CN107929329B - Thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule and preparation method thereof - Google Patents
Thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule and preparation method thereof Download PDFInfo
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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
[ field of technology ]
The invention relates to the field of medicines, in particular to a composite bacteria-containing traditional Chinese medicine granule with thrombolytic and lipid-lowering probiotic effects and a preparation method thereof.
[ background Art ]
Cholesterol is a necessary nutrient for human body, but due to the improvement of living standard, the content of cholesterol in serum of human body is often too high, and even becomes a primary factor for death. At present, nearly 1/3 of the adults in China have high blood fat, and hyperlipidemia becomes the first invisible killer for human health, and along with the improvement of living standard, obesity becomes another important disease seriously harming the health of people. The existing overweight and obese people in the whole country are 2.0 hundred million and 6000 hundred thousand respectively. Obesity affects the normal physiological functions of the whole organism, can increase the risks of diseases such as atherosclerosis, hypertension, diabetes, coronary heart disease, tumor, sexual dysfunction, osteoarthritis and the like, and a great deal of epidemiology and clinical observation show that obese patients often suffer from hyperlipidemia at the same time, and the hyperlipidemia is one of the main risk factors for inducing cardiovascular and cerebrovascular diseases. The medical community has called death five-fold "as hypertension, hyperlipidemia, diabetes, coronary heart disease and cerebral apoplexy which are frequently accompanied by obesity, and the patients with hyperlipidemia are mostly complicated with diseases such as hypertension, coronary heart disease, cerebral arteriosclerosis and the like.
With the progress of society, people's health consciousness is enhanced, and weight and lipid reduction is urgent. At present, the hyperlipidemia is mainly controlled by statin drugs, but the drugs have serious toxic and side effects, and are easy to cause side effects such as myalgia, headache, gastrointestinal discomfort, blood lipid disorder, liver and kidney function damage and the like. Therefore, the development and utilization of the health care medicine with the functions of losing weight and reducing blood fat and no toxicity and harm have important practical significance for protecting the health of human bodies. Probiotics, which are used as a living green medicine, have been researched to prove that the probiotics have the advantages of good effect, safety, ecological compliance and the like, and are increasingly applied to various fields of human diseases.
There are a large number of papers and patent achievements at home and abroad on the research of Guan Yisheng bacteria in cholesterol reduction. CN101864375A lactobacillus plantarum (C88), CN102604851A lactococcus lactis, CN101559082A lactobacillus (MA 2), CN101724589A lactobacillus, CN1186855A streptococcus ureus (DM 891128) and lactobacillus rhamnosus (grx 10) have certain effects on cholesterol reduction, but the cell density is 10 8 ～10 10 The effect of a single strain is poor between CFU/mL, and the effect of health care can be achieved, so that the aim of auxiliary treatment cannot be achieved; CN101361506A adopts three bacteria of lactobacillus (GL-03), lactobacillus bulgaricus and Streptococcus thermophilus, and has thallus density of 10 6 CUF/mL; CN101406489A is prepared from bacillus natto, lactobacillus, yeast and lactobacillus acidophilus, and has a cell density of 10 7 ～10 10 CUF/mL; CN101293001A is a microorganism selected from the group consisting of photosynthetic bacteria, lactic acid bacteria, yeast and actinomycetes, and has a cell density of 10 6 ～10 8 CUF/mL; although the compound bacterial colony has better cholesterol-reducing effect than single bacterial species, the bacterial density is lower, and the auxiliary treatment effect can not be achieved, so that the lipid-reducing effect is not ideal. In addition, specific strains in terms of triglyceride reduction are reported to be few.
[ invention ]
Aiming at the defects of the prior art, the invention provides a traditional Chinese medicine granule containing complex bacteria for eliminating thrombus and reducing lipid and probiotics and a preparation method thereof.
The technical scheme of the invention is as follows:
a preparation method of thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles is characterized in that the composite bacteria contain two strains of lactic acid bacteria, namely lactobacillus pentosus (La.Pentosus) separated from the intestinal tracts of Tibetan mastiffs, and the strains are preserved in the China general microbiological culture collection center (CGMCC No. 7426) on the 04 th month 07 of 2013, and are classified and named as: lactobacillus pentosus Lactobacillus pentosus and lactobacillus plantarum; the yarrowia lipolytica strain separated from beer residue is preserved in China general microbiological culture Collection center (CGMCC) No.7427 in the following classification names: yarrowia lipolytica Yarrowia lipolytica. The preservation address is No. 1 and No. 3 of four-way North Chen in the Chaoyang area of Beijing city of China.
