Coral symbiotic streptomyces sourced unsaturated fatty acid compound and application thereof

Abstract

The invention discloses an unsaturated fatty acid compound derived from coral symbiotic streptomyces and an application of the unsaturated fatty acid compound. The chemical structural formula of the sisa coral acid A is as shown in formula (I). The invention further discloses application of the unsaturated fatty acid compound derived from the coral symbiotic streptomyces in preparation of a medicine for preventing and treating streptococcus diseases. The cisacolinic acid A provided by the invention is derived from natural organisms, and has low side effects and excellent activity. The sisaxacoral acid A can be produced by fermenting marine streptomyces sp.SHEa3, fermentation raw materials are easy to obtain, the production process is green and environment-friendly, the cost is low, and the sisaxacoral acid A has excellent anti-streptococcus activity and is suitable for being used for preparing a medicine for preventing and treating streptococcus diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an unsaturated fatty acid compound derived from coral symbiotic Streptomyces and its application, particularly a novel unsaturated fatty acid compound derived from coral symbiotic Streptomyces and its application in the preparation of drugs for the prevention and control of streptococcal diseases, especially in the preparation of drugs for the prevention and control of streptococcal diseases in aquatic animals. Background Technology

[0002] Streptococcal disease is a serious bacterial disease affecting aquaculture, impacting not only marine fish but also brackish and freshwater fish such as eels and tilapia. The main pathogens include *Streptococcus iniae* and *Streptococcus agalactiae*. *Streptococcus iniae* is characterized by its wide host range, high infectivity, and high mortality rate, causing significant losses to aquaculture. *Streptococcus agalactiae* infects various fish species and has been a major cause of streptococcal disease in tilapia in my country in recent years, impacting production and resulting in substantial economic losses.

[0003] Currently, the main method for controlling streptococcal diseases in aquaculture is still the use of antibiotics. However, with the overuse of antibiotics, streptococci are becoming increasingly resistant to drugs, leading to the emergence of multidrug-resistant strains. As the problem of pathogen resistance becomes increasingly prominent, the search for novel, highly effective drugs or alternatives to combat streptococcal infections is urgently needed.

[0004] Currently, antibiotics used in the prevention and treatment of human, animal, and plant diseases mainly originate from actinomycete metabolites, with the majority coming from the genus *Streptomyces*. Streptomycin, tetracycline, chloramphenicol, neomycin, erythromycin, and kanamycin are important anti-infective compounds used clinically and are also widely applied in the prevention and treatment of bacterial diseases in aquaculture. Marine-derived actinomycetes produce a greater abundance and more novel metabolites than terrestrial ones. Coral reef ecosystems, in particular, are a crucial type of marine ecosystem. The abundant symbiotic actinomycete communities in healthy coral reefs form a vital defense against various pathogens. Therefore, coral symbiotic actinomycetes hold immense potential in the search for novel antibiotic molecules, offering new opportunities for the green and healthy development of the aquaculture industry. Summary of the Invention

[0005] The purpose of this invention is to provide an unsaturated fatty acid compound derived from coral symbiotic Streptomyces, which exhibits excellent activity against Streptococcus dolphinii and Streptococcus agalactiae.

[0006] The present invention also aims to provide the application of the above-mentioned unsaturated fatty acid compounds derived from coral symbiotic Streptomyces in the preparation of drugs for the prevention and treatment of streptococcal diseases.

[0007] The first objective of this invention can be achieved by the following technical solution: an unsaturated fatty acid compound derived from coral symbiotic Streptomyces, wherein the unsaturated fatty acid compound is xisha coral acid A, and the chemical structural formula of xisha coral acid A is shown in the following formula (Ⅰ):

[0008]

[0009] The structural formula of this compound is C. 12 H 18 O3, with a measured molecular weight of 211.1331 ([M+H]). + The molecular ion peak, [M+H] + The theoretical molecular weight is 211.1329.

