Marine source penicillium as well as fermentation method and application thereof

Abstract

The invention discloses marine-derived penicillium as well as a fermentation method and application thereof. The marine source penicillium is named as Penicillium sp.DSF059, and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation date is November 23, 2023; the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: M The marine-derived penicillium is separated from deep-sea sediments at the depth of 2595m in South China Sea, a fermentation culture medium and culture conditions of the strain are optimized, a culture medium and culture conditions with relatively high yield of antibacterial active compounds are selected for fermentation, more target products can be obtained, and the marine-derived penicillium is simple to operate, low in cost and suitable for industrial production. Meanwhile, the marine-derived penicillium sp. DSF059 can provide an excellent strain for the development of new anti-staphylococcus aureus drugs, and has a relatively good application prospect in the prevention and treatment of staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a marine-derived Penicillium and a fermentation method and application thereof. Background Art

[0002] Staphylococcus aureus ( Staphylococcus aureus ) is an aerobic or facultative anaerobic Gram-positive spherical bacterium with no spores, flagella, and most of them without capsules. It has low nutritional requirements, strong resistance to adverse environments, and is widely distributed. It can be found in soil, air, and water. Staphylococcus aureus is the most common pathogen in human suppurative infections. It can cause local suppurative infections, pneumonia, pseudomembranous enterocolitis, pericarditis, and even systemic infections such as sepsis and septicemia. Staphylococcus aureus is also one of the important pathogens of bacterial food poisoning, mainly contaminating dairy products, eggs and egg products, and various cooked meat products, followed by frozen foods containing milk. According to statistics from the US Centers for Disease Control and Prevention, infections caused by Staphylococcus aureus currently rank first, and it is the main antibacterial target for the development of antibacterial drugs.

[0003] At present, the conventional prevention and control methods of Staphylococcus aureus at home and abroad are as follows: First, antibiotics are the traditional treatment for Staphylococcus aureus infection, among which β-lactam drugs (such as penicillin, etc.) are the most important and most commonly used antibiotics. However, after long-term use, Staphylococcus aureus has developed a high degree of resistance to β-lactam antibiotics. Second, bacteriophages mediate the breakage of peptidoglycan linkages in bacterial cell walls by encoding lytic proteins-endolysins, lysins, etc., leading to cell wall rupture, bacterial death and the release of new phages. Low doses of phages can inhibit the formation of biofilms, and high doses of phages can degrade biofilms, thereby killing pathogens. Third, traditional Chinese medicine can inhibit the formation of bacterial biofilms. Traditional Chinese medicine monomers such as eugenol and quercetin inhibit the formation of biofilms, exocytosis of virulence factors and expression of efflux pumps by inhibiting the sensing system of bacterial populations.

[0004] Although the above methods can effectively inhibit Staphylococcus aureus, the abuse of antibiotics and other drugs has led to the emergence of drug-resistant Staphylococcus aureus. The demand for new active biological resources and active metabolites is constantly increasing, and the need for the development of new drugs is becoming increasingly urgent. Therefore, there is an urgent need to continuously explore unique and novel biological control resources.

[0005] As one of the largest ecosystems on Earth, the ocean occupies 71% of the Earth's total area. From the perspective of Earth science, the area in the ocean with a depth of more than 1000m is called the deep sea, which has the characteristics of high pressure, low oxygen, high salt, and extreme temperature. Researchers at home and abroad have obtained some microbial strains and metabolites from this habitat, and have also discovered potential new microbial classification units. The exploration and development of marine fungi, especially deep-sea fungal resources, has not only expanded people's understanding of the diversity of marine fungi, but also provided the possibility of obtaining new metabolites and new biological functional active substances. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention proposes a marine-derived Penicillium and a fermentation method and application thereof to solve the problems raised by the above-mentioned background technology.

[0007] In order to achieve the above object, the present invention provides the following technical solutions: A marine-derived Penicillium, named Penicillium sp. DSF059, is deposited in the China Center for Type Culture Collection (CCTCC). The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is November 23, 2023. The deposit number is CCTCC NO: M 20232310.

