CN114836357B

CN114836357B - Lactobacillus reuteri strain LN0214 for reducing serum branched-chain amino acid level and derivative product and application thereof

Info

Publication number: CN114836357B
Application number: CN202210646957.8A
Authority: CN
Inventors: 任文凯; 王尤霞; 范丽娟; 刘炳南
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-06-08
Filing date: 2022-06-08
Publication date: 2023-07-21
Anticipated expiration: 2042-06-08
Also published as: CN114836357A

Abstract

The invention provides a lactobacillus reuteri strain LN0214 for reducing the level of serum branched-chain amino acid, a derivative product and application thereof, and belongs to the technical field of microorganisms. According to the invention, the level of branched chain amino acid in serum of a sterile animal is obviously increased, and the level of branched chain amino acid in serum is effectively reduced after the mice are treated by lactobacillus reuteri LN0214 combined with antibiotics and the bacteria-free mice are subjected to colonization. Meanwhile, in vitro researches show that lactobacillus reuteri LN0214 effectively up-regulates the gene expression of the enzyme related to branched-chain amino acid metabolism and increases the content of metabolites. In view of the fact that LN0214 reduces the level of branched chain amino acids, the lactobacillus reuteri LN0214 can be used for preparing medicines for diabetes and insulin resistance, and has wide application prospects.

Description

Lactobacillus reuteri strain LN0214 for reducing serum branched-chain amino acid level and derivative product and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a lactobacillus reuteri strain LN0214 for reducing serum branched chain amino acid level, a derivative product thereof and application thereof.

Background

Branched Chain Amino Acids (BCAAs), including leucine, isoleucine and valine, are essential amino acids for human and animal use. BCAAs not only play an important role in protein metabolism, but also have various physiological metabolic functions, for example, BCAAs can stimulate insulin and glucagon secretion, BCAAs are markedly elevated in blood circulation in obese and type 2 diabetics, and high levels of BCAAs in blood circulation can lead to insulin resistance or type 2 diabetes ^[1-3] . Thus, BCAAs are considered as diabetes risk predictors. Thus, BCAA metabolism plays an important role in affecting insulin resistance and diabetes, impaired BCAT and BCKDH function further exacerbate insulin resistance, and reduced BCAT and BCKDH expression in skeletal muscle of diabetic patients. Therefore, the method increases the metabolism of the body BCAA and reduces the concentration of the BCAA in blood circulation, thereby providing a new idea for treating insulin resistance and diabetes.

In recent years, a great deal of research has shown that insulin resistance and diabetes are closely related to intestinal microorganisms, and intestinal microbiota imbalance is an important factor for the rapid development of insulin resistance and diabetes. In insulin resistant individuals, the potential of many microorganisms to synthesize BCAA increases, and prasugrel bacteria and bacteroides vulgaris are the primary drivers of the biosynthesis of BCAA by microorganisms.

While lactobacillus reuteri is present in the intestinal tract of all mammals and vertebrates, it is an internationally recognized novel probiotic lactic acid bacteria. The research at present shows that lactobacillus reuteri can relieve metabolic syndrome, reduce lipopolysaccharide concentration and the like. However, to date, the effect of lactobacillus reuteri on serum BCAA levels and BCAA metabolism has not been reported.

Reference is made to:

[1]Lynch CJ,Adams SH.Branched-chain amino acids in metabolic signalling and insulin resistance.Nat Rev Endocrinol(2014)10(12):723–36.doi:10.1038/nrendo.2014.171

[2]Zhou M,Shao J,Wu C-Y,Le Shu,Dong W,Liu Y,et al.Targeting BCAA Catabolism to Treat Obesity-Associated Insulin Resistance.Diabetes(2019)68(9):1730–46.doi:10.2337/db18-0927

[3]Nilsen MS,JersinUlvik A,Madsen A,McCann A,Svensson P-A,et al.3-Hydroxyisobutyrate,A Strong Marker of Insulin Resistance in Type 2Diabetes and Obesity That Modulates White and Brown Adipocyte Metabolism.Diabetes(2020)69(9):1903–16.doi:10.2337/db19-1174.

disclosure of Invention

In view of the above, the present invention aims to provide lactobacillus reuteri strain LN0214 with reduced serum branched-chain amino acid levels, and derivatives and applications thereof, which have an effect of reducing serum BCAA and enhancing BCAA metabolism for improving insulin resistance and diabetes.

