Specially selected bacterial strain and application thereof in cardiovascular and cerebrovascular maintenance

Abstract

The invention relates to a specially selected strain and application thereof in cardiovascular and cerebrovascular maintenance. The invention provides a specially selected strain-prebiotic composition for enhancing and maintaining an ozone high-dose blood purification effect and application of the specially selected strain-prebiotic composition in establishment of an intestinal barrier function and health maintenance of heart and cerebral vessels. According to the invention, blood cell metabolism, immune system and antioxidant enzyme system can be activated by autologous blood circulation reinfusion, the state of the blood vessel wall is improved, and the physiological function of blood cells is enhanced; the specially selected targeted strain-prebiotic composition can regulate intestinal flora balance, establish an intestinal barrier function, maintain and accelerate metabolism of a human body, decompose blood fat and toxin garbage accumulated in the body, promote detoxification of the intestinal tract, purify the intestinal tract and reduce intestinal toxin substances entering the blood circulation of the human body through a portal system. According to the combination scheme, the blood purification effect can be remarkably improved, the blood fat index is remarkably improved, the purposes of recovering the blood vessel function and enhancing the human immune function are jointly achieved, a new technical scheme and means are provided for prevention and treatment of cardiovascular and cerebrovascular diseases, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention belongs to the fields of food, health care and medicine, and particularly relates to a selected strain combination for enhancing and maintaining the effect of high-dose ozone blood purification (i.e., autologous blood is activated by ozone and then transfused into the body), and its application in establishing intestinal barrier and maintaining cardiovascular and cerebrovascular health. Background Art

[0002] At present, chronic diseases, especially cardiovascular and cerebrovascular diseases, diabetes and tumors, have become the main killers endangering human health. Modern medical research shows that human aging and a large number of diseases, especially chronic diseases, are caused by the combined action of multiple mechanisms. The main mechanisms are as follows: oxygen free radicals generated by biochemical reactions in the human body react with many substances in the human body, leading to diseases and aging of the body; the decline of the immune system function affects other organs, leading to diseases and aging of the body; errors occur in the genetic material DNA, and the long-term accumulation of repeated errors will lead to changes in the functions of offspring cells and even cell death, thus leading to diseases and aging of the body. The disorder of human microecology, the absorption of poisons produced by harmful intestinal bacteria, causes long-term chronic poisoning, leading to diseases and aging of the body.

[0003] Ozone can improve the body's antioxidant defense ability, anti-aging ability, and remove harmful free radicals in the body, achieving the effects of preventing diseases. After ozone enters the human body, various antioxidant enzymes in human red blood cells are increased, especially superoxide dismutase (SOD) has a significant increase. At the same time, it can reduce blood lipids, especially low-density lipoprotein (LDL) has a significant decrease. Therefore, ozone therapy can prevent the incidence of cardiovascular and cerebrovascular diseases, anti-aging and enhance vitality. In ozone clinical treatment, the most benefited are tissue ischemic diseases. For example, cardiovascular diseases such as myocardial infarction and coronary heart disease, because ozone therapy can immediately improve myocardial ischemia and hypoxia, and quickly relieve symptoms such as angina pectoris and chest tightness. At the same time, ozone therapy for cardiovascular diseases can fundamentally improve the stenosis and infarction problems of the coronary artery. Because ozone has the function of reducing low-density lipoprotein. As is well known, low-density lipoprotein is an important factor causing atherosclerosis.

[0004] The gut microbiota has emerged as an environmental risk factor affecting the thrombotic phenotype in various cardiovascular diseases. Evidence includes the identification of signature species through sequencing studies of the gut microbiome in patients with thrombotic diseases, the impact of antithrombotic therapies on gut microbial diversity, and preclinical studies in murine thrombosis models, which have shown that the gut microbiota has a functional impact on vascular inflammatory phenotypes and thrombosis. In addition to impaired gut barrier function promoting low-grade inflammation, gut microbiota-derived metabolites have been shown to act on vascular cell types and promote thrombosis. A healthy gut microbiota, which can repair damaged gut function, can improve various cardiovascular and cerebrovascular diseases by establishing a gut barrier; in particular, the restoration and maintenance of gut barrier function are crucial for preventing the translocation of microbial components from the intestinal lumen into the bloodstream, including microbial patterns that can activate innate immune pathways and affect thrombotic potential through the gut-liver axis; gut microbial metabolites can affect vascular cells and myeloid cells, influencing platelet responses and cell-vessel wall interactions. Summary of the Invention

