Review Reports - Cryoprotective Effects of Tuna Skin Antifreeze Peptides on the Quality of Salmon Flesh During Low-Temperature Fluctuations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

The submission has reported the use of tuna skin peptides as natural cryoprotectant as established through protein conformation, biochemical indices, and microstructure of salmon cubes model. The manuscript has number of typographical mistakes to be checked throughout the manuscript. I raise the following concerns in the submission.

Major concerns

Boiling of the collagen hydrolysate mixture having different pH (acidic, alkaline) suspected to have the effect on the hydrolysed and unhydrolyzed collagen which could have been manifested the anti-freeze properties investigated. Authors are requested to explain this for validating the claims of the investigation.
After boiling, the reaction mixture was neutralized and the supernatant has been freezing dried and used for investigating the cryoprotective properties. Why don’t the authors think that the effects observed were also due to synergetic effects?
What was the fate of inactivated enzyme residues in the hydrolysate’s supernatant?
In general, many of the methods used are not detailed enough for deriving the contextual understanding.
Many of the investigated parameters have been carried out on the isolated Myofibrillar proteins. But there are no authentic evidences on the MPs such as electrophoresis or protein profiles have been presented.
There is no sufficient information provided on the peptides.
The hydrolysis process has not been characterized.

Comments for author File: Comments.pdf

Author Response

Dear Editor-in-Chief,

Foods

It is my great honor to submit the revised manuscript entitled “Cryoprotective Effects of Tuna Skin Antifreeze Peptides on the Quality of Salmon Flesh During Low-Temperature Fluctuations” (foods-4073652) based on the comments from the editor and reviewers. These comments are all valuable and very helpful for revising and improving our manuscript, as well as guiding our research work. The authors appreciate the work of the editor and reviewers very much. According to the comments of editor, a clean version of the revised manuscript will be uploaded, and the revised portions were described as clear as possible. We have made a careful response to these comments one by one as the following.

Reviewers' comments:

Comments 1: Please rewrite the abstract. Including the results for some of the parameters investigated will add the interest of the readers. For example, preserved α-helix (to what extent, percentage), enhanced ATPase activity (how much?), inhibited protein and lipid oxidation (to what extent).

Response 1: Thanks for the helpful comments. Therefore, the adverb "significantly" has been added before the expression "inhibited protein and lipid oxidation" to clarify the extent of the inhibition; meanwhile, to enhance readers' interest, specific experimental data for "enhanced Ca²⁺-ATPase activity" and "preserved α-helix structure" have also been supplemented.

These changes can be found in the revised manuscript on Page 1, Abstract paragraph, Lines 22 to 26.

“The neutral protease-treated group exhibited the optimal cryoprotective ability and it also limited aldehyde and sulfide accumulation, preserved the retention rate of α-helix structure at 49% which was higher than 39% in controls, and enhanced Ca²⁺-ATPase activity to 1.75 μmol Pi·mg⁻¹·h⁻¹ with a 45.8% increase compared to controls, and significantly inhibited protein and lipid oxidation.”

Comments 2: Please be specific under what circumferences, the quality deterioration is very common. Or is it irrespective of the type of freezing?

Response 2: Thanks for the helpful comments. It helps clarify the context of the study more precisely. Relevant content has been revised to specify the common occurrence of quality deterioration in salmon products, with specific scenarios added: quality deterioration often happens during the transportation of salmon products, under conditions like prolonged freezing and temperature fluctuations during loading and unloading.

This revision can be found in the revised manuscript on Page 1 to 2, Introduction paragraph, Lines 40 to 47.

“Nevertheless, in the transportation of salmon products, quality changes are relatively common due to prolonged freezing and temperature fluctuations during loading and unloading. Such quality deterioration during freezing and storage is primarily caused by the formation of ice crystals, which disrupt tissue and cellular structures [2]. As a result, the salmon exhibits an altered texture, reduced moisture content, denatured myofibrillar protein (MP), and oxidized lipids, which greatly compromise its nutritional, organoleptic and commercial value.”

Comments 3: The values for calories and degree of sweetness can be mentioned.

Response 3: Thanks for the helpful comments. The calorie values of sorbitol and sucrose have been supplemented to address this suggestion.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 50 to 53.

“as demonstrated by the fact that each gram of sorbitol provides approximately 2.4 kilocalories (kcal) of energy, while each gram of sucrose provides approximately 4.0 kilocalories (kcal) of energy, which restrict their further application in aquatic products.”

Comments 4: Please change as ‘showed’

Response 4: Thanks for the helpful comments. The relevant wording has been adjusted as suggested, with the corresponding expression changed to "showed" to comply with proper language usage.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Line 62.

“and showed significant cryoprotective effects on frozen surimi.”

Comments 5: Please roughly indicate the high value realization (USD/kg)

Response 5: Thanks for the helpful comments. The comparative advantages of the proposed tuna skin antifreeze peptides over conventional cryoprotectants have been clarified in the relevant sections of the revised manuscript.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 68 to 69.

“a potential that translates to a value of roughly 15-20 USD/kg for such bioderived antifreeze agents.”

Comments 6: Please rewrite the sentences. Suggested to mention the names of functional proteins.

Response 6: Thanks for the helpful comments. Relevant sentences have been revised as suggested, explicitly naming the functional proteins present in tuna skin.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 70 to 73.

“Tuna skin, a major underutilized by-product of tuna processing, serves as an excellent substrate for the production of antifreeze proteins due to its abundance of collagen and various functional proteins, including antioxidant proteins, antibacterial proteins, and antifreeze proteins [8].”

References cited

Ahmed, R.; Chun, B.-S. Subcritical water hydrolysis for the production of bioactive peptides from tuna skin collagen. The Journal of Supercritical Fluids 2018, 141, 88-96. https://doi.org/10.1016/j.supflu.2018.03.006.

Comments 7: Please provide space before the reference. Also mention the enhanced water holding capacity. Please provide the values.

Response 7: Thanks for the helpful comments. Relevant suggestions have been addressed with corresponding revisions made in the appropriate sections of the revised manuscript, including adding a space before the reference and supplementing the specific value for the enhanced water-holding capacity.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 83 to 87.

“For instance, Ej-AFP peptide from Evynnis japonica scales was reported to control ice crystal sizes by regulating morphology, and also significantly enhanced the water-holding capacity of surimi gels after freeze-thaw cycles, with an improvement of more than 40% compared to traditional commercial cryoprotectants [7].”

Comments 8: With limited number of studies, generalizing that marine by products derived AFP…. seems to be too exaggerating. Please rewrite.

Response 8: Thanks for the helpful comments. The relevant statement has been revised to avoid overgeneralization, making the wording more appropriate and thus enhancing the rigor of the manuscript’s expression.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 89 to 90.

“providing preliminary support for the potential protective role of certain marine by-product-derived AFPs in safeguarding the quality of frozen muscle.”

Comments 9: Boiling for 10 min to deactivate the enzyme activity has been carried out at different pH. How the pH mediated effect on the manipulation of the properties has been accounted? Boiling step could have been carried out after neutralization of the reaction mixture to avoid the effect of pH under boiling. Please explain.

Response 9: Thanks for the helpful comments. Here is the explanation for the design of the enzyme deactivation step: Even under non-optimal pH conditions, proteases retain partial residual activity. If neutralization is performed first, the residual proteases will continue to hydrolyze in the non-optimal pH environment during the brief interval (5-10 minutes) between pH adjustment and boiling. This will lead to uncontrollable variations in peptide length and structure within each group, obscuring the true effect of protease specificity. In contrast, boiling at the initial hydrolysis pH enables rapid and complete inactivation, ensuring the immediate termination of hydrolysis. The effects of boiling under different pH conditions on hydrolyzed and unhydrolyzed collagen are controllable and exhibit intra-group consistency, which will not interfere with the research conclusion that "differences in cryoprotective properties originate from protease-specific hydrolysis." After denaturation, unhydrolyzed collagen loses its original spatial structure and its content is extremely low, rendering it unable to participate in cryoprotective processes such as ice-binding and inhibition of recrystallization. Thus, its impact on the overall cryoprotective properties is negligible and does not compromise the research conclusion. As proteases are inherently proteins, boiling disrupts their spatial structure, resulting in the complete loss of not only catalytic activity but also the ability to bind to ice crystals and inhibit recrystallization, thereby preventing them from participating in cryoprotective processes. Following boiling and neutralization, the reaction mixture is subjected to centrifugation. Inactivated proteases, as macromolecular denatured proteins with low solubility, precipitate together with unhydrolyzed collagen. The sample used for subsequent cryoprotective property analysis is the freeze-dried supernatant, meaning that inactivated proteases do not enter the detection system and therefore cannot exert a synergistic effect with hydrolyzed peptides.

Comments 10: What is the practical relevance of measuring the centrifugal loss?

Response 10: Thanks for the helpful comments. Here is the detailed explanation: Measuring centrifugal loss is critical for evaluating the cryoprotective effects of antifreeze peptides on salmon meat. It quantifies the water retention capacity of muscle tissue under mechanical stress and directly relates to commercial processing efficiency and logistics quality.

In salmon meat processing centrifugal loss simulates mechanical stress during preprocessing stages such as dehydration after washing or mechanical handling for portioning and packaging. High centrifugal loss means freeze-thaw damaged muscle fibers cannot retain water under such forces. This reduces the net yield of usable meat and increases raw material costs.

