CN107929329B - A traditional Chinese medicine granule for reducing blood fat and lowering fat and probiotic compound bacteria and preparation method thereof - Google Patents

Abstract

A Chinese medicinal granule with thrombolytic, lipid-lowering and probiotic effects and its preparation method are provided. According to the invention, 3 strains with thrombus eliminating and lipid reducing effects are separated and purified from samples such as Lanzhou slurry water, tibetan mastiff intestinal tracts, beer residues and the like, and are subjected to high-density culture to prepare mixed bacterial suspension, wherein the bacterial densities in the mixed bacterial suspension are respectively as follows: lactic acid bacteria not less than 10 ¹⁶ CFU/mL, saccharomycetes not less than 10 ¹⁴ CFU/mL and bifidobacterium cell density not less than 10 ¹⁰ CFU/mL; then the traditional Chinese medicine for promoting blood circulation and dredging collaterals is used as an auxiliary material carrier and the acid-resistant sodium carboxymethyl cellulose (CMC-Na) of the adhesive to prepare the traditional Chinese medicine for eliminating thrombus and reducing lipid probioticsAnd (5) preparing a granule finished product. The invention has the functions of high efficiency thrombolysis and lipid lowering, can obviously lower serum cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterol (LDL-C), has strong acid resistance and bile salt resistance, has no toxic or side effect, and simultaneously has the effects of regulating intestinal flora and inhibiting the growth of harmful microorganisms.

Description

A traditional Chinese medicine granule for reducing blood fat and lowering blood fat and probiotic compound bacteria and preparation method thereof

【Technical field】

The invention relates to the field of medicine, in particular to a traditional Chinese medicine granule containing compound bacteria and a preparation method thereof, which have the effect of eliminating thrombosis, lowering blood fat and probiotics.

【Background technique】

Cholesterol is an essential nutrient for the human body, but due to the improvement of living standards, the content of cholesterol in human serum is often too high, and even becomes the primary factor leading to death. At present, nearly one-third of adults in my country have high blood lipids. Hyperlipidemia has become the first invisible killer of human health. With the improvement of living standards, obesity has gradually become another important disease that seriously endangers people's health. . The number of overweight and obese people in the country is 200 million and 60 million respectively. Obesity affects the normal physiological functions of the whole body, and can increase the risk of diseases such as atherosclerosis, hypertension, diabetes, coronary heart disease, tumors, sexual dysfunction, and osteoarthritis. A large number of epidemiological and clinical observations show that obese patients Often suffer from hyperlipidemia at the same time, and hyperlipidemia is one of the main risk factors for inducing cardiovascular and cerebrovascular diseases. Most patients with hyperlipidemia are complicated with hypertension, coronary heart disease, cerebral arteriosclerosis and other diseases. The medical field has called the hypertension, hyperlipidemia, diabetes, coronary heart disease, and cerebral apoplexy often accompanied by obesity as "the death five". Replay".

With the progress of society and the enhancement of people's health awareness, it is imminent to lose weight and reduce fat. At present, statins are mainly used to control hyperlipidemia, but these drugs have serious side effects, which can easily cause side effects such as myalgia, headache, gastrointestinal discomfort, blood lipid disorders, and liver and kidney damage. Therefore, the development and utilization of non-toxic and harmless health-care medicines with fat-reducing and fat-reducing effects have important practical significance for protecting human health. As a "living green drug", probiotics have been proved by research to have many advantages such as high efficacy, safety, and conformity to ecology, and have been increasingly used in various fields of human diseases.

There are a large number of paper reports and patent achievements at home and abroad on the research on probiotics in lowering cholesterol. CN101864375A Lactobacillus plantarum (C88), CN102604851A Lactococcus lactis, CN101559082A Lactobacillus (MA2), CN101724589A Lactobacillus, CN1186855A Urinary Streptococcus (DM891128) and Lactobacillus rhamnosus (grx10) all have certain effects in lowering cholesterol , but the cell density is between 10 ⁸ and 10 ¹⁰ CFU/mL, and the effect of a single strain is not good, and it can play a role in health care but cannot achieve the purpose of adjuvant therapy; CN101361506A adopts lactic acid bacteria (GL-03), Three kinds of bacteria, Lactobacillus bulgaricus and Streptococcus thermophilus, with a cell density of 10 ⁶ CUF/mL; CN101406489A uses four types of bacteria, Bacillus natto, lactic acid bacteria, yeast and Lactobacillus acidophilus, with a cell density of 10 ⁷ to 10 ¹⁰ CUF /mL; CN101293001A uses four types of bacteria: photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes, and the density of the bacteria is 10 ⁶ ~ 10 ⁸ CUF/mL; although the composite bacteria has a better cholesterol-lowering effect than a single strain, However, due to the low density of bacteria, it also fails to achieve the effect of adjuvant therapy, and the lipid-lowering effect is not ideal. In addition, reports on specific strains in lowering triglycerides are rare.

