The Effect of Addition of Different Amounts of Chitosan Powder to the Batter and Chitosan Coating of Tea Sausage (Dry-Fermented Sausage) on the Physico-Chemical, Sensory and Microbiological Quality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “The Effect of Addition of Different Amounts of Chitosan Powder to the Batter and Chitosan Coating of Tea Sausage (Dry Fermented Sausage) on the Physico-chemical, Sensory and Microbiological Quality” addresses the effect of chitosan addition and chitosan coating on the quality characteristics of dry fermented sausages during storage. The topic is relevant for the field of meat science and food preservation, and the study includes a large experimental dataset covering physicochemical, sensory, textural, and microbiological analyses. The work is generally well organized and supported by a broad literature background. However, several aspects should be improved before the manuscript can be considered for the journal.

The main concern relates to the scientific novelty and focus of the study. Although the manuscript includes a very large number of experimental groups and statistical analyses, the actual scientific message remains relatively limited. Similar applications of chitosan in meat systems, including fermented sausages, coatings, and packaging, have already been extensively investigated. Therefore, the novelty of the present work should be more clearly highlighted, particularly explaining what additional knowledge this study provides compared to previous reports.

The experimental design is very complex, involving multiple factors (different chitosan concentrations, coating, packaging systems, and storage times), resulting in a large amount of data and numerous statistical comparisons. However, the biological and technological interpretation of the results is often limited. In several sections, the manuscript focuses excessively on reporting statistical significance without adequately discussing the practical relevance of the observed differences. For example, some sensory differences are statistically significant but numerically very small, and their real industrial or consumer relevance remains unclear.

The manuscript would also benefit from substantial shortening and restructuring. The Results and Discussion sections are excessively long and highly repetitive, particularly regarding comparisons with previous literature. In many cases, the discussion follows a repetitive “similar/different to previous studies” pattern without deeper mechanistic interpretation. The manuscript could be considerably improved by focusing on the most relevant findings and reducing repetitive literature comparisons.

An important methodological limitation concerns the chemical composition data. The manuscript explicitly states that some chemical analyses were performed on single aggregate samples without replication. This weakens the robustness of the analytical results and should be acknowledged more clearly as a limitation of the study.

The microbiological evaluation is relatively limited considering the complexity of the experimental design. Besides TPC and LAB, no detailed evaluation of spoilage microbiota or fungal dynamics during ripening/storage is provided, despite the importance of these groups in fermented sausages. The absence of changes in several microbiological parameters should also be discussed more critically.

The PCA analysis adds relatively limited additional insight beyond the univariate analyses and could potentially be simplified or shortened. Similarly, the statistical section should be clarified to improve readability, especially regarding the interpretation of high-order interactions.

Regarding sensory analysis, more information about panel reproducibility and practical significance of the observed differences would strengthen the manuscript. The scoring system is very detailed but may be difficult for international readers to interpret.

Overall, the manuscript contains a substantial amount of experimental work and may be suitable for publication after major revision. The authors are encouraged to improve the scientific focus of the paper, reduce unnecessary repetition, strengthen the interpretation of the results, and better clarify the practical significance of the findings.

Comments on the Quality of English Language

The manuscript is generally understandable; however, the quality of English language can be improved. Several sentences are excessively long and repetitive, which reduces readability and clarity. In particular, the Results and Discussion sections would benefit from more concise writing and improved flow. Minor grammatical inconsistencies and stylistic issues are present throughout the manuscript. Minor language revision is also recommended throughout the manuscript to improve fluency and reduce redundancy.

Author Response

Dear Reviewer,

Thank you very much for your comments and professional advice. These opinions help to improve our article. Based on your suggestion and request, we have made corrected modifications on the manuscript.

All corrections in the revised version of the manuscript are marked in red.

Furthermore, we would like to show the details as follows:

Comment 1: The manuscript entitled “The Effect of Addition of Different Amounts of Chitosan Powder to the Batter and Chitosan Coating of Tea Sausage (Dry Fermented Sausage) on the Physico-chemical, Sensory and Microbiological Quality” addresses the effect of chitosan addition and chitosan coating on the quality characteristics of dry fermented sausages during storage. The topic is relevant for the field of meat science and food preservation, and the study includes a large experimental dataset covering physicochemical, sensory, textural, and microbiological analyses. The work is generally well organized and supported by a broad literature background. However, several aspects should be improved before the manuscript can be considered for the journal.

Response 1: We sincerely thank the reviewer for the positive assessment of our work and for the valuable suggestions for improvement. We carefully considered all comments and revised the manuscript accordingly. All concerns have been addressed in detail below, and the manuscript has been substantially improved as a result of these revisions.

Comment 2: The main concern relates to the scientific novelty and focus of the study. Although the manuscript includes a very large number of experimental groups and statistical analyses, the actual scientific message remains relatively limited.

Response 2: We thank the reviewer for this important comment. We respectfully disagree that the scientific message is limited and have revised the Introduction section to better emphasize the novelty and significance of the study.