[0010] The coral acid A from Xisha was produced through fermentation by the marine Streptomyces strain SHEa3, which was deposited on July 14, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCCNO.66667. The address of the depository is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China. The depository code is GDMCC-Guangdong Provincial Center for Microbial Culture Collection.

[0011] Therefore, the unsaturated fatty acid compound xisha coral acid A described in this invention is obtained by fermentation and isolation of marine Streptomyces sp. SHEa3.

[0012] The marine Streptomyces sp. SHEa3 was isolated from coral samples collected from the Xisha Islands in Hainan, China.

[0013] The present invention discloses a method for preparing the unsaturated fatty acid compound cisaccharide A, comprising the following steps:

[0014] (1) Inoculate Streptomyces sp. SHEa3 onto ISP2 medium plates and culture at 28℃ for 4 days. Then, pick an appropriate amount of culture and inoculate it into TSB liquid medium. Culture at 26-30℃ and 160-180 r / min for 3-5 days to obtain seed culture solution. Inoculate the seed culture solution into rice medium at an inoculation rate of 8%-10% (V / V) and let it ferment at 26-30℃ for 15-25 days to obtain fermentation product.

[0015] (2) Extract the fermentation product with ethyl acetate, combine the ethyl acetate extracts, concentrate them under reduced pressure to dryness, and obtain the fermentation extract.

[0016] (3) The fermentation extract was subjected to normal-phase silica gel column chromatography with dichloromethane-methanol gradient elution at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, and 100:64 to obtain eight fractions A, B, C, D, E, F, G, and H. Fraction C (100:2 dichloromethane-methanol eluent) was purified by reverse-phase silica gel column chromatography and Sephadex LH-20 gel column chromatography to obtain the novel unsaturated fatty acid compound xisha coral acid A.

[0017] In the preparation method of the above-mentioned unsaturated fatty acid compound, cisaccharic acid A:

[0018] Preferably, the ISP2 culture medium in step (1) consists of the following components: 4-5g yeast extract, 8-10g malt extract, 3-5g glucose, 15-20g sea salt, 15-20g agar powder, 1L deionized water, and pH 7.0-7.4.

[0019] Preferably, the 1L of TSB culture medium in step (1) consists of the following components: 15-18g tryptone, 3-5g soybean papain hydrolysate, 4-6g sodium chloride, 2-4g dipotassium hydrogen phosphate, 2-4g glucose, 15-20g sea salt, and 1L deionized water with a pH of 7.0-7.4.

[0020] Preferably, the rice culture medium in step (1) consists of the following components: 50g rice, 15-20g sea salt, and 60mL water.

[0021] Preferably, in step (2), the fermentation product is extracted 4 to 6 times with 2 times the volume of ethyl acetate, and the temperature for vacuum concentration is 40 to 50°C.

[0022] The present invention provides a method for preparing unsaturated fatty acid compounds derived from coral symbiotic Streptomyces, which is simple, environmentally friendly, and low in cost.

[0023] The second objective of the present invention can be achieved by the following technical solution: the application of the unsaturated fatty acid compounds derived from coral symbiotic Streptomyces in the preparation of drugs for the prevention and treatment of streptococcal diseases.

[0024] The unsaturated fatty acid compounds derived from coral symbiotic Streptomyces in this invention have anti-streptococcal activity and can be used in the preparation of drugs for the prevention and treatment of streptococcal diseases, especially drugs for the prevention and treatment of streptococcal diseases in aquatic animals.

[0025] Preferably, the streptococcal disease includes abnormal fish behavior such as wandering away from the group at the edge of the pond, intermittent spiral swimming, and unbalanced swimming posture; symptoms that appear in fish infected with streptococcus, such as ocular hemorrhage, corneal opacity, unilateral or bilateral eyeball protrusion or detachment, congestion on the inner side of the gill cover, thickened blood vessels, enlarged and hemorrhagic liver, spleen and kidneys, tail fin erosion, anal redness and swelling, and ascites.