[0005] Based on high-dose ozone blood purification (i.e., autologous blood is activated by ozone and infused back into the body), the present invention uses selected strains and prebiotic compositions to repair and restore the balance of the gut microbiota, establish a gut barrier, reduce and delay the entry of harmful substances in the gut into the blood, and enhance and maintain the effect of high-dose ozone blood purification. Specifically:

[0006] First aspect, screening of a selected strain of probiotic bacteria: Milk source samples from different sources are cultured in a specific condition through MRS solid-liquid medium, the bacterial cells are collected by centrifugation, washed with MSM medium, screened, separated through a target substrate, and re-screened and re-separated through continuous enrichment technology, and then purified. After morphological and molecular biological identification, a special strain of Bifidobacterium lactis ( Bifidobacterium lactis ) is obtained, named BBA-60, and the deposit number is: CCTCC M2024160. The freeze-dried powder prepared from this strain has a total viable count of: 6.5×10 10 ~1.3×10 12 CFU / g.

[0007] Second aspect, providing a composition for enhancing and maintaining high-dose ozone blood purification, including Bifidobacterium lactis BBA-60 and various prebiotics. The weight ratio of the addition of Bifidobacterium lactis BBA-60 and the prebiotics is 1:(49-99).

[0008] Preferably, in the above technical solution, when the addition ratio of the selected strain of probiotic bacteria and the prebiotics is within this range, it can ensure the optimal survival and colonization of the selected strain of probiotic bacteria, so as to achieve the best strain efficacy.

[0009] Finally, on the basis of autologous blood activation and reinfusion of high-dose ozone blood purification, the above-mentioned selected strains of Bifidobacterium lactis and prebiotic composition are applied. The high-dose ozone blood purification can not only activate blood cell metabolism, immune system and antioxidant enzyme system, improve the state of blood vessel walls, and enhance the physiological functions of blood cells; the selected targeted strain combination can also regulate the balance of intestinal flora, establish intestinal barrier function, maintain normal physiological metabolism of the human body, accelerate normal metabolism of the human body, promote intestinal detoxification, decompose accumulated blood lipids and toxin waste in the body, prevent and treat various acute and chronic diarrhea and constipation, thus strengthening the effect of high-dose ozone blood purification and maintaining this effect for a relatively long effective time.

[0010] Biological deposit information:

[0011] Probiotic strain BBA-60, classified as Bifidobacterium lactis , deposited with the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, Wuhan, China, the deposit date: January 22, 2024, and the deposit number is: CCTCC NO: M 2024160.

[0012] Advantages of the present invention:

[0013] On the one hand, the selected strain-prebiotic composition has the effect of proliferating beneficial bacteria and inhibiting harmful bacteria. When the number of beneficial bacteria such as Bifidobacterium, Bacteroides, and Lactobacillus in the intestine increases significantly, intestinal microorganisms can secrete a large amount of beneficial substances such as acetic acid, butyric acid, protein, hormones, etc.; when the selected strain-prebiotic composition inhibits harmful bacteria and the number of harmful bacteria decreases significantly, the content of endotoxin produced by harmful bacteria also decreases greatly. In this way, the endotoxin entering the human blood circulation through the portal system along with the blood flow in the intestine also decreases greatly. In this way, the purpose of repairing and restoring the balance of intestinal flora, establishing an intestinal barrier, and reducing and delaying the entry of harmful substances in the intestine into the blood is achieved.