The novelty of the present work does not lie merely in evaluating chitosan as an ingredient, but in the simultaneous investigation of two distinct application strategies—incorporation of chitosan into the sausage batter and application of chitosan as an external coating—and, importantly, in assessing their individual and interactive effects through a factorial experimental design. To the best of our knowledge, studies comparing these two approaches within the same dry fermented sausage system are scarce.

Furthermore, the study provides an integrated evaluation of physicochemical, colour, texture, sensory, and microbiological characteristics, allowing a comprehensive assessment of how different chitosan application methods influence product quality. The results demonstrate that chitosan incorporation and chitosan coating do not affect quality attributes in the same manner. Chitosan addition improved overall sensory quality and reduced total plate counts, whereas chitosan coating altered colour characteristics and reduced lactic acid bacteria counts. Moreover, significant interactions between chitosan concentration and coating treatment were identified for several key quality parameters, indicating that the effects of chitosan cannot be fully understood when each treatment is evaluated separately.

Comment 3: Similar applications of chitosan in meat systems, including fermented sausages, coatings, and packaging, have already been extensively investigated. Therefore, the novelty of the present work should be more clearly highlighted, particularly explaining what additional knowledge this study provides compared to previous reports.

Response 3: We thank the reviewer for this valuable comment. We agree that chitosan has been extensively investigated in meat products, including its use as an ingredient, edible coating, and packaging material. However, the novelty of the present study lies not in the use of chitosan itself, but in the experimental approach and the specific knowledge generated regarding its application in dry fermented sausages.

The revised manuscript now more clearly emphasizes that this study simultaneously evaluated two different modes of chitosan application—incorporation into the sausage batter and application as a surface coating—within the same experimental design. While previous studies have typically focused on either chitosan addition or chitosan coating separately, direct comparisons between these approaches under identical processing and storage conditions are limited.

In addition, the factorial design enabled the evaluation of interaction effects between chitosan concentration and coating treatment. The results demonstrated that the two application methods produced different and, in some cases, opposite effects on colour, texture, sensory acceptance, and microbiological quality. These findings provide new information regarding the selection of the most appropriate chitosan application strategy for fermented meat products.

Furthermore, the study contributes data for tea sausage, a traditional dry fermented sausage for which information on chitosan application remains relatively scarce. The comprehensive assessment of physicochemical, sensory, textural, and microbiological characteristics provides an integrated understanding of the technological consequences of different chitosan treatments.

Comment 4: The experimental design is very complex, involving multiple factors (different chitosan concentrations, coating, packaging systems, and storage times), resulting in a large amount of data and numerous statistical comparisons. However, the biological and technological interpretation of the results is often limited. In several sections, the manuscript focuses excessively on reporting statistical significance without adequately discussing the practical relevance of the observed differences. For example, some sensory differences are statistically significant but numerically very small, and their real industrial or consumer relevance remains unclear.

Response 4: Thank you to the reviewer for this valuable observation. We agree that statistical significance does not necessarily imply practical or industrial relevance, particularly when the numerical differences are small. However, considering the limitations of the present study, particularly the relatively limited number of parameters evaluated, as also noted by Reviewer 1, we believe that it is important to report all statistically significant differences in detail. Although some of these differences may have limited immediate practical relevance, they may indicate emerging trends and provide useful guidance for the design and interpretation of future studies addressing similar research questions. Therefore, we have retained the detailed presentation of statistically significant differences while providing a more cautious interpretation of their practical and technological relevance in the revised Discussion.

Comment 5: The manuscript would also benefit from substantial shortening and restructuring. The Results and Discussion sections are excessively long and highly repetitive, particularly regarding comparisons with previous literature. In many cases, the discussion follows a repetitive “similar/different to previous studies” pattern without deeper mechanistic interpretation. The manuscript could be considerably improved by focusing on the most relevant findings and reducing repetitive literature comparisons.

An important methodological limitation concerns the chemical composition data. The manuscript explicitly states that some chemical analyses were performed on single aggregate samples without replication. This weakens the robustness of the analytical results and should be acknowledged more clearly as a limitation of the study.

Response 5: We thank the reviewer for this comment. Due to the differing opinions of the reviewers—some of whom suggested that the manuscript should be expanded by incorporating results from the Supplementary Materials, while others recommended that the manuscript be substantially shortened—we decided to retain the original structure of the manuscript. We have maintained the detailed presentation and discussion of the main factors investigated in the study, particularly considering the existing limitations of the present research. We believe that this approach provides a more complete presentation of the available findings while avoiding selective reporting of the observed effects.

We would like to clarify that the analyses were not performed as single, non-replicated measurements. For all evaluated parameters, repeated measurements were conducted, and the replication structure was incorporated into the statistical analysis. Specifically, repetitions were treated as a random factor in the statistical model to account for measurement variability and to provide appropriate estimates of experimental error.

We recognize that the description of the experimental design may not have been sufficiently clear in the original manuscript, potentially leading to this misunderstanding. Therefore, the Materials and Methods section has been revised to more explicitly describe the replication procedure and the statistical treatment of repeated measurements. As a result, we do not consider the analytical results to be based on unreplicated observations, nor do we believe that this represents a limitation of the study.

