CN111616149A - Application of 2(5H)-furanone to inhibit the formation of marine bacterial microbial envelope in reducing the attachment of mussel spat - Google Patents

Abstract

The present invention provides an application of 2(5H)-furanone in inhibiting the formation of marine bacterial microbial film in reducing the attachment of mussel juveniles thick-shelled, and the method for reducing the adhesion of mussel juveniles thick-shelled by 2(5H)-furanone comprises the following steps: : After the bacteria are expanded and cultured in a liquid medium, the initial bacterial stock solution is obtained; after centrifugation, the supernatant is discarded, mixed with sterile filtered seawater, and resuspended to obtain the bacterial liquid washed from the medium; the bacterial liquid and 2 (5H)-furanone was added to a sterile petri dish containing sterilized glass slides for bacterial microbial film culture, and then sterilized filtered seawater was added to rinse the glass slides to obtain a bacterial microbial film, which was transferred to a sterile petri dish And add thick-shelled mussel juvenile; the present invention uses quorum sensing inhibitor 2(5H)-furanone by interfering with the formation of microbial film, such as EPS biomass and bacterial metabolism process, and then significantly reduces thick-shelled mussel juvenile attachment, This provides a basis for the field of biological antifouling.

Description

2(5H)-Furanone inhibits marine bacterial microbial envelope formation in reduced mussels Application in Spat Attachment

technical field

The invention belongs to the technical field of marine fouling, and in particular relates to the application of 2(5H)-furanone in inhibiting the formation of marine bacterial microbial film in reducing the adhesion of mussel juveniles, and specifically uses a quorum sensing inhibitor 2(5H)- Furanones directly inhibited the formation of microbial envelopes of marine bacteria (Pseudoaltermonas marina ECSMB14103) and indirectly significantly reduced the attachment of mussel juveniles.

Background technique

In the marine environment, microbial envelopes can rapidly form on various substrate surfaces immersed in water. Microbial membranes are mainly composed of bacteria, diatoms and fungi. Microbial membranes play an important role in the attachment and metamorphosis of marine invertebrates such as mussels. Larvae of different marine species also respond differently to microbial envelopes. Marine microbial envelope is one of the important factors that influence and control marine invertebrate larval attachment metamorphosis. The microbial envelope can promote the attachment and metamorphosis of marine invertebrate larvae such as mussels and sea urchins, and can also inhibit the attachment and metamorphosis of marine invertebrate larvae such as polychaetes, barnacles, and sea squirts.

Biofouling is mainly divided into Microfouling and Macrofouling. Microbial fouling mainly refers to the phenomenon that microorganisms such as bacteria adhere to the surface of materials and form a microbial film. In the ocean, biofouling involves not only the attachment of bacteria, etc., but also the attachment of large marine organisms such as mussels, barnacles and oysters, the latter known as macrobiofouling. Large-scale biofouling will increase the power consumption of ships, reduce the sailing speed, and affect the maneuverability and combat performance of ships. Therefore, controlling biofouling is very important to the national economy and national defense security. Traditional antifouling methods mainly play an antifouling role by killing biofouling with toxic substances such as fungicides, such as the antifouling paint tributyltin, which is banned due to environmental pollution to the ocean. With the enhancement of people's awareness of environmental protection, the development of environment-friendly anti-fouling technology has become a hot spot in the field of marine anti-fouling.

The quorum sensing system is a communication system between bacteria that depends on the concentration of chemical signals and is involved in the formation of microbial envelopes. Bacteria communicate with each other by producing and secreting small molecular weight chemical signal molecules, and sense changes in the cell population density of their own or other bacteria. When the population density reaches a certain threshold, the signal molecules will initiate the expression of specific genes in the bacteria, changing and coordinating cells. This phenomenon is called Quorum Sensing (QS). Bacterial quorum sensing affects the formation of microbial envelopes, changes in community structure, and the attachment of macroorganisms.

