A strain of Lactobacillus reuteri B1-10 and its application

Abstract

The embodiment of the present invention relates to a strain of Lactobacillus reuteri B1-10 and its application, which belongs to the field of microorganisms and has a deposit number of CGMCC NO.26166. The Lactobacillus reuteri in the present invention is isolated from fresh and healthy breast milk, has good blood sugar lowering ability and prebiotic properties, and has certain potential for relieving enteritis, allergic rhinitis, and weight loss. It has strong acid and bile salt resistance and good heat resistance. Therefore, the bacteria has the potential to develop functional foods.

Description

[0001] Cross-references

[0002] The present invention claims the priority of the Chinese patent application filed with the Chinese Patent Office, with application number 202310118684.4, application date February 3, 2023, and application name “A strain of Lactobacillus reuteri B1-10 and its application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The invention relates to the field of microorganisms, in particular to a strain of Lactobacillus reuteri B1-10, a microbial preparation and a composition containing the same, and applications thereof. Background Art

[0004] Diabetes is one of the diseases that threaten human life and health, and is accompanied by related complications that are harmful to the human body. Probiotics have been found in long-term research to have potential therapeutic and preventive effects on many diseases. Most of the probiotics currently studied come from food, such as dairy products, sauerkraut, etc., while there are fewer studies on lactic acid bacteria from breast milk, and even fewer studies on their potential functions.

[0005] Breast milk is an important source of nutrients for infants and young children during their growth period, and is crucial to their growth and development. The screening and research of probiotics in breast milk is of great significance to human nutrition and health.

[0006] The information disclosed in this background technology section is only intended to increase the understanding of the overall background of the invention, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art already known to ordinary technicians in this field. Summary of the invention

[0007] Purpose of the Invention

[0008] The present invention aims to provide a strain of Lactobacillus reuteri B1-10, a microbial preparation, a composition and an application thereof, an application of the Lactobacillus reuteri strain in the preparation of probiotic foods, health products or medicines, and a microbial preparation.

[0009] The Lactobacillus reuteri in the present invention is isolated from fresh and healthy breast milk, has good blood sugar lowering ability and probiotic properties, and also has certain potential for relieving enteritis, allergic rhinitis, weight loss, etc. It has strong acid and bile salt resistance and good heat resistance. Therefore, the bacteria has the potential to develop functional foods. The probiotics in the present invention are derived from breast milk, which can enrich the current probiotic library and expand the relevant functional research on probiotics from different sources.

[0010] Solution

[0011] To achieve the purpose of the present invention, the following technical solution is provided:

[0012] In the first aspect, the present invention provides a strain of Limosilactobacillus reuteri B1-10, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 8, 2022, with the deposit number: CGMCC NO.26166.

[0013] In a second aspect, a microbial preparation is provided, which comprises the Lactobacillus reuteri B1-10 and / or its fermentation product described in the first aspect.

[0014] Furthermore, the amount of viable bacteria of Lactobacillus reuteri B1-10 in the microbial preparation is at least 10 8 CFU / mL.

[0015] Furthermore, the microbial preparation is a liquid or solid preparation.

[0016] In a third aspect, a composition is provided, comprising Lactobacillus reuteri B1-10 as described in the first aspect, or comprising the microbial preparation as described in the second aspect.

[0017] Furthermore, the composition is a pharmaceutical composition, which comprises Lactobacillus reuteri B1-10 as described in the first aspect, or comprises the microbial preparation as described in the second aspect, and a pharmaceutically acceptable carrier.

[0018] Furthermore, the pharmaceutical composition is an oral dosage form.

[0019] Furthermore, the oral dosage form is selected from the group consisting of: solutions, suspensions, emulsions, powders, lozenges, pills, syrups, buccal lozenges, tablets and capsules.

[0020] In a fourth aspect, there is provided a use of the Lactobacillus reuteri B1-10 described in the first aspect, the microbial preparation described in the second aspect, and / or the composition described in the third aspect in the preparation of a medicine for at least one of the following purposes:

[0021] 1) Lower blood sugar;

[0022] 2) preventing and / or treating metabolic diseases and their complications, optionally, metabolic diseases include diabetes and / or obesity;

[0023] 3) preventing, alleviating or treating gastrointestinal dysfunction and its complications, optionally, gastrointestinal dysfunction includes enteritis;

[0024] 4) Prevent, alleviate or treat rhinitis and its complications; optionally, rhinitis includes allergic rhinitis;

[0025] 5) Lose weight and / or reduce fat and / or control weight.