[0014] Furthermore, the present invention uses autologous blood activated by ozone and reinfused into the body in combination with the application of the selected targeted strain composition. This combined treatment plan can significantly improve the effect of blood purification, significantly improve blood lipid indicators. The combined application of the two repairs and restores the balance of intestinal flora, establishes an intestinal barrier, reduces and delays the entry of harmful substances in the intestine into the blood, thereby enhancing and maintaining the effect of high-dose ozone blood purification, and jointly achieving the purpose of preventing and treating cardiovascular and cerebrovascular diseases and enhancing the immune function of the human body, providing a new technical solution for the prevention and treatment of cardiovascular and cerebrovascular diseases, and further achieving the purpose of comprehensively preventing and treating diseases and delaying aging in multiple aspects and by multiple mechanisms. Detailed implementation manners

[0016] Example 1: Isolation, screening, and identification of strains

[0017] 1.1 Isolation and purification of strains

[0018] The collected milk source samples were serially diluted and evenly spread on MRS solid plates, and anaerobically cultured at 37 °C for 48 h. Typical single colonies on the plates were picked and inoculated into MRS broth medium, and anaerobically cultured statically at 37 °C for 48 h as the first-generation activated strains. The first-generation activated strains were streaked on solid plates to obtain single colonies, and the streaking purification was repeated. Through screening and separation, a Bifidobacterium lactis strain was obtained by continuous enrichment technology and named BBA-60.

[0019] Formulation of MRS medium: 10.0 g peptone, 8.0 g beef extract powder, 4.0 g yeast extract powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g trisodium citrate, 5.0 sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, and made up to 1 L with deionized water. 15.0 g agar was added per liter in the solid medium.

[0020] 1.2 Identification of strains

[0021] Using acid production as the preliminary screening criterion for lactic acid bacteria, after culturing the purified strains for 24 h, they were inoculated into sterilized skim milk at an inoculation amount of 4% and anaerobically cultured at 37 °C for 72 h. Strains that coagulated milk within 72 h were regarded as acid-producing strains, and morphological and molecular biological identifications were carried out.

[0022] Morphological identification: The bacterial liquid was made into a slide, and its colony morphology was observed under an oil immersion microscope. The colony surface was convex and the edge was curled. Gram staining and microscopic examination identified it as Gram-positive bacilli.

[0023] Molecular biological identification: Bacterial DNA was extracted (using a rapid bacterial genomic DNA extraction kit, Vazyme). Using the total DNA of the strain as a template, PCR amplification was carried out using the universal primers for 16S rRNA gene (27F, 1492R). The forward primer was 5′-AGAGTTTGATCCMTGGCTCAG-3′, and the reverse primer was 5′-GGTTACCTTGTTACGACTT-3′. The 50 µL PCR reaction system was: 2.0 µL of DNA template, 2.0 µL of each primer (10 μM), 25.0 µL of Green Taq Mix, and 19 µL of ddH2O. The PCR reaction program was: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 90 s, for 35 cycles; final extension at 72 °C for 5 min.

[0024] Take 4.0 µL of the PCR product and perform electrophoresis verification on a 1% agarose gel. The PCR product was recovered by cutting the gel using a recovery kit (Axygen). After TA cloning, it was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rRNA gene sequence was aligned with the EzTaxon-e server to identify that the sequence similarity between strain BBA-60 and Bifidobacterium lactis (Bifidobacterium lactis) JCM1136 T was 99.72%.

[0025] In addition, strain BBA-60 was identified as Bifidobacterium lactis ( Bifidobacterium lactis sp.) by combining the morphological, physiological, and biochemical characteristics of the strain. The strain was deposited at the China Center for Type Culture Collection (CCTCC), with the deposit address being Wuhan University, Wuhan, China. The deposit date was January 22, 2024, and the deposit number was: CCTCC M 2024160.

[0026] Example 2: Determination of the tolerance of the strain to acid and bile salts

[0027] Bifidobacterium lactis BBA-60 was inoculated into MRS medium with different acidities (pH 2, pH 3, pH 4, pH 5) or different concentrations of bile salts (0.1%, 0.2%, 0.3%, 0.5%) at an inoculation amount of 1%. The MRS liquid medium without pH adjustment or without added bile salts was used as a control. After culturing at 37°C and 180 rpm for 12 h, 100 μL of the bacterial solution was taken and diluted and spread on an MRS solid plate, and then cultured at 37°C for 24 h, and the number of colonies was counted. The experiment was repeated three times, with three parallels set each time. The test results are shown in Table 1 and Table 2.

[0028] The results showed that Bifidobacterium lactis BBA-60 had a certain ability to tolerate acid and bile salts.

