CN117417863A - Bifidobacterium lactis and application thereof in preparing products for preventing and treating obesity and/or weight loss - Google Patents

Abstract

The invention belongs to the technical field of microbial agents, in particular to bifidobacterium lactis and further discloses application of bifidobacterium lactis in preparation of obesity prevention and treatment products and/or weight reduction products. The YG2013 strain is obtained by screening in healthy human excrement, and identified bifidobacterium lactis is classified and named Bifidobacterium lactis. Experiments prove that the bifidobacterium lactis YG2013 strain can realize white fat beige and activate brown fat metabolism and heat production through targeted regulation and control of tgr/Il-27, achieves the effect of high efficiency and weight reduction, and has better effects of treating weight loss and weight reduction.

Description

Bifidobacterium lactis and application thereof in preparing products for preventing and treating obesity and/or weight loss

Technical Field

The invention belongs to the technical field of microbial agents, in particular to bifidobacterium lactis and further discloses application of bifidobacterium lactis in preparation of obesity prevention and treatment products and/or weight reduction products.

Background

Overweight or obesity has been reported to significantly increase the risk of developing type 2 diabetes, hypertension, nonalcoholic fatty liver, cardiovascular disease, and cancer, and has become a potential hazard affecting resident health, creating a significant challenge to human health. It can be seen that overweight or obesity has become a global public health problem.

Studies have shown that overweight or obesity is mainly due to an imbalance between energy intake and consumption. Currently, anti-obesity strategies are primarily limiting energy intake and absorption by weight-loss drugs. However, these drugs have not only major side effects, but also some or even liver toxicity, such as diarrhea, endocrine disturbance, insomnia and other adverse reactions easily occurring during the administration of Orlistat, and there are cases where the drug is re-inflated. Thus, there is a need in the art to find natural and safe drugs or functional foods for the purpose of treating obesity.

Brown Adipose Tissue (BAT) is a highly metabolically active tissue that contains a large number of mitochondria, and increases the electrical conductivity of the inner mitochondrial membrane by high expression of uncoupling protein 1 (ucp 1), thereby inducing BAT mitochondria to produce heat. The white fat is brown, and the white fat cell has similar form and function to BATs, and can utilize lipid and blood sugar to improve blood fat and glucose metabolism. Currently, by activating BAT activity and promoting the formation of beige fat, it has become an attractive approach to increase energy expenditure to combat diet-induced obesity and related metabolic diseases.

There is growing evidence that there is a close link between gut microbiome and obesity, and gut microbiota has the effect of modulating adipocyte phenotype and function. Therefore, a natural and safe drug or food capable of regulating intestinal microbiota is sought, so that the activation of metabolic heat production by upregulation of BAT and ucp1 in beige adipose tissue is a novel effective therapeutic approach for the prevention and treatment of obesity. A large number of researches prove that probiotics can improve obesity and related metabolic disorder, and are mainly reflected in aspects of promoting intestinal peristalsis, preventing constipation and the like. However, traditional probiotics have low abundance in human bodies and large individual variability, and the existing probiotic products have unobvious regulation on intestinal flora, single action mechanism, long weight reduction time consumption and unsatisfactory product effect.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide the bifidobacterium lactis with the function of promoting white fat beige, and the strain can reduce white adipose tissue inflammation and induce beige remodeling thereof to realize the anti-obesity function;

the second technical problem to be solved by the present invention is to provide the use of the bifidobacterium lactis described above for the preparation of a product for the prevention or treatment of obesity and/or weight loss.

In order to solve the technical problems, the invention discloses application of a regulator with tgr/Il-27 pathway targeting regulation function in preparing a product with the functions of losing weight and reducing weight; wherein,

the regulator is based on targeting regulation of the tgr/Il-27 pathway, promotes beige coloration of white fat and reduces its inflammatory effects, and activates the metabolic heat generation of brown fat and reduces its inflammatory effects.

The invention also discloses application of the preparation with the effect of targeted improvement of the characteristic intestinal flora of obesity in preparing a product with the effect of losing weight and reducing weight; wherein,

the improving intestinal flora comprises increasing the abundance of beneficial bacteria and/or decreasing the abundance of harmful bacteria;

preferably, the improving the intestinal flora comprises increasing the abundance of unclassified f_ Lachnospiraceae, duncaniella, lactobacillus, limosilactobacil and Eubacterium flora, increasing SCFAs production;

preferably, the improving the intestinal flora comprises increasing the abundance of beneficial bacteria unclassified_f_ Lachnospiraceae, duncaniella, lactobacillus, eubacterium flora and decreasing the abundance of harmful bacteria Ileibacterium, vampirovibrio, clostridium flora.

The invention particularly discloses a bifidobacterium lactis YG2013 which is classified and named as bifidobacterium lactis Bifidobacterium lactis and is preserved in the China general microbiological culture collection center (CGMCC) No.27579, and the preservation date is 2023, 6 and 7.

The invention also discloses application of the bifidobacterium lactis YG2013 and the deactivator and metabolite thereof in preparing probiotic preparations.

The invention also discloses a probiotic preparation, and the active ingredients of the probiotic preparation comprise thalli of the bifidobacterium lactis YG2013 and an deactivation substance and/or a metabolite thereof.

Specifically, in the probiotic preparation, the effective cell number of the bifidobacterium lactis YG2013 is 5.0X10 ⁹ -1.0×10 ¹¹ CFU/day/person.

Specifically, the probiotic preparation comprises at least one of powder, granules, pills, capsules, tablets, ointment, liquid preparation, gel, spray or solid beverage.

The invention also discloses a method for preparing the probiotic preparation, which comprises the step of culturing the bifidobacterium lactis YG2013 and the step of processing selected dosage forms by adding conventional auxiliary materials according to a conventional process.

The invention also discloses the use of the bifidobacterium lactis YG2013 and the deactivation, the metabolite or the probiotic preparation thereof for preparing a functional product with at least one of the following effects (1) - (10):

(1) Preventing and treating obesity;

(2) Has the effect of reducing weight;

(3) White fat beige remodeling;

(4) Activating brown fat metabolism to produce heat;

(5) Targeting modulates the tgr5/Il-27 pathway;

(6) Reducing white adipose tissue inflammation;

(7) Alleviating hepatocyte steatosis and inflammatory responses;

(8) Producing a plurality of short chain fatty acids, regulating energy metabolism;

(9) Increasing the abundance of unclassified f_ Lachnospiraceae, duncaniella, lactobacillus, limosilactobacil and Eubacterium flora by cross feeding, and improving the yield of SCFAs;

(10) Targeted improvement of the characteristic intestinal flora of obesity, improvement of the abundance of beneficial bacteria unclassified_f_ Lachnospiraceae, duncaniella, lactobacillus, eubacterium flora, reduction of the abundance of harmful bacteria Ileibacterium, vampirovibrio, clostridium flora and recovery of the health of the intestinal flora.

Specifically, the functional product comprises food, medicine and/or health care product.

The invention also discloses a probiotic preparation with white fat beige remodelling function, and the active ingredients of the probiotic preparation comprise the bifidobacterium lactis YG2013 and the deactivator and/or metabolite thereof;

preferably, the effective cell number of the bifidobacterium lactis YG2013 is 1.0X10 ¹⁰ -5.0×10 ¹⁰ CFU/day/person.