[0008] Preferably, the ITS gene sequence of the marine-derived Penicillium is the nucleotide sequence shown in SEQ ID NO.1, the BenA gene sequence is the nucleotide sequence shown in SEQ ID NO.2, the CaM gene sequence is the nucleotide sequence shown in SEQ ID NO.3, and the RPB2 gene sequence is the nucleotide sequence shown in SEQ ID NO.4.

[0009] Preferably, a fermentation method of marine-derived Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into a triangular flask containing PDA solid culture medium and cultured at 25-30°C for 3-8 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.03-0.08% was added and the activated strain was shaken at 90-120 r / min for 8-15 minutes to prepare a spore suspension as the seed liquid; (2) Fermentation product: inoculating the seed liquid into a rice solid fermentation medium at an inoculum ratio of 2-7% by volume, and performing static fermentation culture at 25-30° C. and natural light for 25-35 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: Soak the fermentation product obtained in step (2) in ethyl acetate for 8-15 hours, shake and culture at 100-150 r / min for 8-20 minutes, filter to obtain a filtrate, perform vacuum rotary evaporation on the filtrate, and extract the filtrate extract obtained by vacuum rotary evaporation with methanol having a volume concentration of 70-90% for 60-100 minutes to obtain ethyl acetate fermentation broth.

[0010] Preferably, a fermentation method of marine-derived Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into a triangular flask containing PDA solid culture medium and cultured at 28°C for 5 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.05% was added and the activated strain was cultured at 110 rpm for 10 min to prepare a spore suspension as the seed liquid. (2) Fermentation product: The seed liquid was inoculated into a rice solid fermentation medium at an inoculum ratio of 5% by volume, and static fermentation was carried out at 28°C under natural light for 30 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) is soaked in ethyl acetate for 12 h, shaken and cultured at 110 rpm for 10 min, and filtered to obtain a filtrate. The filtrate is subjected to vacuum rotary evaporation, and the filtrate extract obtained by vacuum rotary evaporation is extracted with 80% by volume methanol for 80 min to obtain ethyl acetate fermentation broth.

[0011] Preferably, the PDA solid culture medium in step (1) is composed of the following ingredients per liter: 150-250 g of potato, 15-40 g of glucose, 10-30 g of agar, 300-500 mL of aged sea water, and the balance of deionized water. After the PDA solid culture medium is prepared, the natural pH is 5.5-6.5 and sterilized at 121°C for 15-30 min.

[0012] Preferably, the PDA solid culture medium in step (1) is composed of the following ingredients per liter: 200 g potato, 20 g glucose, 15 g agar, 400 mL aged sea water, and the balance deionized water. After the PDA solid culture medium is prepared, it is sterilized at 121° C. for 20 min at natural pH.

[0013] Preferably, the concentration of spore suspension in the spore suspension in step (1) is 1.0×10 6 -1.0×10 10 cfu / mL.

[0014] Preferably, the concentration of spore suspension in the spore suspension in step (1) is 1.0×10 7 cfu / mL.

[0015] Preferably, the rice solid fermentation medium in step (2) is composed of the following ingredients: 8-15 g of rice, 5-20 mL of aged sea water, and 8-30 mL of distilled water. After the ingredients are evenly mixed, they are sterilized at 120-130° C. for 25-40 min.

[0016] Preferably, the rice solid fermentation medium in step (2) is composed of the following ingredients: 10 g rice, 8 mL aged sea water, and 12 mL distilled water. After the ingredients are evenly mixed, they are sterilized at 120-130° C. for 25-40 min.

[0017] Preferably, the parameters of the reduced pressure rotary evaporation in step (3) are vacuum degree 0.02-0.08 MPa and temperature 30-35°C.

[0018] Preferably, the marine-derived Penicillium or the ethyl acetate fermentation broth prepared by the fermentation method is used in the preparation of anti-Staphylococcus aureus drugs.

