Novel bifidobacterium strain Bifidobacterium faviflora IMAU50987

Abstract

The invention discloses a novel Bifidobacterium strain, namely IMAU50987, and belongs to the technical field of bifidobacteria. The invention further discloses a preparation method of the novel Bifidobacteria strain, namely IMAU50987. The invention provides a novel bifidobacterium strain IMAU50987, the 16S rRNA sequence of the strain is as shown in SEQ ID NO.1, and the strain is separated from cliff black honeycomb honey in Dai nationality, Jingyan nationality, Autonomous Dai nationality, Liangghe County, Dehong, Yunnan Province. The strain is preserved in Guangdong Microbial Culture Collection Center on August 8th, 2024, the address is the 5th floor of the Experimental Building, No.100 Court, Middle Xianlie Road, Guangzhou City, Guangdong Province, and the preservation number is GDMCC No: 64993, and the strain is preserved in Japan Microbial Culture Collection Center on October 3th, 2023, the address is Institute of Physical and Chemical Research, Tsubao City, Japan Shuzhou County, and the preservation number is JCM 36315. The novel bifidobacterium strain IMAU50987 grows well on an mMRS agar culture medium, the optimum growth temperature is 35 DEG C, the optimum pH is 6, the bacterial colony is round, semitransparent and convex after being cultured for 72 hours, and the diameter is about 1 mm; two drug-resistant genes and two virulence genes are retrieved in the genome, hemolysis is avoided, and the gene is sensitive to various antibiotics.

Description

Technical Field

[0001] The invention relates to the technical field of new bifidobacterium strains, in particular to a new bifidobacterium strain Bifidobacterium favimelis IMAU50987. Background Art

[0002] Bifidobacterium belongs to the Actinobacteria, Bifidobacteriales, Bifidobacteriaceae. It is a genus of Gram-positive, non-motile, rod-shaped cells, sometimes bifurcated at one end, and strictly anaerobic bacteria. Bifidobacterium is widely present in the digestive tract, vagina, and oral cavity of humans and animals. It is an important member of the intestinal flora of humans and animals. It is a physiologically beneficial bacteria that has many important physiological functions for human health, such as biological barrier, nutritional effect, anti-tumor effect, immune enhancement effect, improvement of gastrointestinal function, anti-aging, etc. Some species of Bifidobacterium are used as probiotics and are widely used in food, medicine, and feed.

[0003] As of October 28, 2024, a total of 102 validly published Bifidobacterium species have been published on LPSN (List of Prokaryotic Names with Standing in Nomenclature, https: / / lpsn.dsmz.de / search?word=Bifidobacterium, a website dedicated to the classification of prokaryotes), among which Bifidobacterium adolescentis, Bifidum bifidum, and Bifidobacterium animalis are the most studied and reported species. The discovery of new species has many scientific values ​​and social significance for biodiversity, ecosystems, evolutionary biology research, as well as the potential application development of species and disease prevention. Summary of the invention

[0004] The purpose of the present invention is to provide a new species of Bifidobacterium favimelisIMAU50987, so as to provide a new species of Bifidobacterium favimelis for biodiversity, ecosystem, evolutionary biology research, potential application development of strains, disease prevention, etc.

[0005] To achieve the above-mentioned purpose, the present invention provides a new species of Bifidobacterium favimelis IMAU50987, which was deposited in Guangdong Microbiological Culture Collection Center on August 8, 2024, with the address of 5th Floor, Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 64993; it was deposited in Japan Microbiological Collection Center on October 3, 2023, with the address of RIKEN, Tsukuba City, Ibaraki Prefecture, Japan, and the deposit number is JCM 36315.

[0006] Preferably, the sequence of 16SrRNA of a new species of Bifidobacterium favimelis IMAU50987 is shown as SEQ ID NO.1.

[0007] Preferably, the new Bifidobacterium species IMAU50987 can be distinguished from its closely related Bifidobacterium strains by 16SrRNA sequence and housekeeping genes pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp sequences.

[0008] Preferably, the new Bifidobacterium species IMAU50987 belongs to the B. asteroides group.

[0009] Preferably, compared with the type strain of the B. asteroides group, the new species of Bifidobacterium IMAU50987 has a maximum ANI value of 77.0%, a maximum AAI value of 73.2%, and a maximum dDDH value of 23.1%, all of which are lower than the threshold for classification as a new species.