In long-distance logistics centrifugal loss mimics vibration and mechanical shock during intercontinental refrigerated transport of frozen salmon fillets. Such stress causes water seepage from damaged muscle tissue. It leads to hidden quality degradation such as texture hardening or nutrient loss even before thawing. Centrifugal loss quantifies the resilience of salmon muscle structure. It enables evaluation of the ability of antifreeze peptides to maintain product quality during long-haul shipping.

Comments 11: Recommended to report the springiness as an index rather than expressing it as springiness having the unit in mm. Though the cubes dimension has been mentioned, to avoid the contribution of the sample height relative expression is more preferable.

Response 11: Thanks for the helpful comments. In accordance with your suggestion, in standard Texture Profile Analysis (TPA), springiness is a dimensionless relative index defined as the ratio of the sample's recovery height to the compression distance. However, the original data in this study reflect the absolute recovery height (unit: mm) of salmon samples after compression, which does not conform to the strict definition of springiness. To avoid terminology misuse while preserving the authenticity of experimental data, the term springiness has been uniformly replaced with elasticity throughout the manuscript.

A comprehensive check of the terminology in all relevant sections of the paper has been conducted to ensure consistent use of the term elasticity, thereby enhancing academic rigor.

This modification not only addresses the concern regarding avoiding the use of length units with springiness but also fully retains the original experimental data, truthfully reflecting the mechanical recovery capacity of salmon muscle tissue.

Comments 12: Please provide the data/evidence related to ice crystal formation.

Response 12: Thanks for the helpful comments. Relevant evidence related to ice crystal formation has been supplemented to clarify the mechanistic link between ice crystal formation and salmon texture changes in the study.

This revision can be found in the revised manuscript on Page 8, Section 3.1 (Textural properties) paragraph, Lines 314 to 319.

“SEM observations confirm that tuna skin collagen hydrolysates, especially NH, mitigate ice crystal-mediated damage to salmon muscle microstructure by maintaining densely packed fiber bundles with narrow inter-fibrillar gaps and intact sarcolemma while NCon shows disorganized, fragmented fibers with enlarged gaps that directly correlate with the graded textural quality across groups.”

Comments 13: Recommended to provide the molecular weight of peptides obtained in the hydrolysate prepared in the present investigation.

Response 13: Thanks for the helpful comments. Detailed information on the molecular weight range of the peptides, the amino acid composition of the peptide sequences, and other relevant characteristics has been supplemented.

This revision can be found in the revised manuscript on Page 9, Section 3.1 (Textural properties) paragraph, Lines 323 to 326.

“In contrast to the other treatment groups, NH treatment generates peptides with a relatively uniform molecular weight distribution as shown in Table S1 and a high proportion of polar amino acids (e.g., aspartic acid, glutamic acid) [25].”

Comments 14: As the results are relative assessment, for a better understanding the presenting the initial moisture content after the treatment is recommended.

Response 14: Thanks for the helpful comments. Explicit initial moisture content data are valuable for relative assessments, and the moisture loss indices including thawing loss, cooking loss and centrifugal loss presented in Table 2 of the manuscript already reflect the initial moisture content after treatment. These loss values were calculated as the percentage of moisture lost relative to the initial moisture content of each sample post peptide treatment after soaking in antifreeze peptides and before freeze-thaw cycles. Thus, the lower loss rates in peptide-treated groups compared to the NCon group directly indicate that these groups retained higher initial moisture content after treatment. The results also highlight the core focus of this study: the peptide-treated groups not only exhibited significantly enhanced WHC compared to the NCon group without antifreeze peptides but also showed favorable WHC performance relative to the Con group, which aligns with the goal of evaluating the cryoprotective efficacy of the antifreeze peptides.

Comments 15: It may be ideal to present the related data for the peptides used in the present investigation.

Response 15: Thanks for the helpful comments. Detailed information regarding the molecular weight range of the peptides, the amino acid composition of the peptide sequences, and other relevant characteristics has been supplemented, and the ambiguous expression "narrow molecular weight distribution" has been replaced with a specific, well-defined molecular weight range. Peptides within this defined range are characterized by highly homogeneous chain lengths and uniform distribution of hydrophilic and charged groups, enabling dense and uniform adsorption onto ice crystal surfaces. Specifically, the incorporation of water molecules into the ice crystal lattice is blocked via the formation of a continuous hydrogen-bonding network; the ordered arrangement of ice crystals is disrupted by a uniform electrostatic field; and the coalescence of small ice crystals is simultaneously inhibited. Accordingly, highly efficient inhibition of ice crystal growth and recrystallization is achieved.

This revision can be found in the revised manuscript on Page 10, section 3.2.1 Water holding capacity paragraph, Lines 377 to 387.

“The superior WHC of NH was likely related to its peptide composition. Peptides generated under optimal hydrolysis conditions (50 °C, pH 7.0) exhibited a highly concentrated molecular-weight distribution, with details provided in Table S1. Among a total of 869 peptide sequences, 94.8% were concentrated in the range of 702-3000 Da, and 72.7% were even below 2000 Da. These peptides were also rich in polar amino acids such as aspartic acid and glutamic acid [14]. Such peptides not only inhibit ice crystal growth and recrystallization through interfacial adsorption, but also stabilize MP conformation by maintaining the hydrophobic microenvironment of tryptophan residues and retaining α-helix content (49%) via hydrogen bonding with MP backbones [14]. Through these combined effects, these peptides reduced structural damage improved the water-holding capacity of muscle tissues during freezing and thawing process [30,31].”

References cited

Yasemi, M. Prevention of denaturation of freshwater crayfish muscle subjected to different freeze-thaw cycles by gelatin hydrolysate. Food Chemistry 2017, 234, 199-204. https://doi.org/10.1016/j.foodchem.2017.04.183.

Xu, Z.; Cao, S.; Cui, N.; Zhang, R.; Qin, Z.; Liu, H.; Wu, J.; Du, M.; Tan, Z.; Li, T. Screening and characterization of an antifreeze peptide from sea cucumber intestinal protein hydrolysates. Food Chemistry 2025, 463, 141194. https://doi.org/10.1016/j.foodchem.2024.141194.

Comments 16: It is recommended to present the colour changes (L*. a* and b*) after dip treatment before freezing and thawing.

Response 16: Thanks for the helpful comments. The rationale behind this suggestion is noted, and similar to the previous clarification on moisture content, the color data in the current Table 2 already aligns with the core focus of this study and common practice in related literature.

All samples including the NCon group showed highly consistent color characteristics right after soaking pre-freeze-thaw cycles, as the soaking treatment either with antifreeze peptides or the control solution itself does not induce significant color changes in salmon meat. Thus, the color differences presented in Table 2 measured post-freeze-thaw directly reflect the degree of color deterioration caused by freeze-thaw cycles, which is the key endpoint for evaluating the protective effect of antifreeze peptides against freeze-thaw-induced color degradation.

References cited

Xu, Z.; Cao, S.; Zhu, Z.; Hu, B.; Chen, H.; Tu, M.; Tan, Z.; Du, M.; Li, T. Characterization and the mechanism underlying the cryoprotective activity of a peptide from large yellow croaker (pseudosciaena crocea). Food Chemistry 2024, 435, https://doi.org/10.1016/j.foodchem.2023.137512.

Comments 17: Recommended to rewrite. The hydrolysis conditions do not have direct relationship with the structural changes observed in salmon flesh. Please rethink on the explanation provided.

Response 17: Thanks for the helpful comments. Images in this section have been redescribed to ensure the accuracy of microstructural descriptions and alignment with the actual microstructural features shown in the images.

This revision can be found in the revised manuscript on Page 14, Section 3.5. SEM analysis paragraph, Lines 531 to 538.

“The myofibrillar structural damage in the PH group was the most prominent: its inter-fiber gaps were markedly enlarged to 20-30 μm, accompanied by fiber fractures and the formation of local pores. This outcome stems from its relatively low cryoprotective efficacy. During freeze-thaw cycles, it failed to effectively inhibit the growth and recrystallization of ice crystals, which in turn led to gap expansion, fiber fractures, and pore formation. This also mutually corroborates with the significant disruption of the secondary structure of myofibrillar proteins in the PH group (Section 3.4).”

Comments 18: If we assume that the freeze thaw cycle affected the SH group, it is reflected in the MP isolation. The SH content has been assessed on the isolated proteins. How could this be solely attributed to the effect of AFP. Kindly provide the data on the yield of MP isolates. The SH expressed is per g. In this gram refer to the isolate or the protein. The methodologies are not detailed enough for a better clarity.

Response 18: Thanks for the helpful comments. Quantification of (T-SH) in MP was determined with a commercial assay kit (A063-2-1, Nanjing Jiancheng Bioengineering Institute Co. Ltd., Nanjing, Jiangsu, China) following the manufacturer's recommended procedure, which is consistent with the methodology employed in previous similar studies on AFP, more detailed operations have been added in Section 2.7.2 to clarify methodological details, unit definition, MP isolate yield, and result interpretation ambiguities regarding total SH content analysis.

According to the A063-2-1 assay kit instructions, the "gram" in the "-SH content per gram" unit refers to the protein in the MP isolate (denoted as "gprot" in the kit’s formula, where "Cpr" represents the protein concentration of tissue homogenate in gprot/L). The final SH content is calculated as μmol per gram of protein in the MP isolate (μmol/gprot), consistent with the kit’s recommended unit. The relevance of MP isolate yield data is acknowledged. However, in studies focusing on the effect of AFP on MP sulfhydryl groups, the determination of total SH content and protein solubility is a widely accepted and common practice. This is because the core research focus lies in evaluating AFP-induced changes in MP structural stability reflected by SH content and solubility rather than MP extraction efficiency. The experimental design is consistent with this established research paradigm, ensuring comparability with existing literature. Freeze-thaw cycles inherently affect SH groups by inducing MP oxidation and structural damage, as this is a well-recognized phenomenon in frozen meat research. This study does not attribute SH content changes solely to AFP but instead emphasizes the protective role of AFP against freeze-thaw-induced damage.

