AU2020101532A4 - A Lactobacillus plantarum with the function of reducing weight and fat and an application thereof - Google Patents

Abstract

The invention discloses a Lactobacillus plantarum with the function of reducing weight and fat and an application thereof, belonging to the technical field of microorganism, wherein the Lactobacillus plantar LP-KFY04 is lactic acid bacterium (LAB), Deposit number CCGMCC No. 15651; lactobacillus plantarum KFY04 was isolated from yogurt and was used for mice maintaining 45% fat diet; liver and serum samples were then obtained, in order to evaluate the effect of LP-KFY04 on obesity, oxidation and inflammation, the expression of genes related to TG level, oxidation, inflammation and PPAR pathway were detected, which indicated that LP-KFY04 could be used to reduce obesity induced by high-fat diet in mice. LP-KFY04 can effectively reduce the liver damage and fat accumulation caused by obesity. -3/6 Normal Control L-carnitine LLP-KFYO4 HLP-KFY04 LDSB Figure 3

Description

-3/6

Normal Control L-carnitine

LLP-KFYO4 HLP-KFY04 LDSB

Figure 3

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

A Lactobacillus plantarum with the function of reducing weight and fat and an

application thereof

The invention is described in the following statement:-

A Lactobacillus plantarumwith the function of reducing weight and fat and

an application thereof

TECHNICAL FIELD

The invention relates to the technical field of microorganism, in particular to a Lactobacillus plantarum with the function of losing weight and reducing fat and the application thereof.

BACKGROUND

Probiotic microorganisms, including Lactobacillus, Bifidobacterium, Lactococcus, Enterococcus and other lactic acid bacteria, are mainly located in the human intestinal tract and can improve the balance of intestinal microorganisms and play a beneficial role. In food, lactic acid bacteria are used for fermentation, and in recent years, it has been widely concerned because of its function. In addition, when raw milk is fermented with lactic acid bacteria, antibacterial substances such as organic acid, hydrogen peroxide and diacetyl are produced, and that deterioration of the dairy products is prevented by preventing the growth of pathogenic bacteria. In addition, many studies have shown that lactic acid bacteria can improve intestinal function and enhance human immunity, while also showing antioxidant, cholesterol-lowering and anti-tumor effects. Natural dairy products are rich in nutrients, immunoglobulins, lactoferrin, other immune factors, as well as a variety of growth factors. These factors affect immune regulation, promote growth and development, regulate intestinal flora, and affect other physiological functions. Traditional yoghurts, cheeses and dairy products fermented with probiotics, such as lactobacilli and bifidobacteria, are rich in nutrients, including amino acids and organic acids. Obesity is a chronic metabolic disease that seriously affects human health and development, and has become a worldwide epidemic. Modern high-sugar, high-fat diets result in an intake of more energy than the body can consume, leading to excessive accumulation of triglycerides (TG) in the liver, surrounding kidneys and adipose tissues, which leads to obesity. The results suggest that obesity leads to the increase of TG and cholesterol levels in blood and can lead to cardiovascular disease and type 2 diabetes. In addition, excessive TG accumulation, which the body cannot decompose, accumulates in the liver, leading to fatty liver and liver fibrosis. In order to curb obesity, currently available weight loss drugs are lipid absorption inhibitors or diuretics, but their side effects can cause endocrine disorders. Therefore, some non-drug treatments for obesity, including probiotics, are being explored to reduce obesity. Some studies have shown that probiotic supplementation can effectively reduce the development of obesity, possibly by altering the structure of gut microbes. This microbial improvement can subsequently improve the absorption of nutrients, alter lipid metabolism-related pathways in the liver, thereby reducing the accumulation of TG;

It also can affect inflammatory factors and oxidative stress to regulate metabolites in the gut, such as the transport of short chain fatty acids through the blood to the liver to regulate lipid metabolism. It can active some certain pathways, such as the peroxisome proliferator-activated receptor (PPAR) pathway, in order to regulate adipocyte division, reduce the size of adipose tissue and cells, and ultimately reduce obesity. Lactobacillus plantarum is a kind of Gram - positive bacteria, the optimum temperature is 30 - 35 °C, facultative anaerobic. The cells were short rod-shaped and did not produce spores, and presented milky white opaque, round and smooth colonies in MRS agar medium. Lactobacillus plantarum is a kind of homofermentative lactic acid bacteria, which can utilize glucose, maltose and sucrose to produce lactic acid only during the fermentation process, and is a typical facultative anaerobic bacterium, which has a strong capability of fermenting carbohydrates and is salt-tolerant. Lactobacillus plantarum, which is widely used in dairy products, meat, vegetables and fruit juice, is closely related to human life, and can play a probiotic role through the gastrointestinal tract and be planted in the intestinal tract. Lactobacillus plantarum has been widely used in food fermentation, lactic acid fermentation and medical care.