[0010] Preferably, Bifidobacterium sp. IMAU50987 does not utilize glucose compared to its closely related Bifidobacterium strains.

[0011] Preferably, the new species of Bifidobacterium IMAU50987 has unique polar lipids PL and PGL compared with its closely related Bifidobacterium strains, wherein PL is PL2 and PL3, and PGL is PGL2 and PGL3.

[0012] The method for culturing the new species of Bifidobacterium favimelis IMAU50987 as described above can grow under strict anaerobic / microaerobic / aerobic conditions using mMRS agar medium and / or culture solution.

[0013] Preferably, the new species of Bifidobacterium favimelis IMAU50987 grows best under strict anaerobic conditions and is passaged at an inoculum size of 2%.

[0014] Preferably, the mMRS agar medium and / or culture solution contains 0.05% L-cysteine ​​hydrochloride.

[0015] Preferably, the pH of the mMRS agar medium and / or culture solution is 5-9, and the culture temperature is 20-42°C.

[0016] Preferably, the optimum pH of the new species of Bifidobacterium IMAU50987 is 6, and the optimum culture temperature is 35°C.

[0017] Therefore, the present invention provides a new species of Bifidobacterium favimelis IMAU50987, and its specific technical effects are as follows:

[0018] (1) The present invention provides a new species of Bifidobacterium IMAU50987, the 16S rRNA sequence of which is shown in SEQ ID NO. 1, which is isolated and screened from cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, and deposited in Guangdong Microbiological Culture Collection Center on August 8, 2024, with the address: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 64993; and deposited in Japan Microbiological Culture Collection Center on October 3, 2023, with the address: RIKEN Institute, Tsukuba City, Ibaraki Prefecture, Japan, with the deposit number JCM 36315;

[0019] (2) The present invention provides a new strain of Bifidobacterium species IMAU50987, which can be distinguished from the type strain of the Bifidobacterium B.asteroides group by 16S rRNA and housekeeping gene (pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp) sequences;

[0020] (3) The new species of Bifidobacterium IMAU50987 provided by the present invention can be distinguished from the type strain of the Bifidobacterium B.asteroides group by ANI value, AAI value and dDDH value; compared with the type strain of the Bifidobacterium B.asteroides group, its ANI value is up to 77.0%, AAI value is up to 73.2%, and dDDH value is up to 23.1%, all of which are lower than the threshold for classification of new species;

[0021] (4) The new Bifidobacterium species IMAU50987 provided by the present invention does not utilize glucose compared to its closely related Bifidobacterium strains;

[0022] (5) The new Bifidobacterium species IMAU50987 provided by the present invention has unique polar lipids PL (PL2 and PL3) and PGL (PGL2 and PGL3) compared with its closely related Bifidobacterium strains;

[0023] (6) The new species of Bifidobacterium IMAU50987 provided by the present invention grows well on mMRS agar medium, with an optimal growth temperature of 35°C and an optimal pH of 6. After 72 hours of culture, the colonies are round, translucent, convex, and about 1 mm in diameter;

[0024] (7) A total of 2 drug-resistant genes and 2 virulence genes were retrieved from the new Bifidobacterium species IMAU50987 provided by the present invention. It has no hemolytic phenomenon and is sensitive to multiple antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0026] Figure 1 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the 16S rRNA sequence in Example 1 of the present invention;

[0027] Figure 2 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the dnaB housekeeping gene sequence in Example 1 of the present invention;

[0028] Figure 3 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the glnA1 housekeeping gene sequence in Example 1 of the present invention;

[0029] Figure 4 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the pyrG housekeeping gene sequence in Example 1 of the present invention;

[0030] Figure 5 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the recA housekeeping gene sequence in Example 1 of the present invention;

[0031] Figure 6The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the rpoC housekeeping gene sequence in Example 1 of the present invention;

[0032] Figure 7 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the thrS housekeeping gene sequence in Example 1 of the present invention;

[0033] Figure 8 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the tuf housekeeping gene sequence in Example 1 of the present invention;

[0034] Fig. 9 The maximum likelihood tree of the new species of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group constructed based on the xfp housekeeping gene sequence in Example 1 of the present invention;

