Simulated Gastrointestinal Digestion of Tilapia (Oreochromisniloticus) Scale Hydrolysates Enhances ACE-Inhibitory Activity and Reveals Antioxidant Effects in STC-1 Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Following the review of your manuscript, the article requires improvement in accordance with the following suggestions:

L33, L75, L846- in vivo should be written in italics.

L459 - The title of Figure 1 is not positioned below the figure.

L471- The authors should clarify the identity and origin of these higher MW fractions, whether their appearance under gastric conditions is reproducible, and whether they contribute to the biological activities measured in the digests.

L519- The title of Figure 2 is not positioned below the figure.

L587 - The conclusion that the digests are non-cytotoxic is insufficiently supported by the data presented.

L690, L726 - Two different IC₅₀ values for enalapril are cited within the same section: 3.62 µg/mL and 6.6 µg/mL. The authors should either use a single reference value throughout or explicitly justify the discrepancy.

L743 - An LC-MS/MS analysis of the obtained peptides should be performed to confirm and support the hypotheses formulated in the Results and Discussion sections.

L846 - The Conclusions should include a paragraph outlining directions for further research.

The chromatogram figures (Figures 1 and 2) should be provided at higher resolution.

Best regards,

Author Response

Comments 1: L33, L75, L846- in vivo should be written in italics.

Response 1:Dear Reviewer, thank you for your observation. The term in vivo in lines 33, 75, and 846 has been corrected and formatted in italics throughout the manuscript.

Comments2: L459 - The title of Figure 1 is not positioned below the figure.

Response 2: Dear Reviewer, thank you for your observation. It appears that the formatting was displaced from the original manuscript during the editing process. We have repositioned the captions of all figures below their corresponding figures throughout the manuscript.

Comments 3: L471- The authors should clarify the identity and origin of these higher MW fractions, whether their appearance under gastric conditions is reproducible, and whether they contribute to the biological activities measured in the digests.

Response 3: Dear Reviewer, thank you for your valuable observation. The identity, possible origin, and transient nature of the higher MW fractions observed during the gastric phase are clarified in the revised paragraph. Specifically, we explain that these fractions were likely associated with peptide populations not readily detected in the undigested hydrolysate due to the centrifugation and filtration steps prior to chromatographic analysis. Under simulated gastric conditions, non-soluble protein was digested by pepsin and new polypeptides were released. These peptides were seen in the SEC as they eluted after 11 min. We also clarify that this behaviour was consistently observed during the SGID analysis and that the disappearance of this peak after the intestinal phase suggests further hydrolysis into lower MW peptides, which may contribute to the generation of bioactive peptide populations.

Comments 4: L519- The title of Figure 2 is not positioned below the figure.

Response 4: Dear Reviewer, thank you for your observation. The title of Figure 2 has been repositioned below the figure in the revised manuscript.

Comments 5: L587 - The conclusion that the digests are non-cytotoxic is insufficiently supported by the data presented.

Response 5: Thanks for your observation. The digests did not show cytotoxic activity in the STC-1 cell line using the CCK-8 kit. We know that the CCK‑8 assay primarily reflects cellular metabolic activity (via dehydrogenase activity) rather than directly measuring the number of cells or proliferation, and therefore other complementary assays should have been performed. For that reason, in the same paragraph, we included the word “suggest”. In this regard, the paragraph includes something about these complementary assays: “Therefore, complementary endpoints (such as LDH release, apoptosis/necrosis markers, or, in differentiated intestinal models, barrier integrity assays) would further strengthen safety assessments”.

Comments 6: L690, L726 - Two different IC₅₀ values for enalapril are cited within the same section: 3.62 µg/mL and 6.6 µg/mL. The authors should either use a single reference value throughout or explicitly justify the discrepancy.

Response 6: Dear Reviewer, thank you for your observation. An error occurred during the manuscript redaction. The IC₅₀ value of 6.6 µg/mL corresponds to a synthetic peptide, whereas the IC₅₀ value of 3.62 µg/mL corresponds to enalapril used as the positive control. The text has been corrected throughout the manuscript to avoid confusion.

Comments 7: L743 - An LC-MS/MS analysis of the obtained peptides should be performed to confirm and support the hypotheses formulated in the Results and Discussion sections.

Response 7: Dear reviewer, thanks for your observation. We agree that LC-MS/MS analysis of gastrointestinal digests can provide useful information on peptide profiles. However, in the present case, such analysis would present important technical and interpretative limitations. Simulated gastrointestinal digestion produces highly complex mixtures rich in free amino acids, dipeptides, and short oligopeptides, as confirmed by the SEC. The confident identification of these small peptides using Orbitrap-based platforms is challenging due to isomeric ambiguity, mainly for dipeptides (e.g., X–Y vs Y–X). In addition, desalting steps required prior to analysis may lead to significant losses of low-molecular-weight peptides. Moreover, these systems typically generate very large and complex datasets, and the assignment of bioactivity to individual peptides remains highly uncertain without further targeted validation. For these reasons, we consider that LC-MS/MS profiling would not substantially strengthen the interpretation of the results in this study.

Comments 8: L846 - The Conclusions should include a paragraph outlining directions for further research.

Response 8: Dear Reviewer, thank you for your valuable suggestion. A statement outlining future research directions has been incorporated into the Conclusions section. Specifically, we highlighted the need for further in vivo studies to confirm the antihypertensive and antioxidant effects of the hydrolysates, as well as additional investigations focused on the identification and characterization of the specific bioactive peptides responsible for the observed biological activities.

 

Comments 9: The chromatogram figures (Figures 1 and 2) should be provided at higher resolution.

Response 9: Dear Reviewer, both figures have been provided at higher resolution.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents a scientifically relevant study on the valorization of tilapia scale by-products through enzymatic hydrolysis to produce multifunctional bioactive peptides. The inclusion of SGID, antioxidant assessment, and multiple enzyme-inhibitory assays provides broad functional characterization and reflects substantial experimental effort. However, despite its potential, the manuscript still contains several conceptual and methodological limitations.

 

Line 97 Why was STC-1 selected instead of more commonly used intestinal models such as Caco-2 cells?

Line 119 How does the present work substantially advance beyond previous studies reporting antioxidant and ACE-inhibitory peptides from tilapia scales?

Line 161 How many tilapia specimens were used in this study, and were the scales pooled before analysis?

Line 207 How were the activities of alkaline protease and esperase standardized? Why was pH 9 selected for all enzymes, and was this condition optimal for each enzyme individually?

Line 285 Why were antioxidant activities evaluated at a single concentration instead of using concentration-dependent curves or IC50 values?

Line 409 Could the authors better explain how the observed amino acid composition contributes specifically to ACE-, DPP-IV-, PEP-inhibitory, and antioxidant activities?

Line 470 Might peptide aggregation or conformational changes also contribute to this observation?

Line 549 The Amide I discussion is repeated almost identically twice. Revise the FTIR discussion to avoid repetition of the Amide I band interpretation.

Line 566 How do the bioactivities and peptide profiles obtained in this study compare with commercially available fish-derived bioactive hydrolysates?

Line 591 Discuss how reliance solely on CCK-8 assays may limit interpretation of cytotoxicity and peptide safety.

Line 638 Could peptide fractionation strengthen the proposed relationship between low-MW peptides and antioxidant effects?

Line 696 Provide a more systematic comparison with previously reported marine-derived ACE inhibitory hydrolysates to support this claim.

Author Response

Comments 1: Line 97 Why was STC-1 selected instead of more commonly used intestinal models such as Caco-2 cells?

Response 1: Dear Reviewer, thank you for your valuable comment. Caco‑2 cells are widely used to model the intestinal epithelial barrier. In contrast, STC‑1 cells are an enteroendocrine model that can respond to digested nutrients and peptides through the secretion of gastrointestinal hormones such as CCK and GLP‑1.  As our objective was to assess the biological effects of gastrointestinal digests rather than epithelial transport, we considered STC‑1 cells to be a more appropriate and functionally relevant model. The effect of digests on these cells could therefore be physiologically relevant. In addition, their use in this context is still relatively limited, which allowed us to provide complementary insight beyond the extensively studied Caco‑2 system.

Comments 2: Line 119 How does the present work substantially advance beyond previous studies reporting antioxidant and ACE-inhibitory peptides from tilapia scales?

Response 2: Dear Reviewer, thank you for your valuable observation. The novelty and advancement of the present study compared with previous reports on tilapia scale-derived peptides lie in several aspects. Unlike earlier studies that primarily focused on isolated antioxidant or ACE-inhibitory activities, the present work provides a comprehensive evaluation of the influence of enzyme selection on peptide generation, multifunctional bioactivity, and digestive stability after simulated gastrointestinal digestion (SGID). In addition to ACE-inhibitory activity, the study also evaluated DPP-IV and PEP inhibitory activities, antioxidant capacity both in vitro and in the enteroendocrine STC-1 cell model, as well as cytotoxicity. Furthermore, the inclusion of SGID allowed the assessment of peptide stability and the evolution of bioactivity under physiological digestive conditions, which is a critical aspect often overlooked in previous studies. The use of the enteroendocrine STC-1 model also provides additional insight into the potential intestinal biological effects of the hydrolysates beyond conventional chemical antioxidant assays.

Comments 3: Line 161 How many tilapia specimens were used in this study, and were the scales pooled before analysis?