[0026] Preferably, the streptococcus is agalactiae or dolphin streptococcus.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) The unsaturated fatty acid compound cisaccharide A with the structure shown in formula (I) of this invention has never been isolated from nature and its structure has never been reported. It is a novel natural compound.

[0029] (2) The coral acid A provided by this invention is derived from natural organisms, has low side effects and excellent activity;

[0030] (3) The coral acid A provided by the present invention can be produced by fermentation of marine Streptomyces sp. SHEa3. The fermentation raw materials are readily available, the production process is green and environmentally friendly, and the cost is low, making it suitable for drug production.

[0031] (4) The present invention demonstrates through antibacterial activity experiments that compounds with the structure shown in formula (I) have excellent anti-streptococcal activity and can be used as drugs to prevent and treat abnormal fish behaviors such as wandering away from the group at the edge of the pond, intermittent spiral swimming, and unbalanced swimming posture, as well as symptoms of streptococcal infection in fish such as eye hemorrhage, corneal opacity, unilateral or bilateral eye protrusion or detachment, congestion on the inner side of the gill cover, thickened blood vessels, enlarged and hemorrhagic liver, spleen and kidney, tail fin erosion, anal redness and swelling, and ascites. Attached Figure Description

[0032] Figure 1 This is the mass spectrum of compound I in Example 3;

[0033] Figure 2 It is compound I in Example 3. 1 H-dimensional nuclear magnetic resonance spectrum;

[0034] Figure 3 It is compound I in Example 3. 13 C1-dimensional nuclear magnetic resonance spectrum;

[0035] Figure 4 This is the DEPT135 one-dimensional nuclear magnetic resonance spectrum of compound I in Example 3;

[0036] Figure 5 This is the HSQC two-dimensional nuclear magnetic resonance spectrum of compound I in Example 3;

[0037] Figure 6 This is the HMBC two-dimensional nuclear magnetic resonance spectrum of compound I in Example 3;

[0038] Figure 7 It is compound I in Example 3. 1 H- 1 H COSY two-dimensional nuclear magnetic resonance spectrum;

[0039] Figure 8 This is the ROESY two-dimensional nuclear magnetic resonance spectrum of compound I in Example 3. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0041] The marine Streptomyces sp. SHEa3 used in this invention was isolated from corals collected from the Xisha Islands in Hainan.

[0042] Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0043] Example 1: Isolation and Identification of Strains

[0044] Coral samples were collected from the waters of the Xisha Islands in Hainan. After grinding, 1g of the coral grinding solution was serially diluted and spread. The samples were isolated and cultured using Gao's Synthetic No. 1 medium. After 2 weeks, strain SHEa3 was picked and purified on fresh ISP2 medium plates and stored in 20% glycerol tubes at -80℃.

[0045] Composition of Gao's Synthetic No. 1 Culture Medium (g / L): soluble starch 20.0g, NaCl 0.5g, FeSO4 0.01g, KNO3 1g, K2HPO4 0.5g, MgSO4 0.5g, agar 15.0g, potassium dichromate 0.05g, sea salt 17.5g, pH 7.1~7.5.

[0046] ISP2 culture medium composition (g / L): yeast extract 4g, malt extract 10g, glucose 4g, sea salt 17.5g, agar powder 20g, deionized water 1L, pH 7.0~7.4.

[0047] Strain SHEa3 grew well on ISP2 medium, forming tight, white colonies with central wrinkles, and abundant white spores on the surface during the later stages of culture. This strain was identified as *Streptomyces* sp. and named *Streptomycess* p. SHEa3, with accession number GDMCC NO. 66667, accession date July 14, 2025, and deposited at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology.