[0035] Fig.10 This is a heat map of the whole genome analysis of the new species of Bifidobacterium IMAU50987 and the type strain of the Bifidobacterium B.asteroides group in Example 1 of the present invention, wherein (A) is the average nucleotide identity (ANI), (B) is the average amino acid identity (AAI), and (C) is DNA-DNA hybridization (dDDH);

[0036] Fig.11 This is a graph showing the Gram staining result of Bifidobacterium IMAU50987, a new species of Bifidobacterium in Example 2 of the present invention;

[0037] Fig.12 This is a morphological result of the new species of Bifidobacterium IMAU50987 under electron microscope in Example 2 of the present invention;

[0038] Fig.13 The polar lipid map of the new species of Bifidobacterium IMAU50987 and its closely related strains in Example 3 of the present invention; wherein A is Bifidobacteriumfavimelis IMAU50987; B is B.choladohabitans B14384H11 T ;C is Bifidobacterium mellis Bin7N T ;

[0039] Fig.14 This is a COG functional annotation map of the new species of Bifidobacterium IMAU50987 in Example 4 of the present invention;

[0040] Fig.15 This is a CAZy function annotation diagram of the new Bifidobacterium species IMAU50987 in Example 4 of the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0042] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0043] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0044] Embodiment 1

[0045] A new species of Bifidobacterium, Bifidobacteriumfavimelis IMAU50987, was isolated from cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province.

[0046] The molecular identification of the new species of Bifidobacterium favimelis IMAU50987 was carried out as follows:

[0047] (1) Classification and identification of 16S rRNA.

[0048] The strain DNA was extracted using the TIANamp Bacteria DNA Kit. The DNA fragment concentration and purity were detected by 0.8% agarose gel electrophoresis and a micro-UV spectrophotometer. 260 / OD 280 The ratio is between 1.7 and 2.0, indicating that the purity of the DNA is qualified and can be used for subsequent experiments. The DNA that meets the purity requirements is amplified by polymerase chain reaction (PCR), and the universal bacterial primers of the 16S rRNA gene are used for amplification and sequencing.

[0049] The 16S rRNA gene universal bacterial primers selected were: forward primer 27F, reverse primer 1492R. The PCR amplification system was 50 μL: 1.5 μL each of the forward and reverse primers, 4 μL of dNTP, 10× buffer (amplification buffer, containing Mg 2+ ) 5 μL, r-Taq enzyme (Taq DNA polymerase) 0.5 μL, DNA template 2 μL, ddHO 2O 35.5μL. PCR amplification conditions: pre-denaturation at 94℃ for 5min; denaturation at 94℃ for 1min, annealing at 58℃ for 1min, extension at 72℃ for 2min, 30 cycles of denaturation, annealing and extension; terminal extension at 72℃ for 10min, PCR product was terminated at 4℃ and stored.

[0050] 2 μL of PCR amplification product was electrophoresed using 1% agarose gel at 100V for 15 minutes, and the electrophoresis bands were observed under ultraviolet light. The PCR products with bright electrophoresis bands and no tailing were selected and sent to Shanghai Paisono Biotechnology Co., Ltd. for purification and bidirectional sequencing under low temperature storage conditions. The results showed that the sequence of 16S rRNA was as shown in SEQ ID NO.1.

[0051] SEQ ID NO.1:

[0052]

[0053] The sequencing results were analyzed by BLAST in the EzBioCloud database. The model strain sequence with high homology was selected as a reference, and the strain phylogenetic tree was constructed using MEGA7.0. The results are shown in Figure 1 EzBioCloudblast results showed that the closest relative of strain IMAU50987 was Bifidobacterium mellis Bin7N T The similarity of the relationship was 98.5%. The phylogenetic tree constructed by 16S rRNA showed that strain IMAU50987 was different from other closely related Bifidobacterium type strains and formed a separate branch. If the 16S rRNA gene sequence alignment result was less than 98.65% consistent with the known reference sequence, it was considered a new species. The above results preliminarily identified strain IMAU50987 as a new species of Bifidobacterium. In order to further determine the phylogenetic status of strain IMAU50987, housekeeping genes were used for identification.

[0054] (2) Classification and identification of housekeeping genes.

