CN112126604B - Lactobacillus plantarum capable of reducing risk factors for hypertension and application thereof - Google Patents
Lactobacillus plantarum capable of reducing risk factors for hypertension and application thereof Download PDFInfo
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- CN112126604B CN112126604B CN202011060751.4A CN202011060751A CN112126604B CN 112126604 B CN112126604 B CN 112126604B CN 202011060751 A CN202011060751 A CN 202011060751A CN 112126604 B CN112126604 B CN 112126604B
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Abstract
The invention discloses lactobacillus plantarum capable of reducing risk factors of hypertension and application thereof, and belongs to the technical field of microorganisms and medicines. The invention provides a lactobacillus plantarum CCFM1149, wherein the lactobacillus plantarum CCFM1149 can relieve hypertension, and is specifically embodied in that: (1) remarkably reduces O in A7R5 cells after Angiotensin II stimulation2 ·‑Horizontal; (2) remarkably reduces the intracellular H of A7R5 after the stimulation of Angiotensin II2O2Horizontal; (3) the SOD enzyme activity in A7R5 cells is obviously improved; (4) the CAT enzyme activity in A7R5 cells is remarkably improved, and therefore, the lactobacillus plantarum CCFM1149 has great application prospect in preparation of products (such as food or medicines) for preventing and/or treating hypertension.
Description
Technical Field
The invention relates to lactobacillus plantarum capable of reducing risk factors for hypertension and application thereof, and belongs to the technical field of microorganisms and medicines.
Background
With the increase of the global living standard, hypertension is affecting the health of more and more people. The survey found that the number of patients with hypertension worldwide has been on the rise year by year for the past 50 years, and more than 30% of people worldwide today have hypertension. Hypertension is an important risk factor for other cardiovascular diseases, can promote the formation of atherosclerosis and thrombus, increase the morbidity risk of cardiovascular diseases such as stroke, heart failure, myocardial infarction and the like, and seriously threatens the life health of patients. More than 90% of patients with hypertension are essential hypertension. The cause of the primary hypertension is complex, the disease process is long, and the patient is difficult to take measures in advance to effectively prevent the primary hypertension. Once hypertension is developed, patients need to control blood pressure by relying on drugs, and drug dependence is easy to develop.
At present, the intervention aiming at hypertension mainly comprises drug intervention and diet auxiliary intervention. The hypotensive drug mainly comprises Angiotensin Converting Enzyme (ACE) inhibitor, angiotensin receptor antagonist, diuretic, beta-receptor blocker and Ca2+Ion channel blockers and the like, and antihypertensive drugs in each category have toxic and side effects in different degrees, and cause damage to the health of patients after long-term administration. Aiming at the intervention of hypertension, a dietary intervention guideline for hypertension is internationally introduced, the reduction of the intake of sodium and fat is emphasized, the intake of high-fiber foods such as fruits, vegetables and complete grains is increased, vegetable protein is advocated to replace animal protein, and the intake of red meat is reduced. Dietary intervention, while improving patient health, helping to control blood pressure levels and reducing the risk of developing chronic diseases such as metabolic syndrome, is slow to become effective and often difficult to control blood pressure to a significant extent. Therefore, the development of a new hypertension prevention and treatment method has important significance for reducing the incidence of hypertension and improving the national health level.
Studies have shown that Reactive Oxygen Species (ROS) in the tissue of the vascular wall are an important risk factor for the development of hypertension. The production of ROS in cardiovascular tissue can be attributed to the stimulation by vasoconstrictors such as angiotensin ii (Ang ii), aldosterone, endothelin, and the like. Elevated levels of Ang ii and/or aldosterone are observed in both essential hypertension patients and animal models. These hormones, when acting on the vessel wall, activate NADPH oxidase in the vascular endothelium and smooth muscle to produce ROS. ROS are important signal molecules for regulating vascular tone, and can cause smooth muscle contraction, inhibit vasodilation and cause hypertension. In addition to raising blood pressure, ROS can also participate in pathways associated with pathological changes in blood vessels, causing thickening of blood vessel walls and narrowing of blood vessel lumens, as well as promoting vascular fibrosis. These structural lesions of the vessel wall will likely further contribute to the development of hypertension while increasing the risk of development of other cardiovascular diseases such as atherosclerosis. Therefore, reducing ROS in cardiovascular tissues would help prevent the development of hypertension and cardiovascular disease.
