NL2033398B1

NL2033398B1 - Formula of probiotics and metabolites thereof for relieving metabolic syndrome, and use thereof

Info

Publication number: NL2033398B1
Application number: NL2033398A
Authority: NL
Inventors: Xie Liwei
Original assignee: Inst Of Microbiology Guangdong Academy Of Sciences Guangdong Detection Center Of Microbiology
Priority date: 2022-04-15
Filing date: 2022-10-26
Publication date: 2023-11-06
Also published as: CN114480228A; WO2023040426A1; ZA202210422B

Abstract

The present invention discloses a formula of probiotics and metabolites thereof for relieving metabolic syndrome, and use thereof. A probiotic formula for relieving the metabolic syndrome is characterized in that the formula of the probiotics is one or more of (Lactobacillus reuteri) PLBK®1‚ (Lactobacillus reuteri) PLBK®2‚ (Lactobacillus gasseri) PLBK® 3, (Lactobacillus acidophilus) PLBK®4 and (Biﬁdobacterium lactis) PLBK®5‚ a fermentation culture or fermentation cultures of one or more of (Lactobacillus reuteri) PLBK®1‚ (Lactobacillus reuteri) PLBK®2‚ (Lactobacillus gasseri) PLBK®3‚ (Lactobacillus acidophilus) PLBK®4 and (Biﬁdobacterium lactis) PLBK® 5, or a metabolite or metabolites of one or more of (Lactobacillus reuteri) PLBK®1‚ (Lactobacillus reuteri) PLBK®2‚ (Lactobacillus gasseri) PLBK®3‚ (Lactobacillus acidophilus) PLBK®4 and (Bifidobacterium lactis) PLBK®5. The present invention particularly relates to five probiotics which are independently separated, identified and deposited in patents, core components of fermentation metabolites (postbiotics) of the probiotics and use of the probiotics in relieving the metabolic syndrome, in particular to a use mechanism of promoting decomposition of white adipose. By using the probiotics, adipose accumulation can be reduced, adipose decomposition can be promoted, insulin sensitivity can be improved, and the metabolic syndrome can be relieved, and the probiotics are expected to be used in prevention and treatment of the metabolic syndrome.

Description

FORMULA OF PROBIOTICS AND METABOLITES THEREOF

FOR RELIEVING METABOLIC SYNDROME, AND USE THEREOF

TECHNICAL FIELD

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a formula of probiotics and metabolites {postbiotics) thereof for relieving metabolic syndrome, and use thereof.

BACKGROUND

[0002] The occurrence and the development of metabolic syndrome, such as obesity, diabetes and non-alcoholic fatty liver, pose a serious threat to human health, and brings heavy economic burden and medical pressure to the countries and families. Diet and exercise intervention is an effective means for the prevention and early treatment of the metabolic syndrome at present, but this means is difficult for most people to adhere to and has problems of long cycle and rapid rebound. Taking drugs to reduce appetite, undergoing clinical surgeries to lose body weight and other means have strong side effects. Studies have found that the occurrence and development of the metabolic syndrome are often accompanied by intestinal flora disorder and imbalance of systemic glucolipid metabolism homeostasis. The intervention by microecological agents represented by probiotics and synbiotics can improve the microenvironment of intestinal microbiota, promote defecation and assist in improving the systemic glucolipid and energy metabolism homeostasis, which regulates the systemic glucolipid homeostasis in a host mainly by enabling the host to absorb metabolites secreted by the probiotics. The probiotics and mixtures of metabolites of the probiotics (also called postbiotics) were obtained by using pre- screened patented probiotic strains and an optimized fermentation process. Through 16S sequencing, whole genome sequencing and evolutionary tree construction, the strains we isolated are different from those found at present. Metabolites of the five strains were identified by metabonomics, and it was found and identified that the components of metabolites of the five strains can inhibit adipose synthesis and promote adipose decomposition. The present invention mainly provides data to support that taking the probiotics and metabolites thereof can significantly promote beiging of white adipose, and significantly improve and alleviate the course of the metabolic syndrome.

SUMMARY

[0003] An object of the present invention is to provide a formula of probiotics and metabolites (postbiotics) thereof that can inhibit adipose accumulation, promote adipose decomposition, 1 improve insulin sensitivity and relieve fatty liver, and are expected to be used in relieving metabolic syndrome, and use of the formula.

[0004] A first object of the present invention is to provide a formula for probiotics that can inhibit adipose accumulation, promote adipose decomposition, improve insulin sensitivity and relieve fatty liver, and is expected to be used in relieving metabolic syndrome. The formula of the probiotics is one or more of (Lactobacillus reuteri)y PLBK"™1, (Lactobacillus reuteri)

PLBK*2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK*4 and (Bifidobacterium lactis) PLBK®5, a fermentation culture or fermentation cultures of one or more of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, Lactobacillus gasseri)

PLBK™3, (Lactobacillus acidophilus) PLBK*4 and (Bifidobacterium lactis) PLBK®5, or a metabolite or metabolites of one or more of Lactobacillus reuteri) PLBK*1, Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseriy PLBK*3, (Lactobacillus acidophilus) PLBK*4 and (Bifidobacterium lactis) PLBK®S.

[0005] The (Lactobacillus reuteri) PLBK*1 was deposited in Guangdong Microbial Culture

Collection Center (GDMCC) on October 24™ 2019, with the address of the 5% floor, Building 59,

No. 100, Xianlie Middle Road, Guangzhou, the zip code of 510070, and the accession number is

GDMCC No. 60828.

[0006] The (Lactobacillus reuteri) PLBK®2 was deposited in Guangdong Microbial Culture

Collection Center (GDMCC) on October 24" 2019, with the address of the 5" floor, Building 59,

GDMCC No. 60829.

[0007] The (Lactobacillus gasseri) PLBK®3 was deposited in Guangdong Microbial Culture

Collection Center (GDMCC) on October 24% 2019, with the address of the 5 floor, Building 59,

GDMCC No. 60830.

[0008] The (Lactobacillus acidophilus) PLBK*4 was deposited in Guangdong Microbial

Culture Collection Center (GDMCC) on October 24™ 2019, with the address of the 5% floor,

Building 59, No. 100, Xianlie Middle Road, Guangzhou, the zip code of 510070, and the accession number is GDMCC No. 60831.

[0009] The (Bifidobacterium lactis) PLBK®5 was deposited in Guangdong Microbial Culture

GDMCC No. 60832.

[0010] The second object of the present invention is to provide use of the formula of the probiotics in preparation of a product for relieving metabolic syndrome. 2

[0011] Preferably, relieving the metabolic syndrome is reduction of adipose accumulation, promotion of adipose decomposition, improvement of insulin sensitivity and/or remission of fatty liver.

[0012] Preferably, the product is a food product, health product or medicine that relieves the metabolic syndrome.

