Marine streptomyces SH110 and application of source compound thereof

Abstract

The invention discloses an application of marine streptomyces SH110 and a source compound thereof, the marine streptomyces SH110 has a preservation number of GDMCC (China General Microbiological Culture Collection Center) NO.66668, and the preservation date is July 14, 2025; the invention also discloses application of the marine streptomyces SH110-derived compound in preparation of a medicine for preventing and treating streptococcus and vibrio diseases. The marine streptomyces source compound has efficient vibrio and streptococcus resisting activity, the vibrio and streptococcus resisting activity is not reported, and the marine streptomyces source compound has new application. The marine streptomyces source compound is used for preparing the medicine for preventing and treating the vibrio and streptococcus diseases, especially for preparing the medicine for preventing and treating the vibrio and streptococcus diseases of aquaculture animals, the safety is high, and the treatment effect is good.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a marine Streptomyces SH110 and its derived compounds, particularly the application of a cinnamic acid compound derived from a coral-symbiotic Streptomyces in the preparation of drugs for the prevention and treatment of Streptococcus and Vibrio diseases. Background Technology

[0002] Aquaculture is a pillar industry in my country's economic development, providing high-quality animal protein to the Chinese people. However, in recent years, bacterial diseases in farmed aquatic animals have become increasingly frequent, seriously threatening the healthy and sustainable development of the aquaculture industry. Currently, the most harmful pathogenic bacteria are Vibrio spp. and Streptococcus spp. Vibrio is one of the most common opportunistic Gram-negative bacteria in the marine environment, capable of infecting various aquatic animals such as fish, shrimp, crabs, and shellfish, causing serious harm to marine aquaculture and resulting in economic losses of tens of billions of yuan annually in my country's aquaculture industry. More than 20 types of Vibrio are known to cause disease, among which Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus are also major pathogens causing acute gastroenteritis from seafood in coastal areas. Streptococcus not only affects farmed fish in marine waters but also farmed eels and tilapia in brackish and freshwater waters. The main pathogens include *Streptococcus dolphinae* and *Streptococcus agalactiae*. *Streptococcus dolphinae* is characterized by its wide range of hosts, high infectivity, and high mortality rate, causing significant losses to the aquaculture industry. *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] To reduce economic losses caused by various diseases, aquaculture farmers indiscriminately use large quantities of various antibiotics to treat diseases in their aquatic animals. However, with the continuous expansion of aquaculture scale and the excessive use of antibiotics, the pathogenic bacteria in aquatic animals have become more resistant to drugs, posing a significant challenge to the control of bacterial diseases in aquatic animals. In the future, there may be a situation where no drugs are available. Therefore, it is urgent to find new antibiotic alternatives to address the problems facing the green and healthy development of the aquaculture industry. Currently, the rational use of antibiotics and the continued search for new antibiotics remain crucial aspects of effective integrated management of bacterial diseases in aquatic animals. Summary of the Invention

[0004] The purpose of this invention is to provide a marine Streptomyces SH110.

[0005] Another objective of this invention is to provide the application of a marine streptomyces-derived compound in the preparation of drugs for the prevention and treatment of streptococcal and vibrio diseases.

[0006] The first objective of the present invention can be achieved by the following technical solution: a marine Streptomyces sp., classified as Streptomyces sp., with accession number GDMCC No. 66668, accession date July 14, 2025, deposited at Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The marine Streptomyces sp. SH110 described in this invention was isolated from coral samples collected from the Xisha Islands in Hainan, China.

[0008] The second objective of this invention can be achieved through the following technical solution: the application of a marine Streptomyces-derived compound in the preparation of drugs for the prevention and treatment of Streptococcus and Vibrio diseases, wherein the marine Streptomyces-derived compound has the structure of the following formula (I):

[0009]

[0010] The marine Streptomyces-derived compounds were obtained by fermentation and isolation of the marine Streptomyces sp. SH110.

[0011] The structural formula of the marine Streptomyces-derived compound of this invention is C1 18 H 14 O5, with a measured molecular weight of 311.0846 ([M+H]). + The quasi-molecular ion peak, chemically named (1-p-hydroxy-cis-cinnamoyl)cinnamic acid.

