WO2022068545A1 - 预防和/或治疗原发性高血压的长双歧杆菌 - Google Patents

预防和/或治疗原发性高血压的长双歧杆菌 Download PDF

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WO2022068545A1
WO2022068545A1 PCT/CN2021/117241 CN2021117241W WO2022068545A1 WO 2022068545 A1 WO2022068545 A1 WO 2022068545A1 CN 2021117241 W CN2021117241 W CN 2021117241W WO 2022068545 A1 WO2022068545 A1 WO 2022068545A1
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bifidobacterium longum
product
ccfm752
cells
food
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French (fr)
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陆文伟
陈卫
王昱升
翟齐啸
赵建新
张灏
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C11/00—Milk substitutes, e.g. coffee whitener compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C13/00—Cream; Cream preparations; Making thereof
    • A23C13/12—Cream preparations
    • A23C13/16—Cream preparations containing, or treated with, microorganisms, enzymes, or antibiotics; Sour cream
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C15/00—Butter; Butter preparations; Making thereof
    • A23C15/12—Butter preparations
    • A23C15/123—Addition of microorganisms or cultured milk products; Addition of enzymes; Addition of starter cultures other than destillates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C17/00—Buttermilk; Buttermilk preparations
    • A23C17/02—Buttermilk; Buttermilk preparations containing, or treated with, microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/061—Addition of, or treatment with, microorganisms
    • A23C19/062—Addition of, or treatment with, microorganisms using only lactic acid bacteria, e.g. pediococcus, leconostoc or bifidus sp., or propionic acid bacteria; Treatment with non-specified acidifying bacterial cultures
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C20/00—Cheese substitutes
    • A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C21/00—Whey; Whey preparations
    • A23C21/02—Whey; Whey preparations containing, or treated with, microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/51—Bifidobacterium
    • A23V2400/533—Longum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to Bifidobacterium longum for preventing and/or treating essential hypertension, and belongs to the technical field of microorganisms and medical technology.
  • Hypertension is one of the most widespread chronic diseases in the population, which brings a heavy burden to the social medical system. At present, more than 30% of people in the world suffer from high blood pressure, and the number of people with high blood pressure is still increasing year by year in recent years. In hypertensive patients, more than 90% are primary hypertension. Essential hypertension has a long onset period and is difficult to cure. Patients need to rely on drugs to control their blood pressure, which is easy to form drug dependence. In addition, the occurrence of hypertension will greatly increase the risk of other cardiovascular diseases such as atherosclerosis, stroke, and heart failure, and seriously threaten the life and health of patients.
  • ROS reactive oxygen species
  • the core blood pressure regulation system of the body is the renin-angiotensin-aldosterone system (RAAS), which contains two important blood pressure-raising hormones, angiotensin II (Ang II) and aldosterone.
  • RAAS renin-angiotensin-aldosterone system
  • AngII and aldosterone can activate NADPH oxidase in the vascular wall, and generate ROS such as O 2 ⁇ - and H 2 O 2 to increase blood pressure.
  • RAAS imbalance exists in patients with essential hypertension and animal models, resulting in elevated levels of Ang II and/or aldosterone.
  • stimulatory factors such as Ang II and aldosterone continue to stimulate the blood vessel wall, excessive ROS will be generated, which will cause the blood vessels to continue to constrict and cause hypertension.
  • the continuously generated ROS will also activate the mitogen-activated protein kinase (MAPK) pathway related to cell proliferation and Collagen synthesis-related Smad pathway and TGF- ⁇ pathway, etc., cause vascular wall thickening and fibrosis, resulting in vascular wall structural lesions and vascular dysfunction.
  • MAPK mitogen-activated protein kinase
  • ROS in the vascular wall may also activate immune pathways such as IL-1 ⁇ , IL-17, and TNF- ⁇ associated with inflammation, causing vascular inflammation and accelerating the formation of hypertension and atherosclerosis. Therefore, scavenging excessive ROS in the blood vessel wall will help prevent the occurrence and development of cardiovascular diseases such as hypertension.
  • the method of clinical intervention for hypertension is mainly drug intervention.
  • Different types of antihypertensive drugs have different degrees of toxic and side effects, such as electrolyte disturbances, damage to kidney function, and angioedema.
  • Long-term use will damage the health of patients.
  • the supplementary intervention method is mainly dietary intervention. Although dietary intervention is beneficial to improve the health status of patients, the effect is slow and the effect is not significant.
  • probiotics have been extensively reported in recent years, and moderate intake of probiotics has no adverse effects on the host. Studies have shown that some Lactobacillus probiotics have the effect of lowering blood pressure. Therefore, the use of probiotics as dietary supplements will help improve patient health and enhance the effects of non-drug interventions.
  • the existing probiotics with antihypertensive function have a relatively single target, which mainly reduces blood pressure by inhibiting the activity of angiotensin-converting enzyme (ACE), and is not suitable for interfering with hypertension caused by other factors such as aldosterone. Therefore, based on the importance of ROS in the pathogenesis of hypertension, it is urgent to screen probiotics that can reduce the level of ROS in the blood vessel wall as probiotic strains with the potential to prevent hypertension.
  • ACE angiotensin-converting enzyme
  • the technical problem to be solved by the present invention is to provide a strain of Bifidobacterium longum capable of preventing essential hypertension.
  • the present invention provides a strain of Bifidobacterium longum CCFM752, and the Bifidobacterium longum CCFM752 is preserved in the Guangdong Provincial Microorganism Culture Collection Center, the preservation number is GDMCC No.61157, and the preservation date is 2020 On August 21, 2008, the preservation address is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou City.
  • the Bifidobacterium longum CCFM752 is derived from an infant stool sample, and the strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID NO. Bifidobacterium longum, named Bifidobacterium longum CCFM752.
  • the thalline of the Bifidobacterium longum CCFM752 is short rod-shaped; the colony is round, convex, moist, white and shiny.
  • the present invention also provides the application of the above-mentioned Bifidobacterium longum in the preparation of products for preventing and/or treating cardiovascular diseases.
  • the cardiovascular disease is hypertension.
  • the viable count of the above-mentioned Bifidobacterium longum CCFM752 is not less than 1 ⁇ 10 6 CFU/mL or 1 ⁇ 10 6 CFU/g.
  • the product comprises food or medicine.
  • the above-mentioned Bifidobacterium longum is added to the food or the above-mentioned Bifidobacterium longum is used as a fermentation strain for the fermentation of food.
  • the medicine contains the above-mentioned Bifidobacterium longum CCFM752, a pharmaceutical carrier and/or a pharmaceutical excipient.
  • the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
  • the pharmaceutical excipients include excipients and/or additives.
  • the excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculants, filter aids and/or or release blockers.
  • the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
  • the dosage form of the medicine is powder, granule, capsule, tablet, pill or oral liquid.
  • the food is a health food; or the food is a dairy product, a soy product or a fruit and vegetable product produced by using a starter containing the above-mentioned Bifidobacterium longum CCFM752; or the food is a food containing Bifidobacterium longum CCFM752 drink or snack above.
  • the preparation method of the starter is to inoculate the above-mentioned Bifidobacterium longum CCFM752 into the medium according to the inoculum amount accounting for 2-4% of the total mass of the medium, and cultivate at 37°C for 36 hours, to obtain a culture solution; centrifuge the culture solution to obtain a bacterial body; wash the bacterial body with a phosphate buffer with a pH of 7.2 to 7.4 for 2 to 4 times, and then resuspend it with a freeze-drying protective agent to obtain a resuspended solution; Freeze-drying by vacuum freezing to obtain a starter.
  • the mass ratio of the freeze-drying protective agent and the bacterial cells is 2:1.
  • the medium comprises 87.7% water, 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract based on the total mass of the medium .
  • the pH of the medium is 6.8.
  • the protective agent comprises 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
  • the present invention also provides a product for preventing and/or treating cardiovascular disease, the product containing the above-mentioned Bifidobacterium longum CCFM752.
  • the cardiovascular disease is hypertension.
  • the viable count of the above-mentioned Bifidobacterium longum CCFM752 is not less than 1 ⁇ 10 6 CFU/mL or 1 ⁇ 10 6 CFU/g.
  • the product comprises food or medicine.
  • the above-mentioned Bifidobacterium longum is an effective active ingredient in a medicine.
  • the medicine contains the above-mentioned Bifidobacterium longum CCFM752, a pharmaceutical carrier and/or a pharmaceutical excipient.
  • the drug carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
  • the pharmaceutical excipients include excipients and/or additives.
  • the excipients comprise solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, absorbents, diluents, flocculants, deflocculants, filter aids and/or or release blockers.
  • the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose and/or refined lecithin.
  • the dosage form of the medicine is powder, granule, capsule, tablet, pill or oral liquid.
  • the food is a health food; or the food is a dairy product, a soy product or a fruit and vegetable product produced by using a starter containing the above-mentioned Bifidobacterium longum CCFM752; or the food is a food containing Bifidobacterium longum CCFM752 drink or snack above.
  • the preparation method of the starter is to inoculate the above-mentioned Bifidobacterium longum CCFM752 into the medium according to the inoculum amount accounting for 2-4% of the total mass of the medium, and cultivate at 37°C for 36 hours, to obtain a culture solution; centrifuge the culture solution to obtain a bacterial body; wash the bacterial body with a phosphate buffer with a pH of 7.2 to 7.4 for 2 to 4 times, and then resuspend it with a freeze-drying protective agent to obtain a resuspended solution; Freeze-drying by vacuum freezing to obtain a starter.