The invention also discloses a probiotic preparation with the weight-losing and weight-losing effects, and the active ingredients of the probiotic preparation comprise the bifidobacterium lactis YG2013 and the deactivation matters and/or metabolites thereof;

Preferably, the effective cell number of the bifidobacterium lactis YG2013 is 2.0X10 ¹⁰ -8.0×10 ¹⁰ CFU/day/person.

The YG2013 strain is obtained by screening in healthy human excrement, and identified bifidobacterium lactis is classified and named Bifidobacterium lactis. Experiments prove that the bifidobacterium lactis YG2013 strain can realize white fat beige and activate brown fat metabolism and heat production through targeted regulation and control of tgr/Il-27, achieves the effect of high efficiency and weight reduction, and has better effects of treating weight loss and weight reduction.

The strain discovers and verifies that the bifidobacterium lactis has a targeting tgr/Il-27 regulation and control way, and has the effect of promoting white fat beige, so that the white fat beige is realized, brown fat metabolism and heat production are activated, white adipose tissue inflammation is further reduced, beige remodeling is induced, the anti-obesity effect is realized, and the high-efficiency weight reduction effect is realized.

The strain provided by the invention discovers and verifies that the bifidobacterium lactis has the effect of activating brown fat heat generation through targeted regulation of an Il-27 pathway for the first time, so that the effects of relieving inflammation of brown fat tissues and activating metabolic heat generation of the brown fat tissues are exerted, and the anti-obesity effect is realized.

Experiments prove that the bifidobacterium lactis YG2013 has the effect of high-efficiency weight reduction through differentiated new targets. The bifidobacterium lactis YG2013 improves the disorder of high fat Gao Tang through a comprehensive mechanism, and is specifically expressed in the following steps:

a. Targeting regulates the tgr5/Il-27 pathway, promoting white fat beige and reducing its inflammatory effects;

b. targeting the regulation of Il-27 pathway, activating brown fat metabolism to produce heat and reducing its inflammatory effects;

c. alleviating hepatocyte steatosis and inflammatory responses;

d. producing a plurality of short chain fatty acids, regulating energy metabolism;

e. targeted improvement of the characteristic intestinal flora of obesity, improvement of the abundance of beneficial bacteria, reduction of the abundance of harmful bacteria and recovery of the health of the intestinal flora.

The bifidobacterium lactis YG2013 serving as a new generation probiotic agent can be prepared and formed according to a conventional mode based on the bifidobacterium lactis YG2013 serving as an active ingredient, so that the problem of more side effects in the existing medicine weight-losing mode can be solved; the defects that the existing traditional probiotic products are low in abundance in human bodies and large in individual variability to influence the efficacy of the products are also effectively overcome.

The active ingredients of the probiotic preparation comprise the bifidobacterium lactis YG2013 and the deactivation substances and/or metabolites thereof, other active ingredients can be optionally added, or only the bifidobacterium lactis YG2013 is used as the active ingredient, and the activity of the bifidobacterium lactis YG2013 is utilized to play a role in losing weight, fat and weight, so that the probiotic preparation has the advantages of medication safety and remarkable improvement effect.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings, in which,

FIG. 1 shows the results of the test for cholesterol-degrading ability of the strain described in example 2;

FIG. 2 shows the results of the assay for bile salt hydrolase activity of the strain described in example 3;

FIG. 3 shows the results of the test for the tolerance of the strain described in example 4 to the simulated gastrointestinal environment;

FIG. 4 shows the results of the SCFAs-producing ability test of the strain described in example 5;

FIG. 5 shows the results of cell adhesion ability test of the strain described in example 6;

FIG. 6 shows the results of a hemolysis test of the strain described in example 7;

FIG. 7 shows the weight gain and fat accumulation results of HFD mice reduced by YG2013 strain of example 8;

FIG. 8 is a beige remodelling result of YG2013 strain in example 8 to reduce inflammation and induce eWAT;

FIG. 9 is the results of the YG2013 strain of example 8 for reducing inflammation and activating the thermogenic activity of brown fat;

FIG. 10 YG2013 strain in example 8 reduced liver inflammation and improved liver steatosis results; wherein A is a liver HE staining section chart; b is a liver oil red O staining section chart; c is a liver pathology scoring result; d is a liver gene detection result;

FIG. 11 shows the results of the change in SCFAs concentration of HFD-fed mice with YG2013 strain intervention in example 8;

FIG. 12 is a graph showing the results of YG2013 altering the intestinal microbiota diversity and composition of HFD fed mice; wherein A-B are Chao, shannon and alpha-diversity analysis indexes respectively; c is the PCoA graph analysis result of each sample; d is the Venn plot result showing observed ASV overlap; e is the portal level in mouse feces; f is the bacterial community result at the family level;

FIG. 13 shows the effect of YG2013 on the composition of intestinal bacteria; wherein A is a generic heat map analysis result; b is LEfSe analysis (LDA > 3); C-D is a Kruskal-Wallis H test bar graph at the genus level and a test bar graph at the species level (< p <0.05, < p <0.01, < p < 0.001);

FIG. 14 is a correlation analysis between gut microbiota and obesity related index, and pathways between groups; wherein A is the correlation between intestinal flora and biochemical indexes; b is the correlation of intestinal flora with eWAT and BAT gene expression; c is a pathway that predicts differences in abundance between groups based on KEGG pathway analysis.

Detailed Description

In the following examples and the results of the drawings, the strain YG2013 may be expressed as YGMCC2013 or simply as 2013, which represents the strain YG2013 selected according to the present invention, and is described herein.

Example 1 isolation and identification of strains

1. Separation of bifidobacterium lactis

Sample source

The strain used in this example was isolated from healthy adult feces.

Isolation and selection of strains

1g of fecal sample is taken and put into 9mL of PBS buffer solution (land bridge, CM 1022), vortex-mixed uniformly, ten-fold gradient dilution is carried out by using sterile physiological saline, and 10 is selected ^-6 、10 ^-7 、10 ^-8 Three dilution gradients, 100 μl of each of which was uniformly spread on MRS medium (land bridge, CM 188) +0.03% L-cysteine hydrochloride (land bridge, P-63) agar medium, i.e. MRS medium, were anaerobically cultured at 37 ℃ for 48h until distinct single colonies formed, and the colonies were picked and continued to be purified on MRS plates for more than 3 times until the colony morphology on the plates was consistent.

2. Identification of bifidobacterium lactis

Colony characterization

The strain selected was cultured in mMRS medium for 24-48h, the diameter was between 0.5-1.0mm, the colony was milky white and the surface was smooth, and it was named YG2013.

16S rRNA Gene sequencing

The screened bifidobacterium lactis strain is sent to Shanghai to carry out 16S rRNA gene sequencing, and the 16S rRNA gene sequence of the bifidobacterium lactis strain 2013 is shown as SEQ ID No. 1.