[0019] Preferably, the PDA solid culture medium in step (1) can also be replaced by the following culture medium for the cultivation of Penicillium. The reagent components and culture medium information involved are as follows: Cha's mother solution: 150g nitric acid, 25g potassium chloride, 25g magnesium sulfate heptahydrate, 0.5g ferrous sulfate heptahydrate, dilute to 500mL with deionized water; Trace element mother solution: anhydrous copper sulfate 0.25g, zinc sulfate heptahydrate: 0.5g, dilute to 500mL with deionized water; CYA medium: 1g potassium hydrogen phosphate, 5g yeast extract, 30g sucrose, 10mL Czapek's mother solution, 20g agar, 400mL Chenhai water, dilute to 1L with deionized water, sterilize at 121℃ for 20min, natural pH; G25N medium: 10 mL of Cha's mother solution, 1 g of potassium hydrogen phosphate, 5 g of yeast extract, 30 g of sucrose, 250 g of glycerol, 20 g of agar, 400 mL of Chenhai water, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH; MEA medium: 20 g malt extract, 1 g peptone, 20 g glucose, 10 mL trace element stock solution, 20 g agar, 400 mL Chenhai water, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH; YES medium: 20 g yeast extract, 150 g sucrose, 0.5 g magnesium sulfate heptahydrate, 10 mL trace element stock solution, 20 g agar, 400 mL Chenhai water, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH; DG18 medium: Dichloran-Glycerol-agar-base 31.5 g, glycerol 220 g, trace element stock solution 10 mL, chloramphenicol 0.05 g, agar 20 g, Chenhai water 400 mL, deionized water to 1 L, sterilized at 121 ° C for 20 min, natural pH; CREA medium: sucrose 30g, creatine 3g, potassium phosphate heptahydrate 1.6g, magnesium sulfate heptahydrate 0.5g, potassium chloride 0.5g, ferrous sulfate heptahydrate 0.01g, trace element mother solution 10mL, bromocresol purple 0.05g, agar 20g, Chenhai water 400mL, deionized water to 1L, sterilized at 121℃ for 20min, natural pH; OA medium: 30 g oatmeal, 10 mL trace element stock solution, 20 g agar, 400 mL Chenhai water, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH; LBA medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15-18 g agar, dilute to 1 L with deionized water, sterilize at 121°C for 20 min, natural pH 7.2-7.4; LB medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, dilute to 1 L with deionized water, sterilize at 121 °C for 20 min, natural pH 7.2-7.4; Preferably, the rice solid fermentation medium in step (2) can also be replaced by the following medium to prepare the ethyl acetate fermentation broth of Penicillium. The medium information involved is as follows: Oat solid fermentation medium: 10g oats, 12ml distilled water, 8ml Chenhai water, placed in a 250ml Erlenmeyer flask, sterilized at 121℃ for 30min; Potato solid fermentation medium: 10g peeled potato pieces, 12ml distilled water, 8ml old sea water, placed in a 250ml Erlenmeyer flask, sterilized at 121℃ for 30min; Cheriois solid fermentation medium: 10 g of General Mills brand Multi grain Cheriois, 12 ml of distilled water, 8 ml of Chenhai water, placed in a 250 ml Erlenmeyer flask, sterilized at 121°C for 30 min.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention optimizes the fermentation medium and culture conditions of the marine Penicillium sp. DSF059, selects the culture medium and culture conditions with higher yield of antimicrobial active compounds for fermentation, and can obtain more target products, with simple operation and low cost. Meanwhile, the marine Penicillium sp. DSF059 can provide an excellent strain for the development of new anti-Staphylococcus aureus drugs, and has good application prospects in the prevention and treatment of Staphylococcus aureus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The growth of Penicillium sp. DSF059 on six different solid culture media; A shows the front view on CYA, YES, MEA, OA, DG18 and CREA solid culture media from left to right; A shows the back view on CYA, YES, MEA, OA, DG18 and CREA solid culture media from left to right; B shows the optical microscopic structure of conidiophores and conidia of Penicillium; C shows the scanning electron microscopic structure of conidiophores and conidia of Penicillium; Figure 2 This is the multi-gene joint phylogenetic tree of Penicillium sp. DSF059; Figure 3 The inhibition zone of Penicillium sp. DSF059 against Staphylococcus aureus; Figure 4 The growth of Penicillium sp. DSF059 on four solid fermentation media: rice, oats, potatoes and Cheriois. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. A marine-derived Penicillium, named Penicillium sp. DSF059, is deposited in the China Center for Type Culture Collection (CCTCC). The address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit date is November 23, 2023. The deposit number is CCTCC NO: M 20232310.

