CN109207389B - Thrombolytic lipid-lowering probiotic compound bacteria traditional Chinese medicine oral liquid and preparation method thereof - Google Patents
Thrombolytic lipid-lowering probiotic compound bacteria traditional Chinese medicine oral liquid and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a thrombolytic lipid-lowering probiotic compound bacterium traditional Chinese medicineThe oral liquid and the preparation method thereof are characterized in that 3 strains are separated and purified from samples such as Lanzhou slurry, tibetan mastiff intestinal tracts, beer residues and the like, and are subjected to high-density culture to prepare mixed bacterial suspensions, wherein the bacterial densities in the mixed bacterial suspensions 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 extract (such as red sage root, angelica, ligusticum wallichii, notoginseng, safflower, etc.) with the corrective agent for activating blood and dredging collaterals is prepared into the finished product of the thrombolytic lipid-lowering probiotic compound bacteria traditional Chinese medicine oral liquid. The invention has the functions of high efficiency thrombolysis and lipid lowering, can obviously lower serum cholesterol (TC), triglyceride (TG) and low density lipoprotein (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, relates to complex bacteria and application thereof in the field of medicines, and in particular relates to thrombolytic lipid-lowering probiotic traditional Chinese medicine oral liquid containing the complex bacteria 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 higher blood fat, and hyperlipidemia is the first invisible killer for human health, and along with the improvement of living standard, obesity is also gradually another important disease seriously endangering the health of people. The existing overweight and obese people in the whole country are respectively 2.0 hundred million and 6000 hundred thousand. Obesity affects 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 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.
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 effect 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 prepared by optical combinationBacteria, lactobacillus, saccharomycetes and actinomycetes, and the density of the bacteria is 10 6 ～10 8 CUF/mL; although the compound bacterial colony has better cholesterol-reducing effect than single bacterial, 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 ]
The invention aims to solve the problems of the prior art and provides a novel composite bacterium, thrombus-eliminating lipid-lowering probiotic composite traditional Chinese medicine oral liquid and a preparation method thereof.
The invention provides a composite bacterium with thrombolytic and lipid-lowering probiotic effects, which is characterized by comprising two strains of lactic acid bacteria, 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) of the microbial culture collection management committee on the date 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 of the Qiangyang 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, designated EAM-ZL010T in this specification. The preservation address is No. 1 and No. 3 of four-way North Chen in the Chaoyang area of Beijing city of China.
The invention also provides a traditional Chinese medicine oral liquid containing the compound bacteria, wherein each milliliter of the oral liquid contains more than or equal to 400 multiplied by 10 of lactobacillus 13 CFU and saccharomycetes are more than or equal to 600 multiplied by 10 12 CFU, bifidobacteria≥400×10 7 CFU. The Chinese medicinal oral liquid containing the composite bacteria has the effects of eliminating thrombus and reducing blood lipid.
For the purpose of matching curative effect, the Chinese medicinal oral liquid containing the composite bacteria recommended by the invention at least contains Chinese medicaments with the effects of activating blood circulation, removing blood stasis and/or relieving pain, in particular to red sage root, angelica, ligusticum wallichii, pseudo-ginseng and/or safflower components.
The yeast isolated from beer residue is yarrowia lipolytica strain, the beer residue is coated on a McO medium for separation, the bacterial colony of the cultured pure strain is milky white, and the microscopic examination form is oval. The isolated pure strains were molecular identified using 26S rDNA. The 26S rDNA sequence is compared in NCBI (National Center for Biotechnology Information ) database, and the result shows that the gene sequence of the strain has 100 percent of homology with yarrowia lipolytica strain (Yarrowia lipolytica) in all similar sequences, so that the strain is identified as the yarrowia lipolytica strain by combining physiological and biochemical characteristics, and is named EAM-ZT003T in the invention. The details are applied to Chinese invention 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, culture, and the pure bacterial strain colony is milky white, has a diameter of about 2mm, has a smooth surface, regular edges and a central bulge, 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 strain gram staining, flat bacterial colony form, bacterial strain degradation rate of cholesterol and cholesterol standard substances in egg yolk, triglyceride degradation rate, acid resistance test, cholate resistance test, toxicity test (mice) and the like. The molecular identification of the isolated pure strains was carried out using the 16S rDNA method. The 16S rDNA sequence 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 with physiological and biochemical characteristics, and was named EAM-ZL016T in the present invention. The Chinese patent application number: 201710091198.2.
the invention also provides a preparation method of the preferred more specific composite bacteria.
