NL2035207A

NL2035207A - Application of lactobacillus paracasei in preparation for reducing serum cholesterol levels

Info

Publication number: NL2035207A
Application number: NL2035207A
Authority: NL
Inventors: Zhang Feng; Deng Yadan; Deng Tao; Zhang Zhen; Xiong Xin; Wang Jing; Hu Xiaofang; Zhu Xu; Tang Tian; He Miao
Original assignee: Chongqing Tianyou Dairy Co Ltd
Priority date: 2022-11-22
Filing date: 2023-06-28
Publication date: 2023-07-27
Also published as: NL2035207B1; CN116059258A

Abstract

The present application belongs to the technical ﬁeld of microorganism applications, and speciﬁcally, relates to an application of a lactobacillus paracasei in a preparation for reducing serum cholesterol levels. A lactobacillus paracasei is assigned with the accession number of CGMCC No. 24626. By means of applying the lactobacillus paracasei to preparation of 10 preparations for reducing the serum cholesterol levels, the serum cholesterol levels may be regulated by means of signiﬁcant reducing liver cholesterol levels, promoting bile acid excretion, and regulating intestinal ﬂora disorders. In addition, the lactobacillus paracasei has no toxic and side effect, and is high in safety.

Description

APPLICATION OF LACTOBACILLUS PARACASEI IN

PREPARATION FOR REDUCING SERUM CHOLESTEROL LEVELS

TECHNICAL FIELD

3 The present application belongs to the technical field of microorganism applications, and specifically, relates to an application of a lactobacillus paracasei in a preparation for reducing serum cholesterol levels.

BACKGROUND

Cholesterol is the most abundant steroid compound in a human body, serving both as a constituent of a cellular biofilm and as a precursor substance for steroid hormones, bile acids and Vitamin D. For most tissues, it is important to ensure the supply of the cholesterol and maintain metabolic balance of the cholesterol. However, when cholesterol metabolism is imbalanced, and the cholesterol in serum is too high, it may cause intravascular deposition and damage cardiovascular and cerebrovascular vessels, thus forming atherosclerosis and causing cardiovascular and cerebrovascular diseases.

Nowadays, it is generally believed that the content of the cholesterol in the serum is a main factor triggering the cardiovascular and cerebrovascular diseases. Furthermore, research also shows that, for every 0.6 mmol/L increase in total cholesterol content in the serum in

Eastern populations, the risk of developing the coronary heart disease increases by 34%.

Therefore, regulation of serum cholesterol levels is of great significance for the health of contemporary people.

Existing research shows that, some probiotics have the effect of reducing the serum cholesterol levels. In addition, studies have reported that there are differences in intestinal flora in people with elevated serum cholesterol levels compared to normal people, indicating that some probiotics may reduce the serum cholesterol levels by regulating the intestinal flora.

Therefore, it is of great significance for discovering a probiotic that may reduce the serum cholesterol levels.

SUMMARY

In view of the above problems, the present application is intended to provide an application of a lactobacillus paracasei in a preparation for reducing serum cholesterol levels. The lactobacillus paracasei may significantly reduce the serum cholesterol levels in a human body.

In order to achieve the above purpose, the present application may use the following technical solutions.

One aspect of the present application provides an application of a lactobacillus paracasei in a preparation for reducing serum cholesterol levels. The lactobacillus paracasei is assigned with the accession number of CGMCC No. 24626.

The lactobacillus paracasei in the present application is deposited with the China

General Microbiological Culture Collection Center (CGMCC) at No. 3, Yard 1, BeiChen

West Road, Chaoyang District, Beijing on April 1, 2022, and is assigned with the accession number of CGMCC No. 24626, with the classification name being lactobacillus paracasei.

The present application has the following beneficial effects. By means of applying the lactobacillus paracasei in the present application to preparation of preparations for reducing the serum cholesterol levels, the serum cholesterol levels may be regulated by means of significant reducing liver cholesterol levels, promoting bile acid excretion, and regulating intestinal flora disorders. In addition, the lactobacillus paracasei has no toxic and side effect, and is high in safety.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 11s a colonial morphology diagram of lactobacillus paracasei TY-GOS.

Fig. 2 is a gram staining diagram of lactobacillus paracasei TY-GOS.