This revision can be found in the revised manuscript on Page 6-7, Section 2.7.2. Total sulfhydryl (T-SH) content paragraph, Lines 234 to 245.

“Quantification of (T-SH) in MP was determined with a commercial assay kit (A063-2-1, Nanjing Jiancheng Bioengineering Institute Co. Ltd., Nanjing, Jiangsu, China) following the manufacturer's recommended procedure: 10% tissue homogenate was prepared by homogenizing salmon muscle with 0.9% physiological saline (1:9, w/v) under ice bath, centrifuged at 2500 rpm for 10 min to collect the supernatant; 10 mmol/L standard stock solution, 500 μmol/L standard application solution, and two working solutions (working solution 1: Reagent 1: Reagent 2: Reagent 4 = 50:25:25; working solution 2: Reagent 2: Reagent 3 = 1:75) were prepared as instructed; 10 μL of standard/application solution or sample supernatant was added to corresponding wells followed by 150 μL of working solution, incubated for 5 min, and absorbance was determined at 405 nm with a microplate analyzer (Bio-Tek, Paramus, NJ, USA); T-SH content was calculated using the kit’s formula.”

References cited

Xu, Z.; Guan, S.; Cao, S.; Zhang, R.; Liu, H.; Qin, Z.; Dong, X.; Wu, J.; Li, T. Cryoprotective activity of different characterized fractions isolated from sea cucumber intestinal protein hydrolysates against salmon. Food Chemistry 2025, 491, 145136. https://doi.org/10.1016/j.foodchem.2025.145136.

Yang, F.; Jiang, W.; Chen, X.; Wu, J.; Huang, J.; Cai, X.; Wang, S. Investigation on the quality regulating mechanism of antifreeze peptides on frozen surimi: From macro to micro. Food Research International 2023, 163, Article 112299. https://doi.org/10.1016/j.foodres.2022.112299.

Comments 19: Conclusion mentions skin protein hydrolysate, but the study has extracted the collagen and hydrolysed using three different proteases. Why don’t authors name the products as collagen peptides or hydrolysates? Why do the authors prefer to designate the product as skin hydrolysate when the substrate is not as such the skin?

Response 19: Thanks for the helpful comments. Employing collagen-related terminology to enhance the rigor of writing is absolutely correct. Previously, the simplified term “skin hydrolysates” was used to emphasize that the peptides originate from tuna skin; this naming was intended to help readers quickly and intuitively link the product to its raw material source. However, in line with the suggestion to use more precise terminology, the term has now been uniformly revised to “tuna skin collagen hydrolysates” throughout the entire manuscript, ensuring both accuracy and consistency in wording.

Comments 20: Please rewrite for the appropriate language.

Response 20: Thanks for the helpful comments. Relevant suggestions have been carefully implemented, with key revisions as follows: Redundant presentation of numerical results in the results section was trimmed, and only descriptions of core trends were retained to avoid unnecessary repetition and highlight main research findings. Additionally, content was added to note that high-value utilization of tuna skin waste in line with circular economy principles is an aspect insufficiently addressed in previous relevant studies. The practical significance of this study was supplemented by including guidance for the food industry in pursuing natural, safe, and resource-efficient preservation technologies, further clarifying the application value of the research findings. Language expression was also polished to render the conclusion more concise and coherent.

This revision can be found in the revised manuscript on Page 18 to 19, Conclusion paragraph, Lines 679 to 695.

“For AFPs derived from tuna skin collagen hydrolysates prepared with pepsin, trypsin, and neutral protease, NH exhibited the most effective cryoprotective performance. It effectively maintained the texture, color, and WHC of salmon samples after freeze-thaw cycles. NH also reduced MP aggregation and oxidation, stabilized protein conformation. For example, NH’s α-helix content (49%) was the highest among all treatment groups except Con. These protective effects are likely ascribed to the suppression of ice crystal growth and the preservation of protein structural integrity. Notably, NH overcomes key limitations of commercial cryoprotectants including high caloric content, potential off-flavors, and consumer concerns over synthetic additives. As a natural derivative of tuna skin, which is an underutilized seafood processing waste, NH offers a safe and biocompatible alternative that aligns with circular economy principles. This aspect has not received sufficient attention in previous studies on AFPs derived from marine by-products. In conclusion, NH showed strong cryoprotective potential and represents a promising natural preservative for aquatic products. This study provides a viable alternative to synthetic cryoprotectants and establishes a sustainable approach for valorizing tuna processing by-products. It offers practical guidance for the food industry in pursuing natural, safe and resource-efficient preservation technologies.”

Comments 21: The hydrolysis process has not been characterized.

Response 21: Thanks for the helpful comments. The characterization of the hydrolysis process has been supplemented in the revised manuscript by adding the relevant content of SDS‑PAGE analysis.

This revision can be found in the revised manuscript on Page 18, Section 3.6.7. SDS-PAGE paragraph, Lines 633 to 648.

“As shown in Figure 3g, the main visible bands include myosin heavy chain (MHC), actin, tropomyosin and myosin light chain (MLC). All samples exhibit typical MP bands, verifying reliable MP extraction. Con without freezing presents clear, sparse bands with sharp, non-diffused MHC, actin, tropomyosin and trace natural MLC, reflecting native MP structure. NCon without hydrolysate protection shows significant protein degradation, with band broadening, tailing, diffusion, intensified low molecular weight bands and nearly vanished MHC. These changes indicate severe MP degradation, fragmentation, reduced partial protein solubility and impaired structural integrity induced by freezing. Compared with NCon, all the hydrolysate treated groups show varying protective effects, characterized by intact MHC and regular overall band distribution, confirming hydrolysates effectively inhibit freezing-induced MP structural damage. NH group exerts the most prominent protective effect, with its electrophoretic profile closest to that of Con, intact MHC free of obvious degradation, fewer low molecular weight bands and MP composition highly consistent with the native state. TH and PH groups also alleviate the formation of low molecular weight degradation bands, but their protective effects are lower than those of the NH group.”

Comments 22: Many of the investigated parameters have been carried out on the isolated Myofibrillar proteins. But there are no authentic evidences on the MPs such as electrophoresis or protein profiles have been presented.

Response 22: Relevant content regarding SDS‑PAGE analysis has been supplemented in the revised manuscript, as provided in Section 2.7.7 and Section 3.6.7, respectively.

We appreciate these helpful comments and suggestions from the editor and reviewers, which are very important for us to improve our research work in the future. We also greatly appreciate the opportunity of resubmitting given by the editor. Please kindly consider the publication of the manuscript in the Foods. Thank you very much.

Sincerely yours,

Zhe Xu

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study is well structured, properly designed, and clearly written. However, some modifications are necessary.

In the conclusions of the abstract, the authors are advised to better explain the novel contribution of the manuscript and its practical relevance to the food industry, as well as the potential use of AFPs as natural cryoprotectants.

The authors are recommended to strengthen the Introduction through a more critical discussion of the state of the art, more clearly defining the knowledge gap and the novelty of the study. In particular, it would be useful to further explain how tuna skin-derived AFPs differ from or provide advantages over AFPs obtained from other marine by-products, as well as to expand the description of the proposed mechanisms of action. In addition, the study objective should be formulated more precisely.

Lines 74-82: The authors are encouraged to provide more detailed information on the initial characteristics of the salmon used (e.g., exact species, average weight, postmortem state, or time since slaughter), as these factors may significantly influence protein quality and the response to freeze-thaw treatments.

Lines 83-111: Although the extraction and preparation procedures for the hydrolysates are well described, it would be beneficial to include additional information regarding hydrolysate yield and basic characterization (or alternatively include this information in the Results section).

Lines 103-111: The authors are advised to more explicitly justify the selected hydrolysis conditions (enzyme type, pH, temperature, and time), as these parameters directly influence peptide composition and, consequently, the observed antifreeze activity.

Lines 112-123: The authors should justify the selected peptide concentration (2.0 mg/mL) and immersion time (4 h), and clarify whether these conditions were based on preliminary experiments or previous literature.

Lines 120-123: A brief justification linking the number of freeze-thaw cycles and the selected freezing–thawing conditions to realistic commercial storage and transportation scenarios is recommended, as this would enhance the practical relevance of the study.

It would be helpful for readers if, at the beginning of each subsection in Materials and Methods, the authors briefly indicated the purpose of each analyzed parameter.

Lines 221-254: The authors are encouraged to reduce the repetition of numerical results already presented in Table 1 and to emphasize the key findings and overall trends. In addition, the discussion could be strengthened by comparing the obtained values with previous studies evaluating AFPs in similar muscle matrices.

Lines 257-280: The discussion of WHC could be improved by more clearly relating these results to the structural changes in myofibrillar proteins described in subsequent sections.

Lines 282-313: The authors could further highlight the technological and sensory relevance of changes in the L*, a*, and b* color parameters in relation to consumer acceptance.

Lines 437-522: The analysis of multiple oxidation indicators is extensive. The authors are encouraged to condense this section and avoid reiterating similar explanations for each parameter.

In the Conclusions, it is recommended to reduce the repetition of numerical results already presented in the Results section and to emphasize the main findings in relation to the study objectives. Additionally, the novel contribution of the study compared with previous work on antifreeze peptides derived from marine by-products should be highlighted more clearly.

