A composite probiotic preparation for assisting in lowering blood pressure and preparation method thereof

Abstract

The present invention relates to the technical field of probiotic preparations, and in particular to a composite probiotic preparation for assisting blood pressure reduction and a preparation method thereof. The probiotic preparation is prepared by fermentation of Lactobacillus acidophilus and Lactobacillus reuteri inoculated into a fermentation medium, and the fermentation medium includes the following raw materials in parts by weight: 2-4 parts of pretreated corn silk, 4-8 parts of strawberry extract, 1-2 parts of a stabilizing promoter, 10-20 parts of an acidic regulator, 5-10 parts of glucose, 8-16 parts of skim milk powder, 3-6 parts of lactose, and 100-200 parts of deionized water. The present invention reuses corn silk with low long-term utilization rate, uses agricultural products corn silk and strawberry for the preparation of microbial fermentation medium, and the fermentation medium prepared by the present invention can effectively promote the growth and reproduction of probiotics, ensure the vitality of the thalli, and improve the survival rate. The probiotic preparation prepared by the present invention can effectively assist in lowering blood pressure.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic preparations, and particularly relates to a compound probiotic preparation for assisting in reducing blood pressure and a preparation method thereof. Background Art

[0002] Hypertension endangers human health and is a clinical syndrome accompanied by functional or organic damage to organs such as the heart, brain, and kidneys. It is a very common chronic disease and the main risk factor for cardiovascular and cerebrovascular diseases. At present, hypertension is still mainly treated with drugs. Antihypertensive drugs include 5 categories and more than 60 kinds, such as calcium channel blockers (CCB), angiotensin-converting enzyme inhibitors (ACEI), diuretics, receptor blockers, and angiotensin II receptor antagonists (ARB). However, clinically, there are still problems such as easy generation of drug resistance, large drug adverse reactions, low treatment rate and compliance rate of patients, and long-term use of ACEI antihypertensive drugs can significantly increase the incidence of lung cancer in patients. In addition, most hypertensive patients need two or more antihypertensive drugs to effectively reduce blood pressure, and many patients still cannot effectively control blood pressure even so. Therefore, the development of blood pressure-lowering products is of great significance. Research shows that probiotics can regulate blood pressure through various channels: through the hydrolysis of extracellular proteases and peptidases (carboxypeptidase, aminopeptidase), including inhibitory peptides with inhibitory activity against angiotensin-converting enzyme; bacterial components, such as the cell wall components of Lactobacillus, show blood pressure-lowering effects in spontaneously hypertensive rats and hypertensive patients; probiotics reach the intestine in the form of live bacteria, promoting the body's absorption of some minerals that can regulate blood pressure; extracellular polysaccharides produced by some probiotics may have a certain blood pressure-lowering effect. At present, the functionality of probiotic products has become a research hotspot, but there is a lack of clinical research on the function of probiotic products in assisting in reducing blood pressure. In summary, how to provide a compound probiotic preparation for assisting in reducing blood pressure is a technical problem that needs to be solved urgently. Summary of the Invention

[0003] Aiming at the deficiencies of the prior art, the present invention provides a compound probiotic preparation for assisting in reducing blood pressure and a preparation method thereof.

[0004] The present invention is realized through the following technical solutions:

[0005] A compound probiotic preparation for assisting in reducing blood pressure, the probiotic preparation is prepared by inoculating Lactobacillus acidophilus and Lactobacillus mucosae reuteri into a fermentation medium and fermenting. The fermentation medium comprises the following raw materials in parts by weight: 2 - 4 parts of pretreated corn silk, 4 - 8 parts of strawberry extract, 1 - 2 parts of stability promoter, 10 - 20 parts of acid regulator, 5 - 10 parts of glucose, 8 - 16 parts of skim milk powder, 3 - 6 parts of lactose, and 100 - 200 parts of deionized water.

[0006] Preferably, the Lactobacillus acidophilus and Lactobacillus mucosae are purchased from Shenzhen Haisige and are Lactobacillus acidophilus HX-LA15 and Lactobacillus mucosae HX-LRE501.

[0007] Further, the preparation method of the pretreated corn silk is as follows: drying the corn silk at 45 - 55 °C for 2 - 3 h, crushing and passing through a 100-mesh sieve, mixing with distilled water according to a mass ratio of 1:3, stirring at 200 rpm for 20 - 40 min, and spray drying with an inlet air temperature of 145 °C and an outlet air temperature of 125 °C to obtain the pretreated corn silk.