>AAGGAGGTGATCCAGCCGCACCTTCCGGTACGGCTACCTTGTTACGACTTAGTCCCAATCACGAGTCTCACCTTAGACGGCTCCCCCCACAAGGGTCGGGCCACCGGCTTCGGGTGCTACCCACTTTCATGACTTGACGGGCGGTGTGTACAAGGCCCGGGAACGCATTCACCGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGACCGGTTTTCAGCGATCCGCCCCACGTCACCGTGTCGCACCGCGTTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCCCACATGAGTTCCCGGCATCACCCGCTGGCAACATGCGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCGGCCCCGAAGGGAAACCGTGTCTCCACGGCGATCCGGCACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGATGCTTAACGCGTTGGCTCCGACACGGGACCCGTGGAAAGGGCCCCACATCCAGCATCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATTGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAGGCGATGGACTTTCACACCGGACGCGACGAACCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTATTCGAACAATCCACTCAACACGGCCGAAACCGTGCCTTGCCCTTGAACAAAAGCGGTTTACAACCCGAAGGCCTCCATCCCGCACGCGGCGTCGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTATCTCAGTCCCAATGTGGCCGGTCACCCTCTCAGGCCGGCTACCCGTCAACGCCTTGGTGGGCCATCACCCCGCCAACAAGCTGATAGGACGCGACCCCATCCCATGCCGCAAAAGCATTTCCCACCCCACCATGCGATGGAGCGGAGCATCCGGTATTACCACCCGTTTCCAGGAGCTATTCCGGTGCACAGGGCAGGTTGGTCACGCATTACTCACCCGTTCGCCACTCTCACCCCGACAGCAAGCTGCCAGGGATCCCGTTCGACTTGCATGTGTTAAGCACGCCGCCAGCGTTCATCCTGAGCCA

BLAST alignment (https:// BLAST. NCBI. Nlm. Nih. Gov/BLAST. Cgi) was performed on the NCBI database with the 16S rRNA sequence results, and screened strain YG2013 was initially determined to be bifidobacterium lactis (Bifidobacterium lactis) in combination with strain morphological characteristics.

The bifidobacterium lactis YG2013 screened in the embodiment is classified and named as bifidobacterium lactis Bifidobacterium lactis, and is preserved in China general microbiological culture Collection center (CGMCC, address: north Chen West Lu No. 1, 3 of the Korean area of Beijing, and post code 100101 of the institute of microorganisms of the national academy of sciences of China), with a preservation number of CGMCC No.27579 and a preservation date of 2023, 6, and 7 days.

Example 2

This example is based on the HPLC method to test the ability of the strain to reduce cholesterol in vitro.

Cholesterol powder medium preparation: cholesterol emulsion (16.7 mg/mL) was obtained by mixing 0.2g of cholesterol, 2.0mL of Tween-80, 0.2g of sucrose ester, and 10.0mL of glacial acetic acid, and subjecting the mixture to ultrasonic filtration and sterilization (10 min,20% power). Adding the prepared cholesterol emulsion into the mMRS culture medium after split charging to make the final concentration be 0.1mg/mL. The activated strain was subjected to OD= 1.0,4 ×10 ⁷ The amount of CFU was inoculated into cholesterol medium (200. Mu.l of 1.8mL of culture system). The non-inoculated tubes served as blank. Culturing at 37deg.C for 48 hr, centrifuging, and collecting supernatant and storing at-80 deg.C.

Liquid phase detection: sample pretreatment: filtering with 0.22 μm organic filter membrane, and transferring filtrate into upper machine vial for use for high performance liquid chromatography analysis. Detection conditions: chromatographic column: c18 reverse phase chromatography column (4.6mm.times.150mm, 5 μm); mobile phase: 100% methanol (V/V, analytical grade), line B; measurement wavelength: 205nm; flow rate: 2.0mL/min; column temperature: 40 ℃; the sample injection amount was 5. Mu.L.

Cholesterol degradation rate= (Ac-As)/ac×100%;

wherein Ac is the initial cholesterol concentration of the culture solution; as is the concentration of cholesterol remaining in the culture broth after cultivation.

In this example, five different bifidobacteria were selected for screening probiotics with anti-obesity potential using the method for degrading cholesterol in vitro described above, and the results are shown in fig. 1.

As shown in fig. 1, bifidobacterium lactis YG2013 and Bifidobacterium breve YGMCC2007 cholesterol-degrading activities were significantly higher than Lactobacillus rhamnosusGG (16.37%) in the experimental strains; wherein, YGGCC 2007 and YG2013 have higher cholesterol degradation activities, reaching 28.28% and 23.26% respectively. Therefore, the screened strain YG2013 has high-efficiency cholesterol degrading activity, is a bifidobacterium strain with anti-obesity potential, and needs to be further analyzed on the probiotic characteristics.

Example 3

The present example is based on HPLC method to detect and screen the activity of BSH enzyme of the strain and evaluate the ability of degrading bile salts.

pH5.0.1M HAc-NaAc buffer: 14.8mL 0.2M HAc+35.2mL 0.2M NaAc, water was added to a volume of 100mL.

The activated strain was inoculated to MRS or BHIS liquid medium at 1% (v/v) inoculum size, OD600 was measured after overnight culture, each strain was subjected to od=1 (fermentation broth was expanded in advance, about 20 ml), supernatant was centrifuged off, cells were washed 1 time with 0.1M HAcNaAc buffer (pH 5.0), and cells were resuspended at equal volume. After 400. Mu.L of the bacterial suspension and 400. Mu.L of 20mM bile salt mixture (TDCA+GDCA) were mixed, water was used for 1 hour at 37 ℃. The control sample was 400. Mu.L of 20mM bile-salt mixture and 400. Mu.L of HAcNaAc buffer. After the water bath is finished, 800 mu L of 15% TCA stop solution is added to stop the reaction, and after uniform mixing, the supernatant is centrifugally taken and stored at-80 degrees.

Liquid phase detection: sample pretreatment: filtering the supernatant with 0.22 μm nylon membrane, and transferring the filtrate into an upper machine vial for use for high performance liquid chromatography analysis. Detection conditions: chromatographic column: agilent TC-18 type C18 reverse phase chromatography column (5 μm,4.6 mm. Times.250 mm); a detector: a diode array detector; detection wavelength: 200nm; sample injection amount: 20. Mu.L; column temperature: 30 ℃; column flow rate: 1mL/min; mobile phase B:7.5mmol/L of tetrabutylammonium bisulfate in acetonitrile-water (60:40) (pH 2.5); mobile phase C:7.5mmol/L of tetrabutylammonium bisulfate in acetonitrile-water (30:70) (pH 2.5); gradient elution: mobile phase B was 10%, mobile phase C was 90%, and the proportion of mobile phase B increased from 10% to 70% in 30 min.

In this example, five different bifidobacteria were selected for screening probiotics with anti-obesity potential by using the method for in vitro degradation combined with bile salts, and the results are shown in fig. 2.

As shown in fig. 1, YG2013 showed the most excellent bile salt degradation ability in the experimental strain, and the degradation rate of both GDCA and TDCA reached 100%, which is significantly higher than the activity of LGG (6.64% and 2.48%). All tested strains showed a variability in TDCA degrading activity, YG2013 degrading ability significantly higher than the other three bifidobacteria, next to YGMCC2019, while YGMCC2007 and YGMCC2031 were relatively low, only 11.53% and 13.69%, respectively. Therefore, the screened strain YG2013 has high-efficiency bile salt degradation activity, is a bifidobacterium strain with anti-obesity potential, and needs to be further analyzed on the probiotics characteristics.

