Strain of serratia marcescens l4 with high extracellular release of prodigiosin and application thereof

Abstract

The application discloses a high-extracellular-release-proportion-providencia-stutzeri L4 strain and application thereof, and belongs to the technical field of biological fermentation.The application provides a providencia stutzeri strain and a method for producing prodigiosin.The providencia stutzeri L4 strain provided by the application can release a high proportion of prodigiosin produced in a liquid fermentation process to the extracellular, and the proportion of extracellular prodigiosin in the total yield reaches more than 85%.Based on the extracellular release characteristics of the strain, efficient enrichment of prodigiosin in the fermentation broth can be realized, and complex operations such as collection of bacterial cells, cell disruption and intracellular pigment release in the traditional process are avoided, so that the production process is effectively simplified, and the prodigiosin preparation efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a Serratia marcescens strain L4 with high extracellular release of erythromycin and its application. Background Technology

[0002] Prodigiosin is a class of natural tripyrrole secondary metabolites produced by microorganisms. It possesses a unique bright red color and exhibits a variety of biological activities, including antibacterial, antifungal, antitumor, antioxidant, and immunomodulatory effects. It shows broad application prospects in food pigments, pharmaceutical intermediates, feed additives, cosmetics, and functional biomaterials. With the increasing demand for the development of natural bioactive products, the production of prodigiosin through microbial fermentation has become a research hotspot both domestically and internationally.

[0003] Currently, strychnine mainly originates from Serratia marcescens (Serratia marcescens). Serratia marcescens Microorganisms such as actinomycetes and a few marine bacteria are used in the production of squalene. Among them, *Serratia marcescens* is an important source of strains for squalene production due to its rapid growth, relatively simple culture conditions, and short fermentation cycle. However, most existing squalene-producing strains accumulate squalene primarily within the cell during fermentation, with only a small amount released extracellularly. Therefore, downstream processing techniques such as cell disruption and organic solvent extraction are usually required to extract squalene. These processes are not only lengthy but also suffer from high energy consumption, limited extraction efficiency, and increased product purification costs, which to some extent restricts the large-scale production and industrial application of squalene. Furthermore, *Serratia marcescens* strains from different sources exhibit significant differences in squalene synthesis capacity, extracellular release capacity, and fermentation stability.

[0004] Therefore, screening for a novel strain capable of stably releasing styraxin from the extracellular environment and establishing a corresponding method for styraxin preparation is of great significance for simplifying downstream extraction processes, reducing production costs, and promoting the industrial application of styraxin. Summary of the Invention

[0005] The purpose of this invention is to promote the industrialization of strychnine, simplify the extraction process, reduce production costs, and provide a *Serratia marcescens* strain that releases strychnine from outside the cell.

[0006] In a first aspect, the present invention provides a strain of *Serratia marcescens*, named L4, and classified as follows: Serratia marcescens L4 was deposited on May 25, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261058, at Wuhan University, Wuhan, China.

[0007] Preferably, the *Serratia marcescens* can secrete styraxin extracellularly during liquid fermentation, and the extracellular styraxin accounts for more than 85% of the total styraxin.

[0008] Secondly, the present invention provides an application of the above-mentioned Serratia marcescens in the preparation of serotonin.

[0009] Thirdly, the present invention provides a method for producing squalene, comprising the following steps: inoculating the above-mentioned Serratia marcescens into a fermentation medium for liquid fermentation culture; collecting the fermentation broth containing extracellular squalene, and obtaining squalene through extraction and purification.

[0010] Preferably, the fermentation medium comprises: 5 g / L sucrose, 20 g / L fish meal peptone, 0.5 g / L MgSO4·7H2O, 0.6 g / L KH2PO4, and a pH of 6.5~7.5.

[0011] Preferably, the *Serratia marcescens* is a seed culture of *Serratia marcescens*, and the seed culture of *Serratia marcescens* is inoculated into the fermentation medium at a volume percentage of 5-8%.

