High and cold special environment aspergillus thermofuscus KLWNF12 as well as fermentation product and anti-infection application of aspergillus thermofuscus KLWNF12

Abstract

The invention relates to the technical field of microbial pharmacy, and discloses an Aspergillus thermofuscus KLWNF12 strain, a fermentation product of the Aspergillus thermofuscus KLWNF12 strain and an anti-infection application of the Aspergillus thermofuscus KLWNF12 strain, and the preservation number of the Aspergillus thermofuscus KLWNF12 strain is CGMCC No.40716. The Aspergillus pyruvus KLWNF12 provided by the invention has unique secondary metabolite production capacity, and has important application value in the fields of pathogenic bacterium resistance, malaria resistance and the like. In addition, the invention further provides a method for preparing an anti-pathogenic-bacterium and anti-malaria product by using the aspergillus thermofuscus KLWNF12, and a new thought and a new technical means are provided for developing novel anti-infection drugs.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a high-cold environment thermophilic Aspergillus KLWNF12 and its fermentation products and anti-infection applications. Background Art

[0002] Pathogens and malarial parasites have posed a serious threat to human public health. Today, drug resistance in pathogens has become a global problem. Malaria is still a major public health problem in tropical and subtropical regions. It is worth noting that some malarial parasites have shown a certain degree of resistance to the classic antimalarial drugs chloroquine and artemisinin derivatives. Therefore, it is urgent to explore and utilize anti-pathogen and antimalarial medicinal resources to find highly effective lead compounds with anti-infective activity.

[0003] Fungi, especially those in special ecological environments, have become a hot topic in the study of active natural medicines in recent years due to the chemical and biological activity diversity of their secondary metabolites. Aspergillus calidoustus has been reported to contain an austinoid gene cluster, and therefore may produce mixed terpenes of various austinoid types. There are currently only two reports on the study of natural products from wetland-derived Aspergillus calidoustus strains, from which DMOA-derived diterpenoids and drimane terpenoid natural products with 3,5-dimethylorsellinic acid (DMOA) as the key precursor source were isolated, and these compounds showed good anti-inflammatory and cytotoxic activities. Summary of the invention

[0004] The present invention studies the development and utilization of anti-infective medicinal microbial resources of stemless astragalus rhizosphere microorganisms collected from Baima Snow Mountain (4215-4300m above sea level) in Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province, and obtains a strain of Aspergillus calidoustus KLWNF12. The crude ethyl acetate extract of the rice fermentation product of the strain and its terpenoid compounds both show certain antibacterial and antimalarial activities.

[0005] In view of this, the present invention proposes a high-altitude and special environment thermophilic Aspergillus KLWNF12 and its fermentation products and anti-infection applications, aiming to provide a new medicinal microbial resource for treating pathogenic bacteria infections and malaria.

[0006] The present invention proposes a thermophilic Aspergillus KLWNF12, the preservation unit of the thermophilic Aspergillus KLWNF12 is the General Microbiological Center of the China Microbiological Culture Collection Administration, the address of the preservation unit is No. 1 Beichen West Road, Chaoyang District, Beijing, the preservation date is June 30, 2023, the preservation number is CGMCC No. 40716, and the taxonomic name of the thermophilic Aspergillus KLWNF12 is Aspergillus calidoustus.

[0007] The present invention also proposes an application of the thermophilic Aspergillus KLWNF12 in producing secondary metabolites, wherein the secondary metabolites include: sesquiterpenoid 6-epi-ophiobolin G (1), sesquiterpenoid Penicisochroman A (2), Pergillin (3) and 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one (4).

[0008] The present invention also proposes an application of the thermopyroxene Aspergillus KLWNF12 in resisting pathogenic bacteria.

[0009] The present invention also proposes an application of the thermopyroxine Aspergillus KLWNF12 in the preparation of products against pathogenic bacteria.

[0010] Preferably, the pathogen is methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus (S. aureus ATCC 25923), Staphylococcus epidermidis ATCC 12228), Streptococcus agalactiae ATCC13813, Vancomycin resistant Enterococcus faecalis ATCC51299 (VREF), Enterococcus faecalis ATCC 29212 and / or Enterococcus faecium ATCC 35667.

[0011] The present invention also proposes an application of the thermopyroxine Aspergillus KLWNF12 in the preparation of anti-malarial products.