[0029] Table 1 Acid tolerance test results of strain BBA-60 pH CK 2 3 4 5 Colony count <![CDATA[7.45×10 8 > <![CDATA[3.55×10 8 > <![CDATA[3.09×10 8 > <![CDATA[5.65×10 8 > <![CDATA[6.56×10 8 >

[0030] When strain BBA-60 was inoculated into media with different pH values and cultured for 12 h, it could be observed that as the pH value decreased, the viable cell count of strain BBA-60 in the MRS medium also decreased faster. At pH 2, the number of colonies decreased by 50% compared to the CK group, but there was no change in the order of magnitude. When the pH was 5, there was no significant change in the viable cell count compared to CK. This result indicated that strain BBA-60 had a good ability to tolerate gastric acid.

[0031] Table 2 Bile salt tolerance test results of strain BBA-60 Bile salt concentration CK 0.1% 0.2% 0.3% 0.5% Colony count <![CDATA[7.75×10 8 > <![CDATA[2.61×10 8 > <![CDATA[1.29×10 8 > <![CDATA[8.55×10 7 > <![CDATA[5.53×10 6 >

[0032] The strain BBA-60 was inoculated in bile salts with different concentrations. As the concentration increased, the viable cell count decreased faster. Compared with the CK, at a bile salt concentration of 0.5%, the viable cell count decreased by two orders of magnitude. However, at concentrations of 0.1% and 0.2%, compared with the CK, there was no change in the order of magnitude of the viable cell count, indicating that the strain BBA-60 could tolerate a bile salt concentration of 0.2%. This result shows that as a probiotic, after being damaged by gastric acid and bile salts, the strain BBA-60 still retains a very high viable cell count and can enter the intestine to play a role.

[0033] Example 3: Preparation of a selected strain-prebiotic composition

[0034] The above-identified Bifidobacterium lactis BBA-60 was expanded in culture, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the cells were obtained, freeze-dried and pulverized to obtain the freeze-dried powder of Bifidobacterium lactis BBA-60, which is the freeze-dried powder of the selected strain. After testing, the total viable cell count of this freeze-dried powder was 9.5×10 11 CFU / g.

[0035] The freeze-dried powder of Bifidobacterium lactis BBA-60 (with a viable cell count of 9.5×10 11 CFU / g) and prebiotics (mannooligosaccharide, stachyose, L-arabinose, xylooligosaccharide, inulin, etc.) were respectively passed through a 60-mesh sieve and reserved. According to the mass ratio of the freeze-dried powder of the selected strain to the prebiotic in the range of 1:(49 - 99), the corresponding materials were weighed, mixed evenly, and then sealed and reserved.

[0036] Example 4: High-dose ozone blood purification and food trial in humans

[0037] For autologous blood reinfusion, 1000 ml of blood was collected. The blood flowed from the blood bag into a quartz glass jar and was placed in a UBIO therapeutic instrument. It was irradiated with ultraviolet light at a dose of 10 J / cm2 and a wavelength of 300 nm, and the oxygen filling flow rate during irradiation was 5 liters / min. After irradiation, the blood was reinfused into the original sterile blood bag and then intravenously transfused back to the patient through the original blood-drawing infusion set once a month.

[0038] The selected strain-prebiotic composition was made into capsules or solid beverage strip packs, taken 3 - 5 g each time, twice a day, and one course of treatment was 30 days.

[0039] Example 5: Food trial for testing human blood lipid indicators

[0040] A human food trial was conducted using the high-dose ozone blood purification in humans and the selected strain-prebiotic composition in Example 4 to test the human serum lipid indicators. The subjects were aged between 45 and 70 years old.

[0041] Experimental design and grouping: Two control designs, self-control and inter-group control, were adopted. Eighty subjects were selected, with 20 subjects in each group. The subjects were grouped according to the requirements of random double-blind method, divided into a control group, experimental group 1, experimental group 2, and experimental group 3. Main factors affecting the results such as age, gender, diet, etc. were considered as much as possible, and a balance test was conducted to ensure the comparability between groups.

[0042] Experimental group 1 was given the high-dose ozone blood purification for human body and the selected strain-prebiotic composition in Example 4, experimental group 2 was given only the high-dose ozone blood purification for human body, experimental group 3 was given only the selected strain-prebiotic composition, and the control group was given a placebo. They were administered continuously for 30 days.