The preparation method of the composite bacteria is characterized by at least comprising 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 according to the inoculum size of 5-10% of the volume ratio, and carrying out shake fermentation culture at the temperature of 32-37 ℃ and the speed of 120-150 r/min for 72-96 h, wherein the density of saccharomycete 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 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 suspension according to the volume ratio of lactobacillus to saccharomycetes to bifidobacterium=1-3:1-2:1 to obtain the composite bacteria.
The formula and the conditions of the MRS liquid culture medium are preferably 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, 0.58 and g 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 preferably 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 bacterial density of lactobacillus and saccharomycete respectively reach 10 16 CFU/mL、 10 14 CFU/mL, and will be specific toThe 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.
The invention also provides a preparation method of the traditional Chinese medicine oral liquid containing the composite microbial inoculum, which at least comprises the following steps:
(1) Preparation of Chinese medicine extract
a. According to the weight portion, the total weight of the traditional Chinese medicine is 10 portions, 0 to 5 portions of red sage root, 0 to 5 portions of angelica, 0 to 5 portions of hemlock parsley, 0 to 3 portions of notoginseng and 0 to 3 portions of safflower (each traditional Chinese medicine portion can not be taken at the same time), respectively cleaning and selecting, crushing into fine powder, mixing, and carrying out alcohol extraction for 3 to 5 times by using ethanol with the volume percentage of 60 to 80 percent, wherein the amount of the added ethanol for the 1 st time of the alcohol extraction is 3 to 5 times of the weight of the medicine, and the time is 2 to 3 hours; the ethanol is added for the 2 nd time for 1.5-2 hours, wherein the amount of the ethanol is 2-4 times of the weight of the medicine; the ethanol is added for 1-2 hours for 3 rd time, wherein the weight of the ethanol is 1-2 times of that of the medicine; mixing the extracting solutions, recovering ethanol, filtering to obtain filtrate and residue, and concentrating the filtrate by 2-3 times according to volume to obtain concentrated solution B;
b. decocting the residues in the step a with water for 3 times, wherein the water adding amount for the 1 st decoction is 3-5 times of the weight of the medicine, and the decoction lasts for 2-3 hours; the water adding amount of the 2 nd time is 2 to 4 times of the weight of the medicine, and the medicine is decocted for 2 to 3 hours; the water adding amount for the 3 rd time is 1-2 times of the weight of the medicine, and the medicine is decocted for 1-2 hours; mixing the decoctions, filtering to obtain filtrate, and concentrating the filtrate by 2-3 times according to volume to obtain concentrated solution C; and finally, fully and uniformly mixing the concentrated solutions B and C to obtain a traditional Chinese medicine leaching solution;
(3) Preparation of oral liquid
Uniformly mixing the traditional Chinese medicine leaching solution and the mixed bacterial agent according to the volume ratio of 1-5:1, adding the flavor-correcting agent maltitol accounting for 5-8% of the total volume of the mixed solution, fully and uniformly mixing, filtering and packaging under the sterile condition to obtain the thrombus-eliminating lipid-lowering probiotic composite bacterial traditional Chinese medicine oral liquid, and storing in a refrigerator at 0-4 ℃.
The traditional Chinese medicine oral liquid containing the complex bacteria, which is prepared by the preparation method, is the traditional Chinese medicine oral liquid containing the complex bacteria for eliminating thrombus and reducing lipid.
The thrombolytic lipid-lowering probiotic compound bacteria Chinese medicine oral liquid is preferably per milliliterThe oral liquid contains lactobacillus not less than 400×10 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 compound bacteria traditional Chinese medicine oral liquid prepared by the invention has high-efficiency thrombolytic lipid-lowering function, can obviously lower serum cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C), has strong acid resistance and bile salt resistance, simultaneously has the effect of regulating intestinal flora, can effectively inhibit the growth of harmful microorganisms, and has no toxic or side effect.
(2) The cell densities in the mixed microbial inoculum of the invention are respectively as follows: the total number of viable bacteria of the lactic acid bacteria is more than or equal to 10 16 CFU/mL, total number of active saccharomycetes is more than or equal to 10 14 CFU/mL and total viable count of bifidobacteria not less than 10 10 CFU/mL, thus can ensure that a large amount of thalli survive in intestinal tracts.