[0023] Example 1 A fermentation method of marine-derived Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into a triangular flask containing PDA solid culture medium and cultured at 25°C for 3 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.03% was added and the activated strain was shaken at 90 rpm for 8 minutes to prepare a spore suspension as the seed liquid. (2) Fermentation product: The seed liquid was inoculated into a rice solid fermentation medium at an inoculum rate of 2% by volume, and static fermentation was carried out at 25°C under natural light for 25 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 8 h, shaken and cultured at 100 r / min for 8 min, and filtered to obtain a filtrate. The filtrate was subjected to reduced pressure rotary evaporation under the conditions of a vacuum degree of 0.02 MPa and a temperature of 30°C. The filtrate extract obtained by reduced pressure rotary evaporation was extracted with methanol having a volume concentration of 70% for 60 min to obtain ethyl acetate fermentation broth.

[0024] The PDA solid culture medium in step (1) is composed of the following ingredients: 150 g potato, 15 g glucose, 10 g agar, 300 mL Chenhai water, and deionized water to 1 L, with a natural pH of 5.5 and sterilized at 121° C. for 15 min.

[0025] Step (1) The concentration of spore suspension in the spore suspension is 1.0×10 6 cfu / mL.

[0026] The rice solid fermentation medium described in step (2) is composed of the following ingredients: 8 g of rice, 5 mL of aged sea water, and 8 mL of distilled water. After mixing evenly, the mixture is sterilized at 120° C. for 25 min.

[0027] Example 2 A fermentation method of marine-derived Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into a triangular flask containing PDA solid culture medium and cultured at 30°C for 8 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.08% was added and the activated strain was shaken at 120 rpm for 15 minutes to prepare a spore suspension as the seed liquid; (2) Fermentation product: The seed liquid was inoculated into a rice solid fermentation medium at an inoculum ratio of 7% by volume, and static fermentation was carried out at 30° C. under natural light for 35 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 15 h, shaken and cultured at 150 r / min for 20 min, and filtered to obtain a filtrate. The filtrate was subjected to reduced pressure rotary evaporation at a vacuum degree of 0.08 MPa and a temperature of 35°C. The filtrate extract obtained by reduced pressure rotary evaporation was extracted with 90% by volume methanol for 100 min to obtain ethyl acetate fermentation broth.

[0028] The PDA solid culture medium in step (1) is composed of the following ingredients: 250 g potato, 40 g glucose, 30 g agar, 500 mL aged sea water, and deionized water to 1 L, with a natural pH of 6.5 and sterilized at 121° C. for 30 min.

[0029] Step (1) The concentration of spore suspension in the spore suspension is 1.0×10 10 cfu / mL.

[0030] The rice solid fermentation medium described in step (2) is composed of the following ingredients: 15 g rice, 20 mL of aged sea water, and 30 mL of distilled water. After mixing evenly, the mixture is sterilized at 130° C. for 40 min.

[0031] Example 3 A fermentation method of marine-derived Penicillium comprises the following steps: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into a triangular flask containing PDA solid culture medium and cultured at 28°C for 5 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.05% was added and the activated strain was shaken at 110 rpm for 10 min to prepare a spore suspension as the seed liquid. (2) Fermentation product: The seed liquid was inoculated into a rice solid fermentation medium at an inoculum ratio of 5% by volume, and static fermentation was carried out at 28°C under natural light for 30 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) was soaked in ethyl acetate for 12 h, shaken and cultured at 110 r / min for 10 min, and filtered to obtain a filtrate. The filtrate was subjected to reduced pressure rotary evaporation under the conditions of a vacuum degree of 0.06 MPa and a temperature of 32°C. The filtrate extract obtained by reduced pressure rotary evaporation was extracted with 80% by volume methanol for 80 min to obtain ethyl acetate fermentation broth.

[0032] The PDA solid culture medium in step (1) is composed of the following ingredients: 200 g potato, 20 g glucose, 15 g agar, 400 mL aged sea water, dilute to 1 L with deionized water, natural pH 6.0, and sterilized at 121° C. for 20 min.

[0033] Step (1) The concentration of spore suspension in the spore suspension is 1.0×107 cfu / mL.

[0034] The rice solid fermentation medium described in step (2) is composed of the following ingredients: 10 g of rice, 8 mL of aged sea water, and 12 mL of distilled water. After mixing evenly, the mixture is sterilized at 121° C. for 30 min.