[0012] The present invention also proposes an application of the thermopyroxine Aspergillus KLWNF12 in inhibiting malarial parasites.

[0013] Preferably, the falciparum Plasmodium is Plasmodium falciparum 3D7 and / or chloroquine-resistant strain P. falciparum Dd2

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The thermopyrophoric Aspergillus WNF12 of the present invention has a unique ability to produce a variety of secondary metabolites, especially terpenoid compounds, which include 6-epi-ophiobolin G (1), Penicisochroman A (2), Pergillin (3) and / or 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one (4), etc. Among them, such diterpenoid secondary metabolites represented by compound 1 have significant effects in anti-pathogens and anti-malarial, and provide a new solution to the current global health challenges.

[0016] First, the crude extract of the fermentation broth of Aspergillus thermophilus KLWNF12 of the present invention has strong antibacterial activity against a variety of pathogens, especially methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, vancomycin-resistant Enterococcus faecalis, Enterococcus faecalis and Enterococcus faecium. These pathogens often cause serious hospital-acquired infections and pose a serious threat to human health. The secondary metabolite 6-epi-ophiobolin G (1) of Aspergillus thermophilus KLWNF12 of the present invention can effectively inhibit the growth of these pathogens, providing an important microbial resource for the development of new antibacterial drugs.

[0017] Secondly, the secondary metabolites of Aspergillus thermophilus KLWNF12 of the present invention also have anti-malarial potential. The secondary metabolite 6-epi-ophiobolin G (1) of Aspergillus thermophilus WNF12 of the present invention has anti-malarial activity, and its IC values ​​against P. falciparum 3D7 and the drug-resistant strain P. falciparum Dd2 are 2.34~2.13kDa. 50 The values ​​were 3.319 and 4.340 μmol / L, respectively, providing new possibilities for the development of new antimalarial drugs.

[0018] In summary, the thermopyroxenic Aspergillus KLWNF12 of the present invention has a unique ability to produce secondary metabolites, especially terpenoid secondary metabolites, and has important application value in the fields of anti-pathogens and anti-malaria. In addition, the present invention also provides a method for preparing anti-pathogens and anti-malarial products using the thermopyroxenic Aspergillus KLWNF12, which provides new ideas and technical means for the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0020] Figure 1 This is the colony morphology of strain KLWNF12;

[0021] Figure 2 This is the result of Neighbor-joining phylogenetic tree analysis of strain KLWNF12;

[0022] Figure 3 is the chemical structure of the secondary metabolite of strain KLWNF12;

[0023] Figure 4 The SEM images of biofilms of Streptococcus agalactiae formed on the surface of glass slides; A and D are negative controls, B and E are treated with 1 / 2×MIC of compound 1, and C and F are treated with MIC of compound 1; the magnification of A, B, and C is 7500×, and the magnification of D, E, and F is 15000×;

[0024] Figure 5 Blood smears of Plasmodium falciparum 3D7 and chloroquine-resistant strain P. falciparum Dd2; ①②③ are untreated group, 50μmol / L compound 1 treated group, and 25μmol / L compound 1 treated group of P. falciparum 3D7, respectively. ④⑤⑥ are untreated group, 50μmol / L compound 1 treated group, and 25μmol / L compound 1 treated group of P. falciparum Dd2, respectively. Ⅰ: trophozoite, Ⅱ: schizont;

[0025] Figure 6 IC values ​​of compound 6-epi-ophiobolin G(1) against P.falciparum3D7 and chloroquine-resistant strain P.falciparum Dd2 at 72 hours 50 Determination; wherein A is P.falciparum 3D7 and B is P.falciparumDd2. DETAILED DESCRIPTION

[0026] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0027] Example 1 Isolation and identification of strain KLWNF12

[0028] The fungal strain KLWNF12 in this experiment was isolated from the rhizosphere soil of stemless astragalus (identified as stemless astragalus Astragalus acaulis by Professor Jiang Bei from the School of Pharmacy of Dali University) in Baima Snow Mountain in northwestern Yunnan (4300m above sea level).

