CN116059258A - Use of lactobacillus paracasei in preparation for reducing serum cholesterol level - Google Patents
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- CN116059258A CN116059258A CN202211468923.0A CN202211468923A CN116059258A CN 116059258 A CN116059258 A CN 116059258A CN 202211468923 A CN202211468923 A CN 202211468923A CN 116059258 A CN116059258 A CN 116059258A
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- lactobacillus paracasei
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- cholesterol level
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Abstract
The invention belongs to the technical field of microorganism application, and particularly relates to application of lactobacillus paracasei in a preparation for reducing serum cholesterol level. The preservation number of the lactobacillus paracasei is CGMCC No.24626, and the lactobacillus paracasei can be used for preparing a preparation for reducing serum cholesterol level, so that the serum cholesterol level can be regulated by obviously reducing the liver cholesterol level, promoting bile acid excretion and regulating the disorder of intestinal flora; and has no toxic and side effects and high safety.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to application of lactobacillus paracasei in a preparation for reducing serum cholesterol level.
Background
Cholesterol is the most abundant steroid compound in the body, which is a constituent of cellular biological membranes, and is a precursor of steroid hormones, bile acids and vitamin D. For most tissues, it is important to ensure the supply of cholesterol and maintain its metabolic balance. However, when cholesterol metabolism is unbalanced and cholesterol in serum is too high, it may cause intravascular deposition to damage cardiac and cerebral blood vessels, form atherosclerosis, and cause cardiovascular and cerebrovascular diseases, and it is generally considered that the content of cholesterol in serum is a main factor for inducing cardiovascular and cerebrovascular diseases nowadays. Furthermore, the study shows that the risk of coronary heart disease is increased by 34% every 0.6mmol/L of serum total cholesterol content in the eastern population. Therefore, regulating serum cholesterol levels is of great importance to contemporary human health.
The existing researches show that part of probiotics have the effect of reducing serum cholesterol level; in addition, studies have reported that the intestinal flora of people with elevated serum cholesterol levels is different from that of normal people, i.e. that part of the probiotics may reduce serum cholesterol levels by modulating the intestinal flora.
Thus, it is of great importance to develop a probiotic that can lower serum cholesterol levels.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide a use of lactobacillus paracasei (lactobacilli paracasei) for preparing a preparation for lowering serum cholesterol level, which can significantly lower serum cholesterol level in the body.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
in one aspect, the invention provides an application of lactobacillus paracasei (Lactobacillus paracasei) in a preparation for reducing serum cholesterol level, wherein the preservation number of the lactobacillus paracasei is CGMCC No.24626.
The lactobacillus paracasei preservation information in the invention is as follows: preservation mechanism: china general microbiological culture Collection center (CGMCC); preservation address: beijing, chaoyang area, north Chenxi Lu No.1, 3; preservation date: 2022, 4, 1; preservation number: cgmccno.24626; classification naming: lactobacillus paracasei (Lactobacillus paracasei).
The beneficial effects of the invention include: the lactobacillus paracasei (Lactobacillus paracasei) is applied to preparation of a preparation for reducing serum cholesterol level, and can be used for obviously reducing liver cholesterol level, promoting bile acid excretion and regulating intestinal flora disorder, so that the serum cholesterol level is regulated; and has no toxic and side effects and high safety.
Drawings
FIG. 1 is a colony morphology of Lactobacillus paracasei TY-G05;
FIG. 2 is a gram of Lactobacillus paracasei TY-G05;
FIG. 3 is a graph of food intake for each group of mice;
FIG. 4 is a graph of water intake for each group of mice;
FIG. 5 is a graph of body weight of each group of mice;
FIG. 6 shows cholesterol levels in serum from mice of each group;
FIG. 7 shows cholesterol levels in the liver of mice in each group;
FIG. 8 shows bile acid levels in the feces of mice in each group;
FIG. 9 is a chart showing the chao index of the alpha diversity of intestinal flora of mice in each group;
FIG. 10 shows the shannon index of intestinal flora alpha diversity for each group of mice;
FIG. 11 shows the intestinal flora beta diversity of mice in each group;
FIG. 12 is a graph showing the composition of the intestinal flora horizontal community of each group of mice;
FIG. 13 shows the abundance of bifidobacteria in the intestinal flora of mice in each group;
FIG. 14 shows the abundance of Lactobacillus in the intestinal flora of mice in each group.
