Introduction
The aquaculture industry is rapidly expanding and is challenged with improving the gastro-intestinal health of farmed salmon in the face of disease outbreaks and issues arising from novel aquafeed formulations. Prebiotics are non-digestible food additives that improve gut function and overall health by stimulating the growth of commensal microbial species. Mannan oligosaccharide (MOS) prebiotics are known to improve the gut morphology of rainbow trout by modulating microbial communities1. Volatile fatty acids (VFAs), the end products of anaerobic fermentation of non-digestible fiber, are known to exert multiple functional benefits on the host such as maintenance of intestinal barrier integrity2. Recently, we presented an in-vitro gastrointestinal tract model of the farmed Atlantic salmon called SalmoSim3. We demonstrated that the major genera from the salmon gut can be maintained within in-vitro, and the microbial community composition and nutritional biochemical products proliferate in SalmoSim as they do in real fish when faced with a changes in diet3. Here we deploy SalmoSim to assay BioMOS (a commercially available prebiotic) for production of VFAs by anaerobic fermentation.
Method and materials
SalmoSim is comprised of bioreactors where physical and biochemical parameters (pH, oxygen levels, temperature, peristaltic flow rate and addition of biofluids) are maintained to ensure similarity with the gut compartments of real salmon3. Here, the microbiomes of pyloric caeca of adult farmed Atlantic salmon were sampled and pre-grown for 20 days to establish stable communities. A sterilized solution of ground fish meal was pumped through a bioreactor simulating the stomach then pumped into three bioreactors simulating the pyloric caeca. The pre-grown microbial communities of three salmon were inoculated into the pyloric caeca bioreactors and subjected to an static growth period of four days, followed by continuous flow pre-treatment phase of 5 days, then a 20 day BioMOS treatment phase, and finally a wash out phase of 6 days (Fig 1). Samples were taken at three time points during each phase (nine sampling time points in total).
Thirty milliliters of digest culture was taken and centrifuged at 5000 rpm for 10 minutes. The supernatant was filtered through the Costar SpinX centrifugation tube filter and VFAs were analysed by gas chromatography at MS-Omics (Denmark). A linear mixed effect model was used to determine if VFA concentrations were statistically different between the phases. The pelleted material was subjected to DNA extraction and next-generation sequencing as described previously3 using Illumina HiSeq amplicon sequencing of the 16S V1 rDNA locus. Pearson correlation coefficients between the operational taxonomic units and concentrations of VFAs were calculated.
Results
Inclusion of the MOS increased the concentrations of the VFAs formic acid (p=0.001), propionic acid (p=0.037) and 3-methylbutanoic acid (p=0.024) whose levels correlated with increasing abundances of principally anaerobic microbial genera (Fusobacteria, Agarivorans, Pseudoalteromonas, Myroides). Concentrations of the VFAs 2-methyl-propanoic acid, butanoic acid, sulfurous acid, pentanoic acid, 4-methyl-pentanoic acid, hexanoic acid and heptanoic acid were unchanged by the inclusion of MOS.
Discussion
Here we assess a commercially available MOS prebiotic’s effect on the production of nutritionally important VFAs by microbial flora native to farmed Atlantic salmon pyloric caeca. Interestingly, we found the MOS stimulated the production of formic acid, salts of which are often supplemented into the diets of farmed pigs to promote growth and protect from pathogens4. Dietary sodium propionate has also been shown to increase weight gain, increase carcass protein content and modulate immune function in European seabass5. Our results indicate BioMOS is a credible candidate prebiotic capable of promoting the synthesis of beneficial VFAs via the farmed salmon’s native gut flora and our NGS sequencing results select genera of bacteria associated with increases in VFA production.
References
1. Dimitroglou, A. et al. Dietary mannan oligosaccharide supplementation modulates intestinal microbial ecology and improves gut morphology of rainbow trout, Oncorhynchus mykiss (Walbaum)1. J. Anim. Sci. 87, 3226–3234 (2009).
2. Silva, Y. P., Bernardi, A. & Frozza, R. L. The Role of Short-Chain Fatty Acids From Gut Microbiota in Gut-Brain Communication. Front. Endocrinol. (Lausanne). 11, 1–14 (2020).
3. Kazlauskaite, R. et al. Development of a three-compartment in vitro simulator of the Atlantic Salmon GI tract and associated microbial communities: SalmoSim. Microbiome 9, 2020.10.06.327858 (2020).
4. Luise, D., Correa, F., Bosi, P. & Trevisi, P. A review of the effect of formic acid and its salts on the gastrointestinal microbiota and performance of pigs. Animals 10, (2020).
5. Wassef, E. A. et al. Sodium propionate as a dietary acidifier for European seabass (Dicentrarchus labrax) fry: immune competence, gut microbiome, and intestinal histology benefits. Aquac. Int. 28, 95–111 (2020).