[0048] The 16S rRNA gene sequence of this strain is as follows:

[0049] tcgggaggggattagtggcgaacgggtgagtaacacgtgggcaatctgcccttcactctgggacaagccctggaaacggggtctaataccg

[0050] gatacgacctccgaccgcatggtctggtggtggaaagctccggcggtgaaggatgagcccgcggcctatcagcttgttggtggggtgatgg

[0051] cctaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggca

[0052] gcagtggggaatattgcacaatgggcgaaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagca

[0053] gggaagaagcgagagtgacggtacctgcagaagaagcgccggctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttg

[0054] tccggaattattgggcgtaaagagctcgtaggcggcttgtcacgtcggatgtgaaagcccggggcttaaccccgggtctgcattcgatacgg

[0055] gcaggctagagttcggtaggggagatcggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcgga

[0056] tctctgggccgatactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgccgtaaacgttgggaac

[0057] taggtgtgggcgacattccacgtcgtccgtgccgcagctaacgcattaagttccccgcctggggagtacggccgcaaggctaaaactcaaag

[0058] gaattgacgggggcccgcacaagcagcggagcatgtggcttaattcgacgcaacgcgaagaaccttaccaaggcttgacatacaccggaa

[0059] aaccctggagacagggtcccccttgtggtcggtgtacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgca

[0060] acgagcgcaacccttgttctgtgttgccagcatgcccttcggggtgatggggactcacaggagactgccggggtcaactcggaggaaggtg

[0061] gggacgacgtcaagtcatcatgccccttatgtcttgggctgcacacgtgctacaatggccggtacaatgagctgcgataccgcgaggtggag

[0062] cgaatctcaaaaagccggtctcagttcggattggggtctgcaactcgaccccatgaagtcggagttgctagtaatcgcagatcagcattgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggtaacacccgaag (as shown in SEQ ID NO: 1).

[0063] Example 2: Fermentation of Streptomyces sp. SHEa3 and Obtaining Fermentation Extract

[0064] Following standard microbial culture methods, the *Streptomyces* strain SHEa3 (Streptomycessp. SHEa3) obtained in Example 1 was inoculated onto ISP2 medium plates and cultured at 28°C for 4 days. A 1cm × 1cm square piece of *Streptomyces* strain SHEa3 was picked from the culture plate and inoculated into TSB liquid medium. The culture was then shaken at 28°C and 160 rpm for 4 days to obtain a seed culture of *Streptomyces* strain SHEa3. This seed culture was inoculated into rice medium at a 10% (volume percentage) inoculation rate for fermentation. The fermentation was allowed to stand at 28°C for 20 days to obtain the fermented product. Twice the volume of ethyl acetate was added to extract the fermented product, and the extraction was repeated five times. The ethyl acetate extracts were combined and concentrated under reduced pressure at 45°C to obtain the fermentation extract.

[0065] ISP2 culture medium composition (g / L): yeast extract 4g, malt extract 10g, glucose 4g, sea salt 17.5g, agar powder 20g, deionized water 1L, pH 7.0~7.4.

[0066] TSB liquid culture medium composition (g / L): tryptone 17g, soybean papain hydrolysate 3g, sodium chloride 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, sea salt 17.5g, deionized water 1L, pH 7.0~7.4.

[0067] Rice culture medium composition (g / L): 50g rice, 17.5g sea salt, 60mL deionized water.

[0068] Example 3: Isolation and preparation of compound I from marine Streptomyces

[0069] Following conventional methods for separating microbial metabolites, the fermentation extract obtained in Example 2 was subjected to normal-phase silica gel column chromatography with gradient elution using dichloromethane-methanol at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, and 100:64, yielding eight fractions: A, B, C, D, E, F, G, and H. Fraction C (100:2 dichloromethane-methanol eluent) was further purified by reverse-phase silica gel column chromatography and Sephadex LH-20 gel column chromatography to obtain a novel compound (Ⅰ).

