CN115537356A

CN115537356A - Compound probiotic with effect of preventing or relieving blood pressure increase and preparation method and application thereof

Info

Publication number: CN115537356A
Application number: CN202211241806.0A
Authority: CN
Inventors: 方曙光; 王命荣; 郭晓娟; 汪欣; 朱建国
Original assignee: WeCare Probiotics Co Ltd
Current assignee: WeCare Probiotics Co Ltd
Priority date: 2022-10-11
Filing date: 2022-10-11
Publication date: 2022-12-30
Anticipated expiration: 2042-10-11
Also published as: CN115537356B

Abstract

The invention provides a composite probiotic agent with the effect of preventing or relieving blood pressure rise and a preparation method and application thereof, wherein probiotics in the composite probiotic agent comprise a Lactobacillus plantarum Lp05 strain, a Lactobacillus casei LC89 strain and a Lactobacillus helveticus LH76 strain. The three strains are creatively compounded to obtain the composite probiotic preparation, all components are mutually matched and synergized synergistically, and compared with a single strain, the composite probiotic preparation has a more remarkable effect of relieving the increase of blood pressure. The composite probiotic can be further applied to the preparation of food, health-care products or medicines.

Description

Compound probiotic with effect of preventing or relieving blood pressure increase and preparation method and application thereof

Technical Field

The invention belongs to the technical field of probiotics, and relates to a compound probiotic with the efficacy of preventing or relieving blood pressure rise, and a preparation method and application thereof.

Background

Hypertension is the most common cardiovascular disease discovered so far, and is also the main pathogenesis of diseases such as sudden brain death, coronary heart disease and kidney failure, so the prevention and treatment of hypertension can reduce the risk of cardiovascular system diseases. At present, people pay more attention to research, prevention and treatment of hypertension, and the multifunctional food research in the aspect of reducing blood pressure plays an important role. Hypertension has been shown to be caused by a variety of causes, including neurological, genetic, psychiatric, endocrine, and Angiotensin Converting Enzyme (ACE), among others.

The mechanism of lowering blood pressure in the human body is generally regulated and controlled by a renin-angiotensin system and a kinin-regenerating enzyme system, which are antagonistic systems. In human body, after the renin flows into the blood plasma, angiotensinogen in the blood plasma is hydrolyzed to obtain capillary tensin I (AT 1); due to the function of angiotensin converting enzyme ACE, capillary tensin I is converted into capillary tensin II (Ang II), so that capillary tensin II can not only improve the contractile ability of the heart, but also can cause the high pressure increase caused by the contraction of vascular smooth muscle. At the same time, ACE also regulates the kallikrein system, leading to the conversion of bradykinin, which has a bradykinin function, into inactive sustained release peptides. ACE acts synergistically on these two systems, causing the rise of high pressure in the human body. To achieve a decrease in blood pressure, inhibition of ACE activity is necessary.

ACE can also directly activate adrenal cortex to release aldosterone, and the main action mechanism of aldosterone is that the excretion of salt and water by kidney is reduced, so that the extracellular fluid volume and the plasma volume of human body are increased, the venous return current is increased, and hypertension is indirectly induced. The protease produced by the lactobacillus plantarum cuts off partial active fragments and releases the active fragments at the same time, the partial active fragments are fused with the active center of ACE, the activity of the ACE is competitively controlled, angiotensin I cannot be converted into angiotensin II, and the purpose of reducing blood pressure is achieved.

The primary antihypertensive drugs proposed today are five major classes of beta-blockers, diuretics, angiotensin converting enzyme inhibitory drugs (ACEIs), calcium channel blockers, angiotensin ii dual receptor binding inhibitors, and the like. Each drug has its own characteristics and also has certain side effects.

The ACEI has certain curative effect on hypertension patients in all periods, does not cause the increase of heart rate or interfere the regulation effect of a parasympathetic nervous system on vascular smooth muscle, but has the main adverse reaction that proteinuria, rash, leukopenia and the like are easy to generate through synthesized ACEI. Treatment with antihypertensive drugs is responsible for an increased incidence of coronary heart disease; alterations in metabolism, particularly potentially adverse effects on sugar, fat and uric acid metabolism, are affected during drug therapy; the probability of cerebral blood reduction caused by blood pressure reduction caused by drug therapy is increased, and visual disturbance, cerebral ischemia, cerebral arterial thrombosis and the like can be caused at the same time. Therefore, the non-drug treatment of hypertension is gaining more and more attention.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a composite probiotic with the efficacy of preventing or relieving blood pressure rise and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a composite probiotic preparation with the efficacy of preventing or relieving blood pressure increase, wherein probiotics in the composite probiotic preparation comprise Lactobacillus plantarum Lp05 strain, lactobacillus casei LC89 strain and Lactobacillus helveticus LH76 strain.

