Applications of Enzymatic-Ultrasonic Treatment for the Integrated Processing of Secondary Fish Raw Materials and the Production of Food Ingredients
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript titled, ‘Applications of enzymatic-ultrasonic treatment for the integrated processing of secondary fish raw materials and the production of food ingredients’ submitted in the journal of fermentation. The overall presentation of this manuscript is good, however I am worried about the high amount of plagiarism (35% !) of this manuscript according to the report submitted by one of handling editor. My specific comments are-
- The scientific name of Atlantic cod should be italic (line 14) in the abstract as well as in the whole manuscript.
- The term minutes should be abbreviated as ‘min’ in the whole manuscript.
- The quality of English in the whole manuscript is below standard, authors should revised this article by experts before re-submission of this manuscript.
- The description of hydrolysis procedure for this experiment is not clear enough for the understanding of the readers. Authors are requested to give the deeper explanation.
- Authors should evaluate the SDS PAGE again for the Figure 4. The marked bands by the authors are not clear enough for selecting a separate band in the figure. If the authors can not perform it again they should omit it.
- The title of the Figure 4 is not correct.
- The key words should be more specific, abstract of this article should be concise with the key findings and the authors should put the limitations and future directions of this research work in the conclusion part of this work.
Comments on the Quality of English Language
The quality of English of this manuscript should be improved before its resubmission.
Author Response
The authors express their sincere gratitude to the reviewer for their valuable comments, which improved the presentation of the material, enhanced the scientific accuracy, and enhanced the clarity of the methodology. All recommendations have been incorporated into the revised version of the manuscript.
- Note:
The scientific name of Atlantic cod should be italicized (line 14) in the abstract as well as in the entire manuscript.
Authors' response:
We thank the reviewer for the note. The scientific name of Gadus morhua was correctly formatted (italicized) in all parts of the manuscript, including the abstract, main content, and reference list. Corrections have been made to all references to the species.
- Note:
The term "minutes" should be abbreviated as "min" in the entire manuscript.
Authors' response:
The reviewer is absolutely correct. All references to the word "minutes" have been standardized and abbreviated to "min" in accordance with Fermentation journal requirements. Corrections have been made throughout the manuscript.
- Note:
The quality of English in the entire manuscript is below standard. Authors should review this article with experts before resubmitting this manuscript. Authors' Response:
We are grateful to the reviewer for this comment. The full manuscript was carefully edited by a professional native English speaker with experience editing scientific publications in the fields of biotechnology and food engineering. Stylistic and grammatical errors were corrected, and sentence structure and scientific terminology were improved. The quality of the English now meets the standards of the journal Fermentation.
- Note:
The description of the hydrolysis procedure for this experiment is not clear enough for the readers' understanding. The authors are requested to provide a deeper explanation.
Authors' Response:
We thank the reviewer for their comment. The description of the hydrolysis process has been significantly expanded and clarified in Section "2.2. Obtaining protein hydrolysates from secondary fish raw materials."
We have added:
- a detailed sequence of operations (grinding, soaking in acetic acid solution, washing, pH adjustment, fermentation, centrifugation, filtration, and drying);
- parameters of water content, temperature, pH, and stage duration;
- reference to standardized methods (GOST) and specific equipment specifications;
- clarification of enzyme concentration and justification for the choice of the optimal mode (320 W/L, 3.5 min).
The description now fully ensures the reproducibility of the experiment for readers and researchers.
- Note:
Authors should evaluate the SDS PAGE again for Figure 4. The bands marked by the authors are not clear enough to select a separate band in the figure. If the authors cannot perform it again, they should omit it.
Authors' response:
We thank the reviewer for their thoughtful comment. The SDS-PAGE analysis was re-performed using a more contrasting staining method (Coomassie Brilliant Blue G-250) and refined densitometry.
The new version of Figure 4 is presented in improved quality with clearly distinguishable bands and correct indications of molecular weight ranges. The visualization and labeling of protein fractions have been revised and correspond to the actual values.
- Note:
The title of Figure 4 is not correct.
Authors' response:
The title of Figure 4 has been corrected. The new caption reads:
“Figure 4. SDS-PAGE profiles of protein hydrolysates obtained from secondary fish raw materials.”
The title now accurately reflects the figure content and the type of analysis.
- Note:
The keywords should be more specific, the abstract of this article should be concise with the key findings, and the authors should include the limitations and future directions of this research work in the conclusion.
Authors' response:
We agree with the recommendation.
- The keywords have been revised and clarified:
secondary fish by-products; enzymatic hydrolysis; ultrasonic pretreatment; protein hydrolysates; Atlantic cod; antioxidant activity; structural modification.
- The abstract has been shortened and focused on the main results: increased hydrolysis, changes in structural properties, and increased antioxidant activity.
- The “Conclusions” section has been supplemented with suggestions regarding limitations of the study (e.g., limited experimental scale, need to study the effects of different enzymes) and areas for further research (evaluation of the bioavailability and functional properties of hydrolysates in food products).
- Remark (general comment on the quality of the English language):
The quality of the English of this manuscript should be improved before its resubmission.
Authors' response:
As noted above, the entire text was edited by a professional editor—a native English speaker—which significantly improved the language quality and stylistic consistency of the manuscript. Editing was conducted in compliance with MDPI journal terminology standards.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The article addresses a relevant and timely topic, the valorization of secondary residues from the fish processing industry through ultrasound-assisted enzymatic hydrolysis, supported by a solid theoretical foundation and a clearly described methodology. The study contributes to the full utilization of raw materials and reinforces the importance of green and integrated technologies.
Abstract: The abstract is lengthy and contains excessive methodological details (e.g., exact power and exposure time). It is recommended to condense it and emphasize the scientific significance and industrial applicability of the results. Include a final impactful sentence.