The invention provides a lactobacillus reuteri strain LN0214 for reducing the level of serum branched-chain amino acid, wherein the preservation number of the lactobacillus reuteri strain LN0214 is CGMCC No.24586.

The invention provides a microbial inoculum for reducing the level of serum branched-chain amino acid, and an active ingredient comprises the lactobacillus reuteri strain LN0214.

Preferably, the live bacteria concentration of the lactobacillus reuteri strain LN0214 is 1×10 ⁸ CFU/g or more.

The invention provides application of lactobacillus reuteri strain LN0214 or the microbial inoculum in preparation of reducing serum branched-chain amino acid level or improving serum branched-chain amino acid metabolism.

Preferably, the branched-chain amino acid comprises one or more of the following: leucine, isoleucine and valine.

Preferably, the increasing serum branched-chain amino acid metabolism includes increasing the branched-chain amino acid metabolite content and increasing the expression level of a branched-chain amino acid metabolic pathway-related gene.

Preferably, the branched-chain amino acid metabolites include acetyl COA and/or succinyl COA content;

genes associated with branched-chain amino acid metabolic pathways include one or more of the following: BCAT1, BCAT2, BCKDHA, BCKDHB, and PPM1K.

The invention provides application of lactobacillus reuteri strain LN0214 or the microbial inoculum in preparation of a medicament for preventing and treating diabetes and/or insulin resistance.

Preferably, the medicament utilizes lactobacillus reuteri strain LN0214 to reduce the level of branched-chain amino acids, promote the metabolism of the branched-chain amino acids, and realize the prevention and treatment of diabetes and/or insulin resistance.

Preferably, the medicament comprises an oral dosage form.

The invention provides a lactobacillus reuteri strain LN0214 for reducing the level of serum branched-chain amino acid, and the preservation number is CGMCC No.24586. The invention obtains the branched chain amino acid to be obvious by analyzing the serum amino acid level in the aseptic pig, the aseptic mouse and the control group. The method comprises the steps of removing intestinal microorganisms of mice through antibiotics, screening to obtain microorganisms with obviously changed branched chain amino acids, and screening to obtain lactobacillus reuteri strain LN0214 through correlation analysis of the abundance of the microorganisms and the levels of the branched chain amino acids. The lactobacillus reuteri strain LN0214 colonization experiments show that the lactobacillus reuteri strain LN0214 reduces the serum levels of three BCAA, leucine, isoleucine and valine. Meanwhile, lactobacillus reuteri LN0214 has a promoting effect on branched-chain amino acid metabolism, improves the gene expression quantity of branched-chain amino acid metabolic pathway, and improves the contents of acetyl COA and succinyl COA which are the final products of branched-chain amino acid metabolism.

Drawings

FIG. 1 is a graph of sterile porcine serum BCAA levels;

FIG. 2 is serum BCAA levels of sterile mice;

FIG. 3 is serum BCAA levels of five antibiotic-treated mice;

FIG. 4 is an analysis of correlation of ampicillin and colistin treated mice 16S rDNA results with serum BCAA levels.

FIG. 5 shows serum BCAA levels of Lactobacillus reuteri LN0214 in combination antibiotic treated mice;

FIG. 6 shows serum BCAA levels of Lactobacillus reuteri LN0214 in sterile mice;

FIG. 7 shows the gene expression levels of BCAT1, BCAT2 and BCKDHA, BCKDHB, PPMIK of IPEC-J2 cells;

FIG. 8 shows the intracellular BCAA content of IPEC-J2;

FIG. 9 shows the contents of the BCAA metabolites acetyl COA and succinyl COA in IPEC-J2 cells.

Biological material preservation survival information

Lactobacillus reuteri, the lactobacillus reuteri strain of the invention is preserved in China general microbiological culture Collection center (China Committee) for a preservation time of 2022.3.24. The address is North Chen West Lu No. 1, no. 3 of the Chaoyang area of Beijing, and the biological preservation number is CGMCC No.24586.