The Lactobacillus plantarum Lp05 strain, the Lactobacillus casei LC89 strain and the Lactobacillus helveticus LH76 strain are creatively compounded to obtain the composite probiotic agent, all components are mutually matched for synergy, and compared with a single strain, the composite probiotic agent has a more remarkable effect of relieving the increase of blood pressure.

Preferably, the viable count ratio of the Lactobacillus plantarum Lp05 strain, the Lactobacillus casei LC89 strain and the Lactobacillus helveticus LH76 strain is (10-20): (5-10): (15-30).

The specific point value in the (10-20) can be selected from 10, 12, 14, 15, 16, 17, 18, 20 and the like.

The specific point value in (5-10) can be selected from 5, 6, 7, 8, 9, 10 and the like.

The specific point value in (15-30) can be selected from 15, 17, 18, 20, 22, 25, 28, 30 and the like.

Other specific values within the above ranges can be selected, and are not described in detail herein.

Preferably, the complex probiotic also comprises a protective agent.

Preferably, the protectant comprises skim milk and/or skim milk powder.

Preferably, the complex probiotic further comprises a carrier.

Preferably, the carrier comprises any one of microcrystalline cellulose, inulin, magnesium stearate, a sugar alcohol or starch, or a combination of at least two thereof.

Preferably, the carrier is a combination of microcrystalline cellulose, a sugar alcohol, inulin and starch.

The combination of microcrystalline cellulose, sugar alcohol, inulin and starch is more preferable as the complex probiotic agent is more stable in this combination mode, and can maintain the shape and mouthfeel of the product for a long time.

Preferably, the sugar alcohol is selected from any one of xylitol, maltitol, erythritol, sorbitol, isomalt or lactitol or a combination of at least two thereof.

Preferably, the starch is selected from any one of or a combination of at least two of acetate starch, sodium starch octenyl succinate, hydroxypropyl distarch phosphate, phosphate distarch, acid-treated starch, oxidized hydroxypropyl starch, acetylated distarch phosphate or acetylated distarch adipate.

The dosage form of the composite probiotic preparation can be freeze-dried powder, tablets, capsules, solutions, emulsions and the like.

In a second aspect, the present invention provides a method for preparing a complex probiotic with efficacy of preventing or alleviating blood pressure increase according to the first aspect, the method comprising:

(1) Mixing Lactobacillus plantarum Lp05, lactobacillus casei LC89 and Lactobacillus helveticus LH76 with a protective agent according to a ratio to obtain an emulsion;

(2) Mixing the emulsion obtained in the step (1) with a carrier, and carrying out micro-embedding treatment to obtain an embedding mixture;

(3) And (3) carrying out electrostatic spray drying on the embedding mixture obtained in the step (2) to obtain the composite probiotic preparation with the effect of preventing or relieving the blood pressure rise.

In a third aspect, the invention provides the use of the complex probiotic with the effect of preventing or relieving blood pressure increase in the preparation of food, health products or medicines.

In a fourth aspect, the invention provides a probiotic beverage, which comprises the following preparation raw materials in percentage by mass: 0.01-10% of composite probiotic agent, 0.01-5% of prebiotics, 0.1-1% of stabilizing agent, 0.01-0.05% of sweetening agent and water.

The mass percentage of the composite probiotic can be selected from 0.01%, 0.1%, 1%, 2%, 3%, 5%, 7%, 8%, 10% and the like.

The prebiotics can be selected from 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 3.5%, 4%, 5% and the like in percentage by mass.

The mass percentage of the stabilizer can be selected from 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 1% and the like.

The mass percentage of the sweetener can be selected from 0.01%, 0.02%, 0.03%, 0.04%, 0.05% and the like.

Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.

Preferably, the prebiotics are selected from any one or a combination of at least two of stachyose, fructo-oligosaccharide, isomalto-oligosaccharide or galacto-oligosaccharide.

Preferably, the sweetener is selected from any one of acesulfame k, aspartame, sucralose or steviol glycosides or a combination of at least two thereof.

Preferably, the stabilizer is selected from any one or a combination of at least two of sodium carboxymethylcellulose, pectin, soybean polysaccharide, agar or sodium caseinate; a combination of pectin and soy polysaccharide is preferred.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively compounds three strains of Lactobacillus plantarum Lp05 strain, lactobacillus casei LC89 strain and Lactobacillus helveticus LH76 strain to obtain the composite probiotic preparation, and the components are mutually matched for synergism.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

(1) The Lactobacillus plantarum Lp05 related to the following is Lactobacillus plantarum Lp05 strain, the preservation unit is China general microbiological culture Collection center (CGMCC) No.23547, the preservation date is 2021 year, 10 month and 09 day, and the preservation address is No. 3 of Xilu No.1 Beicheng sunward area, beijing.