[0008] Further, the preparation method of the stability promoter includes the following steps:

[0009] (1) Wash and air-dry the Sargassum fusiforme, crush and pass through a 100-mesh sieve to obtain algal powder, add it to distilled water, ultrasonicate at 150 W for 1 h, centrifuge at 5000 rpm for 30 min, take the precipitate, add deionized water, water bath at 100 °C for 1 h, filter by suction, mix the filtrate evenly with an equal volume of ethyl acetate, stand at 4 °C for 4 - 6 h, filter by suction, wash the filter cake with deionized water, and dry at 100 °C for 10 h to obtain product A;

[0010] (2) Mix the product A obtained in step (1) with glycine and deionized water, stir at 300 rpm for 6 - 8 h, dialyze in deionized water using a cellulose dialysis membrane with a cut-off molecular weight of 1000 Da for 24 h, and freeze-dry to obtain the stability promoter.

[0011] Further, the dosage ratio of the algal powder to distilled water in step (1) is 1 g:100 mL.

[0012] Further, the dosage ratio of the precipitate to deionized water in step (1) is 1 g:100 mL.

[0013] Further, the dosage ratio of the product A, glycine and deionized water in step (2) is 1 g:0.8 g:40 mL.

[0014] Further, the preparation method of the acid regulator includes the following steps:

[0015] (a) Add the plantago seed gum to a 1 wt% calcium chloride solution, and add citric acid to adjust the pH value to 6.5 to obtain the outer phase solution;

[0016] (b) Dissolve malic acid in distilled water, add chitosan, react at 100 - 110 °C for 2 - 3 h, stand at 4 °C for 1 h, centrifuge at 5000 rpm for 30 min, wash the precipitate with deionized water, and vacuum freeze-dry to obtain the modified chitosan.

[0017] (c) Add the modified chitosan obtained in step (b) to distilled water, stir at 80 - 90 °C and 200 rpm for 10 - 20 min, cool to 20 °C, add sodium bicarbonate, and stir at 300 rpm for 30 - 40 min to obtain the inner phase solution;

[0018] (d) Stir the outer phase solution obtained in step (a) at 50 °C and 300 rpm, add the inner phase solution obtained in step (c) at a rate of 2 mL / min, continue stirring for 30 min, let stand at 4 °C for 6 - 8 h, pour off the supernatant, and dry in vacuum at 50 °C to obtain the acid regulator.

[0019] Further, the dosage ratio of the psyllium gum to the calcium chloride solution in step (a) is 1 g:10 mL.

[0020] Further, the dosage ratio of malic acid, distilled water to chitosan in step (b) is 1 g:50 mL:3 g.

[0021] Further, the dosage ratio of the modified chitosan, distilled water to sodium bicarbonate in step (c) is 1 g:100 mL:0.5 g.

[0022] Further, the volume ratio of the outer phase solution to the inner phase solution in step (d) is 2:1.

[0023] Further, the strawberry extract is prepared according to conventional techniques.

[0024] Preferably, the preparation steps of the strawberry extract are as follows: Cut strawberries into pieces of 1 cm 3 size and grind them into a paste, keep them in a constant temperature water bath at 45 - 55 °C for 100 - 120 min, add 3 times the mass of deionized water, stir at 200 rpm for 2 - 4 h, centrifuge at 4 °C and 10000 rpm for 10 - 20 min, and vacuum dry the supernatant at 20 °C for 24 h to obtain the strawberry extract.

[0025] The present invention also provides a preparation method of the compound probiotic preparation for assisting in reducing blood pressure, including the following steps:

[0026] S1: Mix the components of the fermentation medium evenly by weight, stir at 180 - 200 rpm for 30 - 40 min, and sterilize at 121 °C for 15 min to obtain the fermentation medium;

