CN113832058A - Application of bifidobacterium lactis BLA80 in preparation of medicines or foods for reducing blood fat and regulating intestinal flora - Google Patents
Application of bifidobacterium lactis BLA80 in preparation of medicines or foods for reducing blood fat and regulating intestinal flora Download PDFInfo
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- CN113832058A CN113832058A CN202111111411.4A CN202111111411A CN113832058A CN 113832058 A CN113832058 A CN 113832058A CN 202111111411 A CN202111111411 A CN 202111111411A CN 113832058 A CN113832058 A CN 113832058A
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- Prior art keywords
- bifidobacterium lactis
- bla80
- bifidobacterium
- intestinal flora
- blood fat
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Abstract
The invention discloses an application of bifidobacterium lactis BLA80 in preparing a medicament or a health-care food for reducing blood fat and regulating intestinal flora, belonging to the technical field of microorganisms. The bifidobacterium lactis BLA80 has strong gastric acid resistance and intestinal juice resistance, has strong inhibiting effect on common intestinal pathogenic bacteria, has no antibiotic resistance and is safe probiotic. The bifidobacterium lactis BLA80 has strong bile salt hydrolase activity, has excellent cholesterol-lowering property, can obviously reduce TC, TG and LDL-C levels and AI values of the serum of a high-fat rat, and can improve the HDL-C level of the serum. The bifidobacterium lactis BLA80 powder can remarkably increase both bifidobacterium and lactobacillus in the intestinal tract, and the bifidobacterium lactis BLA80 powder has the function of regulating the intestinal flora. The bifidobacterium lactis BLA80 can be widely applied to the field of foods and microbial preparations, and can achieve the purposes of regulating intestinal flora and reducing blood fat by daily intake without medicaments.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of bifidobacterium lactis BLA80 in preparation of a medicament or food for reducing blood fat and regulating intestinal flora.
Background
In the Chinese resident nutritional and chronic disease condition report 2020, the overweight and obesity rate of residents in various ages in the town and the country continues to rise, more than 50% of the adult residents are overweight and obese, and the overweight rate and the obesity rate of the residents aged 18 years and above are 34.3% and 16.4% respectively. The overweight and obesity rates of children and adolescents under the ages of 6-17 years and 6 years are respectively 19% and 10.4%. But also presents a development trend that the rising speed is higher, the popularity level is higher and the whole crowd is affected. Hyperlipidemia is a common complication associated with overweight and obesity. Hyperlipidemia is a disease in which abnormal blood lipids (total cholesterol, triglyceride, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol) in the body are caused by lipid metabolism disorder in the human body, thereby seriously harming human health. The commonly used drug therapy mainly controls blood fat within a certain range for a long time, can not completely cure hyperlipidemia, and can cause side effects of nausea, vomiting, liver damage, induced ulcer, aggravation of diabetes, gout and the like. The most commonly used lipid lowering drugs include statins, clofibrate, nicotinic acid and bile acid chelating resins. As health consciousness of people in China increases, more and more people tend to achieve physical health by improving diet, eating probiotics and the like instead of drug therapy. The bifidobacterium lactis is one of common probiotics, and the bifidobacterium lactis BLA80 can reduce blood fat by regulating intestinal flora and achieve the purpose of reducing hyperlipidemia.
Disclosure of Invention
The invention provides an application of bifidobacterium lactis BLA80 in preparing medicines or foods for reducing blood fat and regulating intestinal flora. By taking the medicine or food containing the probiotic BLa80 instead of chemical medicines, the hyperlipidemia patients can fundamentally regulate intestinal flora and reduce blood fat.
The Bifidobacterium lactis BLA80 is classified and named as Bifidobacterium animalis subsp, and is preserved in China general microbiological culture Collection center (CGMCC) at 17 th of 2021 year 05, with the address of No. 3 of Xilu No. 1 of Xingyang district of Beijing, and the preservation number of CGMCC No. 22547. The bifidobacterium lactis BLa80 has excellent cholesterol-lowering and antibacterial properties. Further research shows that the bifidobacterium lactis BLA80 can remarkably reduce TC, TG and LDL-C levels and AI values of high-fat rat serum, can improve HDL-C level, and shows that the bifidobacterium lactis BLA80 has the function of reducing blood fat.