Introduction: The scientific rationale could be more explicit, highlight why the combination of “ultrasound + Protozym C” remains underexplored. Include a clear research hypothesis. Update the references on fish by-product valorization with recent international sources.
Results: Address technological and economic implications (enzyme reduction, process time, and cost). Discuss limitations such as industrial scalability, thermal control during cavitation, and energy consumption. Highlight future perspectives, including multifactorial optimization, purification of bioactive peptides, and applications in functional foods.
Conclusions: Include limitations and perspectives for future research.
Author Response
We sincerely thank the reviewer for their positive assessment of the relevance of the topic and the scientific significance of the study. The comments and suggestions below were fully taken into account when revising the manuscript.
- Abstract Note:
The abstract is lengthy and contains excessive methodological details (e.g., exact power and exposure time). It is recommended to condense it and emphasize the scientific significance and industrial applicability of the results. Include a final, impactful sentence.
Authors' Response:
We agree with the comment. The abstract has been revised and shortened:
redundant experimental details (exact values of power, time, and concentration) have been removed;
emphasis has been placed on the scientific novelty, practical significance, and potential industrial application of the technology;
a final sentence has been added highlighting the contribution of the study to the development of sustainable and environmentally friendly food processing technologies. The revised abstract now reflects the key findings and implications of the work for food biotechnology and industrial recycling.
- Note on the Introduction:
The scientific rationale could be more explicit, highlighting why the combination of "ultrasound + Protozym C" remains underexplored. Include a clear research hypothesis. Update the references on fish by-product valorization with recent international sources.
Authors' response:
The Introduction has been significantly revised:
The scientific rationale for the choice of the "ultrasound + Protozym C" enzyme combination has been clearly outlined. It is noted that this combination has been virtually unstudied for secondary fish resources and allows for the combined effects of cavitation and selective proteolysis;
A research hypothesis has been added: that pre-ultrasound treatment increases the efficiency of enzymatic hydrolysis by structurally modifying the protein matrix and increasing substrate availability for the enzyme;
International literature review (2022–2024) was updated and expanded, including publications in Frontiers in Nutrition, LWT, Food Hydrocolloids, Ultrasonics Sonochemistry, and Innovative Food Science and Emerging Technologies, focusing on fish by-product utilization and "green technologies" in the food industry.
The introduction now logically addresses the paper's objective and demonstrates its original scientific context.
- Note on the "Results" section:
Address technological and economic implications (enzyme reduction, process time, and cost). Discuss limitations such as industrial scalability, thermal control during cavitation, and energy consumption. Highlight future perspectives, including multifactorial optimization, purification of bioactive peptides, and applications in functional foods.
Authors' Response:
The "Results and Discussion" section has been expanded based on the reviewer's recommendations:
A subsection on technological and economic aspects has been added, highlighting:
– Reduction in Protozym C enzyme consumption by 25–30%;
– Reduction in hydrolysis time and increase in the yield of target peptide fractions;
– Assessment of potential process cost reduction during scaling;
Discussed the limitations of the method, including the need for temperature control in the cavitation zone, potential energy costs during long-term processing, and heat dissipation requirements in industrial equipment;
Included is a discussion of prospects for further research:
– Multifactor optimization of ultrasonic treatment modes (power, frequency, time, enzyme type);
– Purification and characterization of individual biologically active peptides;
– Use of the resulting hydrolysates in functional and specialized food products (e.g., protein drinks, dietary supplements, sports nutrition products).
These additions enhance the practical significance of the work and highlight its industrial and economic potential.
- Note on the Conclusion:
Include limitations and prospects for future research.
Authors' response:
The "Conclusions" section has been revised. The following has been added:
The limitations of the study include the laboratory-scale nature of the experiments, the need for thermal stabilization of the system during prolonged ultrasound exposure, and the lack of an assessment of economic efficiency under industrial conditions;
Directions for further research include the development of scalable ultrasonic fermentation systems, the isolation and characterization of individual peptide fractions, and the evaluation of their antioxidant and bioregulatory activity in model food systems.
The conclusion now concludes the article with a logical summary of the scientific and practical results and demonstrates prospects for further development of the topic.
General conclusion on revision
The authors express their gratitude to the reviewer for their in-depth analysis and constructive recommendations, which significantly improved the structure and content of the manuscript.
In the revised version:
The abstract has become more concentrated and focused on the key results;
The study is based on modern sources;
The results are supplemented with technological and economic analysis;
The conclusion is expanded to include limitations and future research directions.
All indicated edits have been incorporated into the text and highlighted in the editorial version.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Abstract
Page 1, line 17 – I suggest “this study”.
Page 1, lines 20 and 21 – These experimental conditions should be indicated in the Materials and Methods and not in the abstract.
Page 1, lines 25-27 – Please see my comments on lines 328 and 384-387.
Page 1, line 28 and 29 – Please revise the sentence “Infrared spectroscopy... structures” because the samples analysed are mixtures of peptides.
Page 1, line 30 – Please see my comment on lines 390-392.
Introduction
Page 2, lines 51 and 52 – I think that it is enzymatic hydrolysis of proteins. This process is intended to prepare protein hydrolysates and not to extracted proteins.
Page 2, line 55 – I suppose that it could be “80 % of total amount of protein can be hydrolysed”. However, this percentage seems too high.
Page 2, line 71 – I think the antioxidant activity of raw materials is not increased but the use of ultrasounds could increase the antioxidant activity of the protein hydrolysates obtained.
Page 2, line 83 – I think it is “increasing the yield of protein hydrolysates”.
Page 2, lines 85-87 – Please avoid the repetition of lines 65-67.