  • the mass ratio of the freeze-drying protective agent and the bacterial cells is 2:1.
  • the medium comprises 87.7% water, 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone, and 0.3% yeast extract based on the total mass of the medium .
  • the pH of the medium is 6.8.
  • the protective agent comprises 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
  • the present invention also provides the application of the Bifidobacterium longum or the product containing the Bifidobacterium longum in preventing and/or treating cardiovascular diseases.
  • the use is the ingestion of the Bifidobacterium longum or a product containing the Bifidobacterium longum into the gastrointestinal tract of a mammal.
  • the present invention provides a strain of Bifidobacterium longum CCFM752, and this Bifidobacterium longum CCFM752 can relieve hypertension, and is embodied in:
  • Bifidobacterium longum (Bifidobacterium longum) CCFM752 has a huge application prospect in the preparation of products (such as food or medicine, etc.) for preventing and/or treating hypertension.
  • Bifidobacterium longum (Bifidobacterium longum) is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can Be Used in Food" issued by the Ministry of Health, and has the effect of regulating intestinal health. Therefore, the longum obtained by the present invention is Bifidobacterium longum CCFM752 is relatively healthy to humans and has no side effects.
  • Figure 1 Comparison of intracellular O 2 -levels in different groups of A7R5 cells.
  • Figure 2 Comparison of H 2 O 2 levels in different groups of A7R5 cells.
  • Figure 3 Comparison of NADPH oxidase activity in different groups of A7R5 cells.
  • Figure 4 Comparison of CAT enzyme activity in different groups of A7R5 cells.
  • Figure 5 Changes of systolic blood pressure in different groups of rats with age.
  • Figure 6 Changes of diastolic blood pressure in different groups of rats with age.
  • Figure 7 Comparison of ROS levels in thoracic aorta tissue in different groups of rats.
  • Figure 8 Comparison of NADPH oxidase activity in thoracic aorta tissue of different groups of rats.
  • Figure 9 Comparison of CAT enzyme activity in thoracic aorta tissue of different groups of rats.
  • Figure 10 Comparison of eNOS enzyme activity in thoracic aorta tissue of different groups of rats.
  • Figure 11 Comparison of serum CAT enzyme activity in different groups of rats.
  • Fig. 12 Scanning pictures of thoracic aorta stained with paraffin sections in different groups of rats.
  • Figure 13 Comparison of collagen levels in thoracic aorta tissue of different groups of rats.
  • Figure 14 Comparison of thoracic aortic vessel wall thickness indices in different groups of rats.
  • the trypsin and HBSS buffers involved in the following examples were purchased from Thermo Fisher Company, Angiotensin II was purchased from MCE Company, skim milk was purchased from Bright Dairy Co., Ltd., glucose and yeast extract were purchased from Sinopharm Chemical Reagent Co., Ltd., pancreatic Peptone was purchased from OXOID Company in the UK, DHE fluorescent probe, DCFH-DA fluorescent probe, DAPI staining solution, BCA protein concentration determination kit and protease inhibitor mixture were purchased from Biyuntian Institute of Biotechnology, Lusterjing was purchased from MCE Company in the United States , NADPH tetrasodium and Sirius scarlet were purchased from Solebao, CAT assay kit and nitric oxide synthase (NOS) typing test kit were purchased from Nanjing Jiancheng Bioengineering Institute.
  • MRS liquid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 PO 4 3H 2 O 2.6g/L, MgSO 4 ⁇ 7H 2 0 0.1g/L, MnSO 4 0.05g/L, Tween-80 1ml/L, pH 7.0.
  • MRS solid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 PO 4 ⁇ 3H 2 O 2.6g/L, MgSO 4 ⁇ 7H 2 0 0.1g/L, MnSO 4 0.05g/L, Tween-80 1ml/L, agar 20g/L, L-cysteine hydrochloride 0.05g/L, 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 Salt 42mg/L, L-Isoleucine 105mg/L, L-Leucine 105mg/L, L-Lysine Hydrochloride 146mg/L, L-Methionine 30mg/L, L-Phenylpropane Amino acid 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 4
  • the detection method of the number of viable bacteria adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection”.
  • Bifidobacterium longum was streaked on MRS solid medium and cultured at 37°C for 48h to obtain a single colony; a single colony was picked and inoculated into MRS liquid medium, cultured at 37°C for 18h for activation, and activated continuously for two generations , to obtain the activation solution; inoculate the activation solution into MRS liquid medium at an inoculum of 2% (v/v), and cultivate at 37°C for 18 hours to obtain a bacterial solution; centrifuge the bacterial solution at 8000g for 10 min, and separate the bacterial cells and Supernatant; adjust the pH of the supernatant to 7.0 with NaOH solution with a concentration of 1 mol/L, then filter and sterilize it with a 0.22 ⁇ m filter to obtain the culture supernatant of Bifidobacterium longum, and wash the cells with PBS buffer twice.
  • the bacterial suspension was obtained by resuspending in PBS buffer containing 30% sucrose, and stored in
  • samples were pretreated and stored in a -80°C refrigerator in 20% glycerol. After taking out and thawing, 0.5mL samples were mixed and added to 4.5mL to contain 0.05g/L cysteine.
  • Acidic 9g/L normal saline for gradient dilution select the appropriate gradient dilution to spread on MRS solid medium, cultivate at 37°C for 48h, pick typical colonies of Bifidobacterium longum to streak purification on MRS solid medium , pick a single colony and transfer it to MRS liquid medium (containing 0.05g/L cysteine) for enrichment, and preserve it in 30% glycerol to obtain strain CCFM752 and strain CCFM666; among them, the typical colony of Bifidobacterium longum is circular , Raised, moist, white and shiny.
  • Extract the genomes of strain CCFM752 and strain CCFM666, and amplify and sequence the 16S rDNA of strain CCFM752 and strain CCFM666 (by Suzhou Jinweizhi Biotechnology Co., Ltd., and the 16S rDNA nucleotide sequences of CCFM752 and CCFM666 are respectively as SEQ ID NO. 1. Shown in SEQ ID NO.2, the upstream primer 27F sequence for bacterial classification identification is shown in SEQ ID NO.3, and the downstream primer 1492R sequence is shown in SEQ ID NO.4), and the sequence is carried out in NCBI for nucleic acid. Sequence alignment showed that both strain CCFM752 and strain CCFM666 were Bifidobacterium longum, named Bifidobacterium longum CCFM752 and Bifidobacterium longum CCFM666, respectively.
  • Example 2 Effect of Bifidobacterium longum on intracellular O 2 -level in A7R5 cells
  • Rat thoracic aortic smooth muscle cells A7R5 were purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, and were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin base for cultivation. Cells were cultured in a cell incubator at 37°C with 5% (v/v) CO 2 in the gas phase. Cells were passaged when they had grown to 70% to 80% density.
  • FBS fetal bovine serum
  • A7R5 cells in good growth state were selected, the A7R5 cells were trypsinized, centrifuged, resuspended in DMEM medium, and the cells were counted to obtain a resuspended liquid; the resuspended liquid was inoculated into a 24-well plate at 20,000 cells/well. , 500 ⁇ L of DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin were added to the 24-well plate at 37°C in a gas phase containing 5% (v/v).
  • FBS fetal bovine serum
  • each group had 3 wells, in which 15 ⁇ L MRS liquid medium was added to each well of the blank control group and the model group, respectively, in the CCFM752 intervention group 15 ⁇ L of Bifidobacterium longum CCFM752 culture supernatant was added to each well of the group, and 15 ⁇ L of Bifidobacterium longum CCFM666 culture supernatant was added to each well of the CCFM666 intervention group, and the 24-well plate was placed in 37 °C, gas phase containing 5% (v/v) CO 2 in a cell incubator for 12 h; after 12 h of intervention, Angiotensin II was added to each well of the model group, CCFM752 intervention group and CCFM752 intervention group to a concentration of 1 ⁇ 10 -7 M, at the same time, the same volume of DMEM medium was added to each well of the blank control group as
  • Bifidobacterium longum (Bifidobacterium longum) CCFM752 culture supernatant intervention can significantly reduce the increase of O 2 - level in A7R5 cells stimulated by Angiotensin II.
  • Example 3 Effect of Bifidobacterium longum on intracellular H2O2 levels in A7R5 cells
  • Rat thoracic aortic smooth muscle cells A7R5 were purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, and were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin base for cultivation. Cells were cultured in a cell incubator at 37°C with 5% (v/v) CO 2 in the gas phase. Cells were passaged when they had grown to 70% to 80% density.
  • FBS fetal bovine serum
  • A7R5 cells in good growth state were selected, the A7R5 cells were trypsinized, centrifuged, resuspended in DMEM medium, and the cells were counted to obtain a resuspended liquid; the resuspended liquid was inoculated into a 24-well plate at 20,000 cells/well. , 500 ⁇ L of DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin were added to the 24-well plate at 37°C in a gas phase containing 5% (v/v).
  • FBS fetal bovine serum
  • each group had 3 wells, in which 15 ⁇ L MRS liquid medium was added to each well of the blank control group and the model group, respectively, in the CCFM752 intervention group 15 ⁇ L of Bifidobacterium longum CCFM752 culture supernatant was added to each well of the group, and 15 ⁇ L of Bifidobacterium longum CCFM666 culture supernatant was added to each well of the CCFM666 intervention group, and the 24-well plate was placed in 37 °C, gas phase containing 5% (v/v) CO 2 in a cell incubator for 12 h; after 12 h of intervention, Angiotensin II was added to each well of the model group, CCFM752 intervention group and CCFM752 intervention group to a concentration of 1 ⁇ 10 -7 M, at the same time, the same volume of DMEM medium was added to each well of the blank control group as
  • Bifidobacterium longum (Bifidobacterium longum) CCFM752 culture supernatant intervention can significantly reduce the level of H 2 O 2 in A7R5 cells stimulated by Angiotensin II.
