CN113564078A - Bifidobacterium lactis BLA80 for reducing cholesterol and application thereof - Google Patents
Bifidobacterium lactis BLA80 for reducing cholesterol and application thereof Download PDFInfo
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- CN113564078A CN113564078A CN202110864979.7A CN202110864979A CN113564078A CN 113564078 A CN113564078 A CN 113564078A CN 202110864979 A CN202110864979 A CN 202110864979A CN 113564078 A CN113564078 A CN 113564078A
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- bifidobacterium lactis
- bla80
- cholesterol
- lactis
- bifidobacterium
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a bifidobacterium lactis BLA80 for reducing cholesterol and application thereof, belonging to the technical field of microorganisms. The bifidobacterium lactis BLA80 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 22547. 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 activity of bile salt hydrolase and excellent cholesterol-lowering property, the cholesterol removal rate can reach 92.4 percent, the content of serum cholesterol can be obviously lowered, and the bifidobacterium lactis BLA80 can be used for preparing medicaments or foods such as cholesterol-lowering medicinal health-care foods or solid beverages.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bifidobacterium lactis BLA80 for reducing cholesterol and application thereof.
Background
Probiotics are defined as living microorganisms that are beneficial to the health of the host when taken in appropriate doses. The bifidobacterium and the lactobacillus are common probiotics in human intestinal tracts, have the effects of improving intestinal micro-ecology, inhibiting the growth of intestinal pathogenic bacteria, reducing serum cholesterol, improving the immunity of organisms and the like, and play a vital role in human health.
Hypercholesterolemia is one of the most main factors causing cardiovascular diseases such as coronary heart disease, hypertension, atherosclerosis and the like, and the morbidity and the mortality rate are in a trend of rising year by year. The reduction of serum cholesterol level is related to human health, and as the health consciousness of people in China is improved, more and more people tend to realize the health by improving diet, eating probiotics and the like instead of drug therapy. Therefore, it is a research focus in recent years to explore the cholesterol lowering properties of probiotics.
Disclosure of Invention
The invention aims to provide a bifidobacterium lactis strain with cholesterol-lowering property and application thereof.
The purpose of the invention is realized by the following technical scheme:
a Bifidobacterium lactis strain with cholesterol lowering property is Bifidobacterium lactis BLA80 with the preservation number of CGMCC No.22547, and is classified and named as follows: bifidobacterium animalis subsp. lactis, preservation time: 2021, 05 month, 17 days; the preservation unit: china general microbiological culture Collection center; and (4) storage address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
The bifidobacterium lactis BLA80 has strong bile salt hydrolase activity and excellent cholesterol-lowering property, can obviously reduce the content of serum cholesterol, and can be used for preparing medicaments and foods for lowering cholesterol, such as health-care foods or solid beverages and the like.
A medicine, health food and solid beverage for reducing cholesterol comprise Bifidobacterium lactis BLA 80.
The bifidobacterium lactis BLa80 also has the following characteristics:
(1) can resist gastrointestinal tract environment, and has strong gastric acid resistance and intestinal juice resistance.
(2) Has strong capability of inhibiting pathogenic bacteria, and has strong bacteriostatic capability on escherichia coli, salmonella and staphylococcus aureus.
(3) The antibiotic-free compound preparation is sensitive to 15 common antibiotics of erythromycin, penicillin, amoxicillin, ampicillin, vancomycin, cefaclor, oxacillin, ceftriaxone, neomycin, cephalothin, clindamycin, azithromycin, methoxypyrimidine and rifampicin, has no antibiotic resistance and is high in safety.
Compared with the prior art, the invention has the following advantages and beneficial effects: 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 excellent cholesterol-lowering property and can remarkably lower the content of serum cholesterol.
Drawings
FIG. 1 shows the colony formed by Bifidobacterium lactis.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 Bifidobacterium lactis screening with bile salt hydrolase Activity
11 different strains of Bifidobacterium lactis were isolated from healthy infants and breast milk samples, and Bifidobacterium lactis having high bile salt hydrolase activity was selected from these strains.
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 be an activated generation.
The components of the L-MRS culture medium are as follows: by mass percentage, 2.0 percent of glucose, 0.5 percent of yeast extract powder, 1.0 percent of peptone, 1.0 percent of beef extract powder, 0.5 percent of anhydrous sodium acetate, 800.1 percent of tween, 0.2 percent of diammonium hydrogen citrate, 0.2 percent of dipotassium hydrogen phosphate and MgSO4·7H2O 0.06%、MnSO40.02%, 0.1% L-cysteine hydrochloride, an initial pH value of 7.0 +/-0.1, and sterilizing at 118 ℃ for 30min for later use.
