Lactobacillus johnsonii and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a lactobacillus johnsonii (Lactobacillus johnsonii EU03) and an application thereof. The lactobacillus johnsonii can be used for improving the abundance of dominant intestinal flora such as lactobacillus (Lactobacillus), Muriaceae (p is less than 0.05), ackermansia (Akkermansia), Allobacter, Bacteroides(Bacteroides)(p is less than 0.05) and Blauria in the intestinal flora, and reducing the abundance of flora such as Romburia and Lacnospiraceae_NK4A136, so that the lactobacillus johnsonii has a positive regulation effect on a structure of the intestinal flora, thereby improving the symptoms such as metabolic syndrome and the like; and the lactobacillus johnsonii has effects of intestinal flora related metabolite regulation, trimethylamine oxide (TMAO) related metabolite choline concentration reduction, betaine concentration increase, short chain fatty acid (SCFAs) concentration increase in the intestinal flora, and cardiovascular disease alleviation.

Description

technical field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Lactobacillus johnsonii EU03 and its application, in particular to a Lactobacillus johnsonii EU03 capable of regulating intestinal flora and its metabolites and its application. Background technique

[0002] Gut flora is closely related to many diseases, mainly including gastrointestinal diseases, immune diseases, osteoporosis, cardiovascular diseases, metabolic diseases, malignant tumors and neurological diseases. Intestinal flora affects the host’s microbial ecosystem mainly by enhancing immune function, preventing or treating intestinal diseases such as diarrhea, constipation, and flatulence, helping to absorb nutrients and regulating metabolic abnormalities caused by intestinal flora imbalance, such as bone disease. Metabolism and Glucolipid Metabolism etc. Among intestinal probiotics, Lactobacillus sp. are Gram-positive bacteria and are classified as la...

Examples

Embodiment 1

[0044] Example 1: Isolation and Identification of Lactobacillus in C57B6 / J Mouse Gut

[0045] Fresh intestinal contents of C57B6 / J mice were diluted with sterile water at room temperature. Under sterile conditions, 100 mg of mouse intestinal content was taken in a test tube. Add 1 mL of sterile water, and centrifuge the sample at 4000 rpm for 1 minute. Every 50 μL supernatant was spread on the MRS plate and incubated at 37°C for 24 hours. By observing the characteristics of the colonies on the plate, colonies were screened from the plate for molecular biological identification.

[0046] Genomic DNA of strain EU03 was extracted by ctab / sds method and used as a template for PCR amplification. The 16s ribosomal DNA sequence of the strain was amplified and sequenced with universal primers 27f: 5'-agagttttgatggctcag-3' (SEQ ID NO: 1) and 1492r: 5'-acggttatccttgttaccgatt-3' (SEQ ID NO: 2). Amplification was performed using polymerase chain reaction in a PCR thermocycler (MyCycl...

Embodiment 2

[0048] Example 2: Regulatory effect of Lactobacillus johnsonii EU03 on the diversity of intestinal flora in healthy and MI rats

[0049] The experimental animals were 6-week-old male healthy SD rats with a body weight of 240±10 g, provided by Beijing Weitong Lihua Animal Experiment Center. The rats were raised in the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (Tianjin), at room temperature (22±2°C), relative humidity of 58-65%, and photoperiods of 12 hours alternately. In this study, 3-5 identically treated rats were tested per group.

[0050] Preparation of Lactobacillus agent: Inoculate Lactobacillus johnsonii EU03 into MRS liquid medium according to the inoculum size of 2%, culture at 37°C for 24h, take out the centrifugation (10000r / min, 10min, 4°C) and discard the supernatant, After washing twice with sterilized water, adjust the concentration of the bacterial solution to 10 with sterilized water. 8 CFU / mL (Colony Count: Transfer ...