【Content of invention】

The problem to be solved by the present invention is aimed at the deficiencies in the prior art, and provides a kind of traditional Chinese medicine granules containing complex bacteria for eliminating embolism, lowering fat and probiotics and a preparation method thereof.

Technical scheme of the present invention is as follows:

A method for preparing traditional Chinese medicine granules of probiotic compound bacteria for reducing blood clots, characterized in that the compound bacteria contain two strains of lactic acid bacteria, which are respectively Lactobacillus pentosus (La.Pentosus) isolated from the intestinal tract of Tibetan Mastiff. Preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on 07th, the preservation number is CGMCC No.7426, and the classification is named: Lactobacillus pentosus, and Lactobacillus plantarum; Yarrowia lipolytica strain isolated from brewer's residue , the strain was deposited in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures on April 07, 2013. The preservation number is CGMCC No.7427, and the classification name is: Yarrowia lipolytica. The preservation address is No. 3, Yard No. 1, Beichen 4th Road, Chaoyang District, Beijing, China.

Among them, the source of Lactobacillus plantarum is not particularly limited in the present invention, but it is preferably isolated from Lanzhou pulp water, preferably the Lactobacillus plantarum isolated from Lanzhou pulp water provided by the present invention, and the bacterial strain has been preserved on April 07, 2013 In the General Microbiology Center of China Microbiological Culture Collection Management Committee, its preservation number is CGMCC No.7425, and its classification is named: Lactobacillus plantarum, which is named EAM-ZL010T in this manual. The preservation address is No. 3, Yard No. 1, Beichen 4th Road, Chaoyang District, Beijing, China

The traditional Chinese medicine granules for reducing blood fat, lowering fat and probiotics contain traditional Chinese medicines for promoting blood circulation and dredging veins, preferably including Danshen, Angelica, Chuanxiong, Panax notoginseng and/or safflower.

The yeast isolated from brewer's dregs is Yarrowia lipolytica strain, which is isolated by spreading brewer's dregs on McFarland's medium. The colony of the pure strain is milky white, and the shape of the microscopic examination is oval. Molecular identification of the purified strains was carried out by 26SrDNA method. Its 26SrDNA sequence was compared in the NCBI (National Center for Biotechnology Information, National Center for Biotechnology Information of the United States) database, and it was found that the gene sequence of the strain, in all similar sequences, was similar to that of Yarrowia lipolytica strain (Yarrowia lipolytica) The homology of the strain is 100%. Therefore, combining the physiological and biochemical characteristics, the strain is identified as Yarrowia lipolytica strain, which is named EAM-ZT003T in the present invention. The detailed content has applied for a Chinese invention patent, application number: 201710091197.8.

Lactobacillus pentosus (La.Pentosus) is a lactic acid bacterium isolated from the intestinal tract of Tibetan Mastiff.

The intestinal contents of the Tibetan mastiff were spread on the MRS medium for isolation and culture. The colonies of pure strains were milky white, about 2mm in diameter, with smooth surfaces, neat edges, and a raised center. There were obvious calcium-dissolving circles around the colonies. Microscopic examination It is short rod-shaped, and the purified strains will be subjected to Gram staining, plate colony morphology, degradation rate of the strains to cholesterol in egg yolk and cholesterol standards, degradation rate to triglycerides, acid resistance test, bile salt resistance test, toxicity Experiment (mice) and other experiments. The purified strains were identified by 16SrDNA method. Its 16SrDNA sequence was compared in the NCBI (National Center for Biotechnology Information, National Center for Biotechnology Information, USA) database, and it was found that the gene sequence of the bacterial strain, in all similar sequences, was similar to that of Lactobacillus pentosus (La.Pentosus). The homology is 99%. Therefore, based on the physiological and biochemical characteristics, the identified strain is Lactobacillus pentosus, which is named EAM-ZL016T in the present invention. The detailed content has applied for a Chinese invention patent, application number: 201710091198.2.

The present invention is not particularly limited to the source of the bifidobacterium, and the source of the bifidobacterium can be obtained commercially.

The invention also provides a preparation method of the mixed bacteria suspension. The preparation of the mixed bacterial suspension includes at least the following steps:

a. Mix the purified two strains of lactic acid bacteria uniformly at a volume ratio of 1 to 3:1 to 3, then inoculate them in the MRS medium with an inoculum of 5% to 10% by volume, and ferment and culture at a constant temperature of 35 to 37°C for 36 to After 90 hours of fermentation, the density of lactic acid bacteria is not less than 10 ¹⁶ CFU/mL; the purified yeast strains are inoculated in McFarland medium with an inoculum of 5% to 10% by volume, at 32 to 37°C, 120 to 150r /min shaking table fermentation culture for 72-96 hours, the yeast cell density after fermentation is not less than 10 ¹⁴ CFU/mL; inoculate the bifidobacteria in the MRS medium at 35-37℃ with an inoculum of 5%-10% by volume Fermentation at constant temperature for 72-96 hours, and the cell density after fermentation is not less than 10 ¹⁰ CFU/mL;

b. Centrifuge the three fermentation broths in a at low temperature at 4-8°C respectively, and resuspend the bacteria with distilled water 1-3 times the volume of the fermentation broth to obtain a single-cell bacterial suspension. The cell bacteria suspension is mixed evenly according to the volume ratio of lactic acid bacteria: yeast: bifidobacteria = 1-3:1-2:1 to prepare a mixed bacteria suspension.