Example 4

Gastrointestinal tolerance is an important evaluation index for evaluating the probiotic properties of strains, and the test of gastrointestinal fluid resistance of the screened strains is performed in the embodiment.

ph3.0 manual simulated gastric fluid configuration: the pepsin solution was prepared by dissolving 0.15g of pepsin in 50mL of sterile physiological saline (0.9% w/v) to a final enzyme concentration of 3g/L, adjusting the pH to 3.0 with hydrochloric acid, and filtering the pepsin solution with a 0.22 μm sterile filter membrane.

pH 8.0 artificial simulated colonic fluid formulation: 0.05g trypsin was dissolved in 50mL sterile physiological saline (0.9% w/v) to a final enzyme concentration of 1g/L, and the pH was adjusted to 8.0 with NaOH solution and sterilized by filtration through a 0.22 μm sterile filter.

Taking activated bacterial liquid, centrifuging, discarding supernatant, washing 0.2mL bacterial liquid with PBS once, suspending in 0.2mL PBS for gradient dilution, and counting N by titration ₀ (treatment 0 h). Taking 1.2mL of bacterial liquid, centrifuging, discarding the supernatant, washing with PBS once, re-suspending in 1.2mL of artificial simulated gastric fluid with pH of 3.0, anaerobic culturing at 37 ℃ for 2h, taking 0.2mL of bacterial liquid, centrifuging, discarding the supernatant, washing with PBS once, re-suspending in 0.2mL of PBS, and counting viable bacteria N ₁ (gastric juice treatment 2 h); 1mL of bacterial liquid is washed once and then resuspended in 1mL of artificial simulated intestinal juice with pH of 8.0, after anaerobic continuous culture at 37 ℃ for 4 hours, supernatant is centrifugally discarded, PBS is washed once and then resuspended in 1mL of PBS, and colony count N is counted after culture at 37 ℃ for 48 hours ₂ (intestinal juice treatment 4 h).

In this example, the selected strains YG2013 and LGG were selected for gastrointestinal tolerability experiments, respectively, and the results are shown in FIG. 3.

As shown in fig. 3, the survival rates of the strain YG2013 screened by the method in gastric juice and intestinal juice are 113.77% and 93.44%, respectively, and no significant difference is found between the strain YG2013 and LGG, which indicates that the strain YG2013 has a similar capability of withstanding gastrointestinal environments.

Example 5

The ability of the strain to produce SCFAs is an important evaluation index for evaluating the probiotic properties of the strain, and in this example, the content of SCFAs produced by the strain is detected based on an HPLC method.

Precisely weighing 1.00g of lactic acid, 1.00g of acetic acid, 0.250g of propionic acid, 0.250g of butyric acid and 0.250g of succinic acid, adding a proper amount of ultrapure water for dissolution, and fixing the volume in a 10mL volumetric flask (100 g/L of lactic acid, 100g/L of acetic acid, 25g/L of propionic acid, 25g/L of butyric acid and 25g/L of succinic acid), shaking uniformly, respectively sucking 1mL of 5 standard substances, mixing, sequentially diluting into 5 concentration gradients (see table 1 below), and filtering by a 0.22 mu m filter membrane to obtain each acid standard control solution.

TABLE 1 5 concentration gradients for Mixed standards

Five organic acid standard substances are mixed and sampled, and are detected according to chromatographic conditions shown in the following table 2, chromatographic peaks of each acid are well separated, and peak outlet time of lactic acid, acetic acid, succinic acid, propionic acid and butyric acid is about 3.24 minutes, 3.53 minutes, 4.21 minutes, 6.27 minutes and 13.84 minutes respectively, so that the detection method can detect 5 organic acids simultaneously.

TABLE 2 chromatographic conditions

Note that: phosphate solution: 20mmol/L NaH ₂ PO ₄ (pH was adjusted to 2.7 with phosphoric acid).

Linear regression was performed with peak area on the ordinate (y) and mass concentration on the abscissa (x, g/L) to obtain linear regression equations for lactic acid, acetic acid, propionic acid, butyric acid and succinic acid, see table 3 below. The results show that 5 organic acids have good linear relation in the concentration range, the correlation coefficient r reaches 0.99, and the linearity is good.

TABLE 3 Linear relationship of short chain fatty acids

Composition of the components	Regression equation	Correlation coefficient r	Linear range (g/L)
				Lactic acid	y＝96500x+4340	0.9999	0.5-20
Acetic acid	y＝113000x+3860	1	0.5-20
				Propionic acid	y＝90700x+386	0.9999	0.125-5
Butyric acid	y＝80700x-780	0.9999	0.125-5
				Succinic acid	y＝10800x+229	0.9999	0.125-5

In the embodiment, the capacity of producing SCFAs by two strains is detected by an HPLC method, the frozen strain at the temperature of minus 80 ℃ is transferred into a corresponding liquid culture medium, the strain is placed in an anaerobic environment for static culture for 24-48 hours, 3 generations are continuously transferred under the same condition, fermentation liquor of the 3 rd generation for 20 hours is centrifuged for 1min at the temperature of 4 ℃ and the speed of 11000r/min on a high-speed centrifuge, the supernatant is taken to pass through a 0.22 mu m filter membrane, and the supernatant is transferred into a liquid phase detection bottle. The content of each organic acid in MRS culture medium was detected by the above method, and the yields of Lactobacillus bifidus YG2013 and Lactobacillus rhamnosus LGG for 20h fermentation were finally calculated, and the results are shown in FIG. 4.

As shown in FIG. 4, LGG only produces one short chain fatty acid, lactic acid, and the lactic acid content is as high as 139.97mM; unlike LGG, the strain YG2013 screened in the present invention can produce acetic acid (60.37 mM) at high yield, while producing only a small amount of lactic acid (19.06 mM).

Example 6

Cell adhesion is an important evaluation index for evaluating probiotic properties of strains, and the self-aggregation and hydrophobicity measurement method is adopted in the embodiment to evaluate the adhesion of two strains.

After incubation at 37℃for 18H, the optical density of the Bacteroides strain in PBS at 600nm (A0 or H0) was 0.60. Briefly, 4mL of cell suspension per strain was incubated at 20℃for 24 hours, and the absorbance of 1mL of the upper phase was measured to be OD 600nm (A1).

The self-polymerizing capacity is calculated as follows: a (%) = (1-A1/A0) ×100%.

1mL of chloroform was added to 3mL of the cell suspension and shaken for 3min. After incubation for 1H at 37℃it was measured at 600nm (H1).

The calculation method of the hydrophobicity value is as follows: h (%) = (1-H1/H0) ×100%.

As shown in fig. 5, it can be seen that the YG2013 strain screened by the invention has higher cell surface hydrophobicity, can reach 74.16%, is significantly higher than LGG (61.02%), and the self-agglutination ability of the YG2013 strain is 75.23%, is slightly lower than LGG (84.03%), which reveals that the YG2013 strain screened by the invention has good cell adhesion and intestinal tract colonization ability.