[0026] Furthermore, the complication is ketoacidosis, hyperosmolar coma, or lactic acidosis.

[0027] Beneficial Effects

[0028] (1) The Lactobacillus reuteri B1-10 of the present invention is isolated from fresh, healthy breast milk and has good blood sugar lowering ability and probiotic properties. It has strong acid and bile salt resistance and good heat resistance. Therefore, the bacteria has the potential to develop functional foods, can enrich the blood sugar lowering probiotic bacteria library and provide a theoretical basis for the subsequent development of functional dairy products.

[0029] (2) The Lactobacillus reuteri B1-10 of the present invention also has certain application potential in improving allergic rhinitis, relieving enteritis, and losing weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0031] Figure 1 The identification results of strain B1-10 in Example 3 of the present invention, wherein (A) is the Gram staining of strain B1-10, which is blue-purple and rod-shaped; B is the colony morphology of strain B1-10; C is the species map of the top 20 similarity comparisons of the whole gene sequencing of strain B1-10.

[0032] Figure 2 This is a graph showing the growth curve measurement results of breast milk-derived Lactobacillus reuteri B1-10 in 4.1 of Example 4 of the present invention.

[0033] Figure 3 This is a graph showing the results of an acid resistance experiment of breast milk-derived Lactobacillus reuteri B1-10 and Lactobacillus reuteri BNCC339385 in 4.2 of Example 4 of the present invention.

[0034] Figure 4 This is a graph showing the bile salt resistance test results of breast milk-derived Lactobacillus reuteri B1-10 and Lactobacillus reuteri BNCC339385 in 4.3 of Example 4 of the present invention.

[0035] Figure 5This is a graph showing the heat resistance results of Lactobacillus reuteri B1-10 derived from human milk in 4.6 of Example 4 of the present invention, wherein ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P less than 0.0001.

[0036] Figure 6 This is a graph showing the hydrophobicity results of 4.7 of Example 4 of the present invention and breast milk-derived Lactobacillus reuteri B1-10, wherein ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P less than 0.0001.

[0037] Figure 7 This is a graph showing the experimental results of the inhibition rates of α-amylase by breast milk-derived Lactobacillus reuteri B1-10 and Lactobacillus reuteri BNCC339385 in Example 5 of the present invention, respectively, wherein ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P less than 0.0001.

[0038] Figure 8 This is a graph showing the experimental results of the inhibition rates of α-glucosidase by breast milk-derived Lactobacillus reuteri B1-10 and Lactobacillus reuteri BNCC339385 in Example 5 of the present invention, respectively, wherein ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P less than 0.0001.

[0039] Fig. 9 It is the initial body weight of the rats before the weight loss intervention in Example 6 of the present invention.

[0040] Fig.10 The weight of rats in each group after the weight loss experiment in Example 6 of the present invention was completed. * indicates P < 0.05.

[0041] Fig.11 This is the weight gain of rats in each group after the weight loss experiment in Example 6 of the present invention. * indicates P < 0.05, ** indicates P < 0.01.

[0042] Fig.12 The body fat weight (including epididymal fat and perirenal fat) of each group after the weight loss experiment in Example 6 of the present invention. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0043] Fig.13 The total food intake of each group after the weight loss experiment in Example 6 of the present invention. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0044] Fig.14 This is the total calorie intake of rats in each group after the weight loss experiment in Example 6 of the present invention. *** indicates P < 0.001. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] In addition, in order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In some embodiments, raw materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail, so as to highlight the main purpose of the present invention.

[0047] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0048] The present invention proposes a strain of Limosilactobacillus reuteri B1-10, which was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101) on December 8, 2022, with a deposit number of CGMCC NO.26166.

[0049] Lactobacillus reuteri, also known as Lactobacillus reuteri.

[0050] In the following examples, all the raw materials used are commercially available, for example,

[0051] The control commercial strain, Lactobacillus reuteri BNCC339385, was purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0052] MRS solid culture medium was purchased from Beijing Luqiao Technology Co., Ltd. with the catalog number CM188.

[0053] MRS broth medium was purchased from Beijing Luqiao Technology Co., Ltd. with the catalog number CM187.

[0054] Nutrient agar was purchased from Beijing Luqiao Technology Co., Ltd. with the product number CM107.

[0055] Escherichia coli ATCC 25922 was purchased from Guangdong Microbiological Strain Collection Center.