Fig. 3 is a curve graph of food intake of mice in each group.

Fig. 4 1s a curve graph of water intake of mice in each group.

Fig. 5 is a curve graph of weights of mice in each group.

Fig. 6 shows serum cholesterol levels of mice in each group.

Fig. 7 shows liver cholesterol levels of mice in each group.

Fig. 8 shows bile acid levels in feces of mice in each group.

Fig. 9 shows a chao index of alpha diversity of intestinal flora of mice in each group.

Fig. 10 shows a Shannon index of alpha diversity of intestinal flora of mice in each group.

Fig. 11 shows beta diversity of intestinal flora of mice in each group.

Fig. 12 shows a community composition of intestinal flora of mice in each group on a genus level.

Fig. 13 shows the abundance of bifidobacterium of intestinal flora of mice in each group.

Fig. 14 shows the abundance of lactobacillus of intestinal flora of mice in each group.

In the drawings, experimental data is expressed as mean + standard error (mean +

SEM). «#7 <> “oss” gnd "55 al] represent that there is a statistical difference between two groups, representing p<0.05, p<0.01, p<0.001, and p<0.0001, respectively.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The embodiments are given to better describe the present application, but the content of the present application is not limited only to the embodiments given. Therefore, non-essential improvements and adjustments to the embodiments made by a person skilled in the art in accordance with the content of the above present application still fall within the scope of protection of the present application.

The terms used herein are only intended to describe specific embodiments and are not intended to limit the present disclosure. Expressions in the singular form include those in the plural form unless the expressions have a distinctly different meaning in the context. As used herein, it is to be understood that terms such as "include", "have", "contain", and the like are intended to indicate the presence of features, figures, operations, components, parts, elements, materials, or combinations. The terms of the present application are disclosed in the specification and are not intended to exclude the possibility that one or more other features, figures, operations, components, parts, elements, materials, or combinations thereof may exist or may be added. As used here, "/" may be interpreted as "and" or "or", as appropriate.

An embodiment of the present application provides an application of a lactobacillus paracasei in a preparation for reducing serum cholesterol levels. The lactobacillus paracasei is assigned with the accession number of CGMCC No. 24626. It is to be noted that, strains of the lactobacillus paracasei are deposited with the CGMCC at No. 3, Yard 1,

BeiChen West Road, Chaoyang District, Beijing on April 1, 2022, and a 16S rDNA sequence of the lactobacillus paracasei is shown as SEQ ID NO. 1.

It is to be noted that, the lactobacillus paracasei in the present application does not specifically refer to viable bifidobacterium lactis, and also includes an inactivated bacterial form, lysis buffer form, fermentation broth form or metabolite form thereof or a mixed form of the above forms. Specific forms may be selected by a person skilled in the art according to specific requirements.

It is further to be noted that, the lactobacillus paracasei provided in the present application has strong gastric juice tolerance and bile salt tolerance; the survival rate of the lactobacillus paracasei in gastric juice may reach 82.19%; and the growth efficiency of the lactobacillus paracasei in bile salt may reach 16.47%, indicating that the lactobacillus paracasei may function well in a gastric environment.

It is further to be noted that, in some specific embodiments, compared with cholesterol levels of a high fat and high cholesterol mouse model that does not use the lactobacillus paracasei, the cholesterol levels of a high fat and high cholesterol mouse model that uses the lactobacillus paracasei provided in the present application are reduced by approximately 27.2%, such that the lactobacillus paracasei in the present application has a significant effect on reducing the serum cholesterol levels.

In some specific embodiments, the application includes applications of one or a combination of a plurality of the following: (a) an application of the lactobacillus paracasei in preparation of preparations for reducing liver cholesterol levels; (b) an application of the lactobacillus paracasei in preparation of preparations for promoting bile acid excretion; and (c) an application of the lactobacillus paracasei in preparation of preparations for regulating intestinal flora disorders.

It is to be noted that, the liver is a main regulatory organ involved in synthesis and decomposition of the cholesterol, and 70%-80% of the cholesterol in the human body is synthesized by the liver, such that measurement of the liver cholesterol levels may reflect the function of liver lipid metabolism. Therefore, the serum cholesterol levels may be controlled by means of controlling the liver cholesterol levels. In some specific embodiments, compared with the liver cholesterol levels of the high fat and high cholesterol mouse model that does not use the lactobacillus paracasei, the liver cholesterol levels of the high fat and high cholesterol mouse model that uses the lactobacillus paracasei in the present application are reduced by approximately 32.2%. Therefore, it indicates that the lactobacillus paracasei in the present application may effectively regulate the serum cholesterol levels by means of regulating the liver cholesterol levels, and has a significant effect.