The formula of the MRS liquid medium is: peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, anhydrous sodium acetate 5g, Tween-801mL, diamine citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, distilled water 1000mL, pH6.6～6.8, 115 ℃, sterilization 20min; Anhydrous sodium acetate 8.2g, distilled water 1000mL, sterilize at 113°C for 20min.

The inventor adopts high-density culture of bacteria, so that the highest density of lactic acid bacteria and yeast can reach 10 ¹⁶ CFU/mL and 10 ¹⁴ CFU/mL respectively, and the specific strains with high cholesterol-lowering and triglyceride-lowering Compatible use of bacterial strains reduces triglyceride content while lowering cholesterol.

In order to cooperate with the curative effect, the antithrombotic and lipid-lowering probiotic compound traditional Chinese medicine granules recommended by the present invention contain at least traditional Chinese medicines with the functions of promoting blood circulation, removing blood stasis and/or relieving pain, especially the ingredients of Danshen, Angelica, Chuanxiong, Panax notoginseng and/or safflower .

The present invention also provides a more specific method for preparing traditional Chinese medicine granules of antithrombotic and lipid-lowering probiotic compound bacteria. The preparation method at least includes the following steps:

In terms of parts by weight, the total weight of traditional Chinese medicine is 10 parts, containing at least 0-5 parts of salvia miltiorrhiza, 0-5 parts of angelica, 0-5 parts of Chuanxiong, 0-3 parts of notoginseng and 0-3 parts of safflower. The number of parts cannot be taken as zero at the same time. The traditional Chinese medicines are selected separately, crushed into fine powder, and then mixed; finally, 3-5% of the total weight of Chinese medicines and 10%-50% of the mixed bacterial suspension are added to the mixed powder of Chinese medicines. , and granulate under aseptic state to obtain the traditional Chinese medicine granules of Xiaoshun, lipid-lowering and probiotic complex bacteria, which are dried in cold air and stored in a refrigerator at 0-4°C.

The binder of the present invention is preferably acid-resistant carboxymethylcellulose sodium (CMC-Na).

The traditional Chinese medicine granules containing complex bacteria prepared by the preparation method are traditional Chinese medicine granules containing antithrombotic and lipid-lowering probiotic compound bacteria.

The present invention also provides the best traditional Chinese medicine granules composed of antithrombotic, lipid-lowering and probiotic compound bacteria, the weight of which is 5-5.9g/100 grains, and every 20 grains contains lactic acid bacteria ≥ 400×10 ¹³ CFU and yeast ≥ 600×10 ¹² CFU , Bifidobacteria ≥ 400×10 ⁷ CFU.

The present invention adopts the main advantages and effects of the above technical solutions as follows:

(1) The antithrombotic and lipid-lowering probiotic compound traditional Chinese medicine granules prepared by the present invention have efficient anti-thrombotic and lipid-lowering functions, and can significantly reduce serum cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL- C), it has strong acid resistance and bile salt resistance, and also has the function of regulating intestinal flora, which can effectively inhibit the growth of harmful microorganisms, and has no toxic and side effects.

(2) The total number of viable lactic acid bacteria in the mixed bacterial preparation of the present invention is ≥10 ¹⁶ CFU/mL, the total number of viable yeast bacteria is ≥10 ¹⁴ CFU/mL, and the total number of viable bifidobacteria is ≥10 ¹⁰ CFU/mL, which can ensure intestinal There are a large number of bacteria living in the tract. The traditional Chinese medicine is used as an auxiliary material for granulation, which can play an auxiliary therapeutic role for the compound bacteria to eliminate blood clots and lower blood lipids.

(3) The probiotic compound bacterium of the present invention can play the role of regulating the intestines, clearing the intestines and defecating in the intestinal tract, and inhibiting the growth and reproduction of harmful bacteria in the intestinal tract.

(4) The antithrombotic and lipid-lowering probiotic compound bacterium traditional Chinese medicine granule of the present invention should not be taken together with antibacterial drugs. This product is live bacterial traditional Chinese medicine granules and should be stored in a refrigerator at 0-4°C. Do not place this product at high temperature.

【Description of drawings】

Accompanying drawing is the experimental data in the concrete embodiment of application of the present invention.

Fig. 1 is the degradation rate of cholesterol in egg yolk by different bacterial strains of the present invention.