(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 invention has the effects of effectively reducing blood fat and blood viscosity, depolymerizing and resisting thrombosis on platelet accumulation, expanding blood vessels, increasing cerebral blood flow and improving microcirculation and cerebral ischemia by reasonably matching the traditional Chinese medicines with the effects of promoting blood circulation, removing blood stasis and relieving pain, in particular to the optimal 5 traditional Chinese medicines of red sage root, angelica sinensis, szechuan lovage rhizome, pseudo-ginseng and safflower. The five Chinese medicines have the effects of promoting blood circulation, removing blood stasis and relieving pain, and can be used for treating coronary heart disease, angina pectoris, arrhythmia, blood stasis pain, traumatic injury, etc.
(5) The thrombolytic lipid-lowering probiotic compound bacteria traditional Chinese medicine oral liquid disclosed by the invention is prevented from being taken together with antibacterial medicines. The product is a live bacteria Chinese medicine oral liquid, 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 the compound bacteria Chinese medicine oral liquid of the invention on the reduction of TG, TC, LDL-C content in mouse serum.
FIG. 6 shows the results of a mouse toxicity test (liver tissue section) using a complex bacterium of the present invention.
FIG. 7 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) with the preservation number of CGMCC 7425; the lactobacillus EAM-ZL016T is classified and named as lactobacillus pentosus (Lactobacillus pentosus), and the preservation number is CGMCC 7426 (the detailed content of the lactobacillus EAM-ZL016 is applied for Chinese patent application number: 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 CGMCC 7427 (the detailed content of the microzyme EAM-ZT003T is applied for Chinese patent application No. 201710091197.8). The above strains were deposited in China general microbiological culture Collection center (China general microbiological culture Collection center) by the China Committee for type culture Collection of microorganisms, which was the subject of 04 and 07.
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 an MRS culture medium for separation and culture, the bacterial colony of the pure strain is milky white, has the diameter of about 2mm, smooth surface, tidy edges and raised center, obvious calcium dissolving rings are arranged around the bacterial colony, and the microscopic strain is in a short rod shape (1-1.2) mu m x (1-1.5) mu m, is arranged singly and in short chains, has positive gram staining and is 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 strains was carried out using the 16S rDNA method. The 16S rDNA 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 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, 0.25 and g 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. Uniformly mixing the purified two lactobacillus strains according to the volume ratio of 1:1, inoculating the two lactobacillus strains into an MRS culture medium according to the inoculum size of 10 percent of the volume ratio, fermenting and culturing for 72 hours at the constant temperature of 37 ℃, and after the fermentation is finished, the density of lactobacillus thallus is not less than 10 16 CFU/mL; inoculating purified yeast strain into a McO medium according to an inoculum size of 10% by volume, performing shake fermentation culture at 32deg.C and 120r/min for 96 hr, and fermentingThe density of the parent strain is 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 saccharomycetes to bifidobacterium=3:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of Chinese medicine extract
a. According to the weight portion, the total weight of the traditional Chinese medicine is 10 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 taken, respectively cleaned and selected, crushed into fine powder, then mixed, and subjected to alcohol extraction for 3 times by using 70 percent ethanol by volume, wherein the amount of the added ethanol for the 1 st time of the alcohol extraction is 3 times of the weight of the medicine, and the time is 2 hours; the amount of ethanol added for the 2 nd time is 2 times of the weight of the medicine, and the time is 1.5 hours; the ethanol amount added for the 3 rd time is 1.5 times of the weight of the medicine, and the time is 1 hour; mixing the extractive solutions, recovering ethanol, filtering to obtain filtrate and residue, and concentrating the filtrate twice by volume to obtain concentrated solution B;
b. decocting the residues in step a with water for 3 times, wherein the water adding amount for the 1 st decoction is 4 times of the weight of the medicine, and the decoction is carried out for 3 hours; the water adding amount for the 2 nd time is 2 times of the weight of the medicine, and the medicine is decocted for 2 hours; the water adding amount for the 3 rd time is 1.5 times of the weight of the medicine, and the medicine is decocted for 1 hour; mixing decoctions, filtering to obtain filtrate, and concentrating the filtrate twice by volume to obtain concentrated solution C; and finally, fully and uniformly mixing the concentrated solutions B and C to obtain a traditional Chinese medicine leaching solution;