It is further to be noted that, the bile acid is the most important conversion product of the cholesterol in an animal body, and is the most important form of the cholesterol excreted outside the human body. Promoting the excretion of the bile acid outside the human body is one of the feasible ways to reduce the serum cholesterol levels in the human body. Generally, a metabolic pool of the bile acid in the human body contains 3g-5g of the bile acid. However, 98%-99% of the bile acid secreted into the intestine each time is reabsorbed, and only 1%-2% of the bile acid is excreted outside the human body with the 5 feces. By means of colonizing the lactobacillus paracasei in the present application in the intestine, the recovery of the bile acid in the intestine may be effectively reduced, and excretion of the bile acid in the feces is increased, thereby reducing the serum cholesterol levels. In some specific embodiments, compared with the bile acid excretion levels of the high fat and high cholesterol mouse model that does not use the lactobacillus paracasei, the bile acid excretion levels of the high fat and high cholesterol mouse model that uses the lactobacillus paracasei in the present application are increased by approximately 22.3%.

Therefore, it indicates that the lactobacillus paracasei in the present application may significantly promote bile acid excretion, so as to reduce the serum cholesterol levels.

It is further to be noted that, there are differences between the intestinal flora in people with elevated serum cholesterol levels and the intestinal flora in normal people; and regulation of the intestinal flora is also a way to reduce the serum cholesterol. In some specific embodiments, the abundance and diversity of the intestinal flora in the high fat and high cholesterol mouse model may be significantly improved by using the lactobacillus paracasei in the present application, so as to regulate intestinal flora disorders, thereby achieving the purpose of reducing the serum cholesterol levels.

In some specific embodiments, in the above application, the application (€) includes an application of the lactobacillus paracasei in preparation of preparations for improving the abundance of the bifidobacterium and/or the lactobacillus in intestinal flora. It is to be noted that, the lactobacillus paracasei in the present application may not only regulate the abundance and diversity of flora in the intestine, but also increase the abundance of beneficial bacteria of the bifidobacterium and/or the lactobacillus. In some specific embodiments, the abundance of the bifidobacterium of the high fat and high cholesterol mouse model that uses the lactobacillus paracasei is about 7 times of the abundance of the bifidobacterium of the high fat and high cholesterol mouse model that does not use the lactobacillus paracasei; and the abundance of the lactobacillus of the high fat and high cholesterol mouse model that uses the lactobacillus paracasei is about 6 times of the abundance of the lactobacillus of the high fat and high cholesterol mouse model that does not use the lactobacillus paracasei.

In some specific embodiments, in the above application, the preparations include a food preparation or a pharmaceutical preparation. It is to be noted that, the preparations in the present application do not specifically refer to the pharmaceutical preparation, that is, the lactobacillus paracasei in the present application may be prepared into the pharmaceutical preparation or an edible food preparation by adding a medicinal carrier or an edible carrier, such that a medicinal purpose or an edible purpose may be specifically selected according to specific clinical situations.

In some specific embodiments, in the above application, the preparations include a solid dosage form, a liquid dosage form, a paste dosage form, or an emulsion dosage form.

Tt is to be noted that, the lactobacillus paracasei in the present application may be prepared into the solid dosage form by adding solid dosage form excipients, such as tablets and pills; and the solid dosage form excipients are known in the art, and generally include a diluent (such as starch, dextrin, sucrose or sugar), an absorbent (such as calcium sulfate, calcium hydrogen phosphate or light magnesium oxide), an adhesive (such as povidone, syrup or hydroxypropyl methylcellulose), a wetting agent (such as water), or a disintegrating agent (such as dry starch, sodium hydroxymethyl starch or cross-linked povidone). In some specific embodiments, the solid dosage form may be probiotic tablets, probiotic pills or probiotic granules. Same as above, the lactobacillus paracasei in the present application may also be prepared into the liquid dosage form (for example, probiotic beverages) by adding liquid dosage form excipients (such as a bulking agent, a suspending agent, an emulsifying agent, and a colorant), or may be prepared into the paste dosage form (for example, probiotic jelly, probiotic milk foam, or set yogurt) by adding paste dosage form excipients, or may be prepared into the emulsion dosage form (for example, stirred yogurt) by adding emulsion dosage form excipients (such as pectin). A specific dosage form may be selected by a person skilled in the art according to specific requirements.

