Method for preparing secondary metabolites by halomonas and application thereof

Abstract

This invention discloses a method and application for preparing secondary metabolites from *Halomonas*, an extreme halophilic bacterium isolated from the saline-alkali soil of Dongdaohaizi, Urumqi, Xinjiang. Halomonas sp. HZ-6, whose 16S rRNA gene is accessed in GenBank as PZ523056. This strain is a non-spore-forming, non-motile, slender rod-shaped single-celled organism; its agar colonies are pale milky yellow and viscous. The strain was developed using activated Halomonas halosa. Halomonas The seed culture of sp. HZ-6 was inoculated into a saline liquid culture medium containing sodium chloride, fermented under constant temperature and shaking, the cells were ultrasonically disrupted, centrifuged, and the supernatant was collected; then extracted with ethyl acetate, the organic phases were combined, concentrated under reduced pressure, and dried to obtain a crude ethyl acetate extract, a secondary metabolite. The obtained crude ethyl acetate extract is used in drugs that inhibit *Escherichia coli*, *Staphylococcus aureus*, or *Candida albicans*. This invention provides a green and low-cost preparation process, and the crude metabolite extract can be used to develop medical antibacterial drugs and agricultural biocontrol agents, providing a high-quality resource for the discovery of novel halophilic antibacterial lead compounds.

Description

Technical Field

[0001] This invention belongs to the field of extreme environment microorganisms and natural antibacterial products, specifically relating to a method and application for preparing secondary metabolites from Halomonas bacteria. Background Technology

[0002] Soil salinization is a core ecological problem in arid and semi-arid regions. Xinjiang, my country, has a wide distribution of saline-alkali soils with high salinity and alkalinity, fostering a large number of unique halophilic microorganisms. Long-term evolution has enabled these halophilic bacteria to develop specialized metabolic pathways, enabling them to synthesize novel and highly active secondary metabolites, representing a core resource for the development of novel antibacterial lead compounds.

[0003] *Halomonas* is a γ-Proteobacterium class, Gram-negative, halophilic bacterium widely distributed in high-salt habitats such as salt lakes and saline-alkali soils. Its natural salt tolerance range is 3%-30% (w / v). Existing strains are primarily used for the synthesis of industrially compatible solutes such as polyhydroxy fatty acid esters and tetrahydropyrimidine. Current research confirms that some *Halomonas* strains can synthesize alkaloids, peptides, and polyketides as antimicrobial substances; however, current technologies have significant limitations. (1) The salt tolerance limit of existing Xinjiang-derived Halomonas bacteria is mostly below 20% sodium chloride, with a narrow range of acid and alkali tolerance and poor environmental adaptability; (2) Most strains have a single type of secondary metabolites, a long fermentation cycle, and a low accumulation of active products. (3) The lack of standardized processes for targeted fermentation and efficient extraction makes it difficult to prepare active extracts on a large scale; (4) Only simple antibacterial screening was carried out, without systematic analysis of metabolic diversity, and there were no clear medical and agricultural application plans.

[0004] Therefore, developing a highly resilient, metabolite-rich, broad-spectrum antibacterial strain of *Halomysium halophilum* native to Xinjiang, and establishing a supporting fermentation and extraction process, has significant industrial and ecological value for the development of novel drug-free natural antibacterial drugs and the resource utilization of microbial resources in saline-alkali land. This will enrich my country's resources for preserving microbial strains from extreme environments; establish a set of efficient fermentation and extraction processes suitable for this strain's secondary metabolites, clarifying the optimal fermentation parameters; confirm the broad-spectrum antibacterial and antifungal activity of the strain's ethyl acetate extract, identifying the target bacterial species for inhibition; and expand the application pathways of this strain's metabolites in medical antibacterial preparations and the biocontrol of agricultural fungal / bacterial diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for preparing secondary metabolites from *Haloxylon ammodendron*, wherein the *Haloxylon ammodendron* strain is named... HalomonasThe 16S rRNA gene sequence of sp. HZ-6 is shown in SEQ ID NO:1, NCBI GenBank accession number PZ523056; the strain was isolated from saline-alkali soil in Dongdaohaizi, Urumqi, Xinjiang, using activated Haloxylon ammodendron. Halomonas The seed culture of sp. HZ-6 was inoculated into a saline liquid culture medium containing 15% sodium chloride and fermented under constant temperature and shaking to obtain a fermentation broth. The fermentation broth was then subjected to ultrasonic disruption of the bacterial cells, centrifugation, and collection of the supernatant. An equal volume of ethyl acetate was then used for extraction. The organic phases were combined, concentrated under reduced pressure, and dried to obtain a crude ethyl acetate extract, a secondary metabolite. The crude ethyl acetate extract is then used in drugs that inhibit *Escherichia coli*, *Staphylococcus aureus*, or *Candida albicans*. This invention provides a green and low-cost preparation process, and the crude metabolite can be used to develop medical antibacterial drugs and agricultural biocontrol agents, providing a high-quality resource for the discovery of novel halophilic antibacterial lead compounds.