Quorum sensing inhibitors can inhibit the attachment of large fouling organisms by interfering with the formation of bacterial microbial envelopes. At the same time, quorum sensing inhibitors have the characteristics of obvious bacteriostatic effect, little environmental pollution and no resistance to bacteria. 2(5H)-Furanone (2(5H)-Furanone) is a compound capable of inhibiting quorum-sensing signaling molecules and inhibits the formation of microbial envelopes by interfering with the quorum-sensing system (Ponnusamy, K., Paul, et al. D., Kim, Y.S., Kweon, J.H., (2010).2(5H)-Furanone: a prospective strategy for biofouling-control in membrane biofilm bacteria by quorum sensinginhibition.Braz.J.Microbiol.41,227-234.doi:10.1590 /S1517-83822010000100032). Studies have shown that 2(5H)-furanone reduces the mobility of Campylobacter jejuni, interferes with microbial envelope formation and quorum sensing activity, and has bacteriostatic effects (Castillo, S., Heredia, N. , García, S., (2015). 2(5H)-Furanone, epigallocatechin gallate, and a citric-based disinfectant disturb quorum-sensing activity and reduce motility and biofilmformation of Campylobacter jejuni.Folia Microbiol.60,89-95.doi: 10.1007/s12223-014-0344-0). 2(5H)-furanone has been studied as a potent quorum-sensing inhibitor, but its attachment activity to macrofouling organisms such as mussels has not been reported. Therefore, it is necessary to study the effect of 2(5H)-furanone in inhibiting the attachment of large fouling organisms, which has important practical significance for the sustainable development of the marine antifouling industry.

Effects of quorum-sensing inhibitors on the formation of marine microbial communities and the attachment of Colicoides genus (Dobretsov, S., Dahms, H.U., Yili, H., Wahl, M., Qian, P.Y., (2007). The effect of quorum- sensingblockers on the formation of marine microbial communities and larvalattachment. FEMS Microbiol.Ecol.60,177-188.doi:10.1111/j.1574-6941.2007.00285.x): quorum sensing inhibitors such as multiple furanone inhibitors 5- hydroxy-3[(1R)-1-hydroxypropyl]-4-methylfuran-2(5H)-one(FUR1), (5R)-3,4-dihydroxy-5-[(1S)-1,2-dihydroxyethyl] Effects of furan-2(5H)-one(FUR2) and Triclosan on microbial envelope formation of marine bacteria and their effects on larval attachment of Hydroides elegans and Bugula neritina. The authors collected the natural marine microbial film formed in the 14-day-old intertidal zone, scraped off the microbial film to make a suspended bacterial liquid, formed a microbial film in the presence of quorum sensing inhibitors, and studied the formed microbial film. Effects on larval attachment of Coliform genus and bryozoans. The results showed that the quorum sensing inhibitor significantly reduced the bacterial density of the microbial envelope (Fig. 1: P<0.05) and changed the bacterial community structure of the microbial envelope, and significantly reduced the attachment rate of Coilworm and Bryozoa larvae (Fig. 2 and Figure 3: P<0.05).

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the purpose of the present invention is to provide an application of 2(5H)-furanone in inhibiting the formation of marine bacterial microbial film in reducing the attachment of mussel juveniles.

In order to achieve the above object, the solution of the present invention is:

Application of a 2(5H)-furanone to inhibit the formation of marine bacterial microbial envelopes in reducing the attachment of mussel juveniles.

Preferably, the method for reducing the attachment of mussel spat by 2(5H)-furanone comprises the steps of:

(1), after the bacterium is expanded and cultivated in the liquid medium, the initial bacterial stock solution is obtained;

(2), after the initial bacterial stock solution is centrifuged, the supernatant is discarded, and mixed with sterilizing filtered seawater to obtain the bacterial liquid that washes away the culture medium;

(3), add the bacteria liquid and 2(5H)-furanone from the washed medium to the sterile petri dish containing the sterilized glass slides for bacterial microbial film culture, and then add sterilized filtered seawater to gently rinse the carrier Glass slides to obtain bacterial microbial envelopes;

(4), transfer the glass slide formed with the bacterial microbial film to a sterile petri dish, and add the thick-shelled mussel juveniles.