After the bifidobacterium lactis BLa80 microbial inoculum is eaten by human bodies for 15 days, compared with the bifidobacterium and lactobacillus in the intestinal tract before eating and the control group, the bifidobacterium and the lactobacillus in the intestinal tract are both obviously increased, and bacteroides, enterobacteria, enterococcus and clostridium perfringens are not obviously changed. Therefore, the bifidobacterium lactis BLA80 microbial inoculum has the function of regulating the intestinal flora.
The fermentation method of the bifidobacterium lactis BLA80 bacterial powder comprises the following steps: preparing 10L of fermentation medium, adding into a 15L anaerobic fermentation tank, sterilizing at 121 deg.C for 20min, cooling, and adjusting pH to 7 with NaOH. 200mL of Bifidobacterium lactis seed solution was inoculated into a fermentation medium and fermented at 37 ℃ and 100 rpm. In the fermentation process, 25% ammonia water is automatically fed to maintain the pH value of the fermentation liquor at 5.5-6.0, and nitrogen is introduced in the whole fermentation process. And when the OD600 of the fermentation liquid is not increased any more after 16 hours of fermentation, the fermentation is stopped.
The preparation method of the bifidobacterium lactis BLA80 bacterial powder comprises the following steps: uniformly mixing the centrifugally collected bacterial sludge and a freeze-drying protective agent according to the mass ratio of 1:1, placing the mixture in a freeze-drying machine, pre-freezing for 2h at the temperature of minus 40 ℃, then heating for 4h under the condition of 10Pa to the temperature of minus 10 ℃, freeze-drying for 15h, then heating for 2h under the condition of 20Pa to the temperature of 25 ℃, and freeze-drying for 10h to obtain the freeze-dried bacterial powder of the bifidobacterium lactis. The freeze-drying protective agent comprises the following components in percentage by mass: trehalose 6%, maltodextrin 8%, sucrose 2%, vitamin C0.02%, glycerol 3%, L-arginine 0.8%, and the balance of water.
The invention provides an application of bifidobacterium lactis BLA80 in preparing food, which particularly comprises the following applications: the application in preparing food for reducing blood fat, the application in preparing food for reducing cholesterol and blood fat, the application in preparing food for regulating intestinal flora, and the application in preparing food for reducing cholesterol, reducing blood fat and regulating intestinal flora.
The food refers to common food and health food, including tablet candy, fermented beverage, soft candy, concocted milk powder, fermented milk, and solid beverage.
The invention also provides an application of the bifidobacterium lactis BLA80 in preparing a medicament, which specifically comprises the following applications: the application in preparing the medicines for reducing blood fat, the application in preparing the medicines for reducing cholesterol and blood fat, the application in preparing the medicines for regulating intestinal flora, and the application in preparing the medicines for reducing cholesterol, reducing blood fat and regulating intestinal flora.
Such drugs include, but are not limited to, probiotics.
The bifidobacterium lactis BLA80 adopted by the invention is separated from breast milk, is safe and free from pathogenicity, has strong gastric acid resistance and intestinal juice resistance, has strong inhibiting effect on common intestinal pathogenic bacteria, has no antibiotic resistance, and is safe probiotic. The bifidobacterium lactis BLA80 also has strong activity of bile salt hydrolase and excellent cholesterol-lowering property, and the cholesterol removal rate can reach 92.4%. The rat test and the human body feeding test prove that the traditional Chinese medicine composition has the effects of regulating intestinal flora and reducing blood fat. The food can be widely applied to the field of food, the possibility of ingestion of consumers is increased, and the purposes of regulating intestinal flora and reducing blood fat can be achieved through daily ingestion. Of course, the bifidobacterium lactis BLa80 can also be used for preparing medicines for reducing blood fat and regulating intestinal flora.
Drawings
FIG. 1 is a graph showing the results of the antagonistic activity of Bifidobacterium lactis BLA80 against various pathogenic bacteria.
FIG. 2 is a graph showing the results of serum cholesterol levels in rats of each group in example 6, and the differences in lower case letters represent significant differences between groups (P < 0.05), as follows.
FIG. 3 is a graph showing the results of serum triglyceride levels of rats of each group in example 6.
FIG. 4 is a graph showing the results of the serum HDL-C levels of rats of each group in example 6.
FIG. 5 is a graph showing the results of the LDL-C levels in serum of rats in each group in example 6.
FIG. 6 is a graph showing the effect of Bifidobacterium lactis BLA80 on rat AI in example 6.
