Lactobacillus reuteri strain and application thereof in preparation of cholesterol-lowering, bacteriostatic or probiotic preparation

Abstract

The invention discloses a lactobacillus reuteri strain and application of the lactobacillus reuteri strain in preparation of cholesterol-lowering, bacteriostatic or probiotic preparations. According to the invention, intestinal bacteria are separated from cecum and colon contents of northeast Min pigs, a bacterial strain MP-11 which can effectively reduce cholesterol and has a bacteriostatic effect is finally screened out, the classification name of the bacterial strain MP-11 is identified by molecular biology and morphology, and the microbial preservation number of the bacterial strain MP-11 is CGMCC NO.31670. The lactobacillus reuteri MP-11 has good acid production performance, can tolerate an acid or choline environment, has good adhesion capacity, is sensitive to most antibiotics, does not detect virulence factors, and is good in safety. Tests prove that the lactobacillus reuteri MP-11 strain has excellent cholesterol lowering capacity or antibacterial capacity, and has application prospects in the aspects of preparation of cholesterol lowering and antibacterial drugs or probiotic preparations and the like.

Description

Technical Field

[0001] The invention relates to probiotics separated from the intestinal tract of an animal, in particular to Lactobacillus reuteri separated from the cecum and colon of a Northeastern pig and use thereof in preparing cholesterol-lowering or antibacterial drugs, belonging to the field of lactic acid bacteria and their application. Background Art

[0002] Probiotics are live microorganisms that, when ingested in adequate amounts, confer health benefits on the host and are currently considered to be suitable alternatives to antibiotics for controlling animal infections and improving animal production performance (Ali MS, Lee EB, Hsu WH, et al. Probiotics and postbiotics as an alternative to antibiotics: An emphasis on pigs [J]. Pathogens, 2023, 12 (7): 874). Lactic acid bacteria (LAB) with significant functional properties have been evaluated as possible probiotic candidates in different studies (Mostafa HS, Hashem MM. Lactic acid bacteria as a tool for biovanillin production: A review [J]. Biotechnol Bioeng, 2023, 120 (4): 903-916). LAB, a class of Gram-positive microorganisms that naturally exist in the intestines of humans and other animals, plants and fermented foods and are used as probiotics, has attracted widespread research interest among researchers for many years (Reuben RC, Roy PC, Sarkar SL, et al. Isolation, characterization, and assessment of lactic acid bacteria toward their selection as poultry probiotics[J]. BMC Microbiol, 2019, 19(1): 253). LAB is an effective source of bioactive compounds with various functions and activities, and its metabolites, such as short-chain fatty acids, exopolysaccharides and bacteriocins, have good antibacterial, antiviral and anticancer potential (Mani-López E, Arrioja-Bretón D, López-Malo A. The impacts of antimicrobial and antifungal activity of cell-free supernatants from lactic acid bacteria in vitro and foods[J]. Compr Rev Food Sci Food Saf, 2022, 21(1): 604-641; Garbacz K. Anticancer activity of lactic acid bacteria[J]. Semin Cancer Biol, 86(Pt 3): 356-366).Studies on the interaction between LAB bioactive metabolites and immune mechanisms have shown that these substances can play a powerful immunomodulatory role, making LAB also have great therapeutic potential (Reziguli Kurban, Zhang Zhidong, Sun Jian, etc. Research progress on probiotic lactic acid bacteria in fresh camel milk and fermented camel milk [J]. Food Science and Technology, 2024, 49 (01): 1-8). Existing studies have confirmed that LAB and its fermented products have the ability to lower serum cholesterol (Wan Jingjing, Luo Man, Huang Shixin, etc. Screening of a marine plant lactobacillus with cholesterol-lowering function and analysis of its probiotic properties [J]. Modern Food Science and Technology, 2020, 36 (2): 122-128.; Khare A, Gaur S. Cholesterol-lowering effects of Lactobacillus species [J]. Current Microbiology, 2020, 77 (4): 638-644). Therefore, it is of great significance to screen LAB that can lower cholesterol and further apply it to the development of probiotic products with cholesterol-lowering function.