Given the large number of physicochemical, structural, and biochemical variables evaluated, the authors are encouraged to include a multivariate analysis, such as Principal Component Analysis (PCA). This approach would allow a more integrated interpretation of the results and further enhance the overall quality of the manuscript.

Author Response

Dear Editor-in-Chief,

Foods

Reviewers' comments:

Reviewer #2:

Comments 1: In the conclusions of the abstract, the authors are advised to better explain the novel contribution of the manuscript and its practical relevance to the food industry, as well as the potential use of AFPs as natural cryoprotectants.

Response 1: Thanks for the helpful comments. Revisions have been made to the abstract’s conclusion accordingly. Key revisions are as follows: the valorization of tuna skin by-products for freeze-thaw preservation of premium seafood (i.e., salmon) has been highlighted, addressing the gap in converting underutilized aquatic processing by-products into high-end seafood preservatives; the work has been directly linked to industrial demands, including meeting the food industry’s need for clean-label, low-calorie preservation solutions and advancing the circular economy in aquatic processing; the natural, healthy attributes of tuna skin AFPs have been emphasized, framing them as superior alternatives to synthetic or sugar-based cryoprotectants hindered by high caloric content and incompatibility with clean-label trends.

These changes can be found in the revised manuscript on Page 1, Abstract paragraph, Lines 30 to 33.

“healthy cryoprotectants for seafood preservation, meeting the growing demand of the food industry for clean-label, low-calorie preservation solutions, while advancing the circular economy of aquatic processing via the valorization of tuna skin by-products for high-value seafood applications.”

Comments 2: The authors are recommended to strengthen the Introduction through a more critical discussion of the state of the art, more clearly defining the knowledge gap and the novelty of the study. In particular, it would be useful to further explain how tuna skin-derived AFPs differ from or provide advantages over AFPs obtained from other marine by-products, as well as to expand the description of the proposed mechanisms of action. In addition, the study objective should be formulated more precisely.

Response 2: Thanks for the helpful comments. Revisions have been made to the Introduction section in line with the comments as follows: Critical discussion of the state of the art has been strengthened, clarifying the knowledge gap and highlighting the novelty of this study. Advantages of tuna skin-derived AFPs over those from other marine by-products have been supplemented: tuna processing generates by-products accounting for 50%–70% of the total fish weight, and tuna skin (4%-6% of total fish weight) provides a more abundant and stable raw material supply. The study objective has been refined to focus on evaluating the cryoprotective effect of tuna skin-derived AFPs on high-value seafood (using salmon as an example) during freeze-thaw cycles and exploring the underlying mechanism.

These changes can be found in the revised manuscript on Page 1, Introduction paragraph, Lines 60 to 69, Lines 73 to 80 and 94.

“For instance, antifreeze peptides have been successfully extracted from aquatic biomass like grass carp skin and Evynnis japonica scales [6,7], and showed significant cryoprotective effects on frozen surimi. However, most existing studies focus on low-value aquatic products such as surimi rather than high-value species like salmon, leaving a critical knowledge gap in understanding AFPs' efficacy in premium seafood with delicate muscle structures and strict quality requirements. Particularly, AFPs derived from aquatic by-products have gained increasing attention due to their biocompatibility, low toxicity, and potential for sustainable, high-value utilization of processing residues, a potential that translates to a value of roughly 15-20 USD/kg for such bioderived antifreeze agents.”

“Compared to AFPs from other marine by-products such as shrimp heads and fish viscera, tuna skin-derived AFPs offer a distinct advantage in sustainable and abundant raw material supply. Tuna processing generates massive amounts of by-products, accounting for 50% to 70% of the total fish weight. Among these by-products, tuna skin is a key component with a stable proportion, ranging from 4% to 6% of the total fish weight, a considerable share that ensures a continuous and sufficient raw material source for AFP production. This advantage addresses the resource scarcity challenge faced by AFPs derived from other marine by-products with limited overall supply.”

References cited

Oliveira, D.; Bernardi, D.; Drummond, F.; Dieterich, F.; Boscolo, W.; Leivas, C.; Kiatkoski, E.; Waszczynskyj, N. Potential use of tuna (thunnus albacares) by-product: Production of antioxidant peptides and recovery of unsaturated fatty acids from tuna head. International Journal of Food Engineering 2017, 13, Article 20150365. https://doi.org/10.1515/ijfe-2015-0365.

Comments 3: The authors are encouraged to provide more detailed information on the initial characteristics of the salmon used (e.g., exact species, average weight, postmortem state, or time since slaughter), as these factors may significantly influence protein quality and the response to freeze-thaw treatments.

Response 3: Thanks for the helpful comments. Detailed and accurate initial characteristics of the salmon have been supplemented in the revised manuscript.

These detailed descriptions are added in the revised manuscript on Page 4, Materials and Methods section 2.1. Materials paragraph, Lines 105 to 107.

“the salmon used was of the specific species Oncorhynchus, freshly slaughtered and transported to the laboratory under a cold chain at -20 °C, each piece of fish flesh (half a fish) had an average weight of 230 g,”

Comments 4: Although the extraction and preparation procedures for the hydrolysates are well described, it would be beneficial to include additional information regarding hydrolysate yield and basic characterization (or alternatively include this information in the Results section).

Response 4: Thanks for the helpful comments. The advice has been carefully followed, and the manuscript has been revised accordingly to supplement information on the yield and basic characteristics of the hydrolysates.

These detailed descriptions are added in the revised manuscript on Page 4, Materials and Methods section 2.2. Preparation of hydrolysates from tuna skin protein paragraph, Lines 147 to 150.

“The yield of each hydrolysate was calculated as the mass of freeze-dried product relative to that of initial collagen used, with yields as follows: PH had a yield of 0.4 grams per gram of collagen, TH 0.8 grams per gram of collagen, and NH 0.85 grams per gram of collagen.”

Comments 5: The authors are advised to more explicitly justify the selected hydrolysis conditions (enzyme type, pH, temperature, and time), as these parameters directly influence peptide composition and, consequently, the observed antifreeze activity.

Response 5: Thanks for the helpful comments. Relevant justifications for the selected hydrolysis conditions have been added.

These detailed descriptions are added in the revised manuscript on Page 4, Materials and Methods section 2.2. Preparation of hydrolysates from tuna skin protein paragraph, Lines 138 to 142.

“These three proteases and their respective hydrolysis conditions were selected to cover a broad range of proteolytic environments, where each combination of temperature, pH value and duration represents the enzyme-specific optimal activity range, consistent with the methodologies established in previous studies on collagen hydrolysis [12].”

References cited

Zhu, K.; Zheng, Z.; Dai, Z. Identification of antifreeze peptides in shrimp byproducts autolysate using peptidomics and bioinformatics. Food Chemistry 2022, 383, Article 132568. https://doi.org/10.1016/j.foodchem.2022.132568.

Comments 6: The authors should justify the selected peptide concentration (2.0 mg/mL) and immersion time (4 h), and clarify whether these conditions were based on preliminary experiments or previous literature.

Response 6: Thanks for the helpful comments. The selected peptide concentration and immersion time were determined based on our team’s previous literature focusing on aquatic cryoprotective peptides: 2.0 mg/mL has been validated as an effective concentration for mitigating freeze-thaw damage in seafood muscle tissues, and the references have been added in the text.

This revision can be found in the revised manuscript on Page 5, Materials and Methods section 2.3. Preparatory treatment of salmon cubes paragraph, Lines 159.

References cited

Comments 7: A brief justification linking the number of freeze-thaw cycles and the selected freezing–thawing conditions to realistic commercial storage and transportation scenarios is recommended, as this would enhance the practical relevance of the study.

Response 7: Thanks for the helpful comments. The three freeze–thaw cycles were designed as an accelerated test to simulate extreme transportation and storage conditions, which represents a widely recognized strategy in the efficacy evaluation of cryoprotectants for aquatic products. This design enables comprehensive evaluation of the protective effect of the cryoprotectant under the most challenging scenarios (e.g., repeated temperature fluctuations in long-distance cold chain logistics), which is consistent with the experimental framework of typical aquatic cryoprotection studies.

This revision can be found in the revised manuscript on Page 5, Materials and Methods section 2.3. Preparatory treatment of salmon cubes paragraph, Lines 162-163.

“This accelerated test enables comprehensive assessment of the protective effect of the cryoprotectant under challenging conditions.”

References cited

Jiang, Z.; Tang, Z.; Liu, M.; Yu, W.; Liao, T.; Qiu, L. Preparation of antifreeze glycopeptides from crayfish shells peptides with dextran by non-enzymatic and its applications for frozen pork. Journal of Future Foods 2025, https://doi.org/10.1016/j.jfutfo.2025.05.002.

Comments 8: It would be helpful for readers if, at the beginning of each subsection in Materials and Methods, the authors briefly indicated the purpose of each analyzed parameter.

Response 8: Thanks for the helpful comments. A brief description of the purpose of each analyzed parameter has been added at the beginning of the relevant subsections in the Materials and Methods section.

These detailed descriptions are added in the revised manuscript on Page 5 to 6, Materials and Methods section 2.4. Determination during freeze-thaw cycles of salmon physical properties paragraph and 2.6. Analysis of MP structure paragraph, Lines 167 to 168, 187 to 188, 193 to 194 and Lines 214 to 215.

“To reflect the effect of freeze-thaw cycles on the muscle structure of salmon and evaluate the textural quality by analyzing texture parameters.”

“To monitor the color changes of salmon during freeze-thaw cycles, as color is a direct indicator of visual quality and muscle deterioration.”