In the above figures, experimental data are expressed as mean ± standard error (mean ± SEM); the designations "," "and", "" each represent a statistical difference between the two groups, and each represents p <0.05, p <0.01, p <0.001, and p <0.0001.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
The embodiment of the invention provides application of lactobacillus paracasei (Lactobacillus paracasei) in a preparation for reducing serum cholesterol level, wherein the preservation number of the lactobacillus paracasei is CGMCC No.24626. The lactobacillus paracasei strain is preserved in China general microbiological culture collection center (CGMCC); preservation address: beijing, chaoyang area, north Chenxi Lu No.1, 3; preservation date: 2022, 4 and 1, the 16S rDNA sequence is shown in SEQ ID No. 1.
The lactobacillus paracasei in the present invention is not particularly limited to live bifidobacterium lactis, but includes an inactivated bacterium form, a lysate form, a fermentation broth form or a metabolite form or a mixture of the above forms, and the specific form may be selected by those skilled in the art according to specific requirements.
It should be further noted that lactobacillus paracasei in the invention has stronger gastric juice tolerance capability and bile salt tolerance capability, the survival rate in gastric juice can reach 82.19%, and the growth efficiency in bile salt can reach 16.47%, which indicates that the lactobacillus paracasei can well play a role in the gastric environment.
In some embodiments, the cholesterol level of the high-fat and high-cholesterol mouse model using lactobacillus paracasei according to the present invention is reduced by about 27.2% compared to the cholesterol level of the high-fat and high-cholesterol mouse model without lactobacillus paracasei, i.e., the lactobacillus paracasei according to the present invention has a remarkable effect in reducing serum cholesterol level.
In some embodiments, the above-described applications include applications that are a combination of one or more of the following: (a) Use of lactobacillus paracasei for the preparation of a formulation for lowering liver cholesterol levels; (b) Use of lactobacillus paracasei for the preparation of a formulation for promoting bile acid excretion; (c) Use of lactobacillus paracasei for the preparation of a formulation for modulating a disorder of intestinal flora.
It should be noted that the liver is a major regulatory organ involved in synthesis and decomposition of cholesterol, and 70% -80% of cholesterol in the human body is synthesized by the liver, so that liver cholesterol level measurement can reflect the function of lipid metabolism of the liver, i.e., can control cholesterol level in serum by controlling liver cholesterol level. In some embodiments, the liver cholesterol level in a high-fat, high-cholesterol mouse model using lactobacillus paracasei in the present invention is reduced by about 32.2% as compared to a high-fat, high-cholesterol mouse model without lactobacillus paracasei. The lactobacillus paracasei provided by the invention can effectively regulate and control the cholesterol level in serum by regulating the cholesterol level of the liver, and has remarkable effect.
It should be further noted that bile acid is the most predominant conversion product of cholesterol in an animal body, is the most predominant form of cholesterol excreted in the body to the outside, and promotion of bile acid excretion to the outside is one of the possible ways to reduce serum cholesterol levels in the human body. Generally, the human bile acid metabolism pool contains 3g-5g bile acid, but 98% -99% of bile acid secreted into intestinal tract is absorbed back, and only 1% -2% of bile acid is excreted along with feces. The lactobacillus paracasei in the invention is colonized in the intestinal tract, so that the recovery of bile acid in the intestinal tract can be effectively reduced, and the excretion of bile acid in excrement can be increased, thereby reducing the serum cholesterol level. In some embodiments, the level of bile acid excretion in a high-fat high-cholesterol mouse model using lactobacillus paracasei in the present invention is increased by about 22.3% as compared to a high-fat high-cholesterol mouse model without lactobacillus paracasei. It is demonstrated that lactobacillus paracasei in the present invention can significantly promote bile acid excretion to reduce cholesterol levels in serum.