The health efficacy of probiotics has been reported in large numbers in recent years, and ingestion of probiotics in appropriate amounts does not have adverse effects on the host. Studies have shown that some probiotics of the genus lactobacillus have the efficacy of lowering blood pressure. Thus, the use of probiotics as dietary supplements would help to improve the health of the patient, enhancing the effect of non-pharmaceutical interventions. However, the existing probiotics with antihypertensive function have single action target, mainly reduce blood pressure by inhibiting Angiotensin Converting Enzyme (ACE) activity, and are not suitable for intervening hypertension caused by other factors such as aldosterone. Therefore, based on the importance of ROS in the pathogenesis of hypertension, there is an urgent need to screen out probiotics that can reduce the level of ROS in the vascular wall as probiotic strains with potential for preventing hypertension.
Disclosure of Invention
[ problem ] to
The invention aims to provide Lactobacillus plantarum (Lactobacillus plantarum) capable of reducing hypertension risk factors such as active oxygen level in vascular smooth muscle cells.
[ solution ]
In order to solve the problems, the invention provides a Lactobacillus plantarum CCFM1149, wherein the Lactobacillus plantarum CCFM1149 is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No.61163, the preservation date of 2020, 08 and 21 days, and the preservation address of Guangzhou city, Jielianzhou No. 100, No. 59, building 5.
The lactobacillus plantarum CCFM1149 is derived from a pickle sample, the 16S rDNA sequence of the strain is shown in SEQ ID NO.1 through sequencing analysis, the sequence obtained through sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is lactobacillus plantarum and is named as lactobacillus plantarum CCFM 1149.
The thallus of the lactobacillus plantarum CCFM1149 is in a short rod shape; the bacterial colony is round, rough and transparent.
The invention also provides application of the lactobacillus plantarum in preparation of products for preventing and/or treating cardiovascular diseases.
In one embodiment of the present invention, the cardiovascular disease is hypertension.
In an embodiment of the present invention, in the product, the viable count of lactobacillus plantarum CCFM1149 is not less than 1 × 106CFU/mL or 1X 106CFU/g。
In one embodiment of the invention, the product comprises a food or pharmaceutical product.
In one embodiment of the invention, the medicine contains lactobacillus plantarum CCFM1149, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and/or an additive.
In one embodiment of the invention, the excipient comprises a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid, and/or a release retardant.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
In one embodiment of the present invention, the pharmaceutical composition is in the form of powder, granule, capsule, tablet, pill or oral liquid.
In one embodiment of the invention, the food is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leavening agent containing the lactobacillus plantarum CCFM 1149; or the food is a beverage or a snack containing the lactobacillus plantarum CCFM 1149.
In one embodiment of the invention, the preparation method of the starter is that the lactobacillus plantarum CCFM1149 is inoculated into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and the culture medium is cultured for 36 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the bacteria with phosphate buffer solution with the pH of 7.2-7.4 for 2-4 times, and then re-suspending with a freeze-drying protective agent to obtain a re-suspension solution; and (4) freeze-drying the heavy suspension by using a vacuum freezing method to obtain the leavening agent.
In one embodiment of the present invention, the mass ratio of the lyoprotectant to the microbial cells is 2: 1.
In one embodiment of the invention, the medium comprises 87.7% water, 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract by total mass of the medium.
In one embodiment of the invention, the pH of the medium is 6.8.
In one embodiment of the invention, the protective agent comprises 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
The invention also provides a product for preventing and/or treating cardiovascular diseases, which contains the lactobacillus plantarum CCFM 1149.
In one embodiment of the present invention, the cardiovascular disease is hypertension.
In an embodiment of the present invention, in the product, the viable count of lactobacillus plantarum CCFM1149 is not less than 1 × 106CFU/mL or 1X 106CFU/g。
In one embodiment of the invention, the product comprises a food or pharmaceutical product.
In one embodiment of the invention, the medicine contains lactobacillus plantarum CCFM1149, a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and/or an additive.
In one embodiment of the invention, the excipient comprises a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an absorbent, a diluent, a flocculant, a deflocculant, a filter aid, and/or a release retardant.