[0027] S2: Inoculate the activated Lactobacillus acidophilus and Lactobacillus mucosae reuteri into the fermentation medium obtained in step S1 respectively, and the inoculation amount is 1×10 6CFU / mL, ferment at 37°C, 60 rpm, 0.05 MPa for 12 h, then cool down to 28°C and ferment for 4 h to obtain the fermentation broths of Lactobacillus acidophilus and Lactobacillus mucosae roigii respectively. Mix them in a volume ratio of 1:1, concentrate under vacuum, and freeze-dry to obtain a compound probiotic preparation for assisting in reducing blood pressure.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The present invention uses agricultural products, corn silk and strawberries, for the preparation of a microbial fermentation medium. Corn silk is a food raw material, but it is usually not reasonably utilized in the public perception and has a low long-term utilization rate. The present invention realizes the recycling of resources, reduces the pressure on the environment, and lowers costs. The pretreated corn silk described in the present invention can provide a nutrient source for the growth and development of microorganisms, and at the same time, active ingredients helpful for assisting in reducing blood pressure can be produced through probiotic fermentation. Synergistically with strawberry extract, it can effectively promote the growth and reproduction of probiotics, improve the survival rate, provide growth promoting factors for the growth and reproduction of probiotics, contribute to the increase in the fermentation density of probiotics and the stability of the cell count. The present invention uses probiotics Lactobacillus acidophilus and Lactobacillus mucosae roigii, which can effectively inhibit the activity of ACE, cause vasodilation reactions, and thus reduce blood pressure. The two bacteria synergistically enhance the effect, and the ACE inhibition rate can reach more than 95%. They have a strong ability to assist in reducing blood pressure, can tolerate gastric acid and bile salts, and have a strong proliferation ability; they have strong antibacterial activity. The present invention prepares a fermentation medium, extracts the effective components of Sargassum fusiforme, forms a complex with glycine to obtain a stable promoter, which can effectively promote the growth and reproduction of probiotics, ensure the growth rate and cell viability of the strains, and improve the survival rate; the present invention uses plantago seed gum and modified chitosan to form a sustained-release microcapsule to encapsulate sodium bicarbonate, which can adjust the pH value and prevent the pH value of the system from dropping too fast due to acid substances produced by metabolism, inhibiting the growth and reproduction of probiotics, and then effectively promoting the reproduction, growth and metabolism of probiotics, resulting in a relatively high cell density after the final culture. Using malic acid to modify chitosan can improve the dispersibility, make the acid regulator evenly dispersed in the medium, make the pH value more uniform, and is beneficial to the growth of the cells. The modified chitosan and plantago seed gum synergistically enhance the effect, can provide the nutrients required by probiotics, promote the growth and reproduction of probiotics, form a microcapsule to encapsulate sodium bicarbonate, and during the fermentation process, the microcapsule gradually breaks under the action of metabolites and microbial utilization, slowly releasing sodium bicarbonate, and long-term regulating the pH value to maintain the cell viability. The fermentation medium prepared by the present invention contains nutrient elements and growth promoting factors that are easily utilized by probiotics. Inoculate the probiotics into the prepared fermentation medium for fermentation and then freeze-dry. The obtained probiotic preparation has strong probiotic viability, effectively improves the antibacterial effect and the effect of assisting in reducing blood pressure, is beneficial to the growth of the cells, increases the cell density, and reduces the production cost. The application of the compound probiotic preparation for assisting in reducing blood pressure in the preparation of products for assisting in reducing blood pressure. After using the probiotic preparation of the present invention to intervene in hypertensive model rats for 15 days, the blood pressure of the rats decreased significantly. Brief Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 Scanning electron micrograph of the acid regulator described in Example 1 of the present invention;

[0032] Figure 2 Test on the growth promotion effect of the fermentation medium described in Example 1 of the present invention on probiotics;

[0033] Figure 3 Test on the gastric acid and bile salt tolerance performance of the probiotic preparations described in Example 1 of the present invention and Comparative Examples 1-6;

[0034] Figure 4 ACE inhibition effect of the probiotics described in Example 1 of the present invention and Comparative Examples 1-6. Detailed Description of the Embodiments

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clear and understandable, the following further details the present invention in combination with specific embodiments, but the present invention is not limited to the following embodiments.

[0036] It should be noted that unless otherwise specified, the chemical reagents and strains involved in the present invention are all purchased through commercial channels.

[0037] Example 1: This example provides a compound probiotic preparation for assisting in reducing blood pressure. The probiotic preparation is prepared by inoculating Lactobacillus acidophilus and Lactobacillus mucosae reuteri into a fermentation medium. The fermentation medium includes the following raw materials in parts by weight: 4 parts of pretreated corn silk, 8 parts of strawberry extract, 2 parts of stability promoter, 20 parts of acid regulator, 10 parts of glucose, 16 parts of skim milk powder, 6 parts of lactose, and 200 parts of deionized water.

[0038] The preparation method of the pretreated corn silk is as follows: The corn silk is dried at 55°C for 3 hours, crushed through a 100-mesh sieve, mixed with distilled water according to a mass ratio of 1:3, stirred at 200 rpm for 40 minutes, and spray-dried. The inlet air temperature is 145°C and the outlet air temperature is 125°C to obtain the pretreated corn silk.