Fig. 2 is the degradation rate of triglyceride by different bacterial strains of the present invention.

Fig. 3 is the acid resistance test result of different bacterial strains of the present invention.

Fig. 4 is the test result of bile salt resistance of different bacterial strains of the present invention.

Fig. 5 is the test result of the traditional Chinese medicine granules of the present invention on the TG content in mouse serum.

Fig. 6 is the test result of the traditional Chinese medicine granules of the present invention on the LDL-C content in mouse serum.

Fig. 7 is the test result of the traditional Chinese medicine granule of the present invention on the TC content in mouse serum.

Fig. 8 is the result of mouse toxicity test (liver tissue section) using a compound bacteria of the present invention.

Fig. 9 is the result of mouse toxicity test (kidney tissue section) using a composite bacteria of the present invention.

【Detailed ways】

The present invention will be described in detail below in conjunction with specific embodiments.

The basic information of the three types of bacteria are shown in Table 1.

Table 1 The basic information of the three types of bacteria with lipid-lowering and cholesterol-lowering effects

The two strains of lactic acid bacteria are: lactic acid bacterium EAM-ZL010T, which is classified as Lactobacillus plantarum (Lactobacillus plantarum), and whose preservation number is CGMCC7425; The content has applied for a Chinese invention patent, application number: 201710091198.2); the yeast strain described is: yeast EAM-ZT003T, which is named as Yarrowia lipolytica (Yarrowia lipolytica), and the preservation number is CGMCC7427 (details have been Apply for a Chinese invention patent, application number: 201710091197.8). The above strains have been preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on April 7, 2013.

Lactobacillus plantarum (Lactobacillus plantarum) isolated from Lanzhou pulp water, the strain has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on April 07, 2013, and its preservation number is CGMCC7425, named as EAM-ZL010T. The specific method is: apply Lanzhou slurry water on the MRS medium for separation and culture, and the colony of the pure strain is milky white, with a diameter of about 2 mm, smooth surface, neat edges, and a raised center. There are obvious calcium-dissolving circles around the colony. The tested strains are short rod-shaped (1-1.2) μm × (1-1.5) μm, single, short-chain arrangement, Gram stain positive, facultative anaerobic. Through the test, it was found that the strain has a very high degradation effect on cholesterol in egg yolk and cholesterol standards, and also has a degradation effect on triglycerides. The toxicity test (mice) found that the strain has no toxic and side effects. The purified strains were identified by 16SrDNA method. Its 16SrDNA sequence was compared in the NCBI (National Center for Biotechnology Information, National Center for Biotechnology Information) database, and it was found that the gene sequence of the bacterial strain, among all similar sequences, was homologous to Lactobacillus plantarum The sexiness was 99%, so combining the physiological and biochemical characteristics, the strain was identified as Lactobacillus plantarum.

The formula of the MRS liquid medium is: peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, anhydrous sodium acetate 5g, Tween-801mL, diamine citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, distilled water 1000mL, pH6.6～6.8, 115 ℃, sterilization 20min; Anhydrous sodium acetate 8.2g, distilled water 1000mL, sterilize at 113°C for 20min.

Example 1

(1) Preparation of mixed bacterial agent

a. Mix the purified two strains of lactic acid bacteria evenly at a volume ratio of 1:1, then inoculate them in MRS medium at a volume ratio of 10%, and ferment and culture at a constant temperature of 37°C for 72 hours. After the fermentation, the density of lactic acid bacteria is not less than 10 ¹⁶ CFU/mL; inoculate the purified yeast strains into McFarland medium at an inoculum volume of 10% by volume, and ferment and culture on a shaker at 32°C and 120r/min for 96 hours. After fermentation, the yeast cell density is not less than 10 ¹⁴ CFU/mL; Bifidobacteria (purchased from pharmacies) were inoculated into MRS medium at a constant temperature of 37°C for 72 hours at an inoculum volume of 10% by volume. After the fermentation, the cell density was not less than 10 ¹⁰ CFU/mL;

b. Centrifuge the three kinds of fermentation broths in a respectively at low temperature, take the bacteria and resuspend them with the same amount of distilled water as the fermentation broth to obtain a suspension of single-cell bacteria, and then divide the obtained three kinds of suspensions of single-cell bacteria according to the volume ratio For lactic acid bacteria: saccharomycetes: bifidobacteria = 3:2:1 mixed uniformly, the mixed bacterial agent is obtained;

(2) Preparation of traditional Chinese medicine granules with Xiaoshuan and lipid-lowering probiotic compound bacteria

In terms of parts by weight, take 5 parts of Salvia miltiorrhiza, 1.5 parts of Angelica sinensis, 1.5 parts of Chuanxiong, 1 part of Panax notoginseng and 1 part of safflower, respectively net selected, crushed into fine powder, and then mixed; finally add Chinese medicine weight 3.5 % of the binder and 50% of the mixed bacterial agent, granulated under aseptic conditions to obtain the traditional Chinese medicine granules of Xiaoshun, lipid-lowering and probiotic complex bacteria, dried in cold air and stored in a refrigerator at 4°C.