Response 3: Dear Reviewer, thank you for your observation. A total of 10 adult tilapia specimens were used in this study, and the collected scales were pooled prior to hydrolysis and subsequent analyses in order to obtain a representative and homogeneous raw material. This clarification has been incorporated into the revised manuscript.

Comments 4: Line 207 How were the activities of alkaline protease and esperase standardized? Why was pH 9 selected for all enzymes, and was this condition optimal for each enzyme individually?

Response 4: Dear Reviewer, thank you for your valuable observation. The enzymatic activities of Alkaline Protease and Esperase were determined using casein as substrate, following the methodology described by Lajmi et al. It was mentioned in Section 2.6, but we have included an additional section to describe the methodology used and clarify this point. The hydrolysis reactions were conducted at pH 9 because both Alkaline Protease and Esperase exhibit optimal catalytic activity around this pH (Esperase 8.0 L from Novozymes at pH 9-10.5, Alkaline protease from BioCat at 9-10), according to the manufacturer specifications. The use of a common pH condition also allowed a more standardized comparison of peptide generation and bioactivity between enzymes. This clarification has been incorporated into the revised manuscript.

Comments 5: Line 285 Why were antioxidant activities evaluated at a single concentration instead of using concentration-dependent curves or IC50 values?

Response 5: Dear Reviewer, thank you for your valuable observation. Antioxidant activities were initially evaluated at a single concentration as a screening approach to compare the relative antioxidant potential of the different hydrolysates and their digests under standardized experimental conditions. This strategy allowed the identification of the most promising samples while reducing variability associated with multiple concentration assays. Furthermore, the primary objective of this section was to evaluate the presence or absence of antioxidant activity and its modulation after simulated gastrointestinal digestion rather than to establish detailed dose–response relationships. Nevertheless, we acknowledge that concentration-dependent analyses and IC₅₀ determination would provide additional quantitative information regarding antioxidant potency and should be considered in future studies.

Comments 6: Line 409 Could the authors better explain how the observed amino acid composition contributes specifically to ACE-, DPP-IV-, PEP-inhibitory, and antioxidant activities?

Response 6: Dear Reviewer, thank you for your valuable observation. Additional clarification has been incorporated into the manuscript regarding the relationship between amino acid composition and the observed biological activities. Specifically, the high content of hydrophobic amino acids such as glycine, proline, alanine, valine, leucine, and phenylalanine may contribute to ACE-, DPP-IV-, and PEP-inhibitory activities by promoting interactions between peptides and the hydrophobic regions of enzyme active sites. In particular, proline-containing peptides are frequently associated with ACE and DPP-IV inhibition due to the affinity of these enzymes for substrates containing proline residues. Similarly, PEP inhibitory activity has also been linked to peptides rich in proline because PEP preferentially hydrolyses peptide bonds adjacent to proline residues.

Regarding antioxidant activity, amino acids such as tyrosine, cysteine, methionine, histidine, and hydrophobic residues may contribute to radical scavenging, electron donation, and metal-chelating properties. Additionally, the abundance of glycine and proline, characteristic of collagen-derived peptides, may favor peptide interactions with biological membranes and improve peptide stability and bioavailability, thereby enhancing their biological functionality. These aspects have been clarified in the revised manuscript.

Comments 7: Line 470 Might peptide aggregation or conformational changes also contribute to this observation?

Response 7: Thanks for the observation. We agree that partial de-aggregation cannot be excluded. However, we consider a purely conformational explanation unlikely. In SEC, conformational effects typically result in shifts of existing peaks rather than the appearance of a new, well-defined signal. Importantly, the peak at 8.5 kDa size is consistent with a discrete proteolytic fragment rather than with the simple dissociation of pre-existing aggregates of the same size. Therefore, we attribute this peak mainly to soluble fragments generated during gastric digestion from larger or aggregated precursor material that was not initially detectable. De-aggregation may contribute, but it is unlikely to be the primary mechanism. We have included a brief discussion about this point in section 3.3.

Comments 8: Line 549 The Amide I discussion is repeated almost identically twice. Revise the FTIR discussion to avoid repetition of the Amide I band interpretation.

Response 8: Thanks for the observation, it is a mistake, one paragraph has been deleted, and the other corrected.

Comments 9: Line 566 How do the bioactivities and peptide profiles obtained in this study compare with commercially available fish-derived bioactive hydrolysates?

Response 9: Thank you for this insightful comment. The discussion section has been expanded to include a comparison with commercially available fish-derived bioactive hydrolysates. The peptide distribution observed in our hydrolysates, particularly the predominance of low molecular weight fractions, is comparable to that reported for commercial marine hydrolysates commonly associated with enhanced bioavailability and antioxidant-related bioactivities. Additionally, the FTIR profiles obtained in this study are consistent with characteristic peptide and carbohydrate-associated functional groups previously described in fish-derived commercial hydrolysates.

Comments 10: Line 591 Discuss how reliance solely on CCK-8 assays may limit interpretation of cytotoxicity and peptide safety.

Response 10: Thank you for this important observation. We have expanded the Discussion to clarify the limitations of the CCK-8 assay, emphasizing that it reflects metabolic activity rather than direct cell viability and may not fully capture subtle cytotoxic or proliferative effects. We also now highlight the relevance of complementary assays (e.g., LDH release, apoptosis markers, barrier integrity) to strengthen safety assessment.

Comments 11: Line 638 Could peptide fractionation strengthen the proposed relationship between low-MW peptides and antioxidant effects?

Response 11: Thank you for this valuable comment. We agree that peptide fractionation would provide stronger evidence regarding the relationship between peptide molecular weight and antioxidant activity. Fractionation approaches, such as ultrafiltration or chromatographic separation, followed by antioxidant evaluation of each fraction, would allow a more precise identification of the peptide populations contributing to the observed activity. This consideration has been incorporated into the revised manuscript as a limitation and future research direction.

Comments 12: Line 696 Provide a more systematic comparison with previously reported marine-derived ACE inhibitory hydrolysates to support this claim.

Response 12: Thank you for this valuable comment. Additional comparisons with previously reported marine-derived ACE inhibitory hydrolysates and peptides have been incorporated into the revised Discussion section. Line 739.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall assessment

This manuscript addresses the valorization of tilapia scales as a source of bioactive protein hydrolysates and evaluates the effects of simulated gastrointestinal digestion (SGID) on enzymatic inhibitory activities and cellular antioxidant responses. The topic is appropriate for Foods and is relevant to by-product valorization, circular economy, functional foods, and nutraceutical ingredient development. The study has several strengths, including the use of two commercial proteases, characterization of molecular-weight profiles before and after SGID, evaluation of multiple in vitro bioactivities, and the inclusion of an STC-1 enteroendocrine cell model.

However, in its current form, the manuscript does not provide sufficient evidence to support its strongest conclusions. The reported ACE-inhibitory IC50 values for crude hydrolysates and digests are remarkably potent, approaching the range of pharmaceutical ACE inhibitors. Such results require rigorous assay validation, appropriate positive controls measured under identical conditions, digestion blanks, and careful normalization. In addition, the manuscript repeatedly interprets bioactivity in terms of peptide sequence motifs, but no peptide sequences are identified. Without LC-MS/MS-based peptide identification of the most active fractions or digests, the study remains a preliminary screening of crude hydrolysates rather than a paper that reveals bioactive peptide mechanisms or clear structure-activity relationships.

I therefore recommend major revision with additional experiments required. At minimum, the authors should include positive controls for the enzymatic inhibition assays, particularly ACE inhibition, and perform LC-MS/MS peptide sequence identification of the most active digest or fraction. If these experiments cannot be added, the manuscript should be substantially reframed as a preliminary screening study and all claims regarding potent antihypertensive, hypoglycemic, nootropic, or nutraceutical effects should be considerably weakened.

Major comments

1. Positive controls are essential for the ACE assay and other enzyme-inhibition assays.

Location: Abstract, lines 22-31; Methods, Section 2.9.3, lines 328-345; Results, Tables 2 and 3, lines 599-684; Discussion, lines 686-729. The reported ACE IC50 values are unusually low for crude protein hydrolysates: approximately 13-14 micrograms/mL before digestion and as low as 4.6 micrograms/mL after intestinal digestion. These values are far more potent than many crude fish-derived hydrolysates reported in the literature and approach the potency range of pharmaceutical inhibitors. The authors compare their results with enalapril values from other studies, but cross-study comparisons are unreliable because ACE source, substrate, enzyme lot, assay format, instrument settings, incubation time, buffer composition, and IC50 calculation procedures can differ substantially. The authors should run a positive control such as captopril and/or enalapril under exactly the same assay conditions and report its IC50. They should also provide dose-response curves, concentration ranges, fitting model, goodness of fit, and replicate-level variability. Without these data, the exceptionally strong ACE-inhibitory potency cannot be properly evaluated.

