Introduction
The escalating global demand for animal proteins has led to a substantial increase in the generation of animal by-products (ABPs) by the meat processing industry. Among these, porcine blood -constituting approximately 3% of the live weight of pigs- emerges as a promising substrate for its valorisation into high-value bioproducts through environmentally sustainable methodologies, thereby contributing to the advancement of circular economy practices (Toldrá et al., 2021). In this regard, s equential enzymatic hydrolysis has been identified as a key methodological procedure, as it not only enhances the digestibility of protein substrates but also facilitates the release of bioactive peptides and free amino acids . Accordingly, this approach holds significant potential for the development of functional feed ingredients tailored for aquaculture applications, with the dual aim of promoting aquatic animal health and welfare and reinforcing the environmental sustainability of aquaculture systems (Boyd et al., 2020). To achieve this goal, we evaluated in vitro the properties of a novel feed ingredient derived from a porcine blood hydrolysate (PBSH), and in vivo its bioactivity in a fish feeding trial with gilthead sea bream as a farmed fish model. Such in vivo testing was based on a multi-layer metabolic, transcriptional, and gut microbiota approach, including measurements of behaviour, swimming performance and dynamics of fasting weight loss and weight regain during a fasting/refeeding challenge.
Materials and Methods
PBSH enzymatic hydrolysate was obtained according to Moreno-Mariscal et al. (2025) and then filtered (<10 kDa) and spray-dried. Antioxidant activity and inhibitory activity against dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), tumor necrosis α-converting enzyme (TACE) and monoacylglycerol lipase (MGL) enzymes were studied . PBSH was fractionated by RP-HPLC and peptide sequences from the most bioactive fraction were identified by mass spectrometry techniques and later synthetised to confirm their biological activity . For the in vivo analysis , juvenile fish of approximately 19 g were fed with control (CTRL) or PBSH-supplemented diets for 10 weeks . At the end of this period, blood, liver, intestine and gut microbiota samples were collected after 24 and 48h of fasting for biochemical, gene expression, histological , and gut microbiota composition analyses . After the feeding trial, the pre-feeding f ish behaviour was monitored by video recording . In parallel, swimming performance was assessed to determine basal and maximum metabolic activity rates (BMR, MMR) and critical swimming speed (Ucrit). Finally, a f asting and refeeding challenge of ten and eight days, respectively, was performed to assess the fasting weight loss and weight regain during refeeding .
Results and Discussion
As summarized in Figure 1, the o ptimised hydrolysis of porcine blood resulted in an extract enriched in free amino acids and bioactive peptides with a molecular size lower than 10 kDa . These peptides conferred antioxidant, hypoglycaemic, anti-inflammatory, and immunomodulatory properties to the extract . In fact, five peptide sequences were identified and confirmed as inhibitory of DPP-IV and NEP enzymes. A synergistic effect of these peptides was observed at a concentration of 1mM with inhibition values of these enzymes of 56.43% and 83.54%, respectively. In agreement with these potential benefits, all fish in the trial showed an optimal growth performance (SGR = 2.3; FCR = 1), which was accompanied in PBSH fish by a lower hepatosomatic index and a down-regulated expression of key hepatic lipogenic enzymes (diminished risk of liver steatosis). While no signs of histopathological damage were found in CTRL and PBSH fish, data on gene expression profiling indicated a transient influence of the experimental diet for both liver and intestine profiles, which was prone to reduc e overall metabolic rates highlight ing an anti-inflammatory appearance in the intestine. PBSH fish also showed an improved welfare condition with lower plasma glucose and cortisol levels in concurrence with a less aggressive behaviour during pre-feeding (30 min before feeding). Otherwise, the PBSH diet enhanced physical performance (20% increase in Ucrit) and reduced basal metabolic rates with a minimis ed weight loss during starvation, and accelerated weight regain during re-feeding. Finally, t he intestinal microbiota was also reshaped by the dietary intervention with a redistribution of dominant bacterial genera associated to the PBSH group.
Conclusions
Enzymatic hydrolysis of pig blood offers a sustainable approach to revaloris e this by-product by producing a new functional ingredient enrich ed in essential amino acids and bioactive peptides . The inclusion of the PBSH hydrolysate into fish diets enhances resilience to environmental stressors and improves overall fish welfare and wellbeing contributing to more sustainable and efficient aquaculture practices.
Funding
This study forms part of the ThinkInAzul program and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/024, IP1: J. Pérez-Sánchez, IATS ; GVA/THINKINAZUL/2021/023, IP1: F. Toldrá, IATA). The Accreditation as Center of Excellence Severo Ochoa CEX2021-001189-S, funded by MCIN/AEI/10.13039/501100011033, is also acknowledged.
References
Toldrá, F. et al. (2021). Meat Science, 181, 108608.
Boyd, et al. (2020). In Journal of the World Aquaculture Society (Vol. 51, Issue 3, pp. 578–633).
Moreno-Mariscal, C. et al . (2025). International Journal of Molecular Sciences, 26(8), 3583.