The increasing global need to find alternative and sustainable protein sources has promoted research in the field of non-conventional feed ingredients, such as single-cell proteins (SCPs). SCPs production is based on the fermentation of microorganisms such as yeast, bacteria, microalgae, and filamentous fungi. They have a high protein content with a suitable amino acid profile1. Specifically, single-cell protein products obtained from fungi contain 30 to 50% of proteins, other nutrients including different vitamins mainly from vitamin B-complex, and a relatively high amount of nucleic acid. To date, various substrates have been utilized to cultivate microorganisms for SCPs production, such as agricultural wastes, fruit processing wastes, methane, and animal wastes2. This study was undertaken to assess the effects of different inclusion levels of SCPs on growth, feed utilization, plasma biochemistry, and gut health of gilthead seabream reared under normal conditions and after a crowding stress period. The product utilized in this trial is a dried, inactive Paecilomyces variotii produced from sustainable industrial wood residual feedstocks to partly or fully replace traditional protein sources in fish feed.
Materials and methods
One trial with gilthead sea bream was conducted in a closed recirculation aquaculture system (RAS). Fish (initial weight: 120.3 ± 0.3 g), were fed over 104 days with four experimental diets containing different inclusion levels of SCPs meal (0% CTRL, 5% SCP5, 7.5% SCP7.5, and 10% SCP10) in substitution of fish meal (FM). After the end of the trial, fish were subjected to crowding stress for two hours (density 80 kg/m3). At the end of the growth trial, distal intestine content was collected for gut microbiota analysis and growth and feed efficiency parameters (specific growth rate, SGR, feed intake, FI, feed conversion rate, FCR), were assessed. Furthermore, blood and a portion of the distal intestine were collected at the end of the growth trial (T0), after the 2 hours (T2) and after 24 hours (T24) from the crowding stress in order to get information on the physiological status of the fish and to investigate on the expression of genes correlated to several gut functions, including immune regulation (interleukin 1b, IL-1b, interleukin 6, IL-6, interleukin 8, IL-8, interleukin 10, IL-10, tumour necrosis factora, TNF-α, T-cell receptorb, TCRβ, histocompatibility complex I, MHC I, Proliferating cell nuclear antigen, PCNA, Lysozyme, Lyz), stress response (glutathione reductase, GR, glucocorticoid receptor, GCR, Heat shock protein 70, Hsp70), nutrient absorption (peptide transporter 1, Pept 1, sodium-glucose transporter, SLC5A1, fatty acid binding protein 2, FABP2, Chemerin Chemokine-Like Receptor 1, cmklr1), and epithelial function (aquaporin8, Aquap8, Claudin15, Cldn15, Aminopeptidase N, AminopN). Data were analyzed by one-way, and a two-way ANOVA followed by Tukey’s multiple comparison test, using GraphPad Prism, version 9.1.1.
At T0, no significant differences (p > 0.05) due to the different treatments were observed in final body weight, FI, SGR, and FCR. Regarding gene expression analysis, genes related to immune regulation showed no differences among diets (p > 0.05). Regarding genes involved in nutrient absorption, Pept 1, showed higher values in SCP7.5 with respect to the control diet (p < 0.05), while SLC5A1 showed higher expression in SCP5 as compared to the control diet. Even if not significant (p > 0.05), the FABP2 showed a similar trend. Among genes with epithelial function, Aquap8 showed a dose-response effect (p < 0.05). In fact, its expression increased with the increasing inclusion of SCPs (Fig.1). Considering T2 and T24, time effect, diet effect, and a significant interaction between diet and time were observed in the expression of several genes including interleukin 8, heat shock protein 70, glutathione reductase, glucocorticoid receptor, and FABP2.
Discussion and Conclusion
The results show that experimental diets containing different levels of SCPs inclusion from Paecilomyces variotii can replace 5%, 7.5% and 10% of FM without compromising the growth and feed utilization. Regarding the immune genes, there is no difference in immune response among treatments at T0, a result that matches growth performances. SCPs inclusion affected genes related to nutrient transport and water absorption indicating a probable higher capacity for nutrient and water uptake. Finally, both diet and time clearly affected gene expression.
1.Marchi, A., Bonaldo, A., Scicchitano, D., Candela, M., De Marco, A., Falciglia, S., Mazzoni, M., Lattanzio, G., Clavenzani, P., Dondi, F., Gatta, P.P., Parma, L., 2023. Feeding gilthead sea bream with increasing dietary bacterial single cell protein level: Implication on growth, plasma biochemistry, gut histology, and gut microbiota, Aquaculture,565, 7391322.
2. Sharif, M., Zafar, M.H., Aqib, A. I.., Saeed, M., Farag, M. R., Alagawany, M., 2023. Single-cell protein: Sources, mechanism of production, nutritional value, and its uses in aquaculture nutrition. Aquaculture.531 (735-885).
This research was undertaken under the NextGenProteins (Next Generation Proteins for food and feed) project, funded by European Union’s Horizon 2020, research and innovation programme. Gran Agreement number 862704, 48 months.