2. LC-MS/MS peptide identification is required to support the sequence-dependent interpretations.

Location: Abstract, lines 18-31; Results and Discussion, molecular-weight profile section, lines 462-511; ACE discussion, lines 730-744; DPP-IV discussion, lines 747-790; PEP discussion, lines 791-835. The manuscript repeatedly attributes bioactivity to peptide features such as low molecular weight, proline-rich sequences, C-terminal proline, hydrophobic or aromatic residues, and collagen-derived dipeptides or tripeptides. These are plausible hypotheses, but the manuscript provides no LC-MS/MS peptide sequence data. Molecular-weight profiles and amino acid composition are insufficient to identify the active peptides or to justify structure-activity conclusions. In 2026, LC-MS/MS-based peptidomics is a standard and expected method for studies claiming bioactive peptide generation and sequence-related functionality. The authors should identify peptides, at least in the most active intestinal digest, preferably the E-hydrolysate intestinal digest showing the strongest ACE inhibition. The peptide list should include sequences, mass-to-charge values, confidence scores, peptide abundance or relative intensity, and comparison with known ACE-, DPP-IV-, or PEP-inhibitory peptides where appropriate. If LC-MS/MS cannot be performed, the authors should remove or greatly soften all sequence-specific claims and clearly state that the active peptides remain unidentified.

3. SGID normalization, digestion blanks, and exogenous enzyme contributions must be clarified.

Location: Methods, Section 2.8, lines 232-245; Tables 2 and 3; Results and Discussion, lines 707-729. The central conclusion is that SGID enhances ACE-inhibitory activity. However, SGID introduces exogenous digestive enzymes, especially pepsin and pancreatin, which contribute additional proteins and peptides to the digest. If IC50 values are expressed per total digest dry mass or total protein without correcting for exogenous enzymes, the apparent enhancement could be partly influenced by pepsin-/pancreatin-derived peptides, altered sample mass, or changes in protein basis. The authors should clarify whether IC50 values after SGID are expressed per original hydrolysate mass, total digest dry mass, soluble peptide content, or total protein. They should include digestion blanks containing pepsin and pancreatin without hydrolysate and test these blanks in ACE, DPP-IV, and PEP assays. The protein or peptide content of the digests should be reported before and after SGID. Without appropriate blanks and normalization, the conclusion that digestion specifically enhances tilapia-scale-derived ACE-inhibitory potency is not fully supported.

4. Functional claims are overstated and should be substantially moderated.

Location: Title, lines 1-4; Abstract, lines 22-32; Introduction, lines 99-112; Conclusions, lines 836-846. The use of terms such as “antihypertensive potency,” “hypoglycemic activity,” and “nootropic activity” is premature. The manuscript measures in vitro ACE, DPP-IV, and PEP inhibition, plus an STC-1 ROS assay. These assays do not demonstrate blood-pressure reduction, glucose-lowering effects, neuropeptide preservation, cognitive improvement, or in vivo nutraceutical efficacy. In particular, PEP inhibition should not be described as nootropic activity unless supported by appropriate cellular, ex vivo, animal, or human evidence. The authors should replace these terms with “ACE-inhibitory activity,” “DPP-IV-inhibitory activity,” and “PEP-inhibitory activity.” The title should also be reconsidered; “antihypertensive potency” should be replaced with a more precise phrase such as “ACE-inhibitory activity.” All claims regarding nutraceutical applications should be framed as potential applications requiring in vivo validation.

5. The INFOGEST SGID protocol is insufficiently described.

Location: Methods, Section 2.8, lines 232-245. The manuscript states that the standardized INFOGEST protocol was used “with minor modifications,” but the modifications are not clearly specified. The INFOGEST protocol requires detailed information on simulated salivary, gastric, and intestinal fluids, electrolyte composition, CaCl2, bile salts, enzyme activities, enzyme activity units, final concentrations, pH adjustments, and sample-to-fluid ratios. The authors report pepsin and pancreatin additions but do not sufficiently clarify whether bile salts were included, how pancreatin activity was defined, whether enzyme activities correspond to INFOGEST final concentrations, or what exact changes were made. Since SGID is central to the manuscript, the digestion protocol must be reproducible. The authors should provide the complete composition of each digestive phase and explicitly justify all modifications.

6. Statistical analysis should be revised and Table 3 should be checked.

Location: Statistical analysis, lines 378-384; Tables 2 and 3; Results and Discussion, lines 679-790. The manuscript uses one-way ANOVA, LSD, and independent Student’s t-tests. However, the experimental design includes at least two major factors: enzyme type and digestion phase. A two-way ANOVA or equivalent model would be more appropriate for ACE, DPP-IV, and PEP activities to test enzyme effects, digestion-phase effects, and their interaction. The current superscript letters in Table 3 appear to compare AP and E within each phase, but they do not clearly support statements about changes across digestion phases. In addition, the gastric-phase ACE IC50 values in Table 3 are 6.0 +/- 2.0 micrograms/mL for AP and 5.5 +/- 1.3 micrograms/mL for E, yet they are marked with different letters. Given the large standard deviations, the statistical basis for this difference should be checked and clearly explained. The relative SD is also high for some values, raising questions about assay reproducibility. The authors should revise the statistical analysis, clarify the comparisons represented by superscript letters, and provide exact p values where possible.

7. Antioxidant assays before and after SGID are not directly comparable.

Location: Table 2, lines 599-602; antioxidant discussion, lines 604-675; Figure 4, lines 650-654. The undigested hydrolysates were evaluated using ABTS, FRAP, and Fe(II)-chelating assays, whereas the digested samples were evaluated using an STC-1 ROS assay. These assays measure different phenomena: chemical radical scavenging/reducing/chelating activity versus cellular modulation of oxidative stress. Therefore, the manuscript cannot directly conclude how SGID changed antioxidant activity unless the same antioxidant assays are applied before and after digestion. The authors should either perform ABTS, FRAP, and Fe(II)-chelating assays on the digests as well, or clearly state that chemical antioxidant capacity before digestion and cellular antioxidant response after digestion are separate endpoints that should not be interpreted as a direct before-after comparison.

8. The STC-1 cell experiments require clearer concentration reporting and more cautious interpretation.

Location: Methods, ROS assay, lines 287-309; cell viability assay, lines 310-327; Figure 3, lines 575-579; Figure 4, lines 650-654. The final concentration used in the STC-1 ROS assay is unclear. The method states that 10 microliters of a 10 mg/mL sample solution was added to 100 microliters of medium, which would give a final concentration of approximately 0.9 mg/mL, not 10 mg/mL. The authors should clarify whether reported concentrations refer to stock solutions or final concentrations in the wells. Similarly, the cell viability assay should specify final well concentrations. The ROS data should preferably be expressed as percentage of the H2O2-stimulated control or percentage ROS inhibition, not only as raw fluorescence units. Only the digested E-hydrolysate appears to significantly reduce ROS, so the conclusion should specify which sample was effective and under what conditions. Mechanistic speculation regarding Nrf2/ARE activation should be clearly presented as hypothetical unless gene or protein expression data are provided.

9. The manuscript structure, section numbering, and proofreading require major correction.

Location: Results and Discussion, lines 385 onward; Sections currently labeled 2.2, 2.3, 2.4, and 2.5; later sections labeled 3.5.4 and 3.5.5; Methods line 340; Discussion line 692. The Results and Discussion section begins as Section 3, but subsequent subsections are incorrectly numbered as 2.2, 2.3, 2.4, and 2.5. Later subsections appear as 3.5.4 and 3.5.5 without a consistent preceding structure. This must be corrected throughout. There are also obvious proofreading errors such as “Fl100 nce was monitored” and “matriµx.” Such errors suggest that the manuscript has not been carefully checked before submission. The authors should thoroughly proofread the entire manuscript and verify all figure/table cross-references.

10. The FTIR section contains duplicated text and overinterprets limited data.

Location: Section 2.4/FTIR, lines 513-566, especially lines 531-550. The discussion of the Amide I band is essentially repeated in two consecutive paragraphs. This appears to be a revision artifact and should be removed. More importantly, FTIR confirms broad protein/peptide functional groups but does not provide strong evidence for specific peptide composition, free amino-group exposure, or bioactivity mechanisms. The authors should shorten this section and limit interpretation to what FTIR can support. If the goal is to explain bioactivity, peptide sequencing and/or more targeted compositional analysis would be much more informative.

11. The manuscript should clearly distinguish screening results from mechanistic conclusions.

Location: Throughout Results and Discussion, especially lines 621-643, 655-675, 730-744, and 791-835. Several mechanistic statements are plausible but not directly demonstrated. For example, the authors infer that low-molecular-weight peptides explain antioxidant activity, that Nrf2/ARE signaling may contribute to ROS reduction, that collagen-derived C-terminal proline peptides explain ACE inhibition, and that specific sequence features drive PEP inhibition. These interpretations should be framed as hypotheses unless supported by peptide identification, purified peptide testing, enzyme kinetics, molecular docking, or cellular signaling assays. The current data support the presence of bioactivity in crude hydrolysates/digests, but they do not yet identify the responsible peptides or mechanisms.

12. The conclusions should be rewritten to match the evidence.

Location: Conclusions, lines 836-846. The conclusion emphasizes “significant ACE-inhibitory potential” and nutraceutical development. This may be acceptable only if positive controls, digestion blanks, normalization, and peptide identification are added. Otherwise, the conclusion should be much more conservative, describing the study as a preliminary in vitro screening of tilapia scale hydrolysates. The final sentence should emphasize that in vivo efficacy, bioavailability, peptide identification, and safety validation remain necessary before any nutraceutical application can be proposed.