[0035] Experimental Example 1 Isolation, morphological identification and molecular biological identification of strains (1) A strain of Penicillium sp. DSF059 was isolated from deep-sea sediments at a depth of 2595 m in the South China Sea using Martin medium. It was inoculated on Martin medium and cultured at 28°C for 7 days. It was then taken out and stored in a refrigerator at 4°C.

[0036] Martin medium composition and preparation steps: 10g glucose, 5g peptone, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 3.3mL 0.1% Bengal rose solution, 15-20g agar, 400mL Chenhai water; preparation steps: distilled water is diluted to 1L, pH is natural, sterilized at 121°C for 20min, and after the temperature of the culture medium drops below 65°C, 100mg / mL streptomycin sulfate, 100mg / mL chloramphenicol, and 100mg / mL ampicillin sterilized by ultrafiltration with a 0.22um filter membrane are added in an ultraclean workbench, 1mL each, to obtain Martin medium; (2) Identification of colony morphology: The strain DSF was inoculated into CYA, YES, MEA, OA, DG18 and CREA culture media using the three-point inoculation method. After culturing in the dark at 28°C for 7 days, the growth status of the strain, the color of aerial hyphae, the color of intrabasal hyphae, the presence of soluble pigments and their color were observed and recorded.

[0037] (3) Microscopic morphological identification: After the strain was cultured at 28°C for 7 days using the glass slide wet chamber method, conidiophores and conidia were observed using a Nikon scientific microscope (Ni-U, NIKON, Tokyo, Japan) and a thermal field emission scanning electron microscope (SEMJSM-7610FPlus, JEOL, Tokyo, Japan).

[0038] (4) The operating steps of the slide wet chamber method are as follows: ① Pour PDA thin layer: Under sterile conditions, invert a PDA thin layer with a thickness of 2-3mm in the culture dish. ② Make a culture chamber: In another new culture dish, make a culture chamber. The bottom of the chamber is covered with a layer of round filter paper slightly smaller than the flat dish. A U-shaped glass rod is placed on the round filter paper, and a clean slide is placed on the glass rod. Cover the culture dish with a lid, wrap it with newspaper and sterilize it. ③ Inoculation: Use a scalpel to cut the PDA thin layer into agar blocks with a side length of about 1.0cm, and transfer them to both ends of the slide in the above chamber. Use a sterile inoculation loop to pick up a small amount of spores or hyphae and inoculate them on the edge of the agar block, and use sterile tweezers to cover the cover glass on the agar block. ④ Cultivation: Use a pipette to transfer 2~3mL of sterilized 20% glycerol onto the filter paper of the plate (to maintain the humidity in the plate), cover the culture medium and the plate cover, seal the plate with a sealing film, and place it in a 28℃ incubator for continuous cultivation for 5-10 days. Before observing the morphology of the strain with a scanning electron microscope, the cover glass that has been observed to have the morphology of Penicillium under an optical microscope should be treated. First, soak the cover glass in 2.5% glutaraldehyde and place it in a refrigerator at 4℃ for overnight fixation. After rinsing the cover glass 3-5 times with phosphate buffer (pH7.4), dehydrate it with 30%, 50%, 70%, 80%, 90% and 100% ethanol gradients, and each gradient treatment is 20 minutes. After that, wrap the cover glass in tin foil and dry it in a vacuum freeze dryer for 10 minutes. Fix the dried material to an aluminum pin with conductive carbon tape, sputter gold, and observe the morphology of the strain and spores with a thermal field emission scanning electron microscope.

