Review Reports - Combined Effects of Gas Composition in Modified Atmosphere Packaging and Chitooligosaccharide-EGCG on Quality Changes in Refrigerated Asian Hard Clam Meat

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article presents the significance of different forms of MAP and pretreatment with chitooligosaccharide-EGCG (CE) conjugate to increase the shelf-life of packed Asian hard clam. The article is interesting and I especially appreciated the presentation of relevant correlations by Correlation heatmap analysis. However, the manuscript has some weaknesses that should be corrected.

In the Abstract: Avoid not defined abbreviations in the abstract, such as MAP4.

Materials and Methods Section: The used chemicals should be mentioned with chemical name (incl. producer/supplier, and state as well as CAS number.

Sample preparation Section: The suppliers of the trays and the bags should be mentioned (name of producer, city, country). The gas barrier properties of the PE bags should be analysed (or stated).

Sample preparation Section: The headspace volume should be mentioned. Since a flexible package was used (PE bags), the headspace volume at start of the experiment, as well as after completed experiment, should be mentioned.

Sample preparation Section (or Results and Discussion Section): Since the permeability coefficient is rather high for transport of N2 and O2 through PE, the gas compositions at the start and end of each experiment should be measured.

Sample preparation Section: The authors should describe how the CE conjugate was applied.

Sample preparation Section: The authors should include something about food approval for CE. I also assume that CE migrate into the food. How much CE is present in 1 gram of meat at the end of the test? The author should discuss this in terms of allowed overall migration.

Section Drip loss and cooking loss: The authors wrote “The initial weight of the sample (Wi) before treatment”. Please specify what kind of treatment the authors have in their mind.

Section Statistical Analysis: The number of times each experimental point was repeated should be mentioned, even if p-values are shown.

Section PV and TBARS Content: The authors write rather speculatively about formation of carbonic acid. References supporting the discussion is missing. It would perhaps be better to separate the Results and Discussion Section in two separate sections (one for Results and one for Discussion). As the article is now written, it is difficult for a reader to distinguish between results and speculation.

Section PV and TBARS Content: References are needed to support the discussion about oxygen concentration and TBARS value.

Conclusion Section: I could not find in the Results and Discussion Section how the criteria for end of shelf-life were defined. Was this solely based on TBARS value or was something else behind the definition of shelf-life?

Author Response

Reviewer 1

***** Thank you for the insightful comments and feedback, which can enhance the clarity of our manuscript. All queries have been answered. The corrections have been made, and additional information has been provided as highlighted in yellow color.

In the Abstract: Avoid not defined abbreviations in the abstract, such as MAP4.

******The abbreviation was explained at its first occurrence in the abstract (line 23-24). Due to the abstract word limit, the abbreviation was retained to ensure conciseness while maintaining clarity.

Materials and Methods Section: The used chemicals should be mentioned with chemical name (incl. producer/supplier, and state as well as CAS number.

******The required information has been added to text. Please see revised section 2.1

Sample preparation Section: The suppliers of the trays and the bags should be mentioned (name of producer, city, country). The gas barrier properties of the PE bags should be analysed (or stated).

******The information has been provided about the suppliers. Please see section 2.1. For the gas barrier properties, we have added oxygen barrier properties. However, other information was not provided by the supplier. Please see line 129-130.

******Thank you for valuable comment. In our study, PE bags remained physically intact throughout the storage period, and no visible swelling, collapse, or leakage was observed. However, we acknowledge that gas transmission can occur due to the inherent permeability of PE films and we don’t have facility to directly measure the gas composition.

******We appreciate the reviewer’s valuable comment regarding the monitoring of gas composition before and after the experiment. We agree that changes in gas composition may occur during storage due permeability of PE. However, we acknowledge that we do not currently have the facility to directly measure the gas composition inside the package at the end of each experiment. The initial gas composition was controlled using gas mixture and PE package filled with sample to gas ratio of 1:3 (w/v).