[0013] Further preferably, the formula of the probiotics is a mixture of solutions and/or metabolites of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK*3, (Lactobacillus acidophilus) PLBK®4 and (Bifdobacterium lactis) PLBK®5; and a probiotic content in the mixture is more than 10% cfu/ml, and further preferably, is 10% cfu/ml

[0014] Preferably, a quantity ratio of the (Lactobacillus reuteri) PLBK™1, (Lactobacillus reuteri)

PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK*4 and

Bifdobacterium lactis) PLBK®5 in the mixture is 0.75: 0.75: 1: 1.5 :1.

[0015] The present invention belongs to the technical field of microorganisms, particularly relates to five probiotics which are independently separated, identified and deposited in patents, core components of fermentation metabolites (postbiotics) of the probiotics and use of the probiotics and the metabolites thereof in relieving metabolic syndrome, and further discloses a use mechanism of the probiotics and the fermentation metabolites (postbiotics) thereof for promoting beiging of white adipose. By using the probiotics and the metabolites thereof, adipose accumulation can be inhibited, adipose decomposition can be promoted, insulin sensitivity can be improved, and fatty liver can be relieved, and the probiotics and the metabolites thereof are expected to be used in prevention and treatment of the metabolic syndrome.

[0016] The (Lactobacillus reuteri) PLBK*1 was deposited in Guangdong Microbial Culture

Collection Center (GDMCC) on October 24" 2019, with the address of the 5® floor, Building 59,

GDMCC No. 60828.

[0017] The (Lactobacillus reuteri) PLBK"2 was deposited in Guangdong Microbial Culture

No. 100, Xianliezho Middle Road, Guangzhou, the zip code of 510070, and the accession number is GDMCC No. 60829.

[0018] The (Lactobacillus gasseri) PLBK®3 was deposited in Guangdong Microbial Culture

No. 100, Xianlie MiddleRoad, Guangzhou, the zip code of 510070, and the accession number is

GDMCC No. 60830. 3

[0019] The (Lactobacillus acidophilus) PLBK*4 was deposited in Guangdong Microbial

Culture Collection Center (GDMCC) on October 24™ 2019, with the address of the 5" floor,

Building 59, No. 100, Xianliezho Middle Road, Guangzhou, the zip code of 510070, and the accession number is GDMCC No. 60831.

[0020] The (Bifidobacterium lactis) PLBK®5 was deposited in Guangdong Microbial Culture

GDMCC No. 60832.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1 is a (COG) function categorie of a whole genome sequencing strain of (Lactobacillus reuteriy PLBK*1;

[0022] Fig. 2 is a (COG) function categorie of a whole genome sequencing strain of (Lactobacillus reuteri) PLBK®2;

[0023] Fig. 3 is a (COG) function categorie of a whole genome sequencing strain of (Lactobacillus gasseri) PLBK"3;

[0024] Fig. 4 is a (COG) function categorie of a whole genome sequencing strain of (Lactobacillus acidophilus) PLBK*4;

[0025] Fig. 5 is a (COG) function categorie of a whole genome sequencing strain of (Bifidobacterium lactis) PLBK®5;

[0026] Fig. 6 (A) is an evolutionary map of (Lactobacillus reuteriy PLBK¥1 and (Lactobacillus reuteri) PLBK®2, (B) is an evolutionary map of (Lactobacillus gasseriy PLBK®3, (C) is an evolutionary map of (Lactobacillus acidophilus) PLBK®4, and (D) is an evolutionary map of (Bifidobacterium lactis) PLBK*5;

[0027] Fig. 7 is an identification diagram of fermentation metabolites of five probiotic strains;

[0028] Fig. 8 is an identification diagram of fermentation metabolites of five purified probiotic strains;

[0029] Fig. 9 is a graph of mouse rectal temperature (A), mouse glucose tolerance (GTT, B), mouse fecal suspension time (C) and mouse body weight gain (D);

[0030] Fig. 10 is a graph showing an improvement in promotion of metabolic phenotype through intervention by probiotics and metabolites thereof’

[0031] Fig. 11 is a graph showing an improvement in prevention and treatment of metabolic syndrome through intervention by probiotics and metabolites thereof; and

[0032] Fig. 12 is a graph showing occurrence and development of prevention and treatment of metabolic syndrome through intervention by probiotics and metabolites thereof. 4

DETAILED DESCRIPTION

[0033] The following examples are to further illustrate, rather than limit the present invention.

The protection scope of the present invention is not limited to the following specific embodiments.

[0034] Embodiment 1

[0035] I. Experimental method

[0036] 1. Probiotic strain category and patent accession number

[0037] In the present invention, five probiotic strains were acquired through screening and separation; and genomic information of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri)

PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK*4 and (Bifdobacterium lactis) PLBK*5 was acquired by 16S sequencing (16S full-length amplification primer, 16S Forw: AGAGTTTGATCCTGGCTCAG, 16S Rev:

GGTTACCTTGTTACGACTT), a (COG) function categorie of a whole genome sequencing (extracting nucleic acid of fermentation sludge of strains, and construction of a database for second-generation sequencing, wherein a sequencing platform is Ilumina Hiseq X10) and phylogenetic tree construction. The specific information is as follows:

[0038] The (Lactobacillus reuteri) PLBK®1 was deposited in Guangdong Microbial Culture

GDMCC No. 60828. The nucleotide sequence of 16s rDNA of (Lactobacillus reuteri) PLBK™1 1s shown in SEQ ID NO. 1; the (COG) function categorie of the whole genome sequencing strain of (Lactobacillus reuteri) PLBK®1 is shown in Fig. 1, and the evolutionary map of (Lactobacillus reuteri) PLBK®1 is shown in Fig. 6.

[0039] The (Lactobacillus reuteri) PLBK*2 was deposited in Guangdong Microbial Culture

GDMCC No. 60829. The nucleotide sequence of 16s rDNA of (Lactobacillus reuteri) PLBK®2 is shown in SEQ ID NO. 2; the (COG) function categorie of the whole genome sequencing strain of (Lactobacillus reuteri) PLBK*2 is shown in Fig. 2, and the evolutionary map of (Lactobacillus reuterij PLBK"2 is shown in Fig. 6.

[0040] The (Lactobacillus gasseri) PLBK*3 was deposited in Guangdong Microbial Culture

GDMCC No. 60830. The nucleotide sequence of 16s rDNA of (Lactobacillus gasseri) PLBK*3 5 is shown in SEQ ID NO. 3; the (COG) function categorie of the whole genome sequencing strain of (Lactobacillus gasseri) PLBK*3 is shown in Fig. 3, and the evolutionary map of (Lactobacillus gasseri) PLBK*3 is shown in Fig. 6.