Example 7

The hemolytic property is an important evaluation index for evaluating the probiotic properties of the strain, and the hemolytic properties of the three strains are measured by using Columbia blood agar plates in this example.

The strain was inoculated on Columbia agar containing 5% sheep blood and incubated at 37℃for 48h. After incubation, hemolytic activity was assessed and classified according to lysis of erythrocytes in the medium surrounding the colonies. On Columbia blood agar plates, there was a clear area around the colony (beta-hemolysis), no area around the colony (gamma-hemolysis), and only strains with gamma-hemolysis were considered safe.

As shown in FIG. 6, the screened strain YG2013 of the present invention did not form any hydrolytic circles around the colonies, compared to S.aureus CICC 10473 (positive reference), indicating that it is a safe probiotic candidate strain.

EXAMPLE 8 anti-obesity Effect study of Bifidobacterium lactis YG2013

Design of animal experiment

Experimental animals: 40C 57BL/6J male mice, 7-8 weeks old, were purchased from Experimental animal technologies Inc. of Beijing.

Bacterial strain preparation: firstly, the strain YG2013 preservation tube preserved at-80 ℃ is taken out, and the strain obtained by the third generation is activated according to the inoculum size of 2 percent for expansion culture. And centrifuging for 10-15min at a rotating speed of 7500rpm/min, adding pre-cooled PBS, repeatedly blowing, washing for one time, and pouring out the supernatant to leave thalli at the bottom. Finally adding precooled 20% glycerol solution for resuspension to prepare 1.9X10 ¹⁰ CFU/mL of frozen stock solution is immediately placed in a refrigerator at-80 ℃ for preservation. Before the stomach is irrigated, the bacterial liquid is diluted by PBS and adjusted to the number of viable bacteria (1 multiplied by 10) required by each group of stomach irrigation ⁹ CFU/mL)。

Experimental grouping: ND group (mice fed maintenance diet, gavage PBS100 ul/day), HFD group (mice fed 48.5% high fat diet, gavage PBS100 ul/day), HFD+Orlistat group (mice fed 48.5% high fat diet, intragastric Orlistat1.2 mg/day), hfd+yg2013 group (48.5% high fat feed, intragastric YG2013 bacteria liquid 1×10) ⁹ CFU/day only).

Mice were acclimatized for one week, after one week each group (except ND group) was replaced with 48.5% high fat diet (beijing botaidada), all mice began to perfuse the stomach for 8 weeks, and body weights were recorded once a week during molding. Mouse feces were collected at week 8 and placed in sterile EP tubes, and stored at-80 ℃ after liquid nitrogen flash freezing for fecal microbiologic diversity detection. After 8 weeks, the mice were fasted for 24 hours, the eyeballs were collected for blood collection, and the treated serum was stored at-80 ℃. Collecting body type pictures of each group of mice, taking out brown fat, epididymal fat, abdominal wall fat, liver and cecum contents under aseptic condition, weighing and collecting pictures, fixing each tissue with 4% cell tissue fixing solution, and preserving at room temperature; and the rest part is put into a freezing tube for freezing and preserving at the temperature of minus 80 ℃.

Measurement method

1. Pathological detection

Fixed livers, epididymal fat, brown fat were paraffin embedded, 5mm sections, stained with hematoxylin eosin (H & E) and stained slides were observed under an optical microscope. Frozen liver samples were processed on a cryostat, fixed and subjected to oil red O staining. Liver the liver was assessed for hepatocyte steatosis and inflammatory infiltrate using the following criteria. Hepatocyte adipose degeneration judgment criteria: hepatic steatosis (0-3 min, based on parenchymal cells/total cell count <5%,5-33%,33-66%, > 66%), lobular inflammation (0-3 min, 2-4, >4 lesions per 200 x field < 2), hepatocyte balloon-like changes (0-2 min, no change, few balloon-like cells, balloon-like multiple/herniation). The maximum cell diameter was measured using software for 10 random fields per piece of white fat.

2. Fecal flora determination and analysis

Fresh mouse manure is taken and conveniently stored in a sterile EP tube at the temperature of minus 80 ℃ after quick freezing by liquid nitrogen. Extracting genome, analyzing dominant species in the sample by a 16S rRNA high-throughput sequencing technology, and obtaining microbial community composition in the sample and relative abundance differences between the microbial community composition and the microbial community composition.

3. Gene detection

The Total RNA extraction kit is used for extracting Total RNA of liver, epididymal fat and brown fat, the liver extraction process is operated according to instructions, and the extraction of adipose tissue is carried out at the temperature of 4 ℃ and at the speed of 12000rpm for 10min after Trizol is cracked, so that the purity of the extracted RNA can be improved.

Reverse transcription is carried out by the Takara reverse transcription kit to obtain cDNA sample, and the fast fluorescent quantitative PCR kit and the fluorescent quantitative PCR amplification instrument of the radix siegesbeckiae are provided480 qPCR assays were performed and primers were designed as shown in table 4 below. The detection indexes comprise heat generation correlation factors tgr, dio, il-27ra, elovl3, prdm16, ucp1, ppar alpha and pgc1 alpha; pro-inflammatory factors Il-1 beta, tnf-alpha, il-6, il-8; anti-inflammatory factors IL-4 and foxp3.

TABLE 4qPCR primers

4. Cecal content SCFAs detection

And (3) preparation of a mixed label: and (3) taking 9840 mu L of n-butanol (HPLC grade), loading into a 15mL centrifuge tube, sequentially adding a proper amount of 8 short-chain fatty acid standard substances, and uniformly vortex mixing to obtain 8 short-chain fatty acid mixed standard stock solution A. Preparation of an internal standard: 9990 mu L of n-butanol (HPLC grade) is taken and put into a 15mL centrifuge tube, 10 mu L of internal standard 2-ethylbutyric acid is added, and the internal standard stock solution B is obtained after vortex mixing. And diluting the mixed standard A, B solution with n-butanol to obtain 7 working solutions with different concentrations, and filling the working solutions into a sample injection vial for GC-MS detection and analysis. Sample treatment: a20 mg sample of the cecal content was weighed into a 2mL grind tube, and 800. Mu.L of 0.5% phosphoric acid water (10. Mu.g/mL containing the internal standard 2-ethylbutyric acid) was added. The sample was freeze-milled for 3min (50 HZ) and then sonicated for 10min,4℃and centrifuged at 13000g for 15min. The supernatant was removed from the tube to a 1.5mL centrifuge tube, and then extracted with 200. Mu.L of n-butanol solvent. Vortex for 10s, ultrasonic at low temperature for 10min, centrifuge at 4 ℃ and 13000g for 5min, take supernatant solution to sample injection vial. GC-MS detection: the analytical instrument for this experiment was an 8890B-7000D GC/MSD gas chromatograph-mass spectrometer of Agilent corporation (Agilent Technologies Inc. CA, UAS). Chromatographic conditions: HP FFAP capillary column (30 m×0.25mm×0.25 μm, agilent J & W Scientific, folsom, calif., USA), carrier gas is high purity helium (purity not less than 99.999%), flow rate 1.0mL/min, and inlet temperature 180 ℃. The sample feeding amount is 1 mu L, the split sample feeding is carried out, the split ratio is 10:1, and the solvent is prolonged for 2.5min. Programming temperature: the initial temperature of the column temperature box is 80 ℃, the temperature is programmed to 120 ℃ at 20 ℃/min, the temperature is programmed to 160 ℃ at 5 ℃/min, and the column temperature box is operated at 220 ℃ for 3min. Mass spectrometry conditions: the electrons bombard an ion source (EI), the ion source temperature is 230 ℃, the quaternary rod temperature is 150 ℃, the transmission line temperature is 230 ℃, and the electron energy is 70eV. The scanning mode is selected from an ion scanning mode (SIM). Data analysis: the target short chain fatty acid ion fragments were automatically identified and integrated using Masshunter quantitation software (Agilent company, usa, version number v10.0.707.0) default parameters and assisted in manual inspection. And calculating the detection concentration of each sample through a standard curve, and converting the actual content of the short chain fatty acid in the sample.