[0003] The first step in screening probiotic strains is to select a suitable isolation source. Probiotics from different sources have certain species specificity. At present, the screening of probiotics for livestock and poultry mainly focuses on isolating from the body of the animal. Min pigs are a very important local pig breed distributed in Northeast China. They have many properties such as excellent meat quality, strong fertility, tolerance to roughage and cold resistance (Liu Di. Research Progress on Cold Resistance of Min Pigs [C] / / National Animal Husbandry Station, National Local Pig Breed Protection and Utilization Cooperation Group, National Pig Industry Technology System. Proceedings of the Tenth Annual Meeting of the China Local Pig Breed Protection and Utilization Cooperation Group. "China Pig Industry" Editorial Department, 2013:3). Therefore, taking Min pigs as the research object, isolating, identifying and functionally analyzing probiotics for them is of great significance for the protection and development and utilization of pig breeds.

[0004] Lactobacillus reuteri is widely present in the intestines of various animals. Studies have confirmed that it has good safety and rich probiotic functions. It is currently widely used in biological preservatives, functional probiotics and therapeutic fields (Tian Zhaohong. Active substances derived from Lactobacillus reuteri and their potential applications [J]. Anhui Agricultural Sciences, 2024, 52 (01): 1-6). Lactobacillus reuteri has the characteristics of acid resistance and bile resistance, cholesterol-lowering and antibacterial effects, and plays an important role in the food and medical fields (Mei Xuefeng, Hua Deping, Tian Yujing, et al. Analysis of the probiotic properties of Lactobacillus salivarius ATCC 11741 [J / OL]. Journal of Microbiology, 1-16). Therefore, screening Lactobacillus reuteri with cholesterol-lowering function is of great significance for the development of drugs with high cholesterol-lowering and antibacterial functions. Summary of the invention

[0005] One of the purposes of the present invention is to provide a strain of Lactobacillus reuteri isolated from an animal;

[0006] The second object of the present invention is to provide a probiotic preparation containing the Lactobacillus reuteri;

[0007] The third purpose of the present invention is to apply the Lactobacillus reuteri to the preparation of cholesterol-lowering, antibacterial or probiotic preparations.

[0008] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0009] One aspect of the present invention is to provide a strain of Lactobacillus reuteri MP-11, whose microbial preservation number is: CGMCC NO.31670; its classification name is: Lactobacillus reuteri; the preservation time is: August 19, 2024; the preservation unit is: General Microbiology Center of China Microbiological Culture Collection Administration; the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The colony and bacterial morphology of Lactobacillus reuteri described in the present invention are as follows:

[0011] After pure culture, Lactobacillus reuteri MP-11 appears as milky white round protrusions on the culture medium with neat edges and smooth surface; after Gram staining, it appears as short rod-shaped, paired or chain-arranged Gram-positive bacteria.

[0012] The Lactobacillus reuteri MP-11 isolated by the present invention has good acid production performance, can tolerate acid or choline environment, has good adhesion ability, is sensitive to most antibiotics and has no toxic factors detected, so it has good safety. The Lactobacillus reuteri MP-11 also has excellent cholesterol-lowering ability or antibacterial ability.

[0013] Another aspect of the present invention is to use the Lactobacillus reuteri or a bacterial agent containing the Lactobacillus reuteri in the preparation of cholesterol-lowering, antibacterial or probiotic preparations.

[0014] In a preferred specific embodiment of the present invention, the Lactobacillus reuteri MP-11 is used to prepare a cholesterol-lowering drug; wherein the cholesterol-lowering drug preferably reduces the content of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) in serum or liver; or increases the content of high-density lipoprotein cholesterol (HDL-C) in serum or liver.

[0015] In a preferred embodiment of the present invention, the Lactobacillus reuteri MP-11 is used in the preparation of antibacterial drugs; wherein the antibacterial drugs are antibacterial drugs, and preferably, the bacteria include Escherichia coli, Shigella flexneri or Salmonella.