It should be noted that the intestinal flora of the population with elevated serum cholesterol levels is different from that of the normal population, and that the regulation of the intestinal flora is also one of the ways to lower serum cholesterol. In some specific embodiments, the lactobacillus paracasei in the invention can significantly improve the richness and diversity of intestinal flora of a high-fat high-cholesterol mouse model, thereby regulating the disturbance of the intestinal flora and achieving the purpose of reducing the cholesterol level in serum.
In some embodiments, in the above application, application (c) includes: use of Lactobacillus paracasei for the preparation of a formulation for increasing the abundance of bifidobacteria (bifidobacteria) and/or Lactobacillus (Lactobacillus) in the intestinal flora. In the present invention, lactobacillus paracasei not only can regulate the abundance and diversity of the flora in the intestinal tract, but also can increase the abundance of beneficial bifidobacteria (bifidobacteria) and/or Lactobacillus (Lactobacillus). In some embodiments, the Bifidobacterium (Bifidobacterium) abundance of the high-fat high-cholesterol mouse model using lactobacillus paracasei is about 7 times greater than the Bifidobacterium (Bifidobacterium) abundance of the high-fat high-cholesterol mouse model without lactobacillus paracasei; the abundance of Lactobacillus (Lactobacillus) in the high-fat high-cholesterol mouse model using Lactobacillus paracasei is about 6 times that in the high-fat high-cholesterol mouse model not using Lactobacillus paracasei.
In some embodiments, the above-described application, the formulation comprises a food formulation or a pharmaceutical formulation. The preparation of the invention is not particularly a pharmaceutical preparation, but the lactobacillus paracasei of the invention can be added with a pharmaceutical carrier or an edible carrier to prepare a pharmaceutical preparation or an edible food preparation, and can be specifically selected for pharmaceutical or edible use according to specific clinical situations.
In some embodiments, the above-described application, the formulation comprises a solid dosage form, a liquid dosage form, a paste dosage form, or an emulsion dosage form. The lactobacillus paracasei of the present invention can be added with solid dosage form auxiliary materials to prepare solid dosage forms, such as tablets and pills, wherein the solid dosage form auxiliary materials are known in the art and generally comprise diluents (such as starch, dextrin, sucrose or glycation, etc.), absorbents (such as calcium sulfate, calcium hydrophosphate or light magnesium oxide, etc.), binders (such as povidone, syrup or hypromellose, etc.), wetting agents (such as water, etc.) or disintegrating agents (such as dry starch, sodium carboxymethyl starch or crospovidone, etc.), etc.; in some embodiments, the solid dosage form may be a probiotic tablet, a probiotic pellet, or a probiotic granule; meanwhile, the lactobacillus paracasei can be added with liquid dosage form auxiliary materials (such as compatibilizer, suspending agent, emulsifying agent or coloring agent and the like) to prepare liquid dosage forms (such as probiotic beverage); paste form adjuvants can also be added to prepare into paste form (such as probiotic jelly, probiotic milk cap or set yogurt); can also be added with emulsion adjuvant (such as pectin) to make into emulsion (such as stirred yogurt). The particular dosage form may be selected by one skilled in the art according to particular needs.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the following examples, the collection number of Lactobacillus paracasei used was CGMCC No.24626, and hereinafter also referred to as Lactobacillus paracasei TY-G05 or TY-G05.
EXAMPLE 1 isolation and purification and characterization of Lactobacillus paracasei TY-G05
(1) Experimental materials
Collecting self-made qula of Qinghai province herdsman: taking self-made triton in the herdsman by using a sterile small spoon, putting the triton into a 15mL sterile cap-screwing centrifuge tube, screwing the cap, putting the cap into a refrigerator, and immediately carrying out purification and separation of lactobacillus after transporting the cap back to a laboratory.