[0039] The preparation method of the stability promoter includes the following steps:

[0040] (1) Wash and dry Sargassum fusiforme, crush it and sieve through a 100-mesh sieve to obtain algal powder. Add it to distilled water with the dosage ratio of algal powder to distilled water being 1 g:100 mL. Sonicate at 150 W for 1 h, centrifuge at 5000 rpm for 30 min, take the precipitate, add deionized water with the dosage ratio of precipitate to deionized water being 1 g:100 mL, heat in a water bath at 100 °C for 1 h, filter by suction. Mix the filtrate evenly with an equal volume of ethyl acetate, let it stand at 4 °C for 6 h, filter by suction, wash the filter cake with deionized water, and dry at 100 °C for 10 h to obtain product A;

[0041] (2) Mix the product A obtained in step (1) with glycine and deionized water with the dosage ratio of product A, glycine and deionized water being 1 g:0.8 g:40 mL. Stir at 300 rpm for 8 h, dialyze in deionized water using a cellulose dialysis membrane with a molecular weight cut-off of 1000 Da for 24 h, and freeze-dry to obtain the stability promoter.

[0042] The preparation method of the acidic regulator includes the following steps:

[0043] (a) Add Plantago seed gum to a 1 wt% calcium chloride solution with the dosage ratio of Plantago seed gum to calcium chloride solution being 1 g:10 mL. Add citric acid to adjust the pH value to 6.5 to obtain the outer phase solution;

[0044] (b) Dissolve malic acid in distilled water, add chitosan with the dosage ratio of malic acid, distilled water and chitosan being 1 g:50 mL:3 g. React at 110 °C for 3 h, let it stand at 4 °C for 1 h, centrifuge at 5000 rpm for 30 min, wash the precipitate with deionized water, and vacuum freeze-dry to obtain modified chitosan;

[0045] (c) Add the modified chitosan obtained in step (b) to distilled water, stir at 90 °C and 200 rpm for 20 min, cool to 20 °C, add sodium bicarbonate with the dosage ratio of modified chitosan, distilled water and sodium bicarbonate being 1 g:100 mL:0.5 g. Stir at 300 rpm for 40 min to obtain the inner phase solution;

[0046] (d) Stir the outer phase solution obtained in step (a) at 50 °C and 300 rpm, and add the inner phase solution obtained in step (c) at a rate of 2 mL / min. The volume ratio of the outer phase solution to the inner phase solution is 2:1. Continue to stir for 30 min, let it stand at 4 °C for 8 h, pour off the supernatant, and vacuum dry at 50 °C to obtain the acidic regulator. The scanning electron micrograph is as Figure 1 shown, presenting a spherical structure. Modified chitosan and Plantago seed gum wrap sodium bicarbonate, which can regulate the pH value for a long time.

[0047] The preparation steps of the strawberry extract are as follows: Cut the strawberries into 1 cm 3After being ground into a paste according to the size, it was placed in a constant temperature water bath at 55°C for 120 min, added to deionized water with a mass three times that of the paste, stirred at 200 rpm for 4 h, centrifuged at 4°C and 10,000 rpm for 20 min, and the supernatant was vacuum dried at 20°C for 24 h to obtain strawberry extract.

[0048] This example also provides a preparation method of the compound probiotic preparation for assisting in reducing blood pressure, including the following steps:

[0049] S1: Mix the components of the fermentation medium evenly by weight, stir at 200 rpm for 40 min, and sterilize at 121°C for 15 min to obtain the fermentation medium;

[0050] S2: Inoculate the activated Lactobacillus acidophilus and Lactobacillus mucosae roebuckii into the fermentation medium obtained in step S1 respectively, with the inoculation amount of 1×10 6 CFU / mL, ferment at 37°C, 60 rpm, and 0.05 MPa for 12 h, cool down to 28°C and ferment for 4 h to obtain the fermentation broths of Lactobacillus acidophilus and Lactobacillus mucosae roebuckii respectively, mix them according to the volume ratio of 1:1, vacuum concentrate, and freeze-dry to obtain the compound probiotic preparation for assisting in reducing blood pressure.

[0051] Example 2: This example provides a compound probiotic preparation for assisting in reducing blood pressure. The probiotic preparation is prepared by inoculating Lactobacillus acidophilus and Lactobacillus mucosae roebuckii into a fermentation medium. The fermentation medium includes the following raw materials in parts by weight: 2 parts of pretreated corn silk, 4 parts of strawberry extract, 1 part of stability promoter, 10 parts of acid regulator, 5 parts of glucose, 8 parts of skim milk powder, 3 parts of lactose, and 100 parts of deionized water.