The lactobacillus plantarum is not particularly limited in source, but is preferably obtained by separating from the Lanzhou pulp, more preferably the lactobacillus plantarum obtained by separating from the Lanzhou pulp, provided by the invention, and the strain is preserved in China general microbiological culture Collection center (CGMCC) No.7425, with the classification named: lactobacillus plantarum Lactobacillus plantarum, which is designated EAM-ZL010T in the present specification. Four-way No. 1 and No. 3 of North Chen in the Chaoyang district of Beijing city of China as a preservation address
The thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule contains traditional Chinese medicines for activating blood and dredging collaterals, and preferably comprises salvia miltiorrhiza, angelica, ligusticum wallichii, pseudo-ginseng and/or safflower components.
The saccharomycete separated from beer residue is yarrowia lipolytica strain, and is separated by coating beer residue onto Maillard culture medium, and the pure strain colony is milky white and has oval microscopic examination. The molecular identification of the isolated pure strain was performed using the 26SrDNA method. The 26SrDNA sequence was aligned in NCBI (National Center for Biotechnology Information ) database, and as a result, the gene sequence of the strain was found to be 100% homologous to yarrowia lipolytica strain (Yarrowia lipolytica) in all similar sequences, so that the strain was identified as yarrowia lipolytica strain in combination with physiological and biochemical characteristics, and was named EAM-ZT003T in the present invention. The Chinese patent application number: 201710091197.8.
the lactic acid bacteria isolated from the intestinal tract of Tibetan mastiff are lactobacillus pentosus (la. Pentosus).
The Tibetan mastiff intestinal contents are distributed on an MRS culture medium for separation and culture, a bacterial colony of the split pure strain is milky white, has a diameter of about 2mm, has a smooth surface, regular edges and raised center, is provided with an obvious calcium dissolving ring around the bacterial colony, is in a short rod shape in microscopic examination form, and is subjected to experiments such as bacterial gram staining, plate bacterial colony form, bacterial degradation rate of cholesterol and cholesterol standard substances in egg yolk, degradation rate of triglyceride, acid resistance test, cholate resistance test, toxicity test (mice) and the like. The molecular identification of the isolated pure strain was performed using the 16SrDNA method. The 16SrDNA sequence thereof was aligned in NCBI (National Center for Biotechnology Information ) database, and as a result, the gene sequence of the strain was found to have 99% homology with Lactobacillus pentosus (La. Pentosus) in all similar sequences, and thus, the strain was identified as Lactobacillus pentosus by combining physiological and biochemical characteristics, which was designated EAM-ZL016T in the present invention. The detailed contents are applied for the patent of the invention in China, and the application number is as follows: 201710091198.2.
the present invention is not particularly limited to the source of bifidobacteria, and may be commercially available.
The invention also provides a preparation method of the mixed bacterial suspension. The preparation of the mixed bacterial 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 Maillard culture medium in an inoculum size of 5-10% of the volume ratio, and carrying out shake fermentation culture at a temperature of 32-37 ℃ and a speed of 120-150 r/min for 72 ultra-high96h, and the density of the saccharomycetes is not less than 10 after fermentation 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.
The formula of the MRS liquid culture medium is as follows: 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of anhydrous sodium acetate, 801mL of tween-801, 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, 115 ℃ and sterilizing for 20min; the formula of the Mahalanobis culture medium is 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.
The inventor adopts high-density culture of bacteria to make the cell density of lactic acid bacteria and saccharomycetes respectively reach 10 at maximum 16 CFU/mL、10 14 CFU/mL, and the specific strain with high cholesterol-reducing effect and the strain with high triglyceride-reducing effect are used together, so that the triglyceride content is reduced while the cholesterol is reduced.
In order to match with curative effects, the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule recommended by the invention at least contains traditional Chinese medicines with the effects of activating blood circulation, removing blood stasis and/or relieving pain, in particular to the components of red sage root, angelica, szechuan lovage rhizome, pseudo-ginseng and/or safflower.