[0056] Staphylococcus aureus ATCC 25923 was purchased from Guangdong Microbiological Strain Collection Center.

[0057] Shigella sonnei ATCC25931 was purchased from Guangdong Microbiological Strain Collection Center.

[0058] Salmonella enteritidis ATCC14028 was purchased from Guangdong Microbiological Strain Collection Center.

[0059] Example 1 Strain Isolation

[0060] Fresh breast milk samples were selected, gradiently diluted with sterile PBS buffer solution, appropriate dilution concentrations were selected, the samples were spread on MRS solid culture medium containing 1% CaCO3, and cultured at 37°C anaerobic conditions for 48 hours. Single white or milky white colonies with obvious transparent circles were picked for Gram staining and microscopic examination, and rod-shaped Gram-positive bacterial strains were selected. Six lactobacillus strains with good growth were selected from numerous strains and named B1-10, B1-26, B1-27, B1-28, B1-29, and B1-30, respectively. The selected strains were enriched and cultured in a 37°C anaerobic incubator; the bacterial solution was mixed with sterilized 30% glycerol in a volume ratio of 1:1 and stored in a -80°C refrigerator for use.

[0061] Example 2 Screening of strains with potential blood sugar lowering ability

[0062] The above strains B1-10, B1-26, B1-27, B1-28, B1-29, and B1-30 were inoculated into MRS broth medium after being preserved in glycerol under sterile conditions, and anaerobically cultured at 37°C for 24 hours, and activated for three generations continuously; after centrifugation of the bacterial solution, the medium was washed 2-3 times with sterilized PBS. The hypoglycemic ability of the strains was tested according to the following method:

[0063] In vitro blood sugar lowering test method:

[0064] 2.1 Preparation of bacterial solution:

[0065] Bacterial suspension: Take 5 ml of the original solution of the bacteria after three generations of activation and centrifuge at 4000 r / min, 4°C, for 10 min. Wash with physiological saline (0.85%) 2-3 times to make 10 ml of bacterial suspension for use. Take 1 ml of the bacterial suspension and add it to 9 ml PBS for use.

[0066] Fermentation supernatant: Centrifuge the bacteria after three generations of activation at 4000r / min, 4°C, for 10min, take 1ml of the fermentation supernatant and add it to 9ml PBS for later use.

[0067] 2.2 Reagents:

[0068] Reagents used in the inhibition experiment of Lactobacillus reuteri on α-glucosidase:

[0069] 1) PBS: 0.1 M, pH 6.8;

[0070] 2) PNPG: 20 mmol / l;

[0071] 3) α-glucosidase: dilute 1 μl of enzyme solution (>700,000 U / ml) in 10 ml PBS for later use;

[0072] 4) Sodium carbonate solution: 1 mol / l.

[0073] Reagents used in the α-amylase inhibition test of Lactobacillus reuteri:

[0074] 1) α-amylase: 2 mg / ml prepared in 0.01 M PBS, pH 7.0;

[0075] 2) Soluble starch: 1%;

[0076] 3) Iodine solution.

[0077] 2.3 Steps:

[0078] 2.3.1 Experimental method for inhibition of α-glucosidase by lactic acid bacteria:

[0079] Group A (sample group)

[0080] 1.25 ml PBS, 1 ml PNPG, 0.25 ml of the sample to be tested (the bacterial suspension or fermentation supernatant prepared in 2.1 above) were mixed, incubated in a 37°C water bath for 10 min, 0.6 ml α-glucosidase was added for 45 min, and then 1 ml sodium carbonate solution was added to terminate the reaction. The OD value of the reaction solution was measured at 405 nm and recorded as A1.

[0081] Group B (sample blank group)

[0082] 1.25 ml PBS, 1 ml PNPG, 0.25 ml of the sample to be tested (the bacterial suspension or fermentation supernatant prepared in 2.1 above) were mixed, incubated in a 37°C water bath for 10 min, 0.6 ml PBS was added to react for 45 min, and then 1 ml sodium carbonate solution was added to terminate the reaction. The OD value of the reaction solution was measured at 405 nm and recorded as A2.

[0083] Group C (control group)

[0084] 1.25 ml PBS, 1 ml PNPG, and 0.25 ml PBS were mixed and placed in a 37°C water bath for 10 min. 0.6 ml α-glucosidase was added to react for 45 min, and then 1 ml sodium carbonate solution was added to terminate the reaction. The reaction solution was taken to detect the OD value at 405 nm and recorded as A3.