[0006] The present invention discloses a method for preparing secondary metabolites from *Haloxylon ammodendron*, wherein the strain is named... Halomonas The 16S rRNA gene sequence of sp. HZ-6 is shown in SEQ ID NO:1, and the NCBI GenBank accession number is PZ523056. The strain was isolated from the saline-alkali soil of Dongdaohaizi, Urumqi, Xinjiang. The specific operations were carried out according to the following steps: a. The activated Halomonas bacteria Halomonas Sp. HZ-6 seed culture was inoculated into a saline liquid medium containing 15% sodium chloride at a volume ratio of 5-8%. Fermentation was carried out at 37 ℃ with constant temperature shaking at 180 rpm for 8 days. The initial pH of the fermentation medium was 8.0. The fermentation broth was obtained by adding the following components to 100 mL of saline liquid medium: 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, and 15 g sodium chloride. The mixture was brought to a final volume with deionized water and sterilized. The pH was then adjusted to 7.1-7.2. b. The fermentation broth obtained in step a is ultrasonically disrupted to break up the bacterial cells, centrifuged at 8000 rpm for 15 min, and the supernatant is collected. c. Extract the supernatant from step b with an equal volume of ethyl acetate 4-5 times, combine the organic phases, concentrate under reduced pressure at 40 ℃, and dry to obtain a crude ethyl acetate extract with a secondary metabolite concentration of 50-100 mg / mL.

[0007] The application of the crude ethyl acetate extract, a secondary metabolite obtained by the method, in drugs that inhibit Escherichia coli, Staphylococcus aureus, or Candida albicans.

[0008] The product dosage forms include aqueous solutions, powders, gels, wettable powders, capsules, or antibacterial sprays.

[0009] This invention discloses a method and application for preparing secondary metabolites from *Haloxylon ammodendron*. The method uses a liquid culture medium containing 15% sodium chloride as the substrate, ferments at 37°C and 180 rpm for 8 days with shaking, and then performs ultrasonic disruption, centrifugation, multi-stage extraction with ethyl acetate, and vacuum concentration to obtain a crude extract. Liquid chromatography analysis shows that an initial pH of 8.0 and 8 days of fermentation achieve the highest abundance and chemical diversity of metabolites. In vitro antibacterial tests show that the crude extract possesses broad-spectrum antibacterial activity against *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans*, with the best inhibitory effect against *Escherichia coli*.

[0010] This invention discloses a method and application for preparing secondary metabolites from *Halomonas*, wherein the *Halomonas* is an extreme halophilic *Halomonas* strain isolated from the extreme saline-alkali soils of Xinjiang. Halomonas sp. HZ-6, whose 16S rRNA gene GenBank accession number is PZ523056. This strain is a non-spore-forming, non-motile, slender rod-shaped single cell, and its agar colonies are light milky yellow and viscous; it can tolerate 16%-30% (w / v) NaCl and pH 5.0-10.0 environments, with the optimal growth salt concentration of 16%-20% and pH 8.0-9.0. The technical solution of this invention is: (1) providing a salt-tolerant halomonas. Halomonas sp. HZ-6, strain 16S rRNA sequence is shown in SEQ ID NO:1, GenBank accession number PZ523056; isolated from saline-alkali soil of Dongdaohaizi, Urumqi, Xinjiang, extremely salt and alkali tolerant, non-spore-forming, non-pathogenic appendages, and highly safe. (2) Provide a complete preparation method for the crude ethyl acetate extract, the secondary metabolite of this strain, specifying the culture medium, fermentation temperature, fermentation cycle, extraction solvent, and post-treatment parameters. (3) Provide the application of the above crude extract in the preparation of medical antibacterial drugs and agricultural biological control agents, specifying the applicable target microorganisms and developable formulations.

[0011] The beneficial effects of this invention are as follows: (1) Strong innovation of strain resources: The strain originates from the extreme saline-alkali habitat of Xinjiang, with a bootstrap value of 99% independent evolutionary branch, which is a new halophilic bacterium resource, filling the gap of local halophilic bacteria with high salt tolerance and high antibacterial activity; (2) Outstanding stress resistance: It can tolerate a wide range of saline-alkali environments of 16%-30% sodium chloride and pH 5.0-10.0, and is suitable for high-salt wastewater and in-situ fermentation in saline-alkali land; (3) High diversity of metabolites: The abundance and number of chromatographic peaks in the ethyl acetate extract are significantly better than those in other solvents and culture media, containing a large number of polar active metabolites, which is conducive to subsequent monomer separation and purification; (4) Efficient and controllable fermentation process: The optimal fermentation cycle is only 8 days, and the initial pH of 8.0 can maximize product accumulation. The conditions are mild and the equipment requirements are low, which is suitable for large-scale scale-up; (5) Broad antibacterial spectrum and strong targeting: It produces inhibition zones against Escherichia coli, Staphylococcus aureus and Candida albicans at the same time. The order of antibacterial activity is: Escherichia coli > Candida albicans > Staphylococcus aureus has the most significant inhibitory effect on Escherichia coli, with the largest and most transparent inhibition zone, exhibiting broad-spectrum antibacterial properties; (6) Green and low-cost process: the raw materials for fermentation culture medium are inexpensive and readily available, the extraction solvent ethyl acetate is low-toxic and easy to recycle, and there are no heavy metals or strong irritating reagents, which meet the standards for green production in medicine and agriculture. This invention discloses a method and application for preparing secondary metabolites from *Haloxylon ammodendron*. The *Haloxylon ammodendron* strain was isolated from the edge of Dongdaohaizi Lake in Midong District, Urumqi City, Xinjiang Uygur Autonomous Region, at geographical coordinates 44°41.7′N, 87°33.5′E, an altitude of approximately 361 m, a soil salinity of 16.7%, and a pH of 8.9. A five-point sampling method was used to collect rhizosphere soil from the 0-20 cm topsoil layer. Equal volumes of soil from the five sampling points were mixed into a single composite sample. After removing impurities such as dead leaves, fallen branches, and stones, the sample was placed in a sterile sealed bag, placed in a low-temperature incubator, and transported back to the laboratory for storage at -80℃ for later use. The strain was named... Halomonas The sp. HZ-6, 16S rRNA gene sequence is shown in SEQ ID NO:1, NCBI GenBank accession number PZ523056. Attached Figure Description