(2) Isolation and purification of TY-G05
Under aseptic condition, 5g of sample is added into sterilized 45mL of skim milk, the mixture is placed in a 37 ℃ incubator for enrichment culture, demulsification is carried out after curdlan, under aseptic condition, 1mL of demulsified sample is sucked into 9mL of sterile normal saline, and vortex is carried out to mix the sample uniformly, thus obtaining 10 -1 Sequentially performing 10-time gradient dilution to 10 -7 Selecting 10 -5 、10 -6 And 10 -7 100 mu L of diluent under dilution is uniformly coated on an MRS plate, and is subjected to inversion culture at 37 ℃ for 48 hours; after the culture is finished, observing colony morphology on the MRS plate, picking medium-sized, raised, slightly white or slightly yellow, moist, clean-edged and circular colonies, and purifying the strain by a plate streaking method, and repeating the streaking operation until the purified strain is obtained.
(3) Morphological structure observations
Inoculating the strain purified in (2) into 5mL MRS liquid culture medium at 37deg.CCulturing for 18h; centrifuging 1mL of bacterial liquid at the rotating speed of 12000r/min for 1min, washing twice with sterile physiological saline, adding the same volume of sterile physiological saline to resuspend the bacterial body, finally uniformly coating a small amount of bacterial liquid on a glass slide by using an inoculating loop, fixing, performing gram staining, microscopic examination and photographing; gram-positive bacteria (G) + ) Cells appeared blue-violet after staining, gram-negative bacteria (G - ) Cells appear red; observing and recording bacterial colony morphology and gram staining results;
the bacterial colony morphology is shown in figure 1, single bacterial colony is formed in a solid culture medium after the bacterial colony is purified, the bacterial colony morphology is consistent, and the bacterial colony is hemispherical, slightly white, smooth and moist in surface and flat in edge;
the results of gram staining are shown in FIG. 2, and after gram staining, the morphology of purple cells was observed under a microscope (grey scale treatment in FIG. 2, original purple) and the shape was rod-like, and it was determined that gram positive bacteria (G) + )。
(4) PCR amplification of 16S rDNA sequence
The 16SrDNA gene amplification was performed using a 25. Mu.L reaction system and the following PCR amplification procedure, 25. Mu.L reaction system: template 1. Mu.L, upstream primer (sequence shown as SEQ ID NO. 2) (10. Mu.M) 1. Mu.L, downstream primer (sequence shown as SEQ ID NO. 3) (10. Mu.M) 1. Mu.L, 2 XTaqPCRMasterMix 12.5. Mu.L, with sterile ultrapure water to 25. Mu.L; PCR amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 1min for 35 cycles; end extension at 72 ℃ for 10min; after the sequence is amplified, sequencing a PCR amplified product which is qualified in detection by entrusting a biological engineering (Shanghai) stock company, and measuring the sequence as shown in SEQ ID No. 1; searches and homology analysis were performed in GenBank using BLAST (http:// www.ncbi.nlm.nih.gov/BLAST), and the homology analysis results showed that TY-G05 was Lactobacillus paracasei.
Example 2 verification of gastrointestinal viability of TY-G05
(1) Experimental materials
Lactobacillus paracasei TY-G05: the strain is separated from Qinghai Laback mountain herdsman homemade Qula, and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24626.
(2) Survival of TY-G05 in pH3.0 artificial gastric juice
The probiotics entering the human body have corresponding functional activities, have good digestive tract tolerance, pass through the stomach (the pH of gastric juice is about 3.0, the retention time is 1-3 h) before entering the human intestinal tract to perform the functions, and the strong acid stomach environment is not beneficial to the survival of the probiotics, so the probiotics have the primary condition of being capable of tolerating the gastric juice in the human body.
Inoculating TY-G05 into MRS liquid culture medium according to an inoculum size of 2%, culturing at 37 ℃ for 18h, taking 10mL, centrifuging at 4000r/min for 10min, collecting bacterial precipitate, washing with sterile physiological saline for 2 times, and re-suspending in an equal volume of sterile physiological saline to obtain bacterial suspension; mixing the prepared bacterial suspension with artificial gastric juice (0.2% NaCl and 0.35% pepsin 1:10000, adjusting pH=3.0 with 1mol/L HCl, filtering and sterilizing for later use) according to the proportion of 1:9, uniformly mixing, placing in a constant temperature oscillator, culturing for 3 hours at 37 ℃ at the rotating speed of 100r/min, and respectively measuring the viable count of 0 hours and 3 hours by adopting a pouring plate method; the survival rate of the strain was calculated to withstand pH3.0 artificial gastric juice according to formula (1).