[0012] Preferably, the method for preparing the Serratia marcescens seed culture includes the following steps: inoculating the Serratia marcescens into a seed culture medium and culturing it at 30°C and 180 rpm until the logarithmic phase.

[0013] Preferably, the seed culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 6.8~7.0.

[0014] Preferably, the temperature of the liquid fermentation culture is 25℃~35℃, and the fermentation time is 96~120 h.

[0015] Preferably, the fermented cells are extracted and ultrasonically treated to obtain styraxin.

[0016] Compared with the prior art, the present invention has the following advantages: The *Serratia marcescens* L4 strain provided by this invention can release a high proportion of squalene into the extracellular environment during liquid fermentation, with extracellular squalene accounting for over 85% of the total yield. Based on the extracellular release characteristics of this strain, efficient enrichment of squalene in the fermentation broth can be achieved, avoiding the complex operations of cell collection, cell disruption, and intracellular pigment release in traditional processes, thereby effectively simplifying the production process and improving the efficiency of squalene preparation.

[0017] The Serratia marcescens L4 strain provided by this invention is used for the fermentation of serotonin, and can be applied to the development of natural active pigments, food additives, feed additives and related functional products, providing a new technical path for the green and efficient biomanufacturing of serotonin. Attached Figure Description

[0018] Figure 1 Example 1: Serratia marcescens ( Serratia marcescens Streak plate of L4 strain; Figure 2 Gram staining (a) and scanning electron microscope (b) images of Serratia marcescens strain L4 from Example 1; Figure 3 This is the phylogenetic tree of Serratia marcescens strain L4 from Example 1; Figure 4 This is the supernatant of the fermentation broth of Serratia marcescens strain L4 in Example 3; Figure 5 The HPLC-MS / MS analysis results and ion chromatogram of pyruvicin in Example 3 are shown below. Figure 6 This is the HPLC analysis chromatogram of styraxin in Example 3; Figure 7 The ultraviolet absorption spectrum of styraxin in Example 3 is shown below. Figure 8 Serratia marcescens ( Serratia marcescens Preservation certificate for strain L4. Detailed Implementation

[0019] To better understand the present invention, the following embodiments are further illustrations of the invention, but the scope of the invention is not limited to these embodiments. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods. The reagents and materials used in the embodiments are all commercially available, and the quantitative experiments involved in the embodiments are all performed in at least three replicates.

[0020] Example 1: Isolation, identification and preservation of Serratia marcescens strain L4 The strain *Serratia marcescens* L4 was obtained by isolation and purification from fresh silkworm excrement produced during silkworm rearing at the Sericulture Research Base of Jiangsu University of Science and Technology. Details are as follows: Prepare LB medium. The LB liquid medium consists of: 10 g / L tryptone (Shanghai Sinopharm Chemical Reagent Co., Ltd.), 5 g / L yeast extract (Thermo Fisher Scientific Co., Ltd.), and 10 g / L NaCl. Adjust the pH to 6.8–7.0 after bringing the volume to a final volume with distilled water. The LB solid medium is prepared by adding 1.5% (…) to the above liquid medium. w / v Preparation of agar powder.

[0021] Add 1 g of fresh silkworm excrement sample to 9 mL of sterile physiological saline, shake thoroughly to mix, and obtain a bacterial suspension. Perform serial dilutions on the obtained bacterial suspension, taking 10 g of each solution. -2 10 -3 and 10 -4 100 μL of the diluted solution was spread onto LB solid medium plates and incubated at 30°C for 48 h.

[0022] After incubation, preliminary screening was conducted based on colony color, morphology, and growth characteristics. Colonies exhibiting bright red color and a smooth, moist surface with noticeable stickiness were selected. Colonies with these characteristics were discarded, and the culture was subjected to three consecutive streak plate purification cultures to obtain a morphologically stable and consistent pure culture strain, designated L4.

[0023] The purified L4 strain was inoculated into LB liquid medium and cultured. Sterile glycerol was added to prepare a culture preservation solution, which was then stored at -80℃ for later use.