[0055] In order to further determine the taxonomic status of the new species of Bifidobacterium IMAU50987, a total of eight housekeeping genes, including pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp, were selected to construct a phylogenetic tree for analysis. The results showed that ( Figure 2-Figure 9 ), IMAU50987 and the type strain of the Bifidobacterium B.asteroides group formed a well-separated close cluster. This was consistent with the phylogenetic tree constructed from 16S rRNA sequences. It was further determined that strain IMAU50987 was a new species of Bifidobacterium.

[0056] (3) Identification of basic genome characteristics.

[0057] After obtaining the pure culture of Bifidobacteriumfavimelis IMAU50987, whole genome sequencing was performed, and the basic characteristics of the strain genome are shown in Table 1.

[0058] Table 1 Basic characteristics of the genome of the new Bifidobacterium species IMAU50987

[0059] Basic Features IMAU50987 Genomesize 1.9Mb Number of scaffolds 12 ScaffoldN50 1.6Mb DNAG+C content (mol%) 63.5％ Number of contigs 34 Genome coverage 150.0x Accessionnumber JBANDZ000000000

[0060] (4) Average nucleotide identity (ANI), average amino acid identity (AAI) and genomic DNA-DNA hybridization analysis.

[0061] The average nucleotide identity (ANI) was calculated using an online analysis platform (https: / / www.ezbiocloud.net / ) to calculate the ANI values ​​of the new Bifidobacterium species IMAU50987 and the type strain of the Bifidobacterium B.asteroides group.

[0062] The ANI value results are shown in Table 2. B. choladohabitans B14384H11 T The comparison value with Bifidobacteriumfavimelis IMAU50987 is the highest, which is 77.0%. This shows that the ANI values ​​of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group are both lower than the same species classification threshold (95-96%). The Bifidobacterium favimelis IMAU50987 provided by the present invention is a new species of Bifidobacterium. The visualized heat map is as follows: Fig.10 shown.

[0063] The average amino acid identity (AAI) was obtained using an online amino acid identity calculator (http: / / enve-omics.ce.gatech.edu / aai / ).

[0064] The AAI values ​​are shown in Table 2. B. choladohabitans B14384H11 T The comparison value with Bifidobacteriumfavimelis IMAU50987 is the highest, which is 73.02%. This indicates that the average amino acid consistency values ​​of Bifidobacterium IMAU50987 and the model strain of the Bifidobacterium B.asteroides group are both lower than the same species classification threshold (95%), further indicating that the Bifidobacteriumfavimelis IMAU50987 provided by the present invention is a new species of Bifidobacterium. The visualized heat map is shown in FIG. Fig.10 shown.

[0065] DNA-DNA hybridization based on genomic information is an important criterion for identifying bacterial species. In order to further clarify the similarity between Bifidobacterium IMAU50987 and the type strain of the Bifidobacterium B.asteroides group, DDH online hybridization was performed. The results are shown in Table 2. Using GG DC (Genome-to-Genome Distance Calculator, http: / / ggdc.dsmz.de / dis tcalc2.php) for comparison, Bifidobacterium IMAU50987 and its closely related species B. chola dohabitans B14384H11 T 、B.mellis Bin7N T 、B.mizhiense S053-2 T 、B.asteroides DSM 20089 T , B.polysaccharolyticum W8117 T 、B.apo usia W8102 T 、B.indicum JCM 1302 T 、B.xylocopaeXV2 T and B. actinocoloniiforme DSM 22766 T The comparison values ​​were 22.7%, 22.8%, 22.6%, 22.8%, 22.8%, 22.5%, 22.8%, 23.0% and 22.3%, all less than 70%. See the heat map for visualization Fig.10 Therefore, the dDDH value showed that the strain was an independent genomic species, which further supported that the strain Bifidobacterium favimelis IMAU50987 was a new species of the genus Bifidobacterium.

[0066] In summary, the ANI, AAI and dDDH matching values ​​of Bifidobacteriumfavimelis IMAU50987 and the type strain of the Bifidobacterium B.asteroides group were lower than the new species classification thresholds of 95%, 95% and 70%, respectively, indicating that Bifidobacteriumfavimelis IMAU50987 did not belong to any known species and was a new species of the genus Bifidobacterium.