Example 2

(1) Preparation of mixed bacterial agent

a. Mix the purified two strains of lactic acid bacteria with a volume ratio of EAM-ZL010T:EAM-ZL016T=3:1, and then inoculate them in MRS medium at an inoculum size of 8% by volume, and ferment at a constant temperature of 37°C for 84 hours. After the end, the density of lactic acid bacteria is not less than 10 ¹⁶ CFU/mL; the purified yeast strain is inoculated in the McFarland medium at an inoculation volume of 10% by volume, and fermented on a shaking table at 35°C and 120r/min for 96h. After fermentation The yeast cell density is not less than 10 ¹⁴ CFU/mL; Bifidobacteria (purchased from pharmacies) are inoculated into MRS medium at a volume ratio of 5% inoculum and fermented at 37°C for 96 hours. Less than 10 ¹⁰ CFU/mL;

b. Centrifuge the three fermentation broths in a at 4°C, take the bacteria and resuspend them with distilled water equal to the fermentation broth to obtain a single-cell suspension, and then divide the obtained three single-cell suspensions according to volume Ratio is lactic acid bacteria: saccharomycete: bifidobacteria=2:2:1 mixes evenly, makes mixed bacterial agent;

(2) Preparation of traditional Chinese medicine granules with Xiaoshuan and lipid-lowering probiotic compound bacteria

In terms of parts by weight, the total weight of traditional Chinese medicine is 10 parts. Take 5 parts of Salvia Miltiorrhiza, 2 parts of Angelica, 1.5 parts of Chuanxiong, and 1.5 parts of Panax notoginseng. 3.5% of the binder and 50% of the mixed bacterial agent by weight of the traditional Chinese medicine are granulated in an aseptic state to obtain the traditional Chinese medicine granules of Xiaoshun Lipid-lowering Probiotic Compound Bacteria, which are dried in cold air and stored in a refrigerator at 4°C.

Example 3

(1) Preparation of mixed bacterial agent

a. Mix the purified two strains of lactic acid bacteria with a volume ratio of EAM-ZL010T:EAM-ZL016T=2:1, and then inoculate them in the MRS medium with an inoculation amount of 10% by volume, and ferment at 37°C for 84 hours. After the end, the density of lactic acid bacteria is not less than 10 ¹⁶ CFU/mL; the purified yeast strain is inoculated in the McFarland medium at an inoculum of 5% by volume, and fermented on a shaking table at 32°C and 120r/min for 90h. After fermentation The yeast cell density is not less than 10 ¹⁴ CFU/mL; Bifidobacteria (purchased from pharmacies) are inoculated into MRS medium at a volume ratio of 10% inoculum and fermented at 35°C for 96 hours. Less than 10 ¹⁰ CFU/mL;

b. Centrifuge the three kinds of fermentation broths in a respectively at low temperature, take the bacteria and resuspend them with the same amount of distilled water as the fermentation broth to obtain a suspension of single-cell bacteria, and then divide the obtained three kinds of suspensions of single-cell bacteria according to the volume ratio For lactic acid bacteria: saccharomycetes: bifidobacteria = 3:2:1 mixed uniformly, the mixed bacterial agent is obtained;

(2) Preparation of traditional Chinese medicine granules with Xiaoshuan and lipid-lowering probiotic compound bacteria

In terms of parts by weight, the total weight of traditional Chinese medicine is 10 parts. Take 4 parts of Salvia miltiorrhiza, 3 parts of Panax notoginseng, 2 parts of angelica, and 1 part of Chuanxiong. The binder of 4% by weight of traditional Chinese medicine and the mixed bacterial agent of 50% are granulated under aseptic state to obtain the traditional Chinese medicine granules of antithrombotic, lipid-lowering and probiotic compound bacteria, which are dried in cold air and stored in a refrigerator at 4°C.

In step (2), the binder is acid-resistant sodium carboxymethylcellulose (CMC-Na).

The prepared traditional Chinese medicine granules with Xiaoshuan, lipid-lowering and probiotic complex bacteria weigh 5-5.9g/100 grains, and every 20 grains contain lactic acid bacteria ≥ 400×10 ¹³ CFU, yeast ≥ 600×10 ¹² CFU, and bifidobacteria ≥ 400 ×10 ⁷ CFU.

The above three embodiments have been proved by experimental tests that the effect of embodiment 1 is better than that of embodiments 2 and 3.

【Lipid-lowering test in mice】

1. Cholesterol-lowering (TC) effect of the strain

Under aseptic conditions, add egg yolk as a cholesterol source in the amount of 5% by volume to sterilized MRS and McFarland liquid medium, and aseptically evenly distribute it into test tubes. The inoculum of lactic acid bacteria and yeast was respectively inserted into the inoculum of 2% by volume (cell concentration of 10 ¹² CFU/mL), cultured at 37°C for 24 hours, centrifuged at 8000r/min for 10 minutes, and the supernatant was taken according to the concentration of phosphorus and sulfur. The iron colorimetric method was used to measure the cholesterol content in the supernatant, and 5 repetitions were set for each experiment.