Test results

1. YG2013 strain for preventing obesity caused by high fat diet

In this example, the specific experimental method was designed for animal experiments. The YG2013 strain reduced the weight gain and fat accumulation in HFD mice as shown in FIG. 7.

As shown in fig. 7, a-D, HFD significantly increased the weight of mice, epididymis, abdominal wall adipose tissue, and liver, respectively, as compared to ND-fed mice. HFD-induced increases in body and liver weight were effectively inhibited by the intervention of YG2013 and Orlistat over 8 weeks and helped to significantly reduce epididymal and abdominal wall adipose tissue weight in HFD mice (see C, D in fig. 7). Wherein, YGGCM 2013 had a lower body weight than the ND group from the first week to the fifth week of the intervention (FIG. 7A).

2. YG2013 strain promotes white fat beige and reduces inflammation

In this example, the specific experimental method was designed for animal experiments. The results of the YG2013 strain reducing inflammation and inducing beige remodeling of epididymal adipose tissue (eWAT) are shown in fig. 8.

As shown in fig. 8, strain YG2013 promoted beige cell development in eWAT and significantly reduced the size of adipose cells in the abdominal wall tissue of HFD mice (see B in fig. 8), resulting in a similar adipocyte morphology (see a in fig. 8) compared to mice fed normal diet. Early studies demonstrated that activation of bile acid G protein-coupled receptor 5 (tgr 5) increased energy expenditure and reduced fat mass, thereby contributing to the weight-loss effect. tgr5 is expressed in white and brown fats, and activation of which by bile acids increases energy expenditure and reduces diet-induced obesity. The Il-27-Il-27 ra signal plays a key role in improving thermogenesis, preventing diet-induced obesity, and improving insulin resistance. Mechanical studies have shown that Il-27 targets adipocytes directly, activates p38 MAPK-PGC-1 alpha signaling and stimulates ucp1 production. Il-27 plays an important role in coordinating metabolic processes and is a very promising target for anti-obesity immunotherapy. As shown in FIG. 8D, the present invention found that the expression of tgr, dio and IL-27, IL-27Rα mRNA was significantly up-regulated in epididymal adipose tissue of YG2013 treated mice. To confirm the thermogenic mechanism associated with energy expenditure of YG2013 treatment, this example examined several markers associated with white adipocyte browning in abdominal wall tissue. Significant upregulation of the ucp1, pgc1 alpha, prdm16 and ppar alpha genes in epididymal tissues after treatment with YG2013 strain, indicated that YG2013 could achieve white adipocyte remodeling to beige by modulating Il-27 and tgr 5. In contrast to ND, HFD-fed mice epididymal adipose tissue had no apparent inflammatory response, whereas YG2013 strain had significantly increased the content of anti-inflammatory factor foxp3 after drying (see C in fig. 8).

3. YG2013 strain reduces inflammation and promotes BAT heat production

In this example, the specific experimental method was designed for animal experiments. The results of the YG2013 strain in reducing inflammation and activating the thermogenic activity of brown fat are shown in FIG. 9.

As shown in fig. 9 a-B, H & E staining indicated that supplementation with YG2013 reversed the HFD-induced enhancement of BAT whitening process and the increase of intracellular lipid vesicles. This example shows that Il-27 and Il-27 ra mRNA expression is significantly upregulated in brown adipose tissue of mice treated with strain YG 2013. To confirm the thermogenic mechanism associated with the energy consumption of YG2013 treatment, several markers associated with brown fat thermogenic activity were further examined. As shown in fig. 9D, expression of thermogenic genes ucp1 and ppar α, elovl3 in brown adipose tissue was significantly upregulated after YG2013 strain treatment, indicating that YG2013 strain can activate amplification of thermogenic metabolic effects of mouse brown adipocytes by modulating Il-27-Il-27 ra.

Brown fat contains more mitochondria, and a large amount of uncoupling protein 1 (ucp 1) is released on the inner membrane surface of the cell mitochondria, so that mitochondrial respiratory chain oxidation and ADP phosphorylation are uncoupled and converted into a thermogenesis process, energy consumption and thermogenesis are increased, and the purpose of fat consumption is achieved, and weight is reduced. HFD fed mice also produced an enhanced inflammatory response in brown adipose tissue, significantly increased the expression of inflammatory genes Il- β, tnf- α, il-6 and Il-8, whereas YG2013 strain dried out, significantly reduced the expression of inflammatory genes and increased the expression of anti-inflammatory genes Il-4 and foxp3 (see C in FIG. 9).

4. YG2013 strain improves liver steatosis and inflammation

In this example, the specific experimental method was designed for animal experiments. The YG2013 strain reduced liver inflammation and improved liver steatosis as shown in FIG. 10.

It can be seen that the YG2013 strain significantly reduced HFD-induced liver lipid accumulation and hepatocyte swelling and inflammatory infiltrates (see a and C in fig. 10), and the YG2013 strain significantly reduced HFD-induced liver lipid accumulation (see B in fig. 10). HFD fed mice also produced an enhanced inflammatory response in liver tissue, increasing the expression of inflammatory genes Il- β and tnf- α compared to ND groups. And after the YG2013 strain is dried, the expression of inflammatory genes IL-beta, IL-6 and tnf-alpha is obviously reduced (see D in figure 10).

5. YG2013 intervention increased SCFAs concentration in HFD fed mice

In this example, the specific experimental method was designed for animal experiments. The results of the changes in SCFAs concentration of the YG2013 strain in the HFD-fed mice were shown in fig. 11.

As shown in FIG. 11, the acetate and lactate levels in YG2013 medium were increased compared to fresh medium, but propionate and butyrate levels were similar between medium and fresh medium (see example 3). Acetate utilization by cross-feeding, such as butyrate producing bacteria, is an important metabolic interaction between intestinal bacteria. This example further measures the concentration of various SCFAs in the cecal content of mice by GC-MS analysis. As shown in fig. 11 a, acetic acid, propionic acid, and butyric acid are the major SCFAs in the cecum content. HFD feeding significantly reduced the acetate, propionate, butyrate and total SCFAs levels in mice compared to ND group (see B in fig. 11).

