Processing fish or by-products, as well as the so-called bycatch of fishing, generates a considerable amount of waste. The by-products of fish (or marine invertebrates) processing can be an interesting source of nutrients with high nutritional value. They can be reused as functional ingredients in the feed industry. Hydrolysates have been used as chemoattractants and fishmeal replacers in aquafeed, due to their low molecular weight compounds and balanced amino acid profiles. Peptides with predicted anti-inflammatory, immunostimulatory/anti-microbial properties were identified in the different fractions of the by-products using state-of-the-art peptidomics and bioinformatics techniques (often referred to as the in silico approach).
The present study was designed to evaluate the effects of dietary levels of bioactive peptides (BPs) derived from salmon processing by-products on the presence and distribution of peptic cells (oxyntopeptic cells, OPs) and enteric endocrine cells (EECs) that contain GHR, NPY and SOM in the gastric mucosa of European seabass and gilthead seabream. In this study, 27 seabass and 27 seabreams were divided into three experimental groups: a control group (CTR) fed a control diet and two groups fed different levels of BP to replace fishmeal: 5% BP (BP5%) and 10% BP (BP10%). The stomach of each fish was sampled and processed for immunohistochemistry. Some SOM, NPY and GHR-IR cells exhibited alternating “open type” and “closed type” EECs morphologies. The BP10% group (16.8 ± 7.5) showed an increase in the number of NPY-IR cells compared to CTR (CTR 8.5 ± 4.8) and BP5% (BP10% vs. CTR p ≤ 0.01; BP10% vs. BP5% p ≤ 0.05) in the seabream gastric mucosa. In addition, in seabream gastric tissue, SOM-IR cells in the BP 10% diet (16.8 ± 3.5) were different from those in CTR (12.5 ± 5) (CTR vs. BP 10% p ≤ 0.05) and BP 5% (12.9 ± 2.5) (BP 5% vs. BP 10% p ≤ 0.01). EEC SOM-IR cells increased at 10% BP (5.3 ± 0.7) compared to 5% BP (4.4 ± 0.8) (5% BP vs. 10% BP p ≤ 0.05) in seabass. The results obtained may provide a good basis for a better understanding of the potential of salmon BPs as feed ingredients for seabass and seabream.
The fishing industry has grown steadily over the last decade. This growth has been accompanied by a high volume of protein-rich by-products. This waste includes whole or parts of fish such as fillets, skin and fins, bones, heads, guts and scales. In the context of a circular economy, these by-products are rich in proteins, which can be recovered/reused as functional ingredients in the feed industry. Recently, several studies have reported the utilization of fish by-products by enzymatic hydrolysis for the recovery of various valuable components, and fish protein hydrolysates, such as capelin, mackerel, hoki frame, jumbo squid, yellow stripe trevally and tuna liver, have been shown to possess antioxidant activity with the ability to scavenge hydroxyl radicals, superoxide anion radicals, hydrogen peroxide and chelate metal ions [1,2,3,4,5,6]. Fishery proteins represent a potential source of biopeptides.
Previous studies have described the biological activity of marine protein hydrolysates produced from different species [7,8,9]. Protein hydrolysates are composed of low molecular weight compounds with a balanced amino acid profile. These characteristics have stimulated research and several studies have reported that these hydrolysates are interesting chemotactic agents and can be used as fishmeal substitutes in aquatic feeds [10,11,12,13,14].
The protein hydrolysates obtained contain peptides. To identify which of these peptides have anti-inflammatory, immunostimulatory or antimicrobial properties, state-of-the-art peptidomic and bioinformatic techniques (often referred to as the in silico approach) were applied. In this context, interest in the in silico approach has increased because it is less costly and time-consuming [15,16].