Sample preparation Section: The authors should describe how the CE conjugate was applied.

****** Thank you for comment. We have revised the sample preparation section for better understanding. Please see line 126-128.

******The approval related to application of CE in food is subject to discuss. The cell culture and animal trails focusing on toxicity and allergenicity is in progress. The results based on those studies will be shared with regulatory authorities for the final decision on CE approval.

******We agreed with the migration of CE in hard clam meat tissue and assumed that there is no loss of CE during treatment. Therefore, 0.6 mg of CE is present in 1 gram of meat.

Section Drip loss and cooking loss: The authors wrote “The initial weight of the sample (Wi) before treatment”. Please specify what kind of treatment the authors have in their mind.

******The method has been revised for better understanding. Please see line 162-163.

Section Statistical Analysis: The number of times each experimental point was repeated should be mentioned, even if p-values are shown.

******All the experiments were conducted in triplicate. Please see line 220-221 and 225.

******References have been added to support discussion. Please see line 451 and 455.

******We appreciate the reviewer’s careful reading of our manuscript and the recommendation to separate the Results and Discussion into two distinct sections. After careful consideration, we have decided to retain a single combined “Results and Discussion” section. Our intention in structuring the manuscript this way was to present each set of results immediately alongside its corresponding interpretation, allowing for a more integrated and coherent narrative. We believe this approach helps readers better understand the significance and implications of the findings in direct context, rather than requiring them to move back and forth between separate sections.

Section PV and TBARS Content: References are needed to support the discussion about oxygen concentration and TBARS value.

******The references have been included to support discussion. Please see line 443, 441, 451 and 455.

******Thank you for this comment. The shelf-life of hard clam (HC) meat in this study was determined primarily based on microbiological criteria, specifically the total viable counts (TVC). The end of shelf-life was defined as the point at which microbial loads reached the acceptable limit for seafood products (6 log CFU/g), which is widely used as a microbiological spoilage threshold. TBARS values were measured to evaluate lipid oxidation; however, they were not used as the primary criterion for determining the end of shelf-life. We have modified the conclusion for better understanding. Please see line 743-745.