SUMMARY

An object of the present invention is to provide a Lactobacillus plantarum having a function of reducing weight and lipid and an application thereof, so as to solve the problems of the prior art. In order to achieve the above object, the present invention provides the following scheme: The present invention provides a Lactobacillus plantarum LP-KFY04, which is a lactic acid bacterium (LAB) and has been deposited in the General Microbiology Center of the Management Committee of China Microbial Culture Collection Center. The date of deposit is 28 April 2018 and the deposit number is CGMCC No. 15651. The invention also provides the application of the Lactobacillus plantarum LP KFY04 in reducing weight and reducing fat. The present invention discloses the following technical effects: In the present invention, lactic acid bacteria (Lactobacillus plantarum KFY04) were isolated from yoghurt and used in mice maintaining a 45% fat diet. Liver and serum samples were then obtained and gene expression associated with TG levels, oxidation, inflammation and the PPAR pathway was examined to assess the effect of LP-KFY04 on obesity, oxidation and inflammation. The invention shows that the strain has good tolerance to acid stress, bile salt stress and oxidation stress, has resistance to pathogenic bacteria infection, and can regulates metabolic disorder, nonalcoholic fatty liver and the like. LP-KFY04 can be used to reduce obesity induced by high-fat diet in mice. The results showed that LP - KFY04 could effectively reduce the liver damage and fat accumulation caused by obesity.

BRIEF DESCRIPTION OF THE FIGURES

In order to more clearly illustrate that embodiments of the present invention or the technical solution in the prior art, a brief description will be given to the drawing which are required to be used in the embodiments, and it is obvious that, the drawings in the following description are merely some embodiments of the present invention, and other drawings may be obtained from these drawings without any creative effort by those of ordinary skill in the art. FIG. 1 is an arrangement of animal experiments; FIG. 2 shows the body weight of mice during the experiment; FIG. 3 is a pathological observation of H & E in mouse liver tissue; FIG. 4 is a pathological observation of H & E in mouse epididymis; FIG. 5 shows the expression of PPAR-a, PPAR-y, CYP7A1, CPT1, LPL, C

/ EBPamRNA (A) and protein (B) in the liver of mice; FIG. 6 shows the expression of PPAR-a, PPAR-y, CYP7A1, CPT1, LPL, C

/ EBPamRNA (A) and protein (B) in mouse epididymis tissues.