[0029] Scrape 2 g of soil sample tightly attached to the root system of stemless Astragalus plants, add 20 mL of sterile water, vortex mix, and then perform gradient dilution. Take 200 μL of dilution solution (10 -3 、10- 4 , 10 -5 g / mL) was evenly coated on the PDA medium. The coated plate was inverted and placed in a constant temperature incubator at 28°C for 2-5 days. After the coated plate grew colonies, single colonies were picked under a stereo microscope according to the color, morphological characteristics and texture of the colonies for continuous purification to obtain the pure culture strain KLWNF12. The strain was placed in 25% glycerol and stored at -80°C for identification.

[0030] Pick a small amount of hyphae and place it in a centrifuge tube containing 50μL lysis buffer (Lysis Buffer for Microorganism to Direct PCR), vortex it, and then place it in a PCR instrument for heat denaturation at 80℃ for 15min. Take 2μL as the PCR reaction template and use ITS1 (5′-TCCGTAGGTGAACCTGCGG3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as primers for PCR amplification. PCR amplification system: 2μL PCR reaction template, 1μL primer, 8.5μL ddH2O, 12.5μL 2×Taq PCR Master Mix.

[0031] PCR process: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 45 s, annealing at 55°C for 30 s, extension at 72°C for 90 s, three cycles, and final extension at 72°C for 5 min.

[0032] Shanghai Sangon Biotechnology Co., Ltd. was commissioned to sequence the PCR amplification products and obtain ITS sequence information. Subsequently, Blast was used for sequence alignment, and the neighbor joining method was used to construct a phylogenetic tree.

[0033] The experimental results showed that at 28°C, the colony growth of strain KLWNF12 was most significant at 3-5 days. When cultured on PDA medium, the colonies showed velvety light gray to brown characteristics, accompanied by the secretion of yellow-brown soluble pigments, making the reverse side of the colonies and the potato medium appear light yellow. Figure 1 The colony morphology of strain KLWNF12 is shown.

[0034] Further molecular biological identification, after PCR amplification, the ITS sequence obtained was 491bp long, and the sequence had 100% homology with the ITS sequence of Aspergillus calidoustus (MZ267040.1). Based on the morphological characteristics and ITS sequence analysis results, the stemless Astragalus rhizosphere fungus KLWNF12 was identified as Aspergillus calidoustus ( Figure 2 ).

[0035] The sequencing data of strain WNF12 has been uploaded to NCBI GenBank with the accession number OR447661. The deposit number of strain KLWNF12 is CGMCC No.40716.

[0036] Example 2 Study on secondary metabolites of strain KLWNF12

[0037] 1. Fermentation of strain KLWNF12 and extraction and separation of secondary metabolites

[0038] The stemless astragalus rhizosphere fungus KLWNF12 was inoculated onto the prepared PDA culture plate under sterile conditions and cultured at a constant temperature of 28°C for 3-5 days. Afterwards, the mycelium was picked up in the clean bench and inoculated into a 500mL conical flask containing 200mL PDB liquid culture medium. Enrichment culture was carried out in a shaker at 28°C and 160r / min for 3-5 days. Subsequently, the bacterial liquid was inoculated into the rice culture medium in an amount of 15mL per bottle and cultured statically for 30 days. After the fermentation process was completed, ethyl acetate was used for extraction and filtration, and concentrated by reduced pressure evaporation to obtain a crude ethyl acetate extract of the strain fermentation liquid, with a total weight of 53.6g, of which 3.6g was retained.

[0039] For the preliminary treatment of the crude extract, we used forward silica gel chromatography to separate the crude extract. The specific operation is to use an appropriate amount of silica gel (80-100 mesh) to mix with the crude extract, and use petroleum ether-ethyl acetate (1:0-0:1) as the eluent for elution. Through this step, we obtained 8 components (W12-A~W12-H), and 1g of each eluted component was retained. After the antibacterial activity test, the results showed that the antibacterial activity of W12-B and W12-C components was the most significant. Therefore, subsequent research will focus on the separation and purification of W12-B and W12-C components.