The experimental and calculation results show that the survival rate of TY-G05 cultured in artificial gastric juice with pH=3.0 for 3 hours is 82.19%, and the gastric juice survival rate is higher.
(3) Growth efficiency of TY-G05 in 0.3% bile salts
After the probiotics which survive gastric juice treatment enter the intestinal tract, the probiotics can be inhibited and poisoned by bile salts in the small intestine, so that the tolerance of the strain to the bile salts is also one of important indexes for screening the probiotics, and the mass concentration of the bile salts of a human body fluctuates within the range of 0.03% -0.3%.
TY-G05 was inoculated into MRS liquid medium at an inoculum size of 2%, cultured at 37deg.C for 18h, and the culture solution was inoculated into MRS-THIO medium containing 0.0% and 0.3% pig bile salts at an inoculum size of 2% (MRS liquid medium was supplemented with0.2% sodium thioglycolate), and culturing at 37 deg.c for 24 hr at 100 r/min. OD of the culture solutions containing 0.0% and 0.3% of pig bile salts were measured with unvaccinated MRS-THIO medium as a blank control 600 A value; the growth efficiency of the strain in bile salts was calculated according to formula (2).
The experimental and calculation results show that the growth efficiency of TY-G05 in bile salt with the concentration of 0.3% is 16.47%, and the bile salt tolerance is good.
Example 3 animal test
(1) Experimental mice grouping and intervention
10 SPF-class wild type male C57BL/6 mice and SPF-class male ApoE were selected -/- The mice were 20, all 8 weeks old. The experiment was started after one week of adaptive feeding in a standardized laboratory at room temperature 25.+ -. 2 ℃ and relative humidity 50.+ -. 5% and 12h light/12 h dark. After the end of the adaptation period, C57BL/6 mice were blank, apoE -/- Mice were randomly divided into model and TY-G05 groups of 10 mice each.
The experimental period was 42 days, during which time the mice of the blank group ingested normal diet, the mice of the model group and the TY-G05 group ingested high-fat high-cholesterol diet (specific formulations are shown in Table 1). The mice of all groups were also subjected to a daily gavage procedure, and the mice of the blank group and the model group were gavaged with physiological saline (200. Mu.L), and the TY-G05 group was gavaged with the above bacterial suspension (200. Mu.L), and the gavage time per day was kept consistent.
TABLE 1 Table of high fat high cholesterol feed formulas
| Composition of the components | Gram (g) | |
| Casein protein | ||
| 200 | 800 | |
| L-photo- |
3 | 12 |
| Corn starch | 212 | 848 |
| Maltodextrin | 71 | 284 |
| Sucrose | 113 | 452 |
| Fiber calculation BW200 | 50 | 0 |
| Soybean oil | 25 | 225 |
| Cocoa butter | 155 | 1395 |
| |
10 | 0 |
| |
13 | 0 |
| Calcium carbonate | 5.5 | 0 |
| Potassium citrate | 16.5 | 0 |
| |
10 | 40 |
| Choline tartrate | 2 | 0 |
| Cholesterol | 11.25 | 0 |
| Blue dye | 0.05 | 0 |
| Yellow dye | 0.05 | 0 |
| |
0 | 0 |
| Total amount of | 897.35 | 4056 |
(2) Growth performance monitoring of mice during experiments
The body weight of the mice was recorded every week within 42 days of the experimental period of the mice, and the food intake and water intake of the mice were recorded every day (the food intake, water intake and body weight changes of the mice reflect the health conditions to some extent).