(2) The Lactobacillus helveticus LH76 is Lactobacillus helveticus LH76 strain, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.18796, the preservation date is 11, 4 and 2019, and the preservation address is No. 3 of Xilu No.1 of the morning district of Beijing.

(3) The Lactobacillus casei LC89 is Lactobacillus casei LC89 strain, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.15409, the preservation date is 03-05 days in 2018, and the preservation address is Beijing City Zhongyang district Xilu No.1 Beijing No. 3.

The following related MRS solid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K ₂ PO ₄ ·3H ₂ O 2g、MgSO ₄ ·7H ₂ O 0.6g、MnSO ₄ Dissolving 0.01g of agar, 20g of agar and 1g of L-cysteine by using deionized water, adding 1mL of Tween 80, diluting to a constant volume of 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use.

The following related MRS liquid culture media: weighing 10g of peptone, 10g of beef extract, 20g of glucose, 10g of lactose, 5g of yeast extract, 2g of diammonium hydrogen citrate and K ₂ PO ₄ ·3H ₂ O 2g、MgSO ₄ ·7H ₂ O 0.6g、MnSO ₄ 0.01g and 1g of L-cysteine, dissolving with deionized water, adding 1mL of Tween 80, diluting to 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use.

The following bacterial suspensions of lactobacillus plantarum Lp05, lactobacillus helveticus LH76 and lactobacillus casei LC89 are involved: inoculating the bacteria in an MRS liquid culture medium, culturing at 38 ℃ for 18h for activation, and continuously activating for 2 times to obtain an activated solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at 38 ℃ for 18h to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000g for 10min, and filtering the supernatant with 0.22 μm sterile filter membrane to obtain supernatant; and (5) resuspending the thallus by using PBS.

The sources of the components in the following examples are as follows:

example 1

This example demonstrates the gastric acid resistance of three bacteria, with the following steps:

(1) Preparing artificial gastric juice:

the artificial gastric juice contains 0.20% of NaCl and 0.30% of pepsin by mass fraction, the pH is adjusted to 3.0 by using HCl, and the artificial gastric juice is filtered and sterilized for later use.

(2) And (3) testing gastric acid resistance:

taking 1.0mL of Lactobacillus plantarum Lp05, lactobacillus helveticus LH76 and Lactobacillus casei LC89 bacterial suspension (the concentration is 1 × 10) ⁹ CFU/mL, adopting the method in national standard GB4789.35-2016 food safety national standard food microbiology detection of lactic acid bacteria detection) to measure the concentration of bacterial liquid), respectively mixing with 9.0mL of artificial gastric juice with pH of 3.0, carrying out anaerobic static culture at 37 ℃, respectively sampling after 0h of starting and 3h of treatment, determining the viable count by a pouring culture method and calculating the survival rate, wherein the formula is as follows:

survival (%) = N1/N0 × 100%,

wherein, N1: viable count after 3h of artificial gastric juice treatment; n0: viable count of 0 h. The test results are shown in Table 1.

TABLE 1

As can be seen from the data in Table 1: the probiotics provided by the invention have good gastric acid resistance, and the good acid resistance creates conditions for preparing products for preventing or relieving hypertension.

Example 2

This example demonstrates the bile salt resistance of three bacteria, as follows:

adjusting the content of the bacterial liquid to 10 ⁸ centrifuging at 3000r/min for 10min at cfu/ml, removing supernatant, adding sterilized MRS liquid culture medium containing 0.3% bile salt into the centrifuged thallus, shaking uniformly, placing into a 37 ℃ constant temperature incubator, continuously taking samples within 0h and 3h, diluting with sterilized PBS, coating on an MRS plate, incubating at 37 ℃ for 36h, and counting. The formula is as follows:

survival (%) = N1/N0 × 100%,

wherein, N1: the number of viable bacteria after 3 hours of treatment; n0: viable count of 0 h. The test results are shown in Table 2.

TABLE 2

Bacterial strains	Viable count N0 (0 h)	Viable count N1 (3 h)	Survival rate (%)
				Lactobacillus plantarum Lp05	(7.98±0.06)×10 ⁸	(7.04±0.10)×10 ⁸	90.3％
Lactobacillus helveticus LH76	(7.25±0.12)×10 ⁸	(6.56±0.08)×10 ⁸	90.5％
				Lactobacillus casei LC89	(7.16±0.09)×10 ⁸	(6.51±0.13)×10 ⁸	91.0％

From the data in table 2 it can be seen that: the probiotics provided by the invention have good bile salt resistance, and the good bile salt resistance creates conditions for preparing products for preventing or relieving hypertension.

Example 3

The embodiment provides a composite probiotic with the efficacy of preventing or relieving blood pressure increase, and the preparation method comprises the following steps:

(1) Mixing 40 parts of bacterial powder (the viable count of Lactobacillus plantarum Lp05, lactobacillus casei LC89 and Lactobacillus helveticus LH76 is 15);

(2) Mixing the emulsion obtained in the step (1) with a carrier (5 parts of microcrystalline cellulose, 5 parts of sugar alcohol, 10 parts of inulin and 10 parts of starch), and carrying out micro-embedding treatment to obtain an embedding mixture;

Example 4

The embodiment provides a composite probiotic with the efficacy of preventing or relieving blood pressure increase, and the preparation method is different from the embodiment 3 only in that 40 parts of Lactobacillus plantarum Lp05 and Lactobacillus casei LC89 are prepared from Lactobacillus plantarum with the viable count of 15. The other steps were kept unchanged.

Example 5

The embodiment provides a composite probiotic preparation with the efficacy of preventing or relieving blood pressure increase, and the preparation method is different from the embodiment 3 only in that 40 parts of Lactobacillus plantarum Lp05 and Lactobacillus helveticus LH76 with the viable count of bacterial powder being 15. The other steps were kept unchanged.

Example 6

The embodiment provides a composite probiotic preparation with the efficacy of preventing or relieving blood pressure increase, and the preparation method is different from the embodiment 3 only in that 40 parts of Lactobacillus casei LC89 and Lactobacillus helveticus LH76 with the viable count of 5. The other steps were kept unchanged.

Example 7

This example provides a complex probiotic with efficacy of preventing or alleviating blood pressure elevation, and the preparation method differs from example 3 only in that the carriers in step (2) are 10 parts of sugar alcohol, 10 parts of inulin and 10 parts of starch. The other steps were kept unchanged.

Example 8

The present example provides a complex probiotic with efficacy of preventing or alleviating blood pressure increase, and the preparation method is different from example 3 only in that the carriers in step (2) are 10 parts of microcrystalline cellulose, 10 parts of inulin and 10 parts of starch. The other steps were kept unchanged.

Example 9

The present example provides a composite probiotic preparation with efficacy of preventing or alleviating blood pressure increase, and the preparation method is different from example 3 only in that the carrier in step (2) is 5 parts of microcrystalline cellulose, 5 parts of sugar alcohol, and 20 parts of starch. The other steps were kept unchanged.

Example 10

The present example provides a composite probiotic preparation with efficacy of preventing or alleviating blood pressure increase, and the preparation method is different from example 3 only in that the carriers in step (2) are 5 parts of microcrystalline cellulose, 5 parts of sugar alcohol, and 20 parts of inulin. The other steps were kept unchanged.

Test example 1

And (3) measuring the blood pressure reducing effect of the composite probiotic:

(1) Experimental animals: male primary Severe Hypertension Rats (SHR), about eight weeks old, with an average weight of 170-210 g, supplied by the laboratory animal management center in shanghai city, department of china, animal certification number: hu Dong He Zheng character 152. Male SD rats, 8 weeks old, weighing 170-210 g, were supplied by the laboratory mammal management center, shanghai city, chinese academy of sciences, and the animals met the qualifications number: hu Dong He Zheng character 152. The indoor illumination of the animals is 12h illumination, indoor ventilation and air conditioning equipment is normal, the room temperature is controlled to be 22 +/-3 ℃, and the relative humidity is about 50-70%.

(2) The experimental animal is adapted to the environment for 7 days, adaptive manometry is carried out, continuous gavage is started after the blood pressure is stable, and the experimental gavage time is 9 a.m. every day. 40 SHR rats were randomly divided into 5 groups: model control, example 3, example 4, example 5, example 6; 8 SD rats were used as SD control group. The normal saline for gastric lavage of the control group, the composite probiotic prepared in each example for gastric lavage of the example group are 10mg/kg.

(3) The rat carries out pressure test one week before administration, and tests are carried out after the pressure reduction is stable, wherein the pressure reduction before administration is firstly measured, then the rat is subjected to intragastric administration, the intragastric administration is carried out once a day, the intragastric administration is continuously carried out for 4 weeks, the high pressure of the tail artery of the SHR is measured by using a BP-98A intelligent noninvasive sphygmomanometer, the steps are repeated for three times, an average value is obtained, and the measured value is displayed in the form of the average value. As shown in table 3.

TABLE 3

From the data results of table 3, it can be seen that: the composite probiotic preparation has the obvious effect of relieving the blood pressure rise, and the lactobacillus plantarum Lp05 strain, the lactobacillus casei LC89 strain and the lactobacillus helveticus LH76 strain in the composite probiotic preparation have the synergistic effect on the effect.

Application example 1

The application example provides a probiotic beverage, and the preparation raw materials of the probiotic beverage comprise the following components in percentage by mass: 5% of the composite probiotic prepared in the embodiment 3, 5% of fructo-oligosaccharide, 0.5% of pectin, 0.5% of soybean polysaccharide, 0.04% of sucralose and the balance of water.

Application examples 2 to 8

The application example provides seven probiotic beverages, the preparation raw materials of the probiotic beverage are different from those of the application example 1 only in that the composite probiotic prepared in the example 3 is replaced by the same amount of the composite probiotic prepared in the examples 4 to 10, and other raw materials and content are kept unchanged.

Test example 2

Testing of stability of probiotic beverages:

the probiotic beverages of application examples 1-8 were stored refrigerated at 4 ℃ for 21 days and the state of the tissue was visually observed. The products of each group are graded, wherein the layering phenomenon is obvious when the product is graded, the layering phenomenon is not obvious when the product is graded, and the product is very uniform when the product is not layered when the product is graded. The results are shown in Table 4.

TABLE 4

Sample (I)	Score the points
		Application example 1	3
Application example 2	3
		Application example 3	3
Application example 4	3
		Application example 5	2
Application example 6	1
		Application example 7	1
Application example 8	2

From the results of table 4, it can be seen that: the beverage product prepared by using the composite probiotic disclosed by the invention has good stability, wherein the stability of the beverage product is obviously influenced by the composition mode of the carrier material in the composite probiotic.

The applicant states that the present invention is described by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of the raw materials of the product of the present invention, and the addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that, in the above embodiments, the various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present invention does not separately describe various possible combinations.

Claims

1. The composite probiotic preparation with the effect of preventing or relieving the blood pressure rise is characterized in that probiotics in the composite probiotic preparation comprise Lactobacillus plantarum Lp05 strain, lactobacillus casei LC89 strain and Lactobacillus helveticus LH76 strain.

2. The complex probiotic preparation with the effect of preventing or relieving blood pressure increase according to claim 1, wherein the viable count ratio of Lactobacillus plantarum Lp05 strain, lactobacillus casei LC89 strain and Lactobacillus helveticus LH76 strain is (10-20): (5-10): (15-30).

3. The complex probiotic with the efficacy of preventing or alleviating blood pressure elevation according to claim 1 or 2, characterized in that the complex probiotic further comprises a protective agent;

preferably, the protective agent comprises skim milk and/or skim milk powder.

4. The complex probiotic with the efficacy of preventing or alleviating blood pressure elevation according to any one of claims 1 to 3, characterized in that the complex probiotic further comprises a carrier;

preferably, the carrier comprises any one or a combination of at least two of microcrystalline cellulose, inulin, magnesium stearate, a sugar alcohol or starch;

5. The complex probiotic preparation with the efficacy of preventing or alleviating blood pressure increase according to claim 4, characterized in that the sugar alcohol is selected from any one or a combination of at least two of xylitol, maltitol, erythritol, sorbitol, isomalt or lactitol;

6. The preparation method of the complex probiotic with the efficacy of preventing or alleviating blood pressure increase according to any one of claims 1 to 5, wherein the preparation method comprises the following steps:

7. Use of the complex probiotic preparation with the effect of preventing or alleviating blood pressure increase according to any one of claims 1 to 5 in the preparation of food, health products or medicines.

8. The probiotic beverage is characterized by comprising the following preparation raw materials in percentage by mass: the complex probiotic preparation of any one of claims 1 to 5, 0.01 to 10 percent of prebiotics, 0.01 to 5 percent of stabilizer, 0.1 to 1 percent of sweetening agent, 0.01 to 0.05 percent of water.

9. The probiotic beverage according to claim 8, characterized in that the prebiotic is selected from any one of stachyose, fructo-oligosaccharide, isomalto-oligosaccharide or galacto-oligosaccharide or a combination of at least two thereof;

10. The probiotic beverage according to claim 8, characterized in that the stabilizer is selected from any one of or a combination of at least two of sodium carboxymethylcellulose, pectin, soy polysaccharide, agar or sodium caseinate; a combination of pectin and soy polysaccharide is preferred.