Preferably, in step (1), the bacterium is Pseudoaltermonas marina ECSMB14103.

Owing to adopting the above-mentioned scheme, the beneficial effects of the present invention are:

In the present invention, the quorum sensing inhibitor 2(5H)-furanone is used to interfere with the formation of the microbial film to reduce the attachment of the thick-shelled mussel juveniles of the fouling biological molluscs, that is, 2(5H) with a concentration of 10 ^-4 mol/L is used. - Furanones significantly reduced the attachment of Mussel juveniles by interfering with microbial envelope formation (such as EPS biomass and bacterial metabolic processes), where the attachment rate of Mussel juveniles was reduced by 54% compared with the control, and The molecular mechanism of 2(5H)-furanone affecting the formation and metabolism of microbial membranes was further verified at the molecular level by high-throughput sequencing technology, thus providing a basis for the field of biological antifouling.

Description of drawings

FIG. 1 is a schematic diagram showing that the quorum sensing inhibitor in the prior art significantly reduces the bacterial density of the microbial envelope.

FIG. 2 is a schematic diagram showing that the quorum sensing inhibitor significantly reduces the attachment rate of bryozoan larvae in the prior art.

FIG. 3 is a schematic diagram showing that the quorum sensing inhibitor in the prior art significantly reduces the attachment rate of Colicoides chinensis.

Fig. 4 is a schematic diagram showing the adhesion rate of 2(5H)-furanone to mussel spat in the embodiment of the present invention.

Figure 5 is a graph showing the difference in biomass produced by the extracellular product EPS (protein, fat, alpha polysaccharide and beta polysaccharide in the bacterial membrane of P. marinaECSMB14103) produced by the microbial envelope in the examples of the present invention.

Fig. 6 is the KEGG pathway enrichment analysis diagram of differential genes after the bacterial membrane of P. marina ECSMB14103 was treated with 2(5H)-furanone in the example of the present invention, * means p<0.05.

Detailed ways

The invention provides an application of 2(5H)-furanone in inhibiting the formation of marine bacterial microbial film in reducing the attachment of mussel juveniles.

The method for 2(5H)-furanone of the present invention to inhibit the formation of marine bacterial microbial film in reducing the attachment of mussel juveniles comprises the following steps:

1. Cultivation of microbial envelopes

(1). The bacterial strain P.marina ECSMB14103 was isolated from the natural microbial envelope formed in the East China Sea (Peng, L.H., Liang, X., Guo, X.P., Yoshida, A., Osatomi, K., Yang, J.L., (2018) ).Completegenome of Pseudoalteromonas marina ECSMB14103, a mussel settlement-inducingbacterium isolated from the East China Sea.Mar.Genom.41,46-49.doi:10.1016/j.margen.2018.04.001).

(2). P. marina ECSMB14103 bacteria were cultured in Zobell 2216E broth liquid medium at 27°C for 24h. The bacterial liquid was obtained by centrifugation at 2250g for 15min, and the supernatant was discarded. In order to remove the excess liquid medium in the bacterial liquid, sterile filtered seawater (seawater with a salinity of 30‰ was sterilized at 121 °C for 20 min, and filtered through a 1.2 μm microporous membrane) to suspend and wash the bacterial clumps and centrifuge ( 2250g, 15min), repeated 3 times. The bacterial mass was finally suspended in 50 mL of sterile filtered seawater. 1 mL of bacterial solution was filtered on a filter membrane with a pore size of 0.22 μm (Whatman, UK), and stained with freshly prepared acridine orange at a concentration of 0.1% for 5 min. After that, the stained filter membrane was fixed on a glass slide. The membrane was randomly selected from 10 fields of view under a 1000x oil lens of a fluorescence microscope, with three biological replicates, and the bacterial density of the bacterial liquid was calculated.

(3). Dilute 2(5H)-furanone (purchased from SIGMA company, article number: 283754) with sterile filtered seawater into a mother liquor of 10 ^{- 2} mol/L, ready to use.