In order to better understand the present application, the content of the present application is further described below with reference to specific embodiments, but is not only limited to the following examples.

It is to be noted that, in the following embodiments, the lactobacillus paracasei used is assigned with the accession number of CGMCC No. 24626, and hereinafter also referred to as lactobacillus paracasei TY-G05 or TY-GOS.

Embodiment 1: Separation, purification and identification of lactobacillus paracasei

TY-G05

(1) Experimental material

Collection of home-made Qula from the herdsmen in Qinghai Province: the home-made Qula in the herdsmen was taken by using a sterile spoon, and was put into a 15 mL sterile capped centrifuge tube; the tube was screwed up, then put into a freezer, and transported back to a laboratory for immediate purification and separation of lactobacillus. (2) Separation and purification of TY-G05

Under an aseptic condition, 5 g of the Qula was added to 45 mL of sterilized skimmed milk, and was then placed in a 37°C incubator for enrichment culture; and after curding, the milk was broken. Under an aseptic condition, 1 mL of a sample after milk breaking was pipetted to 9 mL of sterile saline, and a sample diluent of 107! was obtained after the mixture was well mixed by means of vortex. Then 10-fold gradient dilution was performed to 107. 100 uL of diluent at 10%, 10%, or 107 was selected, and uniformly spread on an

MRS plate for inverted culture for 48h at 37°C. After culture was completed, colonial morphology on the MRS plate was observed. Raised, whitish or yellowish, moist and circular colonies with medium sizes and neat edges were selected for strain purification by using a streak plate method, and this streaking operation was repeated until the purified strains were obtained. (3) Morphological structure observation

The purified strains in (2) were inoculated in the 5 mL MRS liquid culture medium for culture for 18h at 37°C. 1 mL of bacterial fluid was taken and centrifuged for 1 min at 12000 r/min. After the bacterial fluid was washed for two times with sterile saline, isochoric sterile saline was then added to resuspend the bacteria. Finally, a small amount of the bacteria was taken and spread uniformly on a glass slide by using an inoculating loop.

After fixation, Gram staining, microscopic examination, and photographing were performed. Gram-positive bacteria (G*) cells stained were blue-purple, and Gram-negative bacteria (G7) cells were red. The colonial morphology and Gram staining results of the strains were observed and recorded.

The colonial morphology of the strains is shown in Fig. 1. The strains form single colonies in solid medium after purification, and the colonies are consistent in morphology, hemispherical in shape, whitish, smooth and moist in surface, and neat in edge.

The gram staining results are shown in Fig. 2. After gram staining, purple cell morphology (grey processing is performed in Fig. 2, and an original drawing is purple) with a rod-like shape is observed under a microscope, such that it is determined as gram-positive bacteria (G*). (4) PCR amplification of 16S rDNA sequence

A 25 uL reaction system and the following PCR amplification procedure were used for 16S rDNA gene amplification. The 25 uL reaction system included: 1 pL of a template, 1 uL of an upstream primer (a sequence being shown as SEQ ID NO.2) (10 pM), 1 pL of a downstream primer (a sequence being shown as SEQ ID NO.3) (10 uM), and 12.5 pL of 2xTaq PCR Master Mix, and making up to 25 pL with sterile ultrapure water. A PCR amplification procedure included: performing pre-denaturation at 94°C for 5 min, performing denaturation at 94°C for 30 s, performing annealing at 55°C for 30 s, and performing extension at 72°C for 1 min, there being a total of 35 cycles; and performing end extension at 72°C for 10 min. After sequence amplification, Sangon Biotech (Shanghai) Co., Ltd. was entrusted to sequence qualified PCR amplification products, and the obtained sequence was shown as SEQ ID No.1; and searching and homology analysis were performed in GenBank by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST).

Homology analysis results showed that, the TY-G05 was lactobacillus paracasei.