[0024] The L4 strain was inoculated onto LB solid medium and incubated at 30°C for 24 h. Colony morphology was then observed. Figure 1 As shown, strain L4 forms round colonies with neat edges, a smooth and moist surface, a bright red color, and a certain degree of stickiness.

[0025] Gram staining analysis of strain L4 yielded the following results: Figure 2 As shown in Figure a, it can be seen that strain L4 appears red under Gram staining, indicating that strain L4 is a Gram-negative bacterium with short rod-shaped cells.

[0026] The morphology of the bacteria was observed using a scanning electron microscope, and the results are as follows: Figure 2 As shown in b, it can be seen that L4 cells have a typical rod-shaped structure, which is consistent with the morphological characteristics of Serratia spp.

[0027] L4 strain was selected, and its genomic DNA was extracted. PCR amplification was performed using the universal primers 27F (AGAGTTTGATCMTGGCTCAG, SEQ ID NO:1) and 1492R (GGTTACCTTGTTACGACTT, SEQ ID NO:2) for bacterial 16S rRNA.

[0028] After agarose gel electrophoresis and purification, the PCR amplification product was sequenced to obtain a 16S rRNA gene sequence of approximately 1478 bp, as shown in SEQ ID NO.3.

[0029] The 16S rRNA gene sequence was uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), BLAST sequence alignment was performed, and homology analysis was conducted. A phylogenetic tree was constructed using MEGA 7.0 software based on the neighbor-joining method. Figure 3 As shown. The results indicate that strain L4 is related to Serratia marcescens (…). Serratia marcescens The related strains have a high degree of similarity.

[0030] Serratia marcescens L4, classified as Serratia marcescens ( Serratia marcescens L4 was deposited on May 25, 2026, at the China Center for Type Culture Collection (CCTCC M 20261058), located at No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit certificate and viability certificate are as follows: Figure 8 As shown.

[0031] Example 2: Fermentation of Serratia marcescens strain L4 1. Preparation of seed culture for strain L4 The *Serratia marcescens* strain L4 from Example 1 was streaked onto LB agar plates and incubated at 30°C for 24 h. After uniformly morphologically uniform and bright red single colonies formed on the surface of the medium, a single colony was picked and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of LB liquid medium. The flask was then incubated at 30°C and 180 rpm with shaking for 24 h to obtain the L4 strain seed culture. The OD600 value of the obtained seed culture was 1.6–1.8, indicating it was in the optimal growth stage.

[0032] 2. Prepare the culture medium for the production of styrax rubigin. Prepare a culture medium for the production of sucralose with the following composition: 5 g / L sucrose, 20 g / L fish meal peptone (Shanghai Sinopharm Chemical Reagent Co., Ltd.), 0.5 g / L MgSO4·7H2O, and 0.6 g / L KH2PO4. Make up to 1 L with distilled water and adjust the initial pH to 7.0. Dispense the culture medium into 100 mL Erlenmeyer flasks (250 mL each), sterilize at 121℃ for 20 min, and use.

[0033] 3. Fermentation The seed solution from step 1 was prepared at 5% ( v / v The inoculum was added to the styrax red pigment production medium and cultured at 30°C for 120 h. During fermentation, the pH of the culture medium was monitored periodically and adjusted as needed using sterile NaOH or HCl solution to maintain the pH of the culture system within the range of 6.8–7.2.

[0034] Example 3: Extraction and identification of styraxin from the fermentation broth of Serratia marcescens strain L4 Take 5 mL of the fermentation broth from Example 2 after fermentation and centrifuge at 9000 rpm for 10 min to separate the bacterial cells from the culture supernatant, obtaining the supernatant and bacterial cell precipitate.

[0035] The supernatant was collected as the extracellular pyruvic erythrin sample. An equal volume of acidic methanol extract (pH=3.0) was added to it, and the mixture was thoroughly mixed to transfer the extracellular pyruvic erythrin to the organic phase. After standing and separating, the organic phase containing pyruvic erythrin was collected and concentrated under reduced pressure to obtain the extracellular pyruvic erythrin sample.