[0040] After separation by silica gel column chromatography (petroleum ether-ethyl acetate 10:1 to 7:3), five subcomponents were separated from component W12-B (6.7g), named W12-B-1 to W12-B-5. Further, from W12-B-2 (1.9g), using petroleum ether-ethyl acetate 8:2 as eluent, compound 1 was separated by silica gel column chromatography, with a mass of 267mg. Similarly, W12-B-3 (2.4g) was separated by silica gel column chromatography, using chloroform-methanol ratios from 80:1 to 50:1 as eluent, and four subcomponents were obtained, named W12-B-3-1 to W12-B-3-4. Finally, by repeatedly using chloroform-methanol ratios of 60:1 and 50:1 as eluents for silica gel column chromatography, compound 4 was obtained from W12-B-3-3 (347mg), with a mass of 18mg.

[0041] After separation by silica gel column chromatography (petroleum ether-ethyl acetate 9:1 to 1:1), 4 different components were obtained from component W12-C (5.4 g), which were named W12-C-1 to W12-C-4. Subsequently, W12-C-2 (2.1 g) was selected for further separation, and 4 components were obtained by elution with silica gel column chromatography (petroleum ether-acetone 4:1 to 1:1), which were named W12-C-2-1 to W12-C-2-4. W12-C-2-3 (1.3 g) was further separated, and after elution with silica gel column chromatography (chloroform-methanol 50:1 to 40:1), 2 components were obtained, namely W12-C-2-3-1 and W12-C-2-3-2. W12-C-2-3-1 (178 mg) was separated by gel column chromatography (Sephadex LH-20, methanol), and finally compound 2 (31 mg) was obtained. After W12-C-2-3-2 (861 mg) was separated by gel column chromatography (Sephadex LH-20, methanol), a precipitate was precipitated. Compound 3 (238 mg) was successfully separated by repeated washing with methanol.

[0042] 2. Identification of secondary metabolites of strain KLWNF12

[0043] The secondary metabolites of strain KLWNF12 were isolated and purified, and a total of 4 compounds were obtained, namely, 6-epi-ophiobolin G (1), penicisochroman A (2), pergillin (3) and 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one (4). The chemical structures are shown in Figure 3 The spectrum data information is shown below.

[0044] 6-epi-ophiobolin G(1):C 25 H 34 O2, white solid dissolved in methanol. ESI-MS m / z: 367.1244 [M+H] + (calcd.for C 25 H 35 O2367.1244). 1 H-NMR(400MHz,CD3OD),δ:1.19(2H,t,H-1),2.66(1H,m,H-2),5.99(1H,s,H-4),3.46(1H,d,H-6),6.91(1 H,d,H-8),2.93(2H,d,H-9),2.61(1H,m,H-10),1.49(2H,m,H-12),1.52(2H,m,H-12),1.28(2H,m,H-13), 1.87(1H,m,H-14),2.68(1H,m,H-15),5.21(1H,t,H-16),6.13(1H,t,H-17),5.99(1H,t,H-18),2.11(3H, s,H-20),9.19(1H,s,H-21),0.90(3H,s,H-22),0.97(3H,d,H-23),1.76(3H,s,H-24),1.84(3H,s,H-25). 13C-NMR(100MHz,CD3OD)δ:45.33(t,C-1),50.86(d,C-2),181.44(s,C-3),130.61(d,C-4),210.97(s,C-5),51.1 3(d,C-6),141.42(s,C-7),159.76(d,C-8),32.09(t,C-9),44.83(d,C-10),46.68(s,C-11),46.42(t,C-12),2 8.72(t,C-13),50.29(d,C-14),33.80(d,C-15),137.31(d,C-16),124.80(d,C-17),121.44(d,C-18),136.92(s,C-19),17.20(q,C-20),194.78(d,C-21),23.19(q,C-22),21.65(q,C-23),18.16(q,C-24),26.55(q,C-25). The above data are basically consistent with those reported in the literature, so the compound is identified as: 6-epi-ophiobolin G.

[0045] Penicisochroman A(2):C 16 H 18 O4, light yellow solid dissolved in chloroform. ESI-MS m / z: 297.1033 [M+Na] + (calcd.for C 16 H 18 O4Na 297.1033) 1H-NMR(400MHz, CDCl3), δ:7.53(1H,d,H-4),6.85(1H,d,H-5),3.00(1H,dd,H-6),4.93(1H,dd,H-9),1.53(3H,s,H-10) ,2.09(3H,s,H-12),2.36(3H,s,H-13),3.55(3H,s,H-7-OMe).13C-NMR(100MHz,CDCl3)δ:145.34(s,C-2),183.59(s,C- 3),121.28(s,C-3a),122.02(d,C-4),123.16(d,C-5),140.77(s,C-5a),39.38(t,C-6),97.50(s,C-7),57.86(t,C-9),118.29(s,C-9a),160.45(s,C-9b),23.10(q,C-10),131.85(s,C-11),20.31(q,C-12),17.53(q,C-13),49.13(q,OMe). The above data are basically consistent with those reported in the literature, so the compound is identified as: Penicisochroman A.