Embodiment 2 Validation of gastrointestinal survival rate of TY-G05 (1) Experimental material

Lactobacillus paracasei TY-G05: separated from the home-made Qula from the herdsmen in Qinghai Province, deposited with the CGMCC and was assigned with the accession number of CGMCC No.24626. (2) Survival rate of TY-GOS in artificial gastric juice with pH being 3.0

The probiotics entering the human body should have desirable digestive tract tolerance in order to exert corresponding functional activities. Before the probiotics entered the human intestine to exert functions, the probiotics needed to pass through the stomach (the pH of gastric juice was about 3.0, and the retention time is 1-3h). However, the strongly acidic stomach environment was not conducive to the survival of the probiotics.

Therefore, the primary condition for the probiotics was to be able to tolerate the action of gastric juice in the human body.

The TY-G05 was inoculated in the MRS liquid culture medium according to a 2% inoculation amount and cultured at 37°C for 18h; 10 mL was taken and centrifuged for 10 min at 4000 r/min, so as to collect a bacterial precipitate; and the bacterial precipitate was washed for 2 times with sterile saline, and resuspended in isochoric sterile saline, so as to obtain bacterial suspension. The well prepared bacterial suspension and the artificial gastric juice (0.2% NaCl:0.35% pepsin being 1:10000, 1 mol/L of HCI being used to adjust the pH to be 3.0, the mixture being filtered and sterilized for later use) were mixed at a ratio of 1:9, then placed in a thermostatic oscillator after being well mixed, and cultured for 3h at 37°C at 100 r/min. Viable counts at 0 h and 3 h were respectively measured by using a pour plate method. The survival rate of the strains tolerant to the artificial gastric juice with pH being 3.0 was calculated according to a formula (1). } 3h viable count/{CFU/mL }+

Survival rate/%« 100

Oh viable count {CFU/mL}:

Experimental and calculation results showed that, the survival rate of the TY-G05 cultured in the artificial gastric juice with the pH being 3.0 is 82.19%, such that the

TY-G05 had a high gastric juice survival rate. (3) Growth efficiency of TY-G05 in 0.3% bile salt

After the probiotics survived the gastric juice treatment entered the intestine, the probiotics were inhibited and poisoned by bile salts in the small intestine. Therefore, the tolerance of the strain to the bile salt was also one of the important indicators for probiotic screening. The mass concentration of the bile salts in the human body fluctuated within a range of 0.03%-0.3%.

The TY-G05 was inoculated in the MRS liquid culture medium according to a 2% inoculation amount and cultured for 18h at 37°C. Then, the culture solution was taken and separately inoculated in MRS-THIO media containing 0.0% porcine bile salt and 0.3% porcine bile salt according to a 2% inoculation amount (0.2% sodium mercaptoacetate being added in the MRS liquid culture medium). The media were placed into the thermostatic oscillator after being well mixed, and cultured at 37°C and 100 r/min for 24h.

A non-inoculated MRS-THIO medium was used as a blank control. OD600 values of culture solutions containing 0.0% porcine bile salt and 0.3% porcine bile salt were measured. The growth efficiency of the strains in the bile salts was calculated according to a formula (2). © 0.3% bie salt containing culture mediunr ODssoe= - growth: == Denon ODeo «IDO (23 efficiency: 0.0% bile sait containing culture. medium: ODsooen - î os blank control O Daos:

Experimental and calculation results showed that, the growth efficiency of the

TY-G05 in the 0.3% bile salt was 16.47%, such that the TY-G05 had desirable bile salt tolerance.

Embodiment 3 Animal test (1) Experimental mouse grouping and intervention

SPF-grade wild-type male C57BL/6 mice and 20 SPF-grade male ApoE” mice were selected, which were all 8 weeks old. The mice were raised in a standardization laboratory at room temperature of 25+2°C and relative humidity of 50£5% and under a condition of 12h light/12h dark, and an experiment started after a week of adaptive feeding. 10 After the adaptive phase ended, the C57BL/6 mice were grouped in Group Blank; and the

ApoE” mice were randomly grouped in Group Model and Group TY-G05, and each group had 10 mice.