[0036] The bacterial precipitate was washed twice with sterile distilled water to remove residual culture medium and extracellular pigments adsorbed on the bacterial surface. Then, an equal volume of acidic methanol solution (pH=3.0) was added, and after sonication, styraxin was extracted from the inside of the bacterial cells. After standing and separating the layers, the organic phase containing styraxin was collected and concentrated under reduced pressure to obtain the intracellular styraxin sample.

[0037] The structures of the obtained intracellular / extracellular squalene samples were identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS), and the purity of the obtained squalene was determined using HPLC. The UV absorption spectrum of squalene in the 350–700 nm range was then measured using a UV-Vis spectrophotometer to determine the absorption spectrum and maximum absorbance value. The contents of extracellular and intracellular squalene were calculated based on the absorbance values. Finally, the extracellular release ratio was calculated using the following formula: Extracellular release percentage (%) = Extracellular lecithin content / (Extracellular lecithin content + Intracellular lecithin content) × 100%.

[0038] fermentation broth supernatant as Figure 4 As shown, the supernatant is distinctly red, indicating the presence of styraxin produced by strain L4 in the extracellular culture medium.

[0039] The results of high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) are as follows: Figure 5 As shown, based on the chromatographic retention time, ultraviolet absorption characteristics, and mass spectrometry ion peaks of the obtained sample, the molecular weight of the compound can be determined to be 323.2061, and its chemical formula is C. 20 H 25 Based on data from the PubChem database, N3O can be identified as squalene, indicating that the L4 strain can produce squalene through fermentation.

[0040] HPLC detection results are as follows Figure 6 As shown, the purity of styraxin in the L4 strain fermentation system was 99.522%; the purity was measured by UV-Vis spectrophotometer as follows. Figure 7 As shown, squalene has a unique maximum absorption peak at 535 nm. Measurements of the intracellular and extracellular absorbance using a UV spectrophotometer revealed that the total squalene yield in the L4 strain fermentation system reached 75.28 mg / L, with an extracellular squalene content of 68.05 mg / L, representing an extracellular release rate of 90.4%. These data indicate that the L4 strain can effectively synthesize squalene and release most of it into the extracellular culture medium during fermentation.

[0041] Example 4: Release of extracellular erythromycin from Serratia marcescens L4 strain under different culture conditions 1. Effect of fermentation temperature on extracellular release capacity The steps in this procedure differ from those in Example 2 only in that the culture temperatures are set to 25℃, 28℃, 30℃, 32℃, and 35℃, respectively. After fermentation, the total yield and extracellular release ratio of styraxin under different temperature conditions were measured.

[0042] The results are shown in Table 1. The L4 strain was able to produce styraxin in the range of 25-35℃ and maintain a high extracellular release ratio. The total yield of styraxin, the level of extracellular styraxin accumulation and the extracellular release capacity were the highest at 30℃.

[0043] Table 1. Effects of different culture temperatures on the extracellular erythromycin release capacity of Serratia marcescens L4 strain.

[0044] 2. Effect of fermentation pH on extracellular release capacity The steps in this procedure differ from those in Example 2 only in that the fermentation pH was set to 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. After fermentation, the total yield and extracellular release ratio of styraxin were measured under different fermentation pH conditions.

[0045] The results are shown in Table 2. The L4 strain maintained good extracellular release capacity in the pH range of 6.5 to 7.5, with better extracellular release capacity at pH 7.0.

[0046] Table 2. Effects of different initial pH values ​​on the exocarboxyerythrin release capacity of Serratia marcescens L4 strain.

[0047] 3. Effect of inoculum size on extracellular release capacity The difference between this step and that in Example 2 is only that the seed liquid inoculation amount is set to 1%, 3%, 5%, 8%, and 10%, respectively. v / v After fermentation, the total yield and extracellular release ratio of levofloxacin under different inoculum sizes were measured.

[0048] The results are shown in Table 3. As the inoculum size increased, the yield of extracellular erythromycin from strain L4 gradually increased. When the inoculum size reached 5-8%, a higher level of extracellular erythromycin accumulation could be obtained.