[0046] Pergillin(3): C 15 H 16 O4, light yellow solid dissolved in chloroform. ESI-MS m / z: 261.2354 [M+H] + (calcd.for C 15 H 17 O4261.2354) 1 H-NMR(400MHz, CDCl3), δ:7.53(1H,d,H-4),6.87(1H,d,H-5),2.93(1H,dd,H-6 ),4.92(1H,dd,H-9),1.63(3H,s,H-10),2.09(3H,s,H-12),2.36(3H,s,H-13). 13C-NMR (100MHz,CDCl3)δ:145.34(s,C-2),183.40(s,C-3),121.42(s,C-3a),122.14(d,C-4),123.34(d,C-5),140.71(s,C-5a),38.77(t,C-6),94.96(s,C-7),57.77(t,C-9),118.30(s,C-9a),160.52(s,C-9b),29.39(q,C-10),132.22(s,C-11),17.58(q,C-12),20.37(q,C-13). The above data are basically consistent with those reported in the literature, so the compound is identified as: Pergillin.

[0047] 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one(4):C 15 H 14 NO2, light yellow solid dissolved in chloroform. ESI-MS m / z: 241.5813 [M+H] + (calcd.forC 15 H 15 NO2241.5813), 1 H-NMR(400MHz, CDCl3), δ:7.78(1H,d,H-4),7.36(1H,d,H-5),7.55(1H,s,H-6),9.51(1H,s,H-9),,2.75(3H,s ,H-10),2.43(3H,s,H-12),2.25(3H,s,H-13).13C-NMR(100MHz,CDCl3)δ:145.67(s,C-2),182.39(s,C-3),11 9.47(s,C-3a),124.42(d,C-4),119.78(d,C-5),141.48(s,C-5a),120.69(d,C-6),156.66(s,C-7),146.28(d,C-9),114.70(s,C-9a),164.12(s,C-9b),24.74(q,C-10),134.22(s,C-11),17.71(q,C-12),20.59(q,C-13). The above data are basically consistent with those reported in the literature, so the compound is identified as: 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one.

[0048] Example 3 Determination of antibacterial activity of secondary metabolites of strain KLWNF12

[0049] The antibacterial activity was determined by the broth microdilution method. The pathogens used: Methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus (S. aureus ATCC 25923), Staphylococcus epidermidis (S. epidermidis ATCC 12228), Streptococcus agalactiae (Streptococcus agalactiae ATCC 13813), Vancomycin-resistant Enterococcus faecalis ATCC 51299 (VREF), Enterococcus faecalis (E. faecalis ATCC29212), and Enterococcus faecium (E. faecium ATCC 35667) were all donated by Professor Xu Ying's research group from the School of Life and Marine Sciences, Shenzhen University. The above pathogens are currently preserved in the Key Laboratory of Screening and Research on Pathogen-Resistant Plant Resources in Western Yunnan, Dali University.

[0050] First, the activated pathogen was inoculated into a 15 mL centrifuge tube containing 3-5 mL LB medium, and then cultured overnight in a shaker at 28°C and 160 r / min to obtain a bacterial solution. Next, the OD of the bacterial solution was measured using a UV spectrophotometer. 595 The absorbance value was measured and diluted to 5 × 10 5 CFU / mL.

[0051] For the test products (including crude extracts, first elution fractions and monomer compounds), they need to be diluted to 20 mg / mL with DMSO. Then, in a 96-well plate, 2 μL of the test product and 198 μL of the diluted bacterial solution are added to each well to obtain an initial test concentration of 200 μg / mL. By sequentially diluting by half, the following test concentrations can be obtained: 100, 50, 25, 12.5, 6.25 and 3.125 μg / mL.