An experimental period was 42 days, during the experiment, the mice in Group Blank were fed with ordinary feed, the mice in Group Model and Group TY-G05 were fed with high fat and high cholesterol feed (specific recipes were shown in Table 1). In addition, an intragastric administration operation was performed on the mice in all groups every day, the mice in Group Blank and Group Model were intra-gastrically administered with the normal saline (200uL), the mice in Group TY-G05 were intra-gastrically administered with the bacterial suspension (200uL), and the time for intragastric administration was the same every day.

Table 1 High fat and high cholesterol feed recipe table

Clos BWED 50 0

Mived miner SI lop

Calcium bicarbonate 13 DO

Calcium carbonate 530]

Pousiumeivae oso

Mixed vitamin V10001 10 40

Chofnebiarae Bp]

Choeserol IE]

Bede bos pb]

Vellowdye pos bo]

Rede pp] (2) Growth performance monitoring of mice during experiment

The weights of the mice were recorded each week within 42 days of the experimental period, and the food intake and water intake of the mice were recorded every day (the changes in the food intake, water intake, and body weight of the mice could reflect the health status of the mice to some extent).

Statistical diagrams of changes in the food intake, water intake, and body weight of the mice during experiment were shown in Fig. 3, Fig. 4 and Fig. 5. During the entire experiment, compared with the mice in Group Normal, the food intake and water intake of the mice in Group Model and Group TY-G05 showed no significant decrease, and compared with the mice in Group Normal, the weights of the mice in Group Model and

Group TY-G05 were slightly increased. The above results showed that the high fat and high cholesterol feed did not affect the normal feeding of the mice, and lactobacillus paracasei TY-G05 had no side effects. (3) Mouse sample collection and treatment

The experimental period lasted 42 days, and then the mice were sacrificed. The mice were fasted for 16h but could drink water before being sacrificed, feces of the mice were collected into a centrifuge tube, and the centrifuge tube was immediately frozen with liquid nitrogen and stored in a -80°C refrigerator. Eyeball blood was extracted, the blood was allowed to stand for 1h at 4°C and centrifuged for 15 min at 3000r/min, and upper serum was collected carefully. The mice were dissected to separate liver and intestinal tissue, which was immediately frozen with the liquid nitrogen and stored in the -80°C refrigerator. (4) Measurement of serum cholesterol levels of mice

The serum refrigerated in (3) was taken, and the cholesterol levels were detected by using a fully-automatic biochemical analyzer.

A detection result was shown in Fig. 6. The serum cholesterol levels of the mice in

Group Blank were 2.01 mmol/L, and were 27.76 mmol/L in Group Model, such that the serum cholesterol levels of Group Model were significantly increased compared with that of Group Blank (p<0.0001), and the serum cholesterol levels of the mice in Group TY-G05 were 20.20 mmol/L and were significantly reduced (by approximately 27.2%) compared with Group Model (p<0.0001). The above results showed that, the lactobacillus paracasei

TY-G05 had the effect of regulating the serum cholesterol levels. (5) Measurement of liver cholesterol levels of mice

The liver refrigerated in (3) was taken, cut and accurately weighed 0.5g; 5 mL of a mixed solution of methanol and chloroform (a volume ratio being 2:1) was added; a tissue homogenizer (60 HZ, homogenization time being 10s/time, with an interval being 30s, homogenization being performed for 5 times continuously at 4°C) was used for full grinding, so as to homogenize liver tissue; after complete grinding, the ground mixed solution was transferred into a graduated test tube, 4.5 mL of the mixed solution of the methanol and the chloroform (the volume ratio being 2:1) was used to wash a grinder, and the washing solution was transferred to the graduated test tube together; the ground mixed solution that was collected before and after was made up to 10 mL, and placed in a water bath to perform warm bath for 1h at 45°C, and then 8000 g was centrifuged for 10 min at 4°C, and then supernatant was collected; and the cholesterol levels were detected by using the fully-automatic biochemical analyzer.