In one embodiment of the invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
In one embodiment of the present invention, the pharmaceutical composition is in the form of powder, granule, capsule, tablet, pill or oral liquid.
In one embodiment of the invention, the food is a health food; or the food is a dairy product, a bean product or a fruit and vegetable product produced by using a leavening agent containing the lactobacillus plantarum CCFM 1149; or the food is a beverage or a snack containing the lactobacillus plantarum CCFM 1149.
In one embodiment of the invention, the preparation method of the starter is that the lactobacillus plantarum CCFM1149 is inoculated into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and the culture medium is cultured for 36 hours at 37 ℃ to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the bacteria with phosphate buffer solution with the pH of 7.2-7.4 for 2-4 times, and then re-suspending with a freeze-drying protective agent to obtain a re-suspension solution; and (4) freeze-drying the heavy suspension by using a vacuum freezing method to obtain the leavening agent.
In one embodiment of the present invention, the mass ratio of the lyoprotectant to the microbial cells is 2: 1.
In one embodiment of the invention, the medium comprises 87.7% water, 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract by total mass of the medium.
In one embodiment of the invention, the pH of the medium is 6.8.
In one embodiment of the invention, the protective agent comprises 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
Has the advantages that:
1. the invention provides a Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149, wherein the Lactobacillus plantarum CCFM1149 can reduce risk factors of hypertension, and is specifically embodied in that:
(1) remarkably reduces O in A7R5 cells after Angiotensin II stimulation2 ·-Horizontal;
(2) remarkably reduces the intracellular H of A7R5 after the stimulation of Angiotensin II2O2Horizontal;
(3) the SOD enzyme activity in A7R5 cells is obviously improved;
(4) obviously improves the activity of CAT enzyme in A7R5 cells,
therefore, the Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 has great application prospect in preparing products (such as food or medicines and the like) for preventing and/or treating hypertension.
2. Lactobacillus plantarum (Lactobacillus plantarum) is one of probiotics, is included in a strain list available for food issued by Ministry of health at present, and has the effect of regulating intestinal health, so the Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 obtained by the invention is relatively healthy for human bodies and has no side effect.
Biological material preservation
A Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 is classified and named as Lactobacillus plantarum, is preserved in Guangdong province microorganism strain preservation center in 21 days 08 and 2020, has the preservation number of GDMCC No.61163, and has the preservation address of No. 59 building 5 of Mieli Midduo No. 100 college in Guangzhou city.
Drawings
FIG. 1: different groups of A7R5 intracellular O2 ·-And (5) horizontal comparison.
FIG. 2: different groups of A7R5 intracellular H2O2And (5) horizontal comparison.
FIG. 3: comparing the activity of SOD enzymes in different groups of A7R5 cells.
FIG. 4: CAT enzyme activities in different groups of A7R5 cells were compared.
Wherein "# #" indicates a significant difference (p <0.01) compared to the Control group, "# # ##" indicates a significant difference (p <0.001) compared to the Control group, "#" indicates a significant difference (p <0.01) compared to the Model group.
Detailed Description
The following examples refer to trypsin and HBSS buffers from Thermo Fisher, Angiotensin II from MCE, skim milk from Bright milk, Inc., glucose and yeast extract from pharmaceutical group Chemicals, Inc., tryptone from OXOID, UK, DHE fluorescent probe, DCFH-DA fluorescent probe, BCA protein concentration assay kit and protease inhibitor cocktail from Biyunnan Biotech institute, and SOD assay kit and CAT assay kit from Nanjing Biotech institute.
The media involved in the following examples are as follows:
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H200.1 g/L、MnSO40.05g/L, Tween-801 ml/L, pH 7.0.
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2PO4·3H2O 2.6g/L、MgSO4·7H200.1 g/L、MnSO40.05g/L, Tween-801 ml/L, agar 20g/L, L-cysteine hydrochloride 0.05g/L, and pH 7.0.