[0052] 2 μL of DMSO was added to the negative control wells, and vancomycin hydrochloride was added to the positive control wells at a final concentration of 2 μg / mL. The OD of each well was measured using a microplate reader. 595 The initial absorbance under 37°C was measured, and then the 96-well plate was placed in a constant temperature incubator at 37°C for 24 hours. After that, the absorbance of each well was measured again using an ELISA reader and compared with the initial absorbance to determine the growth of the pathogen. Two parallel wells were set for each test sample, and the measurement was repeated three times to ensure the accuracy and reliability of the results.

[0053] The antibacterial activity results of the crude ethyl acetate extract (W12) and eight elution fractions (W12-A to W12-H) of the fermentation broth of strain KLWNF12 are shown in Table 1. Table 1 shows the antibacterial activity results (μg / mL) of the ethyl acetate extract (W12) and eight elution fractions (W12-A to W12-H) of the fermentation broth of strain KLWNF12.

[0054] Table 1

[0055]

[0056] Note: “-”: MIC>100μg / mL.

[0057] The experimental results in Table 1 show that the antibacterial activity of the ethyl acetate extract fractions W12-B and W12-C of the fermentation broth of strain KLWNF12 is better than that of the crude extract, indicating that its active ingredients are mainly concentrated in the two fractions W12-B and W12-C.

[0058] The antibacterial activity of the monomer compounds separated from components W12-B and W12-C was determined, and the results of the compound activity determination are shown in Table 2. Table 2 shows the antibacterial activity results (μg / mL) of compounds 1-4.

[0059] Table 2

[0060]

[0061] Note: “-”: MIC>100μg / mL.

[0062] The experimental results in Table 2 show that among the tested compounds, only compound 1 showed a certain broad-spectrum antibacterial activity, among which the activity against Streptococcus agalactiae was the best (MIC=6.25 μg / mL) and was better than crude extract W12 (25 μg / mL) and isolated segment W12-B (12.5 μg / mL), while the other compounds only showed a certain antibacterial activity against Streptococcus agalactiae.

[0063] Example 4 Observation of the antibacterial effect of compound 6-epi-ophiobolin G using scanning electron microscopy

[0064] The compound 6-epi-ophiobolin G showed antibacterial activity against Streptococcus agalactiae. In order to intuitively reveal the antibacterial effect of the compound on Streptococcus agalactiae, the changes in the surface structure and morphology of the biofilm caused by the compound during the formation of the biofilm of Streptococcus agalactiae were observed using scanning electron microscopy.

[0065] Inoculate Streptococcus agalactiae into LB medium and culture overnight in a shaker at 28°C and 160 r / min. Then dilute to 5 × 10 5CFU / mL, take 400 μL and transfer it to a 1.5mL centrifuge tube, continue to place it in a shaker at 28°C and 160r / min for 10-12h; after the culture is completed, transfer it to a 24-well plate, add LB culture medium to 1mL, and at the same time, add each well according to the 1 / 2MIC, MIC, and 2MIC values ​​of compound 1 for Streptococcus agalactiae and then culture it statically for 8-10h; after the culture is completed, discard the supernatant, wash it three times with 1×PBS, add 500 μL of glutaraldehyde and place it in a 4°C refrigerator for 8-10h; after the end, wash it three times with 1×PBS, and then dehydrate it with 30%, 50%, 70%, 90% and 100% ethanol in sequence; finally, transfer the bacteria to a slide with a diameter of 5mm, make a record and send it to the School of Chemistry and Chemical Engineering of Hainan Normal University at low temperature for electron microscope scanning; analyze the results after they are returned.

[0066] SEM images of Streptococcus agalactiae biofilm formed on the surface of a glass slide Figure 4 Among them, A and D are negative controls, B and E are treated with 1 / 2×MIC of compound 1, and C and F are treated with MIC of compound 1; the magnification of A, B, and C is 7500×, and the magnification of D, E, and F is 15000×.

[0067] Figure 4 The results showed that in the negative control, a large number of S. agalactiae cells were embedded in the extracellular biomaterial matrix, and there was a dense biofilm between the cell clusters. In contrast, after treatment with 1 / 2×MIC of compound 6-epi-ophiobolin G, the biofilm structure collapsed, the polymer matrix was significantly reduced, and small cell clusters were loosely attached to the surface of the slide with almost no fragments. After treatment with compound 6-epi-ophiobolin G at MIC, the biofilm structure was severely damaged, and the biofilm formed by S. agalactiae cells was less. Scanning electron microscopy analysis confirmed that compound 6-epi-ophiobolin G had a destructive effect on S. agalactiae biofilm.