[0039] (5) The colony morphology of Penicillium sp. DSF059 is as follows: After culturing at 28°C for 7 days, DSF059 can grow normally on CYA, YES, MEA, OA, DG18 and CREA media (see Figure 1-A). The colony diameter of DSF059 on CYA medium is 33-37mm, with a raised center, low and narrow edges, irregular radial wrinkles, white mycelium, velvety or slightly flocculent texture, no exudate, no soluble pigment, and yellow back; the colony diameter on YES medium is 3.4-4.2mm, with a significant raised center and a large number of radial wrinkles, velvety or slightly flocculent texture, white mycelium, a large number of conidia, dark green, no exudate, green soluble pigment, and yellow-brown to yellow back; the colony diameter on MEA medium is 25-29mm, with a slightly raised center and flat surroundings, velvety and granular texture, abundant conidia, grass green, white mycelium, and no exudate. Exudate, soluble pigment is green, the back is yellow-brown to yellow; on OA medium, the colony diameter is 3.3-4.7mm, slightly convex in the middle and with concentric lines and slight radial wrinkles, the texture is velvety or slightly flocculent, the conidia structure is rich, dark green, the hyphae is white, there is no exudate, the soluble pigment is green, and the back is yellow-brown to yellow; on DG18 medium, the colony diameter is 1.9-2.0mm, the colony is low, slightly convex in the middle, the edge is low, the mycelium is white, the texture is hairy, the spore formation is of medium density, the conidia are gray-green as a whole, there is no soluble pigment, no exudate, and the back is yellow-brown to yellow; on GREA medium, the colony diameter is 2.8-3.3mm, with weak growth and moderate acid production.

[0040] (6) The microscopic morphological characteristics of Penicillium sp. DSF059 are: the conidiophores are arranged in a single whorl, the conidiophores have smooth walls, and there are 4-8 phials in each whorl, which are ampoule-shaped; the conidia are nearly spherical, with obvious rough walls and a diameter of 1.9-2.5 μm; no sclerotia were observed (see Figure 1 -B, C).

[0041] (7) Molecular biological identification: Pick the hyphae on the MEA plate, use the genomic DNA extraction kit to extract DNA, and use it as a template to amplify the gene interval sequence of the housekeeping gene 5.8S rDNA and 28S rDNA (The nuclear ribosomal internal transcribed spacer region (ITS), β-tubulin gene (BenA), calmodulin gene (CaM), and RNA polymerase II second largest subunit (RPB2) gene fragment.

[0042] The primer sequences for ITS PCR amplification were ITS1: 5′-tccgtaggtgaacctgcgg-3′ (SEQ ID NO.5) and ITS4: 5′-tcctccgcttattgatatgc-3′ (SEQ ID NO.6); the primer sequences for BenA PCR amplification were Bt2a: 5′-ggtaaccaaatcggtgctgctttc-3′ (SEQ ID NO.7) and Bt2b: ​​5′-accctcagtgtagtgacccttggc-3′ (SEQ ID NO.8); the primer sequences for CaM PCR amplification were CF1: 5′-gccgactctttgacygargar-3′ (SEQ ID NO.9) and CF4: 5′-tttytgcatcatragytggac-3′ (SEQ ID NO.10); the primer sequence for RPB2 PCR amplification was 5F: 5′-gaygaymgwgatcayttygg-3′ (SEQ ID NO.11). NO.11) and 7CR: 5′-cccatrgcttgyttrcccat-3′ (SEQ ID NO.12).

[0043] PCR reaction system: 25uL 2×Taq Master Mix, 19uL ddH2O, 2uL template DNA, 2uL each primer, total volume 50uL.

[0044] PCR amplification conditions for ITS, Ben A and CaM genes were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 60 s, 35 cycles; post-extension at 72°C for 7 min.

[0045] PCR amplification conditions for RPB2 gene were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s, annealing at 48°C for 45 s, and extension at 72°C for 60 s, for 5 cycles; denaturation at 94°C for 45 s, annealing at 50°C for 45 s, and extension at 72°C for 60 s, for 5 cycles; denaturation at 94°C for 45 s, annealing at 52°C for 45 s, and extension at 72°C for 60 s, for 30 cycles; post-extension at 72°C for 7 min.

[0046] (8) The PCR amplification products of ITS, BenA, CaM, and RPB2 of the Penicillium sp. DSF059 strain that met the requirements were sent to BGI Group Co., Ltd. for sequencing. The sequencing results of the marine-derived Penicillium were as follows: the ITS gene sequence was the nucleotide sequence shown in SEQ ID NO.1, the BenA gene sequence was the nucleotide sequence shown in SEQ ID NO.2, the CaM gene sequence was the nucleotide sequence shown in SEQ ID NO.3, and the RPB2 gene sequence was the nucleotide sequence shown in SEQ ID NO.4.