DESCRIPTION OF THE INVENTION

Various exemplary embodiments of the present invention will now be described in detail, which should not be construed as limiting the invention, but rather as a more detailed description of certain aspects, features, and embodiments thereof. It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the invention. In addition, for the numerical ranges in the present invention, it is to be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any statement value or range of statements and any other statement values or intermediate values within said range is also included within the present invention. The upper and lower limits of these smaller ranges can be included or excluded independently. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as as those of ordinary skill in the art of the invention. Although only preferred methods and materials have been described herein, any method and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents referred to in this specification are incorporated by reference to disclose and describe methods and / or materials related to the documents. All references referred to in this specification are incorporated by reference to disclose and describe methods and / or materials related to the literature. In case of conflict with any incorporated literature, the contents of this specification shall prevail. It will be apparent to those skilled in the art that various modifications and variations may be made in the specific embodiments of the present description without departing from the scope or spirit of the invention. Other embodiments resulting from the description of the invention will be apparent to the skilled person. The specification and embodiments of the present application are only exemplary. The terms "including", "including", "having", "containing" and so on used in this paper are all open terms, which means including but not limited to. In order for probiotics to be beneficial to that gastrointestinal tract, they must be expose to gastric fluids for survival. When fasting or eating acidic foods, pH3.0 or as low as 1.5, bile salts (0.03 to 0.30%), so lactic acid bacteria with low resistance to acid and bile salts cannot survive and will not be effective probiotics. In this example, two lactic acid bacteria LDSB (a commercial fermentation strain) and LP-KFY04 (laboratory separation) were tested. The results show that, compared with LDSB, LP-KFY04 has a stronger ability to resist gastric acid and bile salts, which may provide a higher degree of probiotic benefits. Weight change is an important measure of obesity in mice, because faster weight gain is usually associated with the accumulation of fat tissue, because it consumes more energy than the body needs. Obesity is also the most common cause of non-alcoholic fatty liver because of excessive accumulation of TG. Therefore, examining the liver and adipose tissue index can help to assess obesity in mice from the inside. Although mice maintained on the 45% fat diet showed significant weight gain and tissue index increase relative to the normal group, the LP-KFY04 group showed a reduced effect from the high-fat diet. Adipose tissue is mainly composed of adipocytes, but also contains collagen fiber matrix, blood vessels, fibroblasts and immune cells. In mammal, there are two types of adipose tissue, white adipose tissue (WAT) and brown adipose tissues (BAT). The WAT, located under the skin and around the viscera, is the most common, is the most common and consists mainly of large spherical fat cells filled with single-cavity lipid droplets. In adults, BAT is mainly distributed in the scapular area, mediastinum and upper scapular area of the neck. During the development of obesity, lipids continue to accumulate, pre adipocytes continue to divide and activate, and mature adipocytes increase in volume. During obesity, the liver will also undergo changes, mainly manifested as a disorder of the vacuoles and sinus nodes in the liver cells. Here, this example found that LP-KFY04 can reduce the increase in volume of mature adipocytes, reduce the number of vacuoles in liver cells, and reduce the obesity-related effects in liver cells and adipocytes. The liver plays an important role in synthesis, storage and lipid metabolism, and TC serum concentration is used as an indicator of lipid metabolism. In the body, a large amount of glycerol is stored as TG, which is an important form of energy, mainly synthesized in the liver, and stored in the liver and adipose tissue. The degree of liver damage can be determined histologically and is based on TC and TG levels. In addition, in plasma, LDL-C is converted from VLDL and is the main form of endogenous cholesterol, which is transported to the liver. In addition, HDL-C is mainly synthesized by liver cells and is part of the reverse cholesterol transport mechanism that removes cholesterol from peripheral tissues to protect blood vessels from LDL damage. When fatty liver exists, the ability of the liver to oxidatively decompose fat is reduced, fat synthesis increases, and phospholipid synthesis is impaired; resulting in an increase in LDL-C and a decrease in HDL-C. Another way to assess liver damage is to examine the liver damage markers ALT and AST. The liver stores ALT, an important metabolic enzyme that catalyzes the transamination process of glutamate and pyruvate in liver cells. Under normal conditions, the serum ALT activity is low, and the ALT is in a state of dynamic balance between cells and circulation. During liver injury, liver cell damage causes large amounts of ALT to be released into the blood. In addition, AST catalyzes the transamination between glutamate and oxaloacetate, and can also provide insight into liver damage. AST has two isozymes, called c-AST and m-AST. Studies have shown that c-AST is released when cell damage is mild, and when cell damage is more severe, even during necrosis, m-AST is released into the blood. Therefore, serum ALT and AST levels can also reflect the severity of liver cell and mitochondrial damage. Here, this example found that LP KFY04 can reduce the increase in TC, TG, LDL-C, ALT and AST caused by the high-fat diet, and the decrease in HDL-C. The presence of obesity also promotes the production of free radicals, including the up-regulation of the renin-angiotensin system, which leads to an increase in the production of superoxide anions, followed by increased uptake of LDL by macrophages and increased lipoprotein oxidation. In addition, obesity is accompanied by a weakened antioxidant defense mechanism, which results in excess reactive oxygen species not being removed, and subsequent oxidative stress. Oxidative stress destroys cellular DNA, ultimately leading to accumulation of protein oxidation products and lipid peroxidation products. Here, it was found that LP-KFY04 can increase antioxidant SOD and CAT, and reduce MDA, and reduce lipid peroxidation reaction product MDA, thereby reduce the oxidative stress reaction caused by obesity. Recent scientific research has shown that obesity can lead to chronic, low inflammatory state of the body for a long time. This chronic low-grade inflammation and non-specific activation of the immune system can promote the occurrence and development of obesity-related metabolic diseases and cardiovascular and cerebrovascular diseases. Adipose tissue not only stores energy, but also produces and secretes various hormones and cytokines, which affect metabolism, neuroendocrine and autoimmune functions. During obesity, macrophage infiltration increases, which leads to a subsequent increase in pro-inflammatory factors, such as TNF-a and IL-6, and a decrease in anti-inflammatory factors, including IL-4 and IL-10. In addition, some studies suggest that obesity may lead to imbalance of intestinal flora, reduce immune tolerance in mice, and increase the number and type of inflammatory transmitters secreted in the body, such as IL-Ibeta, IL-6, and IFN-gamma. In this example, it was found that LP KFY04 can effectively reduce the levels of pro-inflammatory factors, TNF-a, IFN-y, IL 1p and IL-6 in obese mice, and increase the anti-inflammatory factors IL-4 and IL-10. Another important factor regulating energy consumption and various other cellular functions is PPA Rs. PPA Ra is highly expressed in liver, skeletal muscle, kidney, heart and blood vessel wall. It mainly regulates energy consumption, can stimulate pre adipocyte differentiation and fatty acid oxidation, and can reduce liver and circulating lipid concentrations. PPA Ra also prevents the expansion of fat cells and the accumulation of fat in insulin-sensitive tissues by increasing intracellular lipolysis in adipose tissue. PPA Ry is an important cell differentiation transcription factor, which is expressed in mammalian adipose tissue, vascular smooth muscle tissue and myocardial tissue. It also mediates adipocyte pre-differentiation, lipid accumulation and adipocyte specific gene expression. PPA R-y activates enzymes in fat cells, which can increase TG synthesis and increase the volume and number of fat cells; therefore, its activity is proportional to fat content. Another adipogenic factor C/EBPa plays an important role in differentiation and promotes the transcription and expression of genes related to lipid droplet formation. PPA R-y and C/EBPa are considered to be the key markers of adipocyte differentiation. C/EBPa promotes the up-regulation of PPA R-y and maintains the differentiated phenotype. In addition, LPL is a proteolytic enzyme and a key enzyme for lipid metabolism. Its main function is to catalyze the decomposition of TGs in CMS and VLDL into free fatty acids, and promote the transport of proteins, phospholipids and apolipoproteins, thereby promoting serum HDL levels. Increased and decreased TG levels. In the liver, 40% of the cholesterol synthesized daily is converted into bile acids through the CYP7A1 pathway, which is the downstream PPA R target. In addition, CYP7A1 has been shown to be inversely proportional to plasma cholesterol levels and liver fat accumulation. Another downstream PPA R target, CPT1, is a key enzyme for fatty acid P oxidation. In addition, increasing the expression of CPT1 has been shown to increase fatty acid breakdown and reduce body fat content. Here, the present example found that LP-KFY04 promotes the expression of PPAR-a, CYP7A1, CPT1, and LPL in the liver and fat, inhibits PPAR. gamma. and C / EBP-a., thereby reducing fat accumulation and reducing obesity. Example 1 1. Materials and methods 1.1 Experimental strain Lactobacillus plantarum HFY15 was isolated from yak yoghurt in Sichuan province. It was identified as Lactobacteria by 16S rDNA sequence analysis. The strain has been deposited in the General Microbiology Center of the Management Committee of China Microbiological Culture Collection (CGMCC) on October 29, 2018. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), accession number: CGMCC No.16648. Lactobacillus plantarum HFY15 was isolated and purified from yak yoghurt sam ples, and all of them were positive by Gram staining. Most of the colonies were whi te or milky white, with round shape, neat edges, moist and smooth surface, and unde r 100 times oil scope, the cells have long stem, short stem, globular shape, and no budding and reproduction. The 16S rDNA base sequence of the strain HFY15 of the present invention is s hown as SEQ ID No. 1; the sequencing results were aligned on the NCBI website (h ttp: // blast.ncbi.nlm.nih.gov/Blast.cgi). The homology was 100% with Lactobacillus p lantarum, MN368282.1.1. 2. Tolerance test of artificial gastric juice Artificial gastric juice (0.2% NaCl and 0.35% pepsin) was prepared, adjusted to 3.0 with 1 mol / L HCl, and sterilized by filtration (0.22 m). To activate the LP-KFY04 cells, the samples were cultured twice in 5 ml MRS, the bacterial cells were collected by centrifugation at 3000 rpm for 10 min, washed twice with sterile saline, and then resuspended into 5 ml sterile saline. The bacterial broth was then combined with sterile artificial gastric broth (1: lv / v), gently mixed, and incubated at 37°C. The survival rate was measured at Oh and 3h and calculated as follows. Survival (%)= x 100% *3 h viable count (CFU/mL) 0 h viable count (CFU/mL) 1.3 Anti-bile salt 0.3%

