Extracting method and application of p-hydroxybenzoic acid

[0032] Arenibacter sp.6A1 strain fermentation

[0033] The low-temperature-preserved marine bacteria Arenibacter sp.6A1 was revived on the plate medium, and then the revived strain was inoculated into the fermentation medium, 180rpm, 28°C for 96 hours;

[0034] Among them, the plate medium is 2216E liquid medium (recipe: peptone 5g / L, yeast extract 1g / L, ferric phosphate 0.01g / L, sea salt 20g / L) plus 1.5% agar, sterilized under high pressure at 121°C for 20 minutes The fermentation medium is 2216E liquid medium (formulation: peptone 5g / L, yeast extract 1g / L, ferric phosphate 0.01g / L, sea salt 20g / L).

[0035] After fermenting according to the above method, the fermentation liquid of marine bacteria Arenibactersp.6A1 containing p-hydroxybenzoic acid can be obtained.

[0037] Separation of p-hydroxybenzoic acid

[0038] The fermented liquid that embodiment 1 obtains is carried out following steps:

[0039] Ethyl acetate extraction: Use 1mol / L hydrochloric acid to adjust the pH of the fermented liquid to 3-4, extract with ethyl acetate, spin the extracted extract to dryness below 50°C, and repeat the extraction process three times to obtain acetic acid 2 g of the extract of the ethyl ester layer.

[0040] Gel column chromatography: dissolve the extract of the ethyl acetate layer in 2mL methanol, carry out Sephadex LH20 (column specification: diameter=3.5cm, column length=100cm) column chromatography, utilize 400mL volume ratio to be 1:1 The mixture of chloroform and methanol was used as the elution solvent for isocratic elution, each 50mL was collected as a fraction, and a total of 8 fractions were obtained, G3-A, G3-B, G3-C, G3-D, G3-E , G3-F, G3-G and G3-H.

[0041] HPLC separation: the G3-H fraction was concentrated and evaporated to dry

[0049] Identification of algae-dissolving activity of p-hydroxybenzoic acid

[0050] Algae-dissolving experiments were carried out by setting gradient concentrations of p-hydroxybenzoic acid. Count the algae liquid through the plankton box to measure the algal concentration, shake up and divide the algal liquid; dissolve p-hydroxybenzoic acid to a sample concentration of 100 μg / μl with DMSO, and then serially dilute to 50 μg / μl, 25 μg / μl, 12.5μg / μl and 6.75μg / μl, take 1ml / well of the algae liquid into a 24-well plate, add 1ul of the prepared sample to the final concentration of the sample to 100μg / ml, 50μg / ml, 25μg / ml, 12.5μg / ml, 6.75μg / ml, add 1μl DMSO to the control group, set up 3 repetitions for each experimental group and control group; put the 24-well plate in the light incubator for culture; use planktonic counting plate at 15min and 120min Count the number of algae. figure 1 It shows the effect of the samples of various concentrations on the cell