Forty-two bacterial colonies were isolated from the gut of different mullet species captured from a Mediterranean lagoon in autumn and in winter in Sardinia (Table 1).

The application of different screening methods allowed the selection of a “group” of intestinal strains as surfactant producers. The drop-collapse method (Bodour et al., 2003) was used as a first screening test for identifying the “bioactive” microbes. Table 1 shows the results of all the used screening tests. 

 Figure 1 shows the emulsification indexes E-24(%) and the surface tension activity of the detected “bioactive” intestinal bacteria.

The most interesting strains are represented by eight isolates (strain 1, 3, 5, 6, 9, 10, 13, 15), showing an emulsification index E-24(%) from 50.0 to 77.0% and a surface tension from 36.5 to 37.2 (mN·m⁻¹).

Thirty strains, which showed bioactivity, were identified by 16S rRNA gene partial sequencing. Table 1 shows fish origin, phylogenetic affiliation and accession number of the studied intestinal strains. Figure 2 shows the phylogenetic tree reconstructed using the 16S rRNA gene sequences. Different groups and subgroups were obtained with respect to the corresponding reference species type strains and among themselves. The most heterogeneous group was represented by Pseudomonas spp., while Aeromonas spp. and Enterobacter sp. formed well distinguished clusters. The outgroup strain NR074804 Cellvibrio japonicus strain Ueda 107 was separated from all the others.

Figure 3 shows the BSs yield extracts of representative intestinal bacteria. The BS producers which gave the highest yield extracts (values from 6–6.42 g L⁻¹) were strains 6, 13, 15.

The chromatographic analyses (TLC) of the BSs extracted from intestinal bacterial supernatants showed two types of glycolipid compounds (Figure 4). The TLC silica gel glass plates stained by anisaldehyde (carbohydrates) (Figure 4a,c) and cerium sulphate (lipids) (Figure 4b,d) indicate a group of specific TLC fractions (retardation factor Rf = 0.42), which presumably represents the di-rhamnolipid structures, while a group of other fractions (Rf = 0.75) detects the mono-rhamnolipid molecules (Figure 4a,b).  Another type of molecule was also detected by the TLC analyses but not separated using the first solvent system indicated above (strains 19 and 26, Figure 4a,b). These less polar compounds from strains 6, 18, 19, 22, 25 and 26 were separated using a less polar solvent system, as described above, and gave different profiles of the TLC fractions (from the bottom, Rf = 0.20, 0.30, 0.60) (Figure 4c,d).

Cell free supernatans (CFSs) of the bioactive strains exhibited inhibitory activity against the target strains S. aureus H1610 and P. mirabilis H1643, while no inhibition was evidenced against the target strains P. aeruginosa H1328, K. pneumoniae H1637 and A. hydrophila H1563 (Table 2).

Of the tested CFSs, 26.6% exhibited antibacterial activity, with halos ≥10 mm compared to the target strain S. aureus H1610 (Figure 5)

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On the other hand, the crude extracts (CEs), obtained from the bacterial supernatants, evidenced antibacterial activity against more target strains, namely S. aureus H1610, P. mirabilis H1643, K. pneumoniae H1637 and A. hydrophila H1563, while no activity was recorded against P. aeruginosa H1628. Of the CEs, 76.7% and 66.7% were active against S. aureus H1610 and K. pneumoniae H1637, respectively, while 20% of the CEs showed inhibitory activity against P. mirabilis H1643 and A. hydrophila H1563 (Figure 5).