The statistical graphs of the food intake, the water intake and the weight change of the mice during the experiment are specifically shown in fig. 3, 4 and 5, and the model group and the TY-G05 group of mice have no obvious reduction compared with the normal group in the aspects of the food intake and the water intake during the whole experiment; in terms of body weight, the model group and TY-G05 group mice had slightly increased weight compared to the normal group. The results show that the high-fat high-cholesterol feed does not affect the normal feeding of mice, and the lactobacillus paracasei TY-G05 has no toxic or side effect.
(3) Mouse sample collection and handling
The experimental period lasted 42 days, after which mice were sacrificed. The day before the sacrifice of the mice, the mice are fasted without water inhibition for 16 hours, and the feces of the mice are collected in a centrifuge tube and stored in a refrigerator at-80 ℃ after being immediately frozen by liquid nitrogen. The eyeballs were collected and the blood was allowed to stand at 4℃for 1 hour, centrifuged at 3000r/min for 15min, and the upper serum was collected carefully. The mice were dissected to separate liver and intestinal tissues, immediately frozen with liquid nitrogen and stored in a-80 ℃ refrigerator.
(4) Mouse serum cholesterol level determination
Taking out the refrigerated serum in the step (3), and detecting the cholesterol level by using a full-automatic biochemical analyzer.
As shown in FIG. 6, the serum cholesterol level of mice in the blank group was 2.01mmol/L, the model group was 27.76mmol/L, the model group was significantly elevated (p < 0.0001) compared to the blank group, and the serum cholesterol level of mice in the TY-G05 group was 20.20mmol/L significantly reduced (about 27.2% reduced) (p < 0.0001) compared to the model group. The results demonstrate that Lactobacillus paracasei TY-G05 has the effect of regulating serum cholesterol levels.
(5) Mouse liver cholesterol level determination
Taking out the cold-stored liver in the step (3), cutting and accurately weighing 0.5g, adding 5mL of a mixed solution of methanol and chloroform (volume ratio is 2:1), and fully grinding by a tissue homogenizer (60 HZ, homogenizing time is 10 s/time, gap is 30s, and continuous 5 times is carried out at 4 ℃), so that liver tissue is homogenized; after complete grinding, the grinding mixture is transferred into a graduated test tube, a grinder is washed by 4.5mL of mixed solution of methanol and chloroform (volume ratio is 2:1), the washing solution is transferred into the graduated test tube, the volume of the homogenate mixture collected before and after is fixed to 10mL, the homogenate mixture is placed into a water bath for 1 hour at 45 ℃, then, 8000g,10min and 4 ℃ are centrifugally collected, and the cholesterol level is detected by a full-automatic biochemical instrument.
As shown in FIG. 7, the liver cholesterol level of mice in the blank group was 2.24. Mu. Mol/G, the liver cholesterol level of mice in the model group was 7.70. Mu. Mol/G, the liver cholesterol level of mice in the model group was significantly increased (p < 0.0001) as compared to the blank group, and the liver cholesterol level of mice in the TY-G05 group was significantly decreased (about 32.2% decrease) (p < 0.0001) as compared to the model group. The results demonstrate that Lactobacillus paracasei TY-G05 has the effect of regulating liver cholesterol level.
(6) Mouse fecal bile acid level determination
Taking out the refrigerated feces in the above (3), cutting and accurately weighing 0.5g, adding 5mL of absolute ethyl alcohol, fully grinding by a tissue homogenizer (60 HZ for 10 s/time, 30s in a gap, and 5 times in succession at 4 ℃) to homogenize the feces; the supernatant was collected by centrifugation at 8000g for 10min at 4℃and the bile acid level was measured by a fully automatic biochemical analyzer.
As shown in FIG. 8, the fecal bile acid level of mice in the blank group was 0.95. Mu. Mol/G, the model group was 1.30. Mu. Mol/G, the model group was significantly elevated (p < 0.01) compared to the fecal bile acid level of mice in the blank group, and the TY-G05 group was 1.59. Mu. Mol/G, which was significantly elevated (about 22.3% elevated) compared to the model group (p < 0.05). The reason why the model group is remarkably increased compared with the blank group is analyzed that the model group ingests high-cholesterol feed and the blank group ingests common feed, so that the cholesterol uptake is increased and the bile acid excretion is also increased; the TY-G05 group also ingests high-cholesterol feed and the fecal bile acid level is further increased on the basis of the model group, which shows that TY-G05 can promote bile acid discharge under the condition of consistent exogenous cholesterol. The results demonstrate that Lactobacillus paracasei TY-G05 has an efficacy of promoting bile acid discharge.