Activated LP-KFY04 cells (2% v / v) were inoculated into MRS-THIO (0.2% sodium thioglycolate) medium containing 0.0% or 0.3% porcine bile salt (121.degree. C. pre-sterilized for 15 min); blank MRS -THIO medium (not inoculated) was negative control; The samples were incubated at 37C, on a shaker for 24 hours. Blank medium and seeding medium were then loaded into 96-well plates (200 ml / well) and absorbance was measured at an optical density (OD) of 600 nm. The growth efficiency is calculated as follows: medium Growth efficiency(%)= x 100% *OD60 0 of 0.3% bile salt medium-blank OD 6 0 0 of 0.0% bile salt medum-blank medium 1.4 Establishment of animal model Six-week-old SPF male and female C57BL / 6J mice (n = 60) were from Chongqing Medical University, Chongqing, China (SCXK (YU) 2017-0001). After 7 days of adaptive feeding, the mice were randomly divided into 6 groups: Normal group, control group, L-carnitine group, LP-KFY04 low-dose group (LLP-KFY04 group), LLP-kFY04 high-dosage group (HLP-LFP-KFFY04 Group) and LDSB group. Normal mice were fed a 10% fat diet, while the other five groups were fed 45% fat diets (Changzhou Qihang Biotechnology Co., Ltd., Changzhou, Jiangsu Province; Table 1) for 8 weeks. During this period, daily oral treatment, L-carnitine 200 mg / kg (body weight), LP-KFY04 daily 1 x 108 LLP-KFY04 group CFU / kg of body weightx109 HLP-kFY04 group CFR (B W) and LDSB were administered at 1x109. Weight was measured weekly and the volume of gastric lavage was recalculated. On the last day of the experiment, the mice fasted for 24 hours. After administration of ether, whole blood was taken from the inferior vena cava and stored at 4°C for further use. Liver, epididymis fat and perinephric fat were isolated from the body and weighed. Part of the liver and epididymis adipose tissues were fixed in % formalin and H & E stained. The remaining liver and epididymis adipose tissue samples were stored at -80°C for further use (FIG. 1). The organ tissue index was determined as follows: Organ tissues index = organ weight / body weight x 100. This study was approved by the Ethics Committee of Chongqing Functional Food Collaborative Innovation Center (201906015B; Chongqing, China). Table 1 Diet formulation of the mice in this study

