Mink-source lactobacillus plantarum capable of reducing cholesterol and application of mink-source lactobacillus plantarum

Abstract

The invention discloses mink-source lactobacillus plantarum capable of reducing cholesterol and application of the mink-source lactobacillus plantarum, and belongs to the technical field of microorganisms. In order to screen out a strain which is high in cholate tolerance, has a non-BSH-dependent cholesterol lowering mechanism and can be adapted to multiple hosts and is used for replacing a medicine to lower cholesterol, the lactobacillus plantarum ZZM01 is separated from the intestinal tract of a mink, and the lactobacillus plantarum ZZM01 has the advantages that the lactobacillus plantarum ZZM01 can be used for reducing cholesterol; experiments show that the lactobacillus plantarum ZZM01 has broad-spectrum host suitability, a non-BSH-dependent cholesterol lowering mechanism, activity of inhibiting multiple pathogenic bacteria, free radical scavenging capacity and safety, and provides an innovative solution for adjuvant therapy of cross-species hyperlipemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and relates to a mink-derived lactobacillus plantarum capable of reducing cholesterol and an application thereof. BACKGROUND

[0002] Hyperlipidemia is a common metabolic disease, which refers to the content of lipids such as cholesterol and triglyceride in blood exceeding the normal level, and is an important risk factor leading to cardiovascular and cerebrovascular diseases. At present, statins are mainly used in the clinical treatment of hyperlipidemia, but long-term use may cause adverse reactions such as abnormal liver function, muscle pain and indigestion.

[0003] At present, the use of probiotics to treat hyperlipidemia can solve the side effects brought by drug treatment, but there are still many problems: for example, the current stage of blood lipid-lowering probiotics lacks systematic research on the intestinal microbial function of carnivorous animals, especially those microbial populations that have long adapted to high-fat diets and can effectively metabolize lipids; most blood lipid-lowering probiotics have weak survival ability in an environment with high bile salt concentration, and cannot be well applied to the treatment of blood lipid-lowering; the blood lipid-lowering function of existing strains is mostly dependent on traditional pathways such as bile salt hydrolyase (BSH)-mediated cholesterol metabolism or bile acid binding, while ignoring a series of negative effects brought by them, such as lipid malabsorption, increased risk of colon cancer, etc. At present, there is insufficient research on other potential mechanisms (such as cholesterol physical adsorption, cell membrane integration, lipoprotein metabolism regulation or short-chain fatty acid synthesis), which cannot meet the individual needs of different hosts. Therefore, it is urgent to screen out strains with strong bile salt tolerance, non-BSH-dependent cholesterol-lowering mechanisms and the ability to adapt to multiple hosts to replace blood lipid-lowering drugs. SUMMARY

[0004] In order to screen out strains with strong bile salt tolerance, non-BSH-dependent cholesterol-lowering mechanisms and the ability to adapt to multiple hosts to replace blood lipid-lowering drugs, the present application considers that since minks have high-fat diet characteristics, the bile salt concentration in their intestines is usually higher than that of other animals, and the strains isolated from the intestines of minks may have stronger bile salt tolerance. Therefore, a lactic acid bacteria ZZM01 is isolated from the intestines of minks, and the strain is identified as Lactobacillus plantarum (L.plantarum) through morphological, molecular biological and physiological and biochemical characteristics identification. Lactobacillus plantarum), and is preserved in China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2025695 and a preservation date of April 3, 2025, and a preservation unit address of China, Wuhan, Wuhan University. It is found through experiments that the Lactobacillus plantarum ZZM01 has a broad spectrum of host adaptability (survival rate of 84.3% under pH 2.0, survival rate of 92.19% under 0.3% bile salt), a non-BSH-dependent cholesterol-lowering mechanism (removal rate of 44.94% in vitro), an activity of inhibiting a plurality of pathogenic bacteria, an ability of scavenging free radicals, safety (no hemolytic activity, controllable risk of natural drug resistance gene), and provides an innovative solution for the auxiliary treatment of hyperlipidemia across species.

[0005] To solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a Lactobacillus plantarum (Lactobacillus plantarum) ZZM01, with a preservation number of CCTCC NO: M 2025695. Lactobacillus plantarum ) ZZM01, with a preservation number of CCTCC NO: M 2025695.