DMEM medium: glycine 30mg/L, L-arginine hydrochloride 84mg/L, L-cysteine hydrochloride 63mg/L, L-glutamine 584mg/L, L-Histidine hydrochloride 42mg/L, L-isoleucine 105mg/L, L-leucine 105mg/L, L-lysine hydrochloride 146mg/L, L-methionine 30mg/L, L-phenylalanine 66mg/L, L-serine 42mg/L, L-threonine 95mg/L, L-tryptophan 16mg/L, L-tyrosine disodium 104mg/L, L-valine 94mg/L, choline chloride 4mg/L, D-calcium pantothenate 4mg/L, folic acid 4mg/L, nicotinamide 4mg/L, pyridoxine hydrochloride 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, i-inositol 7.2mg/L, CaCl2200 mg/L、Fe(NO3)3·9H2O 0.1mg/L、MgSO4 97.67mg/L、KCl 400mg/L、NaHCO33700 mg/L、NaCl 6400mg/L、NaH2PO4H2O 125mg/L, D-glucose 4500mg/L, phenol red 15 mg/L.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus is adopted.
The preparation of the lactic acid bacteria culture supernatants referred to in the following examples was as follows:
streaking lactobacillus on MRS solid culture medium, and culturing at 37 deg.C for 48 hr to obtain single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, culturing for 18h at 37 ℃ for activation, and continuously activating for two generations to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing for 18h at 37 ℃ to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min, and collecting the supernatant; adjusting the pH value of the supernatant to 7.0 by using a NaOH solution with the concentration of 1mol/L, and filtering and sterilizing by using a 0.22 mu m filter membrane to obtain the culture supernatant of the lactobacillus.
Example 1: acquisition of Lactobacillus plantarum
The method comprises the following specific steps:
1. screening
Pretreating a pickle serving as a sample, storing the pretreated pickle in 20% glycerol in a refrigerator at the temperature of-80 ℃, taking out and unfreezing, uniformly mixing the sample, sucking 0.5mL of the sample, adding the sample into 4.5mL, performing gradient dilution by using 9g/L physiological saline containing 0.05g/L cysteine, selecting proper gradient dilution liquid to coat the gradient dilution liquid on an MRS solid culture medium, culturing for 48 hours at 37 ℃, selecting a typical bacterial colony of lactobacillus plantarum to the MRS solid culture medium, streaking and purifying, selecting a single bacterial colony, transferring the single bacterial colony to the MRS liquid culture medium (containing 0.05g/L cysteine) for enrichment, and preserving by using 30% glycerol to obtain a strain CCFM1149 and a strain L86; wherein, the typical colony of the lactobacillus plantarum is round, rough and transparent.
2. Identification
The genome of the strain CCFM1149 and the genome of the strain L86 are extracted, the 16S rDNA of the strain CCFM1149 and the 16S rDNA of the strain L86 are amplified and sequenced (the 16S rDNA nucleotide determination sequences of the strain CCFM1149 and the strain L86 are respectively shown in SEQ ID NO.1 and SEQ ID NO.2, the upstream primer 27F sequence of the strain identification is shown in SEQ ID NO.3, and the downstream primer 1492R sequence is shown in SEQ ID NO. 4), the sequences are subjected to nucleic acid sequence alignment in NCBI, and the result shows that the strain CCFM1149 and the strain L86 are both Lactobacillus plantarum and are respectively named as Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 and Lactobacillus plantarum (Lactobacillus plantarum) L86.
Example 2: lactobacillus plantarum couple A7R5 intracellular O2 ·-Influence of level
The method comprises the following specific steps:
rat thoracic aortic smooth muscle cells A7R5 were purchased from the China academy of sciences type culture Collection cell Bank and cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin. Cell culture at 37 ℃ with 5% (v/v) CO in gas phase2In a cell culture incubator. And carrying out passage when the cells grow to 70-80% of density.