  • Example 4 Effect of Bifidobacterium longum on NADPH oxidase activity in A7R5 cells
  • Rat thoracic aortic smooth muscle cells A7R5 were purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, and were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin base for cultivation. Cells were cultured in a cell incubator at 37°C with 5% (v/v) CO 2 in the gas phase. Cells were passaged when they had grown to 70% to 80% density.
  • FBS fetal bovine serum
  • A7R5 cells in good growth state were selected, the A7R5 cells were trypsinized, centrifuged, resuspended in DMEM medium, and the cells were counted to obtain a resuspended liquid; the resuspended liquid was inoculated into 6 cm cells at 2 ⁇ 10 5 cells/well After the culture dish, add 4 mL of DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin to the cell culture dish.
  • FBS fetal bovine serum
  • the cells were left standing in the cell incubator for 24 hours; after standing for 24 hours, the cells were divided into blank control group (Control), model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752 ) and Bifidobacterium longum CCFM666 intervention group (CCFM666), 3 dishes in each group, in which 120 ⁇ L of MRS liquid medium was added to each dish of the blank control group and the model group, respectively, in the CCFM752 intervention group 120 ⁇ L of Bifidobacterium longum CCFM752 culture supernatant was added to each well, and 120 ⁇ L of Bifidobacterium longum CCFM666 culture supernatant was added to each well of the CCFM666 intervention group, and the cell culture dishes were kept at 37°C.
  • Each cell homogenate sample was set up for 5 parallel tests. 20 ⁇ L of cell homogenate was added to each well of the well plate. Immediately after the addition of the cell homogenate, a microplate reader was used for chemiluminescence measurement. The chemiluminescence intensity was detected between 30 and 120s after the start of the reaction. The ratio of the homogenate protein concentration was used to characterize the relative NADPH oxidase activity of the cell homogenate, and the control group was used as the comparative processing data, and the results are shown in Figure 3.
  • Bifidobacterium longum CCFM752 culture supernatant intervention can significantly inhibit the activity of NADPH oxidase in A7R5 cells stimulated by Angiotensin II and reduce the production of ROS.
  • Example 5 Effect of Bifidobacterium longum on CAT enzyme activity in A7R5 cells
  • Rat thoracic aortic smooth muscle cells A7R5 were purchased from the cell bank of the Type Culture Collection, Chinese Academy of Sciences, and were cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin base for cultivation. Cells were cultured in a cell incubator at 37°C with 5% (v/v) CO 2 in the gas phase. Cells were passaged when they had grown to 70% to 80% density.
  • FBS fetal bovine serum
  • A7R5 cells in good growth state were selected, the A7R5 cells were trypsinized, centrifuged, resuspended in DMEM medium, and the cells were counted to obtain a resuspended liquid; the resuspended liquid was inoculated into 6 cm cells at 2 ⁇ 10 5 cells/well After the culture dish, add 4 mL of DMEM medium containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin to the cell culture dish.
  • FBS fetal bovine serum
  • the cells were left standing in the cell incubator for 24 hours; after standing for 24 hours, the cells were divided into blank control group (Control), model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752 ) and Bifidobacterium longum CCFM666 intervention group (CCFM666), 3 dishes in each group, in which 120 ⁇ L of MRS liquid medium was added to each dish of the blank control group and the model group, respectively, in the CCFM752 intervention group 120 ⁇ L of Bifidobacterium longum CCFM752 culture supernatant was added to each well, and 120 ⁇ L of Bifidobacterium longum CCFM666 culture supernatant was added to each well of the CCFM666 intervention group, and the cell culture dishes were kept at 37°C.
  • Bifidobacterium longum CCFM752 culture supernatant intervention can increase the CAT enzyme activity in A7R5 cells to about 1.9 times that of the blank control group (p ⁇ 0.01), while Bifidobacterium longum CCFM666 Culture supernatant intervention failed to significantly change the activity of CAT enzyme in A7R5 cells.
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination.
  • the tail artery systolic and diastolic blood pressure levels were measured once a week from the second week of the experiment (5 weeks of age), and the results are shown in Figures 5 and 6 .
  • FIG. 5 shows that Bifidobacterium longum (Bifidobacterium longum) CCFM752 live bacteria intervention can significantly reduce the systolic blood pressure of SHR and delay the formation of hypertension.
  • the systolic blood pressure is reduced by 9.65%, while Bifidobacterium longum ) CCFM666 can only slightly reduce the level of systolic blood pressure, failed to delay the formation of essential hypertension.
  • FIG. 6 shows that Bifidobacterium longum CCFM752 live bacteria intervention can significantly reduce the diastolic blood pressure of SHR and delay the formation of hypertension.
  • CCFM666 failed to significantly reduce diastolic blood pressure nor to delay the development of essential hypertension.
  • Bifidobacterium longum CCFM752 can effectively prevent the occurrence and development of essential hypertension.
  • Example 7 Effect of Bifidobacterium longum on ROS levels in SHR aortic vessel wall
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta was collected.
  • Aortic tissue was collected and dehydrated in PBS (pH 7.4) containing 30% (w/w) sucrose for 1-2 hours. After dehydration, OCT embedding gel was used for tissue embedding. After embedding, it was placed in a -80°C refrigerator. freezing. The frozen tissue was taken out and placed in a cryostat and cut into 10 ⁇ m thick sections. The sections were stained in a physiological saline solution containing 5 ⁇ M DHE for 30 min, and then placed in a physiological saline solution containing 300 ⁇ M DAPI for 30 min, and then the sections were removed and washed. Photographs were taken by an inverted fluorescence microscope within 24 h after the preparation of the sections. The ROS level in aortic tissue was calculated by the ratio of ethidium fluorescence intensity/DAPI fluorescence intensity, and the normal blood pressure control group was used as the comparison data. The results are shown in Figure 7.
  • Bifidobacterium longum CCFM752 has antioxidant effect on SHR and helps to reduce the occurrence of hypertension.
  • Example 8 Effect of Bifidobacterium longum on NADPH oxidase activity in SHR aortic vessel wall
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta tissue was collected.
  • the collected aortic tissue was cut into rings with a thickness of 3-4 mm, placed in a physiological saline solution, and temporarily stored in an ice bath. Before the experiment, they were placed in a constant temperature incubator at 37 °C for 30 min. The aortic ring was placed in a 96-well plate, and 200 ⁇ L of a physiological saline solution containing 100 ⁇ M NADPH and 5 ⁇ M lucigenin was added, and immediately placed in a microplate reader for determination. Read the chemiluminescence intensity between 30-200 s after the start of the reaction.
  • the aortic ring is taken out, dried, and the dry weight of the tissue is measured, and the relative NADPH oxidase activity of the tissue is expressed by reading the total chemiluminescence intensity/tissue dry weight.
  • the normal blood pressure control group is used as the comparison processing data, and the results are shown in the figure. 8 shown.
  • Bifidobacterium longum CCFM752 can inhibit the production of ROS in the aortic tissue of SHR and effectively reduce the risk of hypertension and vascular remodeling.
  • Example 9 Effect of Bifidobacterium longum on CAT enzyme activity in SHR aortic vessel wall
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta tissue was collected.
  • the aortic tissue was weighed, and PBS (pH 7.4, 2% protease inhibitor and phosphatase inhibitor) was added at a mass ratio of 1:9.
  • PBS pH 7.4, 2% protease inhibitor and phosphatase inhibitor
  • the aortic tissue was cut into pieces, added with grinding beads, and crushed in a high-throughput tissue grinder (crushing conditions: shaking frequency 65 Hz, shaking duration 15 s/time, continuous breaking 8-10 times.). After the tissue was disrupted, centrifuge at 3000 ⁇ g for 15 min at 4 °C, take the supernatant and aliquot and store it in a -80 °C refrigerator for later use.
  • Aortic tissue homogenate protein concentration was determined by BCA protein concentration assay kit, CAT activity was determined by Nanjing Jiancheng catalase assay kit, aortic tissue CAT enzyme activity was determined by dividing homogenate CAT activity by homogenate protein concentration The results are shown in Figure 9.
  • Bifidobacterium longum CCFM752 can enhance the CAT activity of SHR aortic tissue, enhance the antioxidant capacity, and help to remove ROS from the vessel wall.
  • Example 10 Effect of Bifidobacterium longum on eNOS activity of SHR aortic vessel wall
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta tissue was collected.
  • the aortic tissue was weighed, and PBS (pH 7.4, 2% protease inhibitor and phosphatase inhibitor) was added at a mass ratio of 1:9.
  • the aortic tissue was minced, added to grinding beads, and disrupted in a high-throughput tissue grinder. Crushing conditions: The module is pre-cooled at -20°C, the oscillation frequency is 65Hz, the oscillation duration is 15s/time, and the crushing is performed continuously for 8-10 times. After the tissue was broken, centrifuge at 3000 ⁇ g for 15 min at 4 °C, take the supernatant and aliquot and store it in a -80 °C refrigerator for future use.