[0068] Example 5 Antimalarial Activity Assay

[0069] The antimalarial activity of the compounds was screened using the SYBR Green I method. The Plasmodium falciparum strains used were Plasmodium falciparum 3D7 and P. falciparum Dd2 (chloroquine-resistant strain), which were kindly donated by Jiang Lubin's research group at the Shanghai Institute of Immunology and Infection, Chinese Academy of Sciences. The above strains are now preserved in the Key Laboratory of Screening of Pathogen-Resistant Plant Resources in Western Yunnan Province, Dali University.

[0070] All samples were prepared into 10mmol / L stock solution with DMSO, and the infection rate of Plasmodium in the final detection system was 1% and the hematocrit was 2%. The experiment set up a blank group (prepared blood without worms and 1% DMSO solution), a positive control group (prepared blood containing worms and positive drug chloroquine diphosphate solution), a negative control group (prepared blood containing worms and 1% DMSO solution) and a sample group (prepared blood containing worms and sample solution). The required mother solution in each group was added to a 96-well plate at a ratio of 1:1. Each drug administration group was set with 6 detection concentrations of 50, 25, 12.5, 6.25, 3.125, and 1.5625μmol / L, and each concentration was repeated 3 times. After the plate was laid, it was incubated in a CO2 incubator for 72h. After the incubation, the fluorescence was measured according to the SYBR Green I method, and the inhibition rate was calculated. The data were input and sorted using Microsoft Office Excel 2021 software, and nonlinear regression curve fitting was performed using GraphPad Prism 8.0 software to obtain the 50% inhibitory concentration (IC 50 ).

[0071] Blood smears of Plasmodium falciparum 3D7 and P. falciparum Dd2 Figure 5 As shown; ①②③ are P.falciparum 3D7 untreated group, 50μmol / L compound 1 treated group, 25μmol / L compound 1 treated group, respectively. ④⑤⑥ are P.falciparum Dd2 untreated group, 50μmol / L compound 1 treated group, 25μmol / L compound 1 treated group, respectively. Ⅰ: trophozoite, Ⅱ: schizont.

[0072] In the antimalarial activity test, only compound 6-epi-ophiobolin G showed antimalarial activity, but it is worth noting that this compound was able to inhibit not only the growth of Plasmodium falciparum 3D7, but also the growth of P. falciparum Dd2, a Plasmodium falciparum strain resistant to chloroquine.

[0073] like Figure 5 As shown in the figure, in the negative control group, trophozoites and schizonts of Plasmodium falciparum were observed, while after treatment with compound 6-epi-ophiobolin G, trophozoites and schizonts of Plasmodium falciparum were not observed, and the red blood cells were normal. GraphPad Prism 8.0 software was used for nonlinear regression curve fitting ( Figure 6), IC of compound 6-epi-ophiobolin G against Plasmodium falciparum 3D7 (A) and P. falciparum Dd2 (B) at 72 h 50 The IC values ​​of compound 6-epi-ophiobolin G against P.falciparum 3D7 and P.falciparum Dd2 were 50 The values ​​were 3.319 and 4.340 μmol / L respectively.