Minor comments and technical corrections

• The corresponding author designation should be checked. The asterisk appears after Oscar Martinez Alvarez, whereas the correspondence information lists Mauricio Mosquera with email and phone number on the title page.
• The abstract should avoid claiming “nootropic activity” and should report PEP inhibition only as an in vitro enzyme-inhibition endpoint.
• The term “hypoglycemic activity” should be replaced with “DPP-IV-inhibitory activity” unless glucose-lowering effects are demonstrated in a biological model.
• The abbreviation DPP-IV should be used consistently; avoid switching between DPPIV and DPP-IV.
• PEP, PO, prolyl endopeptidase, and prolyl oligopeptidase should be defined and used consistently.
• The phrase “simulated gastric intestinal digestion” should be corrected to “simulated gastrointestinal digestion.”
• Table 2 reports Fe(II)-chelating activity as “%Act/microgram,” which is not intuitive. The calculation and unit should be clearly explained.
• Figure 4 would be more interpretable if fluorescence were normalized to the H2O2-stimulated control and presented as percentage ROS level or percentage ROS inhibition.
• The authors should report protein or peptide content of hydrolysates and digests, especially because IC50 values are expressed on a mass basis.
• Dose-response curves for ACE, DPP-IV, and PEP inhibition should be included in supplementary material.
• Line 722 appears to contain “1.15 m/mL”; please check whether this should be mg/mL.
• The funding, data availability, and acknowledgments sections should be checked for journal formatting compliance.
• The manuscript states that Paperpal was used for English checking, but the text still contains numerous grammatical and formatting issues. Professional proofreading is recommended.
• The title is currently too strong because it implies demonstrated antihypertensive potency. Consider revising it to focus on ACE-inhibitory activity and in vitro antioxidant effects.
• The data availability link should be checked and should include all raw or processed data needed to verify IC50 calculations, chromatograms, and cell-assay results.

Suggested decision

Major revision, with additional experiments required. The topic is suitable for Foods and the study has potential, but the present evidence is insufficient to support the strongest claims. Positive controls, digestion blanks, normalization of SGID samples, and LC-MS/MS peptide identification are essential for a scientifically convincing revision.

Author Response

Comments 1: Positive controls are essential for the ACE assay and other enzyme-inhibition assays.

Location: Abstract, lines 22-31; Methods, Section 2.9.3, lines 328-345; Results, Tables 2 and 3, lines 599-684; Discussion, lines 686-729. The reported ACE IC50 values are unusually low for crude protein hydrolysates: approximately 13-14 micrograms/mL before digestion and as low as 4.6 micrograms/mL after intestinal digestion. These values are far more potent than many crude fish-derived hydrolysates reported in the literature and approach the potency range of pharmaceutical inhibitors. The authors compare their results with enalapril values from other studies, but cross-study comparisons are unreliable because ACE source, substrate, enzyme lot, assay format, instrument settings, incubation time, buffer composition, and IC50 calculation procedures can differ substantially. The authors should run a positive control such as captopril and/or enalapril under exactly the same assay conditions and report its IC50. They should also provide dose-response curves, concentration ranges, fitting model, goodness of fit, and replicate-level variability. Without these data, the exceptionally strong ACE-inhibitory potency cannot be properly evaluated.

Response 1: We thank the reviewer for this important comment. We agree that direct comparison of ACE inhibitory potency across studies should be interpreted with caution because assay conditions, ACE source, substrate, incubation conditions, and IC₅₀ calculation procedures may differ substantially.

A pharmaceutical positive control such as captopril or enalapril was not included in the present study. Therefore, we acknowledge that direct comparisons between the IC₅₀ values obtained in this work and those reported for pharmaceutical inhibitors in the literature are limited. In response to the reviewer's comment, we have revised the manuscript to soften these comparisons and avoid overinterpretation of the relative potency of the hydrolysates.

The discussion has been modified to emphasize comparisons with other fish derived hydrolysates rather than with pharmaceutical inhibitors. We have also clarified this limitation in the revised manuscript and acknowledge that future studies should include positive controls evaluated under identical assay conditions, together with detailed dose response analyses, to allow a more robust assessment of inhibitory potency.

Comments 2: 2. LC-MS/MS peptide identification is required to support the sequence-dependent interpretations.

Location: Abstract, lines 18-31; Results and Discussion, molecular-weight profile section, lines 462-511; ACE discussion, lines 730-744; DPP-IV discussion, lines 747-790; PEP discussion, lines 791-835. The manuscript repeatedly attributes bioactivity to peptide features such as low molecular weight, proline-rich sequences, C-terminal proline, hydrophobic or aromatic residues, and collagen-derived dipeptides or tripeptides. These are plausible hypotheses, but the manuscript provides no LC-MS/MS peptide sequence data. Molecular-weight profiles and amino acid composition are insufficient to identify the active peptides or to justify structure-activity conclusions. In 2026, LC-MS/MS-based peptidomics is a standard and expected method for studies claiming bioactive peptide generation and sequence-related functionality. The authors should identify peptides, at least in the most active intestinal digest, preferably the E-hydrolysate intestinal digest showing the strongest ACE inhibition. The peptide list should include sequences, mass-to-charge values, confidence scores, peptide abundance or relative intensity, and comparison with known ACE-, DPP-IV-, or PEP-inhibitory peptides where appropriate. If LC-MS/MS cannot be performed, the authors should remove or greatly soften all sequence-specific claims and clearly state that the active peptides remain unidentified.

Response 2: We thank the reviewer for this valuable comment. We agree that LC-MS/MS peptide identification would be required to confirm the peptide sequences responsible for the observed bioactivities and to establish definitive sequence activity relationships. Since peptide sequencing was not performed in the present study, we have revised the manuscript accordingly.

All sequence specific interpretations have been softened throughout the Abstract and the Results and Discussion sections. Statements referring to specific peptide motifs, C terminal proline residues, collagen derived dipeptides and tripeptides, and other sequence related structure activity relationships have been reformulated as hypotheses based on previous literature rather than direct evidence from the present study. Furthermore, we have explicitly stated that the active peptides remain unidentified and that LC-MS/MS based peptidomic analysis will be required in future studies to identify the bioactive peptides and confirm the mechanisms underlying the observed inhibitory activities.

A statement acknowledging this limitation has also been incorporated into the manuscript.

Comments 3: SGID normalization, digestion blanks, and exogenous enzyme contributions must be clarified.

Location: Methods, Section 2.8, lines 232-245; Tables 2 and 3; Results and Discussion, lines 707-729. The central conclusion is that SGID enhances ACE-inhibitory activity. However, SGID introduces exogenous digestive enzymes, especially pepsin and pancreatin, which contribute additional proteins and peptides to the digest. If IC50 values are expressed per total digest dry mass or total protein without correcting for exogenous enzymes, the apparent enhancement could be partly influenced by pepsin-/pancreatin-derived peptides, altered sample mass, or changes in protein basis. The authors should clarify whether IC50 values after SGID are expressed per original hydrolysate mass, total digest dry mass, soluble peptide content, or total protein. They should include digestion blanks containing pepsin and pancreatin without hydrolysate and test these blanks in ACE, DPP-IV, and PEP assays. The protein or peptide content of the digests should be reported before and after SGID. Without appropriate blanks and normalization, the conclusion that digestion specifically enhances tilapia-scale-derived ACE-inhibitory potency is not fully supported.

Response 3: We thank the reviewer for this important observation. The IC₅₀ values reported after SGID were calculated based on the concentration of the freeze-dried digests used in the bioactivity assays and were not normalized to the original hydrolysate mass, peptide content, or protein content. In addition, digestion blanks containing pepsin and pancreatin without hydrolysate were not evaluated in the ACE, DPP IV, or PEP inhibitory assays.

We agree that exogenous digestive enzymes and their autolysis products may contribute to the composition of the final digests and therefore could potentially influence the measured bioactivities. Consequently, the present experimental design does not allow us to completely exclude the contribution of enzyme derived peptides to the observed inhibitory activities.

To address this concern, we have revised the manuscript and softened the interpretation of the SGID results. The discussion now indicates that the increased ACE inhibitory potency observed after SGID may be associated with peptide generation during digestion, but that the contribution of peptides originating from digestive enzymes cannot be ruled out. We have also included this aspect as a limitation of the study and highlighted the need for future experiments including digestion blanks and peptide normalization procedures.

Comments 4: Functional claims are overstated and should be substantially moderated.

Location: Title, lines 1-4; Abstract, lines 22-32; Introduction, lines 99-112; Conclusions, lines 836-846. The use of terms such as “antihypertensive potency,” “hypoglycemic activity,” and “nootropic activity” is premature. The manuscript measures in vitro ACE, DPP-IV, and PEP inhibition, plus an STC-1 ROS assay. These assays do not demonstrate blood-pressure reduction, glucose-lowering effects, neuropeptide preservation, cognitive improvement, or in vivo nutraceutical efficacy. In particular, PEP inhibition should not be described as nootropic activity unless supported by appropriate cellular, ex vivo, animal, or human evidence. The authors should replace these terms with “ACE-inhibitory activity,” “DPP-IV-inhibitory activity,” and “PEP-inhibitory activity.” The title should also be reconsidered; “antihypertensive potency” should be replaced with a more precise phrase such as “ACE-inhibitory activity.” All claims regarding nutraceutical applications should be framed as potential applications requiring in vivo validation.

Response 4: We thank the reviewer for this important and constructive comment. We agree that the biological assays performed in the present study provide evidence only of in vitro ACE inhibitory, DPP-IV inhibitory, and PEP inhibitory activities, together with antioxidant effects in STC-1 cells. Therefore, these results do not directly demonstrate antihypertensive, hypoglycemic, nootropic, or other physiological effects in vivo.