  • Aortic tissue homogenate protein concentration was determined by BCA protein concentration assay kit, and eNOS activity was determined by Nanjing Jiancheng nitric oxide synthase typing kit. The aortic eNOS activity was expressed by dividing the homogenate eNOS activity by the homogenate protein concentration, and the results are shown in Figure 10.
  • Figure 10 shows that Bifidobacterium longum (Bifidobacterium longum) CCFM752 live bacteria intervention can significantly improve the eNOS activity of SHR aorta, eNOS activity increased by 75.25% compared with the hypertension model group, while Bifidobacterium longum (Bifidobacterium longum) CCFM666 failed to improve eNOS vitality.
  • Bifidobacterium longum CCFM752 can increase the level of NO synthesis in SHR aortic endothelial, effectively promote vasodilation and improve vascular function.
  • Example 11 Effect of Bifidobacterium longum on SHR serum CAT enzyme activity
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water.
  • Serum CAT activity was measured using a catalase assay kit built in Nanjing, and the results are shown in Figure 11.
  • Bifidobacterium longum (Bifidobacterium longum) CCFM752 live bacteria intervention can significantly increase the serum CAT activity of SHR, and the CAT activity is increased by 56.90% compared with the hypertension model group, while Bifidobacterium longum (Bifidobacterium longum) CCFM666 fails to increase serum CAT activity.
  • CAT Vitality Bifidobacterium longum (Bifidobacterium longum) CCFM752 live bacteria intervention can significantly increase the serum CAT activity of SHR, and the CAT activity is increased by 56.90% compared with the hypertension model group, while Bifidobacterium longum (Bifidobacterium longum) CCFM666 fails to increase serum CAT activity.
  • CAT Vitality Bifidobacterium longum (Bifidobacterium longum)
  • Bifidobacterium longum CCFM752 can enhance the antioxidant capacity of SHR system and effectively prevent oxidative stress and oxidative damage.
  • Example 12 Effects of Bifidobacterium longum on collagen levels of SHR aortic vessel wall
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta was collected.
  • aorta of about 0.5 cm length was cut, immediately fixed in PBS solution containing 4% paraformaldehyde, then dehydrated, embedded in paraffin, and made into 10 ⁇ m thick sections.
  • the tissue sections were placed in a Sirius red staining solution (the concentration of Sirius red was 0.1% (w/v), dissolved in a saturated aqueous picric acid solution) for staining. Sirius red stained sections were scanned by a digital slide scanner (as shown in Figure 12).
  • the relative collagen level of the aortic vessel wall was calculated using Image-Pro Plus software, and the normal blood pressure control group was used as the comparison processing data. The results are shown in Figure 13.
  • Bifidobacterium longum CCFM752 can effectively prevent hypertension-related vascular fibrosis and help maintain vascular elasticity.
  • Example 13 Effect of Bifidobacterium longum on SHR aortic vessel wall thickness index
  • the SHRs were randomly divided into 3 groups: hypertension model group (Model), Bifidobacterium longum CCFM752 intervention group (CCFM752) and Bifidobacterium longum CCFM666 intervention group (CCFM666). 7. All WKY were normotensive control group (Control), a total of 7 animals.
  • the experimental period was 13 weeks in total. The first week was the adaptation period, and the second week began to perform gavage intervention until the end of the experiment.
  • the CCFM752 intervention group was given a daily gavage of Bifidobacterium longum CCFM752 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; the CCFM666 intervention group was given a daily gavage of Bifidobacterium longum CCFM666 bacterial suspension at a dose of 1.0 ⁇ 10 9 CFU; normotensive control group and hypertension model group were not intervened with any viable bacteria or drugs, and the same volume of PBS buffer was administered by gavage every day as control. All groups had free access to food and water. After the experiment, all the rats were anesthetized and sacrificed by exsanguination, and the thoracic aorta was collected.
  • aorta of about 0.5 cm length was cut, immediately fixed in PBS solution containing 4% paraformaldehyde, then dehydrated, embedded in paraffin, and made into 10 ⁇ m thick sections.
  • the tissue sections were placed in a Sirius red staining solution (the concentration of Sirius red was 0.1% (w/v), dissolved in a saturated aqueous picric acid solution) for staining. Sirius red stained sections were scanned by a digital slide scanner (as shown in Figure 12).
  • the aortic vessel wall thickness and lumen circumference were measured using Image-Pro Plus software, and the aortic vessel wall thickness index was expressed as the ratio of vessel wall thickness to lumen circumference. The results are shown in Figure 14.
  • Bifidobacterium longum CCFM752 can effectively prevent hypertension-related vascular wall thickening and reduce the risk of vascular stenosis.
  • Bifidobacterium longum CCFM752 can be used to prepare solid beverages.
  • the specific preparation process of solid beverages is as follows:
  • Bifidobacterium longum CCFM752 was inoculated into the medium according to the inoculum amount of 2-4% of the total mass of the medium, and cultured at 37°C for 36 hours to obtain a culture solution; the culture solution was centrifuged to obtain cells; the cells were pH Wash the phosphate buffer solution of 7.2 to 7.4 for 2 to 4 times and then resuspend it with a freeze-drying protective agent to obtain a resuspension; freeze-dry the resuspension by a vacuum freezing method to obtain bacterial powder; freeze-dried protective agent and bacteria
  • the mass ratio of the medium is 2:1; the medium contains 87.7% of the total mass of the medium, 10% of enzymatically hydrolyzed skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract, and the pH is 6.8;
  • the protective agent contains 100g/L skimmed milk powder, 150g/L trehalose and 10g/L L-sodium glutamate.
  • the bacterial powder containing 10 10 CFU of Bifidobacterium longum CCFM752 was mixed with 1 g of maltodextrin to obtain a solid beverage containing Bifidobacterium longum CCFM752.

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Abstract

一株长双歧杆菌(Bifidobacterium longum) CCFM752，其保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61157，保藏日期为2020年08月21日。还涉及该长双歧杆菌在制备预防和/或治疗心血管疾病的产品中的应用。该长双岐杆菌预防原发性高血压具体体现在：(1)显著降低AngiotensinⅡ刺激后A7R5细胞内O 2 -水平、H 2O 2水平和NADPH氧化酶活力；(2)显著提升A7R5细胞内、SHR主动脉和SHR血清的CAT酶活力；(3)显著降低SHR血压水平；(4)显著降低SHR主动脉血管壁ROS水平、NADPH氧化酶活力、胶原质水平和厚度指数；(5)显著提升SHR主动脉血管壁eNOS活力。

Description

预防和/或治疗原发性高血压的长双歧杆菌 技术领域
本发明涉及预防和/或治疗原发性高血压的长双歧杆菌，属于微生物技术领域以及医药技术领域。
背景技术
高血压是发病人群最为广泛的慢性病之一，为社会医疗体系带来了沉重的负担。目前，全球已有超过30％的人患有高血压，且近年来高血压患病人数仍旧呈逐年递增趋势。在高血压患者中，超过90％属原发性高血压。原发性高血压发病周期长且难以治愈，患者需依赖药物控制血压，易形成药物依赖性。此外，高血压的发生将大大增加动脉粥样硬化、卒中、心衰等其他心血管疾病的发生风险，严重威胁患者的生命健康。
原发性高血压的发生主要依赖于环境因素及生活习惯，其成因复杂，具体机制至今仍旧难以解释。近年来，研究表明活性氧(ROS)是促进高血压等心血管疾病的发生发展的重要因素。机体最核心的血压调控系统为肾素-血管紧张素-醛固酮系统(RAAS)，其中包含血管紧张素Ⅱ(AngⅡ)与醛固酮两种重要的升血压激素。AngⅡ和醛固酮能够激活血管壁NADPH氧化酶，产生O 2 ·-、H 2O 2等ROS使血压升高。在原发性高血压患者及动物模型体内均存在RAAS失调的现象，使得体内AngⅡ和/或醛固酮水平升高。当AngⅡ、醛固酮等刺激因子持续刺激血管壁将产生过多的ROS，使血管持续收缩，引起高血压，而持续产生的ROS还将激活与细胞增殖相关丝裂原活化蛋白激酶(MAPK)通路以及胶原质合成相关的Smad通路与TGF-β通路等，造成血管壁增厚与纤维化，引起血管壁结构病变与血管功能异常。此外，血管壁ROS还可能激活与炎症相关的IL-1β、IL-17、TNF-α等免疫通路，造成血管炎症，加速高血压与动脉粥样硬化的形成。因此，清除血管壁内过多的ROS将有助于预防高血压等心血管疾病的发生发展。
目前，临床干预高血压的方法主要为药物干预。不同种类的降压药物均有不同程度的毒副作用，如引起电解质紊乱、损害肾脏功能及引起血管水肿等，长期服用将损害患者健康。辅助干预方法主要为膳食干预。膳食干预虽然有利于改善患者健康状况，但见效慢且效果不显著。
近年来益生菌的健康功效已被大量报道，并且，适量摄入益生菌也不会对宿主产生副作用。研究表明，部分乳杆菌属的益生菌具有降低血压的功效。因此，将益生菌作为膳食补充剂将有助于改善患者健康状况，增强非药物干预的效果。但是，现有的具有抗高血压功能的益生菌作用靶点较为单一，主要通过抑制血管紧张素转换酶(ACE)活性而降低血压，并不适用于干预醛固酮等其他因子引起的高血压。因此，基于ROS在高血压发病通路中的重要性，急需筛选出能够降低血管壁内ROS水平的益生菌，作为具有预防高血压潜力的益生菌菌株。
发明内容
[技术问题]
本发明要解决的技术问题是提供一株能够预防原发性高血压的长双歧杆菌(Bifidobacterium longum)。
[技术方案]
为解决上述问题，本发明提供了一株长双歧杆菌(Bifidobacterium longum)CCFM752，所述长双歧杆菌CCFM752保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61157，保藏日期为2020年08月21日，保藏地址为广州市先烈中路100号大院59号楼5楼。
所述长双歧杆菌CCFM752来源于婴儿粪便样本，该菌株经测序分析，其16S rDNA序列如SEQ ID NO.1所示，将测序得到的序列在GeneBank中进行核酸序列比对，结果显示菌株为长双歧杆菌，命名为长双歧杆菌CCFM752。
所述长双歧杆菌CCFM752的菌体呈短杆状；菌落呈圆形、凸起、湿润、白色有光泽。
本发明还提供了上述长双歧杆菌在制备预防和/或治疗心血管疾病的产品中的应用。
本发明的一种实施方式中，所述心血管疾病为高血压。
本发明的一种实施方式中，所述产品中，上述长双歧杆菌CCFM752的活菌数不低于1×10 6CFU/mL或1×10 6CFU/g。
本发明的一种实施方式中，所述产品包含食品或药品。
本发明的一种实施方式中，所述产品中，将上述长双歧杆菌添加至食品中或将上述长双歧杆菌作为发 酵菌株用于食品的发酵。
本发明的一种实施方式中，所述药品含有上述长双歧杆菌CCFM752、药物载体和/或药用辅料。
本发明的一种实施方式中，所述药物载体包含微囊、微球、纳米粒和/或脂质体。
本发明的一种实施方式中，所述药用辅料包含赋形剂和/或附加剂。
本发明的一种实施方式中，所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
本发明的一种实施方式中，所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
本发明的一种实施方式中，所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
本发明的一种实施方式中，所述食品为保健食品；或所述食品为使用含有上述长双歧杆菌CCFM752的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品为含有上述长双歧杆菌CCFM752的饮料或零食。
本发明的一种实施方式中，所述发酵剂的制备方法为将上述长双歧杆菌CCFM752按照占培养基总质量2～4％的接种量接种到培养基中，于37℃下培养36h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2～7.4的磷酸盐缓冲液清洗2～4次后用冻干保护剂重悬，得到重悬液；将重悬液用真空冷冻法进行冻干，得到发酵剂。
本发明的一种实施方式中，所述冻干保护剂和菌体的质量比为2:1。
在本发明的一种实施方式中，所述培养基包含占培养基总质量87.7％的水、10％的酶水解脱脂乳、0.5％的葡萄糖、1.5％的胰蛋白胨以及0.3％的酵母浸膏。
在本发明的一种实施方式中，所述培养基的pH为6.8。
在本发明的一种实施方式中，所述保护剂包含100g/L脱脂奶粉、150g/L海藻糖以及10g/L L-谷氨酸钠。
本发明还提供了一种用于预防和/或治疗心血管疾病的产品，所述产品含有上述长双歧杆菌CCFM752。
本发明的一种实施方式中，所述心血管疾病为高血压。
本发明的一种实施方式中，所述产品中，上述长双歧杆菌CCFM752的活菌数不低于1×10 6CFU/mL或1×10 6CFU/g。
本发明的一种实施方式中，所述产品包含食品或药品。
本发明的一种实施方式中，所述产品中，上述长双歧杆菌为药品中的有效活性成分。
本发明的一种实施方式中，所述药品含有上述长双歧杆菌CCFM752、药物载体和/或药用辅料。
本发明的一种实施方式中，所述药物载体包含微囊、微球、纳米粒和/或脂质体。
本发明的一种实施方式中，所述药用辅料包含赋形剂和/或附加剂。
本发明的一种实施方式中，所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂。
本发明的一种实施方式中，所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
本发明的一种实施方式中，所述药品的剂型为粉剂、颗粒剂、胶囊剂、片剂、丸剂或口服液。
本发明的一种实施方式中，所述食品为保健食品；或所述食品为使用含有上述长双歧杆菌CCFM752的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品为含有上述长双歧杆菌CCFM752的饮料或零食。
本发明的一种实施方式中，所述发酵剂的制备方法为将上述长双歧杆菌CCFM752按照占培养基总质量2～4％的接种量接种到培养基中，于37℃下培养36h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2～7.4的磷酸盐缓冲液清洗2～4次后用冻干保护剂重悬，得到重悬液；将重悬液用真空冷冻法进行冻干，得到发酵剂。
本发明的一种实施方式中，所述冻干保护剂和菌体的质量比为2:1。
在本发明的一种实施方式中，所述培养基包含占培养基总质量87.7％的水、10％的酶水解脱脂乳、0.5％的葡萄糖、1.5％的胰蛋白胨以及0.3％的酵母浸膏。
在本发明的一种实施方式中，所述培养基的pH为6.8。
在本发明的一种实施方式中，所述保护剂包含100g/L脱脂奶粉、150g/L海藻糖以及10g/L L-谷氨酸 钠。
本发明还提供所述长双歧杆菌或含所述长双歧杆菌的产品在预防和/或治疗心血管疾病中的应用。
在一种实施方式中，所述应用是将所述长双歧杆菌或含所述长双歧杆菌的产品摄入哺乳动物胃肠道中。
有益效果：
1、本发明提供了一株长双歧杆菌(Bifidobacterium longum)CCFM752，此长双歧杆菌CCFM752能够缓解高血压，具体体现在：
(1)显著降低Angiotensin Ⅱ刺激后A7R5细胞内O 2 -水平；
(2)显著降低Angiotensin Ⅱ刺激后A7R5细胞内H 2O 2水平；
(3)显著抑制Angiotensin Ⅱ刺激后A7R5细胞内NADPH氧化酶活力；
(4)显著提升A7R5细胞内过氧化氢酶(CAT酶)活力；