Selecting A7R5 cells with good growth state, digesting the A7R5 cells by trypsin, centrifuging, re-suspending by using a DMEM (DMEM) culture medium, and counting the cells to obtain a re-suspension; after the suspension was inoculated into 24-well plates at 20000 cells/well, 500. mu.L of DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin was added to the 24-well plates and the mixture was evaporated at 37 ℃ in 5% (v/v) CO2Culturing for 48 hours in a cell culture box; after 48h incubation, DM containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin in 24-well platesEM medium was replaced with 500. mu.L of DMEM medium containing 0.1% (v/v) Fetal Bovine Serum (FBS), and 24-well plates were incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 24 hours in the cell culture box; after standing for 24h, dividing the cells into a blank Control group (Control), a Model group (Model), a Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 dry pre-group (CCFM1149) and a Lactobacillus plantarum (Lactobacillus plantarum) L86 dry pre-group (L86) by taking the cells in each well in a 24-well plate as a unit, wherein 3 wells are formed in each group, 15 mu L of liquid culture medium is respectively added into each well of the blank Control group and the Model group, 15 mu L of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 culture supernatant is added into each well of the CCFM1149 dry pre-group, 15 mu L of Lactobacillus plantarum (Lactobacillus plantarum) L86 culture supernatant is added into each well of the L86 intervention group, and the 24-well plate is placed at 37 ℃ and contains 5% (v/v) CO in a gas phase2The cell culture box is intervened for 12 hours; after 12h of intervention, Angiotensin II was added to each well of the model group, CCFM1149 run group and CCFM1149 run group to a concentration of 1X 10- 7M, simultaneously, the same volume of DMEM medium was added to each well of the blank control group as a control, and 24-well plates were incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 4 hours in the cell culture box; after standing for 4 hours, the 24-well plate was taken out of the cell culture chamber, the liquid in each well was aspirated and 500. mu.L of DMEM medium containing 10. mu.M DHE fluorescent probe was added to each well, and the 24-well plate was incubated at 37 ℃ in a gas phase containing 5% (v/v) CO2Incubating for 30min in the cell incubator; after incubation for 30min, taking the 24-well plate out of the cell culture box, sucking out liquid in each well and washing cells in each well for 2 times by using HBSS buffer solution; after the washing is finished, adding 500 mu L of HBSS buffer solution into each hole of a 24-hole plate, observing by an inverted fluorescence microscope, exciting cells to generate red fluorescence by adopting green waveband excitation light, and selecting a proper visual field for shooting; image pro plus was used to calculate the fluorescence density of the pictures to characterize intracellular O2 ·-The data were processed in comparison with the blank control, and the results are shown in FIG. 1.
As can be seen from FIG. 1, 1X 10 is used-7Model group A7R5 intracellular O after 4h of M Angiotensin II stimulation2 ·-Level is remarkably increasedUp to 1.6 times (p) of blank control<0.01), CCFM1149 intervenes in group A7R5 intracellular O2 ·-The level is reduced by 34 percent (p) compared with the model group<0.01), whereas L86 intervenes in group A7R5 intracellular O2 ·-The levels did not change significantly from the model groups.
Therefore, the intervention of the culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can obviously reduce the intracellular O of A7R5 cells after the stimulation of Angiotensin II2 ·-The increased level can reduce the risk factors for hypertension.
Example 3: lactobacillus plantarum couple A7R5 intracellular H2O2Influence of level
The method comprises the following specific steps:
rat thoracic aortic smooth muscle cells A7R5 were purchased from the China academy of sciences type culture Collection cell Bank and cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin. Cell culture at 37 ℃ with 5% (v/v) CO in gas phase2In a cell culture incubator. And carrying out passage when the cells grow to 70-80% of density.
Selecting A7R5 cells with good growth state, digesting the A7R5 cells by trypsin, centrifuging, re-suspending by using a DMEM (DMEM) culture medium, and counting the cells to obtain a re-suspension; after the suspension was inoculated into 24-well plates at 20000 cells/well, 500. mu.L of DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin was added to the 24-well plates and the mixture was evaporated at 37 ℃ in 5% (v/v) CO2Culturing for 48 hours in a cell culture box; after 48 hours of culture, the DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin in the 24-well plate was replaced with 500. mu.L of DMEM medium containing 0.1% (v/v) Fetal Bovine Serum (FBS), and the 24-well plate was incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 24 hours in the cell culture box; after standing for 24 hours, the cells were divided into a blank Control group (Control), a Model group (Model), a Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 stem set (CCFM1149), and a Lactobacillus plantarum (Lactobacillus plantarum) L86 stem set (L86) in units of cells in each well of a 24-well plate, with 3 wells per group, wherein in each wellAdding 15 mu of LMRS liquid culture medium into each well of the blank control group and the model group, respectively, adding 15 mu of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 culture supernatant into each well of the CCFM1149 intervention group, adding 15 mu of Lactobacillus plantarum (Lactobacillus plantarum) L86 culture supernatant into each well of the L86 intervention group, and placing 24-well plates at 37 ℃ and containing 5% (v/v) CO in gas phase2The cell culture box is