In addition, YG2013 intervention can significantly increase acetic acid (p < 0.01), propionic acid (p < 0.01), butyric acid (p < 0.001), isohexanoic acid (p < 0.01), isobutyric acid (p < 0.05) and valeric acid (p < 0.0001) content in the cecum of obese mice. Together, these results demonstrate that the YG2013 strain screened according to the present invention does not produce propionate and butyrate, but can provide acetate and lactate substrates to other gut commensal bacteria, resulting in elevated levels of propionate and butyrate in feces.

6. YG2013 reshaped intestinal microbiota of obese mice

In this study, we studied the effect of YG2013 on the diversity and composition of intestinal microbiota in obese mice. In order to analyze the diversity of microorganisms, the present example uses the 16S rRNA gene sequence.

FIG. 12 shows the results of the HFD-fed mice with altered intestinal microbiota diversity and composition by YG 2013; wherein A-B are Chao, shannon and alpha-diversity analysis indexes respectively; c is the PCoA graph analysis result of each sample; d is the Venn plot result showing observed ASV overlap; e is the portal level in mouse feces; f is the bacterial colony result at the family level.

The results shown in fig. 12 demonstrate that alpha-diversity is significantly reduced in both the high fat diet group and the orlistat-treated group, as measured by Shannon index and Simpson index. Treatment with YG2013, however, can reduce this decrease and maintain the diversity and richness of the intestinal microbiota. To further explore differences in microbial structure, we performed principal coordinate analysis (PCoA) based on unweighted UniFrac distances. All four groups showed different microbiota composition, but the YG2013 treated group clustered more closely to the ND group. This suggests that supplementation with YG2013 may restore gut microbiota composition to levels closer to normal diet mice. Analysis of the Operational Taxon (OTU) showed significantly higher numbers of unique OTUs in the YG 2013-intervention group compared to the ND group and orlistat treatment group. In addition, the YG2013 intervention group shared the most common OTUs (628) with the ND group. This suggests that YG2013 treatment retains the presence of specific microbiota in the normal diet of mice. Further categorical abundance analysis showed that the relative abundance of Firmicutes, verrucomicrobia and actionobacteria was higher for the HFD group and lower for the bacterioidota compared to the ND group. However, after supplementation with YG2013, verrucomicrobia and actionobacteria decreased in abundance, while Firmicutes and bacterioidota increased in abundance compared to HFD group. These changes indicate that YG2013 affects the relative abundance of different mycoplasmas in the gut microbiota of obese mice. In the first order of the family, colony abundance analysis showed that the Muribaculaceae, lachnospiraceae, lactobacillaceae and oscilliraceae abundance were higher for the YG2013 group and the Akkermansiaceae, clostridiaceae and unclassified_o_vamp was lower for the group compared to the HFD group. These differences indicate that YG2013 treatment affects the composition of the gut microbiota at grade one. In summary, our studies indicate that intervention of YG2013 improves the composition and diversity of intestinal microbiota in obese mice. These findings indicate that YG2013 can be a potential therapeutic option for controlling obesity-related disorders of intestinal microbiota.

In this study, the composition of the intestinal microbiota of the experimental mice was continuously analyzed, with a focus on the abundance of 30 specific bacteria. FIG. 13 shows the effect of YG2013 on the composition of intestinal bacteria; wherein A is a generic heat map analysis result; b is LEfSe analysis (LDA > 3); C-D is a Kruskal-Wallis H test bar graph at the genus level and a test bar graph at the species level (< 0.05,

**p<0.01，***p<0.001)。

as shown in fig. 13 a, HFD group mice had higher abundance of certain bacteria, including Allobaculum, ileibacterium, bifidobacterium, akkermansia, vampirovibrio, clostridium, dubosiella and Prevotella, than ND group. In contrast, the number of Lactobacillus, alistipes, ruminococcus, bacteroides, limosilactobacillus, unclassified _f_prevotellaceae and Lawsonibacter of the HFD group is smaller. To further investigate the effect of YG2013 treatment on the gut microbiota of high-fat diet fed mice, we performed a LEfSe analysis (see B in fig. 13). We found different bacteria associated with each group. Among them, dominant bacteria of ND, HFD and YG2013 groups are Muribaculum, akkermansia and allobaculom, respectively. YG2013 treatment resulted in decreased abundance of Akkermansia, ileibacterium, vampirovibrio and Clostridium, while unclassified_f_ Lachnospiraceae, duncaniella, lactobacillus and Eubacterium increased abundance compared to HFD group. Furthermore, we also analyzed changes in abundance of specific bacterial species using the Kruskal-Wallis report (see C-D in FIG. 13). Taking YG2013 results in an increase in beneficial bacteria such as Limosilobacillus reuteri, bifidobacterium animalis and Duncaniella fresteri. At the same time, it reduces the number of harmful bacteria, such as Faecalibaculumrodentium, ileibacteriumvalens, romboutsiailealis, unclassified _g_clostridium, compared to the HFD group. These findings indicate that the composition of the intestinal microbiota of mice fed a high fat diet was altered, while the changes induced by YG2013 administration were beneficial. YG2013 treatment promotes the growth of beneficial bacteria and reduces the number of harmful bacteria, suggesting that it may regulate the intestinal microbiota of obesity related diseases as a therapeutic intervention.

Considering that YG2013 can improve obesity and gut microbiota imbalance in HFD-induced obese mice, we performed a spin correlation analysis to investigate the correlation between dominant gut bacteria and metabolites (SCFAs) and obesity-related parameters (see a-B in fig. 14). Lactobacillus, limosilactobacillus, parabacteroides, unclassified _o_Eubbacteria and unclassified_f_Prevoltellaceae are strongly positively correlated with the levels of at least four SCFAs. These genera are also strongly inversely related to three obesity-related parameters. This suggests that these enterobacteria may play a role in the production of SCFAs and in the regulation of obesity-related parameters. Vampirovibrio, romboutsia, akkermansia, prevotella and Clostridium, on the other hand, are significantly inversely related to the four SCFAs indices. In addition, these genera also show strong positive correlation with four obesity-related parameters. This suggests that these enterobacteria may be associated with reduced SCFA production and increased obesity-related indicators. Based on these findings, we studied specific intestinal microorganisms related to the beneficial effects of YG2013 intervention on hewat browning and BAT energy metabolism related gene expression. We found that Lactobacillus, bacteroides, ruminococcus, alistipes and Parabactoides are positively correlated with eWAT browning, but negatively correlated with BAT energy metabolism and inflammatory factors (e.g., IL-beta, tnf-alpha, IL-6 and IL-8). Vampirovibrio, ileibacterium and Faecalibacterium, on the other hand, exhibit opposite patterns. Notably, allobaculum, eubacterium, dubosiella, bifidobacterium and Romboutsia are positively correlated with eWAT browning and BAT energy metabolism. These findings demonstrate complex interactions between mouse intestinal bacteria, SCFAs, obesity-related parameters and metabolic processes. Understanding these interactions may provide a valuable reference for studying the mechanisms of obesity and related diseases. Further research is needed to fully elucidate these mechanisms and explore potential therapeutic interventions for the gut microbiota and to treat related diseases such as obesity.