Detailed Description
The embodiments of the present invention are described in detail below with reference to specific embodiments, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the description of the present invention.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 isolation, screening and identification of Bifidobacterium lactis BLA80
(1) Separation and screening:
collecting breast milk samples in the Chinese outlying pastoral area, diluting to a proper gradient with sterile normal saline, coating on an MRS agar plate added with 5% (V/V) of mupirocin lithium salt and containing 0.1% of L-cysteine hydrochloride, and culturing for 48h under an anaerobic condition at 37 ℃. Selecting an opaque milky white, round and glossy, uniform-edge, convex-surface and wet monoclonal colony, performing repeated streak purification culture on an MRS solid culture medium, and observing cell morphology and individual morphology to obtain the bifidobacterium strain.
(2) Molecular biological identification:
extracting genome DNA of Bifidobacterium strain with genome DNA extraction kit, and extracting with upstream primer 27F (AGTTTGATCMTGGC)TCAG and a downstream primer 1492R (GGTTACCTTGTTACGACTT), and the 16S rDNA sequence is amplified to obtain a PCR product. And sequencing the PCR product. Wherein the PCR reaction system comprises: 10 XBuffer 20 μ L, primer dNTP 4 μ L, upstream and downstream primers 1 μ L each, DNA template 2 μ L, Taq enzyme 0.5 μ L, ddH2O34.5. mu.L. And (3) PCR reaction conditions: 10min at 95 ℃; 30s at 94 ℃, 30s at 56 ℃, 2min at 72 ℃ and 35 cycles; 10min at 72 ℃. And (3) detecting the PCR product through gel electrophoresis, and then sending the PCR product to Wuhan Jinrui bioengineering company Limited for sequencing. The identified gene sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis alignment. According to the identification result of molecular biology, the strain is determined to be bifidobacterium lactis, and the strain is named as bifidobacterium lactis BLA 80. The 16S rDNA sequence of the bifidobacterium lactis BLA80 is shown in SEQ ID No 1.
Bifidobacterium lactis BLa80 was deposited at the china general microbiological culture collection center (CGMCC) on 2021, 05 and 17 months, address: no. 3 of Xilu No. 1 of Beijing area of the rising of the morning, the strain is classified and named as Bifidobacterium animalis subsp.
Example 2 environmental assessment of Bifidobacterium lactis BLA80 by gastrointestinal tract
Simulated artificial gastric fluid: preparing 0.5% NaCl solution, adding 0.3% pepsin, adjusting pH to 2.5 with 1mol/L HCL, fully dissolving, and filtering with 0.22 μm microporous membrane for sterilization.
Simulating artificial intestinal juice: preparing 0.5% NaCl solution, adding 0.1% trypsin, adjusting pH to 8.0 with 0.1mol/L NaOH, dissolving completely, filtering with 0.22 μm microporous membrane, and sterilizing.
Bifidobacterium lactis BLa80 was activated and cultured for 2 passages under anaerobic conditions. And (3) centrifuging the activated bifidobacterium lactis BLA80 bacterial liquid, and collecting the bacterial cells. 0.4mL of thallus suspension is respectively inoculated into 1.6mL of prepared simulated artificial gastric juice with pH2.5 and simulated artificial intestinal juice with pH8.0, the mixture is uniformly mixed and digested at 37 ℃, meanwhile, 0h and 3h of digestive juice are respectively taken to detect the viable count, the survival rate is calculated, and the results are shown in the following table. Wherein the survival rate (%) of the strain is Nt/N0X 100%, wherein N0 representsViable count of 0h (CFU/mL), N of the StraintThe number of viable bacteria of strain 3h (CFU/mL) was indicated.
Experimental data table for simulating digestive tract environment by bifidobacterium lactis BLA80
The survival rate of the bifidobacterium lactis Bla80 in the artificial gastric juice with the pH value of 2.5 for 3h is 96.7 percent, and the survival rate in the artificial intestinal juice with the pH value of 8.0 for 3h is 93.6 percent. Experiments show that the bifidobacterium lactis BLA80 has strong capability of tolerating the environment of the gastrointestinal tract.