[0006] The second object of the present application is to provide the use of the above-mentioned Lactobacillus plantarum ZZM01 in the preparation of a drug for reducing cholesterol.

[0007] In an embodiment of the present application, the drug has a non-BSH-dependent cholesterol-lowering mechanism.

[0008] In an embodiment of the present application, the content of Lactobacillus plantarum ZZM01 in the drug is 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0009] In an embodiment of the present application, the use of the above-mentioned Lactobacillus plantarum ZZM01 is for the preparation of a health food for auxiliary reduction of cholesterol.

[0010] In an embodiment of the present application, the health food has a non-BSH-dependent cholesterol-lowering mechanism.

[0011] In an embodiment of the present application, the content of Lactobacillus plantarum ZZM01 in the health food is 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0012] The fourth object of the present application is to provide a drug for reducing cholesterol with the above-mentioned Lactobacillus plantarum ZZM01 as an active ingredient.

[0013] In an embodiment of the present application, the content of Lactobacillus plantarum ZZM01 in the drug is 1×10 9 CFU / mL or 1×109 CFU / g.

[0014] A fifth object of the present application is to provide a health care food for assisting in reducing cholesterol with the above-mentioned Lactobacillus plantarum ZZM01 as an active ingredient.

[0015] In an embodiment of the present application, the content of Lactobacillus plantarum ZZM01 in the health care food is 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0016] In an embodiment of the present application, the pharmaceutical product further comprises a pharmaceutically acceptable excipient.

[0017] In an embodiment of the present application, the dosage form of the pharmaceutical product is a tablet, a capsule, a granule, a powder or a liquid preparation.

[0018] Advantages of the present application: The present application isolates a strain of lactic acid bacteria ZZM01 from the intestinal tract of mink. After morphological, molecular biological and physiological and biochemical characteristics identification, it is determined that the strain is Lactobacillus plantarum (Lactobacillus plantarum). Lactobacillus plantarum It is found through experiments that Lactobacillus plantarum ZZM01 has a broad spectrum of host adaptability (survival rate of 84.3% under pH 2.0, survival rate of 92.19% under 0.3% bile salt, good tolerance to artificial gastric and intestinal juice), non-BSH-dependent cholesterol-lowering mechanism (in vitro removal rate of 44.94%), inhibition of a variety of pathogenic bacteria (Escherichia coli, Salmonella, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa), free radical scavenging ability (free radical scavenging rate of 84.62%), safety (no hemolytic activity, controllable risk of natural drug resistance gene) and industrialization potential (optimized fermentation process). The present application provides a universal probiotic strain with high-efficiency hypolipidemic function and suitable for multiple hosts (including humans, pets and economic animals), which provides an innovative solution for the auxiliary treatment of hyperlipidemia across species. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Figure 1 is a morphological identification result of Lactobacillus plantarum ZZM01, wherein, Figure 1 A of figure 1 is a colony morphology diagram of Lactobacillus plantarum ZZM01, Figure 1 B of figure 1 is a cell morphology diagram of Lactobacillus plantarum ZZM01; Figure 2 Figure 2 is a Neighbor-Joining phylogenetic tree analysis diagram of Lactobacillus plantarum ZZM01; Figure 3 Figure 3 is a growth curve diagram of Lactobacillus plantarum ZZM01; Figure 4Figure 1 is a graph of acid production curve of Lactobacillus plantarum ZZM01; Figure 5 Figure 4 is a graph of pH and bile salt tolerance test results of Lactobacillus plantarum ZZM01; Figure 6 Figure 5 is a graph of self-aggregation rate test results of Lactobacillus plantarum ZZM01; wherein, , P <0.01; Figure 7 Figure 6 is a graph of hemolytic test results of Lactobacillus plantarum ZZM01; Figure 8 Figure 7 is a graph of BSH screening results of Lactobacillus plantarum ZZM01. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the drawings of the specification. It should be noted that the following examples are only used to explain the present application but not to limit the scope of the present application. The following examples are only a part of the embodiments of the present application but not all the embodiments. Those skilled in the art can refer to the content herein and appropriately improve the process parameters to realize. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein to realize and apply the present application technology without departing from the content and scope of the present application. Other embodiments obtained by those skilled in the art without creative labor are also protected by the present application.