To gain a deeper understanding of the effect of YG2013 on intestinal bacterial function, we performed KEGG pathway analysis to predict the functional pathways involved. FIG. 14 is a correlation analysis between gut microbiota and obesity related index, and pathways between groups; wherein A is the correlation between intestinal flora and biochemical indexes; b is the correlation of intestinal flora with eWAT and BAT gene expression; c is a pathway that predicts differences in abundance between groups based on KEGG pathway analysis.

As shown in fig. 14C, all of the aforementioned signal paths in the HFD group are significantly suppressed as compared with the ND group. Similar effects were also observed in the group treated with orlistat, a well-known drug for weight loss. However, the group treated with YG2013 completely reversed these signaling pathways, showing similar effects as ND group. More specifically, YG2013 has a significant impact on various metabolic pathways, enhancing their activity. These pathways include energy metabolic pathways such as glycolysis I, II and III, pentose phosphate pathway-non-oxidative branching and glycogen degradation I. YG2013 also affects carbohydrate metabolic pathways such as starch degradation V, fermentation of pyruvate to acetic acid and lactic acid II, fermentation of pyruvate to isobutanol and homolactic. These findings indicate that YG2013 is likely to affect energy metabolism and production of metabolites such as SCFAs by modulating intestinal microflora. This modulation of intestinal bacterial function may help to improve the obese status of mice fed a high-fat diet. In summary, this analysis provides valuable insight into the underlying mechanisms by which YG2013 has a beneficial effect on intestinal bacteria and metabolism. It provides a promising approach for further research and potential therapeutic intervention of obesity-related diseases.

In conclusion, the bifidobacterium lactis YG2013 can promote white fat beige and reduce the inflammatory effect thereof, and activate brown fat metabolism and heat production and reduce the inflammatory effect thereof through targeted regulation and control of a tgr/Il-27 pathway; can also relieve hepatic cell steatosis and inflammatory reaction, generate various short chain fatty acids, and regulate energy metabolism; the strain can target and improve the characteristic intestinal flora of obesity, promote the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, recover the health of the intestinal flora and has great application value.

Example 9

The bifidobacterium lactis YG2013 can be used for preparing various probiotic preparations such as powder, medicinal granules, solid beverages, pressed candies and the like.

The bifidobacterium lactis YG2013 can be added with auxiliary materials or other active ingredients which are conventional in the art, such as beneficial ingredients including galactooligosaccharides, inulin, dietary fibers and the like, and can also be added with flavoring functional sugar alcohol, fruit powder and the like, or with functional ingredients including maltodextrin and the like.

In the probiotic preparation containing bifidobacterium lactis YG2013 as an active ingredient, the number of viable probiotic bacteria is controlled to be more than 5 multiplied by 10 ⁹ CFU/day/person.

The preparation method of the probiotic preparation containing bifidobacterium lactis YG2013 as an active ingredient can be processed by adopting a traditional process in the field.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. Use of a modulator with tgr5/Il-27 pathway targeting modulating effect for preparing a product with weight loss and weight loss effects; wherein,

the regulator is based on targeting regulation of the tgr/Il-27 pathway, promotes beige coloration of white fat and reduces its inflammatory effects, and activates the metabolic heat generation of brown fat and reduces its inflammatory effects.

2. Use of a formulation having a targeted improvement of the intestinal flora characteristic of obesity for the preparation of a product having a weight loss and weight reduction effect; wherein,

the improving intestinal flora comprises increasing the abundance of beneficial bacteria and/or decreasing the abundance of harmful bacteria;

Preferably, the improving the intestinal flora comprises increasing the abundance of unclassified f_ Lachnospiraceae, duncaniella, lactobacillus, limosilactobacil and Eubacterium flora, increasing SCFAs production;

preferably, the improving the intestinal flora comprises increasing the abundance of beneficial bacteria unclassified_f_ Lachnospiraceae, duncaniella, lactobacillus, eubacterium flora and decreasing the abundance of harmful bacteria Ileibacterium, vampirovibrio, clostridium flora.

3. The bifidobacterium lactis YG2013 is characterized by being classified and named as bifidobacterium lactis Bifidobacterium lactis, and is preserved in China general microbiological culture Collection center (CGMCC) No.27579, and the preservation date is 2023, 6, 7 and so on.

4. Use of bifidobacterium lactis YG2013 and its deactivator and metabolite as claimed in claim 3 for the preparation of probiotic formulations.

5. A probiotic preparation, characterized in that the active ingredient of the probiotic preparation comprises the cells of bifidobacterium lactis YG2013 and the deactivators and/or metabolites thereof according to claim 1;

preferably, in the probiotic preparation, the effective cell number of the bifidobacterium lactis YG2013 is 5.0X10 ⁹ -1.0×10 ¹¹ CFU/day/person;

preferably, the probiotic preparation comprises at least one of a powder, a granule, a pill, a capsule, a tablet, a paste, a liquid preparation, a gel, a spray or a solid beverage.

6. A method for preparing the probiotic preparation according to claim 5, which is characterized by comprising the steps of culturing the bifidobacterium lactis YG2013 according to claim 3 and processing selected dosage forms by adding conventional auxiliary materials according to a conventional process.

7. Use of bifidobacterium lactis YG2013 as claimed in claim 3 and its deactivator, metabolite or probiotic formulation as claimed in claim 5 for the preparation of a functional product having the efficacy of at least one of the following (1) - (10):

(1) Preventing and treating obesity;

(2) Has the effect of reducing weight;

(3) White fat beige remodeling;

(4) Activating brown fat metabolism to produce heat;

(5) Targeting modulates the tgr5/Il-27 pathway;

(6) Reducing white adipose tissue inflammation;

(7) Alleviating hepatocyte steatosis and inflammatory responses;

(8) Producing a plurality of short chain fatty acids, regulating energy metabolism;

(9) Increasing the abundance of unclassified f_ Lachnospiraceae, duncaniella, lactobacillus, limosilactobacil and Eubacterium flora by cross feeding, and improving the yield of SCFAs;

(10) Targeted improvement of the characteristic intestinal flora of obesity, improvement of the abundance of beneficial bacteria unclassified_f_ Lachnospiraceae, duncaniella, lactobacillus, eubacterium flora, reduction of the abundance of harmful bacteria Ileibacterium, vampirovibrio, clostridium flora and recovery of the health of the intestinal flora.

8. Use according to claim 7, characterized in that the functional product comprises a food, a pharmaceutical and/or a health product.

9. A probiotic preparation with white fat beige remodeling effect, characterized in that the active ingredients of the probiotic preparation comprise bifidobacterium lactis YG2013 and an deactivation substance and/or a metabolite thereof according to claim 3;

preferably, the effective cell number of the bifidobacterium lactis YG2013 is 1.0X10 ¹⁰ -5.0×10 ¹⁰ CFU/day/person.

10. A probiotic preparation with weight-losing and weight-reducing effects, characterized in that the active ingredients of the probiotic preparation comprise bifidobacterium lactis YG2013 and deactivation substances and/or metabolites thereof according to claim 3;

preferably, the effective cell number of the bifidobacterium lactis YG2013 is 2.0X10 ¹⁰ -8.0×10 ¹⁰ CFU/day/person.