Regarding the results obtained from pooled samples (which was also raised by Reviewer 1), if the inclusion of these data in Tables 1 and 2 causes confusion, they can be omitted from the manuscript. Our intention was solely to provide a basic characterization of the sausage batter and sausage samples; therefore, these data were not subjected to statistical analysis or used for comparisons between experimental groups.

Comment 6: The microbiological evaluation is relatively limited considering the complexity of the experimental design. Besides TPC and LAB, no detailed evaluation of spoilage microbiota or fungal dynamics during ripening/storage is provided, despite the importance of these groups in fermented sausages. The absence of changes in several microbiological parameters should also be discussed more critically.

The PCA analysis adds relatively limited additional insight beyond the univariate analyses and could potentially be simplified or shortened. Similarly, the statistical section should be clarified to improve readability, especially regarding the interpretation of high-order interactions.

Response 6: Thank you to the reviewer for this constructive suggestion. We agree that the main findings of the study are derived from the factorial and univariate analyses, which directly enable the assessment of the effects of chitosan concentration, coating treatment, packaging system, and storage period on the investigated quality parameters.

The purpose of the PCA was not to replace the univariate analyses, but rather to provide an overall multivariate visualization of the relationships between the treatments and the investigated quality characteristics, allowing general patterns and associations that may not be readily apparent when individual variables are considered separately to be identified. The PCA presentation has been focused only on the most relevant findings.

Regarding the microbiological evaluation, we acknowledge that the number of microbiological parameters investigated was limited and that more detailed groups of spoilage microorganisms, as well as the dynamics of moulds and yeasts during ripening and storage, were not assessed. This represents one of the limitations of the present study and has now been clearly stated in a dedicated section entitled “Study Limitations and Future Perspectives.”

Comment 7: Regarding sensory analysis, more information about panel reproducibility and practical significance of the observed differences would strengthen the manuscript. The scoring system is very detailed but may be difficult for international readers to interpret.

Response 7: Thank you to the reviewer for this valuable comment. We agree that information regarding panel reproducibility and the practical significance of the observed sensory differences would strengthen the manuscript. We would like to clarify that the sensory evaluation in the present study was conducted in a single evaluation session; therefore, reproducibility across independent sensory sessions could not be assessed. We acknowledge this as a limitation of the present study, and this limitation has now been included in the newly added “Study Limitations and Future Perspectives” section.

The detailed scoring system was retained because it enabled the evaluation of several individual sensory attributes and provided a comprehensive characterization of the sausages. To improve readability for international readers, the scoring system and the interpretation of the scores have been clarified in the revised Materials and Methods section.

We also agree that statistical significance does not necessarily imply practical or consumer relevance. Therefore, the sensory results have been interpreted more cautiously in the revised Discussion, particularly in cases where statistically significant differences were associated with relatively small numerical changes. We have emphasized that such differences should not automatically be interpreted as meaningful changes in consumer perception. Future studies should include repeated sensory evaluation sessions and, preferably, a larger and appropriately trained panel to provide more robust information on the reproducibility and practical significance of the observed sensory differences.

Comment 8: Overall, the manuscript contains a substantial amount of experimental work and may be suitable for publication after major revision. The authors are encouraged to improve the scientific focus of the paper, reduce unnecessary repetition, strengthen the interpretation of the results, and better clarify the practical significance of the findings.

Response 8: We sincerely thank the reviewer for the overall positive assessment of our work and for the constructive recommendations provided throughout the review process. We appreciate the recognition of the substantial experimental work underlying this study.

In accordance with the reviewer’s comments, the manuscript has been revised. The scientific focus of the study has been further strengthened by more clearly emphasizing the main objectives, novelty, and practical significance of comparing the incorporation of chitosan into the sausage batter with the application of chitosan as a surface coating.

Comment 9: Comments on the Quality of English Language - The manuscript is generally understandable; however, the quality of English language can be improved. Several sentences are excessively long and repetitive, which reduces readability and clarity. In particular, the Results and Discussion sections would benefit from more concise writing and improved flow. Minor grammatical inconsistencies and stylistic issues are present throughout the manuscript. Minor language revision is also recommended throughout the manuscript to improve fluency and reduce redundancy.

Response 9: We thank the reviewer for this valuable comment. A detailed final language and grammatical revision will be performed on the final version of the manuscript if the paper is accepted for publication.