Detailed Description

In the present invention, the breeding method of lactobacillus reuteri strain LN0214 preferably comprises the steps of: resuscitating strains: taking out the strain, re-heating and dissolving at-80 ℃, culturing and breeding in MRS culture medium under the constant-temperature anaerobic condition at 37 ℃ for about 18 hours to reach stability; passaging of the following materials: taking a proper amount of mixed solution of MRS and bacterial strain in the step (A), adding the mixed solution into an MRS culture medium, and culturing and breeding at 37 ℃.

In the present invention, the live bacteria concentration of the Lactobacillus reuteri strain LN0214 is preferably 1X 10 ⁸ CFU/g or more.

The preparation method of the microbial inoculum preferably comprises the following steps:

culturing Lactobacillus reuteri LN0214 at 37deg.C under anaerobic condition to logarithmic phase (about 16 hr), centrifuging at 3000rpm for 5min, collecting thallus, and re-suspending thallus with PBS to obtain thallus concentration of 1×10 ⁸ 。

In the present invention, the branched-chain amino acid preferably includes one or more of the following: leucine, isoleucine and valine.

In the invention, the lactobacillus reuteri strain LN0214 is planted in the intestinal tract of animals, so that the serum branched-chain amino acid level can be remarkably reduced compared with a control group. Cell experiments show that the lactobacillus reuteri strain LN0214 can obviously improve the metabolism of the serum branched-chain amino acid, preferably comprises the steps of increasing the content of the branched-chain amino acid metabolite and improving the expression quantity of the related genes of the branched-chain amino acid metabolism path. The branched-chain amino acid metabolites preferably include acetyl COA and/or succinyl COA content. The branched chain amino acid metabolic pathway related genes comprise one or more of the following genes: BCAT1, BCAT2, BCKDHA, BCKDHB and PPM1K.

In view of BCAA being considered as a diabetes risk predictor, the medicament preferably utilizes lactobacillus reuteri strain LN0214 to reduce the level of branched-chain amino acids, promote the metabolism of branched-chain amino acids, and achieve prevention and treatment of diabetes and/or insulin resistance. The medicament preferably comprises an oral dosage form. The preparation method of the oral preparation is not particularly limited, and the preparation method of oral preparation medicaments related to microorganisms known in the art can be adopted. The effective concentration of the lactobacillus reuteri strain LN0214 is 1×10 ⁷ ～1×10 ⁸ More preferably 1X 10 ⁸ 。

The lactobacillus reuteri strain LN0214 with reduced serum branched-chain amino acid levels, and its derivatives and applications, provided by the present invention, are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.

Example 1

In this example, 11 aseptic newborn piglets (pig engineering center for experiment in demonstration area of modern animal husbandry in Chongqing) without specific pathogen were selected, wherein 5 piglets were kept in aseptic state all the time (GF group), and 6 piglets were subjected to fecal transplantation at 7 days old (pig-derived fecal bacteria, 1 mL/d) for 3 consecutive days as control group. All animals were kept in sterile isolators and serum was collected at 42 days of age and the metabolome was tested for BCAA content.

Metabolome pretreatment is as follows:

to 200. Mu.L of serum sample, 800. Mu.L of protein precipitant methanol-acetonitrile mixture (methanol: acetonitrile=1:1) was added, vortexed and oscillated for 2min, and sonicated in ice water bath for 10min.

Centrifuging for 10min (14500 rpm,4 ℃) and taking the supernatant, and vacuumizing in a vacuumizing centrifuge until volatilizing.

200. Mu.L of methanol water (methanol: water=1:1, volume ratio) was redissolved and precipitated, dissolved by vortex oscillation, sonicated for 10min in ice bath, centrifuged for 10min (14500 rpm,4 ℃), and the supernatant was taken out and put into LC-MS sample injection vials with support liners and stored at-20℃for analysis by an upper machine.

Metabolome analysis conditions:

using the Thermo Fisher Scientific UPLC system, the main parameters are: the mobile phase A is 0.1% formic acid solution, the mobile phase B is 100% acetonitrile, the flow rate is 0.2mL/min, the column temperature is 35 ℃, and the sample injection amount is 2 mu L; the initial proportion of mobile phase B was 5%, then increased to 7%,13% and 50% in sequence, respectively for 3 minutes, 2 minutes and 10 minutes, and finally, the proportion of mobile phase B was rapidly decreased to 5%, for 1 minute, and maintained at the 5% proportion for 2 minutes.