[0085] Group D (blank group)

[0086] 1.25 ml PBS, 1 ml PNPG, and 0.25 ml PBS were mixed and placed in a 37°C water bath for 10 min. 0.6 ml PBS was added to react for 45 min, and then 1 ml sodium carbonate solution was added to terminate the reaction. The reaction solution was taken to detect the OD value at 405 nm and recorded as A4.

[0087]

[0088] 2.3.2 Experimental method for inhibition of α-amylase by Lactobacillus reuteri:

[0089] Group A (sample group)

[0090] Mix 2 ml of α-amylase solution, 1 ml of the sample solution (the bacterial suspension or fermentation supernatant prepared in 2.1 above), and 1 ml of PBS, and incubate in a 37°C water bath for 15 min. Add 2 ml of 1% soluble starch solution and react at 37°C for 30 min. Add iodine solution (40 μl) for color development, and measure the OD value at 660 nm, which is recorded as A1.

[0091] Group B (sample blank group)

[0092] Mix 2 ml PBS, 1 ml of the sample solution (the bacterial suspension or fermentation supernatant prepared in 2.1 above) and 1 ml PBS, and incubate at 37°C for 15 min. Add 2 ml of 1% soluble starch solution, react at 37°C for 30 min, add iodine solution for color development, and measure the OD value at 660 nm, which is recorded as A2.

[0093] Group C (control group)

[0094] Mix 2 ml of α-amylase solution, 1 ml of PBS, and 1 ml of PBS, and incubate in a 37°C water bath for 15 min. Add 2 ml of 1% soluble starch solution, and react at 37°C for 30 min. Add iodine solution for color development, and measure the OD value at 660 nm, which is recorded as A3.

[0095] Group D (blank group)

[0096] Mix 2ml PBS, 1ml PBS and 1ml PBS, incubate in a 37℃ water bath for 15min, add 2ml 1% soluble starch solution, react at 37℃ for 30min, add iodine solution for color development, measure OD value at 660nm, and record it as A4.

[0097]

[0098] According to the above method, the in vitro hypoglycemic ability of strains B1-10, B1-26, B1-27, B1-28, B1-29, and B1-30 was compared. The results are shown in Table 1:

[0099] Table 1 Hypoglycemic ability of strains B1-10 and B1-26-30

[0100]

[0101] According to Table 1, strain B1-10 was screened and obtained.

[0102] Example 3 Morphological identification and full genome analysis of strain B1-10

[0103] Morphological identification results

[0104] The identification results are as follows Figure 1 A, 1B, the results showed that the strain of Lactobacillus reuteri B1-10 was blue-purple and rod-shaped after Gram staining. Its colony morphology was round and smooth.

[0105] Whole genome analysis

[0106] The results of whole genome analysis are as follows Figure 1 As shown in Figure C, it can be seen that the full gene sequence of Lactobacillus B1-10 has 97.6% homology similarity with Lactobacillus reuteri. Therefore, combined with the morphological characteristics of strain B1-10, strain B1-10 was identified as Limosilactobacillus reuteri (also known as Lactobacillus reuteri) and named Lactobacillus reuteri B1-10.

[0107] Example 4: Determination of the probiotic properties of Lactobacillus reuteri B1-10

[0108] 4.1 Growth curve determination

[0109] The growth of bacteria is divided into four periods, namely, the slow phase, the logarithmic phase, the stable phase, and the decay phase. By understanding the growth characteristics of the strain, the strain can be selected to be cultured in the appropriate period. In this embodiment, the growth curve of strain B1-10 was determined by the following method.

[0110] The cultured B1-10 bacterial liquid was activated for three generations, inoculated into MRS broth medium, and placed in a 37°C incubator for anaerobically culture. The absorbance OD of the strain at each period was measured at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h and 48h. 600 The result is as follows Figure 2shown.

[0111] Depend on Figure 2 It can be seen that the lag phase of strain B1-10 is relatively short, being in the logarithmic phase within 2 to 6 hours, entering the stable phase after 8 hours of culture, and reaching a higher bacterial liquid concentration. It is in the stable phase between 8 and 40 hours of culture, and begins to enter the decline phase after 40 hours.

[0112] 4.2 Acid resistance determination

[0113] After probiotics enter the human body, they must pass through the human stomach to work, so the acid resistance of the strain is particularly important. In this example, the acid resistance of strain B1-10 was determined by the following method.