[0041] The (Lactobacillus acidophilus) PLBK*4 was deposited in Guangdong Microbial Culture

Collection Center (GDMCC) on October 24 2019, with the address of the 5! floor, Building 59,

GDMCC No. 60831. The nucleotide sequence of 16s rDNA of (Lactobacillus acidophilus)

PLBK*4 is shown in SEQ ID NO. 3; the (COG) function categorie of the whole genome sequencing strain of (Lactobacillus acidophilus) PLBK*4 is shown in Fig. 4, and the evolutionary map of (Lactobacillus acidophilus) PLBK®4 is shown in Fig. 6

[0042] The (Bifidobacterium lactis) PLBK®5 was deposited in Guangdong Microbial Culture

GDMCC No. 60832. The nucleotide sequence of 16s rDNA of (Bifidobacterium lactis) PLBK®5 is shown in SEQ ID NO. 3; the (COG) function categorie of the whole genome sequencing strain of (Bifidobacterium lactis) PLBK®5 is shown in Fig. 5, and the evolutionary map of (Bifidobacterium lactis) PLBK®5 is shown in Fig. 6

[0043] 2. Fermentation medium:

[0044] An optimized MRS medium containing peptone (10 g/L), beef extract powder (8 g/L), yeast extract powder (4 g/L), glucose (20 g/L), dipotassium hydrogen phosphate (2 g/L), diammonium hydrogen citrate (2 g/L), sodium acetate (5 g/L), magnesium sulfate (0.2 g/L), manganese sulfate (0.04 g/L) and solvent water was used for Lactobacillus, including but not limited to Lactobacillus reuteri, Lactobacillus gasseri and Lactobacillus acidophilus; and a specific preparation method was to mix the above components evenly and sterilize them for later use.

[0045] An optimized Bifidobacterium medium containing peptone (15 g/L), glucose (20 g/L), yeast extract powder (2 g/L), soluble starch (0.5 g/L), sodium chloride (5 g/L), L-Cysteine (0.5 g/L), tomato extract powder (5 g/L), liver extract powder (2 g/L) and solvent water was used for

Bifidobacterium; and a specific preparation method was to mix the above components evenly and sterilize them for later use.

[0046] 3. Fermentation and preparation process of probiotics: Lactobacillus or

Bifidobacterium was inoculated into 5 ml of sterilized MRS liquid culture medium or

Bifidobacterium liquid culture medium respectively, and anaerobic culture for bacteria enrichment was performed for 24 hours; and then 5 ml of the bacteria solution was transferred to

IL of the culture medium for anaerobic fermentation and amplification for 48 hours. After the 6 fermentation was completed, centrifugation was performed to collect bacterial sludge, and a supernatant was filtered with a 0.22 um filter membrane to obtain a filtered supernatant for later use. After counting the probiotics cfu by coating, 10™ cfu/ml single-strain bacterial solution was obtained by resuspending the bacterial sludge with the filtered supernatant. Then, five single- strain bacterial solutions were mixed according to the volume ratio of PLBK*1: PLBK*2:

PLBK®3: PLBK"4: PLBK®5 being 0.75: 0.75: 1: 1.5: 1 to obtain a mixed probiotic solution which was used in the following experiments.

[0047] 4. Preparation process of probiotics metabolites: the fermentation broths of strains were centrifuged, and the supernatants were filtered (0.22 um), so as to obtain a fermentation metabolite of each strain. Then, the fermentation metabolites of the five strains were mixed according to the equal proportion of the volume to obtain the mixed probiotic fermentation metabolites, which were stored at 4°C.

[0048] 5. Purification process of probiotic metabolites:

[0049] (A), pouring 500 ml of probiotic fermentation supernatant obtained by filtering the probiotic metabolites with the 0.22 um filter membrane into a separatory funnel, and then adding 500 ml of ethyl acetate into the separatory funnel,

[0050] (B), horizontally shaking the separatory funnel for 2 min to fully mix the two liquids;

[0051] (C), transferring the separatory funnel to a fuming hood, and standing at room temperature for 5 min to fully stratify the liquid;

[0052] (D), repeating steps B and D for three to four times, wherein at this time, it could be observed that the upper layer liquid was yellowish brown and transparent, and the lower liquid was brown (The upper liquid was an oil phase that was a substance soluble in ethyl acetate in the supernatant, and the lower liquid was a water phase that was a substance insoluble in ethyl acetate in the supernatant);

[0053] (E), placing a 500 ml triangular bottle at the lower opening of the separatory funnel to hold the lower layer liquid;

[0054] (F), opening an upper piston of the separatory funnel to keep the air pressure inside and outside the funnel consistent, then, unscrewing a lower piston of the separatory funnel to discharge the lower layer liquid into the triangular bottle, wherein at this time, less upper layer liquid could also be discharged into the triangle bottle together to eliminate the interference at the stratification of the liquid,

[0055] (G), closing the lower piston after the discharging was completed, wherein the lower layer liquid was poured out from the upper opening into a rotary bottle;

[0056] (H), connecting the rotary bottle to a rotary evaporator, soaking 1/4 of the body of the rotary bottle in a water bath kettle, adjusting the value of the water bath kettle of the rotary evaporator to 45°C, and turning on a main switch of the rotary evaporator;

[0057] (I), turning off the main switch when the liquid in the rotary bottle did not decrease any longer through evaporation, and taking down the rotary bottle; adding 2 to 3 ml of ethyl acetate into the rotary bottle, mixing well, and transferring the liquid in the rotary bottle to the sample bottle by a pipette;

[0058] (J), connecting a sample bottle to the rotary evaporator, soaking 1/4 of the body of the sample bottle in the water bath kettle, adjusting the value of the water bath kettle of the rotary evaporator to 45°C, and turning on the main switch of the rotary evaporator;

[0059] (K), turning off the main switch when the liquid in the sample bottle did not decrease any longer through evaporation, taking down the sample bottle, wherein at this time, the liquid in the bottle is the substance acquired by preliminary extraction on the supernatant of the 500 ml of the bacteria solution, such that a concentrated metabolite (water phase) of each probiotic solution was obtained.

[0060] 6. Experimental model of intervention by probiotics and metabolites thereof after healthy model: 14-week-old C57BL/6J mice were fed a regular diet (rodent chow, Beijing Keao

Xieli Feed Co., Ltd., 2012, rat & mice maintenance feed). After 12 weeks of feeding with the regular diet, the mice were given mixed probiotic solution intervention for 12 weeks. One experimental group (the group of the mixed probiotics) and one control group of pure water were set up. The mixed probiotic solution and the pure water were added into drinking bottles for the mice, respectively, and the mice drank freely. The body weight gains of the mice were monitored every week. After the intervention was completed, the glucose tolerance (GTT) and the insulin tolerance (ITT) of the mice were detected. The beiging of white adipose was evaluated by immunohistochemistry, and the accumulation of the liver adipose was detected by oil red staining.

[0061] 7. Function comparison between single strain and mixed strains on metabolic syndrome: 14-week-old C57BL/6J mice were fed a 60% high-fat diet (HFD, D12492, Research

Diets, Inc., Brunswick, NJ) for 24 weeks, during which the mice were given the single probiotic solution intervention and the mixed probiotic solution intervention for 24 weeks, respectively.

Six experimental groups (Lactobacillus reuteri PLBK*1 group, Lactobacillus reuteri PLBK®2 group, Lactobacillus gasseri PLBK*3 group, Lactobacillus acidophilus PLBK®4 group,

Bifidobacterium lactis PLBK®5 group and a mixed probiotics group) and one control group (MRS, MRS culture medium with the same dose) were set up. The single probiotic solution, the mixed probiotic solution and the MRS culture medium were added into the drinking bottles for 8 the mice, respectively, and the mice drank freely. The body weight gains of the mice were monitored every week. After the intervention was completed, the rectal temperature and the glucose tolerance of the mice as well as fecal suspension time of the mice were detected.