[0052] The preparation method of the pretreated corn silk is as follows: Dry the corn silk at 45°C for 2 h, crush it through a 100-mesh sieve, mix it with distilled water according to the mass ratio of 1:3, stir at 200 rpm for 20 min, and spray dry. The inlet air temperature is 145°C and the outlet air temperature is 125°C to obtain the pretreated corn silk.

[0053] The preparation method of the stability promoter includes the following steps:

[0054] (1) Wash and dry Sargassum fusiforme, crush it through a 100-mesh sieve to obtain algal powder, add it to distilled water, and the dosage ratio of algal powder to distilled water is 1 g:100 mL. Ultrasonic at 150 W for 1 h, centrifuge at 5000 rpm for 30 min, take the precipitate, add deionized water, and the dosage ratio of precipitate to deionized water is 1 g:100 mL. Water bath at 100°C for 1 h, filter by suction, mix the filtrate evenly with an equal volume of ethyl acetate, stand at 4°C for 4 h, filter by suction, wash the filter cake with deionized water, and dry at 100°C for 10 h to obtain product A;

[0055] (2) Mix the product A obtained in step (1) with glycine and deionized water. The dosage ratio of product A, glycine and deionized water is 1 g: 0.8 g: 40 mL. Stir at 300 rpm for 6 h, dialyze in deionized water with a cellulose dialysis membrane with a retention molecular weight of 1000 Da for 24 h, and freeze-dry to obtain a stable promoter.

[0056] The preparation method of the acidic regulator includes the following steps:

[0057] (a) Add plantain seed gum to a 1 wt% calcium chloride solution. The dosage ratio of plantain seed gum to the calcium chloride solution is 1 g: 10 mL. Add citric acid to adjust the pH value to 6.5 to obtain an outer phase solution;

[0058] (b) Dissolve malic acid in distilled water, add chitosan. The dosage ratio of malic acid, distilled water and chitosan is 1 g: 50 mL: 3 g. React at 100 °C for 2 h, stand at 4 °C for 1 h, centrifuge at 5000 rpm for 30 min, wash the precipitate with deionized water, and vacuum freeze-dry to obtain modified chitosan;

[0059] (c) Add the modified chitosan obtained in step (b) to distilled water, stir at 80 °C and 200 rpm for 10 min, cool to 20 °C, add sodium bicarbonate. The dosage ratio of modified chitosan, distilled water and sodium bicarbonate is 1 g: 100 mL: 0.5 g. Stir at 300 rpm for 30 min to obtain an inner phase solution;

[0060] (d) Stir the outer phase solution obtained in step (a) at 50 °C and 300 rpm, and add the inner phase solution obtained in step (c) at a rate of 2 mL / min. The volume ratio of the outer phase solution to the inner phase solution is 2:1. Continue to stir for 30 min, stand at 4 °C for 6 h, pour off the supernatant, and vacuum dry at 50 °C to obtain the acidic regulator.

[0061] The preparation steps of the strawberry extract are as follows: Cut the strawberries into pieces of 1 cm 3 in size and then grind them into a paste. Keep them in a constant temperature water bath at 45 °C for 100 min, add 3 times the mass of deionized water, stir at 200 rpm for 2 h, centrifuge at 4 °C and 10000 rpm for 10 min, and vacuum dry the supernatant at 20 °C for 24 h to obtain the strawberry extract.

[0062] This example also provides a preparation method of the compound probiotic preparation for assisting in reducing blood pressure, including the following steps:

[0063] S1: Mix each component of the fermentation medium evenly by weight, stir at 180 rpm for 30 min, and sterilize at 121 °C for 15 min to obtain the fermentation medium;

[0064] S2: Inoculate the activated Lactobacillus acidophilus and Lactobacillus mucosae reuteri into the fermentation medium obtained in step S1 respectively, with the inoculation amount being 1×10 6 CFU / mL. Ferment at 37°C, 60 rpm, and 0.05 MPa for 12 h, then cool down to 28°C and ferment for 4 h to obtain the fermentation broths of Lactobacillus acidophilus and Lactobacillus mucosae reuteri respectively. Mix them according to the volume ratio of 1:1, concentrate under vacuum, and freeze-dry to obtain the compound probiotic preparation for assisting in reducing blood pressure.

[0065] The difference between Comparative Example 1 and Example 1 is only that Sargassum fusiforme is not added.

[0066] The difference between Comparative Example 2 and Example 1 is only that glycine is not added.

[0067] The difference between Comparative Example 3 and Example 1 is only that gelatin is used to replace Plantago seed gum.

[0068] The difference between Comparative Example 4 and Example 1 is only that chitosan is used to replace modified chitosan.

[0069] The difference between Comparative Example 5 and Example 1 is only that strawberry extract is used to replace pretreated corn silk.