Reviewer 2 Report

Comments and Suggestions for Authors

The mention that "HC is widely consumed in Thailand due to its nutritional and price advantages" lacks the Latin name (Meretrix lusoria) and does not specify the industry pain point regarding the short shelf life of fresh HC.
When explaining the principle of MAP, the rationale for selecting specific gas ratios (e.g., why 40%/60%/80% CO₂ gradients were chosen) is not provided.
When introducing CE conjugates, only their bioactivity from previous studies is cited. The preparation method, purity, and structural characterization of CE conjugates in this study are not clarified, leading to a disconnect with the Materials and Methods section.
The research objective only states "evaluating the effects of CE combined with different MAP gas compositions on HC quality and shelf life," without specifying core research questions (e.g., the mechanism of O₂/N₂ selection under high CO₂ in regulating lipid oxidation, or the synergistic targets of CE and MAP).
In section 2.2, "vacuum-sealed and blanched in boiling water for 1 min" does not explain the purpose of blanching. Baseline physicochemical indicators (e.g., pH, water activity) after blanching are not measured, lacking data on how pretreatment affects HC's initial state.
In section 2.3, the concentration of CE conjugates used is 600 ppm, but the basis for selecting this concentration (e.g., whether concentration gradient experiments were conducted) is not provided.
In section 2.4.1, the culture conditions (temperature, time) for assessing H₂S-producing bacteria on TSI agar and Vibrio spp. on TCBS agar are not specified.
In section 2.5, the formula is written incorrectly. The correct formula should be: Drip loss (%) = (Wi - Wd) / Wi × 100.
In section 2.5.2, only "firmness and toughness" are measured, but the testing parameters for the TA-XT2 texture analyzer (e.g., probe speed, trigger force, test distance) are not specified.
In section 2.5.3, only day 0 and day 18 samples from MAP4/MAP4-CE and CON/CON-CE groups were tested. Intermediate storage time points (e.g., day 6, day 12) were not analyzed, preventing observation of dynamic changes in fatty acids.
In section 2.6, the time points for 16S rRNA sequencing were "CON-day6, CON-CE-day12, all groups-day18." The selection criteria are inconsistent, and the rationale for choosing different time points for different groups is not explained.
In section 2.7, only one-way ANOVA and Tukey’s multiple comparison test are mentioned. The results of normality and homogeneity of variance tests, which are prerequisites for ANOVA, are not provided.
In section 3.1, the statement "CON-CE remained below the acceptable limit until day 18" does not cite the reference for the seafood microbial acceptability limit. The conclusion that Pseudomonas is the main spoilage bacterium in HC lacks species identification validation—relying solely on selective medium counts is insufficient.
In section 3.3, the lower TBARS value in the 80% CO₂ + 20% O₂ group compared to the 80% CO₂ + 20% N₂ group is attributed only to "N₂ inhibiting endogenous antioxidant enzymes." Other factors, such as O₂ promoting CO₂ dissolution or microbial community differences affecting lipid oxidation, are not considered.
In section 3.4, the discussion of higher drip and cooking losses in MAP groups only attributes it to CO₂ forming carbonic acid, without analyzing muscle structure or protein denaturation (e.g., myofibrillar protein solubility, sulfhydryl content). The analysis lacks depth.
In section 3.5, the heatmap (Fig. 5A) has unclear axis labels. Only Pearson correlation analysis was performed; partial correlation analysis to account for confounding factors (e.g., CO₂ and O₂ interaction) was not conducted.
In section 3.7, only relative abundances at the family, genus, and species levels are analyzed. Differential species analysis to identify biomarkers for different treatment groups is lacking. The statement "MAP shifts the microbial community toward CO₂-tolerant taxa" does not include functional annotation of these taxa, limiting mechanistic insight.
In section 3.7, PCoA and NMDS analyses only show clustering results. Significance tests for between-group differences (e.g., ANOSIM, Adonis) were not performed, so statistical significance of microbial community differences between CON and MAP groups cannot be confirmed.
Throughout the Results and Discussion sections, figure legends are inconsistent (e.g., CON-CE written as CONCE, MAP4-CE as MAPACE). The meaning of error bars (e.g., SD or SE) is not specified in some figure captions, violating chart standards.
The suggestion to "add water-retaining additives to mitigate drip loss in MAP" is not supported by specific additive types or concentrations based on the study’s findings.

Author Response

Reviewer 2

******Authors would like to express our gratitude for the insightful comments and suggestions. All queries have been responded, and the corrections have been highlighted in green color.

The mention that "HC is widely consumed in Thailand due to its nutritional and price advantages" lacks the Latin name (Meretrix lusoria) and does not specify the industry pain point regarding the short shelf life of fresh HC.

******The Latin name of hard clam has been included in the text and clarified the industry challenge related to the short shelf-life of fresh HC. Please see green highlights in introduction.

When explaining the principle of MAP, the rationale for selecting specific gas ratios (e.g., why 40%/60%/80% CO₂ gradients were chosen) is not provided.

******Thank you for your valuable comment. In this study, the CO₂ gradients of 40%, 60%, and 80% were selected based on ranges commonly reported in previous MAP research for seafood preservation. CO₂ concentrations between 20% and 60% are generally considered effective for inhibiting aerobic spoilage microorganisms due to their bacteriostatic activity. Therefore, 40% and 60% CO₂ were chosen to represent moderate and commonly applied antimicrobial levels in MAP systems. In addition, a higher concentration of 80% CO₂ was included to evaluate whether a stronger antimicrobial atmosphere could further enhance microbial inhibition and extend shelf life. The rationale for changing CO₂ concentrations has been included in the text. Please see line 58-64.

When introducing CE conjugates, only their bioactivity from previous studies is cited. The preparation method, purity, and structural characterization of CE conjugates in this study are not clarified, leading to a disconnect with the Materials and Methods section.

******CE conjugate was prepared and characterized according to our previous study (Mittal et al. 2022). The preparation method of CE conjugate has been added to the text. Please see line 115-124. However, for purity, authors focused on the conjugation efficiency, which is related to total phenolic contents. Thus, in direct we did not measure the purity.

The research objective only states "evaluating the effects of CE combined with different MAP gas compositions on HC quality and shelf life," without specifying core research questions (e.g., the mechanism of O₂/N₂ selection under high CO₂ in regulating lipid oxidation, or the synergistic targets of CE and MAP).

******Thank you for your comment. The research objective has been revised. Please see line 84-90.

In section 2.2, "vacuum-sealed and blanched in boiling water for 1 min" does not explain the purpose of blanching. Baseline physicochemical indicators (e.g., pH, water activity) after blanching are not measured, lacking data on how pretreatment affects HC's initial state.

******Thank you for the comment. The blanching step in Section 2.2 was performed solely to facilitate shell opening and shucking, not as a physicochemical pretreatment. All samples underwent the same brief blanching (1 min) followed by rapid cooling to minimize thermal effects.

In section 2.3, the concentration of CE conjugates used is 600 ppm, but the basis for selecting this concentration (e.g., whether concentration gradient experiments were conducted) is not provided.

******Thank you for this valuable comment. The concentration of CE conjugate (600 ppm) used in Section 2.3 was selected based on results obtained from preliminary experiments conducted prior to the main study. In these initial trials, a range of concentrations (200, 400, and 600 ppm) was evaluated to assess their effectiveness, and 600 ppm was identified as the optimal concentration for achieving consistent and reproducible results without compromising system performance.

In section 2.4.1, the culture conditions (temperature, time) for assessing H₂S-producing bacteria on TSI agar and Vibrio spp. on TCBS agar are not specified.

******H₂S-producing bacteria and Vibrio spp. were assessed on triple sugar iron (TSI) agar and thiosulfate citrate bile salts sucrose (TCBS) agar. The plates were incubated at 37 °C for 18-24 h. Please see line 150-152.

In section 2.5, the formula is written incorrectly. The correct formula should be: Drip loss (%) = (Wi - Wd) / Wi × 100.

******Formula for drip loss has been revised.

In section 2.5.2, only "firmness and toughness" are measured, but the testing parameters for the TA-XT2 texture analyzer (e.g., probe speed, trigger force, test distance) are not specified.

******Testing parameters for analysis has been added. Please see line 174-176.

In section 2.5.3, only day 0 and day 18 samples from MAP4/MAP4-CE and CON/CON-CE groups were tested. Intermediate storage time points (e.g., day 6, day 12) were not analysed, preventing observation of dynamic changes in fatty acids.

******In fatty acid analyses, based on our objectives of extension of shelf-life to maximum, we focused on the initial (day 0) and final (day 18) time points for MAP4/MAP4-CE and CON/CON-CE groups to assess the overall effect of storage on fatty acid composition. While intermediate time points (day 6 and day 12) were not analyzed, the primary objective was to evaluate the net change over the storage period.

In section 2.6, the time points for 16S rRNA sequencing were "CON-day6, CON-CE-day12, all groups-day18." The selection criteria are inconsistent, and the rationale for choosing different time points for different groups is not explained.

******We appreciate the reviewer’s comment. The time points for 16S rRNA sequencing were selected based on the samples showing the highest viable bacterial counts for each group. Specifically, for the CON group, day 6 represented the peak bacterial viability, for the CON-CE group, day 12 showed the highest counts, and for all groups, day 18 was chosen to capture the later stage of microbial dynamics. In case of day 0, it could be possible that all the samples might have similar type of populations, which we have noticed in our previous research. Thus, to understand this, bacterial growth till the acceptable limit (6 log) was achieved and determined for the diversity. We have revised section 2.6. Please see line 206-207.

In section 2.7, only one-way ANOVA and Tukey’s multiple comparison test are mentioned. The results of normality and homogeneity of variance tests, which are prerequisites for ANOVA, are not provided.

******Thank you for this important comment. Prior to performing one-way ANOVA, the assumptions of normal distribution and homogeneity of variance were evaluated using the Shapiro-Wilk test and Levene’s test, respectively. The section 2.7 has been updated. Please see line 222-224.

In section 3.1, the statement "CON-CE remained below the acceptable limit until day 18" does not cite the reference for the seafood microbial acceptability limit. The conclusion that Pseudomonas is the main spoilage bacterium in HC lacks species identification validation—relying solely on selective medium counts is insufficient.

******We thank the reviewer for pointing this out. The acceptable limit for seafood microbial quality, 6 log CFU/g, is provided in line 261.

******Regarding Pseudomonas as main spoilage bacteria, author did not claim the same. Although, some of the previous reports available confirmed that the Pseudomonas is main spoilage bacteria in the seafoods. We do agree that selective medium counts alone are insufficient for definitive species assignment. We modified the text to state to presumptive Pseudomonas spp (line 147).

In section 3.3, the lower TBARS value in the 80% CO₂ + 20% O₂ group compared to the 80% CO₂ + 20% N₂ group is attributed only to "N₂ inhibiting endogenous antioxidant enzymes." Other factors, such as O₂ promoting CO₂ dissolution or microbial community differences affecting lipid oxidation, are not considered.

******Thank you for your valuable comment. The discussion has been modified. Please see line 443-445.

In section 3.4, the discussion of higher drip and cooking losses in MAP groups only attributes it to CO₂ forming carbonic acid, without analyzing muscle structure or protein denaturation (e.g., myofibrillar protein solubility, sulfhydryl content). The analysis lacks depth.

******We appreciate the reviewer’s observation regarding the mechanisms underlying the higher drip and cooking losses in MAP groups. This analysis could be another full-fledged study and adding to this it could exceed the amount of data. In this study, we primarily focused on the effect of different gas compositions on physical properties of HC meat during storage. We acknowledge that a more detailed analysis of muscle structure and protein denaturation-such as myofibrillar protein solubility or sulfhydryl content-would provide deeper mechanistic insight. In our ongoing work, we are doing histological changes, along with other structural analysis of HC during the frozen storage.

******We have updated the conclusion with future recommendation. Please see line 760-761.

In section 3.5, the heatmap (Fig. 5A) has unclear axis labels. Only Pearson correlation analysis was performed; partial correlation analysis to account for confounding factors (e.g., CO₂ and O₂ interaction) was not conducted.

******Thank you for your comment. Axis labels have been added for Fig. 5 in the figure caption. Please see revised figure caption. Concerning the statistical approach, we acknowledge that partial correlation analysis could provide additional insights by accounting for potential confounding factors like CO₂-O₂ interactions. However, our primary objective was to identify overall relationships between gas compositions and quality parameters. The Pearson correlation analysis we employed is widely accepted in food science literature for such exploratory analyses and provides a clear, interpretable overview of the main effects.

In section 3.7, only relative abundances at the family, genus, and species levels are analyzed. Differential species analysis to identify biomarkers for different treatment groups is lacking. The statement "MAP shifts the microbial community toward CO₂-tolerant taxa" does not include functional annotation of these taxa, limiting mechanistic insight.