中进行P.marina ECSMB14103微生物被膜的培养。首先在培养皿中加入适量2(5H)-呋喃酮母液和10⁸个悬浮的细菌细胞(来自步骤2)，定容至20mL，使得2(5H)-呋喃酮的终浓度为10^-3mol/L、10^-4mol/L和10^-5mol/L，在28℃黑暗条件下培养48h。之后每次加入20mL灭菌过滤海水将未附着在载玻片上的细菌洗去，重复3次，得到2(5H)-呋喃酮处理后在载玻片上形成的细菌微生物被膜。(4). In a sterile glass petri dish containing a sterile glass slide (38mm x 26mm) and sterile filtered seawater

The culture of P. marina ECSMB14103 microbial envelopes was carried out in . First add an appropriate amount of 2(5H)-furanone stock solution and 10 ⁸ suspended bacterial cells (from step 2) to a petri dish, and make up to 20 mL, so that the final concentration of 2(5H)-furanone is 10 ^-3 mol /L, 10 ^-4 mol/L and 10 ^-5 mol/L, cultured at 28°C in the dark for 48h. Then, 20 mL of sterile filtered seawater was added each time to wash away the bacteria that did not adhere to the glass slide, and the procedure was repeated 3 times to obtain a bacterial microbial film formed on the glass slide after 2(5H)-furanone treatment.

(5) Thick-shelled mussel juveniles with a shell length of about 0.77±0.15mm and a shell width of about 0.52±0.09mm were selected for attachment experiments. The slides formed with the microbial coating were transferred to a sterile petri dish containing 20 mL of AFSW, and 10 thick-shelled mussels were added to the petri dish (9 biological replicates), and the thick-shelled mussels were recorded after 24 h. The attachment of juveniles, the thick-shelled mussel juveniles attached to the bacterial microbial membrane were considered to be attached juveniles.

2. Bacterial extracellular product staining

(1) Four kinds of dyes were used: fluorescein isothiocyanateisomer I [fluorescein isothiocyanateisomer I (FITC; Sigma F7250)], Calcofluor white M2R (CFW; Sigma 18909) )], 1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate [DiIC18(5)oil,1,1'-dioctadecyl-3 ,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD'oil; Invitrogen, D307)] and concanavalin A [concanavalin A, tetramethylrhodamineconjugate (ConA-TMR; Invitrogen, C860)] on P. marina ECSMB14103 bacterial microorganisms Proteins, fats, alpha polysaccharides and beta polysaccharides in the envelope were stained. The working solution concentrations of FITC, DiD'oil, ConA-TMR and CFW stains were: FITC (2mg FITC dissolved in 100μL 100% ethanol) was 46.6μg/mL; DiD'oil (25mgDiD'oil dissolved in 2.5mL 100% ethanol) middle) was 79.4 μg/mL; ConA-TMR (10 mg ConA-TMR and 168 mg NaHCO dissolved in ₂ mL distilled water) was 944.8 μg/mL; CFW was 189 μl/mL. The working solutions of the four dyes were all prepared with ultrapure water.

(2). P.marina ECSMB14103 bacterial membrane was first rinsed with 150mmol/L sodium chloride solution, and then 50μL of the working solution of stain was added dropwise for 20min in the dark. The dyed biofilm was rinsed with 150 mmol/L sodium chloride solution and air-dried naturally.

(3). The stained biofilms were imaged with a confocal scanning microscope (Leica TCS SP8) 63x oil lens. Biomass analysis of protein, fat, alpha polysaccharide and beta polysaccharide was performed using Image J software.

3. High-throughput sequencing of bacterial membranes

(1). Scrape the cultured biofilm into a sterile 1.5mL centrifuge tube with a sterile glass slide, and add RNAiso Plus solution according to the instructions for RNA extraction (Takara, Japan) and DNase treatment.