Best Regards,

Authors

Reviewer 2 Report

Comments and Suggestions for Authors

This study evaluates the effects of chitosan (0%, 0.25%, and 0.50%) and chitosan coating on some physicochemical, textural, microbiological, and sensory properties of Tea Sausage (a dry-fermented sausage specific to Serbia) in different packaging environments (vacuum and MAP) and at different storage times (0, 45, 90 days). The introduction section should be expanded to provide a clearer understanding of the study's background. The objective is well-defined. However, there are some methodological uncertainties, and some experimental points need to be addressed in more detail. Some statistical analyses and data presentation are quite weak. Overall, the discussion section does not meet expectations. Compared to more comprehensive studies in the literature evaluating the effects of chitosan and chitosan coating on on the overall quality characteristics of sausages, the present study does not offer a novel or innovative perspective. The characteristics of the raw meat and sausage batters used have not been defined. The effects of chitosan on key quality characteristics of sausages such as oxidative stability, microstructure, fatty acid composition have not been evaluated. The language of the article is very complex and its readability is poor. The fact that discussions of certain key interactions are presented in a ‘Supplementary Material’ file detracts from its comprehensibility. Nevertheless, some important changes that could improve the scientific quality and readability of the manuscript are listed below.

- Lines 48-49: Instead of this sentence, please describe the basic ingredients of tea sausage and how it differs from other dry-fermented sausages.

- Line 56: Which polysaccharide is most abundant after cellulose: chitin or chitosan? Please verify this statement with references.

- Lines 68-69: Contrary to the authors' claims, there are many studies in the literature on dry fermented sausages treated with chitosan. In this context, please revise the introduction focusing on how the present study fills a gap in the literature, its innovative aspects, and its significance for the meat industry.

- Lines 76-77: Please share the properties of chitosan and the details of how the coating is prepared.

-Lines 78-80: Please detail the characteristics of the meat used (animal and muscle specific characteristics, pH, fat content, proximate composition, etc.).

- Lines 81-83: On what basis were these amounts of chitosan chosen? How were the chitosan incorporated into the meat mixture? Also, specify how the homogeneous distribution of these powders was ensured. Describe the production process in detail, leaving no room for doubt.

- Lines 102-115: To improve the presentation and better understand the treatments, please provide high-resolution images of the sausages.

- Lines 114-115: Please check the total number of samples according to Table S1 and verify the mathematical calculation.

- Line 115: Why was this storage temperature chosen? Please explain in detail.

- Lines 126-127: How were 15 readings taken from 10 sausage samples? Please explain in detail.

- Lines 174-176: Which statistical model was used in the analysis? Please elaborate.

- Lines 196-198: Chemical composition analyses were performed using only one measurement, and it is unclear which of the three independent sausage batters it represents. Is it acceptable to perform a single measurement from only one of the three independent batters for the presentation and understanding of the results?

- Lines 218-219: Please discuss whether medium-level chitosan (0.25%) treatment resulted in lower TPC and LAB in sausages compared to high-level chitosan (0.50%) treatment.

 

- Lines 233-253: Why were the repetitions significant? Was the meat source or the chemical composition of the meat different? To evaluate the effect of repetitions on the parameters, please first provide the chemical composition and specific characteristics of the three independent meat and fat sources.

- Lines 260-261: Which of the three independent batches do these chemical analysis findings belong to? Given that difference tests were performed between treatments or groups, and the main results of the study are absolutely related to these chemical composition data, determining chemical composition parameters based on a single measurement without repetition is statistically weak.

- Lines 272-275: Throughout the manuscript, only the significance levels of the main factors and their interactions on the tested parameters are presented. The effects and interactions of chitosan amount, coating, packaging, and storage are not discussed or are discussed very little. Please expand the discussion sections throughout the manuscript, taking into account the significant interaction effects. Additionally, to improve readability, please incorporate discussions of some key interactions presented in the "Supplementary Materials" file into the main text.

-Lines 450-482: In the conclusion section, please also present the shortcomings of the current study and your suggestions for future studies.

-Please rewrite the Abstract and Results sections, taking into account the key findings of the current study.

-Please prepare a graphical abstract to improve the presentation of the study and facilitate understanding of the methodology.

Author Response

Dear Reviewer,

Thank you very much for your comments and professional advice. These opinions help to improve our article. Based on your suggestion and request, we have made corrected modifications on the manuscript.

All corrections in the revised version of the manuscript are marked in red.

Furthermore, we would like to show the details as follows:

Comment 1: This study evaluates the effects of chitosan (0%, 0.25%, and 0.50%) and chitosan coating on some physicochemical, textural, microbiological, and sensory properties of Tea Sausage (a dry-fermented sausage specific to Serbia) in different packaging environments (vacuum and MAP) and at different storage times (0, 45, 90 days).

Response 1: This study investigates how different levels of chitosan (0%, 0.25%, and 0.50%) and chitosan coating affect the quality and shelf life of Tea Sausage, a traditional Serbian dry-fermented sausage. The researchers evaluated physicochemical, textural, microbiological, and sensory characteristics under two packaging conditions (vacuum packaging and modified atmosphere packaging, MAP) over a storage period of 0, 45, and 90 days.

Comment 2: The introduction section should be expanded to provide a clearer understanding of the study's background. The objective is well-defined.

Response 2: Thank you for your comment. The Introduction has been revised and expanded to provide a clearer and more comprehensive background to the study.

Comment 3: However, there are some methodological uncertainties, and some experimental points need to be addressed in more detail.