The results are shown in fig. 1, in which the serum leucine content of GF group was significantly increased, and neither valine nor isoleucine was significantly different from that of the control group.

Example 2

BCAA level changes in serum of sterile mice

The experiment of example 1 was repeated with sterile mice taking into account the differences in different species, and SPF mice and sterile (GF) mice without specific pathogen were both KM mice from the experimental animal center of third army university (Chongqing in China), 10 mice each for each group at 4 weeks of age, with SPF group as a control group. The GF group and the SPF group are uniformly fed with sterile diet, and serum is collected at the age of 8 weeks. Metabolome assay BCAA content, metabolome pretreatment was as in example 1.

The results are shown in figure 2, where the serum leucine and valine content of GF group is significantly increased compared to SPF group, while isoleucine is not significantly different.

Example 3

Mice treated with different antibiotics

Examples 1 and 2 demonstrate an increase in BCAA content in serum of sterile animals. To investigate the cause of this change, mice were treated with five antibiotics (neomycin, ampicillin, colistin, metronidazole, 1mg/mL, vancomycin, 0.5 mg/mL) respectively. The mice used in this example were ICR females (6 week old, liaoning long Biotechnology Co., ltd.) divided into 6 groups of 12 replicates each, five antibiotics were added with water at a period of 2 weeks, and feces and serum were collected at 8 weeks of age.

Serum metabolome detection

The BCAA content of the serum samples was measured using the metabolome, and the metabolome pretreatment was the same as in example 1.

The results are shown in figure 3, in which the serum three BCAA levels were significantly increased in the ampicillin and colistin treated mice group, whereas there was no significant change in BCAA levels in the serum of the neomycin treated mice group, and no significant change in leucine and isoleucine levels in the serum of the metronidazole and vancomycin treated mice group, and valine levels were significantly increased, as compared to the control group. Microorganisms associated with ampicillin and colistin were shown to be associated with changes in serum BCAA levels.

16S rDNA sequencing

The control, colistin and ampicillin fecal samples were sent to Beijing No and source company for 16SrDNA sequencing and correlation analysis of the sequencing results with serum BCAA levels.

The results are shown in figure 4, where colistin-treated group had significantly reduced abundance of lactobacillus murine, lactobacillus johnsonii and lactobacillus reuteri compared to the control group, where lactobacillus murine was significantly inversely correlated with serum BCAA levels. Ampicillin-treated groups showed significantly reduced abundance of lactobacillus murine, lactobacillus johnsonii, lactobacillus reuteri and lactobacillus enterica and were all significantly inversely correlated with serum BCAA levels.

Example 4

Separation method of lactobacillus reuteri LN0214 strain

Collecting common ICR mouse faeces, and adding 1ml PBS (phosphate buffered saline) into each 100mg faeces to prepare faecal fungus suspension;

dilution of faecal suspension to 1X 10 ^-5 、1×10 ^-6 And 1X 10 ^-7 Three gradients, respectively coating and inoculating on MRS solid culture medium plates, and culturing for 48h under anaerobic condition at 37 ℃;

selecting single colony, streaking and purifying on MRS solid culture medium plate, repeating for three times, and performing strain identification (Optimago);

the above procedure was repeated, and 16S identification was performed on the screened strain to obtain lactobacillus reuteri LN0214 strain (SEQ ID NO: 13).

Example 5

Treatment of mice with antibiotics Lactobacillus plantarum and Lactobacillus reuteri

Based on example 3, we selectColonization was performed with lactobacillus murine F5 and lactobacillus reuteri LN0214. The mice used in this example were 6-week-old ICR rats, to which were added in 5 consecutive days of drinking water in combination with antibiotics (neomycin, ampicillin, streptomycin, metronidazole, 1mg/mL, vancomycin, 0.5 mg/mL), and the animals were stopped for 2 days, and the treatment groups were fed with Lactobacillus plantarum F5 and Lactobacillus reuteri LN0214 (1X 10) ⁸ CFU), PBS was fed to the control group, serum was collected on day 14 and BCAA content was detected by the metabolome, following 5 days of feeding.

The results are shown in FIG. 5, in which serum valine levels were significantly reduced after colonization with Lactobacillus reuteri LN0214.

