Isolation and selection of probiotic candidates from gastrointestinal tracts of teleosts and molluscs

Abalone aquaculture is increasingly important because of the progressive decline of wild catch abalone overtime, yet the world's demand for the seafood is continuously increasing. However, slow growth rates and disease are two major constraints in the development of abalone aquaculture. One way to deal with these issues is the use of beneficial bacteria, generally known as probionts. This approach has been confirmed to enhance the growth and disease resistance of many cultured terrestrial and aquatic animals. However, viability is still regarded as a major challenge when probionts are applied in aquaculture animals. This is probably because the probiotic strains are isolated from terrestrial organisms, which in fact have very different environmental conditions compared to aquatic species. Given these issues, it has been suggested that probionts for aquaculture species should be isolated from aquatic animals. Therefore, this study aimed at isolation of bacteria associated with gastrointestinal tracts (GITs) of aquatic teleosts and molluscs, and screening for probiotic candidates. The GIT was selected as a source of bacterial isolation, because GITs are important sites where digestion and absorption of feed occurs, as well as one of the most common entry ports for bacterial infections. The study was started by isolating 230 endogenous bacteria from GITs of 155 aquatic teleosts and molluscs collected from different aquatic environments (recirculating aquaculture systems, flow-through aquaculture systems and wild environments). Out of the 230 intestinal bacteria, 24 isolates displayed a capacity to synthesize digestive enzymes, either protease, alginate lyase or cellulase. The other 22 isolates produced antimicrobial compounds against at least one of eight bacterial pathogens (Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio proteolyticus, Yersinia ruckeri, Aeromonas hydrophila, and Listeria monocytogenes). Then, five bacteria having either the highest enzymatic or the broadest antimicrobial activity were selected for studying their viability in simulated GITs' environments. The results showed that the five bacteria displayed good viability in the simulated GITs of abalone, being tolerant to a low pH, to gastric enzymes (trypsin and pepsin) and to surfactants like bile-salt in a simulated intestinal juice. These results indicate that the five bacteria have a good viability and potential capacity to colonize the GITs of abalone. To confirm these in vitro results in live animals, three bacteria were selected to be used as feed supplement in abalone in a mixture of 2 bacteria (Bacillus amyloliquefaciens subsp. plantarum and Enterobacter ludwigii; 2P) or three bacteria (B. amyloliquefaciens subsp. plantarum, E. ludwigii and Pediococcus acidilactici; 3P). These bacteria were incorporated onto Gracillaria sp., which is a natural feed of abalone and was fed to juvenile abalone, Haliotis asinina, for 62 days. The results showed that abalone fed on 2P or 3P-supplemented diets had significantly higher growth rates (both wet weight and shell length) compared to the abalone receiving unsupplemented diets (control). Abalone receiving the 2P or 3P supplemented diet showed similar survival to the abalone in the control, which was >72 %. These results suggest that it is feasible to use a mixture of B. amyloliquefaciens subsp. plantarum, and E. ludwigii to increase production yield of abalone, H. asinina. However, further studies such as the use of different probionts and cell concentrations are required to enhance survival rate of abalone and appropriately commercialise the findings.