(5)显著降低原发性高血压大鼠(SHR)的血压水平；
(6)显著降低SHR主动脉血管壁ROS水平；
(7)显著降低SHR主动脉血管壁NADPH氧化酶活力；
(8)显著提升SHR主动脉血管壁CAT酶活力；
(9)显著提升SHR主动脉血管壁内皮NO合酶(eNOS)活力；
(10)显著提升SHR血清CAT酶活力；
(11)显著降低SHR主动脉血管壁胶原质水平；
(12)显著降低SHR主动脉血管壁厚度指数；
可见，长双歧杆菌(Bifidobacterium longum)CCFM752在制备预防和/或治疗高血压的产品(如食品或药品等)中具有巨大的应用前景。
2、长双歧杆菌(Bifidobacterium longum)是益生菌的一种，目前已被纳入卫生部下发的《可用于食品的菌种名单》，具有调节肠道健康的功效，因此，本发明得到的长双歧杆菌(Bifidobacterium longum)CCFM752对人体而言，相对健康，无副作用。
生物材料保藏
一株长双歧杆菌(Bifidobacterium longum)CCFM752，分类学命名为Bifidobacterium longum，已于2020年08月21日保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61157，保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1：不同组别A7R5细胞内O 2 -水平对比。
图2：不同组别A7R5细胞内H 2O 2水平对比。
图3：不同组别A7R5细胞内NADPH氧化酶活力对比。
图4：不同组别A7R5细胞内CAT酶活力对比。
图5：不同组别大鼠收缩压随周龄的变化。
图6：不同组别大鼠舒张压随周龄的变化。
图7：不同组别大鼠胸腔主动脉组织ROS水平对比。
图8：不同组别大鼠胸腔主动脉组织NADPH氧化酶活力对比。
图9：不同组别大鼠胸腔主动脉组织CAT酶活力对比。
图10：不同组别大鼠胸腔主动脉组织eNOS酶活力对比。
图11：不同组别大鼠血清CAT酶活力对比。
图12：不同组别大鼠胸腔主动脉天狼猩红染色石蜡切片扫描图。
图13：不同组别大鼠胸腔主动脉组织胶原质水平对比。
图14：不同组别大鼠胸腔主动脉血管壁厚度指数对比。
其中，“##”表示与Control组相比具有显著性差异(p<0.01)，“*”表示与Model组相比具有显著性差异(p<0.05)，“**”表示与Model组相比具有显著性差异(p<0.01)。
具体实施方式
具体实施方式
下述实例中涉及的胰蛋白酶和HBSS缓冲液购自Thermo Fisher公司，Angiotensin Ⅱ购自MCE公司，脱脂乳购自光明乳业股份有限公司，葡萄糖与酵母浸膏购自国药集团化学试剂有限公司，胰蛋白胨购自英国OXOID公司，DHE荧光探针、DCFH-DA荧光探针、DAPI染色液、BCA蛋白浓度测定试剂盒与蛋白酶抑制剂混合物购自碧云天生物技术研究所，光泽精购自美国MCE公司，NADPH四钠与天狼猩红够自索莱宝，CAT测定试剂盒、一氧化氮合成酶(NOS)分型测试盒购自南京建成生物工程研究所。
下述实施例中涉及的培养基如下：
MRS液体培养基：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K 2PO 4·3H 2O 2.6g/L、MgSO 4·7H 20 0.1g/L、MnSO 4 0.05g/L、吐温-80 1ml/L，pH 7.0。
MRS固体培养基：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K 2PO 4·3H 2O 2.6g/L、MgSO 4·7H 20 0.1g/L、MnSO 4 0.05g/L、吐温-80 1ml/L、琼脂20g/L、L-半胱氨酸盐酸盐0.05g/L，pH 7.0。
DMEM培养基：甘氨酸30mg/L、L-精氨酸盐酸盐84mg/L、L-半胱氨酸盐酸盐63mg/L、L-谷氨酰胺584mg/L、L-组氨酸盐酸盐42mg/L、L-异亮氨酸105mg/L、L-亮氨酸105mg/L、L-赖氨酸盐酸盐146mg/L、L-甲硫氨酸30mg/L、L-苯丙氨酸66mg/L、L-丝氨酸42mg/L、L-苏氨酸95mg/L、L-色氨酸16mg/L、L-酪氨酸二钠104mg/L、L-缬氨酸94mg/L、氯化胆碱4mg/L、D-泛酸钙4mg/L、叶酸4mg/L、烟酰胺4mg/L、盐酸吡哆醇4mg/L、核黄素0.4mg/L、盐酸硫胺素4mg/L、i-肌醇7.2mg/L、CaCl 2 200mg/L、Fe(NO 3) 3·9H 2O 0.1mg/L、MgSO 4 97.67mg/L、KCl 400mg/L、NaHCO 3 3700mg/L、NaCl 6400mg/L、NaH 2PO 4·H2O 125mg/L、D-葡萄糖4500mg/L、酚红15mg/L。
下述实施例中涉及的检测方法如下：
活菌数的检测方法：采用国标《GB 4789.35-2016 食品安全国家标准 食品微生物学检测 乳酸菌检测》。
下述实施例中涉及的长双歧杆菌培养上清与菌悬液的制备方法如下：
将长双歧杆菌划线于MRS固体培养基上，37℃条件下培养48h，得到单菌落；挑取单菌落接种于MRS液体培养基中，37℃条件下培养18h进行活化，连续活化两代，得到活化液；将活化液按2％(v/v)的接种量接种于MRS液体培养基中，37℃条件下培养18h，得到菌液；将菌液经8000g离心10min，分离菌体与上清；将上清用浓度为1mol/L的NaOH溶液调pH值至7.0后经0.22μm滤膜过滤除菌，得到长双歧杆菌培养上清，将菌体用PBS缓冲液清洗两遍后用含30％蔗糖的PBS缓冲液重悬得到菌悬液，保存于-80℃冰箱备用。
实施例1：长双歧杆菌的获取
具体步骤如下：
1、筛选
以婴儿粪便为样本，将样本经预处理后，在20％甘油中保存于-80℃冰箱，取出解冻后，混匀样本吸取0.5mL样本加到4.5mL，以含有0.05g/L半胱氨酸的9g/L生理盐水进行梯度稀释，选择合适的梯度稀释液涂布在MRS固体培养基上，于37℃培养48h，挑取长双歧杆菌的典型菌落至MRS固体培养基上划线纯化，挑取单菌落转接至MRS液体培养基(含有0.05g/L半胱氨酸)增菌，30％甘油保藏，得到菌株CCFM752和菌株CCFM666；其中，长双歧杆菌的典型菌落呈圆形、凸起、湿润、白色有光泽。
2、鉴定
提取菌株CCFM752和菌株CCFM666的基因组，将菌株CCFM752和菌株CCFM666的16S rDNA进行扩增和测序(由苏州金唯智生物科技有限公司进行，CCFM752、CCFM666的16S rDNA核苷酸测定序列分别如SEQ ID NO.1、SEQ ID NO.2所示，菌种鉴定的上游引物27F序列如SEQ ID NO.3所示，下游引物1492R序列如SEQ ID NO.4所示)，将该序列在NCBI中进行核酸序列比对，结果显示菌株CCFM752和菌株CCFM666均为长双歧杆菌，分别命名为长双歧杆菌(Bifidobacterium longum)CCFM752和长双歧杆菌(Bifidobacterium longum)CCFM666。
实施例2：长双歧杆菌对A7R5细胞内O 2 -水平的影响
具体步骤如下：
大鼠胸主动脉平滑肌细胞A7R5购自中国科学院典型培养物保藏委员会细胞库，以含有10％(v/v)胎 牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基进行培养。细胞培养于37℃、气相含5％(v/v)CO 2的细胞培养箱中。细胞生长至70％～80％密度时进行传代。
选取生长状态良好的A7R5细胞，将A7R5细胞经胰蛋白酶消化后离心、用DMEM培养基重悬，进行细胞计数，得到重悬液；将重悬液按照20000细胞/孔接种至24孔板中后，在24孔板中继续添加500μL含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基，于37℃、气相含5％(v/v)CO 2的细胞培养箱中培养48h；培养48h后，将24孔板中的含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基替换为500μL含有0.1％(v/v)胎牛血清(FBS)的DMEM培养基，并将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置24h；静置24h后，以24孔板中每个孔内细胞为单位，将细胞分为空白对照组(Control)、模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组3个孔，其中，在空白对照组与模型组的每个孔中分别加入15μLMRS液体培养基，在CCFM752干预组的每个孔中加入15μL长双歧杆菌(Bifidobacterium longum)CCFM752培养上清，在CCFM666干预组的每个孔中加入15μL长双歧杆菌(Bifidobacterium longum)CCFM666培养上清，将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中干预12h；干预12h后，在模型组、CCFM752干预组以及CCFM752干预组的每个孔内分别添加Angiotensin Ⅱ至浓度为1×10 -7M，同时，在空白对照组的每个孔内添加同样体积的DMEM培养基作为对照，将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置4h；静置4h后，将24孔板从细胞培养箱中取出，吸去每孔内液体并在每孔中加入500μL含10μM DHE荧光探针的DMEM培养基，将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中孵育30min；孵育30min后，将24孔板从细胞培养箱中取出，吸去每孔内液体并用HBSS缓冲液清洗每孔内细胞2遍；清洗结束后，在24孔板的每孔加入500μLHBSS缓冲液，通过倒置荧光显微镜进行观察，采用绿色波段激发光激发细胞产生红色荧光，选择合适的视野进行拍摄；采用Image pro plus计算图片荧光密度以表征胞内O 2 -水平，以空白对照组为对比处理数据，结果如图1所示。
由图1可知，用1×10 -7MAngiotensin Ⅱ刺激4h后，模型组A7R5细胞内O 2 ·-水平显著升高至空白对照组的1.6倍(p<0.01)，CCFM752干预组A7R5细胞内O 2 ·-水平较模型组降低27％(p<0.01)，而CCFM666干预组A7R5细胞内O 2 -水平较模型组未发生明显变化。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752培养上清干预能够显著降低Angiotensin Ⅱ刺激后A7R5细胞内O 2 -水平的上升。
实施例3：长双歧杆菌对A7R5细胞内H 2O 2水平的影响
具体步骤如下：
大鼠胸主动脉平滑肌细胞A7R5购自中国科学院典型培养物保藏委员会细胞库，以含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基进行培养。细胞培养于37℃、气相含5％(v/v)CO 2的细胞培养箱中。细胞生长至70％～80％密度时进行传代。