[0021] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art. The molecular biology experimental operations involved in the present application are all conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.

[0022] The culture medium and its composition involved in the present application are as follows: The separation culture medium is MRS culture medium, and the specific composition is as follows: Tryptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, diammonium citrate 2.0 g / L, K2HPO4 2.0 g / L, NaAc 2.0 g / L, MnSO4·4H2O 0.25 g / L, MgSO4·7H2O 0.58 g / L, agar 20.0 g / L (liquid medium without agar), the rest is water, pH 6.2-6.5, sterilized at 121℃ for 30 min.

[0023] The data analysis method used in the present application is as follows: The data is preliminarily processed by Excel, and the significance of the data is analyzed by single factor variance analysis in spss27.0, and the results are expressed as mean ± standard deviation, wherein P <0.01 indicates that the difference is extremely significant; 0.01≤ P <0.05 indicates that the difference is significant, and P≥0.05 indicates that the difference is not significant.

[0024] Example 1: Isolation and identification of mink-derived Lactobacillus plantarum ZZM01 1. Isolation of strain ZZM01 (1) Collection of samples and isolation of microorganisms: Ten healthy adult short-haired black minks from a mink farm in Shandong were selected, and their intestinal contents were taken and quickly placed in sterilized screw-cap test tubes containing liquid MRS medium. After being brought back to the laboratory, gradient dilution was immediately performed, with three plates per gradient, and the culture was separated and cultured by pouring method at 37℃.

[0025] (2) Isolation and purification: Take the above plates with various bacteria, select lactic acid bacteria single colonies, and use continuous streaking method for isolation and purification. Single colonies showing different colony morphology were purified. Repeat 3-4 times, and observe under a microscope until pure strains are obtained.

[0026] 2. Identification of strain ZZM01 A strain of lactic acid bacteria was obtained using the above isolation and purification method, and was designated as ZZM01. Its morphological, molecular biological and physiological and biochemical characteristics were identified.

[0027] (1) Morphological identification of strain ZZM01 The colony morphological characteristics of strain ZZM01 on MRS solid medium were observed, and the cell morphology of strain ZZM01 was observed by Gram staining and microscopic examination. The results are shown in Figure 1 After 24 h of culture on MRS solid medium, the colony of strain ZZM01 was convex in the middle, with neat edges and smooth surface (see Figure 1(A in the text); cells of strain ZZM01 exhibit facultative anaerobic behavior, Gram-positive, and rod-shaped (see A in the text). Figure 1 (B in the middle).

[0028] (2) Molecular biological identification of strain ZZM01 Select vigorous bacterial strains and inoculate purified single colonies into MRS liquid medium, then culture on a shaker. Genomic DNA of strain ZZM01 was extracted using the DNA extraction kit (TakaRa 2.0) from Takara Bio Inc. (Dalian), following the kit instructions. Conserved sequences of the extracted genomic DNA were amplified by PCR using universal primers 27F and 1492R. The total volume of the 16S rDNA amplification system was 50 μL, including: 10×PCR Buffer, DNA plate, Primer F, Primer R, dNTP Mixture (2.5 mM), TaKaRa Taq® (5 U / μL), and ddH2O. The PCR reaction program was: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 52℃ annealing for 45 s, 72℃ extension for 90 s, 25 cycles; 72℃ extension for 8–10 min; storage at 4℃. PCR products were detected by 1% agarose gel electrophoresis (120 V, 100 mA, 20 min), purified, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The 16S rRNA sequence of strain ZZM01 obtained by sequencing is shown in SEQ ID NO.1. The sequencing results were compared with the BLAST tool in NCBI (National Center for Biotechnology Information), and the 16S rRNA sequence of strain ZZM01 was found to be consistent with that in the database. Lactobacillus plantarum The highest similarity was found in strain Y409, reaching 99.74%. Using MEGA software and the adjacent linking method, [the following information was obtained]. Escherichia coli DSM 30083 was used to construct a Neighbor-Joining phylogenetic tree for the outer branches. Figure 2 The results showed that strain ZZM01 was related to *Lactobacillus plantarum* (…). Lactobacillus plantarum They are closely related.