In response to this comment, we have carefully revised the manuscript to avoid overstatement of the biological significance of the findings. Terms such as "antihypertensive potency", "hypoglycemic activity", and "nootropic activity" have been replaced by the more precise expressions "ACE inhibitory activity", "DPP-IV inhibitory activity", and "PEP inhibitory activity", respectively. In addition, statements implying direct physiological or therapeutic effects have been reformulated to emphasize that the observed activities were measured in vitro and should be considered indicative of potential biological relevance only.

Furthermore, all references to nutraceutical applications have been revised to clearly indicate that such applications remain potential and require confirmation through additional studies, including peptide identification, bioavailability assessment, and in vivo validation.

The title, abstract, introduction, discussion, and conclusions have been modified accordingly.

Comments 5:  The INFOGEST SGID protocol is insufficiently described.

Location: Methods, Section 2.8, lines 232-245. The manuscript states that the standardized INFOGEST protocol was used “with minor modifications,” but the modifications are not clearly specified. The INFOGEST protocol requires detailed information on simulated salivary, gastric, and intestinal fluids, electrolyte composition, CaCl2, bile salts, enzyme activities, enzyme activity units, final concentrations, pH adjustments, and sample-to-fluid ratios. The authors report pepsin and pancreatin additions but do not sufficiently clarify whether bile salts were included, how pancreatin activity was defined, whether enzyme activities correspond to INFOGEST final concentrations, or what exact changes were made. Since SGID is central to the manuscript, the digestion protocol must be reproducible. The authors should provide the complete composition of each digestive phase and explicitly justify all modifications.

Response 5: We thank the reviewer for this valuable comment. We agree that a detailed description of the SGID procedure is necessary to ensure reproducibility. In the revised manuscript, Section 2.9 has been expanded to clarify the digestion procedure, including the sample to fluid ratios, enzyme activities, incubation conditions, pH adjustments, and the specific modifications introduced relative to the original protocol. We have also clarified that the digestion procedure was performed according to a previously reported adaptation of the INFOGEST harmonized protocol. Furthermore, a digestion blank containing all digestive fluids and enzymes but no protein substrate was prepared and processed under identical conditions, and this information has now been included in the Methods section.

Comments 6: Statistical analysis should be revised and Table 3 should be checked.

Location: Statistical analysis, lines 378-384; Tables 2 and 3; Results and Discussion, lines 679-790. The manuscript uses one-way ANOVA, LSD, and independent Student’s t-tests. However, the experimental design includes at least two major factors: enzyme type and digestion phase. A two-way ANOVA or equivalent model would be more appropriate for ACE, DPP-IV, and PEP activities to test enzyme effects, digestion-phase effects, and their interaction. The current superscript letters in Table 3 appear to compare AP and E within each phase, but they do not clearly support statements about changes across digestion phases. In addition, the gastric-phase ACE IC50 values in Table 3 are 6.0 +/- 2.0 micrograms/mL for AP and 5.5 +/- 1.3 micrograms/mL for E, yet they are marked with different letters. Given the large standard deviations, the statistical basis for this difference should be checked and clearly explained. The relative SD is also high for some values, raising questions about assay reproducibility. The authors should revise the statistical analysis, clarify the comparisons represented by superscript letters, and provide exact p values where possible.

Response 6: We thank the reviewer for this valuable comment. We agree that a two-way ANOVA could provide additional information regarding the effects of enzyme type, digestion phase, and their potential interaction. However, the statistical approach adopted in the present study was designed to evaluate specific comparisons between treatments within each experimental condition. For this purpose, one-way ANOVA followed by the LSD test was considered appropriate and remains a valid statistical approach for the comparisons performed.

We acknowledge that a more comprehensive factorial analysis could provide further insight into the interaction between enzyme type and digestion phase. Such analyses will be considered in future studies specifically designed to investigate these factors in greater detail.

Following the reviewer's suggestion, we carefully re-examined the statistical analysis and the superscript letters presented in Table 3. The table legend and the manuscript have been revised to clarify the comparisons represented by the superscript letters. In addition, the statistical outputs were rechecked to ensure consistency between the reported significance groupings and the experimental data.

Comments 7: Antioxidant assays before and after SGID are not directly comparable.

Location: Table 2, lines 599-602; antioxidant discussion, lines 604-675; Figure 4, lines 650-654. The undigested hydrolysates were evaluated using ABTS, FRAP, and Fe(II)-chelating assays, whereas the digested samples were evaluated using an STC-1 ROS assay. These assays measure different phenomena: chemical radical scavenging/reducing/chelating activity versus cellular modulation of oxidative stress. Therefore, the manuscript cannot directly conclude how SGID changed antioxidant activity unless the same antioxidant assays are applied before and after digestion. The authors should either perform ABTS, FRAP, and Fe(II)-chelating assays on the digests as well, or clearly state that chemical antioxidant capacity before digestion and cellular antioxidant response after digestion are separate endpoints that should not be interpreted as a direct before-after comparison

Response 7: We thank the reviewer for this important observation. We agree that the antioxidant assays performed before and after SGID evaluate different biological endpoints and therefore do not allow a direct assessment of how SGID affected antioxidant activity.

The antioxidant capacity of the undigested hydrolysates was evaluated using chemical assays (ABTS, FRAP, and Fe(II)-chelating activity), whereas the digested samples were assessed using a cellular ROS assay in STC-1 cells. Consequently, these results should not be interpreted as a direct before and after comparison of antioxidant activity.

In the revised manuscript, we have clarified this distinction throughout the Results and Discussion sections and removed any statements that could imply that SGID increased or decreased antioxidant activity. The discussion now emphasizes that the chemical antioxidant capacity of the hydrolysates and the cellular antioxidant response of the digests represent complementary but distinct endpoints.

Comments 8: The STC-1 cell experiments require clearer concentration reporting and more cautious interpretation.

Location: Methods, ROS assay, lines 287-309; cell viability assay, lines 310-327; Figure 3, lines 575-579; Figure 4, lines 650-654. The final concentration used in the STC-1 ROS assay is unclear. The method states that 10 microliters of a 10 mg/mL sample solution was added to 100 microliters of medium, which would give a final concentration of approximately 0.9 mg/mL, not 10 mg/mL. The authors should clarify whether reported concentrations refer to stock solutions or final concentrations in the wells. Similarly, the cell viability assay should specify final well concentrations. The ROS data should preferably be expressed as percentage of the H2O2-stimulated control or percentage ROS inhibition, not only as raw fluorescence units. Only the digested E-hydrolysate appears to significantly reduce ROS, so the conclusion should specify which sample was effective and under what conditions. Mechanistic speculation regarding Nrf2/ARE activation should be clearly presented as hypothetical unless gene or protein expression data are provided.

Response 8: We thank the reviewer for this valuable observation. We agree that the manuscript did not clearly distinguish between stock concentrations and final concentrations in the cell culture wells.

The concentrations reported in the original manuscript corresponded to the stock solutions added to the wells. Following the reviewer's suggestion, the Methods section has been revised to clarify the final concentrations to which STC-1 cells were exposed. In the ROS assay, addition of 10 µL of a 10 mg/mL stock solution to 100 µL of medium resulted in a final concentration of approximately 0.91 mg/mL. Similarly, the final concentrations used in the cell viability assay were recalculated and clarified in the revised manuscript.

We also agree that expression of ROS data as percentage ROS inhibition relative to the H₂O₂-stimulated control improves data interpretation. Therefore, the Results section has been revised to clearly indicate that only the digested Esperase hydrolysate significantly reduced ROS levels under the experimental conditions tested. Finally, references to potential involvement of the Nrf2/ARE pathway have been revised to emphasize that such mechanisms remain hypothetical, as no gene or protein expression analyses were performed in the present study.

 

Comments 9: The manuscript structure, section numbering, and proofreading require major correction.

Location: Results and Discussion, lines 385 onward; Sections currently labeled 2.2, 2.3, 2.4, and 2.5; later sections labeled 3.5.4 and 3.5.5; Methods line 340; Discussion line 692. The Results and Discussion section begins as Section 3, but subsequent subsections are incorrectly numbered as 2.2, 2.3, 2.4, and 2.5. Later subsections appear as 3.5.4 and 3.5.5 without a consistent preceding structure. This must be corrected throughout. There are also obvious proofreading errors such as “Fl100 nce was monitored” and “matriµx.” Such errors suggest that the manuscript has not been carefully checked before submission. The authors should thoroughly proofread the entire manuscript and verify all figure/table cross-references.

Response 9: Dear Reviewer, thank you for your valuable observation. The section numbering and manuscript structure were thoroughly revised and corrected to ensure consistency throughout the document. All subsection numbering errors were fixed accordingly.

In addition, the manuscript was carefully proofread to correct typographical and formatting errors, including those indicated by the reviewer (e.g., “Fl100 nce” and “matriµx”). Figure and table cross-references were also thoroughly checked and corrected where necessary to improve the overall clarity and consistency of the manuscript.

Comments10: The FTIR section contains duplicated text and overinterprets limited data.

Location: Section 2.4/FTIR, lines 513-566, especially lines 531-550. The discussion of the Amide I band is essentially repeated in two consecutive paragraphs. This appears to be a revision artifact and should be removed. More importantly, FTIR confirms broad protein/peptide functional groups but does not provide strong evidence for specific peptide composition, free amino-group exposure, or bioactivity mechanisms. The authors should shorten this section and limit interpretation to what FTIR can support. If the goal is to explain bioactivity, peptide sequencing and/or more targeted compositional analysis would be much more informative.

Response 10:

We thank the reviewer for this valuable observation. We agree that FTIR spectroscopy primarily provides information regarding the presence of characteristic functional groups and overall structural features of proteins and peptides, but it does not allow detailed characterization of peptide composition, peptide sequences, free amino-group exposure, or bioactivity-related mechanisms.

Following the reviewer's recommendation, the FTIR section has been substantially shortened and revised. Repetitive descriptions have been removed, and interpretations that were not directly supported by FTIR data have been eliminated. The discussion is now limited to the identification of the main protein-related absorption bands (Amide A, I, II, and III) and the general spectral differences observed between hydrolysates.

We agree that more detailed characterization of peptide composition and structure would require complementary approaches such as LC-MS/MS peptide sequencing, which has been acknowledged as a limitation of the present study.

Comments 11:  The manuscript should clearly distinguish screening results from mechanistic conclusions.

Location: Throughout Results and Discussion, especially lines 621-643, 655-675, 730-744, and 791-835. Several mechanistic statements are plausible but not directly demonstrated. For example, the authors infer that low-molecular-weight peptides explain antioxidant activity, that Nrf2/ARE signaling may contribute to ROS reduction, that collagen-derived C-terminal proline peptides explain ACE inhibition, and that specific sequence features drive PEP inhibition. These interpretations should be framed as hypotheses unless supported by peptide identification, purified peptide testing, enzyme kinetics, molecular docking, or cellular signaling assays. The current data support the presence of bioactivity in crude hydrolysates/digests, but they do not yet identify the responsible peptides or mechanisms.

Response 11:

We thank the reviewer for this important observation. We agree that the present study was designed as a screening study to evaluate the biological activities of crude hydrolysates and their gastrointestinal digests, rather than to identify the specific peptides or mechanisms responsible for the observed effects.

Following the reviewer's recommendation, the Results and Discussion section has been carefully revised to clearly distinguish experimental observations from mechanistic interpretations. Statements linking bioactivities to specific peptide characteristics, peptide sequences, molecular mechanisms, or signaling pathways have been softened and are now presented as hypotheses or possible explanations supported by previous literature rather than direct conclusions derived from the present data.

In particular, references to low-molecular-weight peptides as the cause of antioxidant activity, the involvement of Nrf2/ARE signaling in ROS reduction, the role of collagen-derived C-terminal proline-containing peptides in ACE inhibition, and sequence-related explanations for PEP inhibition have been reformulated to emphasize that these mechanisms remain speculative in the absence of peptide identification, purified peptide testing, molecular docking, enzyme kinetics, or cellular signaling analyses.

The revised manuscript now clearly states that the observed biological activities were demonstrated at the hydrolysate and digest level, whereas the specific bioactive peptides and mechanisms responsible remain to be identified in future studies.

Comments 12:  The conclusions should be rewritten to match the evidence.

Location: Conclusions, lines 836-846. The conclusion emphasizes “significant ACE-inhibitory potential” and nutraceutical development. This may be acceptable only if positive controls, digestion blanks, normalization, and peptide identification are added. Otherwise, the conclusion should be much more conservative, describing the study as a preliminary in vitro screening of tilapia scale hydrolysates. The final sentence should emphasize that in vivo efficacy, bioavailability, peptide identification, and safety validation remain necessary before any nutraceutical application can be proposed.

Response 12: We thank the reviewer for this valuable comment. We agree that the conclusions should accurately reflect the scope and limitations of the present study. Accordingly, the Conclusions section has been substantially revised and rewritten to avoid overstatement of the findings.

The revised conclusions now emphasize that the study represents an in vitro evaluation of bioactive properties in tilapia scale hydrolysates and their gastrointestinal digests. Statements implying direct nutraceutical efficacy or physiological effects have been moderated, and the conclusions are now limited to the ACE-inhibitory, DPP-IV-inhibitory, PEP-inhibitory, and antioxidant activities observed under the experimental conditions employed.

Furthermore, the revised text explicitly acknowledges that the bioactive peptides responsible for the observed activities were not identified and that additional studies are required to establish peptide identity, bioavailability, safety, and in vivo efficacy. The need for further validation before proposing any nutraceutical application has also been clearly stated.

Minor comments and technical corrections

Comments 13: The corresponding author designation should be checked. The asterisk appears after Oscar Martinez Alvarez, whereas the correspondence information lists Mauricio Mosquera with email and phone number on the title page.

Response 13: Dear Reviewer, thank you for your valuable observation. The corresponding author designation was corrected in the revised manuscript to ensure consistency between the asterisk indication and the correspondence information provided on the title page.

Comments 14: The abstract should avoid claiming “nootropic activity” and should report PEP inhibition only as an in vitro enzyme-inhibition endpoint.

Response 14: Dear Reviewer, thank you for your valuable observation. The abstract was revised to avoid overstating the biological implications of the results. PEP inhibition is now described strictly as an in vitro enzyme-inhibition endpoint rather than as evidence of nootropic activity

Comments 15: The term “hypoglycemic activity” should be replaced with “DPP-IV-inhibitory activity” unless glucose-lowering effects are demonstrated in a biological model.

Response 15: Dear Reviewer, thank you for your valuable observation. The term “hypoglycemic activity” was corrected and replaced with “DPP-IV-inhibitory activity” throughout the manuscript to avoid overinterpretation of the results, since no biological glucose-lowering model was evaluated in this study.

Comments 16: The abbreviation DPP-IV should be used consistently; avoid switching between DPPIV and DPP-IV.

Response 16: Dear Reviewer, thank you for your valuable observation. The abbreviation was standardized throughout the manuscript, and “DPP-IV” is now used consistently in all sections.

Comments 17: PEP, PO, prolyl endopeptidase, and prolyl oligopeptidase should be defined and used consistently.

Response 17: Dear Reviewer, thank you for your valuable observation. The terminology and abbreviations related to prolyl oligopeptidase were revised and standardized throughout the manuscript. The terms PEP, PO, prolyl endopeptidase, and prolyl oligopeptidase are now clearly defined and used consistently in all sections.

Comments 18: The phrase “simulated gastric intestinal digestion” should be corrected to “simulated gastrointestinal digestion.”

Response 18: Dear Reviewer, thank you for your valuable observation. The phrase was corrected to “simulated gastrointestinal digestion” throughout the manuscript.

Comments 19: Table 2 reports Fe(II)-chelating activity as “%Act/microgram,” which is not intuitive. The calculation and unit should be clearly explained.

Response 19: Thank you for your valuable observation. The formula used to calculate the Fe(II)-chelating activity and the corresponding unit are described in the Materials and Methods section (Section 2.10.1.3).

Comments 20: Figure 4 would be more interpretable if fluorescence were normalized to the H2O2-stimulated control and presented as percentage ROS level or percentage ROS inhibition.

Response 20: We thank the reviewer for this valuable suggestion. We agree that expressing the results as percentage ROS level or percentage ROS inhibition relative to the H₂O₂-stimulated control could facilitate data interpretation and comparison among treatments.

However, the ROS data available for the present study were recorded and processed as fluorescence intensity values and are presented in this format throughout the analysis. Therefore, it is not possible to reliably recalculate the dataset as percentage ROS inhibition at this stage. Nevertheless, we have revised the manuscript to clarify the interpretation of the fluorescence data and to explicitly indicate that only the intestinal digest obtained from the Esperase hydrolysate produced a statistically significant reduction in intracellular ROS levels compared with the H₂O₂-treated control.

We appreciate the reviewer's suggestion and will consider this approach in future studies to improve the presentation of cellular antioxidant activity data.

Comments 21: The authors should report protein or peptide content of hydrolysates and digests, especially because IC50 values are expressed on a mass basis.

Response 21: Thank you for your valuable observation. IC50 values were calculated based on peptide/protein concentration rather than total dry mass.

Comments 22: Dose-response curves for ACE, DPP-IV, and PEP inhibition should be included in supplementary material.

Response 22: Thank you for your valuable observation. The dose-response curves for ACE, DPP-IV, and PEP inhibition are included in the link containing all the data provided.

Comments 23: Line 722 appears to contain “1.15 m/mL”; please check whether this should be mg/mL.

Response 23: Dear Reviewer, thank you for your valuable observation. he typographical error was corrected, and the unit was revised to “mg/mL” in the manuscript.

Comments 24: The funding, data availability, and acknowledgments sections should be checked for journal formatting compliance.

Response 24: Dear Reviewer, thank you for your valuable observation. e funding, data availability, and acknowledgments sections were carefully revised and formatted according to the journal guidelines to ensure compliance with the required style and structure.

Comments 25: The manuscript states that Paperpal was used for English checking, but the text still contains numerous grammatical and formatting issues. Professional proofreading is recommended.

Response 25: We thank the reviewer for this observation. Following the reviewer's recommendation, the manuscript has undergone an additional round of English language editing and proofreading by a professional English editor. Grammatical, stylistic, and formatting issues identified throughout the manuscript have been carefully revised to improve clarity, readability, and overall language quality. We believe that the revised version presents a substantially improved level of English expression.

Comments 26: The title is currently too strong because it implies demonstrated antihypertensive potency. Consider revising it to focus on ACE-inhibitory activity and in vitro antioxidant effects.