With its high nutritional value and potential pharmacological applications, farmed Atlantic salmon is a popular food around the world. The fileting operation generates large amounts of by-products consisting of head, backbone, skin and viscera. Mechanically separated salmon muscle is readily available as a low-cost by-product of the filleting process and can account for up to 60% (w/w) of filleting waste [17]. Several studies have evaluated the biochemical functional properties of salmon muscle protein hydrolysate (and its respective peptides) [18,19,20] and there is growing industrial interest in the utilization of bioactive peptides (BPs) within the fish feed and pet food industry. Recently, in European seabass, the dietary inclusion levels of bioactive peptides from farmed Atlantic salmon have shown the possibility of an almost total replacement of fish meal in a plant-based diet in terms of growth and feed efficiency [21].
The regulation of food intake relies heavily on the gut–brain axis. Several studies have highlighted the important role played by gut hormones in response to food digestion [22,23]. These hormones are involved in appetite regulation as short-term peripheral satiety signals. They promote satiety, i.e., a decrease in appetite and a reduction in food intake, through endocrine and nervous pathways by activating various signaling pathways [24,25,26,27]. In vertebrates, appetite and digestion are controlled by the enteroendocrine system [28,29]. Gut–brain hormones, such as ghrelin (GHR), neuropeptide Y (NPY), somatostatin (SOM), cholecystokinin, etc., are important factors in the control of feeding behaviours [28,29]. GHR, NPY and SOM have been found in a large number of fish species. Their tissue distribution supports the idea that GHR has an integrative role in the regulation of energy balance at both the central nervous system and systemic levels [30]. NPY and GHR are reported to correlate positively with feed intake [28]. GHR, a small peptide hormone secreted by the stomach, is an appetite stimulator [31]. GHR levels increase before a meal and decrease postprandially [32] and are involved in the regulation of appetite, energy balance and body weight [33]. NPY is a potent, highly conserved, multi-functional peptide found in vertebrates, including fish. It plays an important role in the regulation of feeding behavior, energy metabolism and digestive processes [34,35,36,37]. In fish, SOMs have many direct and indirect effects on intermediary metabolism and feeding behavior. In general, SOMs inhibit food intake and promote catabolic processes (e.g., mobilizing stored lipids and carbohydrates) [36,38].
Certain hormone-like peptides obtained by protein hydrolysis have the potential to affect gastrointestinal (GI) motility, endocrine metabolism, intake and animal performance [39]. Most bioactive peptides (BPs) share some structural features. These features are represented by the length of the peptide residue (from 2 to 20 amino acids) [40], the presence of proline, lysine or arginine, and the presence of hydrophobic amino acids [41]. Regulatory peptides with hormone-like activity (hormone peptides) or the ability to modulate blood levels of certain hormones could also be obtained by enzymatic hydrolysis. The biological activity of hormone-like peptides is typically mediated by their interaction with G protein-coupled receptors (GPCRs) on the cell surface and further activation of the ligand–receptor signaling pathway to regulate various physiological functions of the body [42]. Fish proteins may also serve as a reservoir of hormone-like peptides. Interestingly, some authors have found neuropeptide immuno-related molecules and molecules capable of binding to specific hormone receptors on cell membranes in fish protein hydrolysates [43,44,45]. In this study, we investigated the effect of supplementing salmon by-products derived from enzymatic hydrolysis on the presence and distribution of oxytocic (OP) and enteroendocrine (EEC) cell subpopulations expressing GHR, NPY and SOM in the gastric mucosa of European seabass and seabream.
Atlantic salmon (Salmo salar) processing waste consisting of fresh heads and backbones was purchased from Biomega (Øygarden, Norway). The material was then blended in tap water (1:1) heated to 50 °C, to which chymotrypsin (0.1% w/w; Enzyme Supplies, Oxford, UK) was added. Hydrolysis was performed for 60 min, followed by deactivation of enzyme activity at T > 90 °C for 10 min. The hydrolyzate obtained (in the aqueous phase) was subsequently separated from the lipid and bone phases and dried to a dry powder by means of a NIRO P-6.3 spray dryer (GEA, Skanderborg, Denmark). The inlet and outlet temperatures were 200 and 92 °C, respectively.