Diet composition (%) 10% fat diet 45% fat diet

Corn flour 24.89 7.79 Blain 15.00 27.00 Wheat flour 7.00 /

Soy milk 18.50 22.00 sucrose 16.38 20.00 Ladd 1.02 19.50 Preliminary preparation 17.21 23.71 Premix: Trace minerals, elements, vitamins, synthetic amino acids. 1.5 The levels of TC, TG, ALT and AST in liver of mice were measured Liver tissue (1g) homogenate : Tissue homogenate was isolated at 4 °C for 10 minutes at 4000 rpm. Supernatants were collected and total TC, TG, ALT and AST levels were determined with the kit according to the manufacturer's instructions (Nanjing Institute of Bioengineering, Nanjing, China). 1.6 Determination of serum HDL-C, LDL-C, TC, TG, ALT and AST levels in mice

Whole blood was centrifuged at 4000 rpm for 10 min at 4 °C. The sera were collected and quantified with a kit for HDL-C, LDL-c, TC, TG, ALT and AST levels according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute). 1.7 The levels of TNF-a, IFN-y, IL-1j, IL4, IL6 and IL- 10 in serum and T-SOD, CAT and MDA in serum were measured Serum TNF-a, IFN-y, IL P, IL-4, IL6 and IL-10 (ABCAM, Cambridge, MA, USA) cytokine levels and T-SOD, CAT and MDA levels were measured with a kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute). 1.8 Histopathological examination of liver and adipose tissue in mice Mouse liver and epididymis adipose tissues were fixed in 10% formalin (v / v) for 24 h, then dehydrated, cleared, paraffin embedded, sectioned and stained (H & E). The samples were then imaged under an optical microscope (B X43; Olympus, Tokyo, Japan) and examined for histology. 1.9 Quantitative PCR analysis of mouse liver and adipose tissue RNA was extracted from liver and epididymis adipose tissue samples using TRIzol (Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using 1 L diluted RNA (1 pg / pL). The resulting cDNA was then combined with 10 pL SYBR green PCR master mix (Thermo Fisher Science, Waltham, MA, USA), 1 L of each forward / reverse primer (Table 2) and distilled water (7 pL). Quantitative real-time PCR was performed in an automated thermocycler for 60 s at 95 0 C; then cycles 40 times at 95 °C for 15 s and at 55 ° C for 30s, and at 72 °Cfor 35 s; the last step was using GAPDH as internal reference gene to calculate the relative mRNA transcription level - ACT for 30s at 95°C, and for 35s at 55°C. Table 2 Primer sequence gene Advance order Reverse sequence

PPAR-a 5'-cctcagggtaccactacgggt-3' 5'gccgaatagttcgccgaa-3'

PPAR-y 5'agccgagaaggagcTGtTG-3' 5'TGgccaccttTGTGTGctc-3'

CYP7A1 5'agcaactaaacaccTGccagtacta-3' 5'gtccggatattcaaggaTGca-3'

cptl 5'aaaagatcaatcggaccctagaca-3' 5'cagcgagtagcgcatagtca-3'

LPL 5'agggcTGccTGagtTGta-3' 5'agaatctcgaaggccTGgt-3'

C/EBPa 5'TGgacagaacagcaacgagtac-3' 5'gcagtTGcccaTGgcctTGac-3'