[0114] The activated Lactobacillus mucosa B1-10 strain and Lactobacillus reuteri BNCC339385 (commercial strain) were washed with sterile PBS and added to sterile MRS broth medium (referred to as acidic medium) at pH 1.5 and pH 3.0 respectively, and cultured anaerobically at 37°C. Samples were taken at 0h, 1h, and 3h for gradient dilution, and spread on MRS solid medium plates. After anaerobic culture at 37°C for 48h, the colonies were counted, and the colony counts before and after the addition of the acidic medium were calculated. The survival rate of the strains under various acidic conditions was calculated according to the following formula:

[0115] Survival rate (%) = (N t / N0)×100(1)

[0116] In the above formula (1), N t N0 is the number of colonies 1h (or 3h) after adding acidic culture medium; N0 is the number of colonies 0h (i.e. before adding acidic culture medium).

[0117] Acid resistance test results Figure 3 As shown, the result shows that Lactobacillus reuteri B1-10 and Lactobacillus reuteri BNCC339385 (commercial strain) can not survive under the condition of pH 1.5. The survival rate of strain B1-10 under the condition of culturing 1h and 3h at pH 3.0 is significantly higher than that of Lactobacillus reuteri BNCC339385 (commercial strain), and B1-10 is hatched for 1h and 3h under the condition of pH 3.0, and its survival rate is all above 100%, while Lactobacillus reuteri BNCC339385 (commercial strain) is all lower than 10%. It is shown that Lactobacillus reuteri B1-10 has good adaptability to acid, can have more inventory after entering the human body through the special environment such as stomach and intestinal tract, and can play the role of probiotics.

[0118] 4.3 Bile salt tolerance assay

[0119] Bile salts have antibacterial effects, so adapting to the bile salts of the small intestine in the human body is an important prerequisite for probiotics to play a probiotic function. In this example, the bile salt tolerance of strain B1-10 was determined by the following method.

[0120] The activated Lactobacillus reuteri B1-10 strain and Lactobacillus reuteri BNCC339385 (commercial strain) were washed with sterile PBS and resuspended in sterile MRS broth medium (referred to as bile salt medium) containing 0.3% bile salts, cultured anaerobically at 37°C, sampled at 0h and 3h for gradient dilution, spread on MRS solid culture medium plates, and counted after anaerobic culture at 37°C for 48h. The survival rate of the strain under various bile salt conditions was calculated according to the following formula.

[0121] Survival rate (%) = (N t / N0)×100(2)

[0122] In the above formula (2), N t N0 is the number of colonies 1h (or 3h) after adding bile salt medium, and N0 is the number of colonies 0h (i.e. before adding bile salt medium).

[0123] The results of bile salt tolerance test are as follows Figure 4 As shown, the results show that under the condition of culturing in MRS broth containing 0.3% bile salt for 1 hour, the survival rate of strain B1-10 is about 15%, while the survival rate of Lactobacillus reuteri BNCC339385 is 11%, which is lower than B1-10. Under the condition of culturing for 3 hours, the survival rate of B1-10 is about 8%, while the survival rate of Lactobacillus reuteri BNCC339385 is about 6.5%, which is still lower than B1-10. Therefore, B1-10 has better bile salt tolerance than Lactobacillus reuteri BNCC339385.

[0124] 4.4 Determination of antibacterial ability

[0125] Antibacterial ability is an important evaluation index of probiotics. In this example, the inhibitory effect of strain B1-10 on Escherichia coli, Staphylococcus aureus, Shigella sonnei and Salmonella enteritidis was determined by the following method.

[0126] Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Shigella sonnei ATCC25931, and Salmonella enteritidis ATCC14028 were used as indicator bacteria. The activated indicator bacteria were spread on nutrient agar, and the agar diffusion method was used to perform a preliminary determination of antibacterial activity. The Oxford cup was placed on the plate with sterile tweezers, and sterile nutrient agar was poured into the Oxford cup to fix the Oxford cup. After it solidified, the Oxford cup was taken out, and 100 μL of the supernatant of Lactobacillus reuteri B1-10 was respectively drawn and added to the wells. The plate was placed in a 4°C refrigerator for diffusion for about 3 hours, and then placed at 37°C for constant temperature culture for 24 hours. The diameter of the inhibition zone was observed and the size was recorded. The results were expressed as mean ± standard deviation. The results of the antibacterial ability determination are shown in Table 2.