(3) Preparation of oral liquid
Mixing the Chinese medicinal leaching solution and the mixed bacterial agent uniformly in a volume ratio of 1:1, adding the corrective maltitol accounting for 6% of the total volume of the mixed solution, fully mixing, filtering and packaging under a sterile state to obtain the thrombus-eliminating lipid-lowering probiotic compound bacteria Chinese medicinal oral liquid, and storing 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, inoculating into MRS culture medium according to 5% of inoculum size, fermenting at 37deg.C for 84 hr, and fermenting 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% 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 72 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 suspensions according to the volume ratio of lactobacillus to saccharomycetes to bifidobacterium=2:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of Chinese medicine extract
a. According to the weight portion, the total weight of the traditional Chinese medicine is 10 portions, 5 portions of red sage root, 2 portions of angelica, 1.5 portions of hemlock parsley and 1.5 portions of pseudo-ginseng are respectively cleaned and selected, crushed into fine powder, then mixed, and subjected to alcohol extraction for 4 times by using ethanol with the volume percentage of 60%, wherein the amount of the added ethanol for the 1 st time is 4 times of the weight of the medicine, and the time is 2 hours; the amount of ethanol added for the 2 nd time is 3 times of the weight of the medicine, and the time is 1.5 hours; the ethanol amount added for the 3 rd time is 2 times of the weight of the medicine, and the time is 1 hour; adding ethanol 1.5 times of the weight of the medicine for 1 hr, mixing the extractive solutions, recovering ethanol, filtering to obtain filtrate and residue, and concentrating the filtrate three times by volume to obtain concentrated solution B;
b. decocting the residues in step a with water for 3 times, wherein the water adding amount for the 1 st decoction is 5 times of the weight of the medicine, and the decoction is carried out for 3 hours; the 2 nd water adding amount is 3 times of the weight of the medicine, and the medicine is decocted for 2 hours; the water adding amount for the 3 rd time is 1.5 times of the weight of the medicine, and the medicine is decocted for 1 hour; mixing decoctions, filtering to obtain filtrate, and concentrating the filtrate three times by volume to obtain concentrated solution C; and finally, fully and uniformly mixing the concentrated solutions B and C to obtain a traditional Chinese medicine leaching solution;
(3) Preparation of oral liquid
Mixing the Chinese medicinal leaching solution and the mixed bacterial agent uniformly in a volume ratio of 2:1, adding the corrective maltitol accounting for 6% of the total volume of the mixed solution, fully mixing, filtering and packaging under a sterile state to obtain the thrombus-eliminating lipid-lowering probiotic compound bacteria Chinese medicinal oral liquid, and storing 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 saccharomycetes to bifidobacterium=3:2:1 to obtain a mixed microbial inoculum;
(2) Preparation of Chinese medicine extract
a. According to the weight portion, the total weight of the traditional Chinese medicine is 10 portions, 4 portions of red sage root, 3 portions of notoginseng, 2 portions of angelica and 1 portion of hemlock parsley are respectively cleaned and selected, crushed into fine powder, then mixed, and subjected to alcohol extraction for 3 times by using ethanol with the volume percentage of 75%, and the amount of the ethanol added for the 1 st time is 4 times of the weight of the medicine for 3 hours; the amount of ethanol added for the 2 nd time is 2 times of the weight of the medicine, and the time is 1.5 hours; the ethanol amount added for the 3 rd time is 1.5 times of the weight of the medicine, and the time is 1 hour; mixing the extractive solutions, recovering ethanol, filtering to obtain filtrate and residue, and concentrating the filtrate twice by volume to obtain concentrated solution B;
b. decocting the residues in step a with water for 3 times, wherein the water adding amount for the 1 st decoction is 4 times of the weight of the medicine, and the decoction is carried out for 3 hours; the water adding amount for the 2 nd time is 2 times of the weight of the medicine, and the medicine is decocted for 2 hours; the water adding amount for the 3 rd time is 1.5 times of the weight of the medicine, and the medicine is decocted for 1 hour; mixing decoctions, filtering to obtain filtrate, and concentrating the filtrate twice by volume to obtain concentrated solution C; and finally, fully and uniformly mixing the concentrated solutions B and C to obtain a traditional Chinese medicine leaching solution;
(3) Preparation of oral liquid
Mixing the Chinese medicinal leaching solution and the mixed bacterial agent uniformly in a volume ratio of 2:1, adding the corrective maltitol accounting for 6% of the total volume of the mixed solution, fully mixing, filtering and packaging under a sterile state to obtain the thrombus-eliminating lipid-lowering probiotic compound bacteria Chinese medicinal oral liquid, and storing in a refrigerator at 4 ℃.