[0029] SEQ ID NO.1: GGGGGTCGACGTCTGACGCTGAGGCTCGAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTGAGCACCAAAGGGGGGACCGCGGGAGG (3) Physiological and biochemical characteristics identification The physiological and biochemical characteristics of the strain ZZM01 were detected by using a biolog microorganism identification instrument, and the detection results are shown in Table 1.

[0030] Table 1: Detection results of physiological and biochemical characteristics of strain ZZM01

[0031] Note: +: positive reaction; -: negative reaction As can be seen from Table 1, strain ZZM01 can utilize esculin, cellobiose, maltose, sorbitol, salicin, sucrose, raffinose, mannitol, lactose, inulin and 1% hippuric acid, and the results of indole experiment, V-P test and hydrogen peroxide test are negative.

[0032] According to the results of morphological identification, molecular biological identification and physiological and biochemical characteristic identification of strain ZZM01, the strain ZZM01 is identified as Lactobacillus plantarum, Lactobacillus plantarum ), and the strain is preserved. The strain is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 2025695, the preservation date is April 3, 2025, and the preservation unit address is China, Wuhan, Wuhan University.

[0033] Example 2: Application of mink-derived Lactobacillus plantarum ZZM01 in reducing cholesterol 1. Determination of growth characteristics and acid production performance of strain ZZM01 Take the logarithmic growth phase bacterial liquid, inoculate in MRS culture solution at 2.0% (v / v) inoculation amount, take the un-inoculated MRS culture solution as blank, and culture at 37℃ for 24 h. Take the culture solution every 2 h, and determine the absorbance value (OD 600 ) at 600 nm and pH value by using an enzyme marker and a pH meter respectively. Take time as horizontal coordinate, and take OD 600 and pH of the culture solution as vertical coordinate respectively, and draw growth curve and acid production curve.

[0034] The detection results show that the OD 600 value of strain ZZM01 has no obvious change when the culture time is 0-4 h, at this time, ZZM01 grows slowly and is in the lag phase; the OD 600 value of ZZM01 increases exponentially when the culture time is 4-9 h, at this time, the strain is in the logarithmic growth phase and grows very rapidly; the OD 600 value of the strain gradually tends to be stable when the culture time is 9-24 h, and the strain grows slowly and is in the stationary phase; and the strain enters the decline phase within 24 h, which conforms to the general growth law of bacteria. Figure 3 Correspondingly, the OD 600 value of the bacteria increases and the pH of the fermentation liquor decreases with the extension of the culture time. The pH value of the culture liquor changes little within 4 h of inoculation, corresponding to the lag phase, and the pH value decreases from about 6.13 to about 4.83 within 4-9 h, with a large change, corresponding to the rapid growth phase. The pH value of the culture liquor decreases slowly within 9-24 h, corresponding to the stationary phase in the growth cycle of the strain, which conforms to the general acid production law of lactic acid bacteria. Figure 4

[0035] 2. Drug sensitivity test of strain ZZM01​ The drug sensitivity test was performed by K-B drug sensitivity paper disc agar diffusion method: the bacterial strain ZZM01 was inoculated in MRS culture medium at a 2.0% inoculation amount, and then cultured at 37°C with shaking for 12 h. The bacterial strain was collected by centrifugation at 6000 rpm and 4°C for 10 min, and then washed twice with normal saline. After washing, the bacterial strain was resuspended with normal saline to about 10 8 CFU / mL, and then coated and pasted with drug sensitivity discs. Each drug sensitivity disc was repeated three times, and then cultured at 37°C for 24 h. The diameter of the drug sensitivity circle was measured, and compared with the CLSI standard to determine the drug sensitivity of the bacterial strain.

[0036] The drug sensitivity test was performed according to the CLSI standard for evaluating the antibiotic sensitivity of the tested bacterial strain. As shown in Table 2, the Lactobacillus plantarum ZZM01 was resistant to vancomycin, streptomycin, ciprofloxacin, amikacin and ampicillin, moderately sensitive to penicillin, and highly sensitive to ceftriaxone and chloramphenicol.