Example 3 evaluation of antibiotic susceptibility of Bifidobacterium lactis BLA80
Marking and activating bacteria to be detected on an MRS solid plate containing 0.1 percent of L-cysteine hydrochloride, preparing bacterial suspension and adjusting the concentration of the bacterial suspension to be 108CFU/mL, 100. mu.L of the bacterial suspension is added to a solid plate containing 0.1% of L-cysteine hydrochloride MRS, the bacterial solution is uniformly coated on the plate by using a sterile cotton swab, and an antibiotic drug sensitive tablet is attached to the plate, wherein a paper sheet without antibiotic is used as a blank control. The strain is placed under anaerobic condition and is cultured at 37 ℃ in an upright way, after 24 hours, the diameter of the strain sensitive to antibiotics is measured by a ruler, and the result is shown in the following table.
Bifidobacterium lactis BLA80 sensitivity data to antibiotics (mm)
Bifidobacterium lactis BLA80 is highly sensitive to the 14 antibiotics erythromycin, penicillin, amoxicillin, ampicillin, vancomycin, cefaclor, oxacillin, ceftriaxone, novobiocin, cepacia, clindamycin, azithromycin rifampicin and methoxypyrimidine evaluated, the diameters of the inhibition zones are all greater than 20mm, and the diameter of the inhibition zone of 10 of the antibiotics is greater than 30 mm. Experiments show that bifidobacterium lactis BLa80 is a safe probiotic.
Example 4 evaluation of bacteriostatic Activity of Bifidobacterium lactis BLA80
Inoculating antagonistic strain into anaerobic glass tube containing 0.1% L-cysteine hydrochloride MRS liquid culture medium at 2% (V/V), and standing at 37 deg.C for 12 hr. Respectively inoculating pathogenic strains of Escherichia coli, salmonella and Staphylococcus aureus into liquid beef extract peptone culture medium, culturing at 37 deg.C with rotation speed of 250rpm, and shaking overnight at constant temperature to obtain pathogenic bacteria suspension. Cooling MRS solid culture medium to about 55 deg.C, mixing with the pathogenic bacteria suspension at a certain ratio to make the number of live bacteria in system pathogenic bacteria be 106CFU/mL order of magnitude, then quickly pouring into a flat plate in which an Oxford cup is placed in advance, taking out the Oxford cup after the culture medium is cooled and solidified, injecting 200 mu L of antagonistic strain liquid into each hole, placing the flat plate in a constant-temperature incubator at 37 ℃ after being lightly covered, observing after culturing for a proper time, and measuring the diameter of the inhibition zone by using a vernier caliper.
The results are shown in FIG. 1, where the antagonistic diameters of Bifidobacterium lactis BLA80 against E.coli, Salmonella typhi and Staphylococcus aureus were 42mm, 41mm and 27mm, respectively. Shows extremely strong inhibition effect on intestinal pathogenic bacteria, in particular to the inhibition capability on escherichia coli and salmonella.
Example 5 measurement of Cholesterol removal Rate of Bifidobacterium lactis BLA80
(1) Cholesterol standard curve determination: accurately weighing 0.1g of cholesterol powder, metering the volume of a solution of 1mg/mL by using n-hexane, then respectively sucking 0.02 mL, 0.04 mL, 0.06 mL, 0.08 mL, 0.1 mL, 0.12 mL and 0.14mL into a colorimetric tube, drying by using water bath nitrogen at 60 ℃, adding 4mL of 0.5mg/mL o-phthalaldehyde solution and 2mL of 98% concentrated sulfuric acid, carrying out color development reaction for 20min, measuring the absorbance value at 553nm, and taking the cholesterol content as the horizontal axis and the absorbance value as the vertical axis as a standard curve.
(2) The method for measuring the cholesterol content by o-xylene comprises the following steps: 1mL of sample was added with 3mL of 95% ethanol and 2mL of 50% (w/v) potassium hydroxide, and vortexed and mixed. And (3) carrying out constant-temperature water bath at 60 ℃ for 10min, cooling, adding 5mL of n-hexane, carrying out vortex oscillation extraction for 1-2min, adding 2mL of distilled water, oscillating uniformly, and standing for layering. Taking 2mL of upper-layer n-hexane, drying the upper-layer n-hexane by blowing with nitrogen in a water bath at 60 ℃, adding 4mL of 0.5mg/mL (w/v) o-dicarbaldehyde solution (constant volume by glacial acetic acid) and 2mL of concentrated sulfuric acid, carrying out color reaction for 20min, and measuring the absorbance value at 553 nm.