Adding 0.37g/L CaCI into freshly prepared MRS liquid culture medium25g/L taurodeoxycholic acid and 15g/L agar, sterilizing at 121 ℃ for 15min, taking out, cooling to 40-50 ℃, pouring on a flat plate with an Oxford cup, and carefully taking out the Oxford cup after the culture medium is completely solidified. And inoculating 100 mu L of activated bacterial liquid on a flat plate, carrying out anaerobic culture at 37 ℃ for 48h, observing whether a precipitation ring is formed around the bacterial lawn, wherein if white precipitates are formed, the bile salt hydrolase activity is shown, and the bigger the diameter of the precipitation ring is, the stronger the bile salt hydrolase activity is shown.
The experimental results are as follows: the size of the precipitation circle of the 11 isolated strains of bifidobacterium lactis is shown in table 1, wherein the precipitation circles of the S01, S03 and S09 strains are shown in fig. 1.
TABLE 1 Bifidobacterium lactis precipitation ring size (mm)
As is clear from Table 1, 5 strains S01, S02, S07, S09 and S10 having larger diameters of the colony were selected by comparing the diameters of the colony, and the cholesterol removal rate was measured.
EXAMPLE 2 Cholesterol removal Rate determination
According to the diameter of the precipitation ring, 5 strains of bifidobacterium lactis are selected for measuring the cholesterol removal rate.
(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:
inoculating the strain into an anaerobic tube filled with 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 for 5min at 4000r/min, and taking the supernatant to measure the cholesterol content by using 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.
(4) Results of Bifidobacterium lactis Cholesterol removal Rate
TABLE 2 Bifidobacterium lactis cholesterol removal rate
As can be seen from Table 2, S09 showed the best removal rate of cholesterol, which was 92.4%, and was selected as the strain to be tested.
Example 3 molecular biological identification of Bifidobacterium lactis
S09 cells are cultured and collected, named Bla80 again, strain genome DNA is extracted by a genome DNA extraction kit (TIANGEN company), and a 16S rDNA sequence is amplified by an upstream primer 27F (AGTTTGATCTMTGGCTCAG) and a downstream primer 1492R (GGTTACCTTGTTACGACTT) to obtain a PCR product. And sequencing the PCR product. Wherein the PCR reaction system comprises: 10 XBuffer 20. mu.L, primer dNTP 4. mu.L, upstream and downstream primers 1. mu.L each, DNA template 2. mu.L, Taq enzyme 0.5. mu.L, ddH2O 34.5.5. 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 measured gene sequences were submitted to the NCBI database (www.ncbi.nlm.nih.gov) for BLAST analysis alignment. Through the above identification results, the strain BLa80, named as Bifidobacterium lactis, was identified as Bifidobacterium animalis subsp. lactis, and was deposited in the common microorganism center of the committee for culture collection of microorganisms china at 17 th 2021, CGMCC for short, address: no. 3 of Xilu No. 1 of Beijing, Chaoyang, and the preservation number is CGMCC No. 22547.
Example 4 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 the thallus suspension is respectively inoculated into 1.6mL of prepared simulated artificial gastric juice with the pH value of 2.5 and simulated artificial intestinal juice with the pH value of 8.0 to be mixed uniformly, the mixture is digested at the temperature of 37 ℃, meanwhile, 0h and 3h of digestive juice are respectively taken to detect the viable count, the survival rate is calculated, and the result is shown in a table 3. Wherein, the survival rate of the strain is N%t/N0X 100%, wherein N0 represents the viable cell count (CFU/mL) of 0h, and NtThe number of viable bacteria of strain 3h (CFU/mL) was indicated.
TABLE 3 Experimental data sheet for Bifidobacterium lactis BLA80 simulated digestive tract environment
The results in Table 3 show that Bifidobacterium lactis Bla80 has a survival rate of 96.7% in artificial gastric juice at pH2.5 for 3 hours and 93.6% in artificial intestinal juice at pH8.0 for 3 hours. Experiments show that the bifidobacterium lactis BLA80 has strong capability of tolerating the environment of the gastrointestinal tract.
Example 5 evaluation of bacteriostatic Activity of Bifidobacterium lactis BLA80
Inoculating antagonistic strain into an anaerobic glass tube of L-MRS liquid culture medium according to 2% (V/V), and standing and culturing at constant temperature of 37 ℃ for 12 h. Respectively inoculating pathogenic strains of Escherichia coli, salmonella and Staphylococcus aureus into liquid beef extract peptone medium, culturing at 37 deg.C and 250rpm with constant temperature shaking table overnight, and preparing 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, quickly pouring into a plate with an Oxford cup 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, slightly covering the plate, placing the plate in a constant-temperature incubator at 37 ℃, culturing for a proper time, observing, and using a vernier caliperThe diameter of the zone of inhibition was measured with a ruler and the results are shown in Table 4.