[0037] Table 2 Drug sensitivity test results of Lactobacillus plantarum ZZM01

[0038] Note: R: not sensitive; I: moderately sensitive; S: sensitive 3. Bacteriostatic test of fermentation supernatant of the bacterial strain ZZM01 The bacterial strain ZZM01 was inoculated in MRS culture medium at a 2.0% inoculation amount, and then cultured at 37°C with shaking at 180 rpm for 24 h. The bacterial strain was collected by centrifugation at 6000 rpm and 4°C for 10 min, and then the supernatant was filtered with a 0.2 μm sterile filter membrane for standby use. The bacteriostatic activity was determined by the Oxford cup method (outer diameter x inner diameter x height = 8 mm x 6 mm x 10 mm). Each cup contained 200 μL of the supernatant, and fresh bacterial liquid of Salmonella ATCC 13311, Escherichia coli ATCC 44350, Listeria monocytogenes ATCC 54007, Staphylococcus aureus CMCC 26003 and Pseudomonas aeruginosa ATCC 10104 was coated on LB solid medium, and then cultured at 37°C for 24 h. The diameter of the bacteriostatic circle was measured.

[0039] The ability of the bacterial strain ZZM01 to inhibit harmful indicator bacteria is shown in Table 3. The Lactobacillus plantarum ZZM01 could effectively inhibit the growth and proliferation of five pathogenic bacteria. The inhibition effect of the cell-free supernatant on Salmonella was significantly better than that on Escherichia coli and Listeria monocytogenes (P<0.05), and the inhibition effect on Staphylococcus aureus was significantly better than that on Listeria monocytogenes (P<0.05).

[0040] Table 3 Bacteriostatic test results of the bacterial strain ZZM01

[0041] 4. Artificial gastric juice tolerance and artificial intestinal juice tolerance test of strain ZZM01 Prepare artificial gastric juice with pH 3.0 and artificial intestinal juice with pH 6.8 according to the 2013 edition of Chinese Pharmacopoeia, both of which are filtered with 0.22 μm microporous sterile filter for standby use. Fresh overnight cultured bacterial liquid of strain ZZM01 is centrifuged, washed and resuspended, mixed uniformly according to resuspension liquid: artificial gastric juice or artificial intestinal juice = 1:9 (volume ratio), incubated at 37℃ for 3 h, spread on MRS, and the 0 h, 1.5 h and 3 h mixed liquids are taken for viable count, repeated three times to take average value, and the bacterial survival rate is calculated: ; Wherein N 0 is the viable count at 0 h, N t t (t = 3) h.

[0042] As shown in Table 4, the survival rate of strain ZZM01 is 90.55% after being treated in artificial gastric juice for 3 h, and the survival rate of strain ZZM01 is 92.68% after being treated in artificial intestinal juice for 3 h. It can be seen that the survival rates of strain ZZM01 after being treated in artificial gastric juice and artificial intestinal juice for 3 h are both higher than 85%, indicating that Lactobacillus plantarum ZZM01 has good tolerance to artificial gastric and intestinal juice.

[0043] Table 4 Viable count of Lactobacillus plantarum ZZM01 in artificial gastric and intestinal juice

[0044] 5. pH and bile salt tolerance test of strain ZZM01 In order to determine the acid tolerance of strain ZZM01, the overnight culture of strain ZZM01 is inoculated into MRS broth with pH adjusted to 2 by hydrochloric acid (1.0 mol / L) at a ratio of 1% (v / v). The bile salt tolerance test is detected by inoculating the culture into MRS broth containing 0.3% (w / v) bile at a ratio of 1% (v / v). The culture is placed in a 37℃ environment, and the absorbance at 600 nm is measured after 3 h of culture in MRS broth with pH 2.0 and 4 h of culture in MRS broth containing 0.3% (w / v) bile to evaluate the acid and bile salt tolerance of the cells. The survival rate is calculated according to the following formula: Survival rate (%) = OD final / OD final × 100; Wherein OD Initial and OD final respectively refer to the optical density measured after 0 h and 3 or 4 h of culture.