Cholesterol lowering rate=(C-A)/C×100%

In the formula, A is the OD value at 560 nm of the culture solution after the fermentation of the strain, and C is the OD value of the blank control at 560 nm.

In the experiment of studying the effect of different strains on the degradation of egg cholesterol, the absorbance of cholesterol in eggs was measured to be 0.4958±0.05. The t test shows that after cultivating 24h, the three strains of bacteria can significantly reduce the cholesterol in eggs (p<0.05), as can be seen from Figure 1: bacterial strains EAM-ZL016T and EAM-ZL010T have higher degradation rates for cholesterol in egg yolk, of which Strain EAM-ZL010T had the highest degradation rate, up to 73.65%. For high-purity cholesterol, all lactic acid bacteria strains tested could degrade effectively, and the highest degradation rate of strain EAM-ZL010T was 40.0%.

2. Triglyceride (TG) lowering effect of the strain

Under aseptic conditions, add triglyceride standard substance in the amount of 5% by volume to sterilized MRS and McFarland liquid medium, and aseptically dispense into test tubes. Add lactic acid bacteria and yeast liquids (uninoculated test tubes are used as blank control) at an inoculum volume of 2% by volume (cell concentration: 10 ¹² CFU/mL), incubate at a constant temperature of 37°C for 24 hours, and measure triglycerides at 420nm Degradation rate.

Triglyceride reduction rate=(C-A)/C×100%

In the formula, A is the OD value at 420nm of the culture solution after the fermentation of the strain, and C is the OD value at 420nm of the blank control.

As can be seen from Figure 2, the three strains all have degrading effects on triglycerides, and the EAM-ZT003T strain has the highest degrading effect, with a degradation rate as high as 80%. Triglycerides are fatty molecules formed from long-chain fatty acids and glycerol. Triglyceride is the most abundant lipid in the human body, and the harm of high triglyceride is most directly reflected in atherosclerosis. The consequence of high triglycerides is that it is easy to cause "blood thickening", that is, blood viscosity caused by excessive lipid content in the blood, deposits on the blood vessel wall, and gradually forms small plaques, which is what we usually call atherosclerosis. The consequences of high triglycerides No matter where it occurs, it is very serious to the human body.

3. Acid-resistant and bile-salt-resistant effect of the strain

After activating each strain, make a bacterial suspension, inoculate them in liquid medium with pH 3.0 and pH 2.0 according to the volume ratio of 2%, and incubate at a constant temperature of 37°C for 10 hours, then use the gradient dilution plate method to determine the viable bacteria Count, and do 5 parallels to find the average value. Take the strain name as the abscissa, and the logarithmic value of the total number of colonies per unit as the ordinate.

After activating different strains, make a bacterial suspension (the cell concentration is 10 ¹⁰ CFU/mL), and inoculate them with 0.0% ox bile salt (blank) and 0.3% ox bile Salt complex liquid medium. After incubating at 37°C for 24 hours, measure the OD values respectively, calculate the average value of more than 3 parallels, and calculate the tolerance of the strains to bile salts according to the following formula.

Bile salt tolerance (%) = OD of bile salt-containing medium/OD of blank medium × 100%

One of the first conditions for screening strains as probiotics is that they must have good acid resistance and growth and development characteristics in an acidic environment, so that they can survive in the stomach and small intestine. After 10 hours of constant temperature cultivation at 37°C, the three strains showed strong acid resistance, and their survival rates were relatively high under the condition of pH 3.0. Still can reach 10 ⁵ CFU/mL. The acid resistance test shows that the strain has better adaptability to the acidic environment, and if it enters the intestinal tract of the human body, it can survive more and will not affect the cholesterol-lowering activity (Figure 3). The content of bile salts in the human small intestine fluctuates within the range of 0.03% to 0.3%, and the highest content of bile salts in the human small intestine is 0.3%. Therefore, it is required that the strain not only can tolerate a certain acidity but also have a certain bile tolerance. It can be seen from Figure 4 that the yeast strains have good tolerance to bile salts, and the tolerance of the two lactic acid bacteria to bile salts is relatively poor, but the content of bile salts selected in the test is the highest content in the intestinal tract, so in practical applications, The survival of the strain in the body should be higher.

4. Antibacterial effect of the strain

Antibacterial test was carried out on two strains of lactic acid bacteria by Oxford cup method.