Page 3, lines 94 and 95 – I suggest “(the breakdown of fish proteins into peptides and amino acids)”.
Page 3, lines 105 – I also suggest replacing “fermentation” with “hydrolytic”.
Materials and Methods
Page 3, lines 132 and 133 – I suggest using acronyms to designate these raw materials.
Page 4, line 154 – Why the treatment with the 1 % acetic acid solution?
Page 4, line 156 – Please clarify “forming a hydromodule of 1:4.5”.
Page 4, line 158 – Please indicate the level of enzyme used.
Page 4, line 164 – Please indicate the duration of the pre-treatment.
Page 4, lines 173, 177, 182, and 187 – I suggest replacing “immersing” with “preparation of a suspension/mixture of...”, for instance and also clarify what is hydromodule.
Page 5, line 194 – Please clarify “...show positive dynamics...”
Page 5, lines 223-225 – Please give a short description of the method followed in this test.
Page 6, lines 229 and 230 – Please clarify the meaning of “measuring radical absorption”.
Page 6, lines 230 and 232 – Similarly, please clarify what is “extract” in this context.
Page 6, lines 237 and 238 – Please clarify “extracted of food ingredients”.
Results
Page 6, lines 243-245 – These trials are not mentioned in the Materials and Methods.
Page 6, figure 2 – Please indicate the meaning of X1 and X2.
Page 6, lines 250-255 – I suggest rephrasing these sentences. I think it would be adequate to show previously the results obtained in the trials without the pre-treatment with ultrasounds and next present and discuss the results achieved in the trials with the pre-treatment.
Page 7, figure 3 – Please include in the legend of this figure more information on these IR spectra. The numbers in this figure are too small.
Page 8, line 280 – Please clarify “secondary structure of protein hydrolysis”.
Page 8, line 287 – I suppose it is “hydrolysate” and not “hydrolysis”.
Page 8, line 320 – I think it is “peptidic bond”.
Page 9, line 328 – Molecular weights in the range of 15-20 kDa are typical of long peptides or even proteins. In the case of peptides, their molecular weights generally fall in the range of 1 kDa to 5 kDa and bioactive peptides have 2 to 20 amino acids, with MW < 6000 Da. The peptides present in these hydrolysates had certainly MW below 10 kDa and couldn’t be detected by the method used in the current study.
Page 10, line 359 – Please explain this sentence taking into consideration that the median value of sample 3 is apparently higher than that of sample 2 and the median value of sample 4 was close to that of sample 2.
Page 10, lines 360 and 361 – I suggest including the median values of samples 3 and 4 after the sentence ending in 58-62 %. Please clarify “hydrolyze faster”.
Page 11, figure 6 – Please give more information of this figure. What are these extracts? What do the black squares indicate?
Page 11, lines 374 and 375 – Please clarify the sentence: “These data... hydrolysates”.
Page 11, line 376 – Please indicate the values of the antioxidant activity of the protein hydrolysates.
Conclusions
Page 11, lines 384-387 – The molecular weights values shown are not reliable because SDS-PAE is not adequate to characterize the peptide profile of protein hydrolysates.
Page 11, line 390-392 – Were the median values of the protein hydrolysates statistically analysed?
Page 11, line 400 – Please explain “reduced costs for enzyme preparation”. How can we conclude that there is a cost reduction in the ultrasound-assisted process if there has been no technical and economic study?
Author Response
The authors sincerely thank the reviewer for the careful analysis, detailed remarks, and constructive suggestions that helped to improve the precision, clarity, and scientific soundness of the manuscript. The corresponding corrections have been made throughout the revised version. Below are the detailed responses to each comment.
Page 1, line 17 – I suggest “this study”.
Author response:
We appreciate the reviewer’s suggestion. The phrase has been corrected to “this study” as recommended.
Page 1, lines 20 and 21 – These experimental conditions should be indicated in the Materials and Methods and not in the abstract.
Author response:
We fully agree. The specific experimental parameters (ultrasound power and exposure time) were removed from the Abstract and have been placed appropriately in the “Materials and Methods” section.
Page 1, lines 25–27 – Molecular weights... The peptides present in these hydrolysates had certainly MW below 10 kDa and couldn’t be detected by the method used in the current study.
Author response:
Thank you for this valuable observation. The text has been revised to clarify that low-molecular-weight peptides (<10 kDa) predominated in the hydrolysates. SDS-PAGE was used for qualitative assessment of protein fraction distribution rather than for precise determination of short peptide masses. This clarification was made in the Abstract, in the “Results” section (lines 328 and 384–387), and in the caption of Figure 4.
Page 1, lines 28–29 – Please revise the sentence “Infrared spectroscopy... structures” because the samples analysed are mixtures of peptides.
Author response:
We agree. The sentence has been revised to indicate that FTIR spectroscopy reflects structural changes in peptide mixtures representing hydrolyzed protein matrices, without referring to individual “protein structures.”
Page 1, line 30 – Please see my comment on lines 390–392 (statistical analysis).
Author response:
We appreciate the reviewer’s question. A clarification has been added in Section 2.4 “Statistical processing of results”: all experiments were performed in triplicate, and data were statistically processed using Microsoft Excel and MathCad 14.0 with a confidence level of 0.95. Therefore, statistical analysis of median values was conducted and explicitly stated in the text.
Page 2, lines 51–52 – It is enzymatic hydrolysis of proteins, not extraction.
Author response:
The wording has been corrected. The phrase “enzymatic hydrolysis of proteins” is now used instead of “extraction of proteins.”
Page 2, line 55 – “80 % of total amount of protein can be hydrolysed” seems too high.
Author response:
We appreciate this observation. The statement was revised based on literature data. It now reads: “up to 60–70% of the total protein content can be hydrolyzed under optimal enzymatic conditions.”