Gapd 5'-acccagaagacTGTGgaTGg-3' 5'-acatt ggggg taggaaca-3'

Note: PPAR-a: Peroxisome proliferator-activated receptor a; PPAR-y: Perox proliferator activated receptor gamma; CYP7A1: Cholesterol 7-alpha hydroxylase; CPT1: Camitine palmitoyl transferase 1; LPL: Lipoprotein lipase; C / EBPa: CCAAT/ enhancer-binding protein a; GAPDH: 3-phosphate dehydrogenase. 1.10 Protein Expression Analysis of Liver and Adipose Tissue in Mice

Liver and adipose tissue samples (100 mg) were homogenized in im L RIPA (Thermo Fisher, Wal tham, MA, USA) and 10 L PMS F (Therm Fisher, Waltham, MA.USA), and hearts were isolated at 4°C for 5 min at 12,000 rpm. The proteins were quantified using a BCA protein assay kit (Thermo Fisher, Waltham, MA, USA) and then diluted 4: 1 with a sample buffer (Themo Fisher, Weltham, MA.USA), and heated at °C for 5 minutes. SDS-PAGE was performed by gel electrophoresis at 100 V which were then transferred to PVDF membranes. PVDF membranes were then blocked with % skim milk for 1 h and then incubated with an antibody overnight at 4°C (Thermo Fisher, Waltham, Mass., USA). The samples were then incubated with a secondary antibody (Thermo Fisher, Waltham, MA, USA) for 1 h, and then the bands were visualized with an ECL Western blot substrate (Therno Fisher, Weltham, MA.USA). The imaging was performed using a Tano luminescence imaging workstation (Shanghai Tanon Technology Co., Ltd., Shanghai, China). 1.11 Statistical analysis All samples, serum and tissue were examined in triplicate and averaged. The data were analyzed using the Social Sciences Statistical Software package (version 22; SPSS, Chicago, Illinois, Ill., USA). The difference between the mean values of the individual groups was assessed using a one-way ANOVA and Duncan's multiple-range test. If a p value of< 0.05 is obtained, the difference is considered to be statistically significant. 2. Results 2.1 Resistance of lactic acid bacteria to artificial gastric juice and bile salt The results showed that the survival rate of LDSB in artificial gastric juice (pH3) was 37.69% ±4.52%, while that of LP-KFY04 was 87.21% ±5.12%, which was 2.35 times of that of LDBS (Table 3). The growth efficiency of LDSB was 7.79% ±0.23% in 0.3% bile salt solution, and that of LP-KFY04 was 17.71% 0. 24%, which was 2.27 times of that of LDSB. Table 3 Resistance of lactic acid bacteria to artificial gastric juice and bile salts stain Artificial gastric juice tolerance test Resistance to 0.3% bile (). acids (%). ldsb 37.69+4.52 7.79+0.23

LP-KFY04 87.21+5.12 17.71+0.24

The values given are mean standard deviation (n = 10 /group). LDSB: Delbrueckii, Bulgaria; LP-KFY04: LactobacillusplantarumKFY04. 2.2 Weight of the mouse During the eight-week period, normal mice fed a 10% fat diet increased from an average of 19 grams to 24 grams with a 26.3% increase in body weight. The other five groups maintained a 45% fat diet. Control group (49.1%), L-camitine Group(46.2%) and LDSB Group(44.2%) had the highest weight gain rate. The lowest rate of weight gain was found in group LLP-KFY04 (38.5%) and HLP-KFY04 (35.6%). These findings indicate that LP-KFY04 significantly reduced weight gain in mice (FIG. 2). 2.3 Organ index of mice Of all organ indices examined, the indices of liver, epididymis fat and perinephric fat were the highest in the control group, equal to the lowest in the normal group (Table 4). In the other four groups, the indices associated with HLP-KFY04 group were significantly lower than all other groups except the normal group (p < 0.05). The next lowest group was LLP-KFY04 group, compared with L-carnitine and LDSB group (p < 0.05) , which was significantly reduce. Table 4. organ index of mice in each group Liver index Epididymal fat Perirenal fat group index index normal 2.35±0.02a 1.24±0.06a 0.22±0.01a control 5.78±0.11e 3.28±0.17' 1.28±0.13'