[0070] The difference between Comparative Example 6 and Example 1 is only that pretreated corn silk is used to replace strawberry extract.

[0071] Experimental Example 1: Prepare the probiotic preparations according to the methods of Example 1 and Comparative Examples 1-6, and measure the cell density at 8 h, 12 h, and 16 h of fermentation. The results are as Figure 2 shown.

[0072] Figure 2 The results show that the cell density and the rising rate of cell density in the Example 1 group are higher than those in Comparative Examples 1-6, indicating that the components of the fermentation medium prepared by the present invention have synergistic effects, effectively promote the growth and reproduction of probiotics, and ensure the growth rate and cell viability of probiotics.

[0073] Experimental Example 2: Suspend the Helicobacter pylori (purchased from ATCC, ATCC NO: 43504) cells in Columbia medium (added with 10% sheep serum) to prepare a bacterial suspension of 10 9 cfu / mL; inoculate it into the mixed fermentation broth at the end of fermentation in Example 2 and Comparative Examples 1-6, with the inoculation amount being 2×10 3 cfu / mL, and place it in a triple-gas incubator (O 2 :CO 2 :N 2= 5:10:85, incubated at 37 °C for 48 h, the antibacterial rate against Helicobacter pylori was measured. The antibacterial rate (%) = [(initial cell density of Helicobacter pylori - cell density of Helicobacter pylori after 48 h) / initial cell density of Helicobacter pylori] × 100%. The results are shown in Table 1.

[0074] Table 1 Results of antibacterial rate measurement

[0075]

[0076] The results in Table 1 show that the antibacterial rate of Example 2 is significantly higher than that of Comparative Examples 1-6, and the antibacterial rate can reach more than 85%, indicating that the fermentation medium prepared by the present invention can effectively improve the antibacterial effect of probiotics. The Lactobacillus acidophilus and Lactobacillus mucosae grown on the fermentation medium prepared by the present invention have good antibacterial effects.

[0077] Experimental Example 3: In a sterilized PBS buffer solution with pH 2.5 (adjusted with 1 mol / L HCl), 3.5 g / L pepsin was added, and it was filtered and sterilized through a 0.22 μm microporous membrane to prepare simulated gastric juice; in a sterilized PBS with pH 8.0 (adjusted with 0.1 mol / L NaOH), 0.1% trypsin and 1.8% bile salts were added, and it was filtered and sterilized through a 0.22 μm microporous membrane to prepare simulated intestinal juice. The probiotic preparations of Example 1 and Comparative Examples 1-6 were added to an equal volume of simulated gastric juice as the fermentation medium used in the preparation process, and incubated at 37 °C for 3 h. The viable cell counts in the artificial simulated gastric juice were measured by the MRS agar medium pour plate method at 0 h and 3 h respectively. After being treated with simulated gastric juice for 3 h, it was centrifuged at 10,000 rpm for 30 min, the precipitate was collected, washed with deionized water, and an equal volume of simulated intestinal juice was added and incubated at 37 °C. The viable cell counts were measured again by the MRS agar medium pour plate method at 0 h and 3 h respectively. The survival rate was calculated. The survival rate (%) = [N1 / N0] × 100%, where N0 represents the viable cell count at 0 h; N1 represents the viable cell count after digestion with simulated gastric and intestinal juices. The results are as Figure 3 shown.

[0078] Figure 3 The results show that the survival rate of the group of Example 1 is higher than that of Comparative Examples 1-6, indicating that the composite probiotic preparation for assisting in reducing blood pressure of the present invention has good acid and bile salt tolerance characteristics. After being digested in simulated gastric juice for 3 h and then continued to be digested in simulated intestinal juice for 3 h, its survival rate is as high as more than 90%.

[0079] Experimental Example 4: Before freeze-drying, the cell density in the mixed fermentation broth was measured. The prepared probiotic preparation was added to an equal volume of physiological saline as the fermentation medium used, and the cell density was measured. The survival rate was calculated. The survival rate (%) = (cell density after freeze-drying / cell density before freeze-drying) × 100%. The results are shown in Table 2.