The prepared thrombolytic lipid-lowering probiotic compound bacteria traditional Chinese medicine oral liquid contains lactobacillus which is more than or equal to 400 multiplied by 10 per milliliter of oral liquid 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 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 a cholesterol source into sterilized MRS and McO liquid culture medium according to the volume ratio of 5%, and packaging into test tubes in an aseptic and uniform way. 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 by phosphothioiron colorimetry, 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
Under aseptic condition, adding triglyceride standard substance into sterilized MRS and Maishi liquid culture medium at a volume ratio of 5%, 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 result of high triglycerides is that they tend to cause "blood viscosity", i.e. the blood is viscous due to too high a lipid content in the blood, which deposits on the vessel wall and gradually forms small plaques, i.e. what we talk of atherosclerosis at ordinary times. The high result of triglycerides is severe in 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 mediums with pH 3.0 and pH 2.0 according to the inoculum size of 2 percent of the volume ratio, 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 log of colony count in units is plotted on the abscissa as strain name.
Activating different strains to obtain bacteriaSuspension (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 properties of growing and developing in an acidic environment in order 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 pH 3.0, 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 good adaptability to acid environment, and can survive more after entering human intestinal tracts without affecting 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 inhibition effects on escherichia coli, bacillus subtilis and staphylococcus aureus. Staphylococcus aureus is the most common pathogenic bacteria in human suppurative infection, and can cause local suppurative infection, pneumonia, pseudomembranous enteritis, 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, triglyceride content and 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 total cholesterol content and the triglyceride 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 compound bacteria Chinese medicine oral liquid on mouse hyperlipidemia
A Chinese medicinal extract and a compound bacterium Chinese medicinal oral liquid were prepared according to example 1. The 60 mice successfully molded by high fat are randomly divided into 3 groups, each mouse is filled with 0.2ml of extract every day, each mouse is filled with 0.2ml of compound bacterium Chinese medicinal oral liquid every day, and the control group is not arranged and is normally eaten. The mice in each group were fed 5g of basal feed each day. In order to ensure a certain number of living bacteria in the composite bacteria traditional Chinese medicine oral liquid, the composite bacteria traditional Chinese medicine oral liquid is synthesized by adopting fresh treated bacterial strains every day. After 35d of feeding, the mice were subjected to blood sampling at broken ends, and the Total Cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL-C) cholesterol contents in the serum of the mice were measured, and the results are shown in FIG. 5.
After 35d of feeding, the Total Cholesterol (TC) content, the Triglyceride (TG) content and the low density lipoprotein (LDL-C) cholesterol content in the serum of the mice in the compound bacteria traditional Chinese medicine oral liquid group and the traditional Chinese medicine leaching liquid group are reduced compared with a control group, but each index of the compound bacteria traditional Chinese medicine oral liquid group is superior to that of the traditional Chinese medicine leaching liquid group, which indicates that the cholesterol and triglyceride content in the serum of the mice can be reduced after the compound bacteria traditional Chinese medicine oral liquid is fed.
7. In vivo toxicity test in mice
A composite microbial inoculum was prepared according to the preparation method of the mixed microbial inoculum of examples 1-3. Each mouse is filled with 0.2ml of gastric complex microbial inoculum every day, and in order to ensure a certain number of viable bacteria in the bacterial solution, each gastric complex microbial inoculum is filled with a fresh treated bacterial strain. After the continuous gastric lavage of the compound bacteria 42d, 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 were not significantly different (p > 0.05) from those of the control group, and all the mice were normal after being observed by tissue sections of the liver and kidney of the mice (as shown in fig. 6 and 7), which shows that the strain has no obvious toxic and side effects 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 compound bacteria Chinese medicinal oral liquid has excellent lipid-lowering and weight-losing effects, can inhibit the growth of intestinal harmful flora, has the effect of regulating the intestines, and has no toxic or side effect.
Claims (11)
1. A composite bacterium with thrombolytic and lipid-lowering probiotics is characterized by comprising two strains of lactobacillus, namely lactobacillus pentosus (La. Pentosus) separated from the intestinal tracts of Tibetan mastiffs, wherein the strains are preserved in China general microbiological culture Collection center (CGMCC) No.7426 and 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.
2. The complex bacterium having thrombolytic and lipid-lowering probiotic effects according to claim 1, wherein lactobacillus plantarum is isolated from lan-zhou slurry.
3. The composite bacterium with thrombolytic and lipid-lowering probiotic effects according to claim 2, characterized in that lactobacillus plantarum (Lactobacillus plantarum) has been deposited in China general microbiological culture Collection center with the accession number CGMCC No.7425 on the date of 07 of 04 of 2013.