Response 3: Thank you for your comment. We acknowledge the need for greater methodological clarity and have revised the manuscript accordingly. Additional details and explanations have been incorporated into the relevant sections to address the identified methodological uncertainties and experimental aspects. Specific responses to each point are provided below.

Comment 4: Some statistical analyses and data presentation are quite weak.

Response 4: Thank you for your comment. We appreciate the reviewer's observation regarding the statistical analyses and data presentation. In response, we have carefully reviewed and revised the statistical analysis section to provide clearer descriptions of the methods used and to ensure their appropriateness for the study design. In addition, the presentation of the results has been improved through revisions to the tables, figures, and accompanying text to enhance clarity, readability, and interpretation of the data. We believe these changes have strengthened the overall quality and rigor of the manuscript.

Comment 5: Overall, the discussion section does not meet expectations. Compared to more comprehensive studies in the literature evaluating the effects of chitosan and chitosan coating on on the overall quality characteristics of sausages, the present study does not offer a novel or innovative perspective.

Response 5: Thank you for your valuable comment. We appreciate the reviewer's concerns regarding the depth of the Discussion and the novelty of the study. In response, the Discussion section has been substantially revised and expanded to provide a more comprehensive interpretation of the findings. Additional comparisons with previous studies on the effects of chitosan and chitosan coatings in fermented meat products have been incorporated, and the similarities and differences between our results and those reported in the literature are now discussed in greater detail.

We acknowledge that the use of chitosan in meat products has been extensively investigated. However, the present study contributes additional information by evaluating the combined effects of different chitosan concentrations, chitosan coating, packaging systems (vacuum and MAP), and storage duration on the quality characteristics of Tea Sausage, a traditional Serbian dry-fermented sausage. The revised Discussion now better highlights the relevance of this specific product and experimental design, as well as the practical implications of the findings for the preservation and quality maintenance of fermented meat products.

Comment 6: The characteristics of the raw meat and sausage batters used have not been defined.

Response 6: Thank you for this valuable comment. The characteristics of the raw meat used in the study were not determined, which represents one of the limitations of the present study. However, the characteristics of the sausage batter are presented in Table 1. To provide greater transparency regarding the scope of the study, a new section has been added to the revised manuscript in which the main shortcomings and limitations of the study are clearly stated and discussed.

Comment 7: The effects of chitosan on key quality characteristics of sausages such as oxidative stability, microstructure, fatty acid composition have not been evaluated.

Response 7: Thank you for this valuable comment. The effects of chitosan on certain important quality characteristics, including oxidative stability, microstructure, and fatty acid composition, were not evaluated in the present study. This also represents an important limitation of the study and has been acknowledged in the revised manuscript. In particular, these aspects have been included among the limitations discussed in the newly added section on the shortcomings and limitations of the study.

Comment 8: The language of the article is very complex and its readability is poor. The fact that discussions of certain key interactions are presented in a ‘Supplementary Material’ file detracts from its comprehensibility. Nevertheless, some important changes that could improve the scientific quality and readability of the manuscript are listed below.

Response 8: Thank you for the constructive comments. We appreciate the reviewer’s concerns regarding the complexity of the language and the overall readability of the manuscript. In response to the reviewer’s comment, the manuscript has been carefully revised to improve the clarity, readability, and logical flow of the text.

Considering the limitations of the study, particularly the number of parameters evaluated, we decided not to change the overall structure of the manuscript or the Supplementary Material, as the latter provides a detailed presentation of the characteristics investigated in the study. Moreover, the reviewers have expressed different opinions regarding the length of the manuscript, making it unclear whether the manuscript should be extended by incorporating additional results from the Supplementary Material or substantially shortened. Therefore, we decided to retain the original structure of the manuscript.

Comment 9: - Lines 48-49: Instead of this sentence, please describe the basic ingredients of tea sausage and how it differs from other dry-fermented sausages.

Response 9: Thank you for this valuable suggestion. In accordance with the reviewer’s comment, the sentence has been replaced with a more detailed description of the basic ingredients and technological characteristics of Tea Sausage. The revised Introduction now provides information on the typical formulation of Tea Sausage, including meat, pork fat, salt, spices, and other relevant ingredients, as well as its characteristic fermentation and drying process. In addition, the differences between Tea Sausage and other dry-fermented sausages have been clarified, with particular emphasis on its traditional production in Serbia, characteristic formulation, sensory properties, and specific technological characteristics. These additions provide a clearer background for the selection of Tea Sausage as the model product in the present study.

Comment 10: - Line 56: Which polysaccharide is most abundant after cellulose: chitin or chitosan? Please verify this statement with references.

Response 10: Thank you for pointing this out. We agree that the original statement was inaccurate. The statement has been corrected to indicate that chitin, rather than chitosan, is considered the second most abundant polysaccharide/biopolymer after cellulose. Chitosan is a derivative of chitin obtained primarily through deacetylation. The corresponding sentence in the revised manuscript has therefore been corrected and supported with appropriate references.

Comment 11: - Lines 68-69: Contrary to the authors' claims, there are many studies in the literature on dry fermented sausages treated with chitosan. In this context, please revise the introduction focusing on how the present study fills a gap in the literature, its innovative aspects, and its significance for the meat industry.