******Thank you for this insightful comment. In this study, our primary objective was to evaluate how MAP at different gas compositions, with and without the CE conjugate, influences physicochemical properties and overall bacterial community structure during refrigerated storage. Therefore, the microbial analysis focused on describing relative abundance patterns at the family, genus, and species levels to identify broad compositional shifts associated with the treatments. Therefore, biomarker identification was beyond the scope of the present study, which aimed to characterize general community dynamics rather than to establish diagnostic microbial biomarkers for each treatment group. Similarly, functional annotation of taxa was not performed because the sequencing approach used (16S rRNA V3-V4 amplicon sequencing) primarily supports taxonomic profiling and provides limited resolution for reliable functional inference.

In section 3.7, PCoA and NMDS analyses only show clustering results. Significance tests for between-group differences (e.g., ANOSIM, Adonis) were not performed, so statistical significance of microbial community differences between CON and MAP groups cannot be confirmed.

******Thank you for this insightful comment. PERMANOVA using adonis2 did not detect a significant treatment effect on community composition (p = 0.1012). ANOSIM similarly was not significant (R = 0.9938, p = 0.1013), although interpretation is limited because one group contained only a single sample. The discussion has been revised 715-717 and 730-732.

Throughout the Results and Discussion sections, figure legends are inconsistent (e.g., CON-CE written as CONCE, MAP4-CE as MAPACE). The meaning of error bars (e.g., SD or SE) is not specified in some figure captions, violating chart standards.

******We checked text and figure captions carefully for consistent use of abbreviations. The error bars indicating standard deviation. The information has been included in the figure captions. Please see revised figure captions.

The suggestion to "add water-retaining additives to mitigate drip loss in MAP" is not supported by specific additive types or concentrations based on the study’s findings.

******Thank you for this valuable comment. We proposed a practical direction to address the increased drip and cooking losses observed under high CO₂ conditions (particularly MAP4 and MAP4-CE), which are consistent with CO₂-induced protein denaturation and reduced water-holding capacity in seafood muscle. However, the present study did not experimentally evaluate specific additives or concentrations. We have revised the text to clarify that this is a suggested direction for future research rather than a conclusion based on our data. Please see line 748-750.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

See attached PDF.

Comments for author File: Comments.pdf

Author Response

The authors have made significant improvements in the latest revision, but fundamental errors still remain. Until these are addressed, my previous recommendation for a "Major revision" remains unchanged. A recommendation for a major revision generally means that additional experiments are required.

Firstly, it is my comment that the gas composition and volume of gas before and after the end of the experiment must be stated. None of this has been addressed. I do not think it would be particularly difficult for the authors to perform additional tests where the volume change is measured. Furthermore, it is well known that PE does not function as a barrier for O2 and CO2. In MAP, plastics that exhibit sufficient barrier properties against these gases are advantageously selected. The authors should perform additional experiments where the gas composition is measured before and at the end of the experiment (preferably with some intermediate measurement point). As an alternative, the authors can try to theoretically calculate the gas composition from permeability coefficients. If the authors do not have access to permeability coefficients for the substances included in the gas mixture, these should be obtained experimentally.

*****We do understand the reviewer’s curiosity on gas composition in the package during storage period. Such measurements would provide additional insight into the dynamics of the modified atmosphere within the package. In the present study, polyamide/polyethylene (PA/PE) plastic bags with a thickness of 35 µm for each layer were used. This information has now been added to Section 2.1, and the corresponding text has been highlighted in the revised manuscript. PA/PE multilayer films are widely used in modified atmosphere packaging (MAP) applications because the polyamide layer provides relatively good gas barrier properties while polyethylene offers sealing and mechanical strength. Similar packaging materials have been used in previous research evaluating MAP for seafood and meat products

Solinho, J., Santos, J., Vázquez, M., & Pinheiro, R. (2025). Comparative study of preservation techniques for refrigerated Atlantic bonito Fillets: Effects of modified atmosphere packaging, vacuum packaging, and alginate coating on shelf life and quality. Food Packaging and Shelf Life, 50, 101556.
Qian, Y. F., Shi, C. J., Liu, C. C., Zhang, J. J., & Yang, S. P. (2025). Effects of CO2 and O2 in modified atmosphere packaging on water retention, protein stability, and microbial growth in atlantic salmon fillets. Fishes, 10(4), 141.
Latou, E., Mexis, S. F., Badeka, A. V., Kontakos, S., & Kontominas, M. G. (2014). Combined effect of chitosan and modified atmosphere packaging for shelf life extension of chicken breast fillets. LWT-Food science and Technology, 55(1), 263-268.

****** We acknowledge the reviewer’s suggestion to measure the gas composition during storage. However, analysis of gas composition requires specialized equipment such as a gas analyzer or gas chromatography system equipped with a thermal conductivity detector (GC-TCD). Unfortunately, we currently do not have access to these instruments in our laboratory and nearabout, and therefore performing additional gas composition analyses is not feasible at this stage.

*****The reviewer also suggested theoretically estimating gas composition based on permeability coefficients. While this approach is valuable, such calculations require accurate information on initial headspace gas composition, headspace volume, film permeability coefficients, and storage conditions. In our experiment, the headspace volume was not recorded because the focus of the study was on evaluating the antioxidant and antibacterial efficacy of the CE conjugate under MAP conditions. Furthermore, fresh hard clam samples are currently out of season and unavailable in the local market, which makes it impossible to repeat the experiment to determine the headspace volume under the same experimental conditions.

*****We would also like to clarify that the primary objective of this study was to evaluate the antioxidant and antibacterial efficacy of the CE conjugate under modified atmosphere storage conditions, rather than to investigate the gas permeability characteristics of the packaging material or the evolution of headspace gas composition. For this reason, commercially available PA/PE bags commonly used in MAP applications were selected to simulate practical packaging conditions.

*****Previous MAP studies have been conducted and published without determining the gas composition changes during the storage, particularly when using PA/PE bags where comparative treatment efficacy is the primary focus rather than material characterization. Please see the references.

Yang, Z., Yan, J., & Xie, J. (2023). Effect of vacuum and modified atmosphere packaging on moisture state, quality, and microbial communities of grouper (Epinephelus coioides) fillets during cold storage. Food Research International, 173, 113340.
Pan, C., Zhang, X., Chen, S., Xue, Y., Wu, Y., Wang, Y., & Wang, D. (2024). Analysis of quality-related proteins in golden pompano (Trachinotus ovatus) fillets with modified atmosphere packaging under superchilling storage. Food Science and Human Wellness, 13(4), 2253-2265.
Wang, S., Liu, Z., Zhao, M., Gao, C., Wang, J., Li, C., ... & Zhou, D. (2023). Chitosan-wampee seed essential oil composite film combined with cold plasma for refrigerated storage with modified atmosphere packaging: A promising technology for quality preservation of golden pompano fillets. International Journal of Biological Macromolecules, 224, 1266-1275.
Carrión-Granda, X., Fernández-Pan, I., Rovira, J., & Maté, J. I. (2018). Effect of antimicrobial edible coatings and modified atmosphere packaging on the microbiological quality of cold stored hake (Merluccius merluccius) fillets. Journal of Food Quality, 2018(1), 6194906.

Secondly, it must be stated whether chitooligosaccharide-EGCG (CE) is approved as a food additive or approved for contact with food. My comment on this has not been addressed. EGCG has health-promoting properties but can be toxic at high doses. In my first review, I intended that the authors should refer to the FDA, Commission Regulation (EU), or similar. The authors replied "The approval related to application of CE in food is subject to discussion. The cell culture and animal trails focusing on toxicity and allergenicity is in progress. The results based on those studies will be shared with regulatory authorities for the final decision on CE approval." They did not enter any amendment to the manuscript. If EGCG and CE are not approved for use in food packaging (including edible packaging), this must be clearly stated.

*****We have provided the statement as suggested. Please see line 125-126.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This has been revised according to the reviewers' suggestions. The article can now be accepted.

Comments on the Quality of English Language

Author Response

Thank you.