[0012] Figure 1 The present invention is a Halomonas bacterium. Halomonas Colony morphology of sp. HZ-6 on a 15% sodium chloride-containing solid medium; Figure 2 The present invention is a Halomonas bacterium. Halomonas Scanning electron micrograph of sp. HZ-6 (scale bar 2 μm), showing the slender rod-shaped morphology of single cells; Figure 3 The present invention is a Halomonas bacterium. HalomonasPhylogenetic tree of sp. HZ-6 based on the 16S rRNA gene (neighbor-join method, bootstrap=1000); Figure 4 The present invention is a Halomonas bacterium. Halomonas Growth curve of sp. HZ-6; Figure 5 The present invention is a Halomonas bacterium. Halomonas Growth curves of sp. HZ-6 under different pH conditions; Figure 6 The present invention is a Halomonas bacterium. Halomonas Growth diagram of sp. HZ-6 at different salt concentrations; Figure 7 This is a high-performance liquid chromatography comparison analysis of secondary metabolites from different strains of the present invention; Figure 8 This is a high-performance liquid chromatography (HPLC) chromatogram of secondary metabolites extracted from different polarity solvent fractions of the strain of this invention. Figure 9 High-performance liquid chromatography (HPLC) chromatograms of secondary metabolites of the strain of this invention in different culture media; Figure 10 This is a high-performance liquid chromatography (HPLC) analysis of secondary metabolites from the strain of this invention at different fermentation times. Figure 11 This is a high-performance liquid chromatography (HPLC) analysis of the secondary metabolites of the strain of this invention under different initial pH conditions; Figure 12 This image shows the inhibition zone effect of the secondary metabolites of the strain of this invention on Escherichia coli, Staphylococcus aureus, and Candida albicans. Detailed Implementation

[0013] All technical and scientific terms used in this invention, unless otherwise stated, generally have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, culture media, drugs, instruments, etc., used in the following embodiments are all conventional products that can be purchased through legitimate channels; each experiment was conducted with 3 biological parallel groups and 2 technical parallel groups, and the results were averaged; all operations not specified were performed under standard aseptic conditions in a microbiology laboratory.

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] The present invention will now be described in detail with reference to specific embodiments. These embodiments are for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements made based on the principles of the present invention should be included within the scope of protection of the present invention. Example 1

[0016] Strain naming Halomonas The 16S rRNA gene sequence of sp. HZ-6 is shown in SEQ ID NO:1, and the NCBIGenBank accession number is PZ523056. The strain was isolated from the saline-alkali soil of Dongdaohaizi, Urumqi, Xinjiang. The specific operations were carried out according to the following steps: a. The activated Halomonas bacteria Halomonas Sp. HZ-6 seed culture was inoculated at a volume ratio of 5% into a saline liquid culture medium containing 15% sodium chloride. Fermentation was carried out at 37 ℃ with constant temperature shaking at 180 rpm for 8 days. The initial pH of the fermentation medium was 8.0. The fermentation broth consisted of 100 mL of the following saline liquid culture medium components: 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, and 15 g sodium chloride. The medium was brought to a final volume with deionized water, and the pH was adjusted to 7.1-7.2 with 1 mol / L sodium hydroxide. The mixture was then autoclaved at 120 ℃ for 20 min. b. The fermentation broth obtained in step a is ultrasonically disrupted to break up the bacterial cells, centrifuged at 8000 rpm for 15 min, and the supernatant is collected. c. Extract the supernatant from step b five times with equal volumes of ethyl acetate, combine the organic phases, concentrate under reduced pressure at 40 °C, and dry to obtain a crude ethyl acetate extract with a secondary metabolite concentration of 50 mg / mL. Example 2

[0017] The strain was named Halomonas sp. HZ-6, with the 16S rRNA gene sequence shown in SEQ ID NO:1 and NCBIGenBank accession number PZ523056. The strain was isolated from saline-alkali soil in Dongdaohaizi, Urumqi, Xinjiang. The specific procedures were as follows: a. The activated Halomonas bacteria Halomonas Sp. HZ-6 seed culture was inoculated at a volume ratio of 6% into a saline liquid culture medium containing 15% sodium chloride. Fermentation was carried out at 37 ℃ with constant temperature shaking at 180 rpm for 8 days. The initial pH of the fermentation medium was 8.0. The fermentation broth consisted of 100 mL of the following saline liquid culture medium components: 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, and 15 g sodium chloride. The medium was brought to a final volume with deionized water, and the pH was adjusted to 7.1-7.2 with 1 mol / L sodium hydroxide. The mixture was then autoclaved at 120 ℃ for 20 min. b. The fermentation broth obtained in step a is ultrasonically disrupted to break up the bacterial cells, centrifuged at 8000 rpm for 15 min, and the supernatant is collected. c. Extract the supernatant from step b four times with equal volumes of ethyl acetate, combine the organic phases, concentrate under reduced pressure at 40 °C, and dry to obtain a crude ethyl acetate extract with a secondary metabolite concentration of 80 mg / mL. Example 3