The invention also provides a more specific preparation method of the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule, which at least comprises 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 thrombus-eliminating lipid-lowering probiotic composite bacteria 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 ℃.
The binder of the present invention is preferably sodium carboxymethyl cellulose (CMC-Na) which is resistant to acid.
The traditional Chinese medicine granule containing the composite bacteria prepared by the preparation method is the traditional Chinese medicine granule of the thrombolytic lipid-lowering probiotic composite bacteria.
The invention also provides the optimal composition of the thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule, the weight of the granule is 5-5.9 g/100 granules, and each 20 granules contain 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。
The invention adopts the technical proposal that the main advantages and effects are as follows:
(1) The thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles prepared by the invention have high-efficiency thrombolytic lipid-lowering functions, can obviously lower serum cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), have strong acid and bile salt resistance, have the effect of regulating intestinal flora, can effectively inhibit the growth of harmful microorganisms, and have no toxic or side effect.
(2) The total number of the viable lactic acid bacteria in the mixed microbial inoculum is more than or equal to 10 16 CFU/mL, total number of live saccharomycetes is more than or equal to 10 14 CFU/mL, total viable count of bifidobacterium is more than or equal to 10 10 CFU/mL, thus can ensure that a large amount of thalli survive in intestinal tracts. The Chinese medicine is adopted as auxiliary materials for granulation, and can play an auxiliary treatment role on thrombolysis and lipid-lowering of the compound bacteria.
(3) The probiotic composite bacteria can play roles in regulating the intestines, cleaning the intestines and relaxing the bowels in the intestines, and inhibit the growth and reproduction of harmful bacteria in the intestines.
(4) The thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule disclosed by the invention is prevented from being taken together with antibacterial medicines. The product is live bacteria Chinese medicine granule, which is preferably stored in a refrigerator at 0-4 ℃ and is not placed at high temperature.
[ description of the drawings ]
The accompanying drawings are experimental data in specific examples of the application of the invention.
FIG. 1 shows the degradation rate of cholesterol in egg yolk by different strains according to the invention.
FIG. 2 shows the degradation rate of triglycerides by different strains of the invention.
FIG. 3 shows the results of acid resistance tests of different strains of the invention.
FIG. 4 shows the results of the cholate resistance test of different strains of the present invention.
FIG. 5 shows the test results of TG content in mouse serum of the Chinese medicinal granules of the present invention.
FIG. 6 shows the results of the test of LDL-C content in mouse serum by using the Chinese medicinal granules of the present invention.
FIG. 7 shows the results of the test of TC content in mouse serum by using the Chinese medicinal granule of the present invention.
FIG. 8 shows the results of a mouse toxicity test (liver tissue section) using a complex bacterium of the present invention.
FIG. 9 shows the results of a mouse toxicity test (kidney tissue section) using a complex bacterium of the present invention.
[ detailed description ] of the invention
The present invention will be described in detail with reference to specific examples.
The basic conditions of the three species are shown in Table 1.
Table 1 three general categories of strain basic conditions table with lipid-lowering and cholesterol-lowering effects
The two strains of lactic acid bacteria are: the lactobacillus EAM-ZL010T is classified and named as lactobacillus plantarum (Lactobacillus plantarum), and the preservation code number is CGMCC7425; the lactobacillus EAM-ZL016T is classified and named as lactobacillus pentosus (Lactobacillus pentosus) with a preservation number of CGMCC7426 (the detailed content of which is applied for Chinese patent application No. 201710091198.2); the strain of saccharomycete is as follows: the microzyme EAM-ZT003T is classified and named as yarrowia lipolytica (Yarrowia lipolytica), and the preservation number is CGMCC7427 (the detailed content of which has been applied for Chinese patent application No. 201710091197.8). The above strains were deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the following 04/07 date 2013.