[0067] Table 2 ANI, AAI and dDDH values ​​of Bifidobacteriumfavimelis IMAU50987 and the type strain of Bifidobacterium asteroides

[0068]

[0069]

[0070] Embodiment 2

[0071] The morphological observation of the obtained new species of Bifidobacterium IMAU50987 was carried out as follows:

[0072] After obtaining the pure culture of Bifidobacteriumfavimelis IMAU50987, the colony morphology of Bifidobacteriumfavimelis IMAU50987 was observed after 72 hours of culture on mMRS solid medium under strict anaerobic conditions. The colonies on mMRS solid medium were round, translucent, convex, and about 1 mm in diameter. The Gram staining results were as follows Fig.11 As shown in the figure, Bifidobacteriumfavimelis IMAU50987 is a Gram-positive bacterium that does not move. Fig.12 As shown, under electron microscopy, the bacteria showed typical characteristics of Bifidobacterium: individual bacteria were arranged in short rods, without spores and flagella.

[0073] Embodiment 3

[0074] The obtained new species of Bifidobacterium IMAU50987 was identified by biochemical experiments, as follows:

[0075] (1) Investigate the pH tolerance of Bifidobacteriumfavimelis IMAU50987.

[0076] The inoculum was inoculated into mMRS liquid medium at a rate of 2%, and the culture solution of the strain was propagated to the third generation under strict anaerobic conditions, and then inoculated into mMRS liquid medium with pH values ​​of 4, 5, 6, 7, 8, and 9 at a rate of 2%, respectively, and cultured at 37°C for 24 hours before streaking on a plate. After 48 hours of plate culture, it was observed whether it grew on the plate. The results are shown in Table 3.

[0077] Table 3 pH tolerance of the new species of Bifidobacterium IMAU50987

[0078] pH 4 5 6 7 8 9 10 IMAU50987 - + + + + + -

[0079] Note: "+" indicates that the plant can grow under this condition, "-" indicates that the plant cannot grow under this condition

[0080] (2) Investigate the temperature tolerance of Bifidobacteriumfavimelis IMAU50987.

[0081] The culture solution of the strain was inoculated into mMRS liquid medium after three generations at an inoculum volume of 2%, and the test tubes were placed at 4°C, 15°C, 25°C, 35°C, 37°C, 45°C, and 60°C for culture. After 24 hours, the plate was streaked. After 48 hours, it was observed whether it grew on the plate. The results are shown in Table 4.

[0082] Table 4 Temperature tolerance results of the new species of Bifidobacterium IMAU50987

[0083] temperature 4℃ 15℃ 25℃ 35℃ 37℃ 45℃ 60℃ IMAU50987 - - - + + + -

[0084] Note: "+" means it can grow under this condition, "-" means it cannot grow under this condition

[0085] (3) API 50CHL biochemical reaction identification of Bifidobacteriumfavimelis IMAU50987.

[0086] Use a sterile inoculation loop to pick up a colony that grows well on the mMRS solid medium, connect it to a PBS buffer solution tube for thorough mixing and dilution, and compare it with the standard bacterial suspension turbidity bottle (McFarland turbidity 2.0) in the kit. Use a pipette to draw 180 μL of the diluted bacterial solution and mix it with the culture medium in the kit. First add sterile triple distilled water to the culture box of the API 50CHL identification card to maintain the humidity of the cell culture environment, then add the mixed bacterial solution to the sugar plate, and finally use sterilized glycerol to isolate oxygen. Place it in a 37°C incubator for anaerobically culture, and observe the color change of the API 50CHL identification card at 24h and 48h respectively and record the color change of the identification card. Because bacteria use carbohydrates to ferment and produce acid, the pH drops, causing the indicator to change color. A positive result refers to the acid production indicated by the yellowing of the bromocresol purple indicator contained in the culture, and a negative result refers to the absence of a reaction in the culture. The results are shown in Table 5.

[0087] Table 5 API 50CHL identification results of the new species of Bifidobacterium IMAU50987

[0088]

[0089]

[0090]

[0091] Note: 1. "-" means negative, "+" means positive, "w" means delayed reaction; 2. A: BifidobacteriumfavimelisIMAU50987; B: B.choladohabitans CGMCC 1.18892 T ; C: B.mellisBin7NT

[0092] (4) API ZYM biochemical reaction identification of Bifidobacteriumfavimelis IMAU50987.