(3) Determination of cholesterol removal rate of bifidobacterium lactis BLa 80:
inoculating bifidobacterium lactis BLA80 into an anaerobic tube filled with an L-MRS liquid culture medium, standing and culturing at 37 ℃ for 12h, transferring for 3 times of activation, inoculating into 5mL of culture solution MRSO-CHOL according to the inoculation amount of 2% (v/v), shaking uniformly, immediately sampling for 1mL, centrifuging at 4000r/min for 5min, and taking the supernatant to measure the cholesterol content by an o-phthalaldehyde colorimetric method. After the inoculated fermentation liquor is statically cultured for 48 hours at 37 ℃, the cholesterol content in the supernatant is measured by the same method, and a plurality of groups of parallel averages are made. Wherein the composition of the culture solution MRSO-CHOL is as follows: consists of MRS liquid culture medium, sodium thioglycollate, cholesterol and ox bile salt; the concentration of sodium thioglycolate is 2g/L, the concentration of cholesterol is 200mg/L, the concentration of the ox bile salt is 0.3% (mass fraction), and the sterilization is carried out for 20min at 115 ℃.
in the formula: a is the absorbance value at 553nm of the culture medium after fermentation of each strain, and B is the absorbance value at 553nm of the blank control.
The result shows that the cholesterol removal rate of the bifidobacterium lactis BLA80 can reach 92.4%.
Example 6 evaluation of the ability of Bifidobacterium lactis BLA80 to reduce blood lipid
Strain activation: taking out the glycerol tube from a refrigerator at minus 80 ℃, unfreezing, inoculating the glycerol tube into an anaerobic tube filled with an L-MRS culture medium in an inoculation amount of 2%, culturing for 12-24 h at 37 ℃, and growing until the bacterial liquid is turbid to obtain an activated first-generation strain.
The components of the L-MRS culture medium are as follows: 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder and K2HPO42g, 2g of triammonium citrate, 5g of sodium acetate, 20g of glucose, 801 mL of Tween and MgSO4·7H2O 0.58g、MnSO4·4H20.25g of O, 1g of L-cysteine hydrochloride and 1000mL of distilled water; the pH value is 6.2 +/-0.2.
(1) Experimental grouping and feeding mode
Collecting cultured Bifidobacterium lactis BLA80, and adding physiological salineWashing for 3 times, adjusting the concentration to 1.0 × 109CFU/mL was used for rat gavage. SD rat raising conditions: the temperature of the animal room is (22 +/-2) ° C, the relative humidity is (50 +/-10)%, the circulation of light/dark for 12 hours is strictly followed, and rats can feed and intake water freely. One week after adaptive feeding, 30 SD rats of 5 weeks old were divided into 3 groups according to no significant difference in mean body mass between groups: normal group (ND): basal feed, control group (HFD): high fat diet + normal saline, experimental group (HFD + BLa 80): feeding high fat diet and Bifidobacterium lactis BLA 80. Each rat was gavaged 1 mL/time/day for 42 days.
High-fat feed: 78.8% basal feed, 1% cholesterol, 10% egg yolk powder, 10% lard, 0.2% bile salt.
Measurement of body mass increase and food intake: weighing the body quality of each group of rats at the beginning and the end of the experiment, and calculating the body quality increment of the rats; the food intake of the rats was recorded every 2d and the food utilization rate was calculated according to the following formula.
Food utilization rate (weight gain/feed intake) × 100%
(2) Determination of serum cholesterol (TC), serum Triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) content in serum
After 42 days of gastric lavage, the rat is fasted for 12 hours, blood is taken for 1mL after tail breaking, centrifugation is carried out for 10min at 2500r/min, and serum is taken. And respectively referring to the kit for description, measuring the contents of TC, TG and HDL-C in the serum by using an enzyme-labeling instrument, and comparing the difference of serum indexes among groups. The Arteriosclerosis Index (AI) is calculated according to the following formula
In the formula: cTCThe serum cholesterol concentration/(mmol/L); cHDL-CThe concentration of serum HDL-C/(mmol/L).
(3) Data processing
Significance was tested by analysis of variance using SPSS 26 statistical software, and data analysis was performed by Least Significant Difference (LSD) test for multiple comparisons.
(4) Effect of Bifidobacterium lactis BLA80 on body quality and food intake of rats with high fat diet
Note: in comparison to the same column, different letters indicate significant differences (P < 0.05).
