Introduction
In aquaculture, finding alternative ingredients to fish meal (FM) is urgently needed, since the depletion of fish wild stocks has led to a decrease in FM availability, thus increasing FM price. Plant feedstuffs (PF) may partially replace FM in aquafeeds (Tacon et al. 2011). However, PF use has its own drawbacks. In terms of resources management, PF are in direct competition with human food and have a high demand for land and water for production. Moreover, the presence of anti-nutritional factors, and, imbalanced amino acid profile, diminishes its effectiveness as an FM substitute in aquafeeds (Gatlin et al. 2007; Henry et al. 2015). Insect meal (IM), recently authorized for use in aquafeeds, positions itself as a promising aquafeed commodity due to its high protein content, balanced amino acid profile and high lipid contents. However, insects are also rich in chitin, a structural polysaccharide present in the insect’s exoskeleton, whose non-digestibility has been linked with lower fish performance and nutrient digestibility (Henry et al. 2015; Gasco et al. 2019). To overcome the chitin impairments of IM inclusion in aquafeeds, IM can be submitted to chemical or enzymatic treatments to promote chitin breakdown before being added to diets, but these processes have high costs, low yield, residual acidity, and may result in serious environmental pollution (Harish Prashanth and Tharanathan 2007). Alternatively, probiotic (PRO) bacteria, knowing to possess chitinase activity (e.g. Bacillus subtilis, (Askarian et al. 2013)), could be a solution. As such, this proposal aims to isolate, from European sea bass gastrointestinal tract, PRO bacteria capable of producing chitinases to improve the use of high IM-containing diets, supporting cost-effective fish growth without compromising welfare and health status.
Materials and methods
Based on the adaptability of gut microbial communities, chitin-containing diets were fed to promote microbial shifts towards chitin-metabolizing bacteria. Five isoproteic (45%) and isolipidic (18%) diets were formulated: a control diet (CTR); a diet similar to the CTR with 5% chitin supplementation (CHIT5); and 3 other diets with 25% inclusion of either Hermetia illucens (HI) larvae meal (HM25), Tenebrio molitor larvae meal (TM25) or HI exuviae meal (HEM25). After 8 weeks feeding trial, whole guts were aseptically excised and squeezed to collect the digesta contents. To select for aerobic bacterial sporeforming isolates, serial dilutions were prepared and heat-treated before inoculation on Luria Bertani (LB) medium (Nicholson 1990). The potential PRO were comprehensively screened in vitro, concerning their putative PRO traits, namely: sporeforming ability; chitinolytic activity; antibiotic resistance; haemolytic activity; and gut-survival aptitude.
Results
From the obtained microorganisms, from each diet and dilution, colonies presenting different morphologies were randomly selected for further analysis, resulting in a total of 363 isolates. Sporulation ability was confirmed by phase-contrast microscopy of cultures submitted to nutrient exhaustion by cultivation on DSM. These isolates were sequentially gauged for their chitin metabolization ability, resulting in a group of 40 isolates with promising chitinolytic activities. The remaining 40 isolates were subjected to full taxonomic identification followed by haemolytic activity characterization narrowing the isolate number to 20. Antibiotic resistance screening of the 20 candidates revealed that all isolates, but one, were resistant to at least one of the tested antibiotics and, as such, total chitinolytic activity was determined for all 20 isolates. Within these, the 4 isolates presenting the highest chitinolytic activity were selected and assessed regarding their gut-survival aptitude and total chitinolytic activity in gut-like conditions. All the selected isolates performed well in the tested conditions with 2 isolates performing better than the rest, namely FI645 and FI658. The best PRO candidates will be subsequently evaluated in vivo, considering their potential to modulate fish gut microbiota, aiming to improve chitin utilization in IM-based diets.
Acknowledgements
This research was supported by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. P.E. and C.R.S. had scientific employment contracts supported by national funds through FCT. F.R., R.A.S, and M.M. were supported by a PhD grant from FCT, Portugal, respectively SFRH/BD/138375/2018, SFRH/BD/131069/2017, and SFRH/BD/114995/2016.
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