intervened for 12 hours; after 12h of intervention, Angiotensin II was added to each well of the model group, CCFM1149 run group and CCFM1149 run group to a concentration of 1X 10- 7M, simultaneously, the same volume of DMEM medium was added to each well of the blank control group as a control, and 24-well plates were incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 4 hours in the cell culture box; after standing for 4 hours, the 24-well plate was taken out of the cell culture chamber, the liquid in each well was aspirated and 500. mu.L of DMEM medium containing 8. mu.M DCFH-DA fluorescent probe was added to each well, and the 24-well plate was incubated at 37 ℃ with 5% (v/v) CO in gas phase2Incubating for 20min in the cell incubator; after incubation for 20min, the 24-well plate was taken out of the cell incubator, the liquid in each well was aspirated and the cells in each well were washed 2 times with HBSS buffer; after the washing is finished, adding 500 mu L of HBSS buffer solution into each hole of a 24-hole plate, observing by an inverted fluorescence microscope, exciting cells to generate green fluorescence by adopting blue waveband excitation light, and selecting a proper visual field for shooting; image pro plus was used to calculate the fluorescence intensity of the pictures to characterize intracellular H2O2The data were processed in comparison with the blank control, and the results are shown in FIG. 2.
As can be seen from FIG. 2, 1X 10 is used-7Model group A7R5 intracellular H4H after M Angiotensin II stimulation2O2The level was significantly increased to 1.8 times (p) of the blank control group<0.001), CCFM1149 intervention group A7R5 intracellular H2O2The level is reduced by 30 percent (p) compared with the model group<0.01), whereas L86 intervenes in group A7R5 intracellular H2O2The levels did not change significantly from the model groups.
Therefore, the intervention of the culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can obviously reduce the intracellular H of A7R5 cells after stimulation of Angiotensin II2O2The rise in the level of the water is,can reduce risk factors of hypertension.
Example 4: effect of Lactobacillus plantarum on enzymatic Activity of SOD enzyme in A7R5 cells
The method comprises the following specific steps:
rat thoracic aortic smooth muscle cells A7R5 were purchased from the China academy of sciences type culture Collection cell Bank and cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin. Cell culture at 37 ℃ with 5% (v/v) CO in gas phase2In a cell culture incubator. And carrying out passage when the cells grow to 70-80% of density.
Selecting A7R5 cells with good growth state, digesting the A7R5 cells by trypsin, centrifuging, re-suspending by using a DMEM (DMEM) culture medium, and counting the cells to obtain a re-suspension; the resuspension solution was adjusted to 2X 105After the cells/well were seeded into 6cm cell culture dishes, 4mL of DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin was added to the cell culture dishes, and 5% (v/v) CO in gas phase at 37 deg.C2Culturing for 48 hours in a cell culture box; after 48h of culture, the DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin in the cell culture dish was replaced with 4mL of DMEM medium containing 0.1% (v/v) Fetal Bovine Serum (FBS), and the cell culture dish was incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 24 hours in the cell culture box; after standing for 24h, dividing the cells into a blank Control group (Control), a Model group (Model), a Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 dry pre-group (CCFM1149) and a Lactobacillus plantarum (Lactobacillus plantarum) L86 dry pre-group (L86) by taking each dish of the cells as a unit, wherein 3 dishes are arranged in each set, 120 mu L of MRS liquid culture medium is respectively added into each dish of the blank Control group and the Model group, 120 mu L of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 culture supernatant is added into each hole of the CCFM1149 dry pre-group, 120 mu L of Lactobacillus plantarum (Lactobacillus plantarum) L86 culture supernatant is added into each hole of the L86 intervention group, and the cell culture dish is subjected to gas phase containing 5% (v/v) CO at 37 DEG C2The cell culture box is intervened for 12 hours; after 12h of intervention, the cell culture dish is taken out of the cell culture box,sucking out the liquid in each dish, placing the dish on ice, and washing the dish for 5 times by using PBS (phosphate buffer solution) precooled by an ice bath; after 5 times of washing, transferring each dish of cells into different 15mL centrifuge tubes, and washing for 2 times by using PBS (phosphate buffer solution) precooled by an ice bath; after washing for 2 times, adding PBS buffer solution (1 mL/tube cell) containing 2.0% (v/v) protease inhibitor mixture into a centrifuge tube to resuspend cells to obtain cell suspension; transferring the cell suspension into a new sterile 1.5mL centrifuge tube, and placing the centrifuge tube on an ice bath for ultrasonic disruption (the ultrasonic power is 300W, the single disruption time is 3-5 seconds, the time interval is 30 seconds, and the process is repeated for 3-5 times) to obtain a cell disruption solution; observing the cell disruption solution under a microscope, and confirming complete disruption if no intact cell exists, thereby completing preparation of cell homogenate; the BCA protein concentration determination kit is adopted to determine the concentration of the cell homogenate protein, the SOD activity of the cell homogenate is determined by establishing a total superoxide dismutase test kit by Nanjing, the intracellular total SOD enzyme activity is expressed by determining the SOD activity/the concentration of the homogenate protein, a control group is used as comparison processing data, and the result is shown in figure 3.