TABLE 4 antagonistic activity of Bifidobacterium lactis BLA80 against pathogenic bacteria data Table
The results in table 4 show that bifidobacterium lactis BLa80 has strong inhibitory effect on common enteropathogenic bacteria of escherichia coli, salmonella and staphylococcus aureus, especially on escherichia coli and salmonella.
Example 6 evaluation of antibiotic susceptibility of Bifidobacterium lactis BLA80
Marking and activating bacteria to be detected on an L-MRS solid plate, preparing a bacterial suspension and adjusting the concentration of the bacterial suspension to be 108CFU/mL, 100. mu.L of the bacterial suspension is added on an L-MRS solid plate, the bacterial liquid is uniformly coated on the plate by using a sterile cotton swab, and an antibiotic drug sensitive sheet is attached, and a paper sheet without antibiotic is used as a blank control. The strain was cultured in an anaerobic condition at 37 ℃ in an upright manner, and after 24 hours, the antibiotic-sensitive diameter of the strain was measured with a ruler, and the results are shown in Table 5.
TABLE 5 Bifidobacterium lactis BLA80 sensitivity data to antibiotics (mm)
The results in table 5 show that bifidobacterium lactis BLa80 is highly sensitive to the 14 antibiotics erythromycin, penicillin, amoxicillin, ampicillin, vancomycin, cefaclor, oxacillin, ceftriaxone, novobiocin, cephamine, clindamycin, azithromycin rifampicin and trimethoprim evaluated, the zone diameters are all greater than 20mm, and the zone diameters for 10 of these antibiotics are greater than 30 mm. Experiments show that bifidobacterium lactis BLa80 is a safe probiotic.
Example 7 Bifidobacterium lactis BLA80 ability to reduce serum cholesterol in mice
(1) Experimental grouping and feeding mode
Collecting cultured milk bifidobacteriaThe strain BLA80 was washed 3 times with physiological saline to adjust the concentration to 1.0X 109CFU/mL was used for mouse gavage. 30 8-week-old BALB/c mice were reclassified into 3 groups with no significant difference in mean body mass between groups: normal group: basal feed was fed, control group: feeding high-fat feed and physiological saline, and performing experiment groups: high fat diet + strain BLa80 was fed, 10 per group. Each mouse was gavaged for 1 mL/day for 14 days.
(2) Determination of TC, TG and HDL-C content in serum
And (4) fasting for 12h at 14d after gastric lavage of the mice, respectively collecting blood, centrifuging for 10min at 2500r/min, and taking serum. 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.
(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) Analysis of results of Bifidobacterium lactis BLA80 on reduction of serum cholesterol in mice
TABLE 6 Effect of Bifidobacterium lactis BLA80 on mouse serum TC, TG, HDL-C (mmol/L)
Note: different letters in the same column indicate significant differences (P < 0.05).
As can be seen from table 6, when the mice were fed with the high-fat diet for 14 days, the serum TC levels of the mice in the control group and the experimental group were both significantly increased, and the levels of the mice in the experimental group were significantly decreased compared to the control group; the content of serum TG in the experimental group has no significant difference with that of the control group, and the level of the control group is significantly increased; the serum HDL-C levels of the control and experimental groups were significantly higher than those of the normal group, while the difference between the two groups was not significant. The TC, TG and HDL-C in the serum of the mice in the experimental group which eat the bifidobacterium lactis BLA80 are respectively reduced by 24.3%, 20.3% and 9.2% compared with the control group, which indicates that the bifidobacterium lactis BLA80 can obviously reduce the serum cholesterol level of the mice.
Claims (7)
1. A strain of Bifidobacterium lactis (Lactobacillus lactis) having cholesterol lowering properties, characterized in that: is Bifidobacterium lactis BLA80 with the preservation number of CGMCC No. 22547.
2. Use of a bifidobacterium lactis as claimed in claim 1 in the manufacture of a medicament for use in lowering cholesterol.
3. A drug for reducing cholesterol, which is characterized in that: comprising bifidobacterium lactis BLa80 as claimed in claim 1.
4. Use of bifidobacterium lactis as claimed in claim 1 in the preparation of a health food for lowering cholesterol.
5. A health food for reducing cholesterol is characterized in that: comprising bifidobacterium lactis BLa80 as claimed in claim 1.
6. Use of bifidobacterium lactis as claimed in claim 1 for the preparation of a solid beverage for lowering cholesterol.
7. A solid beverage for reducing cholesterol is characterized in that: comprising bifidobacterium lactis BLa80 as claimed in claim 1.
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