[0016] Another aspect of the present invention is to provide a probiotic preparation, wherein the probiotic preparation contains the Lactobacillus reuteri MP-11.

[0017] Those skilled in the art can prepare various conventional probiotic preparations from the Lactobacillus reuteri MP-11 provided by the present invention according to conventional preparation methods of probiotic preparations, which are all technical means well known to those skilled in the art; accordingly, various conventional probiotic preparations prepared from Lactobacillus reuteri MP-11 can be used in the preparation of cholesterol-lowering or antibacterial drugs, etc.

[0018] The present invention isolates and identifies LAB strains from the intestinal contents of Northeast Min pigs and analyzes the probiotic properties of these isolates. LAB is isolated and purified from the cecum and colon contents of Min pigs using MRS liquid culture medium, and the acid production capacity, acid and bile salt tolerance, adhesion ability, antibacterial activity and antibiotic sensitivity of LAB are further tested. The probiotic properties and safety of different strains are compared and analyzed.

[0019] LAB can regulate environmental pH and inhibit pathogenic microorganisms by secreting organic acids such as lactic acid. Therefore, the acid production ability of LAB is an important indicator for evaluating whether it has probiotic uses. After identifying the acid production ability and acid and bile salt resistance, a total of 5 LAB strains were screened, including 2 strains of Lactobacillus reuteri, 1 strain each of Lactobacillus salivarius, Lactobacillus fermentum and Lactobacillus paracasei.

[0020] LAB plays a role in regulating the structure of the bacterial flora and promoting the health of the host through adhesion in the host's intestine. Therefore, adhesion ability is usually regarded as an important indicator for measuring the quality of probiotics. After further analysis of probiotic functions and safety evaluation, these five LAB strains have different degrees of adhesion characteristics and antibacterial ability, and have good safety.

[0021] Studies have shown that LAB has the ability to lower serum cholesterol, and microecological preparations have become a safe and effective strategy for lowering serum cholesterol (Khare A, Gaur S. Cholesterol-lowering effects of Lactobacillus species [J]. Current Microbiology, 2020, 77 (4): 638-644). Therefore, screening LAB strains with cholesterol-lowering function is of great significance for the development of efficient cholesterol-lowering functional probiotic products. Determining the reduction of cholesterol in the supernatant of the fermentation broth after culturing LAB strains in high-cholesterol medium is the main method to determine whether LAB has cholesterol-lowering properties. The present invention uses this method to determine the screened LAB strains, among which MP-7 and MP-11 have a high in vitro cholesterol degradation rate. Further selection of these two strains for in vivo lipid-lowering studies in mice found that after 5 weeks of in vivo feeding of MP-7 and MP-11 strains, the body weight and liver index of mice fed a high-fat diet were significantly reduced, and the changes in total cholesterol, triglycerides, high-density and low-density lipoprotein cholesterol levels in the serum and liver of mice induced by a high-fat diet were improved, playing a strong lipid-lowering role. The five strains of LAB screened in the present invention have multiple probiotic functions, but overall, Lactobacillus reuteri MP-11 has better characteristics in all aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the colony morphology of Lactobacillus reuteri MP-11;

[0023] Figure 2 Histogram of the adhesion rate of Lactobacillus reuteri MP-11 to Caco-2 cells;

[0024] Figure 3 Bar graph of in vitro cholesterol-lowering rate of Lactobacillus reuteri MP-11. DETAILED DESCRIPTION

[0025] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0026] Example 1 Isolation and Identification of Lactobacillus reuteri MP-11

[0027] 1. Strain Isolation

[0028] The samples were taken from the cecum and colon of a 30-day-old male pig raised at the experimental base of the Institute of Animal Husbandry of Heilongjiang Academy of Agricultural Sciences. The intestinal contents were aseptically separated in a clean bench, collected and placed in a 15mL centrifuge tube, and then an appropriate amount of MRS culture medium was added for preliminary culture at 37°C. 200μL of the preliminary cultured bacterial solution was spread on an MRS plate and cultured at 37°C for 48h; the gradient dilution method was used to spread the plate again to isolate the Lactobacillus reuteri MP-11 strain, and the strain was purified by continuous streaking.