As can be seen from fig. 3, intervention of culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can increase the enzymatic activity of SOD in A7R5 cells to 1.9 times that of blank control group (p <0.001), while intervention of culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) L86 cannot significantly change the enzymatic activity of CAT in A7R5 cells.
Therefore, intervention of the culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can improve the oxidation resistance of cells by improving the activity of SOD enzyme in A7R5 cells, and can reduce risk factors of hypertension.
Example 5: effect of Lactobacillus plantarum on CAT enzyme Activity in A7R5 cells
The method comprises the following specific steps:
rat thoracic aortic smooth muscle cells A7R5 were purchased from the China academy of sciences type culture Collection cell Bank and cultured in DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin. Cell culture at 37 ℃ with 5% (v/v) CO in gas phase2In a cell culture incubator. And carrying out passage when the cells grow to 70-80% of density.
Selecting A7R5 cells with good growth state, digesting the A7R5 cells by trypsin, centrifuging, re-suspending by using a DMEM (DMEM) culture medium, and counting the cells to obtain a re-suspension; the resuspension solution was adjusted to 2X 105After the cells/well were seeded into 6cm cell culture dishes, 4mL of DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, and 100mg/mL streptomycin and containing 5% (v/v) CO in gas phase at 37 ℃ was added to the cell culture dishes2Culturing for 48 hours in a cell culture box; after 48h of culture, the DMEM medium containing 10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin in the cell culture dish was replaced with 4mL of DMEM medium containing 0.1% (v/v) Fetal Bovine Serum (FBS), and the cell culture dish was incubated at 37 ℃ with 5% (v/v) CO in gas phase2Standing for 24 hours in the cell culture box; after standing for 24h, dividing the cells into a blank Control group (Control), a Model group (Model), a Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 dry pre-group (CCFM1149) and a Lactobacillus plantarum (Lactobacillus plantarum) L86 dry pre-group (L86) by taking each dish of the cells as a unit, wherein 3 dishes are arranged in each set, 120 mu L of MRS liquid culture medium is respectively added into each dish of the blank Control group and the Model group, 120 mu L of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 culture supernatant is added into each hole of the CCFM1149 dry pre-group, 120 mu L of Lactobacillus plantarum (Lactobacillus plantarum) L86 culture supernatant is added into each hole of the L86 intervention group, and the cell culture dish is subjected to gas phase containing 5% (v/v) CO at 37 DEG C2The cell culture box is intervened for 12 hours; after 12h of intervention, taking out the cell culture dish from the cell culture box, sucking out the liquid in each dish, placing the dish on ice, and washing the dish for 5 times by using PBS (phosphate buffer solution) precooled by an ice bath; after 5 times of washing, transferring each dish of cells into different 15mL centrifuge tubes, and washing for 2 times by using PBS (phosphate buffer solution) precooled by an ice bath; after washing for 2 times, adding PBS buffer solution (1 mL/tube cell) containing 2.0% (v/v) protease inhibitor mixture into a centrifuge tube to resuspend cells to obtain cell suspension; transferring the cell suspension into a new sterile 1.5mL centrifuge tube, and placing the centrifuge tube on an ice bath for ultrasonic disruption (the ultrasonic power is 300W, the single disruption time is 3-5 seconds, the time interval is 30 seconds, and the process is repeated for 3-5 times) to obtain a cell disruption solution; observing the cell disruption solution under microscope, and confirming that the cell disruption is finished without intact cellCompletely, completing the preparation of cell homogenate; the BCA protein concentration determination kit is adopted to determine the concentration of the cell homogenate protein, the CAT enzyme activity of the cell homogenate is determined by establishing a catalase determination kit by Nanjing, the intracellular CAT enzyme activity is represented by dividing the activity of the measured homogenate CAT by the concentration of the homogenate protein, a control group is used as comparison processing data, and the result is shown in figure 4.