[0062] 8. Experimental model of treatment of metabolic syndrome by probiotics and metabolites thereof: 14-week-old C57BL/6J mice were modeled with the 60% high-fat diet (HFD, D12492, Research Diets, Inc., Brunswick, NJ) (12 weeks). The mixed probiotic solution intervention was performed for 12 weeks starting from the 13" week. One experimental group (the group of the mixed probiotics) and one control group of pure water were set up. The mixed probiotic solution and the pure water were added into drinking bottles for the mice, respectively, and the mice drank freely. The body weight gains of the mice were monitored every week. After the intervention was completed, the glucose tolerance (GTT) and the insulin tolerance (ITT) of the mice were detected. The beiging of white adipose was evaluated by immunohistochemistry, and the accumulation of the liver adipose was detected by oil red staining.

[0063] 9. Experimental model of prevention of metabolic syndrome by probiotics and metabolites thereof: 14-week-old C57BL/6]J mice were fed the 60% high-fat diet (HFD,

D12492, Research Diets, Inc, Brunswick, NJ) and simultaneously, were given the mixed probiotic solution intervention for 24 weeks. One experimental group (the group of the mixed probiotics) and one control group (the MRS culture medium added with 30% of glucose) were set up. The mixed probiotic solution and the MRS culture medium added with the 30% of glucose were added into drinking bottles for the mice, respectively, and the mice drank freely.

The body weight gains of the mice were monitored every week. After the intervention was completed, the glucose tolerance (GTT) and the insulin tolerance (ITT) of the mice were detected. The beiging of white adipose was evaluated by immunohistochemistry, and the accumulation of the liver adipose was detected by oil red staining.

[0064] 10. Glucose Tolerance Test (GTT)

[0065] (A), weighing and calculating the injection amount of glucose, wherein after the mice were weighed, the glucose injection volume for each mouse was calculated according to 2 mg/g body weight; and in order to identify the mice easily, the tail roots or tips of the mice were marked with a marker pen after weighing;

[0066] (B), determining basic blood glucose, wherein after the tail of each mouse was wiped with sterilized alcohol cotton, a small section of the tail was cut to determine a baseline blood glucose level (0 min), and the first drop of blood was discarded;

[0067] (C), injecting glucose, wherein timing began immediately after intraperitoneal injection of the glucose solution, and the blood glucose levels were determined at 15 min, 30 min, 60 min, 90 min and 120 min after injection; and the measurement method includes reopening the tail 9 wound, discarding the first drop of blood and measuring the blood glucose level of the second drop of blood; and

[0068] (D), determining the blood glucose levels of other mice at each time point after the glucose injection successively, and making records.

[0069] 11. Insulin Tolerance Test (ITT):

[0070] (A), weighing and calculating the injection amount of glucose, wherein after the mice were weighed, the insulin injection volume of each mouse was calculated according to 2 mg/g body weight; and in order to identify the mice easily, the tail roots or tips of the mice were marked with a marker pen after weighing;

[0071] (B), determining basic blood glucose, wherein after the tail of each mouse was wiped with sterilized alcohol cotton, a small section of the tail was cut to determine a baseline blood glucose level (0 min);

[0072] (C), insulin injection, wherein timing began immediately after intraperitoneal injection of the glucose solution, and the blood glucose levels were determined at 15 min, 30 min, 60 min, 90 min and 120 min after injection; the measurement method includes reopening the tail wound, discarding the first drop of blood and measuring the blood glucose level of the second drop of blood; and attentions should be paid to the mental state of the mice always, and if it was found that the mice were in a poor condition, glucose should be injected into the mice immediately.

[0073] 12. Oil red O staining of frozen section of liver tissue:

[0074] (A), taking the frozen slice of liver (6 um) out of -80°C refrigerator and leaving the frozen section of liver at room temperature for 1 hour to dry the slide;

[0075] (B), separating the tissue slice with a water-blocking pen;

[0076] (C), fixing the tissue, wherein the slice is fixed with 1% of PFA for 30 min at room temperature;

[0077] (D) washing the tissue slice with PBS for three times for 5 min each time;

[0078] (E), staining, wherein staining with diluted oil red dye was performed at room temperature for 1 hour (adding 600 pl of undiluted oil red O staining solution into 400 u of distilled water, and filtering it with 0.45 um filter membrane before use),

[0079] (F), discarding the staining solution, and then washing the tissue slice with PBS for three times;

[0080] (G), staining the nucleus, wherein staining with hematoxylin was performed for 2 minutes; and

[0081] (H), sealing the slice with glycerin gelatin, and observing under a microscope.

[0082] 13. Hematoxylin-eosin (HE) staining of tissue: after dewaxing and hydration, the slice was soaked in hematoxylin for 2 minutes; an empty staining box was prepared, filled with tap 10 water, and was washed for 3-5 times until it was colorless. This was immediately followed by differentiation with 0.3% hydrochloric acid alcohol and rapid rinsing for 3s. Finally, the empty staining box was refilled with tap water and pulled up and down, hydrochloric acid alcohol was diluted, and the reaction was terminated.

[0083] II. Experimental results

[0084] 1. Identification results of fermentation metabolites of five probiotic strains are shown in

Fig. 7. After fermentation, the five probiotic strains were filtered by the 0.22 pm filter membrane to obtain fermentation supernatants. The MRS culture medium was used as a blank control. The metabolites in the supernatants were analyzed by LC-MS/MS mass spectrometry. The components and contents of the metabolites were compared with the blank control to obtain the metabolites produced by probiotic fermentation.

[0085] 2. The identification of the fermentation metabolites of the five purified probiotic strains is shown in Fig. 8. After fermentation, the five probiotic strains filtered by the 0.22 pM filter membrane, and the filtered five probiotic strains were concentrated with ethyl acetat. Metabolites in the lower layer (water phase) were collected. After low-temperature concentration and evaporation, 1000x metabolites were obtained. The metabolites were analyzed by LC-MS/MS mass spectrometry. The components and contents of the metabolites were compared with the blank control to obtain the metabolites produced by probiotic fermentation.

[0086] 3. As shown in Fig. 9, the bars in Figs. 9A, 9B and 9C from left to right are HFD+MRS,

HFD+Postbiotics, HFD-+(Lactobacillus reuteri) PLBK"™ 1, HFD+(Lactobacillus reuteri) PLBK*2,

HFD+(Lactobacillus gasseriy PLBK*3, HFD+(Lactobacilhes acidophilus) PLBK®4 and

HFD+(Bifdobacterium lactis) PLBK®*5. C57BL6/] mice were fed the 60% HFD, and simultaneously, were given the intervention by the mixed strains and metabolites (postbiotics metabolites) thereof. The detected rectal temperature of the mice was as shown in Fig. 9A, glucose tolerance (GTT) of the mice was as shown in Fig. 9B, fecal suspension time of the mice was as shown in Fig. 9C, and body weight gains of the mice was as shown in Fig. 9D. The results showed that the basal metabolic rate of the mice intervened by the mixed strains and the metabolites thereof was significantly higher than that of the mice intervened by any single strain, and the body glucose tolerance of the mice intervened by the mixed strains and the metabolites thereof was significantly better than that of the mice intervened by any one of the single strain.