The diets were formulated with FM and with a mixture of vegetable ingredients that are currently used in aquafeed for European seabass and gilthead seabream [46,47]. The control diet (CTR) was formulated to resemble a commercial feed for European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) (Table 1). Two experimental feed mixes were formulated with an exchange of the fishmeal with the experimental fish protein hydrolysate at 5% (BP5) and 10% (BP10) levels, respectively [21]. The composition of the fish protein hydrolysate is reported in Table 2. Diets were preconditioned in an atmospheric double differential preconditioner (Wenger Manufacturing Inc., Sabetha, KS, USA) prior to extrusion on a TX-52 twin screw extruder (Wenger) and expanded through 2.5 mm dies to 3.2 mm pellets. The pellets were dried in a hot-air double-layer carousel dryer (model 200.2, Paul Klockner GmbH, Nistertal, Germany) at a constant air temperature and were coated with oil in a vacuum to achieve the final lipid content. Diets were formulated with FM and a mixture of vegetable ingredients currently used in aquafeed for European seabass and seabream [46,47].
The nitrogen content was analyzed by the Kjeldahl method (ISO 5983-2, 2009 [49]) and the crude protein content was estimated on the basis of N × 6.25. The ash was determined by combustion of the raw material at 550 °C (ISO 5984-2, 2002 [50]). The dry matter content was determined by drying at 103 °C (ISO 6469-2, 2002 [51]). The fat content of the protein hydrolysate was analyzed by the EU method (Commission Directive 98/64/EC), while the fat content of the raw material was analyzed based on ethyl acetate extraction (NS 9402). Peptide size distributions were measured by HPLC size exclusion chromatography (SEC) (1260 series HPLC Agilent Technologies, Santa Clara, CA, USA) using a Superdex Peptide 10/300GL column (GE Healthcare, Uppsala, Sweden). The eluent was acetonitrile with TFA and UV detection at 190–600 nm [52].
Juvenile European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) were collected from an Italian commercial hatchery and reared in recirculating aquaculture systems (RAS) at the Aquaculture Laboratory of Cesenatico, Department of Veterinary Sciences, University of Bologna, Italy. At the beginning of the experimental procedures, sixty seabass and sixty seabreams were individually weighted and allocated, based on the species, in each of eighteen conical-bottomed tanks with a volume of 800 L connected to an RAS supplied with natural seawater (overall water volume: 22 m 3; oxygen level 8.0 ± 1.0 mg L−1; temperature 24 ± 0.5 °C, salinity 28–32 g L−1) according to Busti et al. [53]. Over a period of 58 days, each experimental diet was administered twice daily until full satiety using an overfeeding approach employed for both species as described in Parma et al. [46,47]. At the end of the trial, at 12 h after a meal, three fish per tank were randomly selected from the 120 fish (60 gilthead seabream and 60 seabass) used for the performance studies, giving a total of 27 seabass (mean weight 147.1 g) and 27 gilthead seabreams (mean weight 168.76 g). Subsequently, the previously selected fish were sacrificed under anesthesia (excess of anesthetic MS222, 300 mg L−1). The GI tract from the esophagus to the posterior intestine was gently removed from each seabass and seabream and isolated from the coelomic cavity. The stomachs were isolated and fixed in formalin (pH 7.2) for 48 h at room temperature. After fixation, the stomachs were divided symmetrically by cutting along the long axis to obtain two equal halves. The stomach samples were dehydrated in a graded alcohol series, cleared in xylene and paraffin embedded. Sections (6 µm thick) placed on polylysine slides were obtained from each block of paraffin.
The Ethical-Scientific Committee for Animal Experimentation of the University of Bologna, Italy (ID 113/2020-PR) evaluated and approved all experimental procedures.
The calculations used to determine the various performance parameters were as follows.
Specific growth rate (SGR) (% day − 1) = 100 ∗ (ln FBW − ln IBW)/days (where FBW and IBW are the final and initial body weights, respectively);
Feed intake (FI) (g kg ABW-1 day-1) = ((100 ∗ total intake)/(ABW))/days) (where average body weight, ABW = (IBW + FBW)/2);
Feed conversion ratio (FCR) = feed intake/weight gain
The stomach sections were processed for double labeling immunofluorescence. Table 3