(7) Mouse fecal flora diversity analysis
The frozen intestinal tissue in (3) above was removed, and cecum content was isolated. According to
The instructions of the soil DNA kit carry out extraction of the total DNA of the cecum content microbiota, 1% agarose gel electrophoresis is used for detecting the extraction quality of the DNA, and NanoDrop 2000 is used for determining the concentration and purity of the DNA; the 16S rRNA gene V3-V4 variable region was PCR amplified using 338F (5'-ACTCCTACGGGAGGCAG CAG-3' (SEQ ID NO. 4)) and 806R (5 '-GGACTACHVGGGTWTCTAAT-3' (SEQ ID NO. 5)) as follows: pre-denatured at 95℃for 3min,25 cycles (denaturation at 95℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 45 s), then stable elongation at 72℃for 10min, and finally preservation at 4℃ (PCR instrument: ABI)
Model 9700); the PCR reaction system is as follows: 5X TransStart FastPfu buffer 4. Mu.L, 2.5mM dNTPs 2. Mu.L, upstream primer (5. Mu.M) 0.8. Mu.L, downstream primer (5. Mu.M) 0.8. Mu.L, transStart FastPfu DNA polymerase 0.4. Mu.L, template DNA 10ng, ddH 2 O was made up to 20. Mu.L.
The PCR products of the same sample were mixed and then recovered using 2% agarose gel, purified using AxyPrep DNA Gel Extraction Kit, detected by 2% agarose gel electrophoresis, and purified using Quantus TM The Fluorometer detects and quantifies the recovered product; library construction was performed using the NEXTflexTM Rapid DNA-Seq Kit: (1) a linker linkage; (2) removing the adaptor self-ligating segment using magnetic bead screening; (3) enrichment of library templates using PCR amplification; (4) magnetic beads recovery PCR products to obtain the final library. Sequencing was performed using NovaSeq PE250 platform from Illumina, inc., and was performed by Shanghai Meiji Biotechnology Co.
alpha diversity analysis is an important component in microbial diversity analysis, and is mainly used for researching the flora diversity of a certain sample. The Chao index is one of the alpha diversity indexes, which uses the Chao1 algorithm to estimate the number of OTUs contained in the sample and reflect the community abundance. As shown in fig. 9, the chal index of mice in the blank group was 458.1, the model group was 107.9, the model group was significantly reduced (p < 0.0001) compared to the blank group, and the chal index of mice in the TY-G05 group was 154.8, which was significantly increased (p < 0.05) compared to the model group; the Shannon index is an alpha diversity index reflecting the uniformity of each species of the sample, and reflects the community diversity, and the detection calculation result is shown in fig. 10, wherein the Shannon index of the mice in the blank group is 3.47, the model group is 2.34, the model group is significantly reduced (p < 0.0001) compared with the blank group, the Chao index of the mice in the TY-G05 group is 2.67, and the Chao index is significantly increased (p < 0.05) compared with the model group. The above results demonstrate that elevated serum cholesterol levels lead to a reduction in the richness and diversity of the intestinal flora of mice, while lactobacillus paracasei TY-G05 restored to some extent the richness and diversity of the intestinal flora.
beta diversity analysis is an important component in microbial diversity analysis, and is mainly used for researching microbial diversity among different samples. The analysis quantitatively analyzes the species abundance distribution difference degree among samples through the distance in statistics, calculates the distance between every two samples by using a statistical algorithm, obtains a distance matrix, and performs visual statistical analysis on the distance matrix. In the embodiment of the invention, the Bray-Curtis algorithm is adopted, the calculation result is shown in figure 11 (data points with different shapes in the figure represent intestinal flora samples of mice in each group, the closer the data points are, the more similar the intestinal flora representing two samples), the blank group and the model group are far apart and have no overlapping part at all, which indicates that compared with the blank group, the composition of the intestinal flora of the mice in the model group is changed to a great extent, and compared with the model group, the TY-G05 group is also separated by a certain distance, which indicates that the Lactobacillus paracasei TY-G05 has a certain degree of regulation effect on the intestinal flora of the mice.