The weight gain of the rats in the control group is obviously higher than that of the rats in the other two groups (P < 0.05), and the weight gain of the experimental group is lower than that of the rats in the control group, so that the weight gain is reduced probably because the bifidobacterium lactis influences the lipid metabolism of the rats. The normal group of rats received significantly higher food intake than the other two groups (P < 0.05), probably as a result of a decrease in appetite of the rats due to cholesterol and lipids in the high-fat diet. The feed utilization rate of the control group and the experimental group is obviously higher than that of the normal group of rats.
(5) Effect of Bifidobacterium lactis BLA80 on hyperlipidemic rats
When the rat mice are fed with the high-fat feed for 42d, the serum TC level of the rat in the experimental group is obviously lower than that of the control group, and has no obvious difference with the serum TC level of the rat in the normal group (figure 2), which shows that the serum TC level of the high-fat rat can be obviously reduced by feeding the bifidobacterium lactis BLA80, and the reduction effect is close to that of the rat in the normal group.
The serum TG content of the rats in the experimental group is obviously lower than that of the control group and is obviously higher than that of the rats in the normal group (figure 3), which shows that the serum TG level of the high-fat rats can be obviously reduced by feeding the bifidobacterium lactis BLA 80.
The TC and TG levels of the rats in the control group are obviously higher than those of the rats in the normal group, so that the establishment of a rat model with hyperlipidemia can be determined.
The serum HDL-C of the rats in the normal group is obviously higher than that of the rats in the control group, and the serum HDL-C content of the rats in the experimental group has no obvious difference with the normal group and also has no obvious difference with the control group (figure 4). Feeding bifidobacterium lactis BLa80 reduced serum HDL-C levels to some extent in high-fat rats, but was not significant.
The serum LDL-C content of rats in the experimental group is obviously lower than that of the control group and is obviously higher than that of rats in the normal group (figure 5), which shows that the serum LDL-C level of the rats with high fat can be obviously reduced by feeding bifidobacterium lactis BLA 80.
The AI of rats in the experimental group was significantly lower than that in the control group and was not significantly different from that in the normal group (fig. 6), indicating that feeding bifidobacterium lactis BLa80 significantly reduced the AI value of high-fat rats.
Feeding Bifidobacterium lactis BLA80 can significantly reduce serum TC, TG and LDL-C levels of high-fat rats, increase HDL-C level, and significantly reduce AI value, and Bifidobacterium lactis BLA80 has blood lipid reducing effect.
Example 7 human feeding trial test
1) Subject recruitment criteria: (1) the patient is examined to have hyperlipidemia in the last year; (2) gastrointestinal diseases are not suffered within one month; (3) antibiotics are not taken within one month; (4) the article related to the tested function is not taken in a short time; (5) women under age 65, non-pregnant or lactating.
2) The experimental method comprises the following steps: dividing the subjects meeting the standard into a control group and a test group at random, wherein each group contains 30 persons, and the test group takes 1g (containing 100 hundred million CFU) of Bifidobacterium lactis BLA80 microbial inoculum for 2 times per day; the control group was administered with starch in an equal amount daily for 14 consecutive days without changing the original dietary habits. Feces 1.0g before and after administration to the subject was collected aseptically. And (3) performing 10-fold serial dilution, selecting proper dilution to inoculate on each culture medium, counting colonies, and calculating the number of bacteria in each gram of wet excrement. The media and culture conditions for various intestinal flora are shown in the following table.
3) And (4) processing a result: the test data is measured data and analyzed by t test. The self-control data is analyzed by paired t test, and the two groups of mean values are compared by grouped t test.
4) Results and analysis of human feeding trials
Effect of Bifidobacterium lactis BLA80 on human intestinal flora (Lg CFU/g)
Note: the capital letters are compared with a control group after test feeding, and P is less than 0.05; the lower case letters indicate that P is less than 0.05 in the test group compared with the test group before the test.
From the above table, it can be seen that after the test feeding, the bifidobacteria and lactobacilli are significantly increased, and the bacteroides, enterobacteria, enterococci and clostridium perfringens are not obviously changed in the test feeding group compared with the control group; after the bifidobacterium lactis BLa80 microbial inoculum is tried, compared with the bifidobacterium and the lactobacillus before the lactobacillus is tried, the bacteroides, the enterobacter, the enterococcus and the clostridium perfringens are not obviously changed. Therefore, the bifidobacterium lactis BLA80 microbial inoculum has the function of adjusting intestinal flora.