[0074] In summary, this study first found that the crude ethyl acetate extract of rice fermentation products of Aspergillus calidoustus WNF12, a rhizospheric fungus from the high-altitude and cold environment of northwest Yunnan, has good antibacterial and antimalarial activities. Four compounds were successfully isolated and identified from the extract, including a dipterpenoid 6-epi-ophiobolin G (1) and three dipterpenoid compounds penicisochroman A (2), pergillin (3) and 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one (4). All of the above compounds were first discovered from the secondary metabolites of A. calidoustus. Although the habitat sources of A. calidoustus in this study are different from those reported in previous studies, they all come from extreme environments such as the deep sea and high altitude areas. Therefore, we propose the following hypothesis: these harsh environmental conditions pose a great challenge to the survival of microorganisms, prompting these microorganisms to develop special metabolic mechanisms and produce compounds with novel structures and significant activities. Among the above compounds, the dipterpenoid compound 6-epi-ophiobolin G (1) has the most significant anti-infective activity. This compound belongs to the ophiobolins class and was first isolated in 2004 from the MG medium fermentation product of Emericellavariecolor GF10 from marine sediments in the Japanese Sea (-70m), but its related biological activity was not reported at that time. In 2020, researchers isolated Aspergillus sp. WHU0154 from marine samples in the South China Sea (-3197m), and also isolated compound 1 from the fermentation product of the strain, and reported its anti-inflammatory activity. This study isolated the ophiobolin compound 6-epi-ophiobolin G from the rice fermentation culture of Aspergillus calidoustus for the first time, and reported the antibacterial and antimalarial activities of this compound for the first time. Specifically, at a concentration of 6.25-50 μg / mL, the compound can completely inhibit the growth of 7 tested Gram-positive bacteria, including two drug-resistant bacteria: Methicillin-resistant Staphylococcus aureus (MRSA, ATCC43300) and Vancomycin-resistant Enterococcus faecalis (VREF, ATCC51299).Scanning electron microscopy observations revealed that the compound was able to destroy the biofilm of Streptococcus agalactiae, an important pathogen that causes infections in newborns and adults. The presence of the biofilm provides a survival advantage for pathogens and reduces the penetration of antimicrobial agents, thereby enhancing drug resistance. Therefore, the compound can achieve an antibacterial effect by destroying the biofilm attached to the surface of Streptococcus agalactiae and reducing its resistance to drugs. In addition, the compound 6-epi-ophiobolin G also showed good anti-plasmozoal activity. Specifically, the half inhibitory concentration (IC50) of the compound against Plasmodiumfalciparum 3D7 and P.falciparum Dd2d. 50 ) were 3.319 and 4.340 μmol / L, respectively, which also provides new candidate substances for the research and development of lead compounds for antimalarial drugs. Therefore, the results of this study not only further enriched the biological activity connotation of the ophiopogonin compound 6-epi-ophiobolin G, but also confirmed at the theoretical and practical levels the discovery of active compounds from the secondary metabolites of microorganisms in high-altitude special environments, providing a practical path to solve the problems faced by the clinical frontline. This discovery not only provides a valuable technical reference for subsequent in-depth research on the secondary metabolites of microorganisms in high-altitude special environments, but also provides useful reference and inspiration for the rational development and utilization of characteristic microbial resources in high-altitude special environments.

[0075] The embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the specific implementation modes of the present invention can still be modified or replaced by equivalents, and any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A thermopyroxene Aspergillus KLWNF12, characterized in that The depository of the thermopyroblast KLWNF12 is the General Microbiology Center of the China Microbiological Culture Collection Administration, the address of the depository is No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is June 30, 2023, the deposit number is CGMCC No.40716, and the taxonomic name of the thermopyroblast KLWNF12 is Aspergillus calidoustus.

2. A use of the Aspergillus thermophilus KLWNF12 according to claim 1 in producing secondary metabolites, characterized in that: The secondary metabolites include: 6-epi-ophiobolin G, Penicisochroman A, Pergillin and 7-methyl-2-(1-methylethylethlidene)-furo[3,2-H]isoquinoline-3-one.

3. Use of the thermopyroxene Aspergillus KLWNF12 according to claim 1 in resisting pathogenic bacteria.

4. Use of the thermopyroxene Aspergillus KLWNF12 according to claim 1 in the preparation of products against pathogenic bacteria.

5. The use according to claim 3 or 4, characterized in that: The pathogenic bacteria are methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Staphylococcus aureus (S. aureus ATCC 25923), Staphylococcus epidermidis (S. epidermidis ATCC 12228), Streptococcus agalactiae (Streptococcus agalactiae ATCC 13813), Vancomycin-resistant Enterococcus faecalis ATCC 51299 (VREF), Enterococcus faecalis (E. faecalis ATCC 29212) and / or Enterococcus faecium (E. faecium ATCC 35667).

6. Use of the Aspergillus thermophilus KLWNF12 according to claim 1 in the preparation of antimalarial products.

7. Use of the Aspergillus thermophilus KLWNF12 according to claim 1 in inhibiting malarial parasites.

8. The use according to claim 7, characterized in that: The malarial parasite is Plasmodium falciparum 3D7 and / or P. falciparum Dd2.

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