L-carnitine 4 . 5 6 ±0. 0 7 d 2 . 7 6 ±0. 0 5 d 0.68±0.05d

LLP-KFY04 3.79±0.06c 2.21±0.05c 0.46±0.03c

HLP-KFY04 3.2610.05' 1. 9 9 ±0. 0 5b 0.35±0.05'

ldsb 4 . 5 0 ±0.0 7d 2.93±0.06e 0.75±0.02e

The values given are mean standard deviation (n = 10 / group). According to Duncan's multi-range test, the average values of different letters in the same column were significantly different (p < 0.05). L-Carnitine: Mice treated with 200 mg / kg (b.w) L carnitine; LLP-KFY04: Mice treated with 1.0 x 10 8 CFU / kg of Lactobacillus plantarum KFY04 (b.w); HLP-KFY04: Mice treated with 1.0 x 10 9CFU / kg of Lactobacillus plantarum KFY04 (b.w); LDSB: Mice treated with 1.0 x 10 9CFU / kg of Lactobacillus delbrueckii (b.w) 2.4 Levels of TG, TC, ALT and AST in liver tissue of mice The analysis showed that TG, TC, ALT and AST levels were the lowest in the normal group and the highest in the control group (Table 5). After intragastric administration of LP-KFY04, either Group LDSB or L-carnitine decreased to a certain extent compared with the control group, and the decrease of Group HLP-KFY04 was the largest. Group LLP-KFY04 was the next group that showed the largest decline, followed by LDSB and L-camitine with a much smaller decline. Table 5 TC, TG, ALT and AST Levels in Mice Liver TG tc Alternative text a group (mmol/gprot) (nmol/gprot) (U/gprot) (U/gprot) normal 0.29±0.02a 35.19±2.05a 10.46±0.28a 3.64±0.21a

control 1.15±0.07' 89.76+5.11' 19.27 0.35' 9.18 0.16'

L-camitine 0.78 0. 0 2 d 65.33 3. 4 3 d 14.96 0. 1 3 d 6.76 0. 2 5 d

LLP-KFY04 0.52 0.03c 53.97 3.27c 13.26 0.26c 5.17 0.22c

HLP-KFY04 0.48 0. 0 5 ' 43.12 2. 1 9 ' 11.76 0. 2 1 ' 4.67 0. 3 5 ' ldsb 0.95 0.10e 75.88 3.55° 15.10±0.24e 7.26±0.38e

2.5 Serum levels of HDL-C, LDL-C, TC, TG, ALT and AST in mice In the control group, the levels of LDL-C, TC, TG, ALT and AST were the highest, but the levels in the normal group were the lowest; HDL-C of the control group was the lowest, but that of the normal group was the highest (Table 6). LP-KFY04, LDSB and L carnitine all increased the index values, but the effect of HLP-KFY04 was the most significant, and the next was LLP-KFY04, L-Carnitine and LDSB. Table 6 Serum levels of HDL-C, LDL-C, TC, TG, ALT and AST in mice HDL-c LDL-c TG tc Alternative a group (u/l) (Mmol / L) (Mmol / L) (Mmol / L) text (u/l) normal 1.33±0.12' 1.56±0.06a 1.62±0.02a 4.76±0.17a 2.35±0.12a 17.24±1.06a

control 0.49±0.08a 5.98±0.14e 6.69±0.13' 9.53±0.11' 5. 7 8±0.3I' 33.28±1.17'

L-carnitine 0.86±0.06c 4 .2 7 ±0.0 8 d 3 . 5 6 ±0. 0 7 d 7 .5 6 ±0. 0 7 d 4.56+0. 17 d 27.76+1. 7 5d

LLP-KFY04 1. 0 5 ±0.0 6 d 3.29+0.11c 2.96±0.06c 5.94±0.16 3.79±0.26 25.21+1.22c

HLP-KFY04 1.17+0.1oe 2 .6 6 ±0.0 7 2 .4 6 ±0. 0 5b 5 .2 6 ±0. 0 5 3.26+0. 1 5b 23.99+1. 35

' ldsb 0 .7 8 ±0.0 4 4 .9 5 ±0.0 8 d 3.87±0.04e 8.59±0.07e 5.15±0.28e 30.93±1.38e

2.6 Serum T-SOD, CAT and MDA levels in mice The results showed that the values of T-SOD and CAT were the highest in the normal group, and the lowest in the control group. MDA was the lowest in the normal group and the highest in the control group (Table 7). All the four intervention groups showed a more similar serum oxidation index to the normal group, and the effect was the best in HLP-KFY04 group. Table 7 Serum T-SOD, CAT and MDA levels in mice Thor-Solder Cat Ministry of group (Unit/milli) (Unit/milli) Agricultureml)

normal 185.33±7.18' 7.99±0.15' 7.33±0.16a

control 122.23±3.45a 3.18+0.11a 15.65±0.20'