Response 11: Thank you for this important comment. We agree that the application of chitosan in dry-fermented sausages has been extensively investigated and have revised the Introduction accordingly. We have removed/modified statements that could imply that this topic has not been sufficiently studied. The revised Introduction now provides a more balanced overview of previous studies and more clearly identifies the specific research gap addressed by the present work. In particular, we emphasize the combined evaluation of different chitosan concentrations, chitosan coating, packaging systems (vacuum and MAP), and storage duration in Tea Sausage, a Serbian dry-fermented sausage. We have also clarified the potential significance of these findings for maintaining product quality and extending shelf life in the meat industry. Relevant references to previous studies have been added to better support the revised discussion of the research gap and novelty.

Comment 12: - Lines 76-77: Please share the properties of chitosan and the details of how the coating is prepared.

Response 12: Thank you for this valuable comment. In accordance with the reviewer’s suggestion, the requested information regarding the physico-chemical properties of chitosan and the detailed procedure used for the preparation of the chitosan coating has been added to the revised manuscript. These details have been included in the Materials and Methods section to provide a clearer and more complete description of the experimental procedure.

Comment 13: - Lines 78-80: Please detail the characteristics of the meat used (animal and muscle specific characteristics, pH, fat content, proximate composition, etc.).

Response 13: Thank you for this important comment. The animal species and type of meat used for the production of Tea Sausage have already been specified in the manuscript. However, the requested characteristics of the raw meat, such as pH, fat content, and proximate composition, were not determined at the time of the experiment. Therefore, these parameters for the raw meat cannot be provided retrospectively, which represents one of the limitations of the present study and is now acknowledged in a dedicated section on the study limitations. On the other hand, the characteristics of the fresh sausage batter were determined and are presented in Table 1. We believe that these data provide useful information on the initial characteristics of the material used for sausage production and allow a better understanding of the changes occurring during fermentation and storage.

Comment 14: - Lines 81-83: On what basis were these amounts of chitosan chosen? How were the chitosan incorporated into the meat mixture? Also, specify how the homogeneous distribution of these powders was ensured. Describe the production process in detail, leaving no room for doubt.

Response 14: Thank you for this valuable comment. The chitosan concentrations used in the present study (0.25% and 0.50%) were selected based on concentrations reported in the cited literature, while taking into consideration the combined application of chitosan incorporation into the meat batter and chitosan coating. In this context, the selected concentrations were considered appropriate for evaluating the potential effects of chitosan addition in combination with surface coating on the quality characteristics of Tea Sausage. In response to the reviewer’s comment, the description of the production process has been revised and expanded to provide clearer details on the incorporation of chitosan into the meat mixture and the procedure used to ensure its homogeneous distribution. Specifically, the required amount of chitosan was accurately weighed, initially mixed with a smaller portion of the meat batter to obtain a homogeneous premix, and subsequently incorporated into the remaining batter, followed by thorough mixing. These details have been added to the revised manuscript to ensure a clear and reproducible description of the experimental procedure.

Comment 15: - Lines 102-115: To improve the presentation and better understand the treatments, please provide high-resolution images of the sausages.

Response 15: Thank you for this valuable suggestion. Unfortunately, high-resolution images of the sausages were not recorded during the experiment. We acknowledge that the absence of photographic documentation represents another limitation of the present study. We have taken this limitation into consideration and will ensure that appropriate high-resolution images are included in future studies to provide better visual documentation of the treatments and product characteristics.

Comment 16: - Lines 114-115: Please check the total number of samples according to Table S1 and verify the mathematical calculation.

Response 16: Thank you for your careful observation. We would like to clarify that Table S1 presents the ripening conditions of the sausages, whereas Table S2 presents the different sausage groups included in the experiment. The experimental design comprised three sausage batter formulations and a total of 27 sausage groups. We have carefully rechecked the calculations and confirmed that the total number of samples is consistent with the experimental design presented in the manuscript. We appreciate the reviewer’s comment, which helped us to clarify the presentation of the experimental groups.

Comment 17: - Line 115: Why was this storage temperature chosen? Please explain in detail.

Response 17: Thank you for your valuable comment. The storage temperature used in this study was selected in accordance with the Serbian regulation on the quality and other requirements for meat products, which specified a maximum storage temperature of 15 °C for dry-fermented sausages, including Tea Sausage. We have clarified the basis for the selected storage temperature in the revised manuscript and added the appropriate regulatory reference.

Comment 18: - Lines 126-127: How were 15 readings taken from 10 sausage samples? Please explain in detail.

Response 18: Thank you for pointing this out. We apologize for the lack of clarity in the description of the colour measurements. The description of the colour measurement procedure has been revised in the manuscript to clarify this procedure and avoid any misunderstanding.

Comment 19: - Lines 174-176: Which statistical model was used in the analysis? Please elaborate.

Thank you for this comment. We agree that the statistical model was not described with sufficient detail in the original manuscript. Therefore, the Statistical Analysis section has been revised to provide a clearer explanation of the analytical approach.