The strains EAM-ZL010T and EAM-ZL016T had obvious inhibitory effects on Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, which can cause local suppurative infection, pneumonia, pseudomembranous enteritis, pericarditis, etc., and even systemic infection such as sepsis and sepsis. Some special serotypes of Escherichia coli are pathogenic to humans and animals, especially to infants and young animals (poultry), often causing severe diarrhea and sepsis. The isolated strains have a certain inhibitory effect on these pathogenic bacteria, which undoubtedly can play a role in protecting human health (see Table 2).

Table 2 The inhibitory effect of different strains of fermented liquid on various bacteria (diameter/mm)

5. Establishment of hyperlipidemia mouse model

Mice were fed a high-fat and high-sugar diet to establish the model.

During the high-fat modeling period, it was found that the high-fat diet had a very obvious effect on the weight gain of the mice, and the weight of the mice all increased. After fattening for 28 days, the mouse body weight increased by an average of 11g, and it can be seen that the fattening goal has been preliminarily reached. After feeding the high-fat diet, the total cholesterol content, triglyceride content, and low-density lipoprotein cholesterol content in the serum of the mice in the high-fat diet group were significantly higher than those in the basal diet group (p<0.05), and the high-density lipoprotein cholesterol content was two The difference was not significant (p>0.05). It shows that after feeding the high-fat diet, the lipid metabolism of the mice is disturbed, the cholesterol level in the serum increases, and hyperlipidemia occurs.

Table 3 Changes of serum indicators in mice after high-fat modeling

6. Therapeutic effect of compound bacterial agent traditional Chinese medicine granules on hyperlipidemia in mice

According to Example 1, the compound bacterial traditional Chinese medicine granules were prepared. The 60 mice successfully established high-fat models were randomly divided into 3 groups, the control group was fed with 5g of basal feed every day, and each mouse of the pure Chinese medicine group was fed with Chinese medicine feed (mixed 1g of mixed Chinese medicine with 4g of basal feed) every day. In the feed, pulverize and mix and granulate.) A total of 5g, each mouse in the Chinese medicine granule group is fed with compound bacteria and Chinese medicine granules every day (mixing 1g of Chinese medicine granules in 4g of basic feed, pulverize and mix and granulate.) A total of 5g. In order to ensure a certain number of viable bacteria in the compound bacterium traditional Chinese medicine pellet feed, freshly processed bacterial strains are used to synthesize the bacterium-carrying feed every day. After feeding for 35 days, the mice were decapitated to collect blood, and the content of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL-C) cholesterol in the serum of the mice was determined.

After feeding compound bacteria and Chinese medicine granules for 35 days, the content of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL-C) cholesterol in the serum of mice decreased compared with those of the control group, and were lower than those of the control group. In the pure Chinese medicine group, it shows that after feeding the compound bacteria and traditional Chinese medicine granules, it can reduce the cholesterol and triglyceride levels in the serum of mice. Although the mice had certain immune regulation, but because the high-fat feed contained a considerable amount of cholesterol, in the absence of drug treatment, the measured values of the mice were higher than those of the compound bacteria and traditional Chinese medicine granule group.

7. In vivo toxicity test in mice

According to embodiment 1, method (1) preparation method of mixed bacterial agent prepares composite bacterial agent. Every day, each mouse was fed with 0.2ml of compound bacterial preparation, and in order to ensure a certain number of viable bacteria in the thalline solution, freshly processed bacterial strains were used for each oral gavage. After 42 days of continuous gavage of the compound bacterial agent, observe the signs of the mice, sacrifice them, observe the characteristics of the viscera, and observe the liver and kidney tissue sections of the mice.

In the toxicity test, the coat color of the mice in each group was smooth, the appetite and stool were normal, and no abnormal signs and abnormal deaths were found after 42 days of administration. After 42 days of intragastric administration, the body weight of the mice all increased, and the mice did not appear to become thin or lose weight. The heart/body weight, liver/body weight, spleen/body weight, and kidney/body weight of the mice in each group had no significant difference compared with the control group (p>0.05), and everything was normal through the observation of the tissue sections of the mouse liver and kidney (as shown in Figure 8 , FIG. 9 ), indicating that the bacterial strain has no obvious toxic and side effects on the mice during the feeding of the mice.

Conclusion: The in vitro and in vivo tests of Xiaoshuan and lipid-lowering prove that the compound bacteria and traditional Chinese medicine granules have an excellent effect of reducing fat and losing weight, and can inhibit the growth of harmful intestinal flora and play a role in regulating the intestines, and the compound bacteria agent has no toxicity. side effect.

It should be understood that the protection scope of the present invention is not limited to the above-mentioned embodiments, and those skilled in the art can make improvements or changes according to the above-mentioned descriptions.