Lactobacillus plantarum (Lactobacillus plantarum) obtained by separating lactobacillus plantarum from Lanzhou slurry is preserved in China general microbiological culture Collection center (CGMCC 7425) of China general microbiological culture Collection center (China general microbiological culture collection center) in the 04 th month 07 of 2013, and is named EAM-ZL010T. The specific method comprises the following steps: the Lanzhou slurry is coated on MRS culture medium for separation and culture, the bacterial colony of the pure strain is milky white, has a diameter of about 2mm, smooth surface, tidy edges and raised center, and has obvious calcium dissolving ring around the bacterial colony, and the microscopic strain is short-rod-shaped (1-1.2) mu m x (1-1.5) mu m, single and short-chain arrangement, gram positive staining and facultative anaerobic. Experiments show that the strain has extremely high degradation effect on cholesterol in egg yolk and cholesterol standard substances and also has degradation effect on triglyceride, and toxicity experiments (mice) show that the strain has no toxic or side effect. The molecular identification of the isolated pure strain was performed using the 16SrDNA method. The 16SrDNA sequence thereof was aligned in NCBI (National Center for Biotechnology Information ) database, and as a result, the gene sequence of the strain was found to be 99% homologous to Lactobacillus plantarum (Lactobacillus plantarum) in all similar sequences, and thus, the strain was identified as Lactobacillus plantarum in combination with physiological and biochemical characteristics.
The formula of the MRS liquid culture medium is as follows: 10g of peptone, 10g of beef extract, 5g of yeast powder, 20g of glucose, 5g of anhydrous sodium acetate, 801mL of tween-801, 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, 115 ℃ and sterilizing for 20min; the formula of the Mahalanobis culture medium is 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.
Example 1
(1) Preparation of mixed microbial inoculum
a. Mixing the purified two lactobacillus strains uniformly in a volume ratio of 1:1, and then mixing the two lactobacillus strains according to volumeInoculating 10% of the inoculum size into MRS culture medium, fermenting and culturing at 37deg.C for 72 hr, and culturing at a constant temperature until the lactobacillus density is not less than 10 16 CFU/mL; inoculating purified yeast strain into a Mahalanobis culture medium according to an inoculum size of 10% of the volume ratio, performing shake fermentation culture at 32deg.C and 120r/min for 96 hr, and fermenting to obtain yeast with thallus density not less than 10 14 CFU/mL; inoculating Bacillus bifidus (purchased from pharmacy) at a volume ratio of 10% into MRS culture medium, fermenting at 37deg.C for 72 hr, and culturing at a cell density of not less than 10 10 CFU/mL；
b. C, respectively centrifuging the 3 fermentation liquids in the step a at low temperature, taking thalli, re-suspending the thalli by distilled water with the same quantity as the fermentation liquids to obtain single-cell bacterial suspension, and uniformly mixing the three single-cell bacterial suspension according to the volume ratio of lactobacillus to microzyme to bifidobacterium=3:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles
According to the weight portions, 5 portions of red sage root, 1.5 portions of angelica, 1.5 portions of hemlock parsley, 1 portion of notoginseng and 1 portion of safflower are respectively cleaned and selected, crushed into fine powder and then mixed; finally, adding a binder accounting for 3.5 percent of the weight of the traditional Chinese medicine and a mixed microbial inoculum accounting for 50 percent of the weight of the traditional Chinese medicine into the traditional Chinese medicine mixed powder, granulating in a sterile state to obtain the thrombolytic lipid-lowering probiotic composite bacteria 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 4 ℃.
Example 2
(1) Preparation of mixed microbial inoculum
a. The purified two lactobacillus strains were combined in a volume ratio of EAM-ZL010T: mixing EAM-ZL016 T=3:1 uniformly, inoculating into MRS culture medium according to the inoculum size of 8% by volume, fermenting at 37deg.C for 84 hr, and culturing to obtain lactobacillus with thallus density not less than 10 16 CFU/mL; inoculating purified yeast strain into a Mahalanobis culture medium according to an inoculum size of 10% by volume, performing shake fermentation culture at 35deg.C and 120r/min for 96 hr, and fermenting to obtain yeast with thallus density not less than 10 14 CFU/mL; inoculating Bifidobacterium (purchased from pharmacy) in an inoculum size of 5% by volume into MRS culture medium, fermenting at 37deg.C for 96 hr, and culturing to obtain thallus with density of not less than 10 10 CFU/mL；
b. C, respectively centrifuging the 3 fermentation liquids in the step a at the temperature of 4 ℃, taking thalli, re-suspending the thalli by distilled water with the same quantity as the fermentation liquids to obtain single-cell bacterial suspension, and uniformly mixing the three single-cell bacterial suspension according to the volume ratio of lactobacillus to microzyme to bifidobacterium=2:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles
The total weight of the traditional Chinese medicines is 10 parts by weight, 5 parts of red sage root, 2 parts of angelica, 1.5 parts of ligusticum wallichii and 1.5 parts of pseudo-ginseng are respectively cleaned and selected, crushed into fine powder and then mixed; finally, adding a binder accounting for 3.5 percent of the weight of the traditional Chinese medicine and a mixed microbial inoculum accounting for 50 percent of the weight of the traditional Chinese medicine into the traditional Chinese medicine mixed powder, granulating in a sterile state to obtain the thrombolytic lipid-lowering probiotic composite bacteria 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 4 ℃.