[0127] Table 2 Antibacterial results of Lactobacillus reuteri B1-10

[0128]

[0129] According to Table 2, Lactobacillus reuteri B1-10 had no inhibitory ability against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC25923, but had inhibitory ability against Shigella sonnei ATCC 25931 and Salmonella enteritidis ATCC 14028, with the diameters of the inhibition zones reaching 5.00±0.32mm and 3.89±0.07mm, respectively. This indicates that Lactobacillus reuteri B1-10 has a certain antibacterial ability, can function as a probiotic in the human body, and has the potential to relieve enteritis.

[0130] 4.5 Determination of antibiotic resistance

[0131] Excessive use of antibiotics can increase the drug resistance of pathogenic bacteria and destroy the balance of the human intestinal tract, so it is particularly important to determine the antibiotic sensitivity of probiotics. In this example, the drug resistance of strain B1-10 was determined by the following method.

[0132] The concentration of the third generation bacterial solution was diluted to 10 7 CFU / mL, take 100μL of diluted bacterial solution, evenly spread it on MRS solid medium with a coating rod, let it stand for 20 minutes, take the drug-sensitive paper and blank paper and place them in MRS solid medium at equal distances, gently compact to ensure that they will not slide. After anaerobic culture at 37℃ for 18-24h, use a vernier caliper to measure its diameter (mm), and the results are expressed as mean ± mean.

[0133] The strains were tested for their sensitivity to six antibiotics from six categories, namely:

[0134] (1) Cephalosporins: ceftriaxone; (2) Tetracyclines: tetracycline; (3) Penicillins: penicillin; (4) Aminoglycosides: gentamicin; (5) Fluoroquinolones: enrofloxacin; (6) Peptides: polymyxin B.

[0135] The measurement results are shown in Table 3.

[0136] Table 3 Drug resistance results of Lactobacillus reuteri B1-10

[0137]

[0138] According to Table 3, Lactobacillus reuteri B1-10 was most sensitive to ceftriaxone, with a drug sensitivity zone diameter of 32.03±2.55 mm, and was resistant to polymyxin B. It was moderately sensitive to tetracycline, penicillin, gentamicin, and enrofloxacin, with drug sensitivity zone diameters of 8.92±0.32 mm, 12.41±0.44 mm, 4.14±0.14 mm, and 8.31±0.65 mm, respectively. This indicates that Lactobacillus reuteri B1-10 has moderate sensitivity to antibiotics, which is an important prerequisite for probiotics to play a probiotic role in the human body.

[0139] 4.6 Heat resistance determination

[0140] By understanding the heat resistance of probiotics, appropriate probiotics can be selected for production and application according to different environments. In this example, the heat resistance of strain B1-10 was determined by the following method.

[0141] After the activated bacterial solution was cultured overnight for 12 hours, it was inoculated into sterile MRS broth at a 2% inoculum, and then kept in a constant temperature water bath at 37°C, 50°C, 60°C, and 70°C for 20 minutes. After the water bath, it was cultured at 37°C and 180r / min under anaerobic conditions for 8 hours. The OD of the bacterial solution cultured at 37°C for 8 hours was 600 As the control group, the OD values ​​of the bacterial solution cultured at 50℃, 60℃, and 70℃ for 8h were 600 The survival rate of the bacterial solution after water bath at different temperatures was determined as the experimental group. The formula is as follows:

[0142]

[0143] Heat resistance results such as Figure 5 As shown in the figure, the results show that the survival rate of Lactobacillus reuteri B1-10 is about 90% under the condition of 50℃ water bath temperature. As the temperature rises, its survival rate gradually decreases. At 60℃, its survival rate is about 60%, and at 70℃, the survival rate drops to about 12%. Compared with the survival rate under 50℃ water bath conditions, the survival rate of B1-10 is significantly reduced under 60℃ and 70℃ water bath conditions. Related products can be developed based on its heat resistance characteristics.

[0144] 4.7 Hydrophobicity determination

[0145] Hydrophobicity refers to the unstable state of bacteria in water, which will cause the redistribution and arrangement of bacteria. The hydrophobicity judgment standard is: 71% to 100% is strong hydrophobicity, 36% to 70% is medium hydrophobicity, and less than 35% is weak hydrophobicity. In this example, the hydrophobicity of strain B1-10 to chloroform, toluene, and xylene was determined by the following method.