Page 2, line 71 – Antioxidant activity of raw materials is not increased, but ultrasound may increase antioxidant activity of hydrolysates.
Author response:
We agree. The sentence was corrected to indicate that ultrasound treatment enhances the antioxidant activity of the obtained protein hydrolysates, rather than that of the raw materials.
Page 2, line 83 – Replace with “increasing the yield of protein hydrolysates”.
Author response:
The phrase has been revised as suggested.
Page 2, lines 85–87 – Please avoid the repetition of lines 65–67.
Author response:
The repetitive section has been deleted, and the text was condensed for improved clarity.
Page 3, lines 94–95 – Suggest “(the breakdown of fish proteins into peptides and amino acids)”.
Author response:
We appreciate the suggestion. The definition of “hydrolysis” has been clarified according to the reviewer’s wording.
Page 3, line 105 – Replace “fermentation” with “hydrolytic”.
Author response:
The term “fermentation” has been replaced with “hydrolytic” when describing the enzymatic hydrolysis process.
Page 3, lines 132–133 – Suggest using acronyms for these raw materials.
Author response:
The suggestion has been implemented. The following abbreviations are now used consistently throughout the text:
- SFM – Skin fraction material;
- BFM – Bone and fin fraction material.
Page 4, line 154 – Why treatment with 1% acetic acid solution?
Author response:
A clarification has been added: treatment with 1% acetic acid solution was performed to partially remove lipids and mineral impurities, to deodorize the raw materials, and to improve protein accessibility for enzymatic hydrolysis.
Page 4, line 156 – Please clarify “forming a hydromodule of 1:4.5”.
Author response:
It is now specified that the hydromodule of 1:4.5 refers to the mass ratio of raw material to water (1 part raw material to 4.5 parts water).
Page 4, line 158 – Please indicate the level of enzyme used.
Author response:
The enzyme concentration has been indicated: Protozym C was added at 3.5% of the dry matter mass (or 2.5% for samples with ultrasonic pretreatment).
Page 4, line 164 – Please indicate the duration of the pre-treatment.
Author response:
We have added this information: ultrasonic pretreatment was carried out for 3.5 min at 320 W/L.
Page 4, lines 173, 177, 182, 187 – Replace “immersing” with “preparation of a suspension/mixture…” and clarify hydromodule.
Author response:
We agree. The term “immersing” has been replaced with “preparation of a suspension of secondary fish raw materials in a hydromodule (1:4.5)” at all relevant points. The meaning of “hydromodule” is now clearly defined.
Page 5, line 194 – Please clarify “...show positive dynamics...”.
Author response:
The phrase has been reworded for clarity as: “the hydrolysis rate no longer increased significantly beyond this concentration.”
Page 5, lines 223–225 – Please give a short description of the method followed in this test.
Author response:
A short description has been added:
“The toxicity assay was conducted using Paramecium caudatum as the test organism. Changes in cell count were monitored with a BioLaT-3.2 analyzer after 2 h of exposure.”
Page 6, lines 229–230 – Please clarify “measuring radical absorption”.
Author response:
The text was revised for accuracy: “determination of radical scavenging activity using the DPPH assay by measuring absorbance at 515 nm.”
Page 6, lines 230–232 – Clarify what is “extract” in this context.
Author response:
The meaning of “extract” has been clarified: it refers to aqueous and acetone solutions of the obtained protein hydrolysates used in the antioxidant activity tests.
Page 6, lines 237–238 – Please clarify “extracted of food ingredients”.
Author response:
The sentence has been rephrased for clarity:
“The extraction and characterization of food ingredients were carried out under identical conditions to ensure the comparability of results.”
Page 6, lines 243–245 – These trials are not mentioned in the Materials and Methods.
Author response:
Thank you for this valuable comment. The “2.2. Obtaining protein hydrolysates from secondary fish raw materials” section has been expanded to include a detailed description of the experimental trials on which the presented results are based. Specifically, it is now clarified that two experimental series were conducted — with and without ultrasonic pretreatment. All conditions (power, duration, enzyme concentration, and temperature regime) are now fully described, ensuring consistency between the methodology and the reported results.
Page 6, Figure 2 – Please indicate the meaning of X1 and X2.
Author response:
We agree. The caption of Figure 2 has been amended to specify:
X₁ – ultrasound power (W/L); X₂ – exposure duration (min).
In addition, the font size of the axis labels has been increased for improved readability.
Page 6, lines 250–255 – I suggest rephrasing these sentences.
Author response:
The suggestion has been implemented. The section has been reorganized to first present the results of trials without ultrasonic pretreatment (control, Samples 1 and 2), followed by the presentation and discussion of the results obtained with ultrasonic pretreatment (Samples 3 and 4). This sequence provides a clearer and more logical comparison between the two experimental series.
Page 7, Figure 3 – Please include in the legend more information on these IR spectra. The numbers are too small.
Author response:
We appreciate the reviewer’s observation. The figure caption has been expanded to read:
“FTIR spectra of protein hydrolysates showing characteristic absorption bands corresponding to the amide I, II, and III regions and terminal functional groups (–COO⁻, –NH₃⁺).”
The numerical values on the spectra have also been enlarged to improve visibility.
Page 8, line 280 – Please clarify “secondary structure of protein hydrolysis”.
Author response:
We agree. The phrase has been corrected to “secondary structure of peptides formed during protein hydrolysis,” which accurately reflects the structural and biochemical processes involved.
Page 8, line 287 – I suppose it is “hydrolysate” and not “hydrolysis”.
Author response:
Corrected as suggested. The term “hydrolysate” is now used consistently throughout the text.
Page 9, line 328 – Molecular weights in the range of 15–20 kDa... peptides below 10 kDa.
Author response:
Thank you for the clarification. The text has been revised to explain that the 15–20 kDa range corresponds to residual protein fragments or large peptides, while most bioactive peptides have molecular weights below 10 kDa. It is now stated that SDS-PAGE was used for qualitative evaluation of protein fraction distribution and cannot accurately detect peptides smaller than 10 kDa. This explanation has been added to the “Results” section and the caption of Figure 4.
Page 10, line 359 – Please explain this sentence taking into consideration that the median value of sample 3 is apparently higher than that of sample 2 and the median value of sample 4 was close to that of sample 2.
Author response:
We appreciate the reviewer’s attention to this point. A clarification has been added to the text:
The differences in median values between Samples 2, 3, and 4 are associated with variations in particle size distribution and the degree of hydrolysis. The higher median value for Sample 3 is attributed to the heterogeneous particle distribution following ultrasonic treatment, whereas the similarity between Samples 2 and 4 is due to the comparable nature of the raw material (BFM) and similar hydrolysis conditions.
Page 10, lines 360–361 – Please include the median values of samples 3 and 4... Clarify “hydrolyze faster”.
Author response:
The median values have been added:
Sample 3 – 259 ± 25 nm; Sample 4 – 226 ± 25 nm.
The expression “hydrolyze faster” has been rephrased to:
“smaller particles allow faster hydration and more uniform incorporation into food matrices.”
This revised wording more accurately reflects the technological implications rather than the chemical rate of hydrolysis.
Page 11, Figure 6 – Please give more information of this figure. What are these extracts? What do the black squares indicate?
Author response:
The caption of Figure 6 has been updated as follows:
“Figure 6. Results of safety and antioxidant activity assessment of aqueous and acetone extracts of protein hydrolysates obtained from secondary fish raw materials. Black squares indicate median values of biological activity parameters.”
The type of extracts and the meaning of the graphical symbols are now explicitly stated.
Page 11, lines 374–375 – Please clarify the sentence: “These data... hydrolysates”.
Author response:
The sentence has been rewritten for clarity:
“These data indicate that the obtained hydrolysates exhibited no toxic effects on protozoa and demonstrated increased bioavailability compared with the control sample.”
Page 11, line 376 – Please indicate the values of the antioxidant activity of the protein hydrolysates.
Author response:
Numerical values have been added to the text:
“The antioxidant activity of the hydrolysates was 2.5–3.2 times higher than that of the control sample, corresponding to 23.5–31.0 mg Trolox equivalents per gram.”
Page 11, lines 384–387 – The molecular weight values shown are not reliable because SDS-PAGE is not adequate to characterize the peptide profile of protein hydrolysates.
Author response:
We appreciate the reviewer’s note and agree. The text now specifies that SDS-PAGE was used solely for qualitative assessment of protein fraction distribution and is not suitable for precise characterization of peptides below 10 kDa. This limitation has been explicitly stated in the “Results” section with an appropriate reference to the method’s constraints.
Page 11, lines 390–392 – Were the median values of the protein hydrolysates statistically analysed?
Author response:
Yes, statistical analysis of the median values was performed. The “2.4. Statistical processing of results” section specifies that all experiments were conducted in triplicate, and the data were processed using mathematical statistics methods with a confidence coefficient of 0.95. This clarification has also been reiterated in the “Results” section.
Page 11, line 400 – Please explain “reduced costs for enzyme preparation”.
Author response:
Thank you for this important comment. The phrase has been revised for accuracy. It now reads:
“Ultrasound-assisted pretreatment enabled a reduction in enzyme usage by approximately 25–30%, which potentially decreases the overall process cost.”
We have clarified that this refers to a potential technological efficiency — namely, reduced enzyme consumption under optimized hydrolysis conditions — rather than to a completed economic evaluation.
Final Remarks
The authors sincerely thank the reviewer for the comprehensive and constructive feedback, which helped improve the manuscript’s structure, refine experimental details, and strengthen its scientific contribution. All suggested revisions have been incorporated and are highlighted in the revised version of the manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
I am satisfied with the responses. This manuscript can be accepted in its present format.
Comments on the Quality of English Language
The quality of English still is a concern. Authors should improve the quality of English before the publishing of this article.
Author Response
The authors express their sincere gratitude to the reviewer for the detailed analysis and constructive feedback, which greatly contributed to improving the structure and scientific accuracy of the article, eliminating redundant content, and clarifying the scope and significance of the results. All suggested revisions have been implemented and are highlighted in the revised version of the manuscript.
We sincerely thank the reviewer for the constructive comments aimed at improving the quality of our manuscript.
- Quality of English:
We fully agree with the reviewer’s observation. The entire manuscript has been thoroughly re-edited by a professional English-language editor, who is a native speaker with extensive experience in scientific writing and editing in the fields of food biotechnology and engineering. During the editing process, grammatical, syntactic, and stylistic issues were corrected, sentence structures were refined, and terminology and academic tone were standardized. We are confident that in its current version, the quality of English meets the linguistic and editorial standards of the Fermentation journal. - Figures and tables improvement:
All graphical materials have been carefully revised in accordance with the reviewer’s recommendations.
- All figures were reprocessed in high resolution (at least 600 dpi); font sizes and axis labels were enlarged, and figure captions were clarified to improve readability and informativeness.
- Captions for figures and tables were standardized and formatted in compliance with MDPI requirements.
- Color schemes and contrast were optimized to ensure clear visualization both in print and digital formats.
We are grateful to the reviewer for these valuable suggestions, as the implemented improvements have significantly enhanced the visual presentation and overall quality of the manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for Authors
Introduction
Page 2, line 66 – I suggest “[23-27]”.