[0043] The grouping situation is shown in Table 3. Before the trial diet, there were no significant differences in age, gender, total serum cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) among the four groups of subjects (P>0.05), and they were comparable.

[0044] Table 3 Comparison of general information of subjects before the trial diet Item Control group Experimental group 1 Experimental group 2 Experimental group 3 Number of cases 20 20 20 20 Male / Female 11 / 9 10 / 10 11 / 9 9 / 11 Average age 56.4±9.5 56.5±9.4 57.2±8.9 58.1±8.8 TC (mmol / L) 5.12±0.53 5.11±0.56 5.16±0.56 5.10±0.48 TG (mmol / L) 1.53±0.35 1.49±0.51 1.51±0.47 1.57±0.37 LDL-C (mmol / L) 3.10±0.25 3.09±0.32 3.14±0.36 3.09±0.35

[0045] The changes in blood lipid indexes after the trial diet are shown in Table 4.

[0046] Table 4 Changes in blood lipid indexes of the experimental group and the control group after the trial diet Item Control group Experimental group 1 Experimental group 2 Experimental group 3 TC (mmol / L) 5.21±0.53 4.17±0.54 4.12±0.53 4.17±0.50 TG (mmol / L) 1.47±0.35 1.03±0.40 1.33±0.41 1.34±0.41 LDL-C (mmol / L) 3.11±0.24 1.88±0.31 2.43±0.32 2.46±0.34

[0047] As can be seen from Table 3 and Table 4, after 30 days, the total serum cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in the blood of experimental group 1 were significantly decreased before and after the trial diet (P<0.01), and were significantly better than those of the control group and experimental groups 2 - 3, and the inter-group differences were extremely significant (P<0.01).

[0048] The results showed that after 30 days of applying the combination of the present invention, when comparing experimental group 1 before and after the trial diet and comparing experimental group 1 with the control group and experimental groups 2 - 3 after the trial diet, the total serum cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) in the blood were significantly decreased (P<0.01). Therefore, the combination of the present invention has the efficacy of regulating blood lipid, maintaining the effect of high-dose ozone blood purification, and maintaining the function of cardiovascular and cerebrovascular system.

Claims

1. A selected strain-prebiotic composition for enhancing and maintaining the effect of high-dose ozone blood purification and its application in establishing intestinal barrier function and cardiovascular and cerebrovascular maintenance, characterized in that, The selected strain is Bifidobacterium lactis BBA-60, which is deposited in the China Center for Type Culture Collection with the deposit number: CCTCC M 2024160; the selected strain-prebiotic composition is a composition of freeze-dried powder of Bifidobacterium lactis BBA-60 and one or more of prebiotics such as mannan oligosaccharide, stachyose, L-arabinose, xylo-oligosaccharide, inulin, etc.

2. The selected strain according to claim 1, characterized in that, The identified Bifidobacterium lactis BBA-60 is expanded in culture, centrifuged at 3000 rpm for 10 min, the supernatant is discarded to obtain the bacterial cells, which are then freeze-dried and pulverized to obtain the freeze-dried powder of Bifidobacterium lactis BBA-60.

3. The freeze-dried powder of Bifidobacterium lactis BBA-60 according to claim 2, characterized in that, The total viable count is 6.5×10 10 ~1.3×10 12 CFU / g.

4. The composition according to claim 1, wherein The weight ratio of the freeze-dried powder of Bifidobacterium lactis BBA-60 to one or more of prebiotics such as mannan oligosaccharide, stachyose, L-arabinose, xylo-oligosaccharide, inulin, etc. is 1:(49-99).

5. According to claim 1, the application of triple-oxygen high-dose blood purification combined with the selected strain-prebiotic composition and its application in establishing intestinal barrier function and maintaining cardiovascular and cerebrovascular health, the specific technical solution is to use autologous blood transfusion of 150-2000 ml, 1-2 times per month; the selected targeted strain-prebiotic composition is 1-5 g each time, taken 2 times a day, and one course of treatment is every 15-30 days.

6. The application of the composition according to any one of claims 1 to 4 in the preparation of foods, drugs and health products for preventing and treating various acute and chronic diarrhea and constipation, as well as improving blood lipid indexes and restoring vascular function.