Phytobacterium plantarum YM-7 and application thereof

Abstract

The invention discloses a lactobacillus plantarum YM-7, the preservation number of the lactobacillus plantarum YM-7 in Guangdong Microbial Culture Collection Center is GDMCC No: 65465, and the lactobacillus plantarum YM-7 obtained from fermented food is high in safety, has an inhibition effect on staphylococcus aureus, escherichia coli and candida albicans, and can be used for preparing a food additive. The strain TN-7 has the effect of removing DPPH, hydroxyl free radicals and superoxide anion free radicals, so that the strain TN-7 can be used as a food additive, and a new way is provided for development of antioxidant and antibacterial food additives.

Description

Technical Field

[0001] The present invention belongs to the technical field of food microorganisms and specifically relates to a strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum )YM-7 and its applications. Background Art

[0002] With the accelerated pace of modern life, oxidative stress has become one of the important factors affecting human health. Oxidative stress refers to cell damage caused by excessive production of reactive oxygen species (ROS) and imbalance of antioxidant defense system in the body. Long-term oxidative stress is closely related to the occurrence and development of a variety of chronic diseases, including cardiovascular disease, cancer, diabetes, and neurodegenerative diseases. Therefore, finding effective antioxidants to reduce the impact of oxidative stress has become an important strategy for the prevention and treatment of related diseases.

[0003] Lactobacillus plantarum ( Lactiplantibacillus plantarum ), as a class of probiotics widely found in fermented foods, has received widespread attention due to its multiple benefits to host health. In addition to improving intestinal health and enhancing immunity, Lactobacillus plantarum also shows potential antioxidant activity, which can scavenge free radicals and reduce oxidative damage. In recent years, research on the antioxidant activity of Lactobacillus plantarum has gradually increased, revealing its important role in protecting cells from oxidative stress. Summary of the invention

[0004] The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ) YM-7, deposited in Guangdong Microbiological Culture Collection Center on November 11, 2024, with the deposit number GDMCC No: 65465; the deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0005] Another object of the present invention is to use the above-mentioned Lactobacillus plantarum YM-7 in the preparation of a method for inhibiting Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli )、Candida albicans( Candida albicans ) preparations, or in the preparation of antioxidant preparations.

[0006] The ingredients (or active ingredients) of the preparation of the present invention are the above-mentioned Lactobacillus plantarum YM-7, and one or more excipients acceptable in food production may be added to improve the absorption effect of the active ingredients or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms for use.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: 1. Strain isolation and purification Sterilize a test tube containing 5 mL of MRS liquid culture medium with high-pressure steam at 121°C for 20 min, take 3-5 samples of fermented black beans (traditional fermented black beans from Yunnan) and quickly inoculate them into the test tube, immediately plug it with a rubber stopper, and culture the test tube at 37°C and 150 r / min for 24 h. Pipette 100 μL of culture solution on a sterile operating table and add it to 900 μL of sterile saline that has been dispensed for gradient dilution. Spread the dilution on MRS solid culture medium and culture at 37°C. Pick a single colony and streak it on MRS solid culture medium for purification to obtain strain YM-7. 2. Identification of strain YM-7 (1) The colonies of strain YM-7 on MRS solid medium are round. Gram staining and microscopic identification show that the strain is a Gram-positive bacterium, rod-shaped, milky white, with a smooth and moist surface, regular edges, opaque and shiny.

[0008] (2) DNA extraction: The genomic DNA of strain YM-7 was extracted using QIAamp genomic DNA and RNA kits. The extracted genomic DNA was used as a template and 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') were used for PCR amplification. The PCR product was sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were compared with the sequences on NCBI. Combined with the morphological characteristics and molecular identification results, the strain was finally identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ); 3. Antibacterial experiment The Oxford cup method was used to detect three pathogenic bacteria Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli )、Candida albicans( Candida albicans ), the experimental results showed that strain YM-7 had inhibitory effects on the three pathogens.

[0009] 4. Antioxidant test The activated strain YM-7 was crushed and the supernatant was taken by centrifugation. The DPPH scavenging rate, hydroxyl radical scavenging rate and superoxide anion radical scavenging rate of the supernatant were tested. The results showed that the DPPH scavenging rate of strain YM-7 was 70.56%, the hydroxyl radical scavenging rate was 25.34%, and the superoxide anion radical scavenging rate was 21.35%. Therefore, strain YM-7 has good antioxidant activity.