Embodiment 3

[0061] Example 3: Regulatory effect of Lactobacillus johnsonii EU03 on the intestinal flora structure of healthy and MI rats

[0062] Use the Uparse software (Uparse v7.0.1001) to cluster all the Effective Tags of all samples. By default, the sequences are clustered into OTUs with 97% identity (Identity), and the representative sequences of OTUs will be selected, according to its algorithm principles , and select the sequence with the highest frequency in OTUs as the representative sequence of OTUs. Using the Mothur method and the SSUrRNA database of SILVA (http: / / www.arb-silva.de / ) to perform species annotation analysis on the representative sequences of OTUs (set the threshold value as 0.8-1), obtain taxonomic information and analyze them at each taxonomic level. : phylum (phylum) genus (genus) counts the community composition of each sample. The Kruskal-Wallis H test, referred to as Krusk's rank sum test, was used to analyze the significant differences among the species ...

technical field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a Lactobacillus johnsonii EU03 and its application, in particular to a Lactobacillus johnsonii EU03 capable of regulating intestinal flora and its metabolites and its application. Background technique

[0002] Gut flora is closely related to many diseases, mainly including gastrointestinal diseases, immune diseases, osteoporosis, cardiovascular diseases, metabolic diseases, malignant tumors and neurological diseases. Intestinal flora affects the host’s microbial ecosystem mainly by enhancing immune function, preventing or treating intestinal diseases such as diarrhea, constipation, and flatulence, helping to absorb nutrients and regulating metabolic abnormalities caused by intestinal flora imbalance, such as bone disease. Metabolism and Glucolipid Metabolism etc. Among intestinal probiotics, Lactobacillus sp. are Gram-positive bacteria and are classified as la...

Examples

Embodiment 1

[0044] Example 1: Isolation and Identification of Lactobacillus in C57B6 / J Mouse Gut

[0045] Fresh intestinal contents of C57B6 / J mice were diluted with sterile water at room temperature. Under sterile conditions, 100 mg of mouse intestinal content was taken in a test tube. Add 1 mL of sterile water, and centrifuge the sample at 4000 rpm for 1 minute. Every 50 μL supernatant was spread on the MRS plate and incubated at 37°C for 24 hours. By observing the characteristics of the colonies on the plate, colonies were screened from the plate for molecular biological identification.

[0046] Genomic DNA of strain EU03 was extracted by ctab / sds method and used as a template for PCR amplification. The 16s ribosomal DNA sequence of the strain was amplified and sequenced with universal primers 27f: 5'-agagttttgatggctcag-3' (SEQ ID NO: 1) and 1492r: 5'-acggttatccttgttaccgatt-3' (SEQ ID NO: 2). Amplification was performed using polymerase chain reaction in a PCR thermocycler (MyCycl...

Embodiment 2

[0048] Example 2: Regulatory effect of Lactobacillus johnsonii EU03 on the diversity of intestinal flora in healthy and MI rats

[0049] The experimental animals were 6-week-old male healthy SD rats with a body weight of 240±10 g, provided by Beijing Weitong Lihua Animal Experiment Center. The rats were raised in the Experimental Animal Center of Tianjin University of Traditional Chinese Medicine (Tianjin), at room temperature (22±2°C), relative humidity of 58-65%, and photoperiods of 12 hours alternately. In this study, 3-5 identically treated rats were tested per group.

[0050] Preparation of Lactobacillus agent: Inoculate Lactobacillus johnsonii EU03 into MRS liquid medium according to the inoculum size of 2%, culture at 37°C for 24h, take out the centrifugation (10000r / min, 10min, 4°C) and discard the supernatant, After washing twice with sterilized water, adjust the concentration of the bacterial solution to 10 with sterilized water. 8 CFU / mL (Colony Count: Transfer ...

Embodiment 3

[0061] Example 3: Regulatory effect of Lactobacillus johnsonii EU03 on the intestinal flora structure of healthy and MI rats

[0062] Use the Uparse software (Uparse v7.0.1001) to cluster all the Effective Tags of all samples. By default, the sequences are clustered into OTUs with 97% identity (Identity), and the representative sequences of OTUs will be selected, according to its algorithm principles , and select the sequence with the highest frequency in OTUs as the representative sequence of OTUs. Using the Mothur method and the SSUrRNA database of SILVA (http: / / www.arb-silva.de / ) to perform species annotation analysis on the representative sequences of OTUs (set the threshold value as 0.8-1), obtain taxonomic information and analyze them at each taxonomic level. : phylum (phylum) genus (genus) counts the community composition of each sample. The Kruskal-Wallis H test, referred to as Krusk's rank sum test, was used to analyze the significant differences among the species ...

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