Protein digestibility of aquafeeds in Sparus aurata: A model for in vitro bioaccessibility validation in fish

The in vitro assessment of nutrient digestibility in aquaculture offers a fast, affordable, and ethical alternative enabling to optimize the nutritional value of feeds for improved fish health. Although commonly used in human studies, these in vitro models are still scarce for fish. This study aimed to validate an in vitro digestion method for marine fish using both fish crude digestive extracts and commercial digestive enzymes through a comparison with an in vivo assay using gilthead seabream (Sparus aurata) as a biological model. Additionally, the effects of temperature, digestion time and feed amount (enzyme-to-substrate [E:S] ratio) influenced the in vitro protein digestibility, either individually or through their interactions. Only commercial enzymes lead to similar protein bioaccessibilities to those obtained in vivo (90.8 ± 1.7 %) under the following factorial design: i) 20 °C, 24 h, 250 mg of feed (88.1 ± 2.6 %); ii) 37 °C, 6 h, 136.5, 250 and 500 mg (85.9 ± 2.5 %, 90.1 ± 3.0 % and 87.4 ± 1.0 %, respectively); and iii) 37 °C, 24 h, 500 and 1000 mg (86.0 ± 2.1 %, 86.6 ± 5.2 %, respectively). The distinct action mechanisms of non-fish commercial enzymes seem to significantly enhance protein bioaccessibility compared to fish digestive extracts. Moreover, an optimal balance between temperature and digestion time plays a crucial role in maximizing digestibility, supporting efficient nutrient breakdown and absorption. The validated in vitro digestibility method using commercial enzymes for S. aurata provides a cost-effective, fast alternative and free of ethical constraints. We also propose a standardized E:S ratio to be applied in future studies using this methodology.