The results of fecal suspension experiment showed that the adipose absorption efficiency of the mice intervened by the mixed strains and the metabolites thereof was the lowest, and a large amount of adipose remained in their feces, so the suspension time was the longest. In conclusion, the mixed strains and the metabolites thereof were better than single strains in activating energy 11 metabolism and enhancing the body's resistance to metabolic disorders caused by the 60% high- fat diet (HFD).

[0087] 4. As shown in Fig. 10 (in all the bar graphs in Fig. 10, the two bars in each group are

NCD+H:0 on the left and NCD+Postbiotics on the right), the mice were fed the NCD (normal diet group) and pure water for 12 weeks. Then, obese mice were intervened by postbiotics (the mixed probiotic solution). After 12 weeks of the intervention by the postbiotics, the body weight gains of the mice increased slowly and the body sizes of the mice were smaller (as shown in Figs. 10A and 10C). The ratio of liver and white adipose tissue to the body weight of each of the mice decreased, proving that the postbiotics reduced the accumulation of lipid in the body (as shown in Fig. 10B). Insulin sensitivity of the healthy mouse was enhanced by the intervention by the postbiotics (as shown in Figs. 10D and 10E). By HE staining, the area of oil bubbles in brown and white adipose tissues significantly decreased after the intervention by the postbiotics, indicating that the postbiotics promoted adipose tissue thermogenesis (as shown in Fig. 10F).

Meanwhile, serum total cholesterol, low-density lipoprotein cholesterol and alanine aminotransferase levels were significantly lower than those in the control group, and high- density lipoprotein cholesterol content was significantly higher than that in the control group, which proved that the postbiotics could further relieve blood lipid (as shown in Fig. 10H).

Relative increases in the expressions of some thermogenic markers could also be observed in the mRNA and protein levels of the brown adipose tissue and the white adipose tissue (as shown in

Figs. 101 to 10L), suggesting that the reduction in the area of the oil bubbles in the adipose tissue might be caused by the increase of tissue thermogenic energy consumption. In conclusion, the healthy diet for the mice and the probiotics and the metabolites thereof promoted the improvement of the metabolic phenotype.

[0088] 5. As shown in Fig. 11 (in all the bar graphs in Fig. 11, the two bars in each group are

NCD+HO on the left and NCD+Postbiotics on the right), differences showed between the body weights of the mice fed with the HFD freely for 12 weeks and the body weights of the mice fed with pure water freely for 12 weeks, proving that the modeling of diet-induced obesity mice was successful. In order to prove whether the postbiotics could control body weight and improve glucose and lipid metabolism in HFD-induced obese mice, the postbiotics were used to intervene the obese mice successfully modeled for 12 weeks. After 12 weeks of the intervention by the postbiotics, compared with the control group, the mice in the experimental group gained body weight slowly and were smaller in size (as shown in Figs. 11A and 11C), and the ratio of liver and three adipose tissues (brown adipose, white adipose and groin adipose) to the body weight of the mouse decreased, which proved that the postbiotics could reduce the accumulation of body lipid (as shown in Fig. 11B). After the intervention by the postbiotics, the insulin resistance of 12 the HFD-induced obese mice could be improved, and the blood glucose could be reduced to a level similar to that of normal mice (as shown in Figs. 11D and 11E). High-fat diet aggravated lipid accumulation in liver, brown and white adipose tissues of the mice. After the intervention by the postbiotics, the situation of the accumulation of the lipid in liver and adipose tissues was improved, and the area of oil bubbles in the white adipose tissue was significantly reduced and could return to the normal state (as shown in Figs. 11F and 11G). The expressions of peroxisome proliferator-activated receptor y costimulator (Ppargcta) and uncoupling protein 1(Ucp!) of thermogenic markers were significantly increased in the white adipose tissue of the HFD- induced obese mice after intervention by the postbiotics (as shown in Fig. 11H). Meanwhile, the levels of total triglyceride, total cholesterol, low-density lipoprotein cholesterol and glutamic- pyruvic transaminase in serum decreased significantly, demonstrating that the postbiotics ameliorated dyslipidemia caused by the high-fat diet (as shown in Fig. 111). Relative increases in the expression of some thermogenic markers could also be observed in the mRNA and protein levels of the brown adipose tissue and the white adipose tissue (as shown in Figs. 11J to 11M), suggesting that the reduction in the area of the oil bubbles in the adipose tissue might be caused by the increase of tissue thermogenic energy consumption. In conclusion, the 60% high-fat diet induced the development of the metabolic syndrome, and the intervention by the probiotics and the metabolites thereof could treat the metabolic syndrome.

[0089] 6. As shown in Fig. 12 (in all the bar graphs in Fig. 12, the two bars in each group are

HFD+30%GlucosetMRS on the left and HFD+Postbiotics (metabolites) on the right), another animal model was designed to prove whether the postbiotics could inhibit the body weight gain and maintain the stability of energy metabolism in potentially obese mice. That is, wild

C57BL/6J mice were fed the HFD freely, and simultaneously, were intervened by the postbiotics (a potential obesity model); while the MRS broth medium with 30% sugar content (through the detection of a kit, it was found that the sugar content in a bacterial solution of the MRS broth medium after the fermentation was 30% of that of the original MRS broth medium) was used in the control group. The experimental results showed that the mice after the intervention by the postbiotics were slower in body weight gain and smaller in body size and had no obvious fatty liver compared with the mice in the control group (as shown in Figs. 12A and 12C). The ratio of white adipose and groin adipose to the body weight of the mice decreased, which proved that the postbiotics could reduce the accumulation of body lipid (as shown in Fig. 12B). The possible reason for the absence of this phenomenon in the brown adipose tissue and the liver tissue might be that the body weight of the mice after the intervention by the postbiotics was lower, and the weights of the brown adipose tissue and liver tissue did not increase significantly, but decreased relative to the control group. After the intervention by the postbiotics, insulin resistance of the 13 potentially obese mice was improved, and blood glucose of the potentially obese mice was reduced to a level similar to that of normal mice (as shown in Figs. 12D and 12E). Through HE staining, it was found that the area of oil bubbles in the brown adipose tissue and white adipose tissue in the control group increased obviously; while the area of the oil bubbles in the adipose tissues decreased after the postbiotic treatment (as shown in Figs. 12F and 12G). For the oil red

O staining of frozen sections of the liver tissue, it could be observed that the oil red staining in the experimental group was significantly less than that in the control group, and no fusion of lipid droplets existed. In addition, it could be found that the postbiotics could reduce the accumulation of lipids in the liver tissue through the area stained by oil red (as shown in Fig. 12G). The blood lipid spectrum also showed an obvious improvement trend (as shown in Fig. 12H). Relative increases in the expressions of some thermogenic markers were also observed in the mRNA and protein levels of the brown adipose tissue (as shown in Figs. 121 and 12J).