[0029] 2 Identification of strain MP-11

[0030] The strain MP-11 was identified by 16S rDNA molecular biology method, using universal primers and the strain genomic DNA extracted by the kit as a template for PCR amplification. The amplified products were preliminarily analyzed by agarose gel electrophoresis, and the samples that met the expected size were sequenced. The sequencing results were further compared for homology using NCBI's BLAST software. Finally, the strain MP-11 was determined to be classified as Lactobacillus reuteri.

[0031] Test Example 1 Test on the Acid Production Capacity of Lactobacillus reuteri MP-11

[0032] 1 Test method

[0033] The concentration of the strain to be tested was adjusted to 1×10 9 cfu / mL, inoculated into MRS medium, cultured at 37°C for 48 h, centrifuged at 4°C and 10,000 rpm for 5 min, and the pH value of the supernatant was measured.

[0034] 2 Test results

[0035] The results of the acid production ability test of the supernatant of Lactobacillus reuteri MP-11 are shown in Table 1. The acid production value is 3.64, and the acid production performance is good.

[0036] Table 1 Analysis of the acid production ability of the supernatant of lactic acid bacteria strains

[0037]

[0038] Test Example 2 Test on acid and bile salt tolerance of Lactobacillus reuteri MP-11

[0039] 1 Test method

[0040] The OD of the strain to be tested 600 The values ​​were uniformly adjusted to 1.00, and MRS liquid culture media with a pH value of 4.0 and a bile salt concentration of 0.3% were prepared respectively. The bacteria were inoculated at a 4% inoculation rate. After culturing at 37°C for 4 hours, the bacterial suspension was aspirated, gradient dilutions were spread on the plates, and the cultures were cultured at 37°C for 48 hours, and the colonies were counted.

[0041] Strain survival rate (%) = number of viable bacteria in 4h culture medium (cfu / mL) / number of viable bacteria in 0h culture medium (cfu / mL) × 100%.

[0042] 2 Test results

[0043] The results of the acid and bile salt tolerance test of Lactobacillus reuteri MP-11 are shown in Table 2. Lactobacillus reuteri MP-11 has a relatively stable growth state under the conditions of pH 4.0 and 0.3% bile salt, and can tolerate acid or choline environment.

[0044] Table 2 Results of the acid and bile salt tolerance test of lactic acid bacteria strains

[0045]

[0046] Test Example 3 Safety Evaluation Test of Lactobacillus reuteri MP-11

[0047] 1 Test method

[0048] The antibiotic susceptibility of Lactobacillus reuteri MP-11 isolates was analyzed by filter paper diffusion method. The genomic DNA of Lactobacillus reuteri MP-11 was used as template and multiple pairs of primers corresponding to virulence factors were used for PCR amplification.

[0049] 2 Test results

[0050] The results of the safety evaluation test are shown in Table 3. Except for being resistant to kanamycin, Lactobacillus reuteri MP-11 is sensitive to cefotaxime, amoxicillin, ampicillin, streptomycin and gentamicin. The result of the PCR detection of virulence factors was that no virulence factors were detected.

[0051] Table 3 Antibiotic sensitivity analysis of 5 lactic acid bacteria strains

[0052]

[0053] Note: S means sensitive, I means moderately sensitive, and R means resistant.

[0054] Test Example 4: Adhesion ability test of Lactobacillus reuteri MP-11

[0055] 1 Test method

[0056] The cell lines used in this experiment were maintained by the Key Laboratory of Integrated Breeding and Farming of the Ministry of Agriculture and Rural Affairs, Institute of Animal Husbandry, Heilongjiang Academy of Agricultural Sciences.