A detection result was shown in Fig. 7. The liver cholesterol levels of the mice in

Group Blank were 2.24 umol/g, and were 7.70 umol/g in Group Model, such that the liver cholesterol levels of Group Model were significantly increased compared with that of

Group Blank (p<0.0001), and the liver cholesterol levels of the mice in Group TY-G05 were 5.22 umol/g and were significantly reduced (by approximately 32.2%) compared with

Group Model (p<0.0001). The above results showed that, the lactobacillus paracasei

TY-G05 had the effect of regulating the liver cholesterol levels. (6) Measurement of bile acid levels in feces of mice

Feces refrigerated in (3) was taken, cut and accurately weighed 0.5 g; 5 mL of anhydrous ethanol was added; the tissue homogenizer (60 HZ, homogenization time being 10s/time, with an interval being 30s, homogenization being performed for 5 times continuously at 4°C) was used for full grinding, so as to homogenize the feces; 8000 g was centrifuged for 10 min at 4°C, and then supernatant was collected; and the bile acid levels were detected by using the fully-automatic biochemical analyzer.

A detection result was shown in Fig. 8. The bile acid levels in the feces of the mice in

Group Blank were 0.95 umol/g, and were 1.30 mmnol/g in Group Model, such that the bile acid levels in the feces of Group Model were significantly increased compared with that of

Group Blank (p<0.01), and the bile acid levels in the feces of the mice in Group TY-G05 were 1.59 umol/g and were significantly reduced (by approximately 22.3%) compared with

Group Model (p<0.05). The reason for the significant increase in Group Model compared with Group Blank was that the high cholesterol feed was fed for Group Model while the ordinary feed was fed for Group Blank, and the cholesterol intake increased and therefore the bile acid excretion also increased. The high cholesterol feed was also fed for Group

TY-G05, and the bile acid levels in the feces were further increased on the basis of Group

Model, indicating that the TY-G05 may promote the bile acid excretion under consistent conditions of exogenous cholesterol. The above results showed that, the lactobacillus paracasei TY-G05 had the effect of promoting the bile acid excretion. (7) Bacterial diversity analysis of feces of mice

Intestinal tissue refrigerated in (3) was taken, and cecal contents were separated. The total DNA of microbial flora in the cecal contents was extracted according to the specification of an EZ N. A ® soil DNA kit; electrophoresis was performed by using 1% agarose gel so as to detect the extracted mass of DNA; and NanoDrop2000 was used to measure the concentration and purity of the DNA. PCR amplification was performed on

V3-V4 variable regions of 16S rRNA genes by using 338F (5’-ACTCCTACGGGAGGCAGCAG-3’ (SEQ ID NO.4)) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’ (SEQ ID NQO.5)). An amplification procedure included: performing pre-denaturation for 3 min at 95°C, there being 25 cycles (denaturation for 30s at 95°C, annealing for 30s at 55°C, and extension for 45s at 72°C); then performing stable extension for 10 min at 72°C; and finally, performing storage at 4°C (a PCR instrument: ABI GeneAmp® 9700). A PCR reaction system included: 4 uL of a

SxTransStart FastPfu buffer solution, 2 uL of 2.5mM dNTPs, 0.8 uL (5 uM) of an upstream primer, 0.8 uL (5 uM) of a downstream primer, 0.4 uL of TransStart FastPfu

DNA polymerase, 10 ng of template DNA, and making up to 20 uL with ddH20.

After PCR products of the same sample were mixed, 2% agarose gel was used to recycle the PCR products, and an AxyPrep DNA Gel Extraction Kit was used to purify the recycled products; then electrophoresis detection was performed by using the 2% agarose gel, and a QuantusTM Fluorometer was used to detect and quantify the recycled products; and a NEXTflexTM Rapid DNA-Seq Kit was used for library building, including: (1)

linking connectors; (2) using magnetic beads for screening, so as to remove connector self-connecting fragments; (3) performing enrichment of library templates by means of

PCR amplification; and (4) using the magnetic beads to recycle the PCR products, so as to obtain a final library. A NovaSeq PE250 platform of Illumina was used for sequencing, and the sequencing was completed by entrusting Shanghai Majorbio Technology Co., Ltd. alpha diversity analysis was an important component of microbial diversity analysis and was mainly used to study the bacterial diversity of a particular sample. A Chao index was one of alpha diversity indexes, which used a chaol algorithm to estimate the number of OTUs contained in the sample, reflecting the abundance of a colony. Results were shown in Fig. 9. The Chao index for the mice in Group Blank was 458.1, and was 107.9 for Group Model, such that compared with Group Blank, Group Model was significantly reduced (p<0.0001); and the Chao index for the mice in Group TY-G05 was 154.8, which was significantly increased compared with Group Model (p<0.05). A Shannon index was the alpha diversity index that reflects the homogeneity of species in each sample, and reflected community diversity; and detection calculation results were shown in Fig. 10.