[0049] Table 3. Effects of different inoculum sizes on the extracellular erythromycin release capacity of Serratia marcescens L4 strain.

[0050] 4. Effect of culture time on extracellular release capacity The steps in this procedure differ from those in Example 2 only in that the fermentation times are set to 48 h, 72 h, 96 h, 120 h, and 144 h, respectively. After fermentation, the total yield and extracellular release ratio of styraxin at different fermentation times were measured.

[0051] The results are shown in Table 4. As the culture time increased, the extracellular erythromycin of strain L4 gradually accumulated, reaching a high level at 96-120 h.

[0052] Table 4. Effects of different culture times on the extracellular erythromycin release capacity of Serratia marcescens L4 strain.

[0053] Comparative Example 1: Extracellular erythromycin release capacity of Serratia marcescens The only difference between this comparative example and Example 2 is the use of *Serratia marcescens* CCTCC AB 90046, a strain that produces erythromycin, as the fermentation strain. The CCTCC AB 90046 strain is classified and named as (…). Serratia marcescens The strain (CCTCC AB 90046) was deposited at the China Center for Type Culture Collection (CCTCC) on April 12, 1990, with accession number CCTCC AB 90046, located at Wuhan University, Wuhan, China. It can be obtained through purchase. CCTCC AB 90046 belongs to the Serratia marcescens species and possesses the ability to synthesize squalene. After fermentation, the total squalene content, extracellular squalene content, and extracellular release ratio were measured.

[0054] Table 5. Extracellular erythropoietin release capacity of Serratia marcescens CCTCC AB 90046

[0055] The results are shown in Table 5. It can be seen that the total production of styrax rubra var. rubra is higher than that of Serratia rubra var. rubra CCTCC AB90046. Moreover, the extracellular release rate of the L4 strain reached 90.4%, which is much higher than that of Serratia rubra var. rubra CCTCC AB 90046.

[0056] SEQ ID NO:1 AGAGTTTGATCMTGGCTCAG SEQ ID NO:2 GGTTACCTTGTTACGACTT SEQ ID NO:3 The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A strain of Serratia marcescens, characterized in that, The *Serratia marcescens* strain was named L4, and its classification was named... Serratia marcescens L4 was deposited on May 25, 2026, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20261058, at Wuhan University, Wuhan, China.

2. The *Serratia marcescens* according to claim 1, characterized in that, During liquid fermentation, the *Serratia marcescens* can secrete lecithin into the extracellular space, and the extracellular lecithin accounts for more than 85% of the total lecithin.

3. The use of Serratia marcescens as described in claim 1 or 2 in the preparation of serotonin.

4. A method for producing styraxin, characterized in that, Includes the following steps: The *Serratia marcescens* as described in claim 1 or 2 is inoculated into a fermentation medium for liquid fermentation culture. Fermentation broth containing extracellular lecithin was collected, and lecithin was obtained through extraction and purification.

5. The method according to claim 4, characterized in that, The fermentation medium comprises: 5 g / L sucrose, 20 g / L fish meal peptone, 0.5 g / L MgSO4·7H2O, 0.6 g / L KH2PO4, and pH 6.5~7.

5.

6. The method according to claim 4, characterized in that, The *Serratia marcescens* is a seed culture of *Serratia marcescens*, which is inoculated into the fermentation medium at a volume percentage of 5-8%.

7. The method according to claim 6, characterized in that, The method for preparing the Serratia marcescens seed culture includes the following steps: inoculating the Serratia marcescens into the seed culture medium and culturing it at 30°C and 180 rpm until the logarithmic phase.

8. The method according to claim 7, characterized in that, The seed culture medium comprises: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH 6.8-7.

0.

9. The method according to claim 4, characterized in that, The liquid fermentation culture temperature is 25℃~35℃, and the fermentation time is 96~120 h.

10. The method according to claim 4, characterized in that, After fermentation, the bacterial cells were extracted and ultrasonically treated to obtain styraxin.

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