[0045] As Figure 5As shown, the survival rate of strain ZZM01 at pH 2.0 was 84.3%; in terms of bile salt tolerance, the survival rate of strain ZZM01 at 0.3% bile salt was 92.19%. This indicates that Lactobacillus plantarum ZZM01 has good survival ability under gastrointestinal conditions.

[0046] 6. Detection of surface hydrophobicity and self-aggregation of strain ZZM01 The bacterial carbon hydrocarbon compound adhesion method was used to determine the hydrophobic properties of the strain by the affinity of lactic acid bacteria to carbon hydrocarbon compounds. Fresh bacterial liquid of strain ZZM01 cultured overnight was washed with PBS, and the OD 600 =0.25±0.05 (A0) was adjusted as the test liquid, 3 mL of the test liquid was mixed with an equal volume of dimethylbenzene, vortexed for 3 min, incubated at 37°C for 1 h and 2 h respectively, and the water phase (forming a two-phase system, the water phase in the lower layer) was aspirated, and the OD 600 (A t ) was measured, with 3 repeats at each time point. The hydrophobicity calculation formula is as follows: ; In the formula, A t is the absorbance at 1 h or 2 h; A0 is the absorbance at t=0.

[0047] Fresh bacterial liquid of strain ZZM01 cultured at 37°C for 16 h was centrifuged at 4°C at 6000 rpm for 10 min, washed twice with PBS, resuspended the bacteria with 2 volumes of PBS, took 2 mL of the above test liquid, vortexed for 10 s, and measured the absorbance at 600 nm (A0), then let it stand at 37°C for 4 h, 6 h, 10 h and 24 h respectively, carefully aspirated 200 μL of supernatant, and measured the absorbance (A t ). Each time point was repeated 3 times. The self-aggregation rate calculation formula is as follows: ; In the formula, A t is the absorbance at 4 h, 6 h, 10 h or 24 h; A0 is the absorbance at t=0.

[0048] The surface hydrophobicity detection results of strain ZZM01 showed that the surface hydrophobicity of strain ZZM01 treated with dimethylbenzene for 1 h was 33.19%. The self-aggregation rates of strain ZZM01 at 4 h, 6 h, 10 h and 24 h were 35.97%, 50.88%, 73.04% and 93.85% respectively, with extremely significant differences between groups P (<0.01), and it was found that the self-aggregation rate increased with the increase of culture time Figure 6 .

[0049] 7. Determination of DPPH free radical scavenging activity of strain ZZM01 The DPPH scavenging capacity of strain ZZM01 was determined using a DPPH free radical scavenging capacity assay kit (Suzhou Grace). Specifically, 10 μL of bacterial extract was mixed with 190 μL of DPPH methanol working solution, mixed thoroughly for 60 s, and incubated at room temperature in the dark for 30 min. The absorbance was then measured at 517 nm. The calculation formula is as follows: ; In the formula, A sample A represents the absorbance of the sample-working solution mixture. control A represents the absorbance of the mixture of the sample and 80% methanol solution. blank The absorbance is the value of the mixture of the working solution and 80% methanol solution.

[0050] The test results showed that the DPPH free radical scavenging rate of strain ZZM01 was 84.62%±1.66, indicating that it has good DPPH free radical scavenging ability.

[0051] 8. Hemolytic test of strain ZZM01 The bacterial culture of strain ZZM01, which had been cultured overnight, was streaked onto a blood agar plate containing 7% (v / v) sheep blood. The plate was then incubated at 37°C for 2-3 days, and the hemolytic area around the colony was observed to detect hemolytic activity.

[0052] like Figure 7 As shown, *Lactobacillus plantarum* ZZM01 showed negative hemolytic activity, while *Staphylococcus aureus* was the positive control. This indicates that ZZM01 meets safety standards.