[0010] Advantages and technical effects of the present invention: The plant lactobacillus YM-7 obtained from fermented food of the present invention has high safety, has an inhibitory effect on Staphylococcus aureus, Escherichia coli and Candida albicans, and has a scavenging effect on DPPH, hydroxyl free radicals and superoxide anion free radicals. Therefore, the strain TN-7 of the present invention can be used as a food additive, providing a new way for the development of food additives with antioxidant and antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is the colony morphology of strain YM-7; Figure 2 is the phylogenetic tree of strain YM-7; Figure 3 These are the results of the antioxidant experiment of strain YM-7. DETAILED DESCRIPTION

[0012] The technical scheme of the present invention is further described in detail below through examples, but the content of the present invention is not limited thereto. The methods in the present examples are conventional methods unless otherwise specified, and the materials and reagents used are obtained from commercial channels or prepared according to conventional methods unless otherwise specified; The MRS medium (g / L) in the embodiment is: peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, sodium acetate 5g, diammonium citrate 2g, Tween 80 1mL, magnesium sulfate 0.58g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2g, add 1000mL of water and mix well, and sterilize at 110°C for 20min; Example 1: Acquisition of strain YM-7 Take 4 samples of fermented fermented black beans collected from Kunming, Yunnan, add them to a test tube containing 5 mL of MRS liquid culture medium, plug it with a rubber stopper, and incubate the test tube at 37°C and 150 rpm / min for 24 h. Take 100 μL of the cultured bacterial solution on a sterile operating table and add it to 900 μL of sterile saline for gradient dilution. Then take 100 μL of 10 -5 , 10 -6 , 10 -7 The diluted solution was evenly spread on the MRS solid medium, and each gradient was repeated three times. The coated solid medium was placed in a 37℃ water-proof constant temperature incubator for 24 hours. After a single colony grew, a single colony was picked up with a sterilized toothpick and streaked on the MRS solid medium. The single colony was placed in a 37℃ water-proof constant temperature incubator for 24 hours. The single colony was picked up with a toothpick and streaked 2-3 times until a purified single colony grew to obtain strain YM-7. 2. Identification of strain YM-7 (1) Morphological identification The colonies of the purified strain YM-7 on MRS solid medium were round, milky white, smooth and moist, with regular edges, opaque and shiny ( Figure 1 ), which is a Gram-positive bacterium, rod-shaped under a microscope; (2) 16S rRNA molecular biological identification DNA extraction kit QIAamp genomic DNA and RNA kits was used to extract genomic DNA of strain YM-7. The extracted genomic DNA was used as a template and PCR amplification was performed with 16S rRNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'); the PCR reaction system was 2×TaqPCR Master Mix 10μL, 0.5μL of upstream and downstream primers each, 1μL of DNA template, and 8μL of ddH2O; the reaction program was 95℃ 5 min; 94℃ 1 min, 55-58℃ 1 min, 72℃ 90 s, 30 cycles; 72℃ 10 min.

[0013] The PCR product was sent to Shanghai Biotechnology Co., Ltd. for gene sequencing. The 16S rRNA gene sequence was then Blast compared in GenBank of the NCBI database. The phylogenetic tree is shown in Figure 2 Combined with the morphological characteristics and molecular identification results, strain YM-7 was finally identified as Lactobacillus plantarum ( Lactiplantibacillus plantarum ).