Example 6

Lactobacillus plantarum F5 and Lactobacillus reuteri LN0214 are transplanted into sterile mice

Based on example 5, further validation was performed with sterile mice. The present example used 6 week old sterile mice, KM mice (6 male and female animals per group) from the university of agriculture (Wuhan, china) laboratory animal center. On the first and third days of the beginning of the experiment, the treatment groups were fed with Lactobacillus murine F5 and Lactobacillus reuteri LN0214, respectively (1X 10) ⁸ CFU), control group was perfused with PBS, serum was collected on the seventh day, metabolome was tested for BCAA content, and metabolome sample pretreatment was the same as in example 1.

As a result, as shown in FIG. 6, after the colonization of Lactobacillus reuteri F5 and Lactobacillus reuteri LN0214, the serum levels of the three BCAA were significantly reduced, and as can be seen from a combination of example 5 and this example, lactobacillus reuteri LN0214 was selected for the subsequent examples.

Example 7

Effect of Lactobacillus reuteri LN0214 on BCAA metabolism

Based on examples 5 and 6, lactobacillus reuteri LN0214 reduced serum BCAA levels in mice, in this example we performed a study of the effect of lactobacillus reuteri LN0214 on BCAA metabolism. In this example, porcine small intestine epithelial cells (IPEC-J2), lactobacillus reuteri LN0214 and 10% of Lactobacillus reuteri LN021424h culture supernatants were used to co-culture with IPEC-J2 cells for 4h, and the RNA was collected for cell extraction to detect BCAA metabolism-related enzyme gene expression. Lactobacillus reuteri LN021424h culture supernatant was co-cultured with IPEC-J2 cells for 4h, the cells were collected and the metabolome was examined for intracellular BCAA level changes. Lactobacillus reuteri LN021424h culture supernatant was co-cultured with IPEC-J2 cells for 4h, and cell detection ELISA (acetyl COA and succinyl COA) was collected.

RT-PCR analysis:

cell samples were collected, cellular RNA was extracted according to the total RNA extraction kit (EZBioscience) instructions, and cdnas were prepared and reverse transcribed according to the EZBioscience reverse transcription kit instructions, essentially as follows:

removal of genomic DNA reaction:

1. Mu.g of total RNA was taken, 2. Mu.L of gDNA Remover was added thereto, and the mixture was thoroughly mixed, centrifuged briefly at the bottom of the tube by using a centrifuge tube, allowed to stand at room temperature for reaction for 5 minutes, and then placed on ice after the reaction was completed.

Reverse transcription reaction:

after the completion of the reaction in the above step, a reverse transcription reaction system was prepared by adding 5. Mu.L of 4 XRTMasterMix and then adding ddH to the gDNARemover-treated total RNA ₂ O is made up to 20 μl:

the reaction procedure was 15min at 37℃for 5s at 95℃and stored at 4 ℃.

qRT-PCR detection

The procedure of qRT-PCR was performed with reference to previous studies, beta-actin was used as a reference gene to normalize the transcription level of the target gene. The specific primer sequences are shown in Table 1. The relative expression level of the gene is referred to formula 2 ^-(ΔΔCt) The process is carried out:

TABLE 1 primer sequences

Cell metabolome detection

After the cells were collected, the cells were resuspended in physiological saline and disrupted by repeated freeze thawing, centrifuged at 2000 Xg for 10 minutes, and the supernatant was subjected to metabolome pretreatment. The metabolome pretreatment and metabolome analysis conditions were the same as in example 1.

Enzyme-linked immunosorbent assay (ELISA):

ELISA samples were cell-like and the sample preparation method was as follows: cells were gently washed with cold PBS, then digested with trypsin, centrifuged at 1000×g for 5min and the cells were collected; the collected cells were washed 3 times with cold PBS. Every 1×10 ⁶ 150-200. Mu.LPBS was added to each cell for resuspension and cells were disrupted by repeated freeze thawing. The extract was centrifuged at 1500 Xg for 10 minutes, and the supernatant was collected and examined.

1) Quantitative succinyl-coa (SCoA) assay

The detection method refers to a kit instruction book for quantitative detection of pig succinyl-CoA (SCoA) by Rui Xin bio (Quanzhou, fujian) and comprises the following specific operations:

reagent preparation

Before use, all components are rewarmed for at least 60min, so that the room temperature is fully rewarmed.