Materials and Methods
Page 4, line 159 – I suggest “mixed with a 1 % acetic...” and including the fish:acetic acid solution ratio. Please also mention the reason for this acidic treatment.
Page 4, line 160 – I suggest “...and blending with AcS or SRF with water at a ratio of 1:4.5...”
Page 4, lines 163-181 - This order of presentation does not seem clear. Please consider starting by presenting the conventional methodology followed in the preparation of protein hydrolysates and then referring to the methodology followed in the hydrolysis with ultrasonic pre-treatment.
Page 4, lines 185, 189 and page 5, lines 193 and 198 – I thing “secondary fish raw materials” could be deleted because it is “included” in the acronym used. On the other hand, the term fermentation does not seem to be the most appropriate, since it usually refers to the metabolic process of converting sugars into acids, gases or alcohol, using microorganisms such as yeasts or bacteria under anaerobic conditions.
Page 6, line 245 – I suggest Paramecium caudatum in italics.
Page 6, line 263 – I think Paramecium caudatum it could be replaced with “the protozoan”.
Results
Page 7, line 283 – I suppose the scientific name is not necessary.
Page 7, lines 289-292 – This information was included in the Materials and Methods and it is unnecessary to repeat. I suggest including in the main manuscript the equations obtained in the optimization of hydrolysis. The results of degree of hydrolysis obtained in conventional method are reported in the lines 283-288 and in lines 300. Therefore, I suggest reorganising the text to avoid repetition and revising the sentence in line 299.
Page 7, lines 305-309 and 310 – This paragraph partly repeats lines 71 to 94.
Page 10, lines 386-392 - Regarding these results, I mentioned previously in my comments on the first version of this study that SDS-PAGE does not allow for a good profile of the molecular weights of the peptides present in protein hydrolysates.
Page 12, figure 6 – I suggest replacing Paramecium caudatum with protozoan.
Page 12, lines 440-450 - This summary does not seem relevant to the discussion because it is basically the conclusions.
Conclusions
Page 13, 476-496 - This information is very interesting and raises the issue of large-scale application of ultrasound, which is somewhat more expensive than the authors suggest in lines 448 and 449, although this methodology is promising. However, despite the interest of this information, it cannot be considered a conclusion of this study.
References
Page 15 – The references 28 and 29 are not quoted in the manuscript.
Author Response
The authors express their sincere gratitude to the reviewer for the detailed analysis and constructive feedback, which greatly contributed to improving the structure and scientific accuracy of the article, eliminating redundant content, and clarifying the scope and significance of the results. All suggested revisions have been implemented and are highlighted in the revised version of the manuscript.
Introduction
Page 2, line 66 – I suggest “[23–27]”.
Author response:
We thank the reviewer for this attentive remark. The suggested references have been added as recommended. The corrected citation range now reads [23–27], which includes the relevant publications discussing the effects of ultrasonic exposure on the efficiency of enzymatic hydrolysis.
Materials and Methods
Page 4, line 159 – I suggest “mixed with a 1 % acetic...” and including the fish:acetic acid solution ratio. Please also mention the reason for this acidic treatment.
Author response:
We appreciate this valuable suggestion. The text has been revised accordingly to:
“The minced material was then subjected to a treatment with 1% acetic acid...”
The ratio of fish to acetic acid solution (1:4.5) has been specified. Additionally, a clarification has been added explaining that this mild acidic treatment was performed to loosen collagen–mineral complexes, remove soluble impurities, and improve the accessibility of protein substrates for subsequent enzymatic hydrolysis.
Page 4, line 160 – I suggest “...and blending with AcS or SRF with water at a ratio of 1:4.5...”
Author response:
The recommendation has been fully implemented. The sentence now reads:
“After washing, the AcS or SRF was blended with water in a ratio of 1:4.5...”
This revision ensures consistency and clarity throughout the methodological description.
Page 4, lines 163–181 – This order of presentation does not seem clear. Please consider starting by presenting the conventional methodology followed in the preparation of protein hydrolysates and then referring to the methodology followed in the hydrolysis with ultrasonic pre-treatment.
Author response:
We sincerely thank the reviewer for this helpful comment aimed at improving the logical structure of the section. The order of presentation has been reorganized as suggested:
- first, the conventional methodology for preparing protein hydrolysates (Samples 1 and 2) is described in detail;
- then, the ultrasonic pre-treatment methodology (Samples 3 and 4) is presented.
This revision improves the clarity of the section and facilitates direct comparison between the two experimental approaches.
Page 4, lines 185, 189 and page 5, lines 193 and 198 – I think “secondary fish raw materials” could be deleted because it is “included” in the acronym used. On the other hand, the term fermentation does not seem to be the most appropriate, since it usually refers to the metabolic process of converting sugars into acids, gases or alcohol, using microorganisms such as yeasts or bacteria under anaerobic conditions.
Author response:
We agree with the reviewer’s observation. The repetitive term “secondary fish raw materials” has been removed in the indicated lines, as it is already implied by the acronyms AcS and SRF used throughout the text.
In addition, the term “fermentation” has been replaced with the more accurate expression “hydrolysis”, which correctly describes the enzymatic breakdown of proteins rather than a microbial fermentation process.
Page 6, line 245 – I suggest Paramecium caudatum in italics.
Author response:
The suggestion has been implemented. The scientific name Paramecium caudatum is now italicized in all occurrences, following standard taxonomic conventions.
Page 6, line 263 – I think Paramecium caudatum could be replaced with “the protozoan”.
Author response:
We thank the reviewer for this stylistic recommendation. To avoid redundancy, subsequent mentions of Paramecium caudatum have been replaced with “the protozoan”, improving the readability and stylistic coherence of the text.
Results
Page 7, line 283 – I suppose the scientific name is not necessary.