The Shannon index for the mice in Group Blank was 3.47, and was 2.34 for Group Model, such that compared with Group Blank, Group Model was significantly reduced (p<0.0001); and the Chao index for the mice in Group TY-G05 was 2.67, which was significantly increased compared with Group Model (p<0.05). The above results showed that, elevated serum cholesterol levels resulted in reduction of the abundance and diversity of the intestinal flora of the mice; and the lactobacillus paracasei TY-G05 recovered the abundance and diversity of the intestinal flora to a certain extent. beta diversity analysis was an important component of microbial diversity analysis and was mainly used to study microbial diversity among different samples. The analysis quantified the degree of variation in species abundance distribution among samples by means of distances in statistics, and used a statistical algorithm to calculate the distance between two samples, so as to obtain a distance matrix, and visual statistical analysis was performed on the distance matrix. In the embodiments of the present application, a

Bray-Curtis algorithm was used, and calculation results were shown in Fig. 11 (data points with different shapes in the figure represented intestinal flora samples of the mice in each group, and the closer the data points were, the more similar the intestinal flora of the two samples were). Group Blank and Group Model were far apart and did not overlap at all, indicating that intestinal flora compositions of the mice in Group Model were changed to a great extent compared with Group Blank; and Group TY-G05 was also separated from

Group Model by a certain distance, indicating that the lactobacillus paracasei TY-G05 had a certain degree of regulatory effect on the intestinal flora of the mice.

Community composition analysis was further performed on the intestinal flora of the mice in each group, so as to study changes of the TY-G05 in compositions of the intestinal flora of the mice. Results were shown in Fig. 12. Compared with Group Blank, the compositions of the intestinal flora of the mice in Group Model were obviously changed on a genus level. Specific manifestations included an increase in the abundance of

Allobaculum, reduction of the abundance of Corynebacterium, etc. Therefore, the TY-G05 effectively inhibited a rising and falling trend. In particular, as shown in Fig. 13 and Fig. 14, the TY-GOS included the abundance of bifidobacterium and the lactobacillus. These two genera were recognized as beneficial to health, and the abundance of the two genera was reduced in Group Model compared with Group Blank. The above results showed that, the lactobacillus paracasei TY-G05 had the effect of regulating the intestinal flora.

The above results showed that, the lactobacillus paracasei TY-G05 in the present application may simultaneously reduce the liver cholesterol levels of the human body, promote the bile acid excretion of the human body, and regulate intestinal flora disorders of the human body, such that the purpose of reducing the serum cholesterol levels in the human body was achieved by means of synergism.

It is finally to be noted that, the above embodiments are merely for describing and not intended to limit the technical solutions of the present application. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application may be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and shall all fall within the scope defined by the claims of the present application.

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Tan

Claims

CONCLUSIONS

Use of lactobacillus paracasei (Lactobacillus paracasei) in a preparation for lowering serum cholesterol levels, wherein the collection number of the lactobacillus paracasei is CGMCC No.24626.

Use according to claim 1, characterized in that the use comprises one or a combination of a plurality of the following uses: (a) an use of the lactobacillus paracasei in the preparation of the preparations which lower the cholesterol level in the liver; (b) a use of the lactobacillus paracasei in the preparation of the preparations promoting the secretion of bile acids; (c) an application of the lactobacillus paracasei in the preparation of the preparations regulating intestinal flora disorders.

Use according to claim 2, characterized in that the use (c) comprises an use of the lactobacillus paracasei in the preparation of the preparations to improve the abundance of biidobacterum (Bifidobacterium) and/or lactobacillus (Lectobacias) in the intestinal flora. .

Use according to any one of claims 1 to 3, characterized in that the preparations comprise a food preparation or a pharmaceutical preparation.

Use according to any one of claims 1 to 3, characterized in that the preparations comprise a solid dosage form, a liquid dosage form, a paste dosage form or an emulsion dosage form.

Use according to claim 4, characterized in that the preparations comprise a solid dosage form, a liquid dosage form, a paste dosage form or an emulsion dosage form.