“To detect changes in the odor profile of salmon during freeze-thaw cycles, identifying the generation of off-flavors related to quality deterioration.”

“To explore the effects of freeze-thaw cycles on the secondary and microstructures of MP, revealing the correlation between structural changes and functional deterioration.”

Comments 9: The authors are encouraged to reduce the repetition of numerical results already presented in Table 1 and to emphasize the key findings and overall trends. In addition, the discussion could be strengthened by comparing the obtained values with previous studies evaluating AFPs in similar muscle matrices.

Response 9: Thanks for the helpful comments. Redundant presentation of data already shown in the table within this paragraph has been removed, the analysis of trends has been retained, an analysis associated with SEM has been added, and a horizontal comparison with data from other experiments has been incorporated.

This revision can be found in the revised manuscript on Page 9, Section 3.1 (Textural properties) paragraph, Lines 314 to 319 and 340 to 345.

“In other freeze-thaw studies on meat [17], the hardness increase of the optimal treatment group compared to the worst group was approximately 36.2% for the hardness index. In contrast, the hardness increase of our NH group compared to the NCon group reached 106.7%, which is significantly higher. Furthermore, the improvements in other textural indicators in our study were also significantly higher than those in the reference study.”

Comments 10: The discussion of WHC could be improved by more clearly relating these results to the structural changes in myofibrillar proteins described in subsequent sections.

Response 10: Thanks for the helpful comments. Following the suggestions, targeted revisions have been made, with the core focus on explicitly linking the WHC results to the changes in MP structure described in subsequent sections.

These changes can be found in the revised manuscript on Page 9 to 10, 3.2.1. Water holding capacity paragraph, Lines 359 to 364, 369 to 371 and 382 to 385

“Results of thawing, cooking, and centrifugal losses are shown in Table 2. The Con showed highest water holding capacity, with minimal cooking and centrifugal losses of 11.10% and 6.42%, respectively. This was attributed to its intact MP network, which is characterized by a stable tertiary structure (tryptophan residues in a hydrophobic microenvironment) and high α-helix content (57%), and which retained water through hydrogen bonding and hydrophobic interactions [26,27].”

“The other is MP denaturation induced by freeze-thaw, which causes structural alterations such as exposure of tryptophan residues to polar environments and α-helix unfolding to 39% [29].”

“Such peptides not only inhibit ice crystal growth and recrystallization through interfacial adsorption, but also stabilize MP conformation by maintaining the hydrophobic microenvironment of tryptophan residues and retaining α-helix content (49%) via hydrogen bonding with MP backbones [14].”

Comments 11: The authors could further highlight the technological and sensory relevance of changes in the L*, a*, and b* color parameters in relation to consumer acceptance.

Response 11: Thanks for the helpful comments. The advice has been followed, and relevant content has been added to the revised manuscript; changes in L*, a*, and b* have been clearly linked to consumer acceptance. As suggested, this revision strengthens the technological and sensory relevance between color parameters and consumer responses.

This revision can be found in the revised manuscript on Page 10, Section 3.2.2. Color paragraph, Lines 391 to 398.

“In general, the market price of frozen salmon depends largely on its color. As mentioned in previous studies [33], consumers' willingness to pay (WTP) for salmon increases significantly with improved color characteristics. Salmon with vivid and uniform redness corresponding to optimal a* values and minimal yellowness with low b* values can attract consumers to purchase at a premium. Conversely, salmon with faded redness, excessive yellowness or dull appearance with low L* values is difficult to gain consumer acceptance and even hard to sell at a reasonable price.”

Comments 12: The analysis of multiple oxidation indicators is extensive. The authors are encouraged to condense this section and avoid reiterating similar explanations for each parameter.

Response 12: Thanks for the helpful comments. The relevant sections have been carefully revised as requested, with specific adjustments focused on Sections 3.6.1, 3.6.2, 3.6.4, 3.6.5, and 3.6.6.

The core revisions are as follows: All key experimental data, group comparisons, indicator significance, and citations have been retained without altering the main content or core conclusions. For each revised paragraph, the redundant reiteration of mechanisms (e.g., ice crystal inhibition, structural stabilization) at the end has been removed, eliminating unnecessary repeated elaboration. By condensing the concluding statements, the content has been rendered more concise while maintaining logical coherence and academic rigor.

Comments 13: In the Conclusions, it is recommended to reduce the repetition of numerical results already presented in the Results section and to emphasize the main findings in relation to the study objectives. Additionally, the novel contribution of the study compared with previous work on antifreeze peptides derived from marine by-products should be highlighted more clearly.

Response 13: Thanks for the helpful comments. The suggestions on revising the conclusion section have been carefully implemented, with key revisions as follows: Redundant numerical result presentations in the Results section were trimmed, and only core trend descriptions retained to avoid unnecessary repetition and highlight main research findings. To highlight the study’s innovation, content was added noting that high-value utilization of tuna skin waste in line with circular economy principles is an aspect insufficiently addressed in previous relevant studies. The practical significance of this work was supplemented by including guidance for the food industry in pursuing natural, safe, and resource-efficient preservation technologies, further clarifying the application value of the research findings. Language expression was also polished to render the conclusion more concise and coherent.

This revision can be found in the revised manuscript on Page 18 to 19, Conclusion paragraph, Lines 679 to 695.

Comments 14: Given the large number of physicochemical, structural, and biochemical variables evaluated, the authors are encouraged to include a multivariate analysis, such as Principal Component Analysis (PCA). This approach would allow a more integrated interpretation of the results and further enhance the overall quality of the manuscript.

Response14: Thanks for the helpful comments. The recommendation to incorporate multivariate analysis, such as Principal Component Analysis (PCA), is highly insightful. The advice has been followed, and a PCA biplot has been created. This analysis, based on the first two principal components, visually presents the distribution of the experimental groups and the associations between variables, and these two principal components together capture 91.1% of the core variation in the dataset. Through this PCA biplot, the systematic separation between different experimental groups and the positive correlations between relevant variables are clearly demonstrated, allowing for a more comprehensive and intuitive interpretation of the research findings.

“Principal Component Analysis（PCA）

This figure is constructed using key experimental data, presenting the PCA biplot of physicochemical, structural and biochemical variables across experimental groups. This plot visualizes the distribution of experimental groups and the associations between variables, based on the first two principal components. PC1 serves as the horizontal axis and explains 80.9% of the total variance, while PC2 acts as the vertical axis and accounts for 10.2% of the total variance, and together they capture 91.1% of the core variation in the dataset.

The distribution trend shows clear systematic separation: the Con group (purple points) clusters in the negative direction of PC1, aligning with arrows for protein functional and quality indicators such as Ca²⁺-ATPase activity, protein solubility, and total sulfhydryl content, which reflects higher levels of these traits in the Con group; the NCon group (red points) locates distinctly in the positive direction of PC1, associated with arrows for oxidative damage and structural alteration indicators such as W2S, dityrosine content, and surface hydrophobicity, corresponding to elevated levels of these indices in the NCon group; the NH group (cyan triangles) distributes between Con and NCon along PC1 and is closer to the Con group, while the PH group (yellow inverted triangles) is located in the positive direction of PC1 and negative direction of PC2, and the TH group also falls between Con and NCon. This means that overall, the protein function and oxidative damage levels of NH, PH and TH groups are intermediate between the two extreme groups.

Clustered arrows, such as those for W2S and dityrosine content or for Ca²⁺-ATPase activity and protein solubility, also indicate positive correlations between variables. Compared to individual variable comparisons, this biplot intuitively visualizes both group-level systematic differences and variable interrelationships, enabling a more straightforward and integrated interpretation of the results.”

Sincerely yours,

Zhe Xu

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigated the effects of tuna skin–derived antifreeze peptides (AFPs) on the quality of frozen–thawed salmon. Various physicochemical properties were analyzed, including color, odor, texture, microstructure (SEM), and moisture loss.

However, the characteristics of the AFPs themselves were not described in this manuscript, and the authors focused exclusively on their effects. For better understanding, information regarding the physicochemical characteristics of the AFPs should be provided.

In addition, many explanations throughout the manuscript are not logically structured. Careful revision is required from the Introduction through the References.

L41
In the Introduction, the authors point out the high caloric content and excessive sweetness of commercially available cryoprotectants. How do the caloric content and taste attributes of the AFPs obtained in this study compare with those of existing commercial additives?

L117
During sample preparation, the salmon skin was discarded (L117). What was the antifreeze effect of this discarded salmon skin?
From an industrial perspective, it would be more beneficial for the salmon industry if antifreeze substances could be obtained from salmon skin itself rather than from tuna skin.

L117–118
Except for microscopic observations, the salmon samples were prepared as cubes with dimensions of 1 × 1 × 1 cm³ and soaked in peptide solutions (2 mg/mL) at 4 °C for 4 h. These sample dimensions appear extremely small.
Are the AFPs applicable to salmon fillets under practical conditions? In particular, is it possible for the peptide solution to penetrate into the center of a salmon fillet?

L147–161
E-nose experiments require careful control of storage conditions during analysis. If an autosampler equipped with a cooling unit was used, odor changes could be minimized. In contrast, if no cooling unit was employed, the sample odor may have changed during the waiting period in sealed 50 mL flasks prior to analysis.
A detailed experimental procedure should be added to Section 2.4.4.
(Comment: PEN3 is not originally provided by Insent Co., Ltd.)

L194
Please add the word “content”.