选取生长状态良好的A7R5细胞，将A7R5细胞经胰蛋白酶消化后离心、用DMEM培养基重悬，进行细胞计数，得到重悬液；将重悬液按照20000细胞/孔接种至24孔板中后，在24孔板中继续添加500μL含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基，于37℃、气相含5％(v/v)CO 2的细胞培养箱中培养48h；培养48h后，将24孔板中的含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基替换为500μL含有0.1％(v/v)胎牛血清(FBS)的DMEM培养基，并将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置24h；静置24h后，以24孔板中每个孔内细胞为单位，将细胞分为空白对照组(Control)、模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组3个孔，其中，在空白对照组与模型组的每个孔中分别加入15μLMRS液体培养基，在CCFM752干预组的每个孔中加入15μL长双歧杆菌(Bifidobacterium longum)CCFM752培养上清，在CCFM666干预组的每个孔中加入15μL长双歧杆菌(Bifidobacterium longum)CCFM666培养上清，将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中干预12h；干预12h后，在模型组、CCFM752干预组以及CCFM752干预组的每个孔内分别添加Angiotensin Ⅱ至浓度为1×10 -7M，同时，在空白对照组的每个孔内添加同样体积的DMEM培养基作为对照，将24孔板于37℃、气相含5％(v/v)CO 2 的细胞培养箱中静置3h；静置3h后，将24孔板从细胞培养箱中取出，吸去每孔内液体并在每孔中加入500μL含8μM DCFH-DA荧光探针的DMEM培养基，将24孔板于37℃、气相含5％(v/v)CO 2的细胞培养箱中孵育20min；孵育20min后，将24孔板从细胞培养箱中取出，吸去每孔内液体并用HBSS缓冲液清洗每孔内细胞2遍；清洗结束后，在24孔板的每孔加入500μL HBSS缓冲液，通过倒置荧光显微镜进行观察，采用蓝色波段激发光激发细胞产生绿色荧光，选择合适的视野进行拍摄；采用Image pro plus计算图片荧光密度以表征胞内H 2O 2水平，以空白对照组为对比处理数据，结果如图2所示。
由图2可知，用1×10 -7MAngiotensin Ⅱ刺激3h后，模型组A7R5细胞内H 2O 2水平显著升高至空白对照组的1.8倍(p<0.001)，CCFM752干预组A7R5细胞内H 2O 2水平较模型组降低30％(p<0.01)，而CCFM666干预组A7R5细胞内H 2O 2水平较模型组未发生明显变化。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752培养上清干预能够显著降低Angiotensin Ⅱ刺激后A7R5细胞内H 2O 2水平的上升。
实施例4：长双歧杆菌对A7R5细胞内NADPH氧化酶活力的影响
具体步骤如下：
大鼠胸主动脉平滑肌细胞A7R5购自中国科学院典型培养物保藏委员会细胞库，以含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基进行培养。细胞培养于37℃、气相含5％(v/v)CO 2的细胞培养箱中。细胞生长至70％～80％密度时进行传代。
选取生长状态良好的A7R5细胞，将A7R5细胞经胰蛋白酶消化后离心、用DMEM培养基重悬，进行细胞计数，得到重悬液；将重悬液按照2×10 5细胞/孔接种至6cm细胞培养皿中后，在细胞培养皿中继续添加4mL含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基，于37℃、气相含5％(v/v)CO 2的细胞培养箱中培养48h；培养48h后，将细胞培养皿中的含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基替换为4mL含有0.1％(v/v)胎牛血清(FBS)的DMEM培养基，并将细胞培养皿于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置24h；静置24h后，以每皿细胞为单位，将细胞分为空白对照组(Control)、模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组3个皿，其中，在空白对照组与模型组的每个皿中分别加入120μL MRS液体培养基，在CCFM752干预组的每个孔中加入120μL长双歧杆菌(Bifidobacterium longum)CCFM752培养上清，在CCFM666干预组的每个孔中加入120μL长双歧杆菌(Bifidobacterium longum)CCFM666培养上清，将细胞培养皿于37℃、气相含5％(v/v)CO 2的细胞培养箱中干预12h；干预12h后，在模型组、CCFM752干预组以及CCFM752干预组的每个皿内分别添加Angiotensin Ⅱ至浓度为1×10 -7M，同时，在空白对照组的每个皿内添加同样体积的DMEM培养基作为对照，将细胞培养皿于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置4h；静置4h后，将细胞培养皿从细胞培养箱中取出，吸去每皿内液体并置于冰上，用冰浴预冷的PBS缓冲液清洗5遍；清洗5遍后，将每皿细胞转移至不同的15mL离心管中，以冰浴预冷的PBS缓冲液清洗2遍；清洗2遍后，在离心管中添加含有2.0％(v/v)蛋白酶抑制剂混合物的PBS缓冲液(1mL/管细胞)重悬细胞，得到细胞悬液；将细胞悬液转移至新的无菌1.5mL离心管中，置于冰浴上超声破碎(超声功率：300W，单次破碎时长：3～5秒，间隔30秒，重复3～5次)，得到细胞破碎液；将细胞破碎液于显微镜下观察，无完整细胞即可确认破碎完全，完成细胞匀浆的制备；采用碧云天BCA蛋白浓度测定试剂盒进行细胞匀浆蛋白浓度测定，细胞匀浆中NADPH氧化酶活力测定采用化学发光法进行：在96孔板中加入180μL 50mM磷酸盐缓冲液(pH 7.0)，在缓冲液中添加1mmol/L EGTA、150mmol/L蔗糖、500μmol/L光泽精以及100μmol/LNADPH，每个细胞匀浆样品分别设置5个检测平行，于96孔板的每个孔中分别加入20μL细胞匀浆，加入细胞匀浆后立即采用酶标仪进行化学发光测定，检测反应开始后30～120s间的化学发光强度，将每孔测定的发光强度与该匀浆蛋白浓度的比值用以表征该细胞匀浆的相对NADPH氧化酶活力，以Control组为对比处理数据，结果如图3所示。
由图3可知，用1×10 -7MAngiotensin Ⅱ刺激4h后，模型组A7R5细胞内NADPH氧化酶活性显著升高，提升为空白对照组的2.4倍(p<0.001)，CCFM752干预组A7R5细胞内NADPH氧化酶活力较模型组降低18％(p<0.05)，而CCFM666干预组A7R5细胞内NADPH氧化酶活力较模型组未发生明显变化。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752培养上清干预能够显著抑制Angiotensin Ⅱ刺激 后A7R5细胞内NADPH氧化酶的活性，减少ROS的产生。
实施例5：长双歧杆菌对A7R5细胞内CAT酶活力的影响
具体步骤如下：
大鼠胸主动脉平滑肌细胞A7R5购自中国科学院典型培养物保藏委员会细胞库，以含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基进行培养。细胞培养于37℃、气相含5％(v/v)CO 2的细胞培养箱中。细胞生长至70％～80％密度时进行传代。
选取生长状态良好的A7R5细胞，将A7R5细胞经胰蛋白酶消化后离心、用DMEM培养基重悬，进行细胞计数，得到重悬液；将重悬液按照2×10 5细胞/孔接种至6cm细胞培养皿中后，在细胞培养皿中继续添加4mL含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基，于37℃、气相含5％(v/v)CO 2的细胞培养箱中培养48h；培养48h后，将细胞培养皿中的含有10％(v/v)胎牛血清(FBS)、100U/mL青霉素、100mg/mL链霉素的DMEM培养基替换为4mL含有0.1％(v/v)胎牛血清(FBS)的DMEM培养基，并将细胞培养皿于37℃、气相含5％(v/v)CO 2的细胞培养箱中静置24h；静置24h后，以每皿细胞为单位，将细胞分为空白对照组(Control)、模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组3个皿，其中，在空白对照组与模型组的每个皿中分别加入120μL MRS液体培养基，在CCFM752干预组的每个孔中加入120μL长双歧杆菌(Bifidobacterium longum)CCFM752培养上清，在CCFM666干预组的每个孔中加入120μL长双歧杆菌(Bifidobacterium longum)CCFM666培养上清，将细胞培养皿于37℃、气相含5％(v/v)CO 2的细胞培养箱中干预12h；干预12h后，将细胞培养皿从细胞培养箱中取出，吸去每皿内液体并置于冰上，用冰浴预冷的PBS缓冲液清洗5遍；清洗5遍后，将每皿细胞转移至不同的15mL离心管中，以冰浴预冷的PBS缓冲液清洗2遍；清洗2遍后，在离心管中添加含有2.0％(v/v)蛋白酶抑制剂混合物的PBS缓冲液(1mL/管细胞)重悬细胞，得到细胞悬液；将细胞悬液转移至新的无菌1.5mL离心管中，置于冰浴上超声破碎(超声功率：300W，单次破碎时长：3～5秒，间隔30秒，重复3～5次)，得到细胞破碎液；将细胞破碎液于显微镜下观察，无完整细胞即可确认破碎完全，完成细胞匀浆的制备；采用碧云天BCA蛋白浓度测定试剂盒进行细胞匀浆蛋白浓度测定，细胞匀浆CAT酶活力采用南京建成过氧化氢酶测定试剂盒进行测定，胞内CAT酶活力以测定匀浆CAT活力除以匀浆蛋白浓度表示，以对照组为对比处理数据，结果如图4所示。
由图4可知，长双歧杆菌(Bifidobacterium longum)CCFM752培养上清干预能够提高A7R5细胞内CAT酶活力至空白对照组的1.9倍左右(p<0.01)，而长双歧杆菌(Bifidobacterium longum)CCFM666培养上清干预未能显著改变A7R5细胞内CAT酶活力。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752培养上清干预能够通过提升A7R5细胞内CAT酶活力，帮助清除胞内ROS。
实施例6：长双歧杆菌对SHR血压水平的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死。
自实验第二周(5周龄)起每周测量一次尾动脉收缩压与舒张压水平，结果如图5与图6所示。
由图5可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著降低SHR的收缩压并 延缓高血压的形成，收缩压较高血压模型组降低了9.65％，而长双歧杆菌(Bifidobacterium longum)CCFM666仅能够轻度降低收缩压水平，未能延缓原发性高血压的形成。由图6可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著降低SHR的舒张压并延缓高血压的形成，舒张压较高血压模型组降低了10.97％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能显著降低舒张压，也未能延缓原发性高血压的形成。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够有效预防原发性高血压的发生发展。
实施例7：长双歧杆菌对SHR主动脉血管壁ROS水平的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉。
主动脉组织采集后置于含30％(w/w)蔗糖的PBS(pH 7.4)中脱水1-2h，脱水后采用OCT包埋胶进行组织包埋，包埋后置于-80℃冰箱中冷冻。冷冻的组织取出后置于低温恒温器中切成厚度10μm的切片。切片置于含5μM DHE的生理盐溶液中染色30min，而后置于含300μM DAPI的生理盐溶液中染色30min，取出清洗后封片。切片制作完成后于24h内通过倒置荧光显微镜进行拍照。主动脉组织ROS水平以乙锭荧光强度/DAPI荧光强度比值进行计算，以正常血压对照组为对比处理数据，结果如图7所示。
由图7可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著降低SHR主动脉血管壁ROS水平，ROS水平较高血压模型组降低了55.45％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能够显著降低主动脉血管壁ROS。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752对SHR具有抗氧化功效，有助于降低高血压的发生。