[0053] 9. Detection of in vitro cholesterol clearance rate of strain ZZM01 Take 0.1 g cholesterol (analytical grade), 0.2 g taurine, 1 mL Tween 80, and 0.1 g sucrose octaacetate, add 5 mL glacial acetic acid, heat to dissolve, and then quickly add to 1000 mL MRS medium. Add the bacterial culture of strain ZZM01 (1×10⁻⁶) to the medium. 9CFU / mL) was inoculated into MRS medium containing cholesterol at a ratio of 1% (v / v), and un-inoculated MRS medium was used as a control. After incubation of the cultures at 37°C for 18 h, the supernatants were collected by centrifugation at 12000 rpm for 10 min at 4°C. Subsequently, the cholesterol concentration in the supernatants and un-inoculated MRS medium was determined using the ortho-phthaldehyde method (OPA) by adding 6 mL of 95% ethanol, 4 mL of 50% (w / v) potassium hydroxide and 1 mL of sample (supernatant) into a test tube, mixing well and heating to 60°C for 10 min, cooling to room temperature; then adding 5 mL of hexane and 3 mL of distilled water, mixing well for 1 min, and allowing the test tube to stand for 15 min to promote phase separation; taking 3 mL of the hexane layer and evaporating the solvent under a stream of nitrogen at 60°C; subsequently, adding 4 mL of freshly prepared ortho-phthaldehyde (0.5 mg of ortho-phthaldehyde per mL of glacial acetic acid), mixing well to dissolve the residue, and allowing to stand at room temperature for 10 min; then carefully adding 2 mL of concentrated sulfuric acid and mixing immediately, and measuring the absorbance at 550 nm after 10 min of standing, with a blank control for correction. A standard curve was prepared by performing the same procedure on 0 mg / mL to 0.2 mg / mL cholesterol standard solutions, and the absorbance values were compared to determine the cholesterol concentration. The formula for calculating the cholesterol removal rate (%) is: Cholesterol removal (%) = (1 - R1 / R0) x 100; R0 and R1 represent the cholesterol content in the MRS medium at 0 h and 24 h, respectively.

[0054] The results of the in vitro cholesterol removal rate detection of strain ZZM01 showed that the in vitro cholesterol removal rate of Lactobacillus plantarum ZZM01 was 44.94 ± 2.18%, indicating that it had a high cholesterol removal function.

[0055] 10. Detection of bile salt hydrolase (BSH) activity of strain ZZM01 According to the method of Nguyen et al. (2007), the activity of the bile salt hydrolase (BSH) of strain ZZM01 was detected. Specifically, the bacterial solution of overnight culture of strain ZZM01 was spread on MRS agar medium added with 0.37 g / L CaCl2 and 0.5% (w / v) taurodeoxycholic acid. Then the plate was placed in a constant temperature incubator at 37°C for 2-3 days. The BSH activity was indicated by observing the precipitation area of hydrolyzed salt around the colonies.

[0056] The detection results showed that strain ZZM01 did not show bile salt hydrolase activity on MRS agar medium added with 0.37 g / L CaCl2 and 0.5% (w / v) taurodeoxycholic acid Figure 8 .

[0057] While the application has been described by way of example and in terms of the preferred embodiment, it is to be understood that certain modifications can be made to the disclosed apparatus without departing from the scope of the application, and the scope of the application should be determined not by the embodiment but by the appended claims.

Claims

1. A type of Lactobacillus plantarum ( Lactobacillus plantarum ZZM01, characterized in that, The preservation number is CCTCC NO: M 2025695.

2. Use of the Lactobacillus plantarum ZZM01 in the preparation of a medicine for reducing cholesterol.

3. Use according to claim 2, characterized in that, The medicine has a non-BSH-dependent cholesterol-reducing mechanism.

4. Use according to claim 2, characterized in that, The content of the B. lactis ZZM01 in the medicine is 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. Use of the Lactobacillus plantarum ZZM01 in the preparation of a health food for assisting in reducing cholesterol.

6. Use according to claim 5, characterized in that, The health food has a non-BSH-dependent cholesterol-reducing mechanism.

7. Use according to claim 5, characterized in that, The content of Lactobacillus plantarum ZZM01 in the health food is 1×10 9 CFU / mL or 1×10 9 CFU / g.

8. A medicine for reducing cholesterol with the Lactobacillus plantarum ZZM01 as an active ingredient.

9. Use according to claim 8, characterized in that, The content of Lactobacillus plantarum ZZM01 in the medicine is 1×10 9 CFU / mL or 1×10 9 CFU / g.

10. A health food for assisting in reducing cholesterol with the Lactobacillus plantarum ZZM01 as an active ingredient.

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