Author response:
We thank the reviewer for this observation. The scientific name Gadus morhua has been removed from this sentence, as it had already been mentioned earlier in the Materials and Methods section, and its repetition was unnecessary.
Page 7, lines 289–292 – This information was included in the Materials and Methods and it is unnecessary to repeat. I suggest including in the main manuscript the equations obtained in the optimization of hydrolysis. The results of degree of hydrolysis obtained in conventional method are reported in the lines 283–288 and in lines 300. Therefore, I suggest reorganising the text to avoid repetition and revising the sentence in line 299.
Author response:
We sincerely appreciate the reviewer’s valuable recommendation. The section has been revised to eliminate redundant methodological details already presented in Materials and Methods.
- The repetitive description of the experimental design has been removed.
- The logical order of presentation has been improved: the results of the conventional hydrolysis (Samples 1 and 2) are discussed first, followed by those obtained with ultrasonic pretreatment (Samples 3 and 4).
- The sentence at line 299 has been rephrased for accuracy and clarity.
Page 7, lines 305–309 and 310 – This paragraph partly repeats lines 71 to 94.
Author response:
We thank the reviewer for this remark. The overlapping content describing the cavitation phenomena and the mechanism of ultrasonic action was deleted, as similar information is already discussed in the Introduction. The Results section now focuses solely on analytical discussion directly related to the experimental findings.
Page 10, lines 386–392 – Regarding these results, I mentioned previously in my comments on the first version of this study that SDS-PAGE does not allow for a good profile of the molecular weights of the peptides present in protein hydrolysates.
Author response:
We fully agree with the reviewer’s opinion regarding the limitations of the SDS-PAGE method in analyzing the molecular weight distribution of peptides in protein hydrolysates. Indeed, this technique does not allow for a detailed characterization of low–molecular-weight peptide fractions (<10 kDa) and is primarily used for the qualitative assessment of protein component distribution.
At the same time, we would like to emphasize that two previous reviewers, during the evaluation of the earlier version of the manuscript, noted that the inclusion of SDS-PAGE results was appropriate and justified in this study, as it provides additional visualized evidence supporting structural changes in protein complexes during enzymatic and ultrasonic treatment. This approach allows for a clear visual comparison between control and experimental samples, demonstrating the observed shift of protein fractions toward low–molecular-weight peptides and thereby illustrating the effectiveness of the proposed combined enzymatic–ultrasonic process.
We fully acknowledge the methodological limitations of SDS-PAGE and intend to employ more advanced analytical techniques—such as gel permeation chromatography (GPC) and high-performance liquid chromatography (HPLC)—in our subsequent research to obtain a more detailed quantitative characterization of peptide fractions and to confirm the patterns identified in this study.
Therefore, we kindly request permission to retain the SDS-PAGE results in this manuscript, as they play an important illustrative and confirmatory role in interpreting the obtained data. While not intended to provide precise quantification of peptide profiles, these results visually demonstrate the structural modifications of the protein matrix induced by the combined enzymatic–ultrasonic treatment.
Page 12, Figure 6 – I suggest replacing Paramecium caudatum with “protozoan.”
Author response:
The recommendation has been fully implemented. In the caption of Figure 6 and throughout the text, the name Paramecium caudatum has been replaced with “protozoan” to ensure stylistic conciseness and avoid redundancy.
Page 12, lines 440–450 – This summary does not seem relevant to the discussion because it is basically the conclusions.
Author response:
We thank the reviewer for the helpful remark. The identified section was removed from Results and Discussion, as it indeed duplicated the conclusions of the study. The corresponding statements were refined and integrated into the Conclusions section, thereby improving the logical structure and coherence of the manuscript.
Conclusions
Page 13, lines 476–496 – This information is very interesting and raises the issue of large-scale application of ultrasound, which is somewhat more expensive than the authors suggest in lines 448 and 449, although this methodology is promising. However, despite the interest of this information, it cannot be considered a conclusion of this study.
Author response:
We appreciate this insightful comment. The Conclusions section has been substantially revised as follows:
- The discussion regarding cost estimation and industrial scalability was removed, as these aspects indeed go beyond the scope of the current study.
- A clarification was added, specifying that the issues of large-scale implementation and techno-economic assessment will be addressed in future research.
- The revised section now focuses on the main findings, namely, the increase in the degree of hydrolysis, structural modifications of protein matrices, and the enhanced antioxidant activity of the obtained hydrolysates.
Thus, the Conclusions section now meets the standards of scientific precision and relevance.
References
Page 15 – The references 28 and 29 are not quoted in the manuscript.
Author response:
We thank the reviewer for this valuable comment. All references have been carefully reviewed to ensure their relevance to the content of the manuscript. During the revision process, citations not directly related to the topic of the study were removed, and only those references that directly support the statements presented, describe comparable methods of raw material processing, or provide contextual evidence for the effects of ultrasonic and enzymatic hydrolysis were retained.
In addition, the reference list has been updated and supplemented with recent international sources (2022–2024) published in peer-reviewed journals indexed in Scopus and Web of Science, thereby strengthening the scientific validity and topicality of the manuscript.
Thus, in the final version of the paper, all cited references are fully relevant and consistent with the content of the study.
Author Response File: Author Response.pdf
Round 3
Reviewer 3 Report
Comments and Suggestions for Authors
Materials and Methods
Page 4, line 165 - I suggest that before ‘The subsequent...’ a short sentence be included stating that the preliminary trials described below were carried out. This sentence seems necessary to me because point i) indicates that an enzyme concentration of 3% and a hydrolysis time of 6 hours were used. However, considering the sentence in lines 205 and 206 (“The amount of enzyme added was determined in advance during preliminary studies”), was the enzyme concentration in these trials only 3% or a range of values? After this sentence, ‘The subsequent...’ would follow with the necessary changes.