Concentrating the washing liquid: the concentrated washing solution taken out of the refrigerator is crystallized, which is a normal phenomenon, and the crystallization is completely dissolved by heating in water bath. The concentrated washing solution and distilled water were diluted 1:20, i.e., 1 part of the concentrated washing solution, and 19 parts of distilled water was added.

A substrate: the substrate solutions A and B were thoroughly mixed in a volume of 1:1 before use, and used within 15 minutes after mixing.

Operating program

All reagents and components were first returned to room temperature, standards, quality controls and samples, and duplicate wells were recommended.

1. Working fluids of the various components of the kit are prepared according to the method described in the specification.

2. The required strips are taken out of the aluminum foil bags, and the rest strips are put back into the refrigerator by sealing with the self-sealing bags. Setting a standard substance hole, a 0-value hole, a blank hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are respectively added into the standard substance hole, 50 mu L of sample diluent is added into the 0-value hole, 50 mu L of sample to be detected is added into the sample hole without adding the blank hole.

3. In addition to the blank wells, standard wells, 0-value wells, and sample wells, 100 μl of horseradish peroxidase (HRP) -labeled detection antibody was added.

4. The reaction plate was covered with a sealing plate membrane and incubated for 60min in a 37℃water bath or incubator.

5. Uncovering the sealing plate film, discarding the liquid, beating the water absorbing paper, filling the washing liquid in each hole, standing for 20S, throwing the washing liquid, beating the water absorbing paper, and repeating the steps for 5 times. And after the plate washing is finished, before the substrate is added, the reaction plate is fully patted on clean paper without scraps.

6. Substrates A and B were thoroughly mixed in a 1:1 volume, and 100. Mu.L of the substrate mixture was added to all wells. The reaction plate was covered with a sealing plate membrane and incubated in a 37 incubator for 15min.

7. All wells were added with 50. Mu.L of stop solution and the absorbance (OD) of each well was read at 450nm using an ELISA reader.

Result calculation

The standard concentration was taken as 6 standard wells on the ordinate (the calibrator concentrations were in order:

160. 80, 40, 20, 10, 5 ng/mL), 10 well, 7 concentration points total), the corresponding absorbance (OD value) as abscissa, using computer software, using four parameter Logistic curve fitting (4 pl)), creating a standard curve equation, calculating the concentration value of the sample using the equation by absorbance (OD value) of the sample.

Quantitative detection of acetyl-CoA (ACA)

The detection method refers to a kit instruction book of a pig Acetyl Coenzyme A (ACA) quantitative detection kit of Rui Xin biology (Fujian spring), and specifically comprises the following steps:

reagent preparation

1. Before use, all components are rewarmed for at least 60min, so that the room temperature is fully rewarmed.

2. Concentrating the washing liquid: the concentrated washing solution taken out of the refrigerator is crystallized, which is a normal phenomenon, and the crystallization is completely dissolved by heating in water bath. The concentrated washing solution and distilled water were diluted 1:20, i.e., 1 part of the concentrated washing solution, and 19 parts of distilled water was added.

3. A substrate: the substrate liquids A and B were thoroughly mixed in a volume of 1:1 before use, and used within 15 minutes after mixing.

Operating program

Result calculation

Taking the standard substance concentration as 6 standard substance holes (the standard substance concentrations are 40, 20, 10, 5, 2.5 and 1.25ng/ml in sequence), adding 1 hole with 0 value and 7 concentration points), taking the corresponding absorbance (OD value) as the abscissa, using computer software, adopting four-parameter Logistic curve fitting (4 pl)), creating a standard curve equation, and calculating the concentration value of the sample by using the equation through the absorbance (OD value) of the sample.