[0014] Example 2: Antibacterial activity test of strain YM-7 using the Oxford cup method Pour 10 mL of MRS solid medium evenly into the culture dish, dry it, place an Oxford cup in the middle of the plate, and add 10 8 CFU / mL of Lactobacillus plantarum YM-7 (the culture medium is MRS liquid culture medium); when the culture medium corresponding to the pathogen is cooled to 40°C, the pathogen is added to the culture medium to make the final concentration of the pathogen 10 6 CFU / mL, the pathogens were Staphylococcus aureus (culture medium was BHI solid medium, Huankai Biotechnology), Escherichia coli (culture medium was LB solid medium, Huankai Biotechnology) and Candida albicans (culture medium was Sabouraud medium, Huankai Biotechnology), and after complete cooling and solidification, they were cultured at 37℃ for 24 h, and the diameter of the inhibition zone was measured. All experiments were repeated 3 times in parallel; At the same time, Lactobacillus plantarum AY04, YM4-2, YM5-4, YM5-3, and YM4-1 were set as controls; The results are shown in the table below. It can be seen from the table that all plant lactobacilli have antibacterial effects on the three pathogens. The strain YM-7 has the strongest antibacterial effect on Escherichia coli and Candida albicans, and also has a good antibacterial effect on Staphylococcus aureus. .

[0015] Example 3: Antioxidant experiment of strain YM-7 Take the activated 10 9 CFU / mL of Lactobacillus plantarum (YM-7, AY04, YM4-2, YM5-4, YM5-3, YM4-1) bacterial liquid was centrifuged at 8000 rpm / min for 5 min, the supernatant was discarded, the bacteria were resuspended with 0.9% saline, and the bacteria were washed twice with saline, and then disrupted with an ultrasonic cell disruptor at 150 Hz for 30 min, and centrifuged at 10000 rpm / min for 5 min, and the cell-free supernatant was collected for antioxidant activity detection; (1) DPPH clearance rate Take 2 mL of DPPH methanol solution (0.5 mmol / L) and mix it with 2 mL of cell-free supernatant of Lactobacillus plantarum. After keeping it away from light at room temperature for 30 min, centrifuge and take the supernatant. Detect the absorbance at 517 nm and calculate the DPPH clearance rate = [1-(A 样 -A 空 ) / A 对 ]×100%; among which A 样 2mL cell-free supernatant + 2mL DPPH; A 空 2mL cell-free supernatant + 2mL methanol; A 对 2mL DPPH + 2mL water; (2) Hydroxyl radical scavenging rate Take 1mL salicylic acid solution (9mmol / L), 1mL FeSO4 (9mmol / L) solution, and 1mL 3% H2O2, mix them and place them in a 37℃ constant temperature incubator for 20min. Detect the absorbance at 510nm. The hydroxyl radical scavenging rate = [1-(A1-A2) / A0] × 100%, where A0 is FeSO4+salicylic acid+3%H2O2+water; A1 is FeSO4+salicylic acid+3%H2O2+cell-free supernatant, A2: FeSO4+salicylic acid+water+cell-free supernatant; (3) Superoxide anion clearance rate Take 4.5mL Tris-diethylenetriaminepentaacetic acid (0.1mol / L Tris, 2mmol / L diethylenetriaminepentaacetic acid in a volume ratio of 1:1, pH = 8.3) and 300μL pyrogallic acid (6mmol / L) and mix them. Incubate in a 25℃ water bath for 10min and detect the absorbance at 325nm. The superoxide anion clearance rate = [1-(A 11 -A 10 ) / (A 01 -A 00 )]×100%, where A 11 : Cell-free supernatant + Tris-diethylenetriaminepentaacetic acid + pyrogallol, A 10 : Cell-free supernatant + Tris-diethylenetriaminepentaacetic acid + water, A 01 :Water + Tris-diethylenetriaminepentaacetic acid + pyrogallol, A 00 : Water + Tris-diethylenetriaminepentaacetic acid; The experimental results are shown in Figure 3 From the figure, it can be seen that the six strains of Lactobacillus plantarum all have good DPPH scavenging rates, among which YM-7 has the highest scavenging rate of 70.56%, and the scavenging rates of other Lactobacillus plantarum are lower than YM-7; in addition, the scavenging rates of hydroxyl radicals and superoxide anion radicals of YM-7 are also higher than those of the other five strains, so strain YM-7 has antioxidant activity.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) YM-7, whose deposit number in Guangdong Microbiological Culture Collection Center is GDMCC No: 65465.

2. The plant lactobacillus YM-7 according to claim 1 is used in the preparation of a method for inhibiting Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli )、Candida albicans( Candida albicans ) application in preparations.

3. Use of the plant lactobacillus YM-7 according to claim 1 in the preparation of an antioxidant preparation.

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