As can be seen from FIG. 4, the intervention of the culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can increase the activity of the CAT enzyme in the A7R5 cell to 3.3 times that of the blank control group (p <0.001), while the intervention of the culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) L86 can not significantly change the activity of the CAT enzyme in the A7R5 cell.
Therefore, intervention of culture supernatant of Lactobacillus plantarum (Lactobacillus plantarum) CCFM1149 can improve the oxidation resistance of cells by improving the activity of CAT enzyme in A7R5 cells, and risk factors of hypertension can be reduced.
Example 6: application of lactobacillus plantarum
The lactobacillus plantarum CCFM1149 can be used for preparing cow milk, and the cow milk is prepared by the following specific preparation process:
inoculating lactobacillus plantarum CCFM1149 into a culture medium according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and culturing at 37 ℃ for 36 hours to obtain a culture solution; centrifuging the culture solution to obtain thalli; cleaning the bacteria with phosphate buffer solution with the pH of 7.2-7.4 for 2-4 times, and then re-suspending with a freeze-drying protective agent to obtain a re-suspension solution; freeze-drying the heavy suspension by a vacuum freezing method to obtain a leavening agent; the mass ratio of the freeze-drying protective agent to the thallus is 2: 1; the culture medium comprises 87.7 percent of water, 10 percent of enzyme hydrolysis skim milk, 0.5 percent of glucose, 1.5 percent of tryptone and 0.3 percent of yeast extract by mass of the total mass of the culture medium, and the pH is 6.8; the protective agent comprises 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
Sterilizing skim milk at 95 deg.C for 20min, cooling to 4 deg.C, adding starter to make the viable concentration of Lactobacillus plantarum CCCFM1149 in skim milk reach 1 × 109And (5) refrigerating and storing the milk at 4 ℃ by CFU/mL to obtain the cow milk containing the viable lactobacillus plantarum CCCFM 1149.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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Claims (10)
1. Lactobacillus plantarum (II)Lactobacillus plantarum) The lactobacillus plantarum is preserved in Guangdong province microorganism strain preservation center, the preservation number is GDMCC No.61163, and the preservation date is 2020, 08 and 21 days.
2. Use of lactobacillus plantarum as defined in claim 1 for the preparation of a medicament for the prevention of hypertension.
3. A product comprising the lactobacillus plantarum strain defined in claim 1.
4. A product according to claim 3, wherein the viable count of Lactobacillus plantarum according to claim 1 is not less than 1 x 106CFU/mL or 1X 106 CFU/g。
5. A product according to claim 3 or 4, wherein the product comprises a foodstuff or a pharmaceutical product.
6. A product according to claim 5, wherein the product comprises Lactobacillus plantarum as defined in claim 1, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
7. A product according to claim 6, wherein the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
8. A product according to claim 6 or 7, wherein the pharmaceutical excipient comprises excipients and/or additives.
9. A product according to claim 8, wherein the excipient comprises a solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, absorbent, diluent, flocculant, deflocculant, filter aid and/or release retardant; the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
10. The product according to claim 5, wherein the food is a dairy product, a soy product or a fruit and vegetable product produced by using a starter culture containing the Lactobacillus plantarum according to claim 1; or the food is a beverage or snack containing the Lactobacillus plantarum strain of claim 1.
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|---|---|---|---|
| CN202011060751.4A CN112126604B (en) | 2020-09-30 | 2020-09-30 | Lactobacillus plantarum capable of reducing risk factors for hypertension and application thereof |
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| CN115747092B (en) * | 2022-07-13 | 2023-07-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Two lactobacillus plantarum SR37-3 and SR61-2 with remarkable blood pressure reducing function and application |
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