Example 3
(1) Preparation of mixed microbial inoculum
a. The purified two lactobacillus strains were combined in a volume ratio of EAM-ZL010T: mixing EAM-ZL016 T=2:1 uniformly, inoculating into MRS culture medium according to 10% of inoculum size by volume, fermenting at 37deg.C for 84 hr, and fermenting until the lactobacillus thallus density is not less than 10 16 CFU/mL; inoculating purified yeast strain into a Male culture medium according to an inoculum size of 5% by volume, performing shake fermentation culture at 32deg.C and 120r/min for 90 hr, and fermenting to obtain yeast with thallus density not less than 10 14 CFU/mL; inoculating Bacillus bifidus (purchased from pharmacy) at a volume ratio of 10% into MRS culture medium, fermenting at 35deg.C for 96 hr, and culturing at a cell density of not less than 10 10 CFU/mL；
b. C, respectively centrifuging the 3 fermentation liquids in the step a at low temperature, taking thalli, re-suspending the thalli by distilled water with the same quantity as the fermentation liquids to obtain single-cell bacterial suspension, and uniformly mixing the three single-cell bacterial suspension according to the volume ratio of lactobacillus to microzyme to bifidobacterium=3:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine particles
The total weight of the traditional Chinese medicines is 10 parts by weight, 4 parts of red sage root, 3 parts of pseudo-ginseng, 2 parts of angelica and 1 part of szechuan lovage rhizome are respectively cleaned and selected, crushed into fine powder and then mixed; finally, adding 4% of binder and 50% of mixed bacteria agent by 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 bacteria traditional Chinese medicine particles, and drying by cold air and storing in a refrigerator at 4 ℃.
In the step (2), the binder is acid-resistant sodium carboxymethyl cellulose (CMC-Na).
The weight of the prepared thrombolytic lipid-lowering probiotic composite bacteria traditional Chinese medicine granule is 5-5.9 g/100 granules, and each 20 granules contain lactobacillus which is more than or equal to 400 multiplied by 10 13 CFU and yeast are not less than 600×10 12 CFU, bifidobacterium not less than 400 x10 7 CFU。
The test results of the three examples prove that the effect of the example 1 is better than that of the examples 2 and 3.
[ mouse lipid-lowering test ]
1. Cholesterol (TC) lowering effect of the strain
Under the aseptic condition, adding egg yolk as cholesterol source into sterilized MRS and Maishi liquid culture medium according to the volume ratio of 5%, and packaging into test tubes. At a volume ratio of 2% (cell concentration of 10) 12 CFU/mL), inoculating lactobacillus and saccharomycete liquid respectively, culturing at 37 deg.C for 24 hr, centrifuging at 8000r/min for 10min, collecting supernatant, and measuring cholesterol content in supernatant according to phosphothioiron colorimetric method, wherein each test is set to 5 times.
Cholesterol reduction rate= (C-se:Sub>A)/c×100%
Wherein A is the OD value of the culture solution at 560nm after the strain is fermented, and C is the OD value of the blank at 560 nm.
In the experiment of researching the influence of different strains on the degradation of egg cholesterol, the absorbance of cholesterol in eggs is measured to be 0.4958 +/-0.05. t-test shows that 3 strains can obviously reduce cholesterol in eggs after 24 hours of culture (p < 0.05), and can be seen from fig. 1: the degradation rate of the strain EAM-ZL016T, EAM-ZL010T on cholesterol in yolk is high, wherein the degradation rate of the strain EAM-ZL010T is highest and can reach 73.65%, and the degradation rate of the strain EAM-ZL010T is highest and can reach 40.0% for high-purity cholesterol.