The composition analysis of the colony is further carried out on the intestinal flora of each group of mice, and the change of TY-G05 on the composition of the intestinal flora of the mice is explored. As a result, as shown in FIG. 12, the intestinal flora composition of the mice in the model group was significantly changed at the genus level, including an increase in the abundance of heterologous bacteria (Allobaculum), a decrease in the abundance of corynebacteria (Corynebacterium), and the like, compared with the blank group, and TY-G05 effectively inhibited the upward trend. In particular, as shown in FIGS. 13 and 14, TY-G05 increased the abundance of Bifidobacterium (Bifidobacterium) and Lactobacillus (Lactobacillus) (p < 0.05), which are recognized healthy bacteria, and the abundance of both species was reduced in the model group compared to the blank group. The results demonstrate that Lactobacillus paracasei TY-G05 has the effect of regulating intestinal flora.
The results show that the lactobacillus paracasei TY-G05 can simultaneously reduce the cholesterol level of the liver of the organism, promote bile acid excretion of the organism and regulate the intestinal flora disorder of the organism, thereby achieving the aim of cooperatively reducing the cholesterol level in serum of the organism.
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (6)
1. Use of lactobacillus paracasei (lactobacillus paracasei) with a preservation number of cgmccno.24626 in a preparation for lowering serum cholesterol level.
2. The application of claim 1, wherein the application comprises an application of one or more of the following combinations: (a) Use of lactobacillus paracasei for the preparation of a formulation for lowering liver cholesterol levels; (b) Use of lactobacillus paracasei for the preparation of a formulation for promoting bile acid excretion; (c) Use of lactobacillus paracasei for the preparation of a formulation for modulating a disorder of intestinal flora.
3. The application of claim 2, wherein application (c) comprises: use of Lactobacillus paracasei for the preparation of a formulation for increasing the abundance of bifidobacteria (bifidobacteria) and/or Lactobacillus (Lactobacillus) in the intestinal flora.
4. Use according to any one of claims 1 to 3, wherein the formulation comprises a food formulation or a pharmaceutical formulation.
5. Use according to any one of claims 1 to 3, wherein the formulation comprises a solid, liquid, paste or emulsion dosage form.
6. The use according to claim 4, wherein the formulation comprises a solid, liquid, paste or emulsion dosage form.
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| CN202211468923.0A CN116059258A (en) | 2022-11-22 | 2022-11-22 | Use of lactobacillus paracasei in preparation for reducing serum cholesterol level |
| NL2035207A NL2035207B1 (en) | 2022-11-22 | 2023-06-28 | Application of lactobacillus paracasei in preparation for reducing serum cholesterol levels |
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| CN101559082A (en) * | 2009-06-01 | 2009-10-21 | 天津科技大学 | Method for preparing probiotic preparation for reducing blood lipid and adjusting intestinal flora |
| CN104605345B (en) * | 2015-01-08 | 2016-06-22 | 鲁东大学 | The health food for treating hyperlipemia containing a kind of Lactobacillus paracasei |
| KR20180075463A (en) * | 2018-06-27 | 2018-07-04 | 명지대학교 산학협력단 | Novel Lactobacillus fermentum with probiotic activities and use thereof |
| EP3822338A4 (en) * | 2018-09-30 | 2021-08-18 | Inner Mongolia Yili Industrial Group Co., Ltd. | Lactobacillus paracasei et-22 and use thereof |
| CN110959865B (en) * | 2018-09-30 | 2021-07-09 | 内蒙古伊利实业集团股份有限公司 | New application of lactobacillus paracasei K56 capable of adjusting gastrointestinal flora balance |
| CN112662791B (en) * | 2020-12-23 | 2022-10-11 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactic acid bacteria with cholesterol lowering function and application thereof |
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