[0093] Use a cotton swab or inoculation loop to pick up a single colony that has been separated and purified from the plate of the new species of Bifidobacterium IMAU50987 isolate and put it into a 2mL PBS buffer solution test tube to prepare a bacterial suspension with a turbidity of 6.0. Take 65μL of the prepared bacterial suspension and inject it into each small cup in the reagent strip, cover the lid of the culture box, and culture it in an anaerobic environment at 37℃ for 4h. After culture, add 1 drop of ZYMA and 1 drop of ZYMB reagent to each small hole. After 5 minutes, color will develop. If it is positive, place the reagent strip under a strong light source (1000W bulb) for 10s, and place the bulb on top of the cup for 4s. After placing the test strip in sunlight for 5 minutes, read the result. The result is determined according to the biochemical reaction table of the kit.

[0094] Table 6 API ZYM identification results of the new species of Bifidobacterium IMAU50987

[0095]

[0096]

[0097] Note: 1. "-" means negative, "+" means positive, "w" means delayed reaction; 2. A: BifidobacteriumfavimelisIMAU50987; B: B.choladohabitans GCMCC 1.18892 T ; C: B.mellisBin7N T

[0098] (4) Polar lipid determination of Bifidobacteriumfavimelis IMAU50987.

[0099] The polar lipids produced by the strain were identified by thin layer chromatography (TLC). The TLC system used in the detection was: first phase: chloroform: methanol: distilled water = 65:25:4 (V / V); second phase: chloroform: glacial acetic acid: methanol: distilled water = 80:18:12:5 (V / V). The color developers used for the detection results were: phosphomolybdate, ninhydrin, molybdenum blue and 1-methylnaphthol.

[0100] The polar lipids diphosphoglycerate (DPG), phosphatidylethanolamine (PE), three phospholipids (PL), phosphatidylglycerol (PG), three unidentified phosphoglycolipids (PGL) and five unidentified glycolipids (GL) were identified in the new species of Bifidobacterium IMAU50987. Fig.13 shown. Bifidobacteriumfavimelis IMAU50987, B.choladohabitansB14384H11 T and B.mellis Bin7N T The major polar lipids of all strains are DPG. However, Bifidobacterium sp. IMAU50987 has unique unidentified polar lipids PL (PL2 and PL3) and PGL (PGL2 and PGL3).

[0101] Embodiment 4

[0102] Functional genomic analysis of the obtained new species of Bifidobacterium IMAU50987 was performed as follows:

[0103] (1) COG functional genomic annotation analysis.

[0104] The rational classification of proteins encoded by bacterial genomes is crucial for the study of their functions and evolution. In order to gain a deeper understanding of the functional gene characteristics in the genome of Bifidobacteriumfavimelis IMAU50987, the genome of the new species of Bifidobacterium favimelis IMAU50987 was functionally annotated and analyzed using the COG database. Fig.14 As shown, the results showed that in the new species of Bifidobacterium IMAU50987, the genes related to metabolism accounted for the highest proportion, which was 41.99%, with 598 genes, which were related to carbohydrate transport and metabolism (G category), amino acid transport and metabolism (E category), etc.; followed by genes involved in information storage and modification (Information Storage and Processing), accounting for 24.43%, with 348 genes, including translation, ribosome structure and biogenesis (J category), transcription (K category), replication, recombination and repair (L category); and genes related to cell growth and signal transduction (Cellular Processes and Signaling) and poorly characterized (Poorly Characterized) also accounted for a high proportion.

[0105] (2) Annotation analysis of carbohydrate-active enzymes.

[0106] The Carbohydrate Active Enzyme Database (CAZy) is a database that displays and analyzes the genomic structure and biochemical information of carbohydrate active enzymes based on the amino acid sequence similarity of protein domains. The database divides carbohydrate active enzymes into six major families, which are mainly composed of five types of catalytic enzymes and one type of non-catalytic modules. Catalytic enzymes include glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), glycosyltransferases (GTs) and auxiliary oxidoreductases (AAs), while non-catalytic modules are carbohydrate-binding modules (CBMs).