[0047] (9) The sequencing results were analyzed by Blast analysis at NCBI and a multi-gene joint phylogenetic tree was constructed (see Figure 2 ), combined with morphological analysis, the strain was identified as Penicillium, and the strain was deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M 20232310.

[0048] Test Example 2 Penicillium sp. DSF059 from marine origin inhibits the growth of Staphylococcus aureus 2.1 Inoculate a single colony of Staphylococcus aureus into liquid LB medium (lysate broth medium, according to the Molecular Cloning Experiment Guide (by J. Sambrook and DW Russell), add 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl to each liter of medium in 950 ml of deionized water, shake the container until the solute is dissolved, adjust the pH to 7.0 with 5 mol / L NaOH, make up to 1 L with deionized water, and sterilize at 15 psi high pressure for 21 min), shake and culture at 37 ° C and 180 r / min for 18 hours to obtain seed liquid. Count with a hemocytometer and adjust the concentration of the bacterial solution to 1×10 7 cfu / mL pathogen solution. Spread 100μL pathogen solution on LB agar plate, place 5mm diameter antagonistic fungus cakes on the plate at equal distances, place in a 28℃ constant temperature incubator for 18h, and observe the presence and size of the inhibition zone. Repeat 3 times for each treatment.

[0049] 2.2 Plate confrontation results showed that marine Penicillium sp. DSF059 could inhibit the growth of Staphylococcus aureus, with an inhibition zone of more than 2.1 cm (see Figure 3 ).

[0050] Experimental Example 3 Screening of Fermentation Conditions of Marine Penicillium sp. DSF059 3.1 Penicillium culture at 37℃ and seed solution preparation: Penicillium sp. DSF059 was inoculated into a conical flask containing PDA solid culture medium and cultured at 28℃ for 5 days. When spores grew on the PDA solid culture medium, 50mL of sterile 0.05% Tween 80 was added and shaken at 110rpm for 10min to obtain a spore suspension as the seed solution.

[0051] 3.2 Preparation of solid fermentation medium: Four solid fermentation mediums of rice, oats, potatoes and Cheriois were prepared according to the above formulas and sterilized at 121°C for 30 min.

[0052] 3.3 Fermentation products: 2 mL of the above seed solution was taken respectively, and four solid fermentation media of sterilized rice, oats, potatoes and Cheriois were added respectively, and then static fermentation culture was carried out at 28°C under natural light for 30 days to obtain fermentation products.

[0053] 3.4 Ethyl acetate fermentation broth: The fermentation product obtained by fermentation culture was soaked in 60 mL of ethyl acetate for 12 h, shaken and cultured at 110 rpm for 10 min, and the filtrate was filtered out. The filtrate was subjected to reduced pressure rotary evaporation at a vacuum degree of 0.06 MPa and a temperature of 32 °C. The filtrate extract obtained by reduced pressure rotary evaporation was extracted with 5 mL of methanol with a volume concentration of 80% to obtain ethyl acetate fermentation broth.

[0054] 3.5 Antibacterial activity test: In a clean bench, place a sterilized 6 mm filter paper on a sterile glass slide, and pipette 5 μL and 10 μL of DSF059 ethyl acetate fermentation broth, 5 μL and 10 μL of positive control onto the filter paper, respectively, and allow the filter paper to air dry naturally. The positive control is a 4 mg / mL chloramphenicol solution prepared with 50% ethanol. Then pipette 100 μL of 1×10 7 cfu / mL of Staphylococcus aureus liquid was evenly spread on the LB agar plate. Use sterile tweezers to stick the above filter paper pieces on the LB agar plate and press them carefully to make them fit the surface of the culture medium. Put the LB culture medium plate upside down in a 37℃ incubator and culture it for 16-18h. Observe the presence and size of the inhibition zone. Repeat each treatment 3 times.