Response 26: We thank the reviewer for this valuable suggestion. We agree that the original title could overstate the physiological implications of the findings. In response, the title has been revised to more accurately reflect the experimental evidence obtained in the present study, focusing on ACE-inhibitory activity and antioxidant effects rather than antihypertensive potency.

Comments 27: The data availability link should be checked and should include all raw or processed data needed to verify IC50 calculations, chromatograms, and cell-assay results.

Response 27: We thank the reviewer for this comment. The data availability statement has been revised, and a repository link containing the raw and processed data supporting the findings of this study will be provided in the revised manuscript. The uploaded dataset will include the information necessary to verify the reported IC₅₀ calculations, chromatographic analyses, and cell-based assay results.

Reviewer 4 Report

Comments and Suggestions for Authors

In this manuscript, the authors studied hydrolysates prepared from tilapia scales to see whether they could lower ACE, DPP-IV, and PEP activity and whether they had any antioxidant activities before and after simulated GI digestion. The study is interesting, but the paper has several problems, for example, contradictions between data described and data presented. Most importantly, the antioxidant results are not strong due to a lack of proper before-and-after comparison. The authors used different tests for the undigested and the digested samples. Overall, the work has potential, but it needs revision before it is ready for publication.

1. ABSTRACT:

• Line 19 in ABSTRACT says “…predominantly 875 Da…” which contradicts “… as evidenced by a major peak at 888 Da…” in line 464 of main text.
• Lines 20-21 in ABSTRACT says “…Esperase hydrolysate exhibiting superior activity in the ferric reducing antioxidant power (FRAP) and Fe(II)-chelating assays. …” which contradicts
  • “… The AP-hydrolysate exhibited markedly higher Fe (II)-chelating activity than the E-hydrolysate (Table 2)…”
  • “…AP-hydrolysate showed a significantly (Student’s t-test) higher FRAP value than the E-hydrolysate (Table 2)…” in lines 622-623 of main text.

1. INTRODUCTION:

• The behavior of the two enzymes used is described. But please justify more clearly why Alkaline Protease and Esperase were selected for the hydrolysis experiments. The choice was based on prior literature, preliminary screening, or industrial relevance, etc?

3. M & M:

• Line 198: “…enzymes were introduced (20 µUnits/g of protein) …” – This sounds not very standard or conventional way of indicating the quantity of ezyme used. Please check whether it should be 20 U instead of 20 µUnits. If it was 20 µUnits/g protein, would it not be too low?
• Line 262: “mEqu Mohr salt per gram” – please recheck whether this unit is correct. “mEqu” especially seems a very untypical way to indicate the unit used for FRAP assay.
• Line 276: “milligrams of ascorbic acid equivalents per gram” should be “mg ascorbic acid Equ/g”
• Line 285: “percentage of Fe(II)-chelating activity per microgram…” – this one also seems very unconventional. Typically, the data would be expressed in IC50 values or as % activity at a given concentration. Not as “%Act/mg” (Table 2).

 

1. RESULTS and DISCUSSION:

• Line 388: “…79% …” should be “…19.70% …” according to Table 1.
• Numbering of subsections is wrong – e.g., section 3.1 is followed by section 2.2. Please check the whole manuscript for this problem.
• Some figures seem to have shifted positions, e.g., see figures in pages 11 and 12.
• The way statistical tests are mentioned seem unconventional/non-standard, please improve them. For example, lines 601-602: “Different letters in the same row (Student’s t-test) indicate…”, and line 653: “Different letters (one-way ANOVA)…”. There is the same issue in caption of Table 3 where “Student’s t-test” is mentioned twice. Please recheck.
• Line 619: “76%” – Based on Table 2, the unit is not %, but “mg Equ ascorbic acid/g” instead.
• There is a disconnect between the antioxidant results before and after GI digestion where in pre-GI stage, in vitro assays were conducted. After GI digestion, cell-based assay was done. This makes it impossible to conclude whether or how GI digestion might have affected the antioxidant activity of the hydrolysates.
• Table 3 caption – “ND: not detected” – this should be removed as it is not used in the table at all.
• “6.0^a ± 2.0” vs. “5.5^b ± 1.3” should be changed to “6.0 ± 2.0 ^a” vs. “5.5 ± 1.3 ^b”. Please recheck all tabulated data.
• For data in Table 3: please recheck whether “6.0 ± 2.0” vs. “5.5 ± 1.3” are significantly different. My concern is that their standard deviations seem quite large, and it seems likely they might have no statistically significant difference between them.
• Line 710: “approximately 5-7 µg/mL” should be amended to “approximately 4.6-6.7 µg/mL” because 4.6 is outside the range of 5-7.
• Line 723: “15 m/mL” – unit is wrong/incomplete. Please check.
•  

 

5. CONCLUSIONS:

• Should be numbered 4.
• Should also mention DPP, PEP, and the pre-GI FRAP, ABTS, or Fe(II)-chelating results at least briefly

Author Response

Comments 1: Line 19 in ABSTRACT says “…predominantly 875 Da…” which contradicts “… as evidenced by a major peak at 888 Da…” in line 464 of main text.

Response 1: Dear Reviewer, thank you for your valuable observation. The value indicated in line 19 of the abstract (875 Da) was incorrect. This has been revised to 888 Da to ensure consistency with the data presented and discussed in the main manuscript.

Comments 2: Lines 20-21 in ABSTRACT says “…Esperase hydrolysate exhibiting superior activity in the ferric reducing antioxidant power (FRAP) and Fe(II)-chelating assays. …” which contradicts

Response 2: Dear Reviewer, thank you for your valuable observation. The statement in the abstract that the Esperase hydrolysate exhibited the highest FRAP and Fe(II)-chelating activities was incorrect. The abstract has been revised to accurately reflect the results presented in the main text, which show that the hydrolysate obtained using Alkaline Protease exhibited significantly higher FRAP and metal-chelating activities.

Comments 3: The behavior of the two enzymes used is described. But please justify more clearly why Alkaline Protease and Esperase were selected for the hydrolysis experiments. The choice was based on prior literature, preliminary screening, or industrial relevance, etc?

Response 3: Dear Reviewer, thank you for your valuable observation. Alkaline Protease and Esperase were selected for the hydrolysis process based on preliminary hydrolysis assays carried out prior to the experimental design. These evaluations were performed to assess the hydrolytic performance of different commercial enzymes under the selected processing conditions. Alkaline Protease and Esperase were chosen because they demonstrated favourable hydrolysis efficiency and peptide production compared to the other evaluated enzymes. Their commercial availability and industrial applicability were also considered during the selection process.

Comments 4:  Line 198: “…enzymes were introduced (20 µUnits/g of protein) …” – This sounds not very standard or conventional way of indicating the quantity of ezyme used. Please check whether it should be 20 U instead of 20 µUnits. If it was 20 µUnits/g protein, would it not be too low?

Response 4: Dear Reviewer, thank you for your valuable observation. The unit was carefully checked, and the correct enzyme dosage was 20 U/g protein. The term “µUnits” was a typographical error and has been corrected throughout the manuscript.

Comments 5: Line 262: “mEqu Mohr salt per gram” – please recheck whether this unit is correct. “mEqu” especially seems a very untypical way to indicate the unit used for FRAP assay.

Response 5: We thank the reviewer for the comment. The unit “mEq Mohr’s salt/g” was used because ammonium iron (II) sulfate (Mohr’s salt) was employed as the calibration standard for the FRAP assay, as described in the Materials and Methods section. In this context, “mEq” refers to milliequivalents of Mohr’s salt per gram of sample. This expression has been previously reported in studies using Mohr’s salt as the reference standard.

Comments 6:  Line 276: “milligrams of ascorbic acid equivalents per gram” should be “mg ascorbic acid Equ/g”

Response 6: Dear Reviewer, thank you for your valuable observation. The corresponding modification has been made, and the unit expression was corrected and standardized throughout the manuscript.

Comments 7: Line 285: “percentage of Fe(II)-chelating activity per microgram…” – this one also seems very unconventional. Typically, the data would be expressed in IC50 values or as % activity at a given concentration. Not as “%Act/mg” (Table 2)

Response 7: Thank you for your valuable observation. The formula used to calculate the Fe(II)-chelating activity and the corresponding unit are described in the Materials and Methods section (Section 2.10.1.3).

Comments 8: Line 388: “…79% …” should be “…19.70% …” according to Table 1.

Response 8: Dear Reviewer, thank you for your valuable observation. The glycine content value that was previously reported in the text has been corrected from 19.79% to 19.70%. This was done to ensure consistency with the data that is presented in Table 1.

Comments 9: Numbering of subsections is wrong – e.g., section 3.1 is followed by section 2.2. Please check the whole manuscript for this problem.

Response 9: Dear Reviewer, thank you for your valuable observation. The manuscript has been carefully revised and the numbering of the subsections corrected throughout to ensure consistency and proper organisation.

Comments 10: Some figures seem to have shifted positions, e.g., see figures in pages 11 and 12.

Response 10: Dear Reviewer, thank you for your valuable observation. The manuscript has been carefully revised for formatting, with the figures repositioned to align with the related sections of the text and improve readability.

Comments 11: The way statistical tests are mentioned seem unconventional/non-standard, please improve them. For example, lines 601-602: “Different letters in the same row (Student’s t-test) indicate…”, and line 653: “Different letters (one-way ANOVA)”. There is the same issue in caption of Table 3 where “Student’s t-test” is mentioned twice. Please recheck.