[0018] The strain was named Halomonas sp. HZ-6, with the 16S rRNA gene sequence shown in SEQ ID NO:1 and NCBIGenBank accession number PZ523056. The strain was isolated from saline-alkali soil in Dongdaohaizi, Urumqi, Xinjiang. The specific procedures were as follows: a. The activated Halomonas bacteria HalomonasSp. HZ-6 seed culture was inoculated at 8% (v / v) into a saline liquid culture medium containing 15% sodium chloride. Fermentation was carried out at 37 ℃ with constant temperature shaking at 180 rpm for 8 days. The initial pH of the fermentation medium was 8.0. The resulting fermentation broth consisted of 100 mL of the following saline liquid culture medium: 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, and 15 g sodium chloride. The mixture was brought to a final volume with deionized water, and the pH was adjusted to 7.1-7.2 with 1 mol / L sodium hydroxide. The mixture was then autoclaved at 120 ℃ for 20 min. b. The fermentation broth obtained in step a is ultrasonically disrupted to break up the bacterial cells, centrifuged at 8000 rpm for 15 min, and the supernatant is collected. c. Extract the supernatant from step b five times with equal volumes of ethyl acetate, combine the organic phases, concentrate under reduced pressure at 40 °C, and dry to obtain a crude ethyl acetate extract with a secondary metabolite concentration of 100 mg / mL. Example 4

[0019] Isolation, purification, and identification of bacterial strains: Soil sample collection: Soil samples were collected in May 2025 from the edge of Dongdaohaizi Lake in Midong District, Urumqi, Xinjiang Uygur Autonomous Region, at geographical coordinates of 44°41.7′N, 87°33.5′E, and an altitude of approximately 361 m. The soil salinity was 16.7% and the pH was 8.9. The rhizosphere soil from the 0-20 cm topsoil layer was collected using a five-point sampling method. Equal amounts of soil from the five sampling points were mixed into a single composite sample. After removing impurities such as dead branches, fallen leaves, and stones, the samples were placed in sterile sealed bags, placed in a low-temperature incubator, and transported back to the laboratory for storage at -80℃ for future use. Isolation and purification of strains: Weigh 10.0 g of pretreated fresh soil, add 90 mL of sterile water and shake for 30 min, then prepare a 10⁻¹ suspension at 180 rpm. Serially dilute to 10⁻¹. 6 Select 10⁻ 2 10⁻ 3 10⁻ 4 10⁻ 5Diluent, 0.2 mL, was spread onto MH solid medium plates containing 15% sodium chloride (w / v). The plates were incubated at 37°C in the dark for 7-14 days, observing colony characteristics daily. Single colonies were picked and inoculated onto the same medium using the streak plate method. The streak plate was repeated 2-3 times to obtain a pure culture. The purified strain was stored in glycerol tubes at -80°C. The raw material composition (100 mL) of the MH solid medium containing 15% sodium chloride (w / v) was as follows: magnesium sulfate heptahydrate 0.96 g, magnesium chloride hexahydrate 0.7 g, potassium chloride 0.2 g, calcium chloride 0.036 g, sodium bicarbonate 0.006 g, sodium bromide 0.0026 g, peptone 0.5 g, yeast extract 1 g, sodium chloride 15 g. The mixture was brought to a final volume of 100 mL with deionized water, and the pH was adjusted to 7.1-7.2 with 1 mol / L sodium hydroxide. The mixture was then autoclaved at 120°C for 20 minutes. min, without agar is liquid culture medium; Identification of strains: Morphological observation and molecular biological identification were performed on the isolated pure strains: Morphological phenotypic observation and identification: Observe the colony characteristics of strain HZ-6 after culturing on plates containing 15% sodium chloride for 5-10 days: This strain is light milky yellow, opaque, with a smooth and moist surface, regular raised areas, and a viscous texture on solid medium containing 15% sodium chloride; Morphological electron microscopy identification: A small amount of bacterial cells cultured on a petri dish was picked up with a sterile 100 μL yellow pipette tip and placed in a centrifuge tube. A fixative solution of 37% formaldehyde (5 mL), glacial acetic acid (5 mL), 50% ethanol (90 mL), and glycerol (5 mL) was added. The solution was fixed overnight at 4 ℃. Subsequently, a gradient ethanol dehydration treatment was performed as follows: 50%, 60%, 70%, 75%, 85%, and 95% ethanol solutions were added sequentially, each for 15 min. Finally, 100% ethanol was used twice, each time for 15 min. Between each dehydration step, centrifugation was required to ensure the bacterial cells settled to the bottom of the tube. After discarding the supernatant, the next concentration of ethanol solution was immediately added to prevent the bacterial cells from deforming due to drying. After dehydration, tert-butanol was added, and the sample was frozen at 4 ℃ for 30 min. If the tert-butanol did not solidify, it was replaced with fresh tert-butanol. After the tert-butanol was completely solidified, the sample was freeze-dried for 12 minutes. h; Take the lyophilized bacterial cells, pick them up with a toothpick and adhere them to conductive tape, then use a hair dryer or bulb syringe to blow away any loose cells; subsequently, place the sample in an ion sputtering system (Hitachi E-1045, Japan) for platinum (Pt) metal sputtering; finally, observe the bacterial morphology and take pictures using a scanning electron microscope (Zeiss Supra 55 VP, Germany); the colony morphology observation was repeated 3 times on plates, and 20 cells were randomly selected for electron microscopy to measure the length and take the average value. The cells were slender rod-shaped, growing dispersedly in single-cell form, with a length of 2.244 μm; the scanning electron microscopy observation showed that the fixative was prepared as follows: 37% formaldehyde 5 mL; glacial acetic acid 5 mL; 50% alcohol 90 mL; glycerol 5 mL, and ultrasonically mixed. Molecular biological identification: Total DNA was extracted from strain HZ-6 using a bacterial genomic DNA extraction kit (Sangon Biotech, Shanghai). Using the extracted DNA as a template, 16S rRNA gene PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction system (50 μL) consisted of: 25 μL of 2×TaqPCR MasterMix, 1 μL each of 10 μM forward and reverse primers, 2 μL of DNA template (50 ng / μL), and ddH2O to a final volume of 50 μL. The amplification program was as follows: 95 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 1 min, 55 ℃ annealing for 1 min, 72 ℃ extension for 1.5 min, for a total of 30 cycles; final extension at 72 ℃ for 10 min. min; the PCR product was detected by 1% agarose gel electrophoresis, purified and sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing; the obtained sequence (SEQ ID NO:1) was submitted to the NCBI GenBank database for BLAST homology comparison, and a phylogenetic tree was constructed using MEGA software with the neighbor-joining method, and the bootstrap value was set to 1000 replicates; NCBI GenBank accession number PZ523056.