L206–212
The results of Ca²⁺-ATPase activity are presented in L450–460 and expressed as μmol Pi/mg/h. However, the method used to determine Ca²⁺-ATPase activity is not clearly described.
How did the authors estimate Ca²⁺-ATPase activity (μmol Pi/mg/h) using a UV spectrophotometer?

L220–254
With regard to the texture profile analysis, the reviewer agrees with the reported results. However, the authors focused only on the NH sample, without sufficient explanation of the PH and TH samples.
For better reader comprehension, additional explanations comparing NH, PH, and TH samples should be included.

L274–279
The authors discuss water-holding capacity in relation to peptide composition. However, detailed supporting results are not clearly presented in the text.
What does “narrow molecular weight distribution” mean in quantitative terms?
Why would peptides with a narrow molecular weight distribution inhibit ice crystal growth and recrystallization through hydrogen bonding and electrostatic interactions?

L276
Please clearly define what is meant by “these peptides,” including their molecular weight range, amino acid composition, and other relevant characteristics.

L281–307
The results of color parameters with and without AFP pretreatment provide valuable information for the frozen food industry.
In general, the market price of frozen salmon strongly depends on color.
Therefore, consumer and/or industrial acceptance of salmon surface color is a key factor for the potential application of this study.
Please include validation or discussion of the obtained L*, a*, and b* values in relation to consumer and/or industrial acceptance of salmon surface color.

Figure 1
According to the Methods section, the samples underwent three freeze–thaw cycles. However, the reviewer could not clearly recognize differences in odor among the frozen–thawed salmon samples.
These freeze–thaw cycles appear to represent an accelerated test corresponding to worst-case transportation conditions. Most of the experimental data were obtained after three freeze–thaw cycles.
If only a single freeze–thaw cycle were applied, would similar results be obtained?

L325–326
“Severe odor deterioration” is not clearly evident from Figure 1. In particular, for the W1C and W1S responses, what does “greatly increased” mean quantitatively?
Only the W1W and W2S values appear to show visually a significant difference.

L416–417
The authors state that “the tuna skin hydrolysate groups exhibited…,” but the NCon group is not with a hydrolysate. Therefore, NCon should be excluded from this description.

L576–
Several references do not conform to the journal’s reference formatting guidelines and should be revised accordingly.

Author Response

Dear Editor-in-Chief,

Foods

Reviewers' comments:

Reviewer #3:

Comments 1: The authors investigated the effects of tuna skin–derived antifreeze peptides (AFPs) on the quality of frozen–thawed salmon. Various physicochemical properties were analyzed, including color, odor, texture, microstructure (SEM), and moisture loss.

In addition, many explanations throughout the manuscript are not logically structured. Careful revision is required from the Introduction through the References

Response 1: Thanks for the helpful comments. Relevant experimental data including SDS-PAGE results, detailed information on the molecular weight range of the target peptides, amino acid composition of the identified peptide sequences, and other related physicochemical characteristics have been supplemented in the revised manuscript. The full manuscript has also been carefully revised and polished from the introduction through to the references section.

Comments 2: In the Introduction, the authors point out the high caloric content and excessive sweetness of commercially available cryoprotectants. How do the caloric content and taste attributes of the AFPs obtained in this study compare with those of existing commercial additives?

Response 2: Thanks for the helpful comments. Calorie values of sorbitol and sucrose have been supplemented to address this suggestion.

This revision can be found in the revised manuscript on Page 2, Introduction paragraph, Lines 50 to 53.

Comments 3: During sample preparation, the salmon skin was discarded (L117). What was the antifreeze effect of this discarded salmon skin?

From an industrial perspective, it would be more beneficial for the salmon industry if antifreeze substances could be obtained from salmon skin itself rather than from tuna skin.

Response 3: Thanks for the helpful comments. Salmon skin being an excellent source of potential antifreeze substances is absolutely correct. In fact, relevant studies on extracting antifreeze components from salmon skin are concurrently being conducted by our research team, aiming to explore its cryoprotective potential systematically.

The view that developing antifreeze agents from salmon skin is more beneficial for the salmon industry from an industrial perspective is fully agreed. This aligns with the goal of high-value utilization of aquatic by-products. It is anticipated that the antifreeze effect will be verified through in-depth experiments and the findings of this parallel research will be presented in future work.

Comments 4: Except for microscopic observations, the salmon samples were prepared as cubes with dimensions of 1 × 1 × 1 cm³ and soaked in peptide solutions (2 mg/mL) at 4 °C for 4 h. These sample dimensions appear extremely small.

Are the AFPs applicable to salmon fillets under practical conditions? In particular, is it possible for the peptide solution to penetrate into the center of a salmon fillet?

Response 4: Thanks for the helpful comments. This insightful comment provides important guidance for follow-up research directions. Attention to the alignment between experimental design and real-world scenarios is acknowledged, and the following clarification is provided:

First, the 4-hour soaking time set in the experiment is to achieve standardized experimental conditions and data reproducibility. In practical applications, the soaking duration can be appropriately extended if necessary, which will further ensure the sufficient penetration of the peptide solution into the center of the fillet.

Second, commercial salmon fillets are usually not thick (generally 2–3 cm in practical production), and this thickness is sufficient for the penetration of small-molecule antifreeze peptides. AFPs have a small molecular weight and good diffusibility, enabling them to effectively penetrate into the center of such thin fillets. The experimental results have also indirectly confirmed the feasibility of this penetration.

In summary, the application of AFPs in practical salmon fillets is feasible. The flexibly adjustable soaking time and the thin thickness of commercial salmon fillets jointly ensure that the peptide solution can penetrate to the center.

Comments 5: E-nose experiments require careful control of storage conditions during analysis. If an autosampler equipped with a cooling unit was used, odor changes could be minimized. In contrast, if no cooling unit was employed, the sample odor may have changed during the waiting period in sealed 50 mL flasks prior to analysis.

A detailed experimental procedure should be added to Section 2.4.4.

(Comment: PEN3 is not originally provided by Insent Co., Ltd.)

Response 5: Thanks for the helpful comments. Revisions have been made to Section 2.4.4 to supplement the sample storage condition and correct the manufacturer information, which enhances the rigor and detail of the methodology based on the valuable feedback on the E-nose experiment. To minimize odor changes during the waiting period, all sealed sample flasks were stored at 4 °C before analysis—this low-temperature environment effectively inhibits the progression of lipid oxidation and volatile compound degradation, ensuring the authenticity of odor detection results.

This revision can be found in the revised manuscript on Page 6, Materials and Methods section 2.4.4. Electronic nose (E-nose) analysis paragraph, Lines 194 to 199.

“Odor assessment was performed with a PEN 3 system (AIRSENSE Analytics GmbH, Schwerin, Germany). Each sample was transferred into a 50 mL hermetically sealed flask and immediately stored in a 4 °C refrigerator until analysis. The carrier gas was supplied at a flow rate of 200 mL/min, and headspace sampling was conducted for 60 s. In a fume hood all analyses were conducted.”

Comments 6: Please add the word “content”.

Response 6: Thanks for the helpful comments. The word "content" has been added as suggested.

Comments 7: The results of Ca²⁺-ATPase activity are presented in L450–460 and expressed as μmol Pi/mg/h. However, the method used to determine Ca²⁺-ATPase activity is not clearly described.

How did the authors estimate Ca²⁺-ATPase activity (μmol Pi/mg/h) using a UV spectrophotometer?

Response 7: Thanks for the helpful comments. In response to the inquiry, a detailed description of the Ca²⁺-ATPase activity determination method has been supplemented in Section 2.7.6 Ca²⁺-ATPase activity of the revised manuscript.

This revision can be found in the revised manuscript on Page 7, Materials and Methods section 2.7.6 Ca²⁺-ATPase activity paragraph, Lines 264 to 279.

“Ca²⁺-ATPase activity of MP was determined with a commercial assay kit (A070-3, Nanjing Jiancheng Bioengineering Institute Co. Ltd., Nanjing, Jiangsu, China) as per the manufacturer's instructions: 0.02μmol/mL standard Pi solution (via gradient dilution of 10mmol/L stock solution), chromogenic agent (Reagent 5A:5B=7:6), substrate solution (Reagent 1:2:3=260:80:80) and Reagent 9 (mixed with its diluent and powder) were prepared; a 10% tissue homogenate was prepared by homogenizing salmon muscle with 0.9% physiological saline (1:9, w/v) under ice bath, centrifuged at 2500 rpm for 10 min to collect the supernatant, which was then diluted to 1% and quantified for protein concentration via Coomassie Brilliant Blue method (avoiding phosphorus-containing reagents); control and test tubes were set up with samples and reagents, incubated at 37℃ for 10 min, terminated with Reagent 4, and centrifuged to collect the supernatant; the supernatant/standard solution and chromogenic agent were added to blank, standard, control and test tubes, incubated at 37℃ for 5-10 min, and absorbance was determined at 636 nm with a UV spectrophotometer (UV-1600, Mapada Instruments, Shanghai, China) using Milli Q water for baseline correction; Ca²⁺-ATPase activity (μmol Pi/mg/h) was calculated using the kit’s formula.”

Comments 8: With regard to the texture profile analysis, the reviewer agrees with the reported results. However, the authors focused only on the NH sample, without sufficient explanation of the PH and TH samples.

For better reader comprehension, additional explanations comparing NH, PH, and TH samples should be included.

Response 8: Thanks for the helpful comments. In response to the suggestions, the overall protective efficacy order as NH > TH > PH has been clarified in the texture profile analysis section (Section 3.1). These differences have been further linked to the ability of each hydrolysate to preserve the secondary structure of myofibrillar proteins, especially the α-helix content. Additionally, comparisons with similar data from other relevant literatures have been added.