(2). RNA library construction was performed using the TruSeqTM RNA sample preparation Kit of Illumina (San Diego, CA). Subsequently, the rRNA was removed using the Ribo-Zero Magnetic kit (epicenter), the mRNA was randomly fragmented into small fragments of about 200 bp, and the mRNA was used as a template for reverse transcription using random primers (Illumina) and SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA). Synthesis of double-stranded cDNA. The synthesized double-stranded cDNA was then added to End Repair Mix to make up blunt ends, phosphorylated at the 5' end, added an A base at the 3' end, and connected to a Y-shaped sequencing adapter. The library was enriched and then PCR amplified with Phusion DNA polymerase (NEB) for 15 cycles. After TBS380 (Picogreen) quantification, RNA-seq paired-end sequencing was performed using Illumina HiSeq X Ten (2 × 150 bp).

(3). Use the data generated by Illumina platform for bioinformatics analysis. All analyses were performed using the I-Sanger cloud platform (www.i-sanger.com) of Shanghai Meiji Bio.

The present invention will be further described below in conjunction with the examples.

Example:

The method for the 2(5H)-furanone of the present embodiment to inhibit the formation of marine bacterial microbial film in reducing the attachment of mussel juveniles includes the following steps:

1. Cultivation of microbial envelopes

(1). The bacterial strain P.marina ECSMB14103 was isolated from the natural microbial envelope formed in the East China Sea (Peng, L.H., Liang, X., Guo, X.P., Yoshida, A., Osatomi, K., Yang, J.L., (2018) ).Completegenome of Pseudoalteromonas marina ECSMB14103, a mussel settlement-inducingbacterium isolated from the East China Sea.Mar.Genom.41,46-49.doi:10.1016/j.margen.2018.04.001), which was inoculated into a Zobell 2216E liquid medium (per liter of medium containing peptone 5.0g, yeast extract 1.0g, ferric citrate 0.01g) in a round-bottom flask, place the round-bottom flask in a constant temperature shaking incubator at 27°C for 24h (dark) , the speed of the shaker was 200 rpm, and the initial bacterial stock solution was formed.

(2). After 24 hours of bacterial culture, the bacterial liquid in the round-bottomed flask was divided into 50 mL sterile centrifuge tubes, placed in a centrifuge (Eppendorf, Germany) and centrifuged at 2250 g for 15 min, and the supernatant was discarded. Add 40 mL of sterile filtered seawater to the centrifuge tube, and thoroughly mix the bacteria at the bottom with sterile seawater. The centrifugation conditions are the same as the previous step, and repeat 3 times. After completion, 50 mL of sterilized filtered seawater was added, and the bacteria were blown and mixed to suspend them to obtain a bacterial liquid from which the culture medium was washed. 1 mL of bacterial solution was filtered on a filter membrane with a pore size of 0.22 μm (Whatman, UK), stained with freshly prepared acridine orange at a concentration of 0.1% for 5 min, and then the stained filter membrane was fixed on a glass slide. The membrane was randomly selected from 10 fields of view under a 1000x oil lens of a fluorescence microscope, with three biological replicates, and the bacterial density of the bacterial liquid was calculated.

中进行P.marina ECSMB14103微生物被膜的培养。首先在培养皿中加入适量2(5H)-呋喃酮母液和10⁸个悬浮的细菌细胞(来自步骤2)，定容至20mL，使得2(5H)-呋喃酮的终浓度为10^-3mol/L、10^-4mol/L和10^-5mol/L，在28℃黑暗条件下培养48h。之后每次加入20mL灭菌过滤海水将未附着在载玻片上的细菌洗去，重复3次，得到2(5H)-呋喃酮处理后在载玻片上形成的细菌微生物被膜。(3). In a sterile glass petri dish containing a sterile glass slide (38mm x 26mm) and sterile filtered seawater

The culture of P. marina ECSMB14103 microbial envelopes was carried out in . First add an appropriate amount of 2(5H)-furanone stock solution and 10 ⁸ suspended bacterial cells (from step 2) to a petri dish, and make up to 20 mL, so that the final concentration of 2(5H)-furanone is 10 ^-3 mol /L, 10 ^-4 mol/L and 10 ^-5 mol/L, cultured at 28°C in the dark for 48h. Afterwards, 20 mL of sterile filtered seawater was added each time to wash off the bacteria that did not adhere to the glass slide, and the procedure was repeated three times to obtain a bacterial microbial film formed on the glass slide after 2(5H)-furanone treatment.