[0020] The sequence listing SEQ ID NO:1 of the strain described in this invention: 1 ctgcggcagctacacatgcagtcgagcggaacgatggaagcttgcttccaggcgtcgagc 61 ggcggacgggtgagtaatgcataggaatctgcccgatagtgggggataacgtggggaaac 121 tcacgctaataccgcatacgtcctacgggagaaagcaggggatcttcggaccttgcgcta 181 tcggatgagcctatgtcggattagctagttggtgaggtaacggctcaccaaggcgacgat 241 ccgtagctggtctgagaggatgatcagccacactgggactgagacacggcccagactcct 301 acgggaggcagcagtggggaatattggacaatgggggaaaccctgatccagccatgccgc 361 gtgtgtgaagaaggccttcgggttgtaaagcactttcagcgaggaagaaggcctgagggc 421 taatacccttcaggaaggacatcactcgcagaagaagcaccggctaactccgtgccagca 481 gccgcggtaatacggagggtgcgagcgttaatcggaattactgggcgtaaagcgcgcgta 541 ggtggcttgataagccggttgtgaaagccccgggctcaacctgggaacggcatccggaac 601 tgtcaggctagagtgcaggagaggaaggtagaattcccggtgtagcggtgaaatgcgtag 661 agatcgggaggaataccagtggcgaaggcggccttctggactgacactgacactgaggtg 721 cgaaagcgtgggtagcaaacaggattagataccctggtagtccacgccgtaaacgatgtc 781 gactagccgttgggttccttgagaactttgtggcgcagttaacgcgataagtcgaccgcc 841 tggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggt 901 ggagcatgtggtttaattcgatgcaacgcgaagaaccttacctacccttgacatcgtgcg 961 aactttccagagatggattggtgccttcgggaacgcacagacaggtgctgcatggctgtc 1021 gtcagctcgtgttgtgaaatgttgggttaagtcccgtaacgagcgcaacccttgtcccta 1081 tttgccagcgattcggtcgggaactctagggagactgccggtgacaaaccggaggaaggt 1141 ggggacgacgtcaagtcatcatggcccttacgggtagggctacacacgtgctacaatggt 1201 cggtacaaagggttgcgataccgcgaggtggagctaatcccataaagccggtctcagtcc 1261 ggatcggagtctgcaactcgactccgtgaagtcggaatcgctagtaatcgtgaatcagaa 1321 tgtcacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgga 1381 ctgcaccagaagtggttagcttaaccttcgggggagcgatcacccagcgggggg.

[0021] The obtained sequences were subjected to BLAST homology alignment in the NCBI GenBank database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA software. The results showed that *Haloxylon ammodendron*... Halomonas sp. HZ-6 formed a highly supported (99% bootstrap value) independent clade within the genus *Haloxylon*, distinct from *Haloxylon*. Halomonas sp. EGI 35026 is most closely related. Figure 3 Therefore, this strain was identified as a member of the genus *Halomonas* and named *Halomonas*. Halomonas sp. HZ-6. Example 5