[0080] Table 2 Survival rate of probiotics

[0081] Before freeze-drying After freeze-drying Survival rate (%) Example 2 <![CDATA[1.18×10 10 > <![CDATA[1.15×10 10 > 97.46 Comparative Example 1 <![CDATA[0.44×10 10 > <![CDATA[0.40×10 10 > 90.91 Comparative Example 2 <![CDATA[0.66×10 10 > <![CDATA[0.61×10 10 > 92.42 Comparative Example 3 <![CDATA[0.85×10 10 > <![CDATA[0.81×10 10 > 95.29 Comparative Example 4 <![CDATA[0.79×10 10 > <![CDATA[0.74×10 10 > 93.67 Comparative Example 5 <![CDATA[0.57×10 10 > <![CDATA[0.52×10 10 > 91.23 Comparative Example 6 <![CDATA[0.73×10 10 > <![CDATA[0.68×10 10 > 93.15

[0082] Table 2 results show that the survival rate of Example 2 is significantly higher than that of Comparative Examples 1-6, indicating that the fermentation medium prepared by the present invention helps the growth, reproduction and survival of probiotics, effectively guarantees the viability of the bacteria, and ensures the survival rate of probiotics.

[0083] Experimental Example 5: Add 10 μL of 0.1 u / mL ACE, 50 μL of 1 mmol / mL FAPGG and 40 μL of HEPESS buffer solution to a 96-well microplate. Measure the absorbance value A1 at 340 nm with a microplate reader, then add 40 μL of the mixed fermentation broth, react at a constant temperature of 37 °C for 40 min, measure the absorbance A2 again, and calculate the change in absorbance value ΔA before and after the reaction (ΔA = A1 - A2). At the same time, use PBS buffer solution to replace the mixed fermentation broth as a control group, calculate the change in absorbance value ΔB before and after the reaction, and calculate the ACE inhibition rate. The ACE inhibition rate (%) = [(ΔB - ΔA) / ΔB] × 100%. The results are as Figure 4 shown.

[0084] Figure 4 The results show that both the Example 1 group and the Comparative Examples 1-6 groups have a high ACE inhibition rate, and Example 1 has the best effect, indicating that the probiotic Lactobacillus acidophilus and Lactobacillus mucosae of the present invention can effectively inhibit the ACE activity. Using the fermentation medium of the present invention can further promote the ACE inhibitory activity. The composite probiotic preparation for assisting in reducing blood pressure of the present invention has a high ACE inhibition rate and good effect on assisting in reducing blood pressure.

[0085] Experimental Example 6: Select genetically hypertensive rats (SHR), randomly divide them into 7 groups with 8 rats in each group as the experimental groups (Groups 1-7), and select another 8 normal rats as a control group. Feed the experimental group and control group rats adaptively for one week in the same environment with the same standard, measure their blood pressure as the initial data and record it. The experimental group rats are fed with the probiotic preparations of Example 2 and Comparative Examples 1-6 at a dose of 20 mg / kg respectively, and the control group is fed with the probiotic preparation prepared in Example 1 at a dose of 20 mg / kg. After 15 consecutive days, measure the blood pressure value again and calculate the blood pressure reduction rate (%). The test results are shown in Table 3.

[0086] Table 3 Effect of probiotic preparation on assisting in reducing blood pressure

[0087]

[0088] Table 3 results show that the blood pressure reduction rate of Group 1 is significantly higher than that of Groups 2 - 7, indicating that the compound probiotic preparation for assisting in reducing blood pressure of the present invention has a good effect on assisting in reducing blood pressure. Moreover, when the probiotic preparation prepared by the present invention is fed to normal rats, there is no obvious change in the blood pressure of the rats, indicating that the probiotic preparation of the present invention has no reducing effect on normal blood pressure and is safer to use. In addition, no death or other adverse reactions occurred in the rats during the experiment. At the same time, the probiotic preparation of the present invention does not have hemolytic activity and has high biological safety.

[0089] In addition, the fermented liquid of Lactobacillus acidophilus and the fermented liquid of Lactobacillus mucosae reuteri prepared in Example 1 were respectively concentrated, freeze-dried, and fed to hypertensive rats, which were Group A and Group B respectively. In addition, Group C fed with the probiotic preparation of Example 1 was set up. After 15 days, the blood pressure was measured, and the blood pressure reduction rate (%) is shown in Table 4.

[0090] Table 4 Effect of probiotic preparation on assisting in reducing blood pressure

[0091]

[0092]

[0093] Table 4 results show that the blood pressure reduction rates of Group A and Group B are significantly lower than those of Group C, indicating that the effect of Group C on assisting in reducing blood pressure is better than that of Group A and Group B, indicating that the two probiotics of the present invention can have a synergistic effect, and the effect of assisting in reducing blood pressure is better than using only one of the probiotics alone.

[0094] Those of ordinary skill in the art should understand that: the discussion of any above embodiment is only exemplary and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; under the idea of the present invention, the technical features between the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, and they are not provided in detail for the sake of brevity.