[0146] The third generation strain B1-10 was inoculated into MRS liquid medium and cultured for 18-24 hours. The suspension was centrifuged at 4000 r / min and 4°C for 10 minutes, the supernatant was discarded, and the suspension was washed 1-2 times with sterile PBS solution. The concentration of the bacterial suspension was adjusted to an OD of 600 The adjusted bacterial suspension was mixed with equal volumes of chloroform, toluene, and xylene, respectively, and mixed with a vortex for 90 seconds, and allowed to stand at room temperature for 1 hour. The aqueous phase of the liquid after stratification was taken and its OD was measured. 600 , as A1; OD in this section 600 PBS was used as a blank control for all measurements. The surface hydrophobicity of the strain was calculated according to the following formula:

[0147] Bacteria surface hydrophobicity (%) = (1-A1 / A0) × 100

[0148] The hydrophobicity results are as follows Figure 6 The results show that the hydrophobicity of B1-10 to chloroform is as high as 70%, which is a strong hydrophobicity, and the hydrophobicity to toluene and xylene is above 50%, which is a medium hydrophobicity.

[0149] Example 5: Comparison of in vitro hypoglycemic ability of Lactobacillus reuteri B1-10 and known Lactobacillus reuteri BNCC339385

[0150] The in vitro hypoglycemic effect of Lactobacillus reuteri was tested according to the in vitro hypoglycemic detection method in Example 2. The experimental results are as follows: Figure 7 , 8 As shown, the results showed that the fermentation supernatant and bacterial suspension of Lactobacillus reuteri B1-10 had an inhibitory effect on α-amylase and α-glucosidase, and were significantly higher than Lactobacillus reuteri BNCC339385. Therefore, the in vitro hypoglycemic ability of Lactobacillus reuteri B1-10 is better than that of the commercial strain Lactobacillus reuteri BNCC339385, and has potential in vivo hypoglycemic ability.

[0151] Example 6: Weight loss effect of Lactobacillus reuteri B1-10

[0152] Experimental animals and feed

[0153] 75 SPF male SD rats weighing 200±20g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. Maintenance feed and high-calorie feed (high-calorie feed is maintenance feed with 15.0% sucrose and 15.0% lard added) were purchased from Huafukang Biotechnology Co., Ltd. L-carnitine lotus leaf cassia seed capsules are products evaluated by Beijing Union University (other products containing L-carnitine lotus leaf cassia seed can also be used as positive controls). Animal experiments were entrusted to the Health Food Function Testing Center of the School of Applied Arts and Sciences, Beijing Union University.

[0154] Dose group

[0155] Blank control group, model control group, positive control group (L-carnitine lotus leaf cassia seed capsule), low-, medium-, and high-dose groups of Lactobacillus reuteri (low dose was 2.5×10 8 CFU / unit; medium dose 5×10 8 CFU / unit; high dose is 1.5×10 9 CFU / piece)

[0156] Experimental methods

[0157] 1) 75 male SD rats were fed maintenance diet in an SPF environment and observed for 5-7 days.

[0158] 2) Screening experiment: After the adaptation period, the rats were randomly divided into two groups according to their body weight: 10 rats were given maintenance feed as the blank control group, and 65 rats were given high-calorie feed as the model group. The amount of food given, the amount of food scattered, and the amount of food left were recorded every week, and the body weight was weighed once. After 2 weeks of feeding, the rats given high-calorie feed were sorted according to weight gain, and the 15 obesity-resistant rats with low weight gain were eliminated.

[0159] 3) Weight loss intervention: The 50 obese-sensitive rats screened were randomly divided into 5 groups according to their body weight, namely the model group, the positive drug control group and the low, medium and high doses of Lactobacillus reuteri B1-10. All 5 groups were given high-calorie model feed. The blank control group was given maintenance feed. The low, medium and high dose groups were inoculated with 2 mL of bacterial solution once a day. The bacterial solution was Lactobacillus reuteri B1-10 resuspended in PBS, with a concentration of 2.5×10 8 CFU, 5 × 10 8 CFU and 1.5 × 10 9 The model control group and the blank control group were given an equal amount of PBS (2 mL) for 6 consecutive weeks. The amount of food given, the amount of food scattered, and the amount of food left were recorded every week, and the body weight was weighed once. The total food intake was also calculated.

[0160] 4) After the experiment, the animals were weighed, anesthetized with 1% sodium pentobarbital (0.5 ml / 100 g BW), and the fat around the kidney and testicles was removed and weighed to calculate the fat / body ratio. The total calories of the blank group (maintenance feed) = total food intake × 16.4 J / g; the total calories of the model group or intervention group (high-calorie feed) = total food intake × 21.5 J / g.