[0022] Growth characteristics of the strain: Growth characteristics of the strains at different culture times: This experiment investigated the effects of different culture durations on *Halomonas*. Halomonas The effects of activated *Haloxylon ammodendron* on the growth characteristics of strain HZ-6 were investigated to clarify the complete growth cycle of the strain and determine the optimal culture time. Halomonas The HZ-6 seed culture was inoculated into a saline liquid medium containing 15% sodium chloride and an initial pH of 7.0 at a rate of 6% (v / v). The culture system consisted of 20 mL of medium per 100 mL Erlenmeyer flask. After inoculation, the flask was continuously cultured at 37°C and 180 rpm under constant temperature shaking conditions. The experiment was set up with a continuous time gradient of 3-51 h, with a total of 17 detection time nodes. Samples were taken at 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 21 h, 24 h, 27 h, 30 h, 33 h, 36 h, 39 h, 42 h, 45 h, 48 h, and 51 h of culture. The biomass of the strain was characterized by measuring the optical density value OD600 (OD, hereinafter referred to as OD). Each time node was set up with 3 parallel replicate experiments. Based on the biomass change pattern of the strain throughout its entire life cycle, *Haloxylon ammodendron* was... Halomonas The growth cycle of sp. HZ-6 can be divided into three stages: the first is the lag phase, during which the growth rate of the strain is slow and the OD600 value remains stable in the range of 0.1-0.3 within 0-9 h of culture; the second is the logarithmic growth phase, during which the metabolic activity of the strain is vigorous and the proliferation rate is significantly increased within 9-36 h of culture, and the OD600 value rises rapidly to 1.6 or above; the third is the stationary phase, after which the growth of the strain tends to be stable, the biomass basically no longer increases, and the OD600 value remains stable in the range of 1.7-1.8. Based on the characteristics of the strain's growth rate and biomass accumulation, the optimal culture time for this strain is determined to be 24 h. Growth characteristics of the strain under different salt concentrations: To clarify the identity of Halomonas Halomonas This study investigated the salt tolerance and halophilic type of strain sp. HZ-6. Salt tolerance verification tests were conducted based on the commonly used classification standards for halophilic bacteria in the field of microbiology. Based on the optimal sodium chloride concentration for growth, the microorganisms were classified into six categories: non-halophilic bacteria, weakly halophilic bacteria, moderately halophilic bacteria, marginal extreme halophilic bacteria, extreme halophilic bacteria, and halophilic bacteria. Specifically, the optimal sodium chloride concentration for non-halophilic bacteria was <0.2 mol / L (<1.170%, w / v); for weakly halophilic bacteria, it was 0.2–0.5 mol / L (1.17%–2.93%, w / v); for moderately halophilic bacteria, it was 0.5–2.5 mol / L (2.93%–14.630%, w / v); for marginal extreme halophilic bacteria, it was 1.5–4.5 mol / L (8.78%–26.34%, w / v); and for extreme halophilic bacteria, it was 2.5–5.2 mol / L. The strain with a sodium chloride concentration of 0.2 mol / L (14.63%-30.40%, w / v) is an obligate high-salt-tolerant strain; salt-tolerant bacteria can tolerate sodium chloride concentrations ranging from 0.2 to 5.2 mol / L (1.17%-30.40%, w / v), and can adapt to a wide range of salt concentration environments. Based on the above classification criteria, this experiment set up gradient salt concentrations to investigate the growth patterns of the strains. The experimental results showed that *Halomonas* exhibited optimal growth within a sodium chloride concentration range of 16%-20%, with an OD value of 1.2-1.4 and the highest biomass accumulation. As the salt concentration increased, the growth rate of the strain gradually decreased. Even at a sodium chloride concentration of 24%, the strain could still proliferate normally, corresponding to an OD value of approximately 0.5. Even in an extreme high-salt environment of 30%, the strain maintained good survival and proliferation capabilities, with an OD value close to 0.4. Based on the classification criteria for halophilic microorganisms, the optimal growth salt concentration and the extreme tolerance salt concentration of this strain both fall within the 14.63%-30.40% (w / v) range for extreme halophiles, conforming to the biological classification characteristics of extreme halophiles. This confirms the effectiveness of the *Halomonas* strain tested in this experiment. Halomonas sp. HZ-6 is a typical extreme halophilic bacterium; Growth characteristics of the strain under different pH conditions: To investigate the effects of environmental pH on Halomonas bacteria Halomonas To investigate the effects of different initial pH levels on the growth and proliferation of strain sp. HZ-6, and to determine the strain's pH tolerance range and optimal growth pH conditions, this study employed a gradient initial pH system for strain culture experiments. A series of saline liquid culture media with initial pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, and 12.0 were prepared. The activated strain seed culture was inoculated into each group of culture media at a 6% (v / v) inoculum. The cultures were then uniformly cultured for 24 h under constant temperature shaking at 37 ℃ and 180 rpm. The OD value of each group of samples was measured to characterize the strain biomass, thereby evaluating the strain's growth performance under different pH conditions. The results showed that *Haloxylon ammodendron*: *Haloxylon ammodendron* Halomonas The sp. HZ-6 strain exhibits strong acid and alkali tolerance, growing and proliferating normally within a wide pH range of 3.0-12.0. The optimal pH range for its growth is 5.0-10.0, within which the strain demonstrates good growth activity and stable biomass accumulation. The optimal pH for growth is 8.0-9.0, under which conditions the strain exhibits the best proliferative and metabolic activity, with an OD value reaching 1.5-1.7 and peak biomass accumulation. These results indicate that this strain prefers a slightly alkaline growth environment while also possessing excellent broad-spectrum acid and alkali tolerance. Example 6