Response 19: Specifically, a mixed-effects ANOVA model was applied, where the amount of chitosan, chitosan coating, packaging type, and storage time were treated as fixed effects, while experimental repetition was included as a random effect to account for variability among independent experimental runs. The model evaluated the main effects of each factor as well as their two-way, three-way, and four-way interactions. When significant effects were detected, differences among treatment means were assessed using Tukey’s HSD post hoc test at a significance level of P < 0.05.

Comment 20: - Lines 196-198: Chemical composition analyses were performed using only one measurement, and it is unclear which of the three independent sausage batters it represents. Is it acceptable to perform a single measurement from only one of the three independent batters for the presentation and understanding of the results?

Response 20: Thank you for this important comment. We agree with the reviewer that a single measurement from only one of the three independent sausage batters is not sufficient for statistical comparison or for drawing conclusions about differences among the experimental groups. We would like to clarify that the chemical composition analysis was performed only for the purpose of characterizing the fresh sausage batter and the final sausage products. These measurements were not intended for statistical evaluation, and the corresponding results were neither statistically compared nor discussed in relation to treatment effects. They were included only to provide basic compositional information about the samples. Nevertheless, we acknowledge the reviewer’s concern regarding the presentation of these results and agree that their inclusion may cause confusion regarding their interpretation. Therefore, for the sake of clarity and to avoid potential misinterpretation, we are prepared to remove these results from the manuscript if deemed necessary. The explanation of which sausage batter/sample these results refer to is provided in Table S1.

Comment 21: - Lines 218-219: Please discuss whether medium-level chitosan (0.25%) treatment resulted in lower TPC and LAB in sausages compared to high-level chitosan (0.50%) treatment.

Response 21: Thank you for this valuable comment. We agree that the significantly lower TPC and LAB values observed in the Ch0.25/B batter compared with both the control (Ch0/B) and Ch0.50/B treatments require further explanation. Accordingly, the Discussion section has been revised to address this concentration-specific response. We have added a discussion of the possible reasons why the intermediate chitosan concentration (0.25%) resulted in significantly lower TPC and LAB values than the higher concentration (0.50%), taking into consideration the complex interactions between chitosan and the meat matrix. We have also clarified that this finding refers specifically to the fresh sausage batter and should not be generalized to the subsequent fermentation and storage stages.

Comment 22: - Lines 233-253: Why were the repetitions significant? Was the meat source or the chemical composition of the meat different? To evaluate the effect of repetitions on the parameters, please first provide the chemical composition and specific characteristics of the three independent meat and fat sources.

Response 22: Thank you for this important comment. The significant effect of repetition is likely attributable to the natural variability among the independent meat and fat batches used in the study. The raw materials originated from different production batches and were not completely homogeneous with respect to their physicochemical characteristics. Such biological variation is common in meat products and may influence several quality parameters measured during processing and storage. For this reason, repetition was included as a random effect in the statistical model to account for variability among independent experimental runs and to avoid confounding treatment effects with batch-to-batch variation.

We agree that additional information regarding the raw materials is important for interpreting the significance of the repetition effect. We have also clarified in the Discussion that the significant repetition effect likely reflects the inherent heterogeneity of the raw materials rather than systematic differences among treatments.

Comment 23: - Lines 260-261: Which of the three independent batches do these chemical analysis findings belong to? Given that difference tests were performed between treatments or groups, and the main results of the study are absolutely related to these chemical composition data, determining chemical composition parameters based on a single measurement without repetition is statistically weak.

Response 23: Thank you for this comment. The sampling procedure used for these analyses was the same as that applied to the sausage batter. Therefore, the analyses were performed on a pooled sample rather than on individual experimental units. For this reason, statistical analysis was not performed for these data. The specific sausage batter/sample to which the results refer is clearly indicated in the table heading and is additionally described in Table S1. If considered necessary, these results, as well as the corresponding results for the sausage batter, can be removed from the manuscript to avoid any potential confusion regarding their statistical interpretation.

Comment 24: - Lines 272-275: Throughout the manuscript, only the significance levels of the main factors and their interactions on the tested parameters are presented. The effects and interactions of chitosan amount, coating, packaging, and storage are not discussed or are discussed very little. Please expand the discussion sections throughout the manuscript, taking into account the significant interaction effects. Additionally, to improve readability, please incorporate discussions of some key interactions presented in the "Supplementary Materials" file into the main text.

Response 24: Thank you for this comment. As previously stated, we decided to retain the original structure of the manuscript and the Supplementary Materials. This decision was made because the reviewers expressed different opinions regarding the appropriate length of the manuscript: some suggested that the manuscript should be expanded by incorporating results from the Supplementary Materials, whereas others considered that the manuscript should be substantially shortened. Nevertheless, all statistically significant differences and interaction effects are presented and discussed either in the main manuscript or in the Supplementary Materials.

Comment 25: - Lines 450-482: In the conclusion section, please also present the shortcomings of the current study and your suggestions for future studies.