[0161] Experimental Results

[0162] After two weeks of feeding and screening, there was no significant difference in the body weight of rats in the high-calorie diet group during the screening experiment before the intervention experiment began ( Fig. 9 ).

[0163] After 8 weeks of experiments, the body weight of rats in each group of high-calorie diet groups increased significantly. The body weight of rats in the blank group (ordinary diet) reached 483.80±35.67g; the body weight of rats in the model group (high-calorie diet) reached 520.80±34.30g. The body weight of rats in the model group was significantly higher than that in the blank group (P<0.05), indicating that the high-calorie diet model of obese rats was successful. After positive drug intervention, the body weight was 481.84±29.53g, and the body weight of rats was significantly lower than that of the model group (P<0.05). High doses of Lactobacillus reuteri significantly reduced the body weight of obese rats (P<0.05). Low and medium doses of probiotic intervention could not reduce the body weight of rats fed a high-calorie diet. Although medium-dose probiotic intervention showed a certain downward trend, there was no statistical difference compared with the model group ( Fig.10 ). This indicates that a certain dose is required for probiotics to exert their weight loss effect. In order to accurately calculate the effect of probiotic intervention on body weight, we calculated the weight gain of rats. The results showed that compared with the model group, the positive control group and the high-dose probiotic intervention group could significantly reduce the weight gain of rats ( Fig.11 Obesity is mainly manifested as an increase in body fat. We separated the epididymal fat and perirenal fat of rats and weighed them. The results showed that a high-calorie diet significantly increased the body fat weight of rats (P<0.001), and positive drugs and high-dose probiotics could significantly reduce the body fat weight of rats ( Fig.12 ). In order to verify whether the weight loss effect of probiotics is caused by reduced food intake, we weighed the rats' food intake. Compared with the control group, the rats fed with high-calorie diet had a significantly reduced food intake, but there was no significant statistical difference in food intake between the groups of rats fed with high-calorie diet ( Fig.13 ). Because high-calorie diets contain more calories, the total energy intake of the high-calorie diet group was significantly higher than that of the maintenance diet group. However, there was no significant statistical difference in the total energy intake of the rats fed high-calorie diets ( Fig.14 ).

[0164] The above results show that the high-calorie feed group had a higher total energy intake than the control group, but positive drugs and high-dose probiotics can significantly reduce the body weight and body fat weight of rats, and have a good weight loss effect.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Limosilactobacillus reuteri B1-10 was deposited in the General Microbiology Center of China National Culture Collection Administration on December 8, 2022, with the deposit number: CGMCCNO.26166.

2. A microbial preparation comprising the Lactobacillus reuteri B1-10 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The amount of viable Lactobacillus reuteri B1-10 in the microbial preparation is at least 10 8 CFU / mL.

4. The microbial preparation according to claim 2, characterized in that The microbial preparation is a liquid or solid preparation.

5. A composition, characterized in that The composition comprises the Lactobacillus reuteri B1-10 as claimed in claim 1, or comprises the microbial preparation as claimed in any one of claims 2 to 4.

6. The composition according to claim 5, characterized in that The composition is a pharmaceutical composition, which comprises the Lactobacillus reuteri B1-10 as claimed in claim 1, or the microbial preparation as claimed in any one of claims 2 to 4, and a pharmaceutically acceptable carrier.

7. The composition according to claim 6, characterized in that The pharmaceutical composition is in oral dosage form.

8. The composition according to claim 7, characterized in that The oral dosage form is selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, tablets and capsules.

9. The composition according to claim 8, characterized in that The tablet is a buccal tablet.

10. Use of Lactobacillus reuteri B1-10 according to claim 1, the microbial preparation according to any one of claims 2 to 4, and / or the composition according to any one of claims 5 to 9 in the preparation of a medicament for at least one of the following uses: 1) Lower blood sugar; 2) Prevent enteritis; 3) Lose weight.

11. Use of Lactobacillus reuteri B1-10 according to claim 1, the microbial preparation according to any one of claims 2 to 4, and / or the composition according to any one of claims 5 to 9 in the preparation of a medicament for the following purposes: treating metabolic diseases, wherein the metabolic diseases are diabetes and / or obesity.

12. Use of Lactobacillus reuteri B1-10 according to claim 1, the microbial preparation according to any one of claims 2 to 4, and / or the composition according to any one of claims 5 to 9 in the preparation of fat-reducing drugs.

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