Claims (10)

1. A preparation method of thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles is characterized in thatThe composite bacteria comprise two strains of lactobacillus, namely lactobacillus pentosus (La.Pentosus) separated from the intestinal tracts of Tibetan mastiffs, wherein the strains are preserved in the China general microbiological culture Collection center (CGMCC) No.7426 and lactobacillus plantarum (Lactobacillus plantarum) in the 04 th month 07 of 2013; a yarrowia lipolytica strain (Yarrowia lipolytica) isolated from beer residue, which has been deposited in China general microbiological culture Collection center (CGMCC) No.7427, on the date of 2013, 04, 07; bifidobacteria; preparing the strain into mixed bacterial suspension, wherein the bacterial density in the mixed bacterial suspension is respectively as follows: lactic acid bacteria not less than 10 ¹⁶ CFU/mL, saccharomycetes not less than 10 ¹⁴ CFU/mL and bifidobacterium cell density not less than 10 ¹⁰ CFU/mL; and then the traditional Chinese medicine for activating blood circulation and dredging collaterals is used as an auxiliary material carrier and a binder to prepare the finished product of the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles.

2. The method for preparing the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to claim 1, wherein the lactobacillus plantarum is separated from Lanzhou serous fluid.

3. The method for preparing the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to claim 2, wherein lactobacillus plantarum (Lactobacillus plantarum) is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.7425 on the 07 th month of 2013.

4. The method for preparing the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to claim 3, which is characterized in that the preparation of the mixed bacteria suspension at least comprises the following steps:

a. uniformly mixing the purified two lactobacillus strains according to the volume ratio of 1-3:1-3, inoculating the mixture into an MRS culture medium according to the inoculum size of 5-10% of the volume ratio, fermenting and culturing at the constant temperature of 35-37 ℃ for 36-90 h, and after fermentation, keeping the lactobacillus cell density to be not less than 10 ¹⁶ CFU/mL; inoculating purified saccharomycete strain into a Mahalanobis culture medium in an inoculum size of 5-10% in volume ratio at 32-37 ℃ and 120-150 r +.Fermenting and culturing in a shaking table for 72-96 h, wherein the density of yeast thallus after fermentation is not less than 10 ¹⁴ CFU/mL; inoculating bifidobacteria in an inoculum size of 5-10% of the volume ratio into an MRS culture medium, fermenting and culturing for 72-96 hours at a constant temperature of 35-37 ℃, wherein the thallus density is not less than 10 after fermentation is finished ¹⁰ CFU/mL；

b. And c, respectively centrifuging the 3 fermentation liquids in the step a at a low temperature of 4-8 ℃, taking thalli, re-suspending the thalli with distilled water with a volume which is 1-3 times that of the fermentation liquids to obtain single-cell bacterial suspension, and uniformly mixing the three single-cell bacterial suspensions according to the volume ratio of lactobacillus to saccharomycetes to bifidobacterium=1-3:1-2:1 to obtain mixed bacterial suspension.

5. The preparation method of claim 4, wherein the MRS liquid culture medium comprises the following formula and conditions: 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of anhydrous sodium acetate, 801mL of tween-801 mL, 2g of diamine citrate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 1000mL of distilled water, pH of 6.6-6.8, and sterilizing for 20min at 115 ℃; the formula and the conditions of the Mahalanobis culture medium are as follows: glucose 1.0g, potassium chloride 1.8g, yeast powder 2.5g, anhydrous sodium acetate 8.2g, distilled water 1000mL,113 ℃ and sterilizing for 20min.

6. The method for preparing the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to any one of claims 1 to 5, wherein the traditional Chinese medicine for activating blood and dredging collaterals comprises the components of red sage root, angelica, ligusticum wallichii, pseudo-ginseng and/or safflower.

7. The method for preparing the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to claim 6, which is characterized by comprising at least the following steps:

the total weight of the traditional Chinese medicines is 10 parts by weight, at least comprises 0-5 parts of red sage root, 0-5 parts of Chinese angelica, 0-5 parts of szechuan lovage rhizome, 0-3 parts of pseudo-ginseng and 0-3 parts of safflower, the parts of the traditional Chinese medicines cannot be simultaneously taken to be zero, the traditional Chinese medicines are respectively cleaned and selected, crushed into fine powder and then mixed; finally, adding a binder accounting for 3-5 percent of the total weight of the traditional Chinese medicine and mixed bacterial suspension accounting for 10-50 percent of the total weight of the traditional Chinese medicine into the traditional Chinese medicine mixed powder, granulating in a sterile state to obtain thrombolytic lipid-lowering probiotic composite bacterial traditional Chinese medicine particles, and drying the traditional Chinese medicine particles by cold air and storing the traditional Chinese medicine particles in a refrigerator at 0-4 ℃.

8. The method for preparing thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles according to claim 7, wherein the binder is acid-resistant sodium carboxymethyl cellulose.

9. The thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule prepared by the preparation method of the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule of claims 1-8.

10. The thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule according to claim 9, which is characterized in that the weight of the granule is 5-5.9 g/100 granules, and each 20 granules contains lactobacillus which is more than or equal to 400 multiplied by 10 ¹³ CFU and saccharomycetes are more than or equal to 600 multiplied by 10 ¹² CFU, bifidobacterium not less than 400 x10 ⁷ CFU。

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