A new species of Bifidobacterium, Bifidobacterium favimelis IMAU50987

Abstract

The present invention discloses a new species of Bifidobacterium favimelis IMAU50987, which belongs to the field of bifidobacterium technology. 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 from the cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province. It was deposited in the Guangdong Provincial Microbial Culture Collection on August 8, 2024, with the address being: 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, with the deposit number being GDMCC No: 64993, and was deposited in the Japan Microbial Culture Collection on October 3, 2023, with the address being: RIKEN Institute, Tsukuba City, Ibaraki Prefecture, Japan, with the deposit number being JCM 36315. The new species of Bifidobacterium IMAU50987 grows well on mMRS agar medium, with the optimal growth temperature of 35°C and the optimal pH of 6. After 72 hours of culture, 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 hemolytic phenomenon and it is sensitive to multiple antibiotics.

Description

Technical Field

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

[0002] Bifidobacterium belongs to the phylum Actinobacteria, order Bifidobacteriales, family Bifidobacteriaceae. It is a genus of Gram-positive, non-motile, rod-shaped bacteria that sometimes have a bifurcated end and are strictly anaerobic. Bifidobacterium is widely found in the digestive tract, vagina, and oral cavity of humans and animals. It is a key component of the intestinal flora of humans and animals and is a physiologically beneficial bacterium. It has multiple important physiological functions for human health, including biological barrier function, nutritional effects, anti-tumor effects, immune enhancement, gastrointestinal function improvement, and anti-aging effects. Some species of Bifidobacterium are used as probiotics in food, medicine, and feed.

[0003] As of October 28, 2024, 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 prokaryotic taxonomic information. Among them, B. adolescentis, B. bifidum, and B. animalis are the most studied and reported species. The discovery of new species has multifaceted scientific value and social significance for biodiversity, ecosystems, evolutionary biology research, as well as potential applications and disease prevention. Summary of the Invention

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

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

[0006] Preferably, the sequence of 16S rRNA 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 16S rRNA 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 Bifidobacterium species 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 to its closely related Bifidobacterium strains, wherein PL is PL2 and PL3, and PGL is PGL2 and PGL3.

[0012] The above-mentioned new species of Bifidobacterium favimelis IMAU50987 can be cultured using mMRS agar medium and / or culture fluid and can grow under strictly anaerobic / microaerobic / aerobic conditions.

[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 optimal pH of the new species of Bifidobacterium IMAU50987 is 6, and the optimal culture temperature is 35°C.

[0017] Therefore, the present invention provides a new species of Bifidobacterium, Bifidobacterium favimelis IMAU50987, which has the following specific technical effects:

[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. The new species was isolated and screened from cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, and was deposited in Guangdong Provincial Microbial 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 was deposited in Japan Microbial Culture Collection Center on October 3, 2023, with the address: RIKEN, Tsukuba City, Ibaraki Prefecture, Japan, with the deposit number JCM 36315;

[0019] (2) The present invention provides a new strain of Bifidobacterium 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 present invention provides a new species of Bifidobacterium IMAU50987, which can be distinguished from the type strain of the Bifidobacterium B.asteroides group by its ANI value, AAI value, and dDDH value. Compared with the type strain of the Bifidobacterium B.asteroides group, its ANI value is as high as 77.0%, its AAI value is as high as 73.2%, and its dDDH value is as high as 23.1%, all of which are lower than the threshold for classification as a 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 two drug-resistant genes and two 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 following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

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

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

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

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

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

[0036] Figure 11 This is a Gram staining result of the new species of Bifidobacterium IMAU50987 in Example 2 of the present invention;

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

[0038] Figure 13 Polar lipid map of the new species IMAU50987 and related strains in Example 3 of the present invention; A is Bifidobacterium favimelis IMAU50987; B is B. coladohabitans B14384H11 T ;C is Bifidobacterium mellis Bin7N T ;

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

[0040] Figure 15 This is a functional annotation diagram of CAZy 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 with reference to the accompanying drawings and embodiments.

[0042] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details 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 application belongs.

[0043] The instruments, equipment, reagents and materials used in the examples were obtained from commercial sources.

[0044] Example 1

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

[0046] Molecular identification of the new species of Bifidobacterium favimelis IMAU50987 was performed using the following steps:

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

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

[0049] The 16S rRNA gene universal bacterial primers used were: forward primer 27F, reverse primer 1492R. The PCR amplification system was 50 μL: 1.5 μL each of 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, ddH2O 35.5 μL. PCR amplification conditions: initial denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, 30 cycles of denaturation, annealing, and extension; terminal extension at 72°C for 10 min. PCR products were terminated at 4°C and stored.

[0050] 2 μL of the PCR amplification product was subjected to electrophoresis on a 1% agarose gel at 100 V for 15 minutes. The electrophoretic bands were observed under UV light. PCR products with bright electrophoretic bands and no tailing were selected and stored at low temperatures. They were then sent to Shanghai Paisonno Biotechnology Co., Ltd. for purification and bidirectional sequencing. The results showed that the 16S rRNA sequence was shown in SEQ ID NO. 1.

[0051] SEQ ID NO.1:

[0052]

[0053] The sequencing results were compared and 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 the figure below. Figure 1 EzBioCloudblast results showed that the closest relative of strain IMAU50987 was Bifidobacterium mellis Bin7N T The similarity between strains was 98.5%. A phylogenetic tree constructed using 16S rRNA sequencing revealed that strain IMAU50987 was distinct from other closely related Bifidobacterium type strains, forming a separate branch. 16S rRNA gene sequence alignments with known reference sequences that showed less than 98.65% identity were considered novel species. These results preliminarily identified strain IMAU50987 as a new species of Bifidobacterium. To further confirm 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 formed a well-separated, closely related cluster with the type strain of the Bifidobacterium asteroides group. This was consistent with the phylogenetic tree constructed from 16S rRNA sequences. This further confirmed that strain IMAU50987 was a new species of Bifidobacterium.

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

[0057] After obtaining pure culture of Bifidobacterium favimelis 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 Accession number JBANDZ000000000

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

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

[0062] The ANI values ​​are shown in Table 2. T The highest comparison value with Bifidobacterium favimelis IMAU50987 is 77.0%. This indicates 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 shown in FIG. Figure 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. T The highest comparison value with Bifidobacterium favimelis IMAU50987 is 73.02%. This indicates that the average amino acid identity between Bifidobacterium IMAU50987 and the type strain of the Bifidobacterium asteroides group is lower than the threshold for classification as a species (95%), further indicating that the Bifidobacterium favimelis IMAU50987 provided by the present invention is a new species of Bifidobacterium. The visualized heat map is shown in FIG. Figure 10 shown.

[0065] DNA-DNA hybridization based on genomic information is an important criterion for identifying bacterial species. To further clarify the similarity between Bifidobacterium IMAU50987 and the type strain of the Bifidobacterium 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), Bifidobacterium IMAU50987 and its closely related species B. chola dohabitans B14384H11 were compared. 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%, respectively, all less than 70%. Figure 10 Therefore, the dDDH value showed that the strain was an independent genomic species, further supporting 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 Bifidobacterium favimelis IMAU50987 with the type strain of the Bifidobacterium asteroides group were lower than the new species classification thresholds of 95%, 95% and 70%, respectively, indicating that Bifidobacterium favimelis IMAU50987 does not belong to any known species and is a new species of the genus Bifidobacterium.

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

[0068]

[0069]

[0070] Example 2

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

[0072] After obtaining a pure culture of Bifidobacterium favimelis IMAU50987, the colony morphology of Bifidobacterium favimelis 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: Figure 11 As shown, Bifidobacterium favimelis IMAU50987 is a Gram-positive bacterium that does not move. Figure 12 As shown in the figure, electron microscopic observation showed that the bacteria exhibited typical characteristics of bifidobacteria: individual bacteria were arranged in short rods, without spores or flagella.

[0073] Example 3

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

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

[0076] A 2% inoculum was inoculated into mMRS liquid medium. The culture was passaged three times under strict anaerobic conditions. Subsequently, a 2% inoculum was inoculated into mMRS liquid medium at pH values ​​of 4, 5, 6, 7, 8, and 9. The inoculum was then inoculated again into mMRS liquid medium at pH values ​​of 4, 5, 6, 7, 8, and 9. The cells were incubated at 37°C for 24 hours and then streaked onto plates. After 48 hours of incubation, the plates were observed for growth. The results are shown in Table 3.

[0077] Table 3 pH tolerance of the new Bifidobacterium species 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] After three passages of culture, the strain was inoculated into mMRS liquid medium at a 2% inoculum. The tubes were incubated at 4°C, 15°C, 25°C, 35°C, 37°C, 45°C, and 60°C. After 24 hours, the plates were streaked. After 48 hours, the plates were observed for growth. 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: "+" indicates that the plant can grow under these conditions, "-" indicates that the plant cannot grow under these conditions.

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

[0086] Use a sterile inoculating loop to pick well-grown colonies on mMRS solid medium, transfer them to a PBS buffer tube, mix thoroughly, and dilute them. Compare the results to the standard bacterial suspension turbidity vial (McFarland turbidity 2.0) provided in the kit. Use a pipette to draw 180 μL of the diluted bacterial suspension and mix it with the culture medium in the kit. Sterile triple-distilled water is first added to the incubator containing the API 50CHL identification card to maintain humidity. The mixed bacterial suspension is then added to the sugar plate, and sterilized glycerol is used to isolate the cells from oxygen. The cells are incubated anaerobically at 37°C. The color change of the API 50CHL identification card is observed and recorded after 24 and 48 hours. Since bacteria ferment carbohydrates to produce acid, the pH decreases, causing the indicator to change color. A positive result indicates acid production, indicated by the yellowing of the bromocresol purple indicator in the culture, while a negative result indicates no reaction. 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. "-" indicates negative, "+" indicates positive, and "w" indicates delayed reaction; 2. A: Bifidobacterium favimelis IMAU50987; B: B. choladohabitans CGMCC 1.18892 T ;C:B.mellisBin7NT

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

[0093] Use a cotton swab or inoculating loop to pick a single, isolated and purified colony from the plate containing the new Bifidobacterium species IMAU50987 and transfer it to a 2mL test tube containing PBS buffer to prepare a bacterial suspension with a McFarland turbidity of 6.0. Inject 65μL of the prepared bacterial suspension into each small cup in the test strip, close the lid of the incubator, and incubate at 37°C for 4 hours under anaerobic conditions. After incubation, add 1 drop of ZYMA and 1 drop of ZYMB reagent to each well. Wait 5 minutes for color to develop. If positive, place the test strip under a strong light source (1000W bulb) for 10 seconds, then place the bulb on top of the cup for 4 seconds. After exposing the test strip to sunlight for 5 minutes, read the result. The result is determined according to the biochemical reaction chart in the test kit.

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

[0095]

[0096]

[0097] Note: 1. "-" indicates negative, "+" indicates positive, and "w" indicates delayed reaction; 2. A: Bifidobacterium favimelis IMAU50987; B: B. choladohabitans GCMCC 1.18892 T ;C:B.mellisBin7N T

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

[0099] 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] 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. Figure 13 shown. Bifidobacteriumfavimelis IMAU50987, B.choladohabitansB14384H11 T and B.mellis Bin7N T The major polar lipids in the strains are DPG. However, the new strain of Bifidobacterium sp. IMAU50987 has unique unidentified polar lipids PL (PL2 and PL3) and PGL (PGL2 and PGL3).

[0101] Example 4

[0102] Functional genomic analysis of the obtained new Bifidobacterium species 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 studying their functions and evolution. In order to gain a deeper understanding of the functional gene characteristics in the genome of Bifidobacterium favimelis IMAU50987, the genome of the new species of Bifidobacterium IMAU50987 was functionally annotated and analyzed using the COG database. The COG gene annotation results are shown in Figure 2. Figure 14 As shown, the results showed that in the new species of Bifidobacterium IMAU50987, genes related to metabolism accounted for the highest proportion, accounting for 41.99%, with a total of 598 genes, which were related to carbohydrate transport and metabolism (G category) and amino acid transport and metabolism (E category); followed by genes involved in information storage and modification (Information Storage and Processing), accounting for 24.43%, with a total of 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 amino acid sequence similarity within protein domains. The database classifies carbohydrate-active enzymes into six major families, primarily composed of five types of catalytic enzymes and one type of non-catalytic module. 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 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: Figure 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, responsible for the hydrolysis and transglycosylation of glycosidic bonds. GHs-encoding genes are distributed across most genomes and are the most abundant. They are primarily used in biotechnology and medicine and represent the best biochemically characterized enzyme group in the existing CAZy database. Seventeen functional subclasses of the GHs family have been annotated. GH13, GH3, GH32, and GH65 have been annotated in the new Bifidobacterium species IMAU50987 and related strains of Bifidobacterium, with GH3 and GH32 being more abundant. GH3s primarily encode glucosidases and xylosidases, both of which are widely found in bacteria, fungi, and plants and primarily involved in cellulose metabolism. GH32s are responsible for the hydrolysis or synthesis of fructan glycosidic bonds and include hydrolases such as inulinase, sucrase, and fructanase, as well as fructosyltransferases.

[0109] Glycosyltransferases (GTs) are enzymes that catalyze the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. They are widely distributed in Lactobacilli and are involved in the biosynthesis of disaccharides, oligosaccharides, and polysaccharides in bacterial life, playing essential roles in microbial adaptability and pathogenicity. Seven GT families were annotated, of which GT2, GT20, GT28, GT39, and GT4 were annotated in all strains.

[0110] Carbohydrate linking modules (CBMs) are a class of enzymes with non-catalytic domains that facilitate the binding of proteins to polysaccharides. Their primary function is carbohydrate attachment, enabling the enzyme to recognize and bind to insoluble substrates, for example, mediating the binding of proteins to starch. This CBM family has only been annotated in the new Bifidobacterium species IMAU50987, as CBM48.

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

[0112] The protein sequence of the new Bifidobacterium species IMAU50987 was compared with the CARD database. The results, shown in Table 7, revealed that two resistance genes, Bado_rpoB_RIF and Bbif_ileS_MUP, were identified with nucleotide identity greater than 70%. The Bbif_ileS_MUP gene mediates mupirocin resistance, while the Bado_rpoB_RI gene mediates rifampicin resistance.

[0113] Table 7 Major drug-resistant 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 Bifidobacterium species IMAU50987 was aligned with the Virulence Factor Database (VFDB). The results are shown in Table 8. With nucleotide identity greater than 70%, two virulence genes were annotated: Rv0440 and sigA / rpoV. Rv0440 binds to short fragments of protein substrates, stabilizing unfolded or partially folded proteins and preventing their aggregation and degradation. sigA / rpoV encodes an RNA polymerase sigma factor, which interacts with the transcriptional activator WhiB3, allowing the expression of genes required for virulence.

[0117] Table 8 Major virulence genes in the genome of the new Bifidobacterium species 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 Kirbv-Bauer method (KB method) was used. Several individual colonies of Bifidobacterium IMAU50987 were selected and directly inoculated into 4.5% saline to create a bacterial suspension. The turbidity of the suspension was adjusted to 0.5 McFarland units using a turbidimeter. After the suspension had adjusted turbidity, it was evenly spread onto MH solid medium. The plate with the bacterial solution was left at room temperature for 5 minutes to allow the agar to absorb excess surface moisture before the drug-containing paper disc was placed. Antimicrobial drug discs were applied to the inoculated agar surface, with each disc pressed down to ensure complete contact with the agar surface. The discs were evenly distributed, with the center distance between the two discs being no less than 24 mm and the edge of the disc no less than 15 mm from the edge of the agar. Within 15 minutes of placing the discs, the MH agar medium was inverted and placed in a 35°C incubator for 24 hours.

[0121] Strain susceptibility was determined according to CLSI standards, with the diameter of the inhibition zone used to report the sensitivity, intermediate, and resistance of the tested bacteria to the tested drugs. The antibiotics used included penicillin, ampicillin, cefazolin, amikacin, gentamicin, erythromycin, neomycin, piperacillin, carbenicillin, oxacillin, kanamycin, tetracycline, doxycycline, minocycline, cefoperazone, ceftazidime, cefuroxime, cefradine, ceftriaxone, and cephalexin. The results are shown in Table 9.

[0122] Table 9 Sensitivity of the new Bifidobacterium species 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. The strain was isolated and screened from cliff black beehive honey in Lianghe County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, and was deposited in the Guangdong Provincial Microbial Culture Collection on August 8, 2024, with the address being: 5th Floor, Laboratory 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 hemolytic phenomenon, and it is sensitive to multiple antibiotics.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. 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 solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A new species of Bifidobacterium favimelis IMAU50987 was deposited on August 8, 2024 in the Guangdong Provincial Microbial Culture Collection, located at 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 on October 3, 2023 in the Japan Microbial Culture Collection, located at RIKEN, Tsukuba City, Ibaraki Prefecture, Japan, with the deposit number JCM 36315.

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

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

4. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 2, characterized in that: The mMRS agar medium and / or culture solution contains 0.05% L-cysteine ​​hydrochloride.

5. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 2, characterized in that: The pH of the mMRS agar medium and / or culture solution is 5-9, and the culture temperature is 20-42°C.

6. The method for culturing a new species of Bifidobacterium favimelis IMAU50987 according to claim 2, characterized in that: The optimum pH and culture temperature of the new Bifidobacterium species IMAU50987 are 6 and 35℃, respectively.

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