The results are shown in fig. 7-9, lactobacillus reuteri LN021424h culture medium supernatant is co-cultured with IPEC-J2 cells for 4h, which significantly improves gene expression of BCAA metabolism related enzymes, significantly reduces intracellular BCAA content, and further increases intracellular BCAA metabolic end product acetyl COA and succinyl COA content. From this, lactobacillus reuteri LN0214 increases BCAA metabolism.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> agricultural university of south China

<120> Lactobacillus reuteri strain LN0214 for reducing serum branched-chain amino acid level, and derivative product and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

gactgcaaga tgggagggaa t 21

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

aatgatgcca tctagcgggg 20

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

tgtaccaagg caagttcctc ca 22

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctcgtgggct tttgttccgt 20

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gcagtcccgc aagaaggtga tg 22

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tcctgataca cgtccgagaa gagc 24

<210> 7

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

actttacaga gcagcagtgg aacag 25

<210> 8

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gcaacctctc ggatcacatg aacc 24

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ggagcagtgc atcctggatt t 21

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tccccaacac tggctacaac 20

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

tggaacggtg aaggtgacag c 21

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gcttttggga aggcagggac t 21

<210> 13

<211> 1327

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

tttaagagat tagcttactc tcgcgagctt gcgactcgtt gtaccgtcca ttgtagcacg 60

tgtgtagccc aggtcataag gggcatgatg atctgacgtc gtccccacct tcctccggtt 120

tgtcaccggc agtctcacta gagtgcccaa cttaatgctg gcaactagta acaagggttg 180

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacga ccatgcacca 240

cctgtcattg cgtccccgaa gggaacgcct tatctctaag gttagcgcaa gatgtcaaga 300

cctggtaagg ttcttcgcgt agcttcgaat taaaccacat gctccaccgc ttgtgcgggc 360

ccccgtcaat tcctttgagt ttcaaccttg cggtcgtact ccccaggcgg agtgcttaat 420

gcgttagctc cggcactgaa gggcggaaac cctccaacac ctagcactca tcgtttacgg 480

catggactac cagggtatct aatcctgttc gctacccatg ctttcgagcc tcagcgtcag 540

ttgcagacca gacagccgcc ttcgccactg gtgttcttcc atatatctac gcattccacc 600

gctacacatg gagttccact gtcctcttct gcactcaagt cgcccggttt ccgatgcact 660

tcttcggtta agccgaaggc tttcacatca gacctaagca accgcctgcg ctcgctttac 720

gcccaataaa tccggataac gcttgccacc tacgtattac cgcggctgct ggcacgtagt 780

tagccgtgac tttctggttg gataccgtca ctgcgtgaac agttactctc acgcacgttc 840

ttctccaaca acagagcttt acgagccgaa acccttcttc actcacgcgg tgttgctcca 900

tcaggcttgc gcccattgtg gaagattccc tactgctgcc tcccgtagga gtatggaccg 960

tgtctcagtt ccattgtggc cgatcagtct ctcaactcgg ctatgcatca tcgccttggt 1020

aagccgttac cttaccaact agctaatgca ccgcaggtcc atcccagagt gatagccaaa 1080

gccatctttc aaacaaaagc catgtggctt ttgttgttat gcggtattag catctgtttc 1140

caaatgttat cccccgctcc ggggcaggtt acctacgtgt tactcacccg tccgccactc 1200

actggtgatc catcgtcaat caggtgcaag caccatcaat cagttgggcc agtgcgtacg 1260

acttgcatgt attaggccac cgccggcgtc tctgaggggg ggaaaaaaaa aaaaaaaaac 1320

caaattt 1327

Claims

1. A lactobacillus reuteri strain LN0214 for reducing the level of serum branched-chain amino acids, characterized in that the lactobacillus reuteri strain LN0214 has the preservation number of cgmccno.24586.

2. A microbial agent for reducing serum branched-chain amino acid levels, characterized in that the active ingredient comprises lactobacillus reuteri strain LN0214 according to claim 1.

3. The microbial agent according to claim 2, wherein the live bacteria concentration of lactobacillus reuteri strain LN0214 is 1×10 ⁸ CFU/g or more.

4. Use of lactobacillus reuteri strain LN0214 according to claim 1 or of a microbial agent according to claim 2 or 3 for the manufacture of a medicament for reducing the level of serum branched-chain amino acids or for increasing the metabolism of serum branched-chain amino acids.

5. The use according to claim 4, wherein the branched-chain amino acids comprise one or more of the following: leucine, isoleucine and valine.

6. The use according to claim 4, wherein said increasing serum branched-chain amino acid metabolism comprises increasing the branched-chain amino acid metabolite content and increasing the expression level of a branched-chain amino acid metabolic pathway-related gene.

7. The use according to claim 6, wherein the branched-chain amino acid metabolites comprise acetyl COA and/or succinyl COA content;