[0023] Preparation and fermentation process optimization of secondary metabolites a. Preparation of strain seed liquid Preservation of glycerin at low temperatures Halomonassp. HZ-6 was inoculated into a saline solid medium containing 15% (w / v) sodium chloride and incubated at 37 ℃ in the dark for 7 days. Single colonies with intact morphology and no contamination were picked and transferred to a 100 mL Erlenmeyer flask (20 mL volume) containing saline liquid medium and incubated at 37 ℃ with shaking at 180 r / min for 24 h to obtain a seed culture in the logarithmic growth phase. The OD of the seed culture was... 600 Keep it at 1.60±0.05 for later use; Salt-containing liquid culture medium formula (100 mL): 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, 15 g sodium chloride, bring to volume with deionized water, adjust pH to 7.1-7.2 with 1 mol / L sodium hydroxide, autoclave and cool before use; b. Standardized fermentation, crushing and extraction processes Will Halomonas The HZ-6 seed culture was inoculated at a rate of 6% into a saline liquid culture medium containing 15% sodium chloride and pH 7.1-7.2, and cultured under constant temperature shaking at 37 ℃ and 180 rpm. After fermentation, the culture was ultrasonically disrupted at 200 W, with a working time of 2 s followed by a 3 s interval, for a total time of 10 min. The mixture was then centrifuged at 8000 rpm for 15 min to separate the supernatant. The supernatant was subjected to multi-stage extraction with an equal volume of ethyl acetate. The single extraction conditions were: shaking at 100 r / min for 150 min, followed by standing for 2 h to separate the organic phase. This extraction was repeated 4 times, and all ethyl acetate organic phases were combined. The organic phase was transferred to a rotary evaporator and concentrated under reduced pressure at a constant water bath temperature of 40 ℃ and a vacuum degree of -0.08 MPa until completely dry to obtain a crude extract of ethyl acetate secondary metabolites. The crude extract was sealed and protected from light and stored at -20 ℃ for subsequent high-performance liquid chromatography analysis and in vitro antibacterial activity determination. c. High-performance liquid chromatography (HPLC) detection conditions Chromatographic column: Agilent ZORBAX Eclipse Plus C18 (4.6 × 250 mm, 5 μm); mobile phase A: water (containing 0.1% formic acid); mobile phase B: acetonitrile (containing 0.1% formic acid); gradient elution program: 0-5 min, 5% B; 5-40 min, 5%-40% B; 40-70 min, 40%-80% B; 70-80 min, 80%-95% B; 80-85 min, 95% B; 85-90 min, 95%-5% B; column temperature: 30 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: full wavelength scan 200-600 nm, chromatogram recording wavelength: 254 nm; d. Single-factor variable optimization experimental design (1) Screening of extraction solvents The fermentation supernatant was obtained and subjected to gradient extraction with petroleum ether, dichloromethane, ethyl acetate, and n-butanol, respectively, 4-5 times with each solvent. The organic phases were combined and concentrated under reduced pressure. Metabolites from each extract fraction were analyzed by high-performance liquid chromatography. Blank control group: uninoculated. Halomonas The blank salt-containing culture medium of sp. HZ-6 was simultaneously fermented, extracted, and concentrated to eliminate interference from chromatographic peaks caused by impurities in the culture medium itself. The results showed that the number of chromatographic peaks and the peak area response values ​​of the ethyl acetate extract were significantly higher than those of the other three solvents, and the variety of polar secondary metabolites was the most abundant. Therefore, ethyl acetate was determined to be the optimal extraction solvent for the secondary metabolites of this strain. (2) Fermentation time gradient optimization Fermentation durations of 4 days, 8 days, 15 days, and 22 days were set. After fermentation, extraction and high-performance liquid chromatography (HPLC) detection were performed. The results showed that on day 8 of fermentation, the sum of the peak areas of the core characteristic metabolic peaks with retention times of 12–22 min reached its peak value, and the response value of the characteristic high-abundance chromatographic peak was the highest at 32 min. Considering both metabolite abundance and time cost, 8 days was determined to be the optimal fermentation period.

[0024] (3) Optimization of initial pH during fermentation With a fixed fermentation period of 8 days, the initial pH of the saline liquid culture medium was adjusted to 6.0, 8.0, and 10.0, respectively, while other conditions remained consistent. High-performance liquid chromatography (HPLC) analysis was performed after fermentation and extraction. Results showed that the initial pH of 8.0 resulted in the highest number of chromatographic peaks and the highest overall peak intensity, maximizing the accumulation of secondary metabolites and representing the optimal initial pH for fermentation.

[0025] (4) Comparison test of culture medium types Fermentation was carried out for 8 days using 15% (w / v) sodium chloride saline liquid medium and rice solid medium, respectively. The high-performance liquid chromatography (HPLC) chromatograms after treatment under the same extraction conditions were compared. Results: The high-salt liquid medium was more conducive to the synthesis of polar antibacterial secondary metabolites and is suitable for the large-scale preparation of active extracts.

[0026] This embodiment establishes Halomonas Standardized preparation process of secondary metabolites of sp. HZ-6: Using a saline liquid culture medium containing 15% (w / v) sodium chloride and an initial pH of 8.0 as the fermentation substrate, fermentation was carried out at 37 ℃ and 180 r / min for 8 days. After ultrasonic disruption and low-temperature centrifugation, the fermentation broth was subjected to multi-stage extraction with ethyl acetate and concentration under reduced pressure at 40 ℃ to obtain crude extract. Under this process, the secondary metabolites of the strain exhibit the best chemical diversity and can stably obtain high-abundance active metabolites, providing a standardized preparation scheme for subsequent separation of active monomers and formulation development. Example 7