[0107] The CAZy annotation of the new species of Bifidobacterium IMAU50987 and the genome of the closely related model strains of Bifidobacterium was performed using the carbohydrate-active enzyme online annotation website, and the annotation information of CAZy enzymes was obtained as follows: Fig.15 As shown, a total of 25 subclass gene families of three major categories of carbohydrate-active enzymes (GHs, GTs, and CBMs) were annotated to the new species of Bifidobacterium IMAU50987.

[0108] Glycoside hydrolases (GHs) consist of glycosidases and transglycosidases, which are responsible for the hydrolysis and transglycosylation of glycosidic bonds. GHs encoding genes are distributed in most genomes and are the most abundant. They are mainly used in biotechnology and medicine, and are also the enzyme group with the best biochemical characteristics in the existing CAZy database. A total of 17 functional subclasses of the GHs family have been annotated. GH13, GH3, GH32 and GH65 have been annotated in the new species of Bifidobacterium IMAU50987 and related strains of Bifidobacterium, among which GH3 and GH32 are more abundant. The GH3 class mainly encodes glucosidases and xylosidases, both of which are widely present in bacteria, fungi and plants, and are mainly involved in cellulose metabolism. GH32 is responsible for the hydrolysis or synthesis of fructan glycosidic bonds, including hydrolases such as inulinase, sucrase, and fructanase, and fructosyltransferases.

[0109] Glycosyltransferases (GTs) are a class of enzymes that catalyze the transfer of sugar groups from activated donor molecules to specific acceptor molecules and form glycosidic bonds. They are widely distributed in lactobacilli and are related to the synthesis pathways of disaccharides, oligosaccharides and polysaccharides in bacterial life activities. They play an essential role in the adaptability and pathogenicity of microorganisms. A total of 7 GTs families were annotated, of which GT2, GT20, GT28, GT39 and GT4 were annotated in all strains.

[0110] Carbohydrate binding modules (CBMs) are a class of non-catalytic domain enzymes that facilitate the binding of proteins to polysaccharides, with the main function being the attachment of carbohydrates, which enables the enzyme to recognize and bind to its insoluble substrates, for example, mediating the binding of proteins to starch. The CBMs family has only been annotated in Bifidobacterium spp. IMAU50987, as CBM48.

[0111] (3) Analysis of drug resistance genes.

[0112] The protein sequence of the new species of Bifidobacterium IMAU50987 was compared with the drug resistance gene database (CARD). The results are shown in Table 7. It was found that when the nucleotide identity was greater than 70%, a total of 2 drug resistance genes were retrieved: Bado_rpoB_RIF and Bbif_ileS_MUP. The Bbif_ileS_MUP gene mediates mupirocin resistance, and the Bado_rpoB_RI gene mediates rifampicin resistance.

[0113] Table 7 Major drug resistance genes in the genome of the new Bifidobacterium species IMAU50987

[0114] Gene type AROID antibiotic Nucleotide identity% rpoB ARO:3004480 Rifampicin 88.98 ileS ARO:3003730 Mupirocin 79.30

[0115] (4) Virulence gene analysis.

[0116] The protein sequence of the new species of Bifidobacterium IMAU50987 was compared with the Virulence Factor Database (VFDB), and the results are shown in Table 8. In the case of nucleotide identity greater than 70%, two virulence genes were annotated: Rv0440 and sigA / rpoV. Rv0440 can bind to short fragments of protein substrates and stabilize unfolded or partially folded proteins, thereby preventing these proteins from aggregation and degradation; the encoding product of sigA / rpoV is the RNA polymerase sigma factor, which interacts with the transcription activator WhiB3 to allow the expression of genes required for virulence.

[0117] Table 8 Major virulence genes in the genome of the new species of Bifidobacterium IMAU50987

[0118] Virulence Factors Coding product Nucleotide identity% Rv0440 Molecular chaperone GroEL 73.66 sigA / rpoV RNA polymerase sigma factor 73.20

[0119] (5) Antibiotic sensitivity test.