This revision can be found in the revised manuscript on Page 9, section 3.1. Textural properties paragraph, Lines 321 to 323and 329 to 336.

“while TH and PH showed graded improvements relative to the NCon group with the overall protective efficacy following the order NH > TH > PH. Hardness and elasticity in NH were 107% and 173% higher than the NCon group, respectively.”

“The differences in textural protective efficacy among the three hydrolysates are closely related to their ability to preserve the secondary structure of myofibrillar proteins, especially the α-helix content that is critical for maintaining muscle structural rigidity and network integrity. NH effectively inhibited the α-helix-to-random coil transition during freeze-thaw cycles, retaining a higher α-helix content than TH and PH which directly translated to superior textural properties. Similarly, other texture parameters, including cohesiveness, gumminess, chewiness, and resilience, exhibited consistent improvements with the NH treatment, followed by TH and PH.”

Comments 9: The authors discuss water-holding capacity in relation to peptide composition. However, detailed supporting results are not clearly presented in the text.

What does “narrow molecular weight distribution” mean in quantitative terms?

Why would peptides with a narrow molecular weight distribution inhibit ice crystal growth and recrystallization through hydrogen bonding and electrostatic interactions?

Response 9: Thanks for the helpful comments. Detailed information regarding the molecular weight range of the peptides, the amino acid composition of the peptide sequences, and other relevant characteristics has been supplemented, and the ambiguous expression "narrow molecular weight distribution" has been replaced with a specific, well-defined molecular weight range. Peptides within this defined range are characterized by highly homogeneous chain lengths and uniform distribution of hydrophilic and charged groups, enabling dense and uniform adsorption onto ice crystal surfaces. Specifically, the incorporation of water molecules into the ice crystal lattice is blocked via the formation of a continuous hydrogen-bonding network; the ordered arrangement of ice crystals is disrupted by a uniform electrostatic field; and the coalescence of small ice crystals is simultaneously inhibited. Accordingly, highly efficient inhibition of ice crystal growth and recrystallization is achieved.

This revision can be found in the revised manuscript on Page 10, section 3.2.1 Water holding capacity paragraph, Lines 377 to 387.

References cited

Comments 10: Please clearly define what is meant by “these peptides,” including their molecular weight range, amino acid composition, and other relevant characteristics.

Response 10: Thanks for the helpful comments. Detailed information regarding the molecular weight range of the peptides, the amino acid composition of the peptide sequences, and other relevant characteristics has been supplemented.

Comments 11: The results of color parameters with and without AFP pretreatment provide valuable information for the frozen food industry.

In general, the market price of frozen salmon strongly depends on color.

Therefore, consumer and/or industrial acceptance of salmon surface color is a key factor for the potential application of this study.

Please include validation or discussion of the obtained L*, a*, and b* values in relation to consumer and/or industrial acceptance of salmon surface color.

Response 11: Thanks for the helpful comments. The advice has been followed, and relevant content has been added to the revised manuscript. Changes in L*, a*, and b* have been clearly linked to consumer acceptance. As suggested, this revision strengthens the technological and sensory relevance between color parameters and consumer responses.

This revision can be found in the revised manuscript on Page 10, Section 3.2.2. Color paragraph, Lines 391 to 398.

Comments 12: According to the Methods section, the samples underwent three freeze–thaw cycles. However, the reviewer could not clearly recognize differences in odor among the frozen–thawed salmon samples.

These freeze–thaw cycles appear to represent an accelerated test corresponding to worst-case transportation conditions. Most of the experimental data were obtained after three freeze–thaw cycles.

If only a single freeze–thaw cycle were applied, would similar results be obtained?

Response 12: Thanks for the helpful comments. The three freeze–thaw cycles were designed as an accelerated test to simulate extreme transportation/storage conditions—a widely recognized strategy in the efficacy evaluation of cryoprotectants for aquatic products. This design enables comprehensive evaluation of the protective effect of the cryoprotectant under the most challenging scenarios (e.g., repeated temperature fluctuations in long-distance cold chain logistics), which is consistent with the experimental framework of typical aquatic cryoprotection studies.

Previous studies on aquatic products have confirmed that freeze–thaw cycle frequency is positively correlated with the degree of quality deterioration. If only one freeze–thaw cycle were used, the response trend of each electronic nose sensor would remain consistent with the three-cycle results, but the extent of odor deterioration would be significantly reduced. For example, a study on pork patties showed that three freeze–thaw cycles led to greater accumulation of volatile oxidation products such as aldehydes than one cycle, with the carbonyl content—a marker of protein oxidation linked to off-odor—being significantly higher in the control group after three cycles. Consistent results were found in chicken breast research: three freeze–thaw cycles caused more severe protein aggregation and exposure of hydrophobic groups, promoting the generation of undesirable volatile substances like trimethylamine and fatty acid oxidation products, and the off-odor intensity was significantly higher than after one cycle.

Compared with the mild single-cycle condition, the three-cycle design is more in line with industrial application practice—especially for products with long cold chain circulation, which often experience multiple temperature fluctuations during transportation. This approach ensures that the evaluated cryoprotectant can provide effective protection in practical scenarios rather than only under ideal conditions.

References cited

Li, Y.; Han, X.; Zhang, Y.; Wang, Y.; Wang, J.; Teng, W.; Wang, W.; Cao, J. Thawed drip and its membrane-separated components: Role in retarding myofibrillar protein gel deterioration during freezing-thawing cycles. Food Research International 2024, 188, Article 114461. https://doi.org/10.1016/j.foodres.2024.114461.

Bai, X.; Shi, S.; Kong, B.; Chen, Q.; Liu, Q.; Li, Z.; Wu, K.; Xia, X. Analysis of the influencing mechanism of the freeze-thawing cycles on in vitro chicken meat digestion based on protein structural changes. Food Chemistry 2023, 399, 134020. https://doi.org/10.1016/j.foodchem.2022.134020.

Comments 13: “Severe odor deterioration” is not clearly evident from Figure 1. In particular, for the W1C and W1S responses, what does “greatly increased” mean quantitatively?

Only the W1W and W2S values appear to show visually a significant difference.

Response 13: Thanks for the helpful comments. Each concern has been carefully addressed by revising the descriptions. Below is the detailed response: Clarification on odor differences among frozen-thawed samples. Sincere apologies for the confusion caused by the initial sensor labeling error (incorrectly referencing W1C; the core odor-related sensors are W1S, W1W, W2S, and W5S). To address the issues of "unclear odor differences" and "lack of quantitative explanation for 'greatly increased'", revisions have been made to the descriptions with key information supplemented. Note on visual clarity: Although there is partial overlap in the radar plot, the response differences of specific sensors confirm that the NCon group exhibits significant odor deterioration, while the NH group exerts an effective protective effect. Explanation for "severe odor deterioration" and "greatly increased": The NCon group shows significant differences in several key sensors compared with the fresh group and the NH group, which confirms that freeze-thaw-induced muscle tissue disruption activates lipases and proteases, thereby accelerating lipid oxidation and protein degradation. Based on previous studies, this wording is consistent with the result description of electronic nose.

References cited

Comments 14: The authors state that “the tuna skin hydrolysate groups exhibited…,” but the NCon group is not with a hydrolysate. Therefore, NCon should be excluded from this description.

Response 14: Thanks for the helpful comments. The word "NCon" has been removed as suggested.

Comments 15: Several references do not conform to the journal’s reference formatting guidelines and should be revised accordingly.

Response 15: Thanks for the helpful comments. The journal name of Reference 12 has been revised, as well as the author name format and spelling error of Reference 34 have been corrected.

Sincerely yours,

Zhe Xu

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Nice and well structured manuscript that investigate cryoprotective effects of tuna skin antifreeze peptides (obtained from protein hydrolysates prepared using trypsin, pepsin, and neutral protease) on salmon cubes subjected to repeated freeze-thaw cycles that mimic repetitive temperature fluctuations during transportation and storage. Authors clearly proved best cryoprotective effects of neutral protease protein hydrolysate, showing high levels of effectiveness.

Some minor comments and suggestions are given i PDF.

Comments for author File: Comments.pdf

Author Response

Dear Editor-in-Chief,

Foods

Reviewers' comments:

Reviewer #4:

Comments 1: Authors need to add in abstract which enzyme hydrolisates were used and which one shoved the best effects

Response 1: Thanks for the helpful comments. The types of enzyme hydrolysates and the optimal performance of NH have been supplemented.

These changes can be found in the revised manuscript on Page 1, Abstract paragraph, Lines 18 to 28.

“Cubes treated with AFPs from three groups of protein hydrolysates prepared using trypsin, pepsin, or neutral protease were evaluated for texture, color, water-holding capacity, volatile odor profiles, protein conformation, biochemical indices, and microstructure.”

Comments 2: Please add the word “organoleptic”.

Response 2: Thanks for the helpful comments. This change can be found in the revised manuscript on Page 2, Introduction Paragraph, Lines 46.

Comments 3: Please add “mixture of what,filtrate with what and supernatant with what”?

Response 3: Thanks for the helpful comments. These changes can be found in the revised manuscript on Page 4, 2.2. Preparation of hydrolysates from tuna skin protein Paragraph, Lines 126 to 128.

“The mixture of acetic acid and tuna skin was filtered through double-layer gauze, and the filtrate containing solubilized collagen was centrifuged at 10,000×g for 30 min to collect the supernatant enriched with crude collagen.”

Comments 4: Please add the word “pH”.

Response 4: Thanks for the helpful comments. This change can be found in the revised manuscript on Page 4, 2.2. Preparation of hydrolysates from tuna skin protein Paragraph, Lines 123.