However, in the white adipose tissue, the increase in the expression of some thermogenic markers could be observed only at mRNA level (as shown in Figs. 12K and 12L). In conclusion, the intervention by the probiotics and the metabolites thereof could prevent the occurrence and development of the metabolic syndrome while the 60% HFD induced the metabolic syndrome. 14 i Alum. overslon=T1.0" encoding=TUTF-Qn Tx 2 <!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing 1.3//EN" "ST265equenceListing V1 3.dtd"> 3 <ST26Sequencelisting drdvVersion="NWL 3% filoName="Fl40834NLO0. xml” soïtwaceNems=*WIPO Segvencen zscoïtwareVension="2 8,05 vrodooricnDeie="2eddrld-2n">

A <Applicatioconidentiiflcatlon> <IPOfficeCode>NL</IPOfficelode> & <ApplicationNumerTezt></ApplicetionNumberText»> 7 <FiliogDate></FilingDaLter 8 </Ppplicationidentification> 3 <ApplicentFilefeference:P12687NL00 </ApplicantFileReference> <BarliiestPricritvipplicationidentification> il <IPOfEiceloderCN</TIPOfflceloderx 12 <ApplicationNumberText>202210392976.2</ApplicaticnNunberText> 13 <“FilingDate>2022-04-15</FilingZete»> 14 </FarliestPriorityApplicationidentification> in <ApplicantNams languagelcde=Ven*>Institute of Microbiology, Guangdong Academy of

Sciences (Guangdong Detection Center of Microbiology)</ApplicantName> ia <Bpplicantiamelstin>Institute of Microbiology, Guangdong Academy of Sciences

Guangdong Detection Center of Microbiology/ApplicantNemeLatin»>

Lj <InventorName languagelode=Ven*rLiwei XIE</InventorName>

Ls <inventorNamelalinsLiwei XIE</InventorNemeLakin>D

Le <IpventionTitle languagyeloede="an">FORMULA OF PROBIOTICS AND METABOLITES THEREOF

FOR RELIEVING METABOLIC SYNDROME, AND USE THEREOF</InventionTitle> <JequanceTotalQuantity>T7</ SaquenceTotaluantity>

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44 <IN3DSeq> 47 <INSDSeq length>1475</INSDSay Lengih> a8 <INSDSeq moltype>DNA</INSDSeq moltype> 43 <IN3DSeq division»PAT</INSD3eq division» 50 <INSDSeq feature-table> 51 <INSDFeaturs>

SE <INSDFeature key>source</INIDFeature key> <INSDFeature location»l..1475</1INSDFeature location»

Sá <INSDFearure gualsr 58 <INSDQualifier»> 54 <INSDQualifier name>mol type</iNSDQualifier name> 57 <IN3DQualifier value>genomic DNA</iN5SDgualifier value> 58 </INSDOQuali fier 58 <INSDQualifler 1d=Vgd®>

En <INSDQualifier namevorganism“/INSDQualifier name>

Sl <INSDQualifier valuerLactobacillus reuteri PLBK 2</INSDgvali fien valuex>

GZ </INSDOualifier> a2 </INSDFeature guals> ad </INSDFeature> ah </INSDSeg features table» <INSDSeq sequencertggcgcggcggtgtgctatacatgcagtcgtacgcacgggcccaactgattgatgg tgettgcacctgattgacgatggatcaccagtgagtggcggacgggtgagtaacacgtaggtaacctgcccegg agcgggggataacatttggaaacagatgctaataccgcataacaacaaaagccacatggcttttgtttgaaaga tggctttggectatcactctgggatggacctgeggtgcattagctagttggtaaggtaacggcttaccaaggcga tgatgcatagccgagttgagagactgatcggccacaatggaactgagacacggtccatactcctacgggaggca gcagtagggaatcttccacaatgggcgcaagcctgatggagcaacaccgcgtgagtgaagaagggtttcggecte gtaaagctctgttgttggagaagaacgtgcgtgagagtaactgttcacgcagtgacggtatccaaccagaaagt cacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttatccggatttattgggcgtaaag cgagcgcaggcggttgcttaggtctgatgtgaaagcctteggcttaaccgaagaagtgcatcggaaaccgggcg acttgagtgcagaagaggacagtggaactccatgtgtagcggtggaatgcgtagatatatggaagaacaccagt ggcgaaggcggctgtctggtctgcaactgacgctgaggctcgaaagcatgggtagcgaacaggattagatacce tggtagtccatgccgtaaacgatgagtgctaggtgtttggagggttteecgccecttcagtgccggagctaacgca ttaagcactccgcctgggagtacgaccgcaaggttgaaactcaaagaattgacgggggcccgcacaagcgtgga gcatgtggttgatttcaagctacgcgaagaccttaccaggtcttgacatcttgegctaaccttagaggataagg cgtteccttcggggacgcaatgacaggtggtgcatggtcgtcgtcagctegtgtecgtgagatgttgggttaagt cccgcaacgagcgcaacccttgttactagttgccagcattaagttgggcactctagtgagactgcecggtgacaa accggaggaaggtggggacgacgtcagatcatcatgccccttatgacctgggctacacacgtgctacaatggac ggtacaacgagtcgcaagctcgcgagagtaagctaatctcttaaagccgttectcagttcggactgtaggctgca actcgcctacacgaagtcggaatcgctagtaatcgcggatcagcatgcecgeggtgaatacgttccegggccttg tacacaccgccegtcacaccatgggagtttgtaacgcccaaagtecggtggcctaacctttatggaggagccget aagcgacagtgcg</INSD3eq sequence’ ad </INSDSeg> an </SeguencaData> eo <SequenceData zaquencalDNumben=n3n> a <INSDSeqd»>

TL <INSDSeq length>1449</IN3DSeqg length»

Td <INSDSeq moltype>DNA</INSDSeg moltype> 73 <INSDSeq divislion»PAT</INSDSeqg division»

Fd <IN3D3eq feature-table>

TA <INSDEeature> ia <IN3DFeature key>source</IN3DFeature key> i <IN3DFeature location>1l..1449</INSDFeature location»

UB <INSDFsature qualsg>

Ta <INSDuuelifier> ai <INSDoualifier name>mol type“ /INSDQualifier name> 81 <INSDQualifier valus>genomic DNA</INSDQuali fier valus> az </INSDOualifier> 82 <INSDOuaiifier id="qg8*> 23 <IN3DQualifier namevorganism“/INSDQuali fier name> <INSDQualifier valuerLactobacillus gasseri PLBK 3</INSDRualifier value» 56 «</INSDOQualifier»> av </INSDFeaturs auals> ss </INSDF=aturex 83 “/INSDSeg feature-table> <IN3D3eq sequencergggatgggeggegtgctatacatgcagtcgagcgagettgcctagatgaatttggt gettgcaccaaatgaaactagatacaagcgagcggcggacgggtgagtaacacgtgggtaacctgcccaagaga ctgggataacacctggaaacagatgctaataccggataacaacactagacgcatgtctagagtttaaaagatgg ttetgctatcactcttggatggacctgcggtgcattagctagttggtaaggtaacggcttaccaaggcaatgat gcatagccgagttgagagactgatcggccacattgggactgagacacggcccaaactcctacgggaggcagcag tagggaatcttccacaatggacgcaagtctgatggagcaacgccgcgtgagtgaagaagggtttcggctegtaa agctetgttggtagtgaagaaagatagaggtagtaactggcctttatttgacggtaattacttagaaagtcacg gctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtceggatttattgggcgtaaagcgag tgcaggcggttcaataagtctgatgtgaaagccttcggctcaaccggagaattgcatcagaaactgttgaactt gagtgcagaagaggagagtggaactccatgtgtagcggtggaatgcgtagatatatggaagaacaccagtggcg aaggcggectctctggtctgcaactgacgctgaggctcgaaagcatgggtagcgaacaggattagataccctggt agtccatgccgtaaacgatgagtgctaagtgttgggaggtttcegcctctcagtgetgcagctaacgcattaag cactccgcctggggagtgcgaccgcaaggttgaaagtcaaaggatttgacgggggcccgcgcgagcggtggagec gtgtggtttagttcgaagcaacgcgaggagcctgtaccagtettgacgtccagtgcaaaccgaggagattagga gtacctgtegtcagctegtgtegtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttge catcattaagttgggcactctaatgagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatc atgccccttatgacctgggctacacacgtgctacaatggacggtacaacgagaagcgaacctgcgaaggcaagc ggatctctgaaagccgttctcagtteggactgtaggctgcaactcgcctacacgaagctggaatcgctagtaat cgcggatcagcacgccgecggtgaatacgtteccegggeccttgtacacaccgccegtcacaccatgagagtctgta acacccaaagccggtgggataacctttataggagtcagcegtctaagtagacagggtgggt/INSDS=q sey vencer

EN </INSDS=eo> 32 </Seguencedata> 32 <SequenceData sequence lDNumben="4%> 84 <INSDSeq> 95 <INSDSeag lengith>1816</INSDSeq lergth>

GE <INSDSeq moltype>DNA</INSDSeg moltype» 7 <INSDSeq division>PAT</INSDSeg division»

Gd <INSDSeq feature-table> 33 <INSDFeature)> 1440 <INSDFeature key>sourcec/INSDFeature key» ijl <IN3DFeature location>l..1816</INSDFeature location» 192 <INSDFeature guals> 103 <INSDOualifien> 134 <INSDQualifier name>mol type</INSDQualiifier name>

LOL <INSDQualifier vaiuergenomic DNA</INSDQualifier value> 108 </INSDOualiLfier»> 107 <INSDQualifler in="g8"> 108 <INSDQualifier name>organism</INSDQualifier name> 143 <IiNSDgualifier value>Lactobacillus acidophilus PLBK 4</INSDQualifier value»

LAO </INSDQuali fier»

ER </INSDFeature quals>

Lid </IN3DFeature>

LLS </INSDSeg feature-table> <IN3D3eq sequence»ggcatggcggegtgctatacatgcagtcgagcgagcgtgaaccaacagattcactt cggtgatgacgttgggaacgcgagcggcggatgggtgagtaacacgtggggaacctgccccatagtctgggata ccacttggaaacaggtgctaataccggataagaaagcagatcgcatgatcagcttataaaaggcggcgtaagct gtecgctatgggatggccccgecggtgcattagctagttggtagggtaacggcctaccaaggcaatgatgcatagc cgagttgagagactgatcggccacattgggactgagacacggcccaaactcctacgggaggcagcagtagggaa tcttccacaatggacgaaagtctgatggagcaacgccgcgtgagtgaagaaggttttcggatcgtaaagctectg ttgttggtgaagaaggatagaggtagtaactggcctttatttgacggtaatcaaccagaaagtcacggctaact acgtgccagcagccgcggtaatacgtaggtggcaagcgttgtcecggatttattgggcgtaaagcgagcgcaggec ggaagaataagtctgatgtgaaagcccteggcttaaccgaggaactgcatcggaaactgtttttcttgagtgca gaagaggagagtggaactccatgtgtagcggtggaatgcgtagatatatggaagaacaccagtggcgaaggcgg ctetetggtctgcaactgacgctgaggctecgaaagcatgggtagcgaacaggattagataccctggtagtccat gccgtaaacgatgagtgctaagtgttggggaggtttcegcctctcagtgctgcagctaacgcattaagcactce gcctggggagtacggaccgcaaggttgagactcaaaggaattgacgggggcccgcacaagcggtggagcgatgt ggtttagtgtcgaagcaacgcgaagagccttaccaggtcttgacgtctagtgcaatccgggagagtggaactce atgtgtagcggtggaatgcgtagatatatggaagaacaccagtggcgaaggcggctctetggtctgcaactgac gctgaggctcgaaagcatgggtagcgaacaggattagataccctggtagtccatgccgtaaacgatgagtgcta agtgttgggaggtttcegcectctcagtgectgcagctaacgcattaagcactcegcctggggagtacgaccgcaa ggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaa gaaccttaccaggtcttgacatctagtgcaatccgtagagatacggagttcccttcggggacactaagacaggt ggtgcatggctgtegtcagctegtgtegtgagatgttgggttaagtccegcaacgagcgcaacccttgtcatta gttgccagcattaagttgggcactctaatgagactgccggtgacaaaccggaggaaggtggggatgacgtcaag tcatcatgcccecttatgacctgggctacacacgtgctacaatggacagtacaacgaggagcaagcctgcgaagg caagcgaatctcttaaagctgttctcagtteggactgcagtctgcaactegactgcacgaagctggaatcgcta gtaatcgcggatcagcacgccgeggtgaatacgttccegggccttgtacacaccgccegtcacaccatgggagt ctgcaatgcccaaagccggtggcctaacctcggaaggagcecgtctaagcagegttgeg/INS2Seq sequen can 115 </INSDSeg> iia </SeguenceDala>

LL? <Sequencebata zecquencelDNumser="Bt>»

Lis <INSDSeq>

LLG <INSDSeq length>1439</INSDSea length»

LAG <INSDSeq moltype>DNA</INSDSea moliype> 173 <INSDSeq divislon»PAT</INSDSey division 1322 <IN3D3eq feature-table> 123 <INSDFeatura> 124 <IN3DFeature key>source</IN3DFeature key» 12% <INSDFeature location>l..1439</INSDFeature location» ze <INSDFeature quals>

Lad <IN3DQualifiers 178 <INSDQualifier name>mol type</INSDQualifier name> 123 <IN3DQualifier value>genomic DNA</INSDQuaiifier valued ijn </INSDQualifier>

LL <INSDOualifier ld="gion> 132 <IN3DQualifier namerorganism“/INSDQuali fier name> 133 <INSDQualifier valuerBifdobacterium lactis PLBK

