4.8.3. Catalase (CAT)

CAT activity was determined in PMS by measuring substrate (H2O2) consumption at 240 nm according to Claiborne [60] adapted to microplate. Briefly, in a microplate well, 0.140 mL of phosphate buffer (0.05 M pH 7.0) and a 0.150 mL H2O2 solution (30 mM in phosphate buffer 0.05 M pH 7.0) were added to 0.01 mL of intestine PMS (0.7 mg mL−<sup>1</sup> total protein). Enzymatic activity was determined in a microplate reader (BioTek Synergy HT, Winooski, VT, USA), reading the optical density at 240 nm for 2 min every 15 s interval.

#### 4.8.4. Superoxide Dismutase (SOD)

SOD activity was measured according to Flohé and Otting [61], adapted to microplate by Lima, et al. [62]. Briefly, in a microplate well, 0.2 mL of the reaction solution [1 part xanthine solution 0.7 mM (in NaOH 1 mM) and 10 parts cytochrome c solution 0.03 mM (in phosphate buffer 50 mM pH 7.8 with 1 mM Na-EDTA)] was added to 0.05 mL of intestine PMS (0.25 mg mL−<sup>1</sup> total protein). Optical density was measured at 550 nm in a microplate reader (BioTek Synergy HT, Winooski, VT, USA) every 20 s interval for 3 min at 25 ◦C.

#### *4.9. Gene Expression*

RNA isolation from target tissue (anterior gut) and cDNA synthesis was conducted with NZY Total RNA Isolation kit and NZY first-strand cDNA synthesis kit (NZYTech, Lisbon, Portugal), following manufacturer's specifications. Real-time quantitative PCR was carried out on a CFX384 Touch Real-Time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). Genes comprised in the assay were selected for their involvement in gut integrity, health and immunity (Table 10). Specific primer pair sequences are listed in Table S1. Controls of general PCR performance were included on each array. Briefly, RT reactions were diluted to obtain the equivalent concentration of 20 ng of total input RNA which were used in a 10 μL volume for each PCR reaction. PCR wells contained a 2× SYBR Green Master Mix (Bio-Rad Laboratories, Hercules, CA, USA) and specific primers were used to obtain amplicons 50–250 bp in length. The program used for PCR amplification included an initial denaturation step at 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C denaturation for 15 s, with primer annealing and extension temperature (Table S1) for 1 min. The efficiency of PCR reactions was always higher than 90%, and negative controls without sample templates were routinely performed for each primer set. The specificity of reactions was verified by analysis of melting curves (ramping rates of 0.5 ◦C/10 s over a temperature range of 55–95 ◦C). Fluorescence data acquired during the PCR extension phase were normalised using the Pfaffl [63] method. The geometric mean of two carefully selected housekeeping genes (elongation factor 1-α (ef1α) and ribosomal protein S18 (rps18)) was used as the normalisation factor to normalise the expression of target genes. For comparing the mRNA expression level of each gene in a given dietary treatment, all data values were in reference to the expression level of CTR fish.


**Table 10.** PCR-array layout for gene expression profiling of anterior gut in sea bream.

#### *4.10. Data Analysis*

All results are expressed as mean ± standard error (mean ± SE). Residuals were tested for normality (Shapiro–Wilk's test) and homogeneity of variance (Levene's test). When residuals did not meet the assumptions, data was transformed by a Log10 or square root transformation. For gene expression data, a log2 transformation was applied to all expression values. Two-way ANOVAs were performed in data arising from both trials one and two, with "dietary treatment and time" as the fixed effects. Analysis of variance was followed by Tukey post-hoc tests. All statistical analyses were performed using the computer package SPSS 26 for WINDOWS. The level of significance used was *p* ≤ 0.05 for all statistical tests.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/md20070407/s1, Table S1. Relative gene expression profiling of anterior intestine in gilthead seabream juveniles fed experimental diets.; Figure S1. Protein/peptide profile of *C. vulgaris* hydrolysate, showing the main molecular weight ranges, the area of the main peak, and the localization of all the 42 identified peaks.

**Author Contributions:** Conceptualization, B.R., J.D., L.C., E.M. and B.C.; data curation, B.R.; formal analysis, B.R. and L.R.-P.; funding acquisition, J.D., L.C. and B.C.; investigation, B.R., L.R.-P. and S.A.C.; project administration, J.D. and B.C.; resources, M.P., J.L.d.S., J.D., E.M. and B.C.; supervision, J.D., L.C. and B.C.; writing—original draft, B.R.; writing—review and editing, L.R.-P., S.A.C., M.P., J.L.d.S., J.D., L.C., E.M. and B.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by Compete 2020, Lisboa 2020, Algarve 2020, Portugal 2020, and the European Union through FEDER in the framework of VALORMAR project (POCI-01-0247-FEDER-024517) and by national funds through the Foundation for Science and Technology (FCT) within the scope of UIDB/50016/2020, UIDB/04423/2020, and UIDP/04423/2020. The views expressed in this work are the sole responsibility of the authors. B. Reis was supported by FCT, Soja de Portugal, SA, and Sparos Lda., through the grant PD/BDE/129262/2017. S.A. Cunha and B. Costas were supported by FCT, through grants SFRH/BD/144155/2019 and 2020.00290.CEECIND, respectively.

**Institutional Review Board Statement:** The experiment was carried out in compliance with the Guidelines of the European Union Council (Directive 2010/63/EU) and Portuguese legislation for the use of laboratory animals. CIIMAR facilities and their staff are certified to house and to conduct experiments with live animals (Group-C licences by the Direção Geral de Alimentação e Veterinária (DGAV), Ministério da Agricultura, Florestas e Desenvolvimento Rural, Portugal). The protocol was approved by the CIIMAR Animal Welfare Committee in 29/04/2020 with the reference 0421/000/000/2020 from DGAV.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