(4) Thick-shelled mussel juveniles with a shell length of about 0.77±0.15mm and a shell width of about 0.52±0.09mm were selected for attachment experiments. The coated slides were transferred to sterile petri dishes containing 20 mL of AFSW, and 10 mussel juveniles were added to the dishes (9 biological replicates). According to the formula of spat attachment rate: spat attachment rate=(number of spat attached to microbial film/total number of spat)×100%, the attachment of spat was calculated.

(5) Extracellular product analysis and transcriptome analysis were performed on the microbial membranes that had a significant inhibitory effect on the addition of 2(5H)-furanone, to explore the effect of 2(5H)-furanone on the formation of microbial membranes and its relationship with mussels thick-shelled. Spat attachment link. Therefore, the results showed that 2(5H)-furanone significantly reduced the attachment rate of mussel juveniles, and the attachment rate of mussel juveniles was reduced by 54% compared with the control (see Figure 4).

2. Bacterial extracellular product staining

(1) Four kinds of dyes were used: fluorescein isothiocyanateisomer I [fluorescein isothiocyanateisomer I (FITC; Sigma F7250)], Calcofluor white M2R (CFW; Sigma 18909) )], 1,1'-octadecyl-3,3,3',3'-tetramethylindole dicarbocyanine perchlorate [DiIC18(5)oil,1,1'-dioctadecyl-3 ,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD'oil; Invitrogen, D307)] and concanavalin A [concanavalin A, tetramethylrhodamineconjugate (ConA-TMR; Invitrogen, C860)] on P. marina ECSMB14103 bacterial microorganisms Proteins, fats, alpha polysaccharides and beta polysaccharides in the envelope were stained. The concentrations of FITC, DiD'oil, ConA-TMR and CFW staining reagent working solutions were: FITC (2mg FITC dissolved in 100μL 100% ethanol) was 46.6μg/mL; DiD'oil (25mg DiD'oil dissolved in 2.5mL 100% ethanol) was 46.6μg/mL. ethanol) was 79.4 μg/mL; ConA-TMR (10 mg ConA-TMR and 168 mg NaHCO dissolved in ₂ mL of distilled water) was 944.8 μg/mL; CFW was 189 μl/mL. The working solutions of the four dyes were all prepared with ultrapure water.

(2) P.marina ECSMB14103 biofilm was first rinsed with 150mmol/L sodium chloride solution, and then 50μL of dye working solution was added dropwise for 20min in the dark. The dyed biofilm was rinsed with 150 mmol/L sodium chloride solution and air-dried naturally.

(3). The stained biofilms were imaged with a confocal scanning microscope (Leica TCS SP8) 63x oil lens. Biomass analysis of proteins, fats, alpha polysaccharides and beta polysaccharides was performed using Image J software (see Figure 5). The results showed that β-polysaccharide was the main component of the biofilm of P. marina ECSMB14103, while the lipid biomass was the least. 2(5H)-furanone significantly inhibited the production of α-polysaccharide (P<0.05) and β-polysaccharide (P<0.001), but had no significant effect on lipid production (P>0.05), but 2(5H)- Furanones significantly promoted the production of extracellular proteins (P<0.01).

3. High-throughput sequencing of bacterial membranes

(1). Scrape the cultured biofilm into a sterile 1.5mL centrifuge tube with a sterile glass slide, and add RNAiso Plus solution according to the instructions for RNA extraction (Takara, Japan) and DNase treatment.