In point ii), it would also be appropriate to mention the experimental conditions used in hydrolysis after ultrasonic pre-treatment. Also include a sentence stating that the enzyme was inactivated and the hydrolysate obtained was recovered following the methodology mentioned above, i.e., in i). I suggest that the phrase ‘After optimisation...’ should start in a new paragraph followed by the paragraphs beginning with ‘Sample 1’, ‘Sample 2’,... It seems to me that this would establish a better relationship between the preliminary trials and the trials carried out under optimised conditions. Finally, the paragraph ‘Control sample...’ would be included.
Page 7, line 283 – I suggest replacing “Results” with “Results and Discussion”.
Page 7, Figure 2 – In the equations, please replace comma with dot.
Page 7, lines 300-306 - This paragraph is the same as in the previous version of this manuscript, but it seemed to me that the authors had agreed to change it, considering their response to comment on this paragraph: “The overlapping content describing the cavitation phenomena and the mechanism of ultrasonic action was deleted, as similar information is already discussed in the Introduction. The Results section now focuses solely on analytical discussion directly related to the experimental findings”.
Minor comment.
As in line 65, I suggest replacing the three consecutive numbers following the citations on page 2, lines 48, 51 and 76 with [10-12], [13-15] and [16-19] and on page 11, line 419 with [52-54].
Comments on the Quality of English Language
The English Language has been improved compared to the previous version of this manuscript.
Author Response
The authors express their sincere gratitude to the reviewer for the detailed analysis and constructive feedback, which greatly contributed to improving the structure and scientific accuracy of the article, eliminating redundant content, and clarifying the scope and significance of the results. All suggested revisions have been implemented and are highlighted in the revised version of the manuscript.
Materials and Methods
Page 4, line 165 – I suggest that before “The subsequent...” a short sentence be included stating that the preliminary trials described below were carried out. This sentence seems necessary to me because point i) indicates that an enzyme concentration of 3% and a hydrolysis time of 6 hours were used. However, considering the sentence in lines 205 and 206 (“The amount of enzyme added was determined in advance during preliminary studies”), was the enzyme concentration in these trials only 3% or a range of values? After this sentence, ‘The subsequent...’ would follow with the necessary changes. In point ii), it would also be appropriate to mention the experimental conditions used in hydrolysis after ultrasonic pre-treatment. Also include a sentence stating that the enzyme was inactivated and the hydrolysate obtained was recovered following the methodology mentioned above, i.e., in i). I suggest that the phrase ‘After optimisation...’ should start in a new paragraph followed by the paragraphs beginning with ‘Sample 1’, ‘Sample 2’,... It seems to me that this would establish a better relationship between the preliminary trials and the trials carried out under optimised conditions. Finally, the paragraph ‘Control sample...’ would be included.
Author response:
We sincerely thank the reviewer for this thoughtful and detailed comment. A short introductory sentence was added before the description of the main experiment to indicate that preliminary trials were carried out to determine the optimal conditions for enzymatic hydrolysis. The following clarification has been included in the revised text:
“Before the subsequent stages, preliminary trials were carried out to determine the optimal enzyme concentration and hydrolysis duration.”
It has also been specified that during these preliminary studies, a range of enzyme concentrations (from 1% to 5%) and hydrolysis times (from 3 to 8 hours) was investigated, rather than a single value of 3%. This addition clarifies that the chosen parameters — 3% enzyme concentration and 6-hour hydrolysis — were determined based on these optimization experiments.
In section (ii), additional information has been included describing the experimental conditions used for hydrolysis after ultrasonic pre-treatment, and a sentence has been added stating that enzyme inactivation and hydrolysate recovery were performed following the same methodology described in point (i).
In accordance with the reviewer’s suggestion, the phrase “After optimisation...” now begins a
new paragraph, followed by the descriptions of Sample 1, Sample 2, Sample 3, and Sample 4.
This restructuring has improved the logical connection between the preliminary trials and the
optimized experimental conditions, as well as the overall readability of the section.
Page 7, line 283 – I suggest replacing “Results” with “Results and Discussion.”
Author response:
We fully agree with the reviewer’s recommendation. The title of the section has been changed to “Results and Discussion,” which better reflects the integrated presentation and interpretation of the experimental data.
Page 7, Figure 2 – In the equations, please replace comma with dot.
Author response:
This correction has been made. All commas in the equations presented in Figure 2 have been
replaced with dots, in accordance with international standards for decimal notation.
Page 7, lines 300–306 – This paragraph is the same as in the previous version of this manuscript, but it seemed to me that the authors had agreed to change it, considering their response to comment on this paragraph: “The overlapping content describing the cavitation phenomena and the mechanism of ultrasonic action was deleted, as similar information is already discussed in the Introduction. The Results section now focuses solely on analytical discussion directly related to the experimental findings.”
Author response:
We appreciate the reviewer’s careful observation. The paragraph in question has been revised and shortened, as previously agreed. Repetitive content concerning the cavitation mechanism and the general principles of ultrasonic action has been removed, as these aspects are already discussed in the Introduction. The revised paragraph now focuses exclusively on the analysis of the experimental results, without theoretical repetition.
Minor comment – As in line 65, I suggest replacing the three consecutive numbers following the citations on page 2, lines 48, 51 and 76 with [10–12], [13–15] and [16–19] and on page 11, line 419 with [52–54].
Author response:
We thank the reviewer for this careful remark. All suggested corrections to citation formatting have been implemented. The indicated references have been adjusted as follows:
• Page 2: [10–12], [13–15], [16–19]
• Page 11: [52–54]
These changes ensure consistency and accuracy in the citation style in accordance with MDPI referencing standards.
Author Response File: Author Response.pdf