2. Triglyceride (TG) lowering effect of the strain
Adding triglyceride standard substance into sterilized MRS and Maishi liquid culture medium at a volume ratio of 5% under aseptic condition, and aseptically packaging into test tubes. At a volume ratio of 2% (cell concentration of 10) 12 CFU/mL), lactobacillus and yeast liquid (non-inoculated test tube is used as blank control), culturing at 37deg.C for 24 hr, and determining triglyceride degradation rate at 420 nm.
Triglyceride reduction = (C-se:Sub>A)/c×100%
Wherein A is the OD value of the culture solution at 420nm after the strain is fermented, and C is the OD value of the blank control at 420 nm.
As can be seen from FIG. 2, 3 strains all have degradation effect on triglyceride, while EAM-ZT003T strain has the highest degradation effect and the degradation rate is as high as 80%. Triglycerides are fatty molecules formed from long chain fatty acids and glycerol. Triglycerides are the most abundant lipids in humans, and the high risk of triglycerides is most directly manifested in atherosclerosis. The consequence of high triglycerides is that they tend to cause "blood viscosity", i.e. a blood viscosity caused by too high a lipid content in the blood, which deposits on the walls of the blood vessels and gradually forms small plaques, i.e. what we speak of atherosclerosis at ordinary times. The high triglycerides have serious damage to the human body no matter where they occur.
3. Acid and bile salt resistant effects of strain
The bacterial strains are activated to prepare bacterial suspension, the bacterial suspension is inoculated into liquid culture media with the pH value of 3.0 and the pH value of 2.0 respectively according to the inoculum size of 2 percent by volume, the bacterial suspension is cultivated for 10 hours at the constant temperature of 37 ℃, the number of the viable bacteria is measured by a gradient dilution plate method, and the average value is calculated for 5 parallel bacteria. The total number of unit colonies is plotted on the ordinate against the strain name on the abscissa.
Activating different strains to obtain bacterial suspension (thallus concentration 10) 10 CFU/mL), and inoculating the seed into a compound liquid culture medium containing 0.0% of ox gall salt (namely blank) and 0.3% of ox gall salt according to the inoculum size of 2% of the volume ratio. After 24h incubation at 37℃the OD values were measured separately, averaged over 3 replicates and the strain tolerance to bile salts was calculated according to the following formula.
Bile salt tolerance (%) = OD of bile salt-containing medium/OD of blank medium×100%
One of the primary conditions for screening strains as probiotics is that they must have good acid resistance and characteristics of growth and development in an acidic environment so as to survive in the stomach and the forepart of the small intestine. After the culture is carried out for 10 hours at the constant temperature of 37 ℃, the three bacteria all show stronger acid resistance, the survival rate is higher under the condition of pH3.0, and the survival rate of the strain is reduced when the pH is 2.0, but the culture time is still 10 hours 5 CFU/mL. Acid resistance experiments show that the strain has better adaptability to acid environment, can survive more after entering the intestinal tract of a human body, and can not influence cholesterol-lowering activity (figure 3). The content of bile salt in the small intestine of human body fluctuates within the range of 0.03% -0.3%, and the maximum content of bile salt in the small intestine of human body is 0.3%, so that the strain is required to not only endure certain acidity but also have certain bile tolerance. As can be seen from FIG. 4, the yeast strain has good resistance to bile salts, and the 2 strains of lactic acid bacteria have relatively poor resistance to bile salts, but the content of bile salts selected in the test is the highest content in intestinal tracts, so that in practical application, the survival amount of the strain in vivo is higher.
4. Bacterial strain antibacterial effect
And (3) performing an antibacterial test on 2 strains of lactic acid bacteria by adopting an oxford cup method.
The strain EAM-ZL010T, EAM-ZL016T has obvious inhibiting effect on escherichia coli, bacillus subtilis and staphylococcus aureus. Staphylococcus aureus is the most common pathogenic bacteria in suppurative infection of human beings, and can cause local suppurative infection, pneumonia, pseudomembranous enteronitis, pericarditis and the like, and even systemic infection such as septicemia, sepsis and the like. Coli of some specific serotypes is pathogenic to humans and animals, particularly infants and young animals (birds), and often causes severe diarrhea and sepsis. The isolated strain has a certain inhibitory effect on these pathogenic bacteria, which can certainly play a role in protecting human health (see table 2).
