Rheinheimera aquimaris strain from ocean sludge and application thereof
A Haimeria, marine technology, applied in the field of marine microorganisms, to achieve the effect of simple fermentation and culture conditions, simple and easy-to-control preparation process
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Embodiment 1
[0020] Example 1: Strains R. aquimaris Enrichment screen for QSI02.
[0021] 1.1 Sample collection: In September 2010, seawater sludge samples were collected from the Qingdao waters of the Yellow Sea and stored in sterile bottles.
[0022] 1.2 Enrichment culture: Take 2g of the sample and add it to 150mL denitrifying bacteria enrichment medium (KNO 3 2g, K 2 HPO 4 0.5g, MgSO 4 5g, sodium acetate 9.5g, H 2 O 1L, pH 7.0-7.4) in a 250mL Erlenmeyer flask, cultured in a shaker at 30°C and 150r / min, sampled every 3-4 days and tested for NO with Griess reagent 2 - changes. Choose NO 2 - The highly variable denitrifying bacteria were introduced into fresh medium. After repeated operations several times, take 1 mL of the bacterial suspension and place it in a test tube filled with 9 mL of sterile water, mix well to make a dilution of 10 -1 For the bacterial suspension, do 5 more dilutions in the same way.
[0023] 1.3 Isolation and purification of bacterial strains: Tak...
Embodiment 2
[0024] Example 2: Strains R. aquimaris QSI02 identification and preservation.
[0025] 2.1 Morphological characteristics: strains R. aquimaris QSI02 was milky white with neat edges on LB medium, and it was Gram-negative bacteria. Scanning results by transmission electron microscopy showed that ( figure 1 ), the bacteria are short rod-shaped, about 1.5-2.0 in size mu m wide, 7.0-9.0 mu m long, contains many flagella, and has transparent inclusions in the cell.
[0026] 2.2 Identification and preservation of strain 16S rDNA: 16S rDNA sequence analysis was performed by colony PCR method, primers were fD1: 27F 5'-AGAGTTTGATCMTGGCTCAG-3' and rD1: 1492R 5'-TACGGYTACCTTGTTACGACTT-3', the PCR reaction conditions were as follows: 94°C Denaturation for 3min, 30 cycles at 94°C for 30s, 55°C for 1min, 72°C for 90s; extension at 72°C for 10min. After PCR amplification, the PCR products were analyzed by 1.0% agarose gel electrophoresis. The PCR product purified by the gel recove...
Embodiment 3
[0032] Example 3: Strains R. aquimaris Quorum sensing inhibitory activity assay of QSI02 metabolites.
[0033] 3.1 Strains R. aquimaris Preparation of QSI02 metabolites: take activated marine bacteria R. aquimaris QSI02, inoculated into the fermentation medium for shaker culture, after the fermentation, centrifuged, concentrated in vacuum at low temperature to a paste, leached with methanol to obtain the methanol extract, and the methanol extract was mixed with 0.22 mu The m organic phase filter was used for biological activity detection after filtration.
[0034] 3.2 Detection of quorum sensing inhibition activity: coating 200 on LB plate mu l diluted 10 times C. violaceum For the fermentation broth, paste a filter paper sheet dipped in 0.5% DMSO aqueous solution in the center as a control, and paste filter paper sheets dipped in the extract of the bacteria to be tested around it in turn, and observe whether bacteria grow around the filter paper sheet but no purp...
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