Response 11: Dear Reviewer, thank you for your valuable observation. The statistical methodology was already described in Section 2.11 (Statistical Analysis) of the Materials and Methods, including the use of one-way ANOVA followed by Fisher’s LSD test for multiple comparisons and Student’s t-test for pairwise comparisons. However, the wording used in tables and figure legends was revised to follow more conventional statistical terminology and improve clarity.

Comments 12: Line 619: “76%” – Based on Table 2, the unit is not %, but “mg Equ ascorbic acid/g” instead.

Response 12: Dear Reviewer, thank you for your valuable observation. To ensure accurate representation of the results, the units used to express the ABTS antioxidant activity were corrected from percentage (%) to milligrams of ascorbic acid equivalents per gram. Additionally, the relevant text was revised for clarity and scientific consistency: In a scale-specific instance, Chen et al. [52] reported an ABTS scavenging activity of 16.41% for papain hydrolysates derived from grass carp scale gelatin. While the present study expressed antioxidant activity using different units (18.76 mg Equ ascorbic acid/g), the results demonstrate similar antioxidant potential for fish scale-derived hydrolysates, even at lower concentrations.

Comments 13: There is a disconnect between the antioxidant results before and after GI digestion where in pre-GI stage, in vitro assays were conducted. After GI digestion, cell-based assay was done. This makes it impossible to conclude whether or how GI digestion might have affected the antioxidant activity of the hydrolysates.

Response 13: We thank the reviewer for this important observation. We agree that the antioxidant assays performed before and after SGID evaluate different biological endpoints and therefore do not allow a direct assessment of how SGID affected antioxidant activity.

The antioxidant capacity of the undigested hydrolysates was evaluated using chemical assays (ABTS, FRAP, and Fe(II)-chelating activity), whereas the digested samples were assessed using a cellular ROS assay in STC-1 cells. Consequently, these results should not be interpreted as a direct before and after comparison of antioxidant activity.

In the revised manuscript, we have clarified this distinction throughout the Results and Discussion sections and removed any statements that could imply that SGID increased or decreased antioxidant activity. The discussion now emphasizes that the chemical antioxidant capacity of the hydrolysates and the cellular antioxidant response of the digests represent complementary but distinct endpoints.

Comments 14:  Table 3 caption – “ND: not detected” – this should be removed as it is not used in the table at all.

Response 14: Dear Reviewer, thank you for your valuable observation. The abbreviation 'ND' (not detected) has been removed from the title of Table 3, since it was not used in the corresponding table.

Comments 15: “6.0^a ± 2.0” vs. “5.5^b ± 1.3” should be changed to “6.0 ± 2.0 ^a” vs. “5.5 ± 1.3 ^b”. Please recheck all tabulated data.

Response 15: Dear Reviewer, thank you for your valuable observation. The suggested correction was implemented, and all tables were revised to ensure consistent statistical notation throughout the manuscript.

Comments 16:  For data in Table 3: please recheck whether “6.0 ± 2.0” vs. “5.5 ± 1.3” are significantly different. My concern is that their standard deviations seem quite large, and it seems likely they might have no statistically significant difference between them.

Response 16: Dear Reviewer, thank you for your valuable observation. After rechecking the statistical analysis, we confirmed that there is no statistically significant difference between the values “6.0 ± 2.0” and “5.5 ± 1.3”. The previous significance annotation was incorrect and has been corrected in the revised version of Table 3.

Comments 17:  Line 710: “approximately 5-7 µg/mL” should be amended to “approximately 4.6-6.7 µg/mL” because 4.6 is outside the range of 5-7.

Response 17: Dear Reviewer, thank you for your valuable observation. The reported range has been amended from 5–7 µg/mL to 4.6–6.7 µg/mL, to accurately reflect the experimental data presented in the manuscript.

Comments 18:  Line 723: “15 m/mL” – unit is wrong/incomplete. Please check.

Response 18: Dear Reviewer, thank you for your valuable observation. The unit was incorrectly reported as '15 m/mL' and has been amended to 'mg/mL', ensuring accuracy and consistency in the reported data.

Comments 19: Conclusions: Should be numbered 4. Should also mention DPP, PEP, and the pre-GI FRAP, ABTS, or Fe(II)-chelating results at least briefly

Response 19: Dear Reviewer, thank you for your valuable observation. The Conclusions section has been revised according to the reviewer’s observation, including the correction of the section numbering to Section 4. In addition, the conclusion has been expanded to briefly include the main findings related to DPP-IV and PEP inhibitory activities, as well as the antioxidant results

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors Dear Authors,   The manuscript has been improved in accordance with the comments. The authors have adequately addressed all the issues identified. The manuscript can be accepted for publication after the following two minor corrections have been made:

1. The term in vitro should be written in italics consistently throughout the entire text.
2. Figure 1 (page 11) should be provided with an appropriate title, placed below the figure.

Best regards,

Author Response

Comments 1: The term in vitro should be written in italics consistently throughout the entire text.

Response 1: We thank the reviewer for this observation. The manuscript has been carefully reviewed, and all occurrences of the term in vitro have been checked and revised to ensure consistent italic formatting throughout the text in accordance with the journal style requirements.

Comments 2: Figure 1 (page 11) should be provided with an appropriate title, placed below the figure.

Response 2: We thank the reviewer for this observation. The figure has been revised accordingly. An appropriate title/caption has been added and placed below Figure 1 in accordance with the journal formatting requirements.

 

Reviewer 2 Report

Comments and Suggestions for Authors

All revisions have been addressed.

Author Response

Comments 1: All revisions have been addressed.

Response 1: We sincerely thank the reviewer for the careful evaluation of our manuscript and for the valuable comments and suggestions provided throughout the review process. We appreciate the time and effort dedicated to improving the quality of this work. We are pleased that all concerns have been satisfactorily addressed.

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript entitled “Simulated gastrointestinal digestion of tilapia (Oreochromis niloticus) scale hydrolysates enhances ACE-inhibitory activity and reveals antioxidant effects in STC-1 cells” has been satisfactorily improved. The authors have addressed the main concerns by moderating the title, abstract, and interpretation of the results, and by presenting the study as an in vitro screening rather than making excessive physiological or nutraceutical claims.

The manuscript now clearly describes the preparation of tilapia scale hydrolysates using Alkaline Protease and Esperase 8.0 L®, followed by simulated gastrointestinal digestion and evaluation of antioxidant activity, ACE-, DPP-IV-, and PEP-inhibitory activities, cytotoxicity, and cellular ROS levels in STC-1 cells. The results are generally well presented and support the conclusion that tilapia scales can be valorized as a potential source of bioactive peptide-rich hydrolysates.

I also appreciate that the authors now acknowledge the key limitation of the study, namely that the specific peptides responsible for the observed bioactivities were not identified. The statement that further peptide characterization, bioavailability assessment, and in vivo validation are required is appropriate and improves the balance of the manuscript.

Only minor editorial checking is still recommended before publication, particularly for formatting, units, superscripts/subscripts, and consistency of terminology.

Overall, the manuscript is scientifically sound and suitable for publication.

Author Response

Comments 1: 

The revised manuscript entitled “Simulated gastrointestinal digestion of tilapia (Oreochromis niloticus) scale hydrolysates enhances ACE-inhibitory activity and reveals antioxidant effects in STC-1 cells” has been satisfactorily improved. The authors have addressed the main concerns by moderating the title, abstract, and interpretation of the results, and by presenting the study as an in vitro screening rather than making excessive physiological or nutraceutical claims.

The manuscript now clearly describes the preparation of tilapia scale hydrolysates using Alkaline Protease and Esperase 8.0 L®, followed by simulated gastrointestinal digestion and evaluation of antioxidant activity, ACE-, DPP-IV-, and PEP-inhibitory activities, cytotoxicity, and cellular ROS levels in STC-1 cells. The results are generally well presented and support the conclusion that tilapia scales can be valorized as a potential source of bioactive peptide-rich hydrolysates.

I also appreciate that the authors now acknowledge the key limitation of the study, namely that the specific peptides responsible for the observed bioactivities were not identified. The statement that further peptide characterization, bioavailability assessment, and in vivo validation are required is appropriate and improves the balance of the manuscript.

Only minor editorial checking is still recommended before publication, particularly for formatting, units, superscripts/subscripts, and consistency of terminology.

Overall, the manuscript is scientifically sound and suitable for publication

Response 1: 

We sincerely thank the reviewer for the careful evaluation of the revised manuscript and for the positive assessment of our work. We greatly appreciate the constructive comments and suggestions provided throughout the review process, which have significantly improved the quality, clarity, and scientific rigor of the manuscript.

We are pleased that the revisions satisfactorily addressed the major concerns raised during the review, including the moderation of the title, abstract, and interpretation of the results, as well as the clarification of the study's scope as an in vitro screening investigation. We also appreciate the reviewer's recognition of our efforts to acknowledge the limitations of the study and to clearly outline the need for further peptide identification, bioavailability assessment, and in vivo validation.

Following the reviewer's recommendation, we have performed a final editorial revision of the manuscript to verify formatting, units, superscripts, subscripts, and terminology consistency throughout the text.

We sincerely thank the reviewer for the valuable feedback and for supporting the publication of our manuscript.