S</INSDQualifier value» 1x4 </INSDOualifiers 135 </INSDFeaturs guals> 1348 </INSDFeature> 127 </INBDSeq feature-table> <INSDSex seduencergaggcatgtggegtgettaccatgcagtcgacgggatccctggcagcttgetgteg gggtgagagtggcgaacgggtgagtaatgecgtgaccaacctgccctgtgcaccggaatagctcctggaaacggg tggtaataccggatgctcegctccatcgcatggtggggtgggaaatgettttgeggcatgggatggggtegegt cctatcagcttgttggcggggtgatggcccaccaaggcgttgacgggtagccggcctgagagggtgaccggcca cattgggactgagatacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcct gatgcagcgacgccgcgtgcgggatggaggccttcgggttgtaaaccgettttgttcaagggcaaggcacggtt teggccgtgttgagtggattgttcgaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggt gegagcgttatccggatttattgggcgtaaagggctcgtaggcggttegtegegtceggtgtgaaagtccateg cctaacggtggatctgcgccgggtacgggecgggctggagtgcggtaggggagactggaattcccggtgtaacgg tggaatgtgtagatatcgggaagaacaccaatggcgaaggcaggtctctgggcegtcactgacgctgaggagcg aaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacggtggatgctggatgtggggce ctttccacgggtccegtgtcggagccaacgegttaagcatccegcctgggagtacggccgcaaggctaaaactc aaagaaattgacggggcccgcacaagcggcggagcatgcggattaattcgatgcaacgcgaagaaccttacctg ggcttgacatgtgccggatcgccgtggagacacggtttcccttcggggeccggttcacaggtggtgcatggtegt cgtcagctegtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctecgeccgcatgttgccagcgggt gatgccgggaactcatgtgggaccgccggggtcaactcggaggaaggtggggatgacgtcagatcatcatgcce cttacgtccagggcttcacgcatgctacaatggccggtacaacgcggtgcgacacggtgacgtggggcggatcg ctgaaaaccggtctcagttcggatcgcagtetgcaactcgactgcgtgaaggcggagtcgctagtaatcgcgga tcagcaacgccgcggtgaatgegttccegggeccttgtacacaccgcecegtcaagtcatgaaagtgggtagcacc cgaagccggtggcccgacccttgtggggggagccgtetaagtagactcatg/INSLSeq sequencer 13% </INSDS=eo> 14h </Seguencedata> 14d <SequenceData semiencelDNunbern=N8#> 142 <INSDSeq> 1473 <IN3DSeqy Leng:th>20</IN5DSeq length idd <INSDSeq moltype>RNA</INSDSeg moltype»

TAL <INSDSeq division>PAT</INSDSeg division» 146 <INSDSeq feature-table> nav <INSDFeature> 148 <INSDFeature key>sourcec/INSDFeature key» 143 <INSDFearure location>l..20</INSDFeature location» 150 <INSDFeature guals> 151 <INSDOualifien>

LE <INSDQualifier name>mol type“/INSDQualifier name>

LES <INSDQualifier valuerother RNA</INSDQualifier value» 154 </INSDOualifiers 155 <INSDQualiifler id=vgle™> 158 <INSDQualifier name>organism</iNSDQualifier name> isd <IiNSDgualifier value>synthetic construct - amplification primer</INsSDGualifier value> 158 </INSDQuali fier» 15% </INSDFeature quals>

LEG </IN3DFeature>

Lel </INSDSeg feature-table> 16d <INSDSeq segquenceragagtttgatectggeteag</INSDSeq sequence» 182 </INSDSeg> 144 </SeguenceData> 18h <SeguenceData semuenceIDNumber=Njn>

TARE <INSDSedg>

Lo? <INSDSeq length>19</INSD3eq lengths

Len <INSDSeq moltype>RNA</INSDSegq moltype> its <INSDSeq division>PAT</INSDSeq divisicn>

LEG <INSDSeq feature-iable> ijd <INSDPFSsarure:» 172 <IN3DFeature key>source</IiN3DFeature key» 173 <IN3DFeature lowation>l..19</INSDFeaturs location»

Lid <INSDFeature guals>

Lin <INSDOualifier> iE <INSDOQualifier name>mol type</INSDQualifier name> 177 <INSDgualifier valuerother RNA</INSDQualifier value» 178 </INSDOualifier> 13 <INSDOualifier id="qL7n> inn <IN3DQualifier namevorganism“/INSDQualifier name>

LEL <INSDQualifier valiuersynthetic construct - amplification primer“/INSDQGuallfier value»

LEE <{INSDQualifier»

LES </INSDFeature quals> 14 </IN3DFeature> u 185 “/INSDSeqg fesature-table> ia <INSDSeq sequencer>ggttaccttgttacgactt</INSD3eg sequence 87 <JINgDseqy u

LEE </SeguenceDala>

LS </ST26SeguencelListing>

LSO

Claims

Conclusions What is described is:

1. Formula of probiotics for alleviating metabolic syndrome, wherein the formula of the probiotic is (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifidobacterium lactis) PLBK®5, or a fermentation culture of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifidobacterium lactis) PLBK®5, or a metabolite of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifidobacterium lactis) PLBK®s; the accession number of (Lactobacillus reuteri) PLBK®1 is GDMCC No.: 60828; the accession number of (Lactobacillus reuteri) PLBK®2 is GDMCC No.: 60829; the accession number of (Lactobacillus gasseri) PLBK®3 is GDMCC No.: 60830; the accession number of (Lactobacillus acidophilus) PLBK®4 is GDMCC No.: 60831; and the accession number of (Bifidobacterium lactis) PLBK®5 is GDMCC No.: 60832.

2. Formula of the probiotic according to claim 1, wherein the formula of the probiotic is a mixture of solutions and/or metabolites of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifdobacterium lactis) PLBK®s.

A formula of the probiotic according to claim 2, wherein the probiotic content in the mixture is more than 10°cfu/ml.

Formula of the probiotic according to claim 2 or claim 3, wherein the amount ratio of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifdobacterium lactis) PLBK®5 in the mixture is 0.75: 0.75: 1: 1.5: 1.

Use of the probiotic formula according to claim 1 in the preparation of a product for alleviating metabolic syndrome.

Use according to claim 5, wherein the alleviation of the metabolic syndrome is reduction of adipose accumulation, promotion of adipose breakdown, improvement of insulin sensitivity and/or remission of fatty liver.

Use according to claim 5 or claim 6, wherein the product is a food product, health product or drug that alleviates metabolic syndrome.

Use according to claim 5, wherein the formula of the probiotic is a mixture of solutions and/or metabolites of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus ) PLBK®4 and (Bifdobacterium lactis) PLBK®s.

Use according to claim 8, wherein the amount is a ratio of (Lactobacillus reuteri) PLBK®1, (Lactobacillus reuteri) PLBK®2, (Lactobacillus gasseri) PLBK®3, (Lactobacillus acidophilus) PLBK®4 and (Bifdobacterium lactis) PLBK® 5 in the formula of the probiotics 0.75: 0.75: 1: 1.5: Iris.

Use according to claim 8 or claim 9, wherein the probiotic content in the formula of the probiotic is more than 10”°cfu/ml.