(2). RNA library construction was performed using the TruSeqTM RNA sample preparation Kit of Illumina (San Diego, CA). Subsequently, the rRNA was removed using the Ribo-Zero Magnetic kit (epicenter), the mRNA was randomly fragmented into small fragments of about 200 bp, and the mRNA was used as a template for reverse transcription using random primers (Illumina) and SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA). Synthesis of double-stranded cDNA. The synthesized double-stranded cDNA was then added to End Repair Mix to make up blunt ends, phosphorylated at the 5' end, added an A base at the 3' end, and connected to a Y-shaped sequencing adapter. The library was enriched and then PCR amplified with Phusion DNA polymerase (NEB) for 15 cycles. After TBS380 (Picogreen) quantification, RNA-seq paired-end sequencing was performed using Illumina HiSeq X Ten (2 × 150 bp).

(3). Use the data generated by Illumina platform for bioinformatics analysis. All analyses were performed using the I-Sanger cloud platform (www.i-sanger.com) of Shanghai Meiji Bio.

In conclusion, high-throughput sequencing technology was used to study the significant differential expression of 61 genes in bacteria treated with 2(5H)-furanone. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis showed that 10 ^-4 mol/L 2(5H)-furanone significantly altered propionic acid metabolism, carbapenem biosynthesis, methane metabolism, arginine compared with control Acid and proline metabolic pathways (P<0.05, see Figure 6). Carbapenems are secondary metabolites produced by bacteria with broad-spectrum antibiotics against Gram-positive and Gram-negative bacteria (McGowan, SJ, Holden, MT, Bycroft, BW, Salmond, GP, ( 1999). Molecular genetics of carbapenem antibiotic biosynthesis. Anton. Leeuw. Int. JG75, 135-141. doi:10.1023/a:1001768428383). Studies have demonstrated that carbapenem production is regulated by the bacterial QS system (Thomson, NR, Crow, MA, McGowan, SJ, Cox, A., Salmond, GPC, (2000). Biosynthesis of carbapenem antibiotic and prodigiosinpigment in Serratia is under quorum sensing control. Mol. Microbiol. 36, 539-556. doi: 10.1046/j.1365-2958.2000.01872.x). In addition, 10 ^-4 mol/L 2(5H)-furanone exposure significantly reduced the expression of aspartokinase genes and flagellar assembly protein genes (fliH; flagellar assembly protein), which are important for bacterial cell wall and motility. . Aspartokinase is an important intermediate in the synthesis of diaminopimelic acid, which is involved in the biosynthesis of lysine, which is a key substance in the synthesis of most bacterial cell walls (Yoshida, A., Tomita , T., Kuzuyama, T., Nishiyama, M., (2007). Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus. Acta. Crystallogr. F. 63, 96-98 doi: 10.1107 /S1744309106055837).

Taken together, the present invention demonstrates that 2(5H)-furanone indirectly reduces mussel attachment by interfering with microbial envelope formation, such as EPS biomass and metabolic processes.

The foregoing description of the embodiments is provided to facilitate understanding and use of the present invention by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications to these embodiments can be readily made, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art according to the principles of the present invention without departing from the scope of the present invention should all fall within the protection scope of the present invention.

Claims (3)

1. Application of 2(5H) -furanone in inhibiting marine bacterial microbial capsule formation in reducing juvenile mytilus coruscus is provided.

2. Use according to claim 1, characterized in that: the method for reducing the adhesion of juvenile mytilus coruscus by 2(5H) -furanone comprises the following steps:

(1) carrying out amplification culture on the bacteria in a liquid culture medium to obtain an initial bacteria stock solution;

(2) centrifuging the initial bacteria stock solution, removing a supernatant, and mixing the initial bacteria stock solution with sterilized filtered seawater to obtain a bacteria solution with a washed culture medium;

(3) adding the bacteria liquid with the washed culture medium and the 2(5H) -furanone into a sterile culture dish containing a sterilized glass slide for bacterial microbial capsule culture, and then adding sterilized filtered seawater to wash the glass slide to obtain a bacterial microbial capsule;

(4) and transferring the slide glass with the formed bacterial microbial envelope into a sterile culture dish, and adding juvenile mytilus coruscus.

3. Use according to claim 2, characterized in that: in step (1), the bacterium is Pseudomonas marina ECSMB 14103.

Images