TABLE 2 inhibition of various bacteria by fermentation broths of different strains (diameter/mm)
5. Establishment of hyperlipidemic mouse model
And feeding the mice with high-fat and high-sugar feed for molding.
During the high-fat molding period, the weight gain effect of the high-fat feed on mice is obvious, and the weight of the mice is increased. The weight of the mice increases by 11g on average after fattening for 28 days, and the initial achievement of the fattening target can be seen. After feeding the high-fat feed, the total cholesterol content, the triglyceride content and the low-density lipoprotein cholesterol content in the serum of the mice in the high-fat feed group are obviously higher than those in the basal feed group (p < 0.05), and the difference between the high-density lipoprotein cholesterol content and the low-density lipoprotein cholesterol content is not obvious (p > 0.05). Indicating that after feeding the high-fat feed, lipid metabolism disorder of mice is caused, the cholesterol level in serum is increased, and hyperlipidemia is generated.
TABLE 3 variation of various indices of mouse serum after high fat modeling
6. Treatment effect of composite microbial inoculum traditional Chinese medicine particles on mouse hyperlipidemia
According to example 1, a composite bacterial traditional Chinese medicine granule was prepared. The 60 mice successfully molded by high fat are randomly divided into 3 groups, each control group feeds 5g of basic feed per day, each mouse of the pure traditional Chinese medicine group feeds 5g of traditional Chinese medicine feed with traditional Chinese medicine feed per day (1 g of mixed traditional Chinese medicine is mixed into 4g of basic feed, crushed, mixed and granulated), and each mouse of the traditional Chinese medicine granule group feeds 5g of compound bacteria traditional Chinese medicine granule feed per day (1 g of traditional Chinese medicine granule is mixed into 4g of basic feed, crushed, mixed and granulated). In order to ensure a certain number of viable bacteria in the compound bacteria traditional Chinese medicine pellet feed, the fresh treated bacterial strain is adopted for carrying out the synthesis of the bacteria-carrying feed every day. After 35d feeding, mice were bled at broken ends and the Total Cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C) cholesterol levels in the serum of the mice were determined.
After the compound bacteria Chinese medicine granule is fed for 35d, the Total Cholesterol (TC) content, triglyceride (TG) content and low density lipoprotein (LDL-C) cholesterol content in the serum of the mice are reduced compared with a control group and are lower than those of a pure Chinese medicine group, which indicates that the cholesterol and triglyceride content in the serum of the mice can be reduced after the compound bacteria Chinese medicine granule is fed. Although the mice have a certain immune regulation, the high-fat feed contains a considerable amount of cholesterol, and under the condition of lack of drug treatment, each measured value is higher than that of the traditional Chinese medicine granule group of the compound bacteria.
7. In vivo toxicity test in mice
A composite microbial inoculum was prepared according to the method of example 1, method (1) for preparing the mixed microbial inoculum. Each mouse is filled with 0.2ml of the gastric complex microbial inoculum every day, and fresh treated strains are adopted every time in order to ensure a certain number of viable bacteria in the bacterial solution. After 42d of continuous gastric lavage of the compound bacteria, various signs of the mice are observed, the mice are sacrificed, visceral features are observed, and tissue sections of the livers and kidneys of the mice are observed.
The mice in each group of toxicity test have clean hair, normal appetite and urination, no abnormal signs and no abnormal death after 42 days of administration. The weight of the stomach 42d is increased, and the mice do not have emaciation, weight loss and the like. The heart/body weight, liver/body weight, spleen/body weight and kidney/body weight of each group of mice have no significant difference (p > 0.05) compared with the control group, and all the mice are normal after being observed through tissue sections of the liver and kidney of the mice (as shown in fig. 8 and 9), so that the strain has no obvious toxic or side effect on the mice during the period of feeding the mice.
Conclusion: proved by in vitro and in vivo experiments of thrombolysis and lipid reduction, the composite bacterial traditional Chinese medicine granule has excellent lipid reduction and weight reduction effects, can inhibit the growth of harmful flora in intestinal tracts, has the effect of regulating intestinal tracts, and has no toxic or side effect.
It is to be understood that the scope of the present invention is not limited to the above embodiments, and that modifications or variations may be made by those skilled in the art in light of the above description.
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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