L-camitine 148.65±5.19c 5.12±0.13c 12.33+0.18 d

LLP-KFY04 153.79±6.75d 5.96±0. 1 7 d 9.16+0.17c

HLP-KFY04 167.85±7.32e 6.54±0.12° 8.46+0. 1 5b

ldsb 139.17±4.98' 4.77±0.16' 10.95±0.23e

2.7 Serum Levels of TNF-a, IFN-y, IL- I, IL4, IL6 and IL- 10 in Mice The levels of pro-inflammatory factors (TNF-a, IFN-y, IL- IPand IL-6) were the highest in the control group, while anti-inflammation factors (IL-4 and IL -10) were lowest. In the normal group, an entirely opposite trend was noted. In the other four groups, the pro-inflammatory factors were significantly lower than those in the control group (P < 0.05), and the anti-inflammatory factors were significantly higher than those in the control group (P, 0.05). In HLP-KFY04 group, the improvement degree was the highest, followed by LLP-KFY04, L-carnitine and LDSB groups (Table 8). Table 8 Cytokine levels of TNF-a, IFN-y, IL- I, IL4, IL6 and IL10 in serum of mice TNF-a IFN-y IL-1j il-4 il-6 il-10 group (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml) normal 48.69±2.76a 33.87±1.54a 20.77±0.53a 57.12±1.54' 43.49±2.21a 12 9 . 3 2 ±8. 1 3

' control 87.63±3.39' 97.62±4.34' 79.82±0.97' 33.21±0.85a 8 9 .6 5 ±3.4 5 ' 49.12±3.66a

L-carnitine 7 8 .3 9 ±3. 0 5 d 73.51±3.43d 41. 6 5 ±0. 6 1d 40.34±0.54c 6 5 .4 5 ±3. 7 5 d 75.16±4.97°

LLP-KFY04 66.37±2.95c 61.38±3.05c 35.76±0.58c 4 6 . 6 5 ±1. 0 4 d 59.05±2.22c 84.59 ±5.94e

HLP-KFY04 56.98 50.66 29.45 50.07 54.12* 90.34 5. 4 6d 2.61P 2.66' 0.47' 1.38e 2.35 ldsb 82.16 75.99 50.55 36.40 70.45± 64.37±4.41 2.89e 3.56e 0.78e 0.75' 3.85e 2.8 Histopathological evaluation of adipose tissue in liver and epididymis of mice Among the liver tissues obtained in the normal group, the structures of the hepatic lobules were organized, the central veins were clear, and the hepatic sinuses were clear (Fig. 3). However, in the control group, the liver lobules were blurred, the hepatic cords were disordered, the sinusoids were narrow, and a large number of fatty vacuoles were found in the hepatocytes. When LP-KFY04 group was compared with control group, the structure of hepatic lobule was basically normal, the hepatic sinusoids were clearer, the arrangement of hepatic cords was more orderly, and the number of hepatic fatty vacuoles was significantly reduced. This effect was more obvious in HLP-KFY04 group than in LLP-KFY04 group. In addition, LP-KFY04 group showed a more significant improvement than L-carnitine and LDSB group, and LP-kFY04 was closer to the morphology of the normal group. In examine that adipose tissue of the epididymis (fig. 4), the normal group was observed to have a regular structure and size of adipocyte, while the control group had the largest adipocytes and the thinnest cell wall. LP-KFY04, LDSB and L-carnitine all inhibited the increase of adipocytes to some extent. The most significant effect was in the HLP-KFY04 group, which showed adipocyte size close to that of the normal group. 1.9 Expression levels of RNA and protein in adipose tissue of liver and epididymis PPAR-a, PPAR-y, CYP7A1, CPT1, LPL and C / EBPa mRNA and protein expressions were examined in liver and epididymal adipose tissue (FIG. 5, FIG. 6). The expression of PPAR-a, CYP7A1, CPT1 and LPL was the highest in normal mouse samples, while PPAR -y and C / EBPa were the lowest. HLP-KFY04 group showed the trend of expression closest to that of normal group, followed by LLP-KFY-04 group among mice that maintained a high fat diet. In addition, LDSB and L-camitine also inhibited the expression of PPAR-y and C / EBPa to some extent, and increased the expressions of PAPR-a, CYP7A1, CPT1 and LPL, but LP-KFY04 had the highest intervention degree. The embodiments described above are only for the description of the preferred mode of the present invention, and are not intended to limit the scope of the invention. Without departing from the design spirit of the invention, various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the protection scope defined in the claims of the invention.

Claims (2)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A kind of Lactobacillus plantarum LP-KFY04 is characterized in that the Lactobacillus plantarum LP-KFY04 belongs to the genus of lactic acid bacteria (LAB), and has been preserved in the general microbiology center of the Management Committee of China microbial species preservation center on April 28, 2018, with the preservation number of CGMCC no.15651.
2. 2. Application of Lactobacillus plantarum LP-KFY04 in weight loss and lipid reduction according to claim 1.

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