**1. Introduction**

Anti-nutritional factors (ANFs) can be defined as substances that, by themselves or through their metabolic products, can exert negative effects on food utilization and interfere with the normal growth, reproduction and health of fish [1]. From a nutritional point of view, the presence of these compounds in diets is responsible for the deleterious effects on the absorption of nutrients and micronutrients, which may interfere with the normal functioning of certain organs [2]. This fact is one of the most important issues derived from using novel dietary ingredients in aquaculture, as well as one of the main drawbacks that limits their practical application in formulated feeds [3,4].

In general, ANFs have been mainly related to plant-derived feedstuffs, and they comprise a wide variety of compounds, such as protease inhibitors, phytohemagglutinin, lectins, phytic acid, saponins, phytoestrogens or antivitamins [1,5]. Although less known, recent studies have also documented the presence of these substances in seaweeds, considered currently a potential alternative ingredient for farmed fish [6,7].

Seaweeds have drawn the attention of researchers not only as an important source of dietary protein, but also as functional ingredients in aquafeeds [8]. More specially, some species of the genus *Ulva* have been successfully evaluated as a dietary ingredient in some farmed fish species, such as gilthead seabream (*Sparus aurata*) [9,10], Senegalese sole (*Solea senegalensis*) [8,11] or seabass (*Dicentrarchus labrax*) [12], with promising results in terms of growth, survival and nutrient utilization. Despite the health benefits reported for seaweeds, some studies have described that the dietary inclusion level of algae above 20% yields detrimental e ffects on fish growth and other zootechnical parameters. It has been suggested that these e ffects could be attributed to the existence of ANFs, which might a ffect the bioavailability and/or digestibility of nutrients [13].

In this regard, Oliveira et al. [14] and Maehre, [6] confirmed the presence of lectins, trypsin and alpha-amylase inhibitors, as well as phytic acid, in some species of marine algae, although polyphenolic compounds are considered the substances most frequently linked to their antinutritional effects [6,14]. It has been described that the presence of lectins in feed alters the intestinal epithelium, resulting in the over-secretion of mucus that may impair the enzymatic and absorptive capacity of fish [15], altogether leading to reduced growth [5]. Phytic acid and polyphenols bind to proteins and polysaccharides producing insoluble high-molecular complexes, a fact that reduces nutrient bioavailability and consequent nutrient deficiency, such as that described for methionine, which is essential for lipid metabolism [15,16]. In addition, other antinutritional compounds of seaweeds, such as phytates, can inhibit the action of gastrointestinal enzymes like tyrosinase, trypsin, pepsin, lipase and amylase [2].

It is also worth mentioning the existence of protease inhibitors, substances that bind to proteolytic enzymes causing not only reduced proteolysis, but also increased pancreatic secretion as an attempt to overcome these antinutritional e ffects [17]. Despite the evidence suggesting the presence of protease inhibitors in seaweeds and their possible e ffects on the digestive physiology of aquacultured fish [7,13,17], scarce research is available regarding the characterization of their e ffects. In this context, this this research aims to assess the presence of protease inhibitors in *Ulva ohnoi*, evaluating the e ffects of such inhibitors on fish digestive proteases, characterizing their mode of inhibition, and exploring potential strategies to reduce their detrimental e ffects on fish digestive enzymes.