[0057] The test bacterial suspension with a multiplicity of infection (MOI) of 50 was added to the Caco-2 cell plate laid the day before, incubated for 30 minutes, washed 3 times with PBS, 100 μL of 0.5% TritonX-100 was added to each well and incubated for 10 minutes to lyse the cells, 900 μL of cell culture medium was added to terminate the reaction, and the sample was diluted and plated to count to determine the amount of adherent bacteria.

[0058] 2 Test results

[0059] Adhesion test results Figure 2 As shown, Lactobacillus reuteri MP-11 has the highest adhesion rate compared with other lactic acid bacteria, with an adhesion rate of more than 75%. Therefore, Lactobacillus reuteri MP-11 has a certain adhesion ability to Caco-2 cells and has the ability to colonize in the host intestinal epithelium.

[0060] Test Example 5 In vitro cholesterol degradation ability test of Lactobacillus reuteri MP-11

[0061] 1 Test method

[0062] A cholesterol standard curve was drawn and the OD value of LAB was determined to evaluate the in vitro cholesterol-lowering ability of LAB.

[0063] 2 Test results

[0064] The test results are as follows Figure 3 As shown, Lactobacillus reuteri MP-11 has the best ability to degrade cholesterol in vitro compared with other lactic acid bacteria, with a cholesterol removal rate of more than 80%.

[0065] Test Example 6 In vivo cholesterol-lowering ability test of Lactobacillus reuteri MP-11

[0066] 1 Test method

[0067] After one week of adaptive feeding, male ICR mice were selected and grouped according to the feeding method in Table 4 and fed for 5 consecutive weeks. The activated Lactobacillus reuteri MP-11 was washed twice with saline and the bacterial concentration was adjusted to 10 9 CFU / mL is used for mouse gavage experiments and is prepared fresh every day.

[0068] Table 4 Grouping and feeding methods of mice

[0069]

[0070] After the start of gavage feeding, the mice's food intake was recorded at a fixed time every day, and the mice were weighed at the same time every week to calculate the mice's feed utilization rate: feed utilization rate / % = (weight gain / food intake) × 100%. The mice were fasted for 12 hours after gavage feeding for 5 weeks. The heart, liver, spleen and kidney were immediately removed after being killed. The separated organs were weighed and the organ index was calculated according to the following formula: organ index / (mg / g) = weight of each organ / weight of the mouse before being killed. At the same time, the blood and liver tissues of the mouse's orbital venous plexus were collected respectively, and the total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (TG) contents in the mouse serum were detected using a kit, and the TC and TG levels in the liver were determined.

[0071] 2 Test results

[0072] The test results of weight gain, food intake and feed utilization rate of each group of mice are shown in Table 5. There were no significant differences in body weight, food intake and feed utilization rate between the two groups of mice fed with LAB and the normal control group (P>0.05), while the body weights of the two groups of mice were significantly lower than those of the high-fat model group (P<0.05), indicating that LAB can effectively inhibit the weight gain of high-fat mice, among which Lactobacillus reuteri MP-11 has a stronger inhibitory effect on body weight.

[0073] Table 5 Weight gain, food intake and feed utilization of mice

[0074]

[0075] Note: Different letters in the same column indicate significant differences (P<0.05), the same below.

[0076] The organ indexes of mice in each group are shown in Table 6. After 5 weeks of high-fat diet feeding, there were no significant differences in the heart, spleen and kidney indexes of mice in each group (P>0.05), while the liver index of mice in the high-fat model group was significantly higher than that in the control group (P<0.05). The liver indexes of mice in the two LAB gavage groups were significantly lower than those in the high-fat model group (P<0.05), indicating that gavage with LAB strains MP-7 and MP-11 can reduce the deposition of lipids in the liver of mice.

[0077] Table 6 Organ index of mice in each group

[0078]

[0079] Further analysis of the effect of Lactobacillus reuteri MP-11 on the blood lipid levels of hyperlipidemia mice showed that the TC, TG and LDL-C levels of the LAB-fed group were significantly lower than those of the high-fat model group (P < 0.05), while the HDL-C level was significantly higher than that of the high-fat model group (P > 0.05), indicating that the LAB strains MP-7 and MP-11 have the effects of inhibiting the increase of TC, TG and LDL-C levels in serum and increasing the HDL-C level in serum, among which the effect of the MP-11-fed group was more obvious.