[0027] Evaluation of the antibacterial activity of secondary metabolites: Test strains: Escherichia coli ( Escherichia coli ATCC11229, EC), Staphylococcus aureus ( Staphylococcus aureus ATCC6538, SA), Candida albicans ( Candida albicans ATCC10231, CA). Preparation of bacterial culture: Cryopreserved *Escherichia coli*, *Staphylococcus aureus*, and *Candida albicans* were inoculated onto their respective solid culture media: LB broth for *E. coli* and *S. aureus*, and Saburg broth for *Candida albicans*. The media were incubated at 37 °C. After colony growth, single colonies were transferred to their respective liquid media: LB broth for *E. coli* and *S. aureus*, and Saburg broth for *Candida albicans*. The media were incubated at 37 °C and 180 rpm for 24 h on a shaker. At a 1% inoculation rate, the media were incubated for 16-19 h to achieve a bacterial concentration of 5 × 10⁻⁶. 5 cfu / mL -5×10 6cfu / mL, for later use; the raw material composition of the LB (Luria-Bertani) solid-based medium by weight / volume percentage is: 1 g peptone, 0.5 g yeast extract, 1 g sodium chloride, 1.5 g agar powder, diluted to 100 mL with deionized water, adjusted to pH 7.0 with 1 mol / L sodium hydroxide, and autoclaved at 120 ℃ for 20 min. The liquid medium is prepared without agar. The raw material composition of the Saburg medium solid-based medium by weight / volume percentage is: 4 g glucose, 1 g peptone, 2 g agar powder, diluted to 100 mL with deionized water, adjusted to pH 5.6 with 1 mol / L hydrochloric acid, and autoclaved at 120 ℃ for 20 min. The liquid medium is prepared without agar. Preparation of bacterial plates: Cool the autoclaved agar medium (LB medium for Escherichia coli and Staphylococcus aureus, and Saburg medium for Candida albicans) to 46±0.5℃, and add fresh bacterial suspension prepared from the bacterial suspension to the medium at the following ratios: 0.2% (v / v) for Escherichia coli, 1.2% (v / v) for Staphylococcus aureus, and 1.6% (v / v) for Candida albicans. Mix well and pour into petri dishes (90 mm Petri dishes), 15-20 mL per dish, and let stand for 20 min to allow the medium to solidify. Drilling and sample loading: Using a sterile agar punch, perforates were made on the plates with 6 mm wells, 4-5 wells per plate, evenly distributed, with a center-to-center spacing of more than 25 mm and a distance of more than 15 mm between the well and the edge of the plate. The crude ethyl acetate extract prepared in Examples 1-3 was prepared into a 100 mg / mL solution using dimethyl sulfoxide (DMSO), and 20 μL was added to each well. Positive controls: 100 mg / mL ampicillin for Escherichia coli, 50 mg / mL kanamycin for Staphylococcus aureus, and 100 μg / mL amphotericin B for Candida albicans. The negative control was an equal volume of dimethyl sulfoxide (DMSO). After adding the samples, the plates were incubated at 37 ℃ for 30-60 min to allow for full absorption of the drug, and then inverted for 16-18 h. The diameter of the inhibition zone was measured using calipers; an inhibition zone diameter >7 mm was considered to have antibacterial effect. The results showed that: Halomonas Halomonas The secondary metabolites of sp. HZ-6 showed antibacterial activity against all three tested strains, with the largest and most transparent inhibition zone against Escherichia coli, indicating the most significant antibacterial effect. This suggests that the secondary metabolites of this strain have broad-spectrum antibacterial activity.

[0028] In summary, the *Haloxymonas* strain provided by this invention... Halomonassp. HZ-6 exhibits strong salt and alkali tolerance, rich chemical diversity of its secondary metabolites, and well-defined optimal fermentation process parameters (ethyl acetate extraction, 8-day fermentation, initial pH 8.0). Furthermore, its metabolites possess broad-spectrum antibacterial activity, making it a novel microbial resource applicable to the development of antibacterial natural products and the ecological improvement of saline-alkali land.

Claims

1. A method for preparing secondary metabolites from Halomonas bacteria, characterized in that: The strain was named Halomonas The 16S rRNA gene sequence of sp. HZ-6 is shown in SEQ ID NO:1, and the NCBI GenBank accession number is PZ523056. The strain was isolated from the saline-alkali soil of Dongdaohaizi, Urumqi, Xinjiang. The specific operations were carried out according to the following steps: a. The activated Halomonas bacteria Halomonas Sp. HZ-6 seed culture was inoculated into a saline liquid medium containing 15% sodium chloride at a volume ratio of 5-8%. Fermentation was carried out at 37 ℃ with constant temperature shaking at 180 rpm for 8 days. The initial pH of the fermentation medium was 8.

0. The fermentation broth was obtained by adding the following components to 100 mL of saline liquid medium: 0.96 g magnesium sulfate heptahydrate, 0.7 g magnesium chloride hexahydrate, 0.2 g potassium chloride, 0.036 g calcium chloride, 0.006 g sodium bicarbonate, 0.0026 g sodium bromide, 0.5 g peptone, 1 g yeast extract, and 15 g sodium chloride. The mixture was brought to a final volume with deionized water and sterilized. The pH was then adjusted to 7.1-7.

2. b. The fermentation broth obtained in step a is ultrasonically disrupted to break up the bacterial cells, centrifuged at 8000 rpm for 15 min, and the supernatant is collected. c. Extract the supernatant from step b with an equal volume of ethyl acetate 4-5 times, combine the organic phases, concentrate under reduced pressure at 40 ℃, and dry to obtain a crude ethyl acetate extract with a secondary metabolite concentration of 50-100 mg / mL.

2. The use of the crude ethyl acetate extract of the secondary metabolite obtained by the method of claim 1 in drugs for inhibiting Escherichia coli, Staphylococcus aureus, or Candida albicans.

3. The application as described in claim 2, wherein the product dosage form includes an aqueous solution, powder, gel, wettable powder, capsule, or antibacterial spray.

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