[0070] Example 4: Structural identification of compound (Ⅰ) derived from marine Streptomyces

[0071] The quasi-molecular ion peak of compound (Ⅰ) in high-resolution mass spectrometry is 211.1331 [M+H]. + (like Figure 1 As shown), the molecular formula is C. 12 H 18 O3, and its [M+H] + The theoretical molecular weight (211.1329) matches. Its structure was confirmed by one-dimensional and two-dimensional nuclear magnetic resonance experiments (e.g., Figures 2-8 The compound was identified using various spectroscopic methods (as shown in the image) as a novel 6,7-epoxy unsaturated fatty acid compound with a previously unreported structure, and named Xisha coral acid A.

[0072] Compound (I) 1 H, 13 The C nuclear magnetic resonance spectral data are shown in Table 1 below:

[0073] Table 1. NMR data and attribution of compound (Ⅰ) in this embodiment.

[0074]

[0075]

[0076] The structural formula is as follows:

[0077]

[0078] Example 5: Test of the anti-streptococcal activity of the marine streptomyces-derived compound, Xisha coral acid A.

[0079] 5.1 Anti-streptococcal activity test

[0080] *Streptococcus agalactiae* and *Streptococcus dolphinae* were used as test strains. The compound sample, cisaccharide A (obtained in Example 3), and the positive control drug kanamycin were dissolved in a small amount of DMSO, and a stock solution with a concentration of 1280 μg / mL was prepared using sterile water. DMSO was used as the negative control. Sterilized 96-well polystyrene plates (8 rows × 12 columns) were used. 160 μL of LB liquid medium was added to the first well of each row, and 100 μL of LB liquid medium was added to each of the remaining wells. 40 μL of the compound sample stock solution was added to the first well and mixed. Then 100 μL was transferred to the second well, mixed, and then another 100 μL was transferred to the third well. This serial dilution was continued until the eleventh well, where 100 μL was discarded. The twelfth well contained no sample and contained pathogenic bacteria. Then, 100 μL of pathogenic bacteria solution (observation value OD) was added to each well. 600=0.6~0.8). The sample concentrations in wells 1 to 11 were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg / mL, respectively. Each sample was repeated three times. LB liquid medium control and bacterial growth control were also included. After sealing, the samples were incubated at 28℃ for 24 hours and the results were observed. When bacterial growth was significant in the control wells, the lowest compound concentration that completely inhibited bacterial growth in the wells was taken as the MIC value of the compound's antibacterial activity.

[0081] 5.2 Results of antibacterial activity

[0082] In Example 3, cisaccharide A exhibited significant inhibitory activity against both *Streptococcus agalactiae* and *Streptococcus dolphinus* (as shown in Table 2), with minimum inhibitory concentrations (MICs) of 2 and 4 μg / mL, respectively. Their activity was stronger than that of the positive control drug kanamycin, indicating that this compound can be used in the preparation and application of drugs for the prevention and control of streptococcal diseases, especially streptococcal diseases in aquatic animals. The novel epoxy-unsaturated fatty acid compound provided by this invention also offers promising lead molecules for the development of naturally derived antimicrobial drugs.

[0083] Table 2 shows the antibacterial activity test results of compound cisaccharide A in Example 3 against two strains of pathogenic streptococci in aquatic animals.

[0084]

[0085] The above examples illustrate specific embodiments of the present invention. It is important to note that these specific embodiments are only for further explanation and do not constitute a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the scope of protection of the present invention.

Claims

1. An unsaturated fatty acid compound derived from coral-symbiotic Streptomyces, characterized in that: The unsaturated fatty acid compound is cisaccharic acid A, and the chemical structural formula of cisaccharic acid A is shown in formula (Ⅰ) below:

2. The unsaturated fatty acid compound derived from coral-symbiotic Streptomyces according to claim 1, characterized in that: The coral acid A from Xisha is produced by fermentation of marine streptomyces strain SHEa3, which has the accession number GDMCC No. 66667 and the accession date of July 14, 2025.

3. The use of the unsaturated fatty acid compounds derived from coral symbiotic Streptomyces as described in claim 1 or 2 in the preparation of drugs for the prevention and treatment of streptococcal diseases.

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