4. A method for preparing the composite bacteria with thrombolytic lipid-lowering probiotic effects according to any one of claim 3, which is characterized by at least comprising 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 according to the inoculum size of 5-10% of the volume ratio, and carrying out shake fermentation culture at the temperature of 32-37 ℃ and the speed of 120-150 r/min for 72-96 h, wherein the density of saccharomycete 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 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 suspension according to the volume ratio of lactobacillus to saccharomycetes to bifidobacterium=1-3:1-2:1 to obtain the composite bacteria.
5. The method for preparing the composite bacteria with thrombolytic and lipid-lowering probiotic effects according to claim 4, wherein the formula and the conditions of the MRS liquid culture medium are 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 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. A Chinese medicinal oral liquid containing the composite bacteria with thrombolytic and lipid-lowering effects of claim 1, wherein each mL of the oral liquid contains lactobacillus not less than 400×10 13 CFU and saccharomycetes are more than or equal to 600 multiplied by 10 12 CFU, bifidobacterium not less than 400 x10 7 CFU。
7. The Chinese medicinal oral liquid containing the composite bacteria with thrombolytic and lipid-lowering effects according to claim 6, wherein the oral liquid contains Chinese medicaments with the effects of activating blood circulation, removing blood stasis and/or relieving pain.
8. The traditional Chinese medicine oral liquid containing the composite bacteria with the thrombolytic and lipid-lowering probiotic effects according to claim 7, which is characterized in that the oral liquid contains traditional Chinese medicines with the effects of activating blood, removing blood stasis and/or relieving pain, respectively: radix Salviae Miltiorrhizae, radix Angelicae sinensis, rhizoma Chuanxiong, radix Notoginseng, and/or flos Carthami.
9. The method for preparing the traditional Chinese medicine oral liquid containing the composite bacteria with the thrombolytic and lipid-lowering probiotic effects of claim 8, which is characterized in that: the preparation method at least comprises the following steps:
(1) Preparation of Chinese medicine extract
a. According to the weight portion, the total weight of the traditional Chinese medicines is 10 portions, 0 to 5 portions of red sage root, 0 to 5 portions of angelica, 0 to 5 portions of hemlock parsley, 0 to 3 portions of pseudo-ginseng and 0 to 3 portions of safflower are taken, the traditional Chinese medicines cannot be simultaneously taken for zero, respectively cleaned and crushed into fine powder, then mixed, ethanol with the volume percentage of 60 to 80 percent is used for 3 to 5 times of ethanol extraction, the ethanol added for the 1 st time of the ethanol extraction is 3 to 5 times of the weight of the medicine, and the time is 2 to 3 hours; the ethanol is added for the 2 nd time for 1.5-2 hours, wherein the amount of the ethanol is 2-4 times of the weight of the medicine; the ethanol is added for 1-2 hours for 3 rd time, wherein the weight of the ethanol is 1-2 times of that of the medicine; mixing the extracting solutions, recovering ethanol, filtering to obtain filtrate and residue, and concentrating the filtrate by 2-3 times according to volume to obtain concentrated solution B;
b. decocting the residues in the step a with water for 3 times, wherein the water adding amount for the 1 st decoction is 3-5 times of the weight of the medicine, and the decoction lasts for 2-3 hours; the water adding amount for the 2 nd time is 2-4 times of the weight of the medicine, and the medicine is decocted for 2-3 hours; the water adding amount for the 3 rd time is 1-2 times of the weight of the medicine, and the medicine is decocted for 1-2 hours; mixing the decoctions, filtering to obtain filtrate, and concentrating the filtrate by 2-3 times according to volume to obtain concentrated solution C; and finally, fully and uniformly mixing the concentrated solutions B and C to obtain a traditional Chinese medicine leaching solution;
(2) The method of claim 4 is used for preparing the composite bacteria with the thrombolytic and lipid-lowering probiotics effects;
(3) Preparation of oral liquid
Mixing the Chinese medicine leaching solution and the composite bacteria in the volume ratio of 1-5 to 1, adding the corrective accounting for 5-8% of the total volume of the mixed solution, fully mixing, filtering and packaging in a sterile state, and storing in a refrigerator at 0-4 ℃.
10. The method for preparing the compound bacteria traditional Chinese medicine oral liquid according to claim 9, which is characterized in that: the flavoring agent is maltitol.
11. The method for preparing the compound bacteria traditional Chinese medicine oral liquid according to claim 8, which is characterized in that: the oral liquid contains lactobacillus not less than 400×10 per mL 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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