Comments 5: Please add the word “one of” and replace "and" with "or"

Response 5: Thanks for the helpful comments. These changes can be found in the revised manuscript on Page 4, 2.2. Preparation of hydrolysates from tuna skin protein Paragraph, Lines 136 to 146.

Comments 6: Please add the word “(WHC)”.

Response 6: Thanks for the helpful comments. This change can be found in the revised manuscript on Page 9, 3.2.1. Water holding capacity paragraph, Lines 360.

Comments 7: Add spaces

Response 7: Thanks for the helpful comments. This change can be found in the revised manuscript on Page 10, 3.2.2. Color, Lines 405.

Sincerely yours,

Zhe Xu

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

After conducting a critical evaluation of the revised version of the manuscript submitted by the authors, I consider that they have made an excellent effort to address the revisions and modifications suggested in my previous evaluation. Overall, the observations and comments were adequately addressed and properly incorporated throughout the manuscript. However, the principal component analysis (PCA) was not included in the Results section nor in the Statistical Analysis subsection within Materials and Methods. This analysis was described by the authors in their response-to-reviewers document, but it was not incorporated into the revised manuscript. Therefore, my recommendation is to return the manuscript to the authors with a suggestion for minor revisions.

Author Response

Dear Editor-in-Chief,

Foods

It is my great honor to submit the revised manuscript entitled “Cryoprotective Effects of Tuna Skin Antifreeze Peptides on the Quality of Salmon Flesh During Low-Temperature Fluctuations” (foods-4073652) based on the comments from the editor and reviewers. These comments are valuable and helpful for revising and improving our manuscript and further research work. The authors appreciate the work of the editor and reviewers very much. According to the comments of editor, a clean version of the revised manuscript will be uploaded, and the revised portions were described as clear as possible. We have made a careful response to these comments one by one as the following.

Reviewers' comments:

Reviewer #2:

Comments 1: After conducting a critical evaluation of the revised version of the manuscript submitted by the authors, I consider that they have made an excellent effort to address the revisions and modifications suggested in my previous evaluation. Overall, the observations and comments were adequately addressed and properly incorporated throughout the manuscript. However, the principal component analysis (PCA) was not included in the Results section nor in the Statistical Analysis subsection within Materials and Methods. This analysis was described by the authors in their response-to-reviewers document, but it was not incorporated into the revised manuscript. Therefore, my recommendation is to return the manuscript to the authors with a suggestion for minor revisions.

Response 1: Thank you for the helpful comments. The principal component analysis (PCA) has been added to Results section and following discussions.

This revision can be found in the revised manuscript on Page 18, 3.6.9. Principal Component Analysis (PCA) paragraph, Lines 672 to 692.

“The PCA biplot is shown in Figure 3h, constructed with key physicochemical, structural and biochemical variables across all experimental groups, and it visualizes group distribution and inter-variable associations. The first two principal components explained 80.9% of total variance with PC1 as the horizontal axis and 10.2% of total variance with PC2 as the vertical axis, collectively contributing to 91.1% of the dataset’s core variation. Clear systematic group separation was observed. Con clustered in the negative region of PC1, aligning with protein quality indicators such as Ca²⁺-ATPase activity, protein solubility and total sulfhydryl content. NCon was localized to the positive region of PC1, associated with oxidative and structural damage indices such as W2S, dityrosine content and surface hydrophobicity. On the PC1 axis, NH was positioned between Con and NCon, though it clustered more closely with Con. PH was located in the lower-right quadrant, characterized by positive PC1 and negative PC2 values. TH was also positioned between the two extreme groups. Clustered loading arrows revealed strong positive correlations between paired variables, such as W2S and dityrosine, Ca²⁺-ATPase activity and protein solubility. This biplot enabled a more intuitive and integrated interpretation of group differences and variable relationships than single-variable comparisons, confirming that NH exerted the most comprehensive myofibrillar protein protective effect. NCon showed severe protein oxidative denaturation from freeze-thaw damage, while PH and TH presented intermediate changes, verifying that tuna skin antifreeze peptides, especially the NH fraction, effectively maintain muscle protein stability during freeze-thaw cycles, supporting their application in frozen preservation of aquatic products.”

Sincerely yours,

Zhe Xu

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised the manuscript line by line in response to the reviewer’s comments and suggestions. Several references have been newly added to the Introduction and Materials and Methods sections, which improves the background information and methodological description and allows readers to gain a deeper understanding of the study.

The presentation of the results has also been substantially updated, and the text is now more reliable than in the initial version of the manuscript. In particular, the results obtained from SDS–PAGE provide useful support for the discussion. However, several formatting issues remain in the manuscript; therefore, a second revision is still required.

Based on the revised PDF version, it is difficult to determine whether Figure 1 is correctly presented. It appears that duplicate figures are overlapping. In addition, the font size used in all figures remains too small and should be increased to improve readability.

The SDS–PAGE image should be placed immediately next to Figure 3f or in its vicinity. In the current PDF version, the SDS–PAGE image is positioned between sections of text, which makes it difficult to interpret in relation to the corresponding results.

Sections 2.7.7 and the Results of SDS–PAGE, Information about the molecular weight markers is missing in the legend of Figure 3g (i.e., the units corresponding to the numbers shown on the left side of the image). Marker information is also unclear in the Materials and Methods section (L288–298) and in the Results section (L680–695). If possible, the authors should add an appropriate reference in Section 2.7.7 to support the discussion.

Section 2.7.8, The analytical conditions for LC–MS/MS are not described clearly. In addition, information about the LC–MS/MS instrument is lacking. Citing an appropriate reference describing the LC–MS/MS procedure would be sufficient for methodological clarification.

New abbreviations should be listed in L764. For example, MHC (L680), MLC (L681), and Ej (L86).

Several references still do not follow the journal’s formatting instructions. For example, reference No. 8 (the scientific name should be written in italics, and the first letter should be capitalized), as well as references Nos. 16, 21, 22, 27, 32, 35, 36, 37, 38, and 43, require correction.

Additional minor corrections include:

L111: “u” should be written as “U”, consistent with L115.

L97: “Salmon” should be written as “salmon.”

In general, minor formatting issues may be corrected during the editorial process; however, authors are fundamentally responsible for ensuring that the manuscript fully conforms to the journal’s formatting requirements.

Author Response

Dear Editor-in-Chief,

Foods

Reviewers' comments:

Reviewer #3:

Comments 1: Based on the revised PDF version, it is difficult to determine whether Figure 1 is correctly presented. It appears that duplicate figures are overlapping. In addition, the font size used in all figures remains too small and should be increased to improve readability.

Response 1: Thank you for the helpful comments. Figure 1 and other figures have been revised to improve the resolution and readability. Both the Word and PDF versions have been uploaded.

Comments 2: The SDS–PAGE image should be placed immediately next to Figure 3f or in its vicinity. In the current PDF version, the SDS–PAGE image is positioned between sections of text, which makes it difficult to interpret in relation to the corresponding results.

Response 2: Thank you for the helpful comments. The manuscript has been reorganized. The figures have been adjusted to the correct positions adjacent to Figure 3f, and Figure 3h has been added. The revised Word and PDF versions have been uploaded.

Comments 3: Sections 2.7.7 and the Results of SDS–PAGE, Information about the molecular weight markers is missing in the legend of Figure 3g (i.e., the units corresponding to the numbers shown on the left side of the image). Marker information is also unclear in the Materials and Methods section (L288–298) and in the Results section (L680–695). If possible, the authors should add an appropriate reference in Section 2.7.7 to support the discussion.

Response 3: Thank you for the helpful comments. Unit information has been added to Figure 3g, and marker information has been clearly specified in Section 2.7.7 and the Results of SDS–PAGE at lines 284 and 644–645.

Comments 4: Section 2.7.8, The analytical conditions for LC–MS/MS are not described clearly. In addition, information about the LC–MS/MS instrument is lacking. Citing an appropriate reference describing the LC–MS/MS procedure would be sufficient for methodological clarification.

Response 4: Thank you for the helpful comments. In Section 2.7.8, detailed descriptions of the analytical conditions have been added, and information on the LC–MS/MS instrument has been supplemented. Appropriate references describing the LC–MS/MS procedure have been cited to clarify the methodology.

Comments 5: New abbreviations should be listed in L764. For example, MHC (L680), MLC (L681), and Ej (L86).

Response 5: Thanks for the helpful comments. New abbreviations have been added, and the abbreviation has been supplemented at the first occurrence of Ej on line 61.

Comments 6: Several references still do not follow the journal’s formatting instructions. For example, reference No. 8 (the scientific name should be written in italics, and the first letter should be capitalized), as well as references Nos. 16, 21, 22, 27, 32, 35, 36, 37, 38, and 43, require correction.

Response 6: Thanks for the helpful comments. Issues regarding the scientific names have been revised. References numbered 7, 9, 10, 11, 15, 16, 18, 21, 22, 24, 26, 27, 30, 32, 33, 35, 36, 37, 38, 40, 43, 47, 48, 50, 51, 52, 53 have been uniformly corrected in line with the required formatting specifications, including italicization and capitalization of the first letter, to ensure the accurate presentation of all scientific names.

Comments 7: Additional minor corrections include:

L111: “u” should be written as “U”, consistent with L115.

L97: “Salmon” should be written as “salmon.”

Response 7: We appreciate your kind observation and comments. Corresponding revisions have been made at lines 94 and 109.

Sincerely,

Zhe Xu

Author Response File: Author Response.pdf