Identification markers of proteus hauseri xbdl-0121 in angelica tibetica and application of flavones production

Abstract

The application belongs to the field of microbial resource development and bio-fermentation technology, discloses identification marks of a strain of XBDL-0121 of Tibetan angelica endophytic proteus and application of flavones, the strain is preserved under the number of CGMCC No.37465, is separated from Tibetan angelica, and can stably synthesize 12 kinds of flavone components with functional isoflavones as the main body, wherein the content of the main 6 kinds exceeds 0.4 ng / mg, and the content of the characteristic core component genistein is significantly dominant.The application simultaneously discloses a strain separation and identification method, a flavone fermentation production and extraction and purification process, and a strain flavone synthesis related functional gene.The application breaks the traditional cognition of the metabolic function of proteus genus, solves the technical problem of the production of flavones by traditional plant extraction method, the strain has excellent fermentation performance, the process is green and low-cost, the product can be widely used in the fields of medicine, food, agriculture and the like, and provides a new resource and technical path for the large-scale green production of flavones.

Description

Technical Field

[0001] This invention relates to the fields of microbial resource development, natural product synthesis and bio-fermentation technology, specifically to an endophytic Proteus XBDL-0121 isolated from Angelica sinensis, and a method for producing flavonoids by fermentation using this strain. The products can be applied in the fields of medicine, health products, food additives and agricultural biostimulants. Background Technology

[0002] Flavonoids are a class of polyphenolic secondary metabolites with broad pharmacological activities. They exhibit clear efficacy in antioxidation, anti-inflammation, regulation of tumor cell proliferation, and improvement of glucose and lipid metabolism, and have become core raw materials in the pharmaceutical, food, and cosmetic industries, with market demand continuing to rise. However, the traditional method of obtaining flavonoids mainly relies on plant extraction, which has several unavoidable limitations: First, flavonoids are present in extremely low concentrations in plants and are mostly bound to cell wall components, resulting in low extraction efficiency and the consumption of large amounts of plant materials; second, plant cultivation is strictly limited by geographical environment, climate conditions, and growth cycle, leading to insufficient stability in raw material supply, and large-scale harvesting can easily cause ecological damage; third, organic solvents are commonly used in the extraction process, which not only increases production costs but may also cause environmental residues and product pollution, severely restricting the large-scale production and application of flavonoids.

[0003] Plant endophytic bacteria, as a group of microorganisms that colonize plant tissues and form symbiotic relationships with their hosts, have gradually acquired secondary metabolic potentials similar to those of their hosts through long-term co-evolution. They are capable of synthesizing various host-related bioactive compounds, making them an important resource for screening novel functional strains. Tibetan Angelica (Angelica pubescens), a rare medicinal plant unique to the Qinghai-Tibet Plateau of my country, grows in a special ecological environment of high altitude and strong ultraviolet radiation. It accumulates abundant flavonoids, volatile oils, and other bioactive components. This unique growth environment may have led to the evolution of unique metabolic mechanisms in the endophytic microbial community of Tibetan Angelica, providing a valuable resource for screening strains with special metabolic functions.

[0004] Proteus sp., belonging to the order Enterobacteriaceae, has been the subject of conventional research on metabolic functions, primarily focusing on urease activity and biofilm formation, with few reports on its synthesis of flavonoids. This invention, for the first time, isolates an endophytic Proteus sp. strain XBDL-0121 from Angelica sinensis. This strain can stably synthesize flavonoids, and its metabolites are consistent with the main medicinal active ingredients of Angelica sinensis. This provides a novel microbial resource to address the problems of traditional flavonoid extraction, such as reliance on plant resources, high cost, and low efficiency, and aligns with the industrial demands of green production and sustainable development. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Proteus xanthipes* XBDL-0121 from *Angelica sinensis* capable of efficiently synthesizing flavonoids. Characteristic genes for strain identification have been screened, and a method for fermenting and producing flavonoids using this strain is provided. This aims to solve the problems of existing flavonoid production methods, which rely on plant extraction, have low yields, and are costly, thereby expanding the resources and technical pathways for microbial synthesis of natural products. To achieve the above objectives, this invention provides the following technical solutions:

[0006] 1. A strain of Proteus xanthipes from Angelica sinensis, wherein the strain is Proteus sp. XBDL-0121, which was deposited on January 19, 2026 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37465.

[0007] 2. The aforementioned Proteus sp. XBDL-0121 was isolated from the tissue of Angelica sinensis plant. Its taxonomic classification is: Kingdom Bacteria, Phylum Pseudomonadota, Class Gammaproteobacteria, Order Enterobacterales, Family Morganellaceae, Genus Proteus. After culturing on TSA solid medium for 24-48 hours, the strain formed smooth, uniformly sized colonies, indicating it is a facultative anaerobic Gram-negative bacterium.

[0008] 3. The 16S rRNA gene of Proteus sp. XBDL-0121 strain, the sequence of which is shown in SEQ ID No.1 of the sequence listing, and its length is 1269 bp.

[0009] 4. Application of the 16S rRNA gene of Proteus sp. XBDL-0121 in the identification of this strain. The gene sequence is shown in SEQ ID No. 1, with a length of 1269 bp. When the 16S rRNA gene sequence of the test strain has more than 99.5% identity with the sequence shown in SEQ ID No. 1, it is identified as Proteus sp. XBDL-0121.

[0010] 5. The fermentation products of *Proteus sp.* XBDL-0121 were analyzed qualitatively and quantitatively for specific flavonoid compounds using metabolomics sequencing. Results showed that six stable high-content flavonoid components were detected, with concentrations ranging from 0.40 to 11.00 ng / mg. These components covered two core categories: free isoflavone aglycones and conjugated isoflavone glycosides, demonstrating a complete and well-defined flavonoid composition. Among the detected high-content flavonoid components, genistein, a core functional glycoside active ingredient in soybean isoflavones, achieved stable high abundance in the sample, reaching 1.77 ng / mg. This was the highest content among all conjugated glycoside flavonoids detected, significantly higher than similar glycoside components such as daidzein (1.26 ng / mg) and genistein (0.64 ng / mg), indicating its dominant position among the functional glycoside flavonoids in the sample. Several other high-abundance flavonoid components were also detected in this test, listed in descending order of content as follows: daidzein (10.91 ng / mg), genistein (0.95 ng / mg), and epicatechin (0.41 ng / mg). Overall, the high-content flavonoid components in this sample are mainly functional isoflavones, with genistein as the characteristic core functional component. Its content level is significantly higher than that of similar glycoside components, providing a crucial material basis for the stable physiological activity of this sample and fully demonstrating the differentiated characteristics and functional advantages of the flavonoid composition in this sample. Figure 1 Table 1 details the specific information on the flavonoid compounds produced.

[0011] 6. Application of Proteus sp. XBDL-0121 strain in the production of flavonoids, wherein the flavonoids are genistein, daidzein, genistein, daidzein, daidzein, and epicatechin.

[0012] 7. The method for isolating and purifying Proteus XBDL-0121 includes the following steps: (1) Pretreatment of Angelica sinensis samples: Select healthy and disease-free Angelica sinensis plants, take their root and stem tissues, rinse the surface impurities with sterile water, soak in 75% ethanol for 30s, disinfect with 0.1% mercuric chloride solution for 5min, and finally rinse with sterile water 5 times to remove the surface disinfectant. Under sterile conditions, cut the tissue into small pieces of 0.5cm×0.5cm; (2) Isolation of endophytic bacteria: Inoculate the treated Angelica sinensis tissue pieces onto TSA solid medium plates, and incubate at 28℃ for 48h. After colony growth appears on the plates, pick single colonies with different morphologies; (3) Purification of strains: Inoculate the picked single colonies onto new TSA solid medium plates, purify them by streak isolation method, repeat streak 3 times to obtain purified single colonies, and then purify them by 16S. rRNA identification clarified its isolation status; (4) Flavonoid yield screening: the purified single colony was inoculated into TSB medium and cultured at 28℃ and 180 r / min for 72h. The fermentation broth was extracted with ethyl acetate and concentrated under reduced pressure. The content of flavonoids was detected by ultraviolet spectrophotometer combined with HPLC. The strain that produces flavonoids, XBDL-0121, was screened.

[0013] 8. The species identification method of Proteus XBDL-0121 includes the following steps: (1) Morphological observation: Strain XBDL-0121 was inoculated on TSA solid medium and cultured at 28℃ for 24-48 h. The colony morphology, color, size and edge characteristics were observed. Gram staining was used. After primary staining with crystal violet, mordant staining with iodine solution, decolorization with 95% ethanol and counterstaining with safranin, the morphology of the cells was observed under an oil microscope. (2) Scanning electron microscopy (SEM) morphological characterization: Strain XBDL-0121 was fixed, dehydrated, dried and sputter-coated with gold and then observed under a scanning electron microscope. The results showed that the cells had a typical rod-shaped morphology with blunt ends. The cells were uniform, with an average length of about 4.560 μm and a diameter of about 657.9 nm (0.658 μm). The length-to-width ratio was about 6.93:1. The surface of the cells had obvious fine wrinkles, indicating that its cell wall structure was complex and layered. Multiple fine filamentous flagella are visible in the polar regions of the bacterial cell, consistent with the motility phenotype of this strain. Figure 2(3) Detection of physiological and biochemical characteristics: Determine the carbon source utilization capacity, nitrogen source utilization capacity, acid and alkali resistance, temperature adaptability and other physiological and biochemical indicators of the strain; (4) 16S rRNA gene sequencing identification: Extract the genomic DNA of the strain, and use the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for PCR amplification and sequencing identification. At the same time, the whole genome was sequenced using PacBio third-generation sequencing technology, the 16S rRNA gene sequence was annotated, and it was compared with the known sequences in the NCBI GenBank database for homology. The phylogenetic tree was constructed using MEGA software to determine its taxonomic position.

[0014] 9. A fermentation method for producing flavonoids, comprising the following steps:

[0015] (1) Preparation of primary seed culture: The preserved strain XBDL-0121 was streaked onto TSA solid medium and incubated at 28℃ for 24-48 h to activate it. Single colonies were picked and inoculated into 10 mL TSB medium and cultured at 28℃ and 180 r / min for 12 h with shaking.

[0016] (2) Preparation of secondary seed culture: The primary seed culture was transferred to 100 mL TSB medium at an inoculation rate of 1%, and cultured at 28 °C and 180 r / min for 2 h with shaking until the bacterial concentration reached the appropriate level;

[0017] (3) Preparation of fermentation medium: Add 15 g / L tryptone, 5 g / L soybean peptone and 5 g / L NaCl, and make up to 1000 mL with ddH2O. Autoclave at 120 °C for 30 min and cool for later use.

[0018] (4) Fermentation culture: The secondary seed culture was inoculated into a 250 mL shake flask containing 100 mL of fermentation medium and fermented for 3 days at an initial pH of 7.0, a temperature of 28 °C and a shaking speed of 180 r / min.

[0019] (5) Fermentation process control: During fermentation, samples are taken regularly to monitor the bacterial concentration, pH changes and flavonoid accumulation to ensure the stability of the fermentation system.

[0020] 10. A method for extracting and purifying flavonoids, comprising the following steps:

[0021] (1) Pretreatment of fermentation broth: After fermentation, the fermentation broth was centrifuged at 5000 r / min for 15 min and the supernatant was collected;

[0022] (2) Extraction: Add 2 times the volume of ethyl acetate to the supernatant, shake and extract 3 times, 15 min each time, and combine the organic phases after the 3 extractions;

[0023] (3) Concentration: The organic phase is concentrated to dryness under reduced pressure to obtain crude flavonoids;

[0024] (4) Purification: The crude product was purified by silica gel column chromatography with petroleum ether-ethyl acetate (volume ratio 5:1) as the eluent. The eluent was collected, and the target components were combined by HPLC. After being concentrated again under reduced pressure, high-purity flavonoids were obtained.

[0025] 11. Genome analysis and flavonoid-related gene screening methods for Proteus sp. XBDL-0121, including: the whole genome of strain Proteus sp. XBDL-0121 contains one chromosome and two plasmids, with an average GC content of 38.66%. Figure 4 As shown in Table 2, a total of 3481 protein-coding genes (CDS) were predicted, and 83 tRNAs and 25 rRNAs were annotated. In the KEGG annotation results, 44 genes were annotated to other secondary metabolite biosynthetic pathways, such as... Figure 5 Note: The genome of strain XBDL-0121 is predicted to contain gene1205 encoding aroA (3-phosphate shikimate-1-carboxyvinyltransferase). This enzyme is a key rate-limiting enzyme in the shikimate pathway, which catalyzes the generation of branched acids, providing core precursors such as phenylalanine and tyrosine for flavonoid synthesis. This pathway is crucial for ensuring the supply of precursors for flavonoid biosynthesis in strain XBDL-0121. Figure 6 The KS (ketoacyl synthase) encoded by genes 3254 and 3255, along with key flavonoid synthesis enzymes such as chalcone synthase (CHS) and type III PKS, belong to the Thiolase-like (Thiolase-like superfamily, IPR016039) group. They share a common ancient α / β-sheet catalytic origin, retain the core KS carbon chain condensation catalytic activity, and possess the structural basis for evolving into aromatic polyketide synthesis functions. See details... Figure 7 The 23 glycosyltransferase and SDR oxidoreductase encoding genes, including gene0023 and gene1160, provide a complete set of genetic safeguards for flavonoid nucleus cyclization, aromatization, and terminal activity modification. Table 3 lists the detailed information of 26 related gene series after alignment with the reference gene at NCBI. The complete sequences can be found in the nucleotide sequence listing.

[0026] 12. A gene related to flavonoid synthesis from Proteus XBDL-0121, said gene comprising the sequences listed in SEQ ID No. 2 to SEQ ID No. 27.

[0027] Compared with the prior art, the outstanding advantages of the present invention are:

[0028] (1) Novel strain resources: For the first time, Proteus XBDL-0121, which can efficiently synthesize flavonoids, was isolated from Angelica sinensis, breaking the traditional understanding of the metabolic function of strains of this genus and filling the resource gap of flavonoid production by endophytic fungi of Angelica sinensis.

[0029] (2) Excellent fermentation performance: Through systematic optimization of fermentation conditions, the yield of flavonoids is significantly improved compared with the initial fermentation, the content of the core component genistein is stable, and the fermentation process does not require expensive raw materials, resulting in low cost;

[0030] (3) Green and environmentally friendly: The production of flavonoids by microbial fermentation technology avoids the dependence on natural resources and environmental damage caused by traditional plant extraction. The reaction conditions are mild and there is no organic solvent residue, which is in line with the concept of green production.

[0031] (4) Plant-microbe interaction model: This study is the first to discover endophytic bacteria in Angelica sinensis that are homologous to and structurally similar to recognized flavonoid-producing strains, providing a novel microbial resource for flavonoid biosynthesis. Its innovation lies in combining the special habitat (plateau plants), the characteristics of endophytic bacteria, and known functional predictions, which not only expands the diversity of flavonoid-producing strains, but also provides a unique model for elucidating the mechanism of plant-microbe interaction in producing active ingredients, and has important resource and theoretical innovation value.

[0032] (5) Wide range of applications: The strain's metabolites are consistent with the active medicinal ingredients of Angelica sinensis and have high safety. In the field of medicine and health, they can be used to develop natural drugs or health products with antioxidant and anti-inflammatory functions. In the field of green agriculture, they have the potential to be used as biostimulants or biopesticides to promote crop health and reduce the use of chemical agents. In the field of industrial biotechnology, it is expected to achieve sustainable and large-scale production of flavonoids through fermentation engineering, and promote the development of the big health industry and biomanufacturing industry. Attached Figure Description

[0033] Figure 1 Metabolomics sequencing of the strain's fermentation broth revealed high levels of six flavonoids, including daidzein and genistein, in the fermentation products.

[0034] Figure 2 The image shows a scanning electron microscope (SEM) image of strain XBDL-0121, revealing its rod-shaped morphology, surface wrinkled texture, and polar flagella structure.

[0035] Figure 3 A phylogenetic tree was constructed based on the highest similarity sequence of the 16S rRNA gene of Proteus sp. strain XBDL-0121 as determined by the NCBI website. Figure 3 A) and gene sequence structure diagram ( Figure 3 B). Among them. Figure 3 In B, the underlined characters represent the start and stop codons.

[0036] Figure 4 This is a diagram showing the complete genome of the strain. Figure 4 A-major chromosome related information loop diagram (3,812,067 bp); Figure 4 B is a related information loop diagram of plasmid A (1,049,59 bp); Figure 4 C is a circle diagram of information related to plasmid B (5,005 bp). Note: From the outside to the inside, the first and fourth circles represent CDS on the positive and negative strands, with different colors indicating different COG functional classifications; the second and third circles represent CDS, tRNA, and rRNA on the positive and negative strands, respectively; the fifth circle represents GC content, with the outer part indicating that the GC content in this region is higher than the average GC content of the whole genome, and the inner part indicating that the GC content in this region is lower than the average GC content of the whole genome, with higher peak values ​​indicating a larger difference from the average GC content; the sixth circle represents the GC-Skew value, where green represents a positive GC skew (G > C), and purple represents a negative GC skew (G < C); the innermost circle is a genome size indicator.

[0037] Figure 5 Annotation diagram of the KEGG pathway for strain.

[0038] Figure 6 The aroA (3-phosphoshikimate-1-carboxyvinyltransferase) contained in the gene1205 can catalyze the generation of branched acid, which is the core guarantee for the supply of precursors for the biosynthesis of flavonoids.

[0039] Figure 7 The ketoacyl synthase (KS) contained in genes 3254 and 3255 shares a common ancient α / β-sheet catalytic origin with key flavonoid synthesis enzymes such as chalcone synthase (CHS) and type III PKS. Figure 7 A: The antiSMASH encoding genes gene3254 and gene3255 contain ketoacyl synthase (KS). Figure 7 B: InterPro single-gene domain analysis; Figure 7 Details of the C:Thiolase-like family. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the invention clearer, specific embodiments of the invention will be described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the drawings. The embodiments of the invention shown in and described with reference to the drawings are merely exemplary, and the invention is not limited to these embodiments. It should also be noted that, in order to avoid obscuring the technical solutions of the invention with unnecessary details, only structures and / or processing steps closely related to the solutions according to the invention are shown in the drawings, while other details that are not closely related are omitted.

[0041] Example 1

[0042] This embodiment provides a method for isolating and purifying *Proteus xanthipes* XBDL-0121 from *Angelica sinensis*, specifically including:

[0043] (1) Sample pretreatment: Select healthy Tibetan Angelica plants from the Qinghai-Tibet Plateau production area, take root tissue, rinse with sterile water, soak in 75% ethanol for 30 s, disinfect with 0.1% mercuric chloride solution for 5 min, rinse with sterile water 5 times, and cut into 0.5cm×0.5cm small pieces under sterile conditions;

[0044] (2) Isolation and culture: Tissue blocks were inoculated onto TSA solid medium plates and incubated at 28°C for 48 h. Milky white, smooth single colonies appeared on the plates.

[0045] (3) Purification and screening: Single colonies were picked and purified three times by streak method to obtain purified strains; the purified strains were inoculated into TSA liquid medium and cultured at 28 ℃ and 180 r / min for 3 days. After the fermentation broth was extracted with ethyl acetate and concentrated, flavonoid-producing strains were screened by HPLC detection.

[0046] Example 2

[0047] This embodiment provides a method for species identification of *Proteus xanthipes* XBDL-0121 from *Angelica sinensis*, specifically including:

[0048] (1) Morphological identification: After strain XBDL-0121 was cultured on TSA solid medium at 28 ℃ for 24-48 h, the colonies were milky white, smooth, with regular edges and a diameter of 1-2 mm; after Gram staining and oil immersion, it was observed to be a Gram-negative bacterium with short rod-shaped cells.

[0049] (2) 16S rRNA sequencing: Genomic DNA of the strain was extracted, and its 16S rRNA gene was amplified by PCR. The amplified product was bidirectionally sequenced, and the full-length sequence obtained by splicing was compared with the NCBI database for homology. The results showed that the sequence had the highest homology with the model strain of Proteus mirabilis (ATCC 29906).

[0050] (3) Whole-genome sequencing and 16S rRNA gene sequence analysis: Genomic DNA was extracted from strain XBDL-0121, and whole-genome sequencing was performed using the PacBio platform. After assembly and annotation, the complete 16S rRNA gene sequence, totaling 1269 bp, was obtained. The target nucleotide sequence was searched for homology in the core_nt database using the NCBI BLASTN tool. The alignment results showed that the sequence had the highest similarity to several previously reported Proteus mirabilis strains, with 100% sequence homology, 100% coverage, and an E value of 0, indicating extremely high reliability. The phylogenetic tree constructed based on the BLAST alignment results showed ( Figure 3 This strain clustered with Proteus mirabilis and belonged to the genus Proteus.

[0051] (4) Physiological and biochemical characteristics: This strain is a facultative anaerobe and can utilize carbon sources such as corn flour and glucose, and nitrogen sources such as ammonium sulfate and potassium nitrate. The optimal growth temperature is 28-33 ℃ and the optimal pH is 6.0-7.0.

[0052] (5) Scanning electron microscopy (SEM) morphological observation: The activated strain XBDL-0121 was fixed overnight in 2.5% glutaraldehyde, washed with phosphate buffer, dehydrated by ethanol gradient, replaced with tert-butanol, freeze-dried, and sputtered with gold. The bacteria were then observed and photographed under a scanning electron microscope. The results showed that the bacteria were rod-shaped with wrinkled surfaces and obvious flagella in the polar regions. The average length was about 4.560 μm and the diameter was about 657.9 nm.

[0053] Example 3

[0054] This embodiment provides a qualitative and quantitative analysis method for flavonoids produced by strain XBDL-0121, specifically including:

[0055] (1) Sample preparation: The fermentation broth of XBDL-0121 in Example 1 was centrifuged, extracted with ethyl acetate, concentrated, and then dissolved in anhydrous ethanol to prepare the sample solution;

[0056] (2) Quantitative analysis: A standard curve was plotted using rutin as a standard, and the absorbance of the sample was measured using an ultraviolet spectrophotometer. The total flavonoid content was calculated by substituting the rutin into the standard curve equation.

[0057] (3) Quantitative and qualitative analysis: The content of each monomer flavonoid was determined by liquid chromatography-mass spectrometry (LC-MS). The results showed that the high content of flavonoids in the sample was mainly daidzein, with a content of 10.91 ng / mg. The contents of the other high content flavonoids, genistein, daidzein, daidzein, and epicatechin were 1.77 ng / mg, 1.26 ng / mg, 0.95 ng / mg, 0.64 ng / mg, and 0.41 ng / mg, respectively.

[0058] Example 4

[0059] This embodiment provides optimization of fermentation conditions, specifically including:

[0060] (1) Single-factor experiment: The effects of carbon source type and concentration, nitrogen source type and concentration, initial pH, fermentation temperature, fermentation time and shaking speed on flavonoid yield were investigated. The results showed that the optimal single-factor conditions were: corn flour 90 g / L, ammonium sulfate 42 g / L, initial pH 6.0, fermentation temperature 33 ℃, fermentation time 4 d, and shaking speed 180 r / min.

[0061] (2) Plackett-Burman design experiment: Six factors were selected: carbon source concentration, nitrogen source concentration, initial pH, fermentation temperature, fermentation time, and rotation speed. Twelve sets of experiments were designed, and three factors that significantly affected flavonoid production were screened out: carbon source concentration, nitrogen source concentration, and fermentation time.

[0062] (3) Box-Behnken design experiment: with significant influencing factors as independent variables and flavonoid yield as response value, a three-factor, three-level optimization was carried out to obtain the regression equation Y=2.36+0.3360A+0.5658B-0.0205C+0.0207AB+0.1213AC-0.0017BC-0.3110A²-0.3985B²-0.2795C² (A is corn flour concentration, B is ammonium sulfate concentration, and C is fermentation time). The model R²=0.9877, and the predicted optimal fermentation conditions are consistent with the results of the single-factor experiment.

[0063] Example 5

[0064] This embodiment provides a method for the extraction and purification of flavonoids, specifically including:

[0065] (1) Fermentation broth treatment: Take 100 mL of fermentation broth, centrifuge at 5000 r / min for 15 min, and collect the supernatant;

[0066] (2) Extraction and concentration: Add 200 mL of ethyl acetate, shake and extract 3 times, combine the organic phases, concentrate under reduced pressure to 5 mL, and obtain crude product solution;

[0067] (3) Silica gel column chromatography purification: The crude product solution was loaded onto a silica gel column (2cm×30cm), eluted with petroleum ether-ethyl acetate (5:1), with a flow rate of 1mL / min, and 1 tube was collected for every 5mL. The combined target components were detected by HPLC.

[0068] (4) Refining: The target component is concentrated to dryness under reduced pressure to obtain high-purity flavonoid compounds with a purity of over 95%.

[0069] Table 1. Specific information on the flavonoid compounds produced

[0070] Table 2. Details of strain whole genome assembly

[0071] Table 3. BLAST homology analysis of flavonoid synthesis-related modification genes.

[0072]

[0073] The above description is only a specific embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A strain of *Proteus sp.* XBDL-0121 from *Angelica sinensis*, characterized in that... This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 19, 2026, with accession number CGMCC No. 37465.

2. An isolated 16S rRNA gene of the Proteus XBDL-0121 strain according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID No.1 in the sequence listing, with a sequence length of 1269 bp.

3. The application of the 16S rRNA gene according to claim 2 in identifying Proteus XBDL-0121 strain, characterized in that, When the 16S rRNA gene sequence of the test strain has more than 99.5% identity with the sequence shown in SEQ ID No.1, it is identified as the Proteus XBDL-0121 strain.

4. The application of the Proteus XBDL-0121 strain according to claim 1 in the fermentation production of flavonoids, characterized in that, The flavonoids are at least one of genistein, daidzein, genistein, daidzein, daidzein, and epicatechin.

5. A fermentation method for producing flavonoids, characterized in that, Fermentation using the Proteus XBDL-0121 strain according to claim 1 includes the following steps: (1) Preparation of primary seed culture: The preserved Proteus XBDL-0121 strain was streaked onto TSA solid medium and activated by static culture at 28℃ for 24-48 h. Single colonies were picked and inoculated into 10 mL TSB medium and cultured at 28℃ and 180 r / min for 12 h with shaking. (2) Preparation of secondary seed culture: The primary seed culture was transferred to 100 mL of TSB medium at an inoculation rate of 1%, and cultured at 28℃ and 180 r / min for 2 h with shaking until the bacterial culture OD was reached. 600 The value reaches 0.6-0.8; (3) Preparation of fermentation medium: Each 1000 mL of fermentation medium contains 15 g of tryptone, 5 g of soybean peptone and 5 g of NaCl. The medium is brought to a final volume of 1000 mL with distilled water, autoclaved at 120°C for 30 min, and cooled for later use. (4) Fermentation culture: The secondary seed culture was inoculated into a 250 mL shake flask containing 100 mL of fermentation medium and fermented for 3 days under the conditions of initial pH 7.0, culture temperature 28℃ and shaker speed 180 r / min; (5) Fermentation process control: Samples were taken every 12 hours during fermentation to monitor bacterial concentration, pH changes and flavonoid accumulation, and to maintain the stability of the fermentation system.

6. A method for extracting and purifying flavonoids from the fermentation broth obtained by the method of claim 5, characterized in that, Includes the following steps: (1) Pretreatment of fermentation broth: After fermentation, the fermentation broth was centrifuged at 5000 r / min for 15 min at room temperature, and the supernatant was collected; (2) Extraction: Add 2 times the volume of ethyl acetate to the supernatant, and extract by shaking at room temperature 3 times, 15 min each time. Combine the organic phases obtained from the 3 extractions. (3) Concentration: The combined organic phases are concentrated to dryness under reduced pressure to obtain crude flavonoids; (4) Purification: The crude product was purified by silica gel column chromatography under normal pressure. A mixture of petroleum ether and ethyl acetate with a volume ratio of 5:1 was used as the eluent. The eluent was collected in fractions and the eluents containing the target flavonoids were detected by high performance liquid chromatography (HPLC). The eluents were then concentrated again under reduced pressure to obtain high-purity flavonoids.

7. A group of genes related to flavonoid synthesis from Proteus strain XBDL-0121, characterized in that, The nucleotide sequences of the gene are shown in SEQ ID No. 2 to SEQ ID No. 24 in the sequence listing.

8. The application of the flavonoid synthesis-related genes of a group of Proteus sp. XBDL-0121 strains according to claim 7 in increasing the yield of flavonoids, characterized in that, The application includes: introducing the flavonoid synthesis-related genes into a host microorganism and expressing them in the host microorganism to increase the flavonoid production of the host microorganism.

9. A method for species identification of Proteus XBDL-0121 as described in claim 1, characterized in that, Includes the following steps: (1) Morphological observation: The strain was inoculated on TSA solid medium and cultured at 28℃ for 24-48h. Colony characteristics were observed. Gram staining was performed and the cell morphology was observed under an oil immersion microscope. (2) Scanning electron microscopy characterization: After the strain was fixed, dehydrated, dried and sputter-coated with gold, the ultrastructure of the bacterial cells was observed under a scanning electron microscope; (3) Physiological and biochemical detection: Determine the physiological and biochemical characteristics of the strain, such as carbon source utilization, nitrogen source utilization, acid and alkali resistance, and temperature adaptability; (4) Molecular biological identification: Genomic DNA of the strain was extracted, and 16S rRNA gene and whole genome were sequenced. Its taxonomic position was determined by homology comparison and phylogenetic tree analysis.

10. A method for isolating and purifying Proteus XBDL-0121 as described in claim 1, characterized in that, Includes the following steps: (1) Pretreatment of Tibetan Angelica sinensis samples: Take the root and stem tissues of Tibetan Angelica sinensis, rinse with sterile water and disinfect the surface, and cut into small pieces under sterile conditions; (2) Isolation of endophytic bacteria: The treated tissue blocks were inoculated onto TSA solid medium and incubated at 28°C for 48 hours. Single colonies with different morphologies were picked. (3) Strain purification: Single colonies were inoculated into new TSA solid medium and purified three times by streak separation method. The purified strain was obtained by 16S rRNA identification. (4) Screening of flavonoid yield: The purified strain was inoculated into TSB medium and cultured at 28℃ and 180r / min for 72h. The fermentation broth was taken to detect the content of flavonoid compounds, and the XBDL-0121 strain that produces flavonoids was screened.

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