[0055] 3.6 The results of the fermentation condition screening showed that the diameter of the inhibition zone produced by 5 μL of 4 mg / mL chloramphenicol solution was 2.5 cm, and the diameter of the inhibition zone produced by 10 μL of 4 mg / mL chloramphenicol solution was 2.7 cm. The ethyl acetate fermentation broth of rice, oats, potatoes and cheriois solid fermentation medium of marine Penicillium sp. DSF059 could inhibit the growth of Staphylococcus aureus. The inhibition zone produced by 5 uL of ethyl acetate fermentation broth prepared from each solid fermentation medium (see Figure 4 ) sizes are 1.9cm, 1.5cm, 1.3cm and 1.6cm respectively. Among them, the fermentation products of rice solid fermentation medium have the most significant inhibitory effect on Staphylococcus aureus, can obtain more target products, and are simple to operate, low cost, and have good application value.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A marine-derived Penicillium, characterized in that: Named Penicillium sp. DSF059, deposited in China Center for Type Culture Collection, the address of the depository is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: November 23, 2023; deposit number: CCTCC NO: M 20232310; The ITS gene sequence of the marine-derived Penicillium is the nucleotide sequence shown in SEQ ID NO.1, the BenA gene sequence is the nucleotide sequence shown in SEQ ID NO.2, the CaM gene sequence is the nucleotide sequence shown in SEQ ID NO.3, and the RPB2 gene sequence is the nucleotide sequence shown in SEQ ID NO.

4.

2. The fermentation method of marine Penicillium according to claim 1, characterized in that: The following steps are involved: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into PDA solid culture medium for activation, and 0.03-0.08% of sterile Tween 80 was added. The activated strain was shaken and cultured at 90-120 rpm for 8-15 min to prepare a spore suspension as seed liquid; (2) Fermentation product: inoculating the seed liquid into a rice solid fermentation medium at an inoculum ratio of 2-7% by volume, and performing static fermentation culture at 25-30° C. and natural light for 25-35 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: Soak the fermentation product obtained in step (2) in ethyl acetate for 8-15 hours, shake and culture at 100-150 r / min for 8-20 minutes, filter to obtain a filtrate, perform vacuum rotary evaporation on the filtrate, and extract the filtrate extract obtained by vacuum rotary evaporation with methanol having a volume concentration of 70-90% for 60-100 minutes to obtain ethyl acetate fermentation broth.

3. The fermentation method of marine Penicillium according to claim 2, characterized in that: The following steps are involved: (1) Penicillium culture and seed liquid preparation: The marine Penicillium strain was inoculated into PDA solid culture medium and cultured at 25-30°C for 3-8 days to activate the strain. Sterile Tween 80 with a volume fraction of 0.05% was added and the activated strain was cultured at 110 rpm for 10 minutes to prepare a spore suspension as the seed liquid. (2) Fermentation product: The seed liquid was inoculated into a rice solid fermentation medium at an inoculum ratio of 5% by volume, and static fermentation was carried out at 28°C under natural light for 30 days to obtain a fermentation product; (3) Ethyl acetate fermentation broth: The fermentation product obtained in step (2) is soaked in ethyl acetate for 12 h, shaken and cultured at 110 rpm for 10 min, and filtered to obtain a filtrate. The filtrate is subjected to vacuum rotary evaporation, and the filtrate extract obtained by vacuum rotary evaporation is extracted with 80% by volume methanol for 80 min to obtain ethyl acetate fermentation broth.

4. The fermentation method of marine Penicillium according to claim 2, characterized in that: The PDA solid culture medium described in step (1) is composed of the following ingredients per liter: 150-250 g of potato, 15-40 g of glucose, 10-30 g of agar, 300-500 mL of aged sea water, and the balance of deionized water.

5. The fermentation method of marine Penicillium according to claim 2, characterized in that: Step (1) The concentration of spore suspension in the spore suspension is 1.0×10 6 -1.0×10 10 cfu / mL.

6. The fermentation method of marine Penicillium according to claim 5, characterized in that: The concentration of spore suspension in the spore suspension is 1.0×10 7 cfu / mL.

7. The fermentation method of marine Penicillium according to claim 2, characterized in that: The rice solid fermentation medium in step (2) is composed of the following ingredients: 8-15 g of rice, 5-20 mL of aged sea water, and 8-30 mL of distilled water.

8. The fermentation method of marine Penicillium according to claim 2, characterized in that: The parameters of the reduced pressure rotary evaporation in step (3) are vacuum degree 0.02-0.08 MPa and temperature 30-35°C.

9. Use of the marine-derived Penicillium according to claim 1 or the ethyl acetate fermentation broth prepared by the fermentation method according to any one of claims 2 to 8 in the preparation of anti-Staphylococcus aureus drugs.

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