Response 25: Thank you for this comment. These aspects have been addressed in a separate section entitled “Limitations of the Study and Future Perspectives,” which has been added to the revised manuscript.

Comment 26: - Please rewrite the Abstract and Results sections, taking into account the key findings of the current study.

Response 26: Thank you for this important comment. Due to the numerous limitations of the present study, particularly the relatively limited number of parameters evaluated, we decided to provide a detailed description of all investigated parameters. This approach was adopted even in cases where the numerical differences were relatively small but statistically significant, as these findings may nevertheless provide useful guidance and a basis for further studies of a similar nature.

Comment 27: - Please prepare a graphical abstract to improve the presentation of the study and facilitate understanding of the methodology.

Response 27: Thank you for this comment. As previously mentioned, the present study has certain limitations, including the lack of photographic documentation of the sausages, which considerably limits the possibility of preparing an appropriate and informative graphical abstract. In addition, graphical abstracts are not mandatory for submission to the journal Foods. Therefore, considering these limitations, we decided not to include a graphical abstract in the revised manuscript.

Best Regards,

Authors

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a study on the use of chitosan powder and chitosan coating in dry fermented tea sausages. The topic is interesting, especially in the context of natural additives and preservation strategies for meat products. The experimental work includes physicochemical, sensory and microbiological analyses. However, the manuscript would benefit from revision to improve clarity, reduce repetition and better explain the experimental rationale.

The Introduction provides sufficient general background on chitosan and fermented sausages, but the novelty of the study should be stated more clearly. In particular, the authors should better explain why chitosan was used both as a powder added to the batter and as a surface coating, as this is not clearly presented.

This issue becomes particularly important when interpreting the results, as the addition of chitosan to the batter appears to provide more consistent benefits than coating.

The Materials and Methods section is generally detailed, but several points require clarification. The coating preparation procedure should be briefly described in the manuscript rather than referring to previous publications.

Similarly, the use of the term “lean pork meat” for pork shoulder is questionable and should be reconsidered or supported with compositional data.

What is the allspice extract?

In addition, the phrase “left to rest overnight” is too vague and should be replaced with precise conditions.

Although smoking, fermentation and ripening conditions are presented in the Supplementary Material, the most important process parameters should also be summarized in the main manuscript in my opinion.

Another important issue concerns the experimental design and statistical analysis. The authors states that three independent production batches were prepared, but it is not entirely clear what constituted the experimental unit. It would be helpful if the authors explicitly stated whether statistical analyses were performed using production batches, individual sausages or analytical measurements as replicates.

The Results and Discussion section contains a large amount of information, but it is often difficult to identify the most important findings. A considerable part of the discussion is repetitive. For example, similar discussion patterns are repeated for pH, colour, texture and microbiological characteristics, where the authors first present their results and then provide long lists of studies reporting similar, contradictory or neutral effects. This approach considerably increases the length of the manuscript without always improving interpretation (the colour section is a good example. The manuscript discusses numerous colour parameters (L*, a*, b*, C*, h and λ), as well as multiple interaction effects between treatment factors. However, the practical significance of many of these differences remains unclear. For instance, several significant differences in colour traits are numerically very small and may not be perceptible to consumers or relevant from a technological perspective). The authors themselves note in the Supplementary Material that some significant differences were not of great magnitude. This issue deserves greater emphasis in the discussion.

A similar situation occurs in the sensory analysis. The reported overall sensory scores ranged approximately from 4.81 to 4.87 depending on treatment. Although some of these differences reached significance, it is questionable whether such small differences would be meaningful from a consumer perspective.

The observed reduction in LAB counts after coating deserves further discussion. Since lactic acid bacteria play an important role in fermented sausage quality, it would be useful to discuss whether this effect should be regarded as beneficial, neutral or potentially undesirable.

The Supplementary Material contains useful information regarding processing conditions and experimental design. However, a substantial part of the Supplementary Material consists of detailed narrative descriptions of ANOVA outputs, which largely repeat information already presented in the Results section. Considerably shortening these descriptions would improve readability and reduce redundancy.

Finally, the Conclusions section should be more focused. At present, it largely summarizes individual analytical results. Instead, the authors should concentrate on the broader message of the study. Moreover, please delete numbering in conclusions.

Author Response

Dear Reviewer,

Thank you very much for your comments and professional advice. These opinions help to improve our article. Based on your suggestion and request, we have made corrected modifications on the manuscript.

All corrections in the revised version of the manuscript are marked in red.

Furthermore, we would like to show the details as follows:

Comment 1: The manuscript presents a study on the use of chitosan powder and chitosan coating in dry fermented tea sausages. The topic is interesting, especially in the context of natural additives and preservation strategies for meat products. The experimental work includes physicochemical, sensory and microbiological analyses. However, the manuscript would benefit from revision to improve clarity, reduce repetition and better explain the experimental rationale.

Response 1: We sincerely thank the reviewer for the positive assessment of the study and for recognizing the relevance of investigating chitosan as a natural additive and preservation strategy in dry fermented sausages.

Furthermore, the Introduction and Materials and Methