Armillobacterium chironomidae and application thereof

Abstract

The invention discloses a strain of Limebacterium chironomidae and application thereof, and belongs to the technical field of microbiology, biochemistry and fermentation engineering. According to the present invention, the Leucobacter chironomi capable of converting the tryptophan into the I3CA is the Leucobacter chironomi I3CA-03 with the preservation number of CCTCC M 20252763, and the strain can convert the tryptophan into the I3CA in the time-dependent manner, has the high sensitivity on a variety of antibiotics, and has the clinical application prospect. According to the invention, the source of a wild strain for producing I3CA is expanded, and a foundation is laid for industrial production and application of I3CA in the future.

Description

Technical Field

[0001] This invention relates to the fields of microbiology, biochemistry and fermentation engineering, and particularly to a strain of Leucobacter chironomid (Bacillus chinensis). Leucobacter chironomi ) and its application in the preparation of products for converting tryptophan into indole-3-carboxylic acid (I3CA). Background Technology

[0002] Indole-3-carboxylic acid (I3CA) is a physiologically important tryptophan metabolite with the molecular formula C9H7NO2, containing an indole ring skeleton and a carboxyl group at the C-3 position. Recent studies have shown that I3CA can exert anti-inflammatory, intestinal barrier-enhancing, and neuroprotective effects by activating aryl hydrocarbon receptors (AhR) and PXR / CAR nuclear receptors, demonstrating great potential in regulating the gut-brain-immune axis. With the continued rise in the prevalence of chronic diseases such as inflammatory bowel disease, depression, and metabolic syndrome, the demand for I3CA as a functional factor in pharmaceuticals, functional foods, and cosmetics is increasing. However, plant-derived I3CA content is extremely low (<0.5 μg g). -1 DW), chemical synthesis requires high temperature and pressure and precious metal catalysis, which has problems such as high cost and heavy pollution, making it difficult to meet market demand.

[0003] Microbial transformation has become a research hotspot for I3CA production due to its advantages such as mild conditions, high selectivity, and environmental friendliness. Currently, only a few recombinant Escherichia coli strains have been reported to synthesize I3CA, but these strains pose safety risks such as dependence on IPTG induction and carrying antibiotic markers, making them unsuitable for direct use in oral probiotics. Therefore, screening for wild-type strains with natural I3CA synthesis capabilities, genetic stability, and tolerance to the gastrointestinal environment remains a technological gap in this field.

[0004] Therefore, it is necessary to provide a strain that can efficiently convert tryptophan into I3CA. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Leucobacterium chironomida* (…). Leucobacter chironomi This strain can effectively convert tryptophan into I3CA and its application in I3CA production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the present invention, a strain of *Leucobacterium chironomida* is provided, the preservation number of which is: CCTCC NO: M 20252763.

[0007] In a second aspect of the invention, the use of the aforementioned *Leucobacterium chironomida* in the preparation of products for converting tryptophan into I3CA is provided.

[0008] In a third aspect of the present invention, a fermentation agent is provided, the fermentation agent comprising: The fermentation broth obtained by fermenting the aforementioned Leucobacterium chironomida; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

[0009] In a fourth aspect of the invention, a product for converting tryptophan into I3CA is provided, the product comprising the aforementioned *Leucobacterium chironomida* or the aforementioned fermentation agent.

[0010] Furthermore, the product for converting tryptophan into I3CA also includes pharmaceutically acceptable excipients.

[0011] The excipients include those required for preparation into oral dosage forms.

[0012] In a fifth aspect of the invention, a method for converting tryptophan to I3CA using the aforementioned *Leucobacterium chironomida* is provided, the method comprising: I3CA was obtained by adding the aforementioned Leucobacterium chironomida or the aforementioned fermentation agent to a culture medium containing tryptophan and fermenting it in vitro. Alternatively, I3CA can be obtained in vivo by gavage or oral administration of the described *Leucobacterium chironomida* or the described fermentation agent.

[0013] Furthermore, the culture medium containing tryptophan is LB medium, with 400 mg / L-600 mg / L tryptophan added.

[0014] Furthermore, the in vitro fermentation culture temperature is 36℃-38℃, the pH range is 6.5-7.5, and the optimal inoculum size is 5%-10%.

[0015] As a specific implementation method, the method for converting tryptophan into I3CA using the aforementioned *Leucobacterium chironomida* specifically includes: The chironomid bacillus was inoculated into a test tube containing LB liquid medium and activated by shaking in a shaker at 35 °C overnight. Centrifuge the bacterial culture after overnight incubation, discard the supernatant, resuspend in sterile water, centrifuge again, discard the supernatant, and resuspend in sterile water; transfer the resuspended bacterial culture with OD600 to a triangular flask containing tryptophan-based LB medium and incubate on a shaker.

[0016] Furthermore, the gas chromatography-mass spectrometry (GC-MS) system is model G1540N, and the chromatographic column used is a TraceGOLDTG-5MS column (30m × 0.25μm). The gas flow rates are: high-purity nitrogen: 1.5 mL / min, hydrogen: 30 mL / min, and air: 300 mL / min. The injection port and detector temperatures are 280℃. The column temperature is: initial temperature 50℃ held for 2 min, increased to 230℃ at a rate of 40℃ / min, increased to 320℃ at a rate of 20℃ / min and held for 20 min. The injection volume is 1 μL. The detector temperature is 325℃.

[0017] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: 1. This invention provides a strain of *Leucobacterium chironomida* and its application. A bacterium capable of converting tryptophan to I3CA was isolated from fresh fecal samples of healthy individuals. This strain, identified as *Leucobacterium chironomida*, can convert tryptophan to I3CA in a time-dependent manner. As described in Example 2, the strain was cultured in LB medium containing 500 mg / L tryptophan at 37°C and 220 rpm for 72 hours. The I3CA yield increased linearly with increasing culture time. Specific data show that the yield began to accumulate at 24 hours, reaching a peak at 72 hours (in mg / L), while the control group without added tryptophan produced no I3CA, demonstrating the specificity of the conversion. Compared to plant extraction methods: plant-derived I3CA content is extremely low (<0.5 μg / g dry weight), while this invention achieves a yield at the mg / L level, increasing efficiency by hundreds of times. Compared to chemical synthesis methods: microbial conversion does not require precious metal catalysis or high temperature and pressure, avoiding byproduct generation and simplifying purification steps.

[0018] 2. The *Leucobacterium chironomida* strain provided by this invention has the ability to produce I3CA, which broadens the production pathways and application fields of I3CA.

[0019] The *Leucobacterium chironomida* strain of this invention was deposited on December 3, 2025, with accession number CCTCC NO: M20252763. Its classification and naming... Leucobacter chironomi I3CA-03, deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, Hubei Province, China, 430072, China. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 Image of Leucobacterium chironomida I3CA-03 isolated in Example 1; Figure 2 This is the phylogenetic tree of Leucobacterium chironomida I3CA-03; Figure 3 The detection results for I3CA produced by Leucobacterium chironomida I3CA-03 isolated in Example 1. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0025] The following will provide a detailed description of a strain of Leucobacterium chironomida and its applications, in conjunction with embodiments and experimental data.

[0026] Example 1: Isolation, purification and identification of Leucobacter chironomid (1) Isolation of Leucobacter chironomid Based on the hypothesis that intestinal bacteria can convert tryptophan into I3CA through degradation, the inventors mixed 0.5 g of fresh fecal sample from healthy individuals with a selective culture medium (tryptophan as the sole carbon source). The medium was incubated in the dark on a rotary shaker at 37°C and 250 rpm. The medium was changed after three to four days of incubation. This process was repeated five times, and the culture suspension was then inoculated onto blood agar plates to isolate the bacterial strains. Figure 1 As shown.

[0027] (2) Identification of Leucobacter chironomid The isolated bacterial strains were inoculated into LB liquid medium containing 500 mg / L tryptophan and cultured at 37 °C, 200 rpm for 24 h on a shaker. The bacterial culture was then mixed with 30% glycerol at a 1:1 volume ratio and sent to Qingke Biotechnology Co., Ltd. for sequencing. The 16S rDNA sequence of the obtained strain (as shown in SEQ ID NO.1) was subjected to BLAST alignment analysis in the NCBI database. Phylogenetic analysis of the strain and the type strain was performed using MEGA11 software, and a phylogenetic tree was constructed using the neighbor-linked method, as shown below. Figure 2 As shown; Based on physiological and biochemical characteristics, as well as matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry, it was identified as *Leucobacterium chironomida*. It was deposited on December 3, 2025, at the China Center for Type Culture Collection (depository address: Wuhan University, Wuhan, China), with accession number CCTCC M 20252763.

[0028] Example 2: Validation of I3CA production by Leucobacterium chironomida Leucobacter chironomid was inoculated into LB medium, with 500 mg / L tryptophan added to the medium as the I3CA group, and the group without added tryptophan as the control group. The culture flasks were placed on a shaker at 37°C and incubated at 220 rpm, with samples taken continuously once daily. Finally, the concentration of I3CA was determined by LC-MS / MS, and the results are as follows: Figure 3 As shown.

[0029] Depend on Figure 3 It is known that the *Leucobacterium chironomida* strain of this application converts tryptophan to I3CA in a time-dependent manner, while the control group without added tryptophan does not produce I3CA. Furthermore, the *Leucobacterium chironomida* strain of this application achieves an I3CA concentration of mg / L after 72 hours. The optimal culture temperature for this strain of *Leucobacterium chironomida* is 37℃, the optimal pH is 6.5-7.5, and the optimal inoculum size is 5%-10%.

[0030] Example 3: Preparation of fermentation inoculum and its application in in vitro transformation I. Experimental Objective: The preparation process of the fermentation agent was demonstrated, and its effect on the in vitro conversion of tryptophan to I3CA was evaluated.

[0031] II. Experimental Methods: 1. Preparation of fermentation broth: Inoculate Leucobacterium chironomidae I3CA-03 into LB liquid medium and ferment at 37°C and 200 rpm for 24 hours to obtain the fermentation broth (as described in claim 3).

[0032] 2. Preparation of dry powder inoculant: Take the above fermentation broth and spray dry it (inlet temperature 150℃, outlet temperature 80℃) to obtain dry powder inoculant (as described in claim 3).

[0033] 3. In vitro transformation experiment: Add the fermentation broth or dry powder inoculum (resuspended in sterile water) to LB medium containing 500 mg / L tryptophan at a 5% inoculation rate.

[0034] Culture conditions: 37℃, 220rpm, pH 7.0, culture for 72 hours.

[0035] The I3CA detection method is the same as in Example 2, using LC-MS / MS analysis.

[0036] III. Experimental Results Both the fermentation broth and the dry powder inoculum can effectively convert tryptophan to I3CA, with yields comparable to those of the strain used directly in Example 2 (reference). Figure 3 (Trend). Dry powder microbial agents maintain stable activity after storage, making them suitable for industrial transportation and application.

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0038] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0039] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0040] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of *Leucobacterium chironomida*, characterized by: The classification name of the aforementioned *Leucobacterium chironomida* is... Leucobacter chironomi I3CA-03 ， The accession number is CCTCC M 20252763.

2. The use of *Leucobacterium chironomida* as described in claim 1 in the preparation of a product for converting tryptophan to I3CA.

3. A fermentation agent, characterized in that, The fermentation agent includes: The fermentation broth obtained by fermenting the *Leucobacterium chironomida* as described in claim 1; Alternatively, the fermentation broth can be spray-dried to obtain a dry powder inoculum.

4. A product for converting tryptophan into I3CA, characterized in that, The product for converting tryptophan into I3CA includes the *Leucobacterium chironomida* of claim 1 or the fermentation agent of claim 3.

5. The product according to claim 4, characterized in that, The product also includes pharmaceutically acceptable excipients.

6. The product according to claim 5, characterized in that, The excipients include carriers required for preparing oral dosage forms, Diluent or stabilizer.

7. A method for converting tryptophan into I3CA, characterized in that, The method includes: I3CA was obtained by adding the *Leucobacterium chironomida* of claim 1 or the fermentation agent of claim 3 to a culture medium containing tryptophan and fermenting it in vitro. Alternatively, I3CA can be obtained in vivo by gavage or oral administration of the *Bryophyte leucobacterium* of claim 1 or the fermentation agent of claim 3.

8. The method according to claim 7, characterized in that, The in vitro fermentation culture temperature is 36℃-38℃.

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