactobacillus reuteri as claimed in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent is liquid, solid or semisolid.

4. Use of the canine-derived Lactobacillus reuteri according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in the preparation of fermented food, characterized in that: The fermented food is solid food, liquid food and semi-solid food, and the food is pet food.

5. Use of the canine-derived Lactobacillus reuteri according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in the preparation of a medicine or feed for any of the following purposes, characterized in that: (1) inhibiting pathogenic microorganisms, wherein the pathogenic microorganism is selected from at least one of Escherichia coli, Staphylococcus aureus and Salmonella; (2) Enhance intestinal immunity.

6. The use according to claim 5, characterized in that: The improvement of intestinal immunity is achieved by enhancing the intestinal barrier function or enhancing the body's antioxidant level.

7. The use according to claim 6, characterized in that: The enhancement of intestinal barrier function comprises at least one of the following: (1) Maintain the integrity of the villus structure of the jejunum and / or ileum; (2) Increase the ratio of villus length to crypt depth of the jejunum and / or ileum; (3) Reduce or eliminate crypt abscesses or ruptures in the jejunum and / or ileum; and (4) Reduce the crypt depth of the jejunum and / or ileum.

8. The use according to claim 6, characterized in that: The said enhancing the body's antioxidant level refers to enhancing the body's free radical scavenging ability.

9. The use according to claim 8, characterized in that: The free radical is selected from at least one of the following: a DPPH free radical, an ABTS free radical and a superoxide anion free radical.

10. The use according to any one of claims 5 to 9, characterized in that: The drug is formulated for oral administration or intragastric administration.

11. A composition, characterized in that It comprises the canine-derived Lactobacillus reuteri described in claim 1 or the bacterial agent described in any one of claims 2-3.

12. The composition according to claim 11, characterized in that The composition is a pharmaceutical composition or a feed composition.

13. The composition according to claim 11, characterized in that The composition is a vaccine composition.

14. The method for preparing the bacterial agent according to any one of claims 2 to 3, characterized in that: It includes: The canine-derived Lactobacillus reuteri is cultured at 20-40°C.

15. The preparation method according to claim 14, characterized in that: The canine-derived Lactobacillus reuteri was cultured at 37°C.

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