[0120] The paper diffusion method, also known as the Kirbv-Bauer method, or KB method for short, is used. Several single colonies of Bifidobacterium IMAU50987 are picked and directly inoculated into 4.5% physiological saline to prepare a bacterial suspension. The turbidity of the suspension is adjusted to 0.5 McFarland units using a turbidimeter. After adjusting the turbidity of the suspension, it is evenly spread on the MH solid culture medium. The plate with the bacterial solution is placed at room temperature for 5 minutes so that the agar can absorb excess surface moisture before placing the paper containing the drug. The antimicrobial drug paper is attached to the agar surface inoculated with bacteria. Each paper must be pressed down to ensure complete contact with the agar surface, and the paper must be evenly distributed. The distance between the centers of the two paper is not less than 24 mm, and the distance between the edge of the paper and the edge of the agar is not less than 15 mm. Within 15 minutes after the paper is placed, the MH agar culture medium is inverted in a 35°C constant temperature incubator and cultured for 24 hours.

[0121] The strain sensitivity was interpreted according to the CLSI standard, and the test bacteria were reported as sensitive, intermediate and resistant to the test drugs according to the diameter of the inhibition ring. The antibiotics used included: penicillin, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, neomycin, piperacillin, carbenicillin, oxacillin, kanamycin, tetracycline, doxycycline, minocycline, cefoperazone, ceftazidime, cefuroxime, cephradine, ceftriaxone and cephalexin. The results are shown in Table 9.

[0122] Table 9 Sensitivity of the new species of Bifidobacterium IMAU50987 to different antibiotics

[0123]

[0124]

[0125] Therefore, the present invention provides a new species of Bifidobacterium favimelis IMAU50987, the 16S rRNA sequence of which is shown in SEQ ID NO.1, which is isolated and screened from cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, and is deposited in Guangdong Microbiological Culture Collection Center on August 8, 2024, with the address of 5th Floor, Compound Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC No: 64993; the new species of Bifidobacterium IMAU50987 grows well on mMRS agar medium, with an optimal growth temperature of 35°C and an optimal pH of 6. After culturing for 72 hours, the colonies are round, translucent, convex, and about 1 mm in diameter; a total of 2 drug resistance genes and 2 virulence genes were retrieved from the genome, there is no hemolysis, and it is sensitive to multiple antibiotics.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A new species of Bifidobacterium favimelis IMAU50987 was deposited in the Guangdong Provincial Microbiological Culture Collection on August 8, 2024, with the address: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number GDMCC No: 64993; it was deposited in the Japan Microbiological Collection on October 3, 2023, with the address: RIKEN Institute, Tsukuba City, Ibaraki Prefecture, Japan, with the deposit number JCM 36315.

2. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: The sequence of 16S rRNA of Bifidobacterium new species IMAU50987 is shown in SEQ ID NO.

1.

3. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: The new Bifidobacterium species IMAU50987 can be distinguished from its closely related Bifidobacterium strains by 16S rRNA sequence and housekeeping genes pyrG, thrS, glnA1, recA, tuf, dnaB, rpoC and xfp sequences.

4. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: The new species of Bifidobacterium IMAU50987 belongs to the B.asteroides group.

5. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: Compared with the type strain of the B. asteroides group, the new species of Bifidobacterium IMAU50987 had the highest ANI value of 77.0%, the highest AAI value of 73.2%, and the highest dDDH value of 23.1%, all of which were lower than the threshold for classification into a new species.

6. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: Compared with its closely related Bifidobacterium strains, the new Bifidobacterium species IMAU50987 does not utilize glucose.

7. The new species of Bifidobacterium favimelis IMAU50987 according to claim 1, characterized in that: Compared with its closely related Bifidobacterium strains, the new species of Bifidobacterium IMAU50987 has unique polar lipids PL and PGL, PL is PL2 and PL3, and PGL is PGL2 and PGL3.

8. A method for culturing the new species of Bifidobacterium favimelis IMAU50987 according to any one of claims 1 to 7, characterized in that: The strain can grow under strict anaerobic / microaerobic / aerobic conditions using mMRS agar medium and / or culture medium.

9. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 8, characterized in that: The new species of Bifidobacterium IMAU50987 grows best under strict anaerobic conditions and is passaged at an inoculum size of 2%.

10. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 8, characterized in that: The mMRS agar medium and / or the culture solution contains 0.05% L-cysteine ​​hydrochloride.

11. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 8, characterized in that: The pH of the mMRS agar medium and / or the culture solution is 5-9, and the culture temperature is 20-42°C.

12. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 8, characterized in that: The optimum pH value and culture temperature of the new Bifidobacterium species IMAU50987 are 6 and 35℃, respectively.

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