实施例8：长双歧杆菌对SHR主动脉血管壁NADPH氧化酶活力的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉组织。
将采集的主动脉组织剪成3-4mm厚度的环置于生理盐溶液中，冰浴条件下暂存。实验前置于37℃恒温培养箱中静置30min。将主动脉环置于96孔板中，加入200μL含有100μMNADPH与5μM光泽精的生理盐溶液，立刻置于酶标仪中测定。读取反应开始后30-200s之间的化学发光强度。测定完成后将主动脉环取出，烘干并测定组织干重，以读取总化学发光强度/组织干重表示组织的相对NADPH氧化酶活力， 以正常血压对照组为对比处理数据，结果如图8所示。
由图8可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够降低SHR主动脉组织NADPH氧化酶活力，NADPH氧化酶活力较高血压模型组降低了53.51％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能够降低NADPH氧化酶活力。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够抑制SHR主动脉组织中ROS的产生，有效降低高血压与血管重构的发生风险。
实施例9：长双歧杆菌对SHR主动脉血管壁CAT酶活力的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉组织。
对主动脉组织进行称重，按照1:9质量比加入的PBS(pH 7.4，添加2％蛋白酶抑制剂与磷酸酶抑制剂)。将主动脉组织剪碎，加入研磨珠，于高通量组织研磨器中破碎(破碎条件：震荡频率65Hz，震荡时长15s/次，连续破碎8-10次。)。组织破碎后，3000×g，4℃条件下离心15min，取上清液分装并冻存于-80℃冰箱备用。主动脉组织匀浆蛋白浓度采用BCA蛋白浓度测定试剂盒进行测定，CAT活力采用南京建成过氧化氢酶测定试剂盒进行测定，主动脉组织CAT酶活力以测定匀浆CAT活力除以匀浆蛋白浓度表示，结果如图9所示。
由图9可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够提升SHR主动脉组织CAT活力，CAT活力较高血压模型组提升了65.80％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能提升CAT活力。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够提升SHR主动脉组织CAT活力，提升抗氧化能力，帮助清除血管壁ROS。
实施例10：长双歧杆菌对SHR主动脉血管壁eNOS活力的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉组织。
对主动脉组织进行称重，按照1:9质量比加入的PBS(pH 7.4，添加2％蛋白酶抑制剂与磷酸酶抑制剂)。将主动脉组织剪碎，加入研磨珠，于高通量组织研磨器中破碎。破碎条件：模块于-20℃条件下预冷，震荡频率65Hz，震荡时长15s/次，连续破碎8-10次。组织破碎后3000×g，4℃条件下离心15min，取上 清液分装并冻存于-80℃冰箱备用。主动脉组织匀浆蛋白浓度采用BCA蛋白浓度测定试剂盒进行测定，eNOS活力采用南京建成一氧化氮合成酶分型测试盒进行测定。主动脉eNOS活力以测定匀浆eNOS活力除以匀浆蛋白浓度表示，结果如图10所示。
由图10可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著提升SHR主动脉eNOS活力，eNOS活力较高血压模型组提升了75.25％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能提升eNOS活力。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够提升SHR主动脉内皮NO合成水平，有效促进血管舒张并改善血管功能。
实施例11：长双歧杆菌对SHR血清CAT酶活力的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)够自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集血液。动物血液在3000×g，4℃条件下离心15min后分离血清，保存于-80℃冰箱备用。
血清CAT活力采用南京建成的过氧化氢酶测定试剂盒进行测定，结果如图11所示。
由图11可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著提升SHR血清CAT活力，CAT活力较高血压模型组提升了56.90％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能提升血清CAT活力。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够提升SHR系统抗氧化能力，有效预防氧化应激与氧化损伤。
实施例12：长双歧杆菌对SHR主动脉血管壁胶原质水平的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉。
截取约0.5cm长度的主动脉，立即置于含4％多聚甲醛的PBS溶液中固定，然后进行脱水、石蜡包埋，制成10μm厚度切片。组织切片置于天狼猩红染色液(天狼猩红浓度为0.1％(w/v)，溶于饱和苦味酸水溶液)中进行染色。天狼猩红染色切片通过数字切片扫描仪进行扫描(如图12所示)。主动脉血管壁相对胶原质水平采用Image-Pro Plus软件进行计算，以正常血压对照组为对比处理数据，结果如图13所示。
由图13可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著降低SHR主动脉血管 壁胶原质水平，主动脉血管壁胶原质水平较高血压模型组降低了24.66％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能降低主动脉胶原质水平。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够有效预防高血压相关的血管纤维化，有助于保持血管弹性。
实施例13：长双歧杆菌对SHR主动脉血管壁厚度指数的影响
具体步骤如下：
4周龄SPF级Wistar Kyoto大鼠(WKY)与原发性高血压大鼠(SHR)购自北京维通利华实验动物有限公司。实验动物饲养于江南大学实验动物中心。动物饲养与实验条件参照江南大学实验动物中心伦理审核，批准编号：JN.No20200630SW0720909[117]。
将SHR随机分为3组，分别为高血压模型组(Model)、长双歧杆菌(Bifidobacterium longum)CCFM752干预组(CCFM752)以及长双歧杆菌(Bifidobacterium longum)CCFM666干预组(CCFM666)，每组7只。WKY全部为正常血压对照组(Control)，共7只。
实验周期共13周。第一周为适应期，第二周开始进行灌胃干预直到实验结束。CCFM752干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM752菌悬液，剂量为1.0×10 9CFU；CCFM666干预组每天灌胃长双歧杆菌(Bifidobacterium longum)CCFM666菌悬液，剂量为1.0×10 9CFU；正常血压对照组与高血压模型组不采用任何活菌或药物进行干预，每天灌胃同样体积PBS缓冲液作为对照。所有分组均自由摄食与饮水。实验结束后将全部大鼠麻醉后放血处死并采集胸腔主动脉。
截取约0.5cm长度的主动脉，立即置于含4％多聚甲醛的PBS溶液中固定，然后进行脱水、石蜡包埋，制成10μm厚度切片。组织切片置于天狼猩红染色液(天狼猩红浓度为0.1％(w/v)，溶于饱和苦味酸水溶液)中进行染色。天狼猩红染色切片通过数字切片扫描仪进行扫描(如图12所示)。主动脉血管壁厚度与内腔周长采用Image-Pro Plus软件进行测量，主动脉血管壁厚度指数表示为血管壁厚度与内腔周长的比值，结果如图14所示。
由图14可知长双歧杆菌(Bifidobacterium longum)CCFM752活菌干预能够显著降低SHR主动脉血管壁厚度指数，主动脉血管壁厚度指数较高血压模型组降低了13.15％，而长双歧杆菌(Bifidobacterium longum)CCFM666未能降低主动脉血管壁厚度指数。
可见，长双歧杆菌(Bifidobacterium longum)CCFM752能够有效预防高血压相关的血管壁增厚，降低血管腔狭窄的发生风险。
实施例14：长双歧杆菌的应用
长双歧杆菌CCFM752可用于制备固体饮料，固体饮料的具体制备过程如下：
将长双歧杆菌CCFM752按照占培养基总质量2～4％的接种量接种到培养基中，于37℃下培养36h，得到培养液；将培养液离心，得到菌体；将菌体用pH为7.2～7.4的磷酸盐缓冲液清洗2～4次后用冻干保护剂重悬，得到重悬液；将重悬液用真空冷冻法进行冻干，得到菌粉；冻干保护剂和菌体的质量比为2:1；培养基包含占培养基总质量87.7％的水、10％的酶水解脱脂乳、0.5％的葡萄糖、1.5％的胰蛋白胨以及0.3％的酵母浸膏，pH为6.8；保护剂包含100g/L脱脂奶粉、150g/L海藻糖以及10g/L L-谷氨酸钠。
将含有10 10CFU长双歧杆菌CCFM752的菌粉与1g麦芽糊精混合，得到含长双歧杆菌CCFM752的固体饮料。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一株长双歧杆菌(Bifidobacterium longum)，其特征在于，所述长双歧杆菌保藏于广东省微生物菌种保藏中心，保藏编号为GDMCC No.61157，保藏日期为2020年08月21日。
  2. 权利要求1所述长双歧杆菌在制备预防和/或治疗心血管疾病的产品中的应用。
  3. 如权利要求2所述的应用，其特征在于，所述产品包含食品或药品。
  4. 如权利要求3所述的应用，其特征在于，所述产品中，权利要求1所述长双歧杆菌的活菌数不低于1×10 6CFU/mL或1×10 6CFU/g。
  5. 如权利要求2～4任一所述的应用，其特征在于，所述产品中，将权利要求1所述长双歧杆菌添加至食品中或将权利要求1所述长双歧杆菌作为发酵菌株用于食品的发酵。
  6. 一种产品，其特征在于，所述产品含有权利要求1所述长双歧杆菌。
  7. 如权利要求6所述的一种产品，其特征在于，所述产品包含食品或药品。
  8. 如权利要求6或7所述的一种产品，其特征在于，所述产品中，权利要求1所述长双歧杆菌的活菌数不低于1×10 6CFU/mL或1×10 6CFU/g。
  9. 如权利要求8所述的一种产品，其特征在于，权利要求1所述的长双歧杆菌为药品中的有效活性成分。
  10. 如权利要求9所述的一种产品，其特征在于，所述药品含有权利要求1所述的长双歧杆菌、药物载体和/或药用辅料。
  11. 如权利要求10所述的一种产品，其特征在于，所述药物载体包含微囊、微球、纳米粒和/或脂质体。
  12. 如权利要求11所述的一种产品，其特征在于，所述药用辅料包含赋形剂和/或附加剂。
  13. 如权利要求12所述的一种产品，其特征在于，所述赋形剂包含溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂和/或释放阻滞剂；所述附加剂包含微晶纤维素、羟丙基甲基纤维素和/或精制卵磷脂。
  14. 如权利要求7所述的一种产品，其特征在于，所述食品包括保健食品；或所述食品为使用含有权利要求1所述长双歧杆菌的发酵剂生产得到的乳制品、豆制品或果蔬制品；或所述食品为含有权利要求1所述长双歧杆菌的饮料或零食。
  15. 一种预防和/或治疗心血管疾病的方法，其特征在于，对哺乳动物施用权利要求1所述的长双歧杆菌或权利要求6～14任一所述的产品。
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