EXAMPLE 8 preparation of Bifidobacterium lactis BLA80 powder
1) Fermentation process of bifidobacterium lactis BLA80 bacterial powder:
inoculating the bifidobacterium lactis strain preserved in the glycerin pipe into 10mL of L-MRS culture medium, and standing at 37 ℃ for anaerobic culture for 18h to obtain first-grade seeds; inoculating the primary seed bacterial liquid into an L-MRS culture medium with the inoculum size of 2% (V/V), culturing for 12h under the anaerobic condition at 37 ℃ to obtain secondary seeds, inoculating the secondary seeds into the L-MRS culture medium with the inoculum size of 2%, and culturing for 12h under the anaerobic condition at 37 ℃ to obtain the tertiary seeds.
The formula of the fermentation medium is as follows: 20/L of lactose, 10/L of beef extract powder, 10/L of yeast extract powder, 10g/L of beef bone peptone, 3g/L of dipotassium phosphate, 3g/L of ammonium hydrogen citrate, 5g/L of sodium acetate, 0.5g/L of magnesium sulfate, 0.1g/L of manganese sulfate and 1g/L of tween-801 mL/L, L-cysteine hydrochloride.
Fermentation: preparing 10L of fermentation medium, adding into a 15L anaerobic fermentation tank, sterilizing at 121 deg.C for 20min, cooling, and adjusting pH to 7 with NaOH. 200mL of the bifidobacterium lactis tertiary seed solution is inoculated into a fermentation medium and fermented at the temperature of 37 ℃ and the rotating speed of 100 rpm. In the fermentation process, 25% ammonia water is automatically fed to maintain the pH value of the fermentation liquor at 5.5-6.0, and nitrogen is introduced in the whole fermentation process. And when the OD600 of the fermentation liquid is not increased any more after 16 hours of fermentation, the fermentation is stopped.
2) The preparation process of the bifidobacterium lactis BLA80 bacterial powder comprises the following steps:
(1) and (3) centrifuging the fermentation liquor obtained by the high-density fermentation method of the bifidobacterium lactis, removing the supernatant, and collecting bacterial sludge. The centrifugation method is preferably centrifugation at 8000rpm for 10min at 4 ℃.
(2) Uniformly mixing the centrifugally collected bacterial sludge and a freeze-drying protective agent according to the mass ratio of 1:1, placing the mixture in a freeze-drying machine, pre-freezing for 2h at the temperature of minus 40 ℃, then heating for 4h under the condition of 10Pa to the temperature of minus 10 ℃, freeze-drying for 15h, then heating for 2h under the condition of 20Pa to the temperature of 25 ℃, and freeze-drying for 10h to obtain the freeze-dried bacterial powder of the bifidobacterium lactis.
The freeze-drying protective agent comprises the following components in percentage by mass: trehalose 6%, maltodextrin 8%, sucrose 2%, vitamin C0.02%, glycerol 3%, L-arginine 0.5-0.8%, and water in balance.
Example 9 use of Bifidobacterium lactis BLA80 in fermented milk
Raw materials in parts by weight (per 1000 mL): 80 g of white granulated sugar; 5g of a stabilizer; 1g of bifidobacterium lactis BLA80 bacterial powder, 0.04 vitality unit of streptococcus thermophilus/lactobacillus bulgaricus starter and the volume of fresh milk is fixed to 1000 mL.
The preparation method of the fermented milk comprises the following steps:
(1) uniformly mixing the stabilizer and the white granulated sugar, adding the mixture into the heated fresh milk, and uniformly stirring and dissolving to obtain a mixture;
(2) homogenizing, sterilizing and cooling the mixture to obtain a cooled base material; wherein the homogenizing pressure is 150-170 bar, the sterilization temperature is 95 +/-3 ℃, the time is 300 seconds, and the temperature is cooled to 42 +/-1 ℃.
(3) Inoculating the bifidobacterium lactis BLA80 bacterial powder and the streptococcus thermophilus/lactobacillus bulgaricus starter to the base material, and standing and fermenting for 10 hours at 43 ℃ to obtain the yoghourt with the functions of regulating intestinal tracts and reducing blood fat.
