Isolation and identification of potential probiotic bacteria from the soil of Saint Martin's island Bangladesh

the marine soil samples were collected from the shore of Saint Martin’s Island (20°37'40.3"N’ 92°19'22.3"E) of the Bay of Bengal, Bangladesh

the samples were collected from 6-15 inch depth using an auger

immediately transferred to a 50 mL sterile falcon tube

1 g of each soil sample was suspended in 9 mL of autoclaved seawater in an individual test tube

100 µL suspension from each diluted stock (10^-2 and 10^-3) was aseptically inoculated on individual Starch Casein Agar (SCA)

After incubation, different colonies were picked up based on their colony characteristics

Individual colonies grown on SCA plates were carefully observed and colony characteristics viz, colony size, shape, color, type and elevation were recorded

Biochemical tests such as oxidase, catalase, motility, oxidative-fermentative (O-F) tests were accomplished

Bacteria were grown in Marine Broth (MB) 2216 (Merck, USA) for 7 days at 28°C

The broth culture was centrifuged at 10,000 ×g for 15 min and the culture supernatant was passed through a 0.22 µm millipore membrane filter

The inhibitory activity of the culture supernatant was determined by agar well diffusion assay

Genomic DNA of the selected isolates was extracted by using a commercial GenJET genomic DNA purification kit (Thermo Fisher Scientific, USA) #K0721

DNA was amplified by using universal primer 8F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGATACCTTGTTACGACTT-3').

The PCR amplification condition was done by an initial denaturation at 94 °C for 5 min; 35 cycles of a denaturation at 94 °C for 1 min, an annealing at 57 °C for 40 sec and an extension at 72 °C for 1 min and a final extension step at 72 °C for 10 min.

Then the PCR amplicons were purified by using a commercial kit (Thermo Fisher Scientific, USA)

16S rRNA gene sequencing was done by Sanger Sequencing

Enzymatic activity such as, protease, lipase, amylase, and cellulose activity of the B. haynesii strain CD223 and A. mimigardefordensis strain SM421were assessed to evaluate their probiotic effects.

The pH of SCA broth was adjusted from pH 3–9. Then, B. haynesii strain CD223 and A. mimigardefordensis strain SM421 were inoculated in this broth and kept for 24 h incubation at 28°C.

The viability of the cells was confirmed by inoculating them onto SCA agar plates (pH 7) by the spread plate method

Bacteria were enriched in MB at 28^oC for 10 days

The ECPs were harvested and mixed with an equal volume of ethyl acetate in a separatory funnel

The air-dried extract was weighted and dissolved in methanol for further use

MIC was measured with different concentrations of ECPs extracts (1000, 500, 250, 125, 62.5, and 31.25 μg mL^-1)

Fish from each treatment were anesthetized with the clove oil (0.05 mL per 500 mL of water) for hematological analysis

Blood was collected from fish using a 3 cc syringe containing 10% blood anti-coagulant (EDTA) inserted into the caudal peduncle region to drag out blood.

The blood was transferred to a test tube coated with EDTA, and stored at -30 oC until use.

Red blood cells (RBCs) and white blood cells (WBCs) were counted using an improved Neubauer hemocytometer (MarienFeld Company Germany) under the light microscope (DM 100; Leica, Wetzlar, Germany)

To measure hemoglobin, fresh blood was collected from fish from each treatment and was poured in the edge of a strip of hemoglobin meter before the coagulation of blood.

Estimation of immunoglobulin (IgM) was carried out by using Humalyzer-3000 analyzer.

Gut microbiome DNA was extracted using a commercial kit

V3 and V4 primer were used for sequencing