[0095] The present invention aims to cover all such substitutions, modifications, and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A composite probiotic preparation for assisting in lowering blood pressure, characterized in that: The probiotic preparation is prepared by inoculating Lactobacillus acidophilus and Lactobacillus reuteri into a fermentation medium and fermenting the medium, wherein the fermentation medium comprises the following raw materials in parts by weight: 2-4 parts of pretreated corn silk, 4-8 parts of strawberry extract, 1-2 parts of a stabilizing promoter, 10-20 parts of an acidity regulator, 5-10 parts of glucose, 8-16 parts of skim milk powder, 3-6 parts of lactose, and 100-200 parts of deionized water; the Lactobacillus acidophilus and Lactobacillus reuteri are Lactobacillus acidophilus HX-LA15 and Lactobacillus reuteri HX-LRE501; The preparation method of the stabilizing accelerator comprises the following steps: (1) Wash and dry Sargassum fusiformis, crush and sieve to obtain algae powder, add it to distilled water, sonicate it, centrifuge it, take out the precipitate, add deionized water, water bath it at 100° C., filter it with suction, mix the filtrate with an equal volume of ethyl acetate, let it stand at 4° C., filter it with suction, and dry the filter cake to obtain product A; (2) mixing the product A obtained in step (1) with glycine and deionized water, stirring, dialyzing, and freeze-drying to obtain a stabilizing promoter; The preparation method of the acidic regulator comprises the following steps: (a) adding psyllium seed gum to a 1 wt % calcium chloride solution, and adding citric acid to adjust the pH value to obtain an outer phase solution; (b) dissolving malic acid in distilled water, adding chitosan, reacting at 100-110° C., standing at 4° C., centrifuging, washing the precipitate, and freeze-drying in vacuum to obtain modified chitosan; (c) adding the modified chitosan obtained in step (b) into distilled water, stirring at 80-90° C., cooling to 20° C., adding sodium bicarbonate, stirring to obtain an inner phase solution; (d) stirring the outer phase solution obtained in step (a) at 50° C. and 300 rpm, adding the inner phase solution obtained in step (c), continuing stirring, standing at 4° C., decanting the supernatant, and vacuum drying at 50° C. to obtain an acidity regulator; The preparation method of the pretreated corn silk is as follows: drying the corn silk, crushing and screening, mixing with distilled water, stirring, and spray drying to obtain the pretreated corn silk; the preparation steps of the strawberry extract are as follows: cutting the strawberry and grinding it into a mud, placing it in a constant temperature water bath of 45-55° C., adding it to deionized water, stirring, centrifuging, and vacuum drying the supernatant to obtain the strawberry extract; The method for preparing the composite probiotic preparation for assisting in lowering blood pressure comprises the following steps: S1: uniformly mixing the components of the fermentation medium according to weight, stirring, and sterilizing to obtain a fermentation medium; S2: Inoculate the activated Lactobacillus acidophilus and Lactobacillus reuteri into the fermentation medium obtained in step S1, respectively, with an inoculation amount of 1×10 6 CFU / mL, ferment at 37°C for 12h, cool to 28°C for 4h, and obtain Lactobacillus acidophilus and Lactobacillus reuteri fermentation broths, respectively. The broths were mixed in a volume ratio of 1:1, vacuum concentrated, and freeze-dried to obtain a composite probiotic preparation for assisting in lowering blood pressure.

2. The composite probiotic preparation for assisting in lowering blood pressure according to claim 1, characterized in that: The ratio of the algae powder to distilled water in step (1) is 1 g:100 mL; the ratio of the precipitate to deionized water in step (1) is 1 g:100 mL.

3. The composite probiotic preparation for assisting in lowering blood pressure according to claim 2, characterized in that: The dosage ratio of the product A, glycine and deionized water in step (2) is 1 g:0.8 g:40 mL.

4. The composite probiotic preparation for assisting in lowering blood pressure according to claim 3, characterized in that: The amount ratio of the psyllium seed gum to the calcium chloride solution in step (a) is 1g:10mL.

5. The composite probiotic preparation for assisting in lowering blood pressure according to claim 4, characterized in that: In step (b), the dosage ratio of malic acid, distilled water and chitosan is 1g:50mL:3g.

6. The composite probiotic preparation for assisting in lowering blood pressure according to claim 5, characterized in that: The usage ratio of the modified chitosan, distilled water and sodium bicarbonate in step (c) is 1 g:100 mL:0.5 g.

7. The composite probiotic preparation for assisting in lowering blood pressure according to claim 6, characterized in that: In step (d), the volume ratio of the outer phase solution to the inner phase solution is 2:1.

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