Sequence listing
<110> Mikang Probiotics (Suzhou) GmbH
<120> application of bifidobacterium lactis BLA80 in preparation of medicines or foods for reducing blood fat and regulating intestinal flora
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1375
<212> DNA
<213> Bifidobacterium animalis subsp. lactis
<400> 1
ccggcttcgg gtgctaccca ctttcatgac ttgacgggcg gggtgtacaa ggcccgggaa 60
cgcattcacc gcggcgttgc tgatccgcga ttactagcga ctccgccttc acgcagtcga 120
gttgcagact gcgatccgaa ctgagaccgg ttttcagcga tccgccccac gtcaccgtgt 180
cgcaccgcgt tgtaccggcc attgtagcat gcgtgaagcc ctggacgtaa ggggcatgat 240
gatctgacgt catccccacc ttcctccgag ttgaccccgg cggtcccaca tgagttcccg 300
gcatcacccg ctggcaacat gcggcgaggg ttgcgctcgt tgcgggactt aacccaacat 360
ctcacgacac gagctgacga cgaccatgca ccacctgtga accggccccg aagggaaacc 420
gtgtctccac ggcgatccgg cacatgtcaa gcccaggtaa ggttcttcgc gttgcatcga 480
attaatccgc atgctccgcc gcttgtgcgg gcccccgtca atttctttga gttttagcct 540
tgcggccgta ctccccaggc gggatgctta acgcgttggc tccgacacgg gacccgtgga 600
aagggcccca catccagcat ccaccgttta cggcgtggac taccagggta tctaatcctg 660
ttcgctcccc acgctttcgc tcctcagcgt cagtgacggc ccagagacct gccttcgcca 720
ttggtgttct tcccgatatc tacacattcc accgttacac cgggaattcc agtctcccct 780
accgcactcc agcccgcccg tacccggcgc agatccaccg ttaggcgatg gactttcaca 840
ccggacgcga cgaaccgcct acgagccctt tacgcccaat aaatccggat aacgctcgca 900
ccctacgtat taccgcggct gctggcacgt agttagccgg tgcttattcg aacaatccac 960
tcaacacggc cgaaaccgtg ccttgccctt gaacaaaagc ggtttacaac ccgaaggcct 1020
ccatcccgca cgcggcgtcg ctgcatcagg cttgcgccca ttgtgcaata ttccccactg 1080
ctgcctcccg taggagtctg ggccgtatct cagtcccaat gtggccggtc accctctcag 1140
gccggctacc cgtcaacgcc ttggtgggcc atcaccccgc caacaagctg ataggacgcg 1200
accccatccc atgccgcaaa agcatttccc acccccccat gcgatggagc ggagcatccg 1260
gtattaccac ccgtttccag gagctattcc ggtgcacagg gcaggttggt cacgcattac 1320
tcacccgttc gccactctca ccccgacagc aagctgccag ggatcccgtt cgact 1375
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
agtttgatcm tggctcag 18
<210> 3
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggttaccttg ttacgactt 19
Claims (8)
1. A Bifidobacterium lactis (B)Bifidobacterium animalis subsp. lactis) The application in preparing the food or the medicine for reducing the blood fat is characterized in that: the bifidobacterium lactis is bifidobacterium lactis BLA80 with the preservation number of CGMCC No. 22547.
2. The application of bifidobacterium lactis in preparing cholesterol-lowering and blood fat-lowering foods or medicines is characterized in that: the bifidobacterium lactis is bifidobacterium lactis BLA80 with the preservation number of CGMCC No. 22547.
3. The application of bifidobacterium lactis in preparing food or medicine for regulating intestinal flora is characterized in that: the bifidobacterium lactis is bifidobacterium lactis BLA80 with the preservation number of CGMCC No. 22547.
4. The application of bifidobacterium lactis in preparing food or medicine for reducing cholesterol, reducing blood fat and regulating intestinal flora is characterized in that: the bifidobacterium lactis is bifidobacterium lactis BLA80 with the preservation number of CGMCC No. 22547.
5. Use according to claim 3 or 4, characterized in that: the intestinal flora is adjusted to promote the growth of bifidobacteria and lactobacilli.
6. Use according to any one of claims 1 to 5, characterized in that: the food is common food or health food.
7. Use according to any one of claims 1 to 5, characterized in that: the food comprises tablet candy, fermented beverage, soft candy, concocted milk powder, fermented milk, and solid beverage.
8. Use according to any one of claims 1 to 5, characterized in that: the medicament comprises a microecological preparation.
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Application publication date: 20211224 |
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