*Article* **Partial Characterization of Protease Inhibitors of** *Ulva ohnoi* **and Their E**ff**ect on Digestive Proteases of Marine Fish**

#### **Antonio Jesús Vizcaíno \*, Alba Galafat, María Isabel Sáez, Tomás Francisco Martínez and Francisco Javier Alarcón**

Departamento de Biología y Geología, Escuela Superior de Ingeniería, Ceimar-Universidad de Almería. La Cañada de San Urbano, 04120 Almería, Spain; albagalafat@gmail.com (A.G.); msc880@ual.es (M.I.S.); tomas@ual.es (T.F.M.); falarcon@ual.es (F.J.A.)

**\*** Correspondence: avt552@ual.es; Tel.: +34-950-015954

Received: 30 April 2020; Accepted: 16 June 2020; Published: 18 June 2020

**Abstract:** This piece of research evaluates the presence of protease inhibitors in the macroalga *Ulva ohnoi* and provides an initial overview of their mode of action. The ability of *Ulva* protease inhibitors to inhibit digestive proteases of three marine fish species, as well as their capacity to hamper the hydrolysis of a reference protein by those fish proteases, were assessed. In addition, thermal stability and the mode of inhibition on trypsin and chymotrypsin were also studied. Dose-response inhibition curves and in vitro protein hydrolysis assays revealed a noticeable inhibition of fish enzymes when *Ulva* concentration increased in the assay. The thermal treatment of *Ulva* reduced markedly the inhibitory effect on fish digestive protease. Finally, Lineweaver–Burk plots indicated that trypsin and chymotrypsin inhibition consisted of a mixed-type inhibition mechanism in which the inhibitory effect depends on *Ulva* concentration. Overall, the results confirmed the presence of protease inhibitors in *Ulva*, though heat treatment was enough for inactivating these compounds.

**Keywords:** inhibitor; macroalgae; marine fish; protease; *Ulva ohnoi*