[0080] Table 7 Serum total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein levels in mice

[0081]

[0082] The results of total cholesterol and triglyceride content in mouse liver are shown in Table 8. After LAB gavage, the TC and TG contents in the liver of the two groups of mice were significantly reduced (P<0.05). The TC and TG contents of the mice in the MP-7 group were reduced to 0.172mmol / g and 0.091mmol / g, respectively, while the TC and TG contents of the mice in the MP-11 group were reduced to 0.136mmol / g and 0.083mmol / g, respectively. This shows that gavage of LAB strains MP-7 and MP-11 can promote cholesterol metabolism in the body and reduce the accumulation of cholesterol in the liver of high-fat mice, and the effect of the MP-11 group is more obvious. This shows that gavage of Lactobacillus reuteri MP-11 can better promote cholesterol metabolism in the body and reduce the accumulation of cholesterol in the liver of high-fat mice.

[0083] Table 8 Total cholesterol and triglyceride content in mouse liver

[0084]

[0085] Test Example 7 Antibacterial ability test of Lactobacillus reuteri MP-11

[0086] 1 Test method

[0087] The three indicator bacteria used in this experiment, Escherichia coli, Shigella flexneri and Salmonella, are preserved by the Key Laboratory of Integrated Breeding and Farming of the Ministry of Agriculture and Rural Affairs, Animal Husbandry Research Institute, Heilongjiang Academy of Agricultural Sciences.

[0088] The Oxford cup method was used to detect the inhibitory effect of Lactobacillus reuteri MP-11 on three indicator bacteria.

[0089] 2 Test results

[0090] Table 9 Results of the inhibition zone diameter determination of 5 lactic acid bacteria strains against pathogenic bacteria

[0091]

[0092] The results of the antibacterial test are shown in Table 9. Lactobacillus reuteri MP-11 has an inhibitory effect on Escherichia coli, Shigella flexneri and Salmonella. The inhibitory activity on Escherichia coli and Shigella flexneri is relatively strong, and the diameter of the inhibition zone is greater than 17 mm, while the diameter of the inhibition zone for Salmonella is also above 14 mm. Therefore, it can be seen that Lactobacillus reuteri MP-11 has a strong antibacterial ability.

Claims

1. A strain of Lactobacillus reuteri MP-11, characterized in that: Its microbiological deposit number is: CGMCC NO.31670.

2. A probiotic preparation containing the Lactobacillus reuteri MP-11 according to claim 1.

3. Use of the Lactobacillus reuteri MP-11 according to claim 1 in the preparation of cholesterol-lowering drugs.

4. Use of the Lactobacillus reuteri MP-11 according to claim 1 in the preparation of cholesterol-lowering health products or probiotic preparations.

5. The use according to claim 3 or 4, characterized in that: The cholesterol-lowering method includes reducing the content of total cholesterol, low-density lipoprotein cholesterol and triglyceride in serum or liver; and increasing the content of high-density lipoprotein cholesterol in serum or liver.

6. Use of the Lactobacillus reuteri MP-11 according to claim 1 in the preparation of antibacterial drugs.

7. Use of the Lactobacillus reuteri MP-11 according to claim 1 in the preparation of antibacterial health products or probiotic preparations.

8. The use according to claim 6 or 7, characterized in that: The antibacterial activity includes inhibiting Escherichia coli, Shigella flexneri or Salmonella.

9. Use of the Lactobacillus reuteri MP-11 according to claim 1 in the preparation of a probiotic product or preparation.

10. A pharmaceutical composition or probiotic preparation for lowering cholesterol or inhibiting bacteria, characterized in that: Contains the therapeutic, health-care or preventive effective amount of Lactobacillus reuteri MP-11 as claimed in claim 1 and a pharmaceutically acceptable carrier thereof.

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