[0012] The marine streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid described in this invention was obtained by fermentation and isolation of Streptomyces sp. SH110, a symbiotic fungus of the Xisha coral.

[0013] This invention has discovered through experiments that the marine Streptomyces sp. SH110 can produce the compound (1-p-hydroxy-cis-cinnamoyl)cinnamic acid, and antibacterial activity tests show that it has significant anti-Vibrio and anti-Streptococcal activity. Currently, there are no reports of microorganisms producing this compound, nor of its antibacterial activity, and no related drugs are available on the market.

[0014] This invention utilizes compounds derived from marine Streptomyces in the preparation of drugs for the prevention and treatment of Vibrio and Streptococcus diseases, particularly in the preparation of drugs for the prevention and treatment of Vibrio and Streptococcus diseases in aquatic animals, exhibiting high safety and good therapeutic effects.

[0015] As a preferred embodiment of the present invention, the method for preparing the marine Streptomyces-derived compound of the present invention includes the following steps:

[0016] (1) The marine Streptomyces sp. SH110 was inoculated onto ISP2 medium plates with added semi-sea salt and cultured at 28°C for 3 days. An appropriate amount of culture was then inoculated into TSB liquid medium with added semi-sea salt and cultured at 26-30°C and 160-180 r / min for 3-5 days to obtain seed culture solution. The seed culture solution was then inoculated into rice medium with added semi-sea salt at an inoculation rate of 10%-15% and fermented at 26-30°C for 25-35 days to obtain fermented product.

[0017] (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.

[0018] (3) The fermentation extract was subjected to normal-phase silica gel column chromatography with gradient elution of dichloromethane-methanol at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, 100:64, and 0:100 to obtain 9 fractions A, B, C, D, E, F, G, H, and I. Fraction F (100:16 dichloromethane-methanol eluent) was subjected to reverse-phase silica gel column chromatography to obtain 11 fractions (F1 to F11). Fraction F1 was subjected to Sephadex LH-20 gel column chromatography and then separated and purified by high-performance liquid chromatography equipped with a semi-preparative C18 column to obtain the marine streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid.

[0019] In the method for preparing the marine Streptomyces-derived compound:

[0020] Preferably, the ISP2 culture medium with added semi-sea salt 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, pH 7.0-7.4.

[0021] Preferably, the TSB culture medium with added semi-sea salt 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, 1L deionized water, and pH 7.0-7.4.

[0022] Preferably, the rice culture medium with added semi-sea salt in step (1) consists of the following components: 50g rice, 15-20g sea salt, and 60mL water.

[0023] Preferably, in step (2), the fermentation product is extracted (fermentation broth) with an equal volume of ethyl acetate 4 to 6 times, and the temperature for vacuum concentration is 40 to 50°C.

[0024] The preparation method of the marine streptomyces-derived compound described in this invention is simple, environmentally friendly, and low in cost.

[0025] Preferably, when marine streptomyces-derived compounds are used to prevent and control streptococcal and vibrio diseases, they are used to prevent and control abnormal fish behaviors such as wandering away from the group at the edge of the pond, intermittent spiral swimming, and unbalanced swimming postures; symptoms that appear in fish infected with streptococci, such as ocular 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 kidneys, caudal fin erosion, anal redness and swelling, and ascites; and to treat diseases of aquatic animals caused by Vibrio harveyi, Vibrio alginolyticus, Vibrio erwinis, and other Vibrio species.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The marine streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid in this invention has highly efficient anti-Vibrio and anti-streptococcal activity, which has not been reported before and has new uses;

[0028] (2) The marine Streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid in this invention is derived from marine Streptomyces sp. SH110, and has low side effects and excellent activity;

[0029] (3) The marine Streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid in this invention can be produced by fermentation of marine Streptomyces sp. SH110. The fermentation raw materials are readily available, the production process is green and environmentally friendly, and the cost is low, making it suitable for use in the production of marine drugs.

[0030] (4) The present invention demonstrates through antibacterial activity experiments that marine streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid with the structure shown in formula (I) has excellent anti-Vibrio and anti-streptococcal activity. It can be used as a drug to treat fish behavioral abnormalities such as wandering away from the group at the edge of the pond, intermittent spiral swimming, and unbalanced swimming posture, as well as ocular hemorrhage, corneal opacity, unilateral or bilateral eyeball protrusion or detachment, congestion on the inner side of the gill cover, thickened blood vessels, hepatosplenomegaly and hemorrhage of the liver, spleen and kidneys, caudal fin erosion, anal redness and swelling, ascites, and other symptoms of streptococcal infection in fish, as well as diseases of aquatic animals caused by Vibrio harveyi, Vibrio alginolyticus, Vibrio erwinis, and other Vibrio species. Attached Figure Description

[0031] Figure 1 This is the mass spectrum of compound I in Example 3;

[0032] Figure 2 It is compound I in Example 3. 1 H-dimensional nuclear magnetic resonance spectrum;

[0033] Figure 3 It is compound I in Example 3. 13 C1-dimensional nuclear magnetic resonance spectrum;

[0034] Figure 4 This is the HSQC two-dimensional nuclear magnetic resonance spectrum of compound I in Example 3;

[0035] Figure 5 This is the HMBC two-dimensional nuclear magnetic resonance spectrum of compound I in Example 3;

[0036] Figure 6 It is compound I in Example 3. 1 H- 1 H COSY two-dimensional nuclear magnetic resonance spectrum. Detailed Implementation

[0037] 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.

[0038] The marine Streptomyces sp. SH110 used in this invention was isolated from corals collected from the Xisha Islands of China.

[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0040] Example 1: Isolation and Identification of Strains

[0041] Using the gradient dilution coating method, the grinding broth of coral samples collected from the Xisha Islands of Hainan was isolated, cultured and purified using Gao's Synthetic No. 1 medium to obtain strain SH110, which was stored in 20% glycerol tubes at -80℃.

[0042] 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.

[0043] Strain SH110 grew well on ISP2 medium, with abundant aerial mycelia and yellowish-brown spores on the surface of the colonies. This strain was identified as *Streptomyces* sp. and named *Streptomyces sp. SH110*, with accession number GDMCC NO.66668, deposit date July 14, 2025, 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.

[0044] The 16S rRNA gene sequence of this strain is as follows:

[0045]

[0046] Example 2: Fermentation of marine Streptomyces sp. SH110 and acquisition of fermentation extract

[0047] Following conventional microbial culture methods, the Streptomyces sp. SH110 strain obtained in Example 1 was inoculated onto ISP2 medium plates containing sea salt and cultured at 28°C for 3 days. An appropriate amount of Streptomyces sp. SH110 culture was picked and inoculated into TSB liquid medium containing half sea salt and cultured in a shaker at 28°C and 160 r / min for 4 days to obtain a seed culture. This seed culture was inoculated into rice medium containing half sea salt at a 10% (volume percentage) inoculation rate for fermentation and allowed to ferment statically at 28°C for 30 days to obtain the fermented product. The fermented product was extracted with an equal volume of ethyl acetate, and the extraction was repeated 3 times with an interval of 48 h between each extraction. The ethyl acetate extracts were combined and concentrated under reduced pressure at 45°C to obtain the fermentation extract.

[0048] 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.

[0049] TSB medium composition (g / L): 17g tryptone, 3g soybean papain hydrolysate, 5g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose, 17.5g sea salt, 1L deionized water, pH 7.0–7.4.

[0050] Rice culture medium composition (g / L): 50g rice, 17.5g sea salt, 60mL deionized water.

[0051] Example 3: Isolation and preparation of compound (I) from marine Streptomyces

[0052] Following conventional methods for separating microbial metabolites, the fermentation extract obtained in Example 1 was subjected to normal-phase silica gel column chromatography using dichloromethane-methanol gradient elution at volume ratios of 100:0, 100:1, 100:2, 100:4, 100:8, 100:16, 100:32, 100:64, and 0:100, yielding nine fractions: A, B, C, D, E, F, G, H, and I. Fraction F (100:16 dichloromethane-methanol eluent) was subjected to reverse-phase silica gel column chromatography to obtain eleven fractions (F1 to F11). Fraction F1 was further purified by Sephadex LH-20 gel column chromatography followed by high-performance liquid chromatography (HPLC) with a semi-preparative C18 column to obtain compound (I).

[0053] Example 4: Structural identification of compound I from marine Streptomyces

[0054] The positive source high-resolution mass spectrometry peak of compound (I) obtained in Example 3 is quasi-molecular ion at m / z 311.0846 [M+H]. + (like Figure 1 As shown), the molecular formula is C. 18 H 14 O5. Its structure is further enhanced by hydrogen ( 1 H)( Figure 2 ),carbon( 13 C)( Figure 3 ) and two-dimensional [HSQC( Figure 4 ), HMBC ( Figure 5 )and 1 H- 1 H COSY( Figure 6 The nuclear magnetic resonance data identified it as (1-p-hydroxy-cis-cinnamoyl)cinnamic acid, marking the first time this compound has been isolated and identified from a microorganism.

[0055] Compound (I) 1 H, 13 The C nuclear magnetic resonance spectral data are shown in Table 1 below:

[0056] Table 1. NMR data and assignments of compound (I) in Example 3.

[0057]

[0058] The structural formula is as follows:

[0059]

[0060] Example 5: Antibacterial activity test of marine Streptomyces-derived compound (1-p-hydroxy-cis-cinnamoyl) cinnamic acid

[0061] 5.1 Antibacterial activity test

[0062] *Vibrio harveyi*, *Vibrio alginolyticus*, *Vibrio owensii*, *Streptococcus agalactiae*, and *Streptococcus nigraecum* were used as test strains. A small amount of dimethyl sulfoxide (DMSO) was used to dissolve the compound sample (1-p-hydroxy-cis-cinnamoyl) cinnamic acid (obtained in Example 3) and the positive control drug kanamycin, and prepared a stock solution with sterile water at a concentration of 1 mg / mL. Using sterile 96-well plates, 160 μL of LB liquid medium (with 3.5% sea salt added when the test strain was *Vibrio*, by mass) was added to the first well of each row, and 100 μL of LB liquid medium (with 3.5% sea salt added when the test strain was *Vibrio*, by mass) was added to each of the remaining wells. Add 40 μL of the stock solution of compound (I) to the first well and mix well. Then dilute sequentially using the two-fold dilution method. Next, add 100 μL of pathogenic bacterial solution (observable value OD of pathogenic bacterial solution) to each well. 600 =0.6~0.8), ensuring 200μL of solution in each well. Each sample was repeated 3 times. After sealing, the samples were placed in a constant temperature incubator at 28℃ for 24h and then observed and recorded. When bacterial growth was obvious in the growth control wells, the lowest concentration of the compound that completely inhibited bacterial growth in the wells was taken as the MIC value of the antibacterial activity of compound (I).

[0063] 5.2 Results of antibacterial activity

[0064] Compound (I) (1-p-hydroxy-cis-cinnamoyl)cinnamic acid in Example 3 exhibited significant inhibitory activity against Vibrio harveyi, Vibrio alginolyticus, Streptococcus agalactiae, and Streptococcus dolphinii, with stronger antibacterial activity than the positive control drug kanamycin. Compound (I) (1-p-hydroxy-cis-cinnamoyl)cinnamic acid in Example 3 also showed some inhibitory activity against Vibrio erwinis (as shown in Table 2), indicating that this compound has broad-spectrum antibacterial activity against various aquatic pathogenic Vibrio and Streptococcus bacteria, and can be used in the preparation and application of drugs for the prevention and control of Vibrio and Streptococcus diseases, especially in aquatic farmed animals. The (1-p-hydroxy-cis-cinnamoyl)cinnamic acid provided by this invention also provides a promising lead molecule for the development of marine-derived antibacterial drugs.

[0065] Table 2 shows the antibacterial activity test results of compound (I) in Example 3 against pathogenic bacteria in aquatic animals.

[0066]

[0067] 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. A marine Streptomyces SH110, characterized in that, Its classification name is Streptomyces sp., its accession number is GDMCC NO.66668, and its accession date is July 14, 2025.

2. The application of a marine Streptomyces-derived compound in the preparation of drugs for the prevention and treatment of Streptococcus and Vibrio diseases, characterized in that, The marine Streptomyces-derived compound has the structure of formula (I): The marine Streptomyces-derived compound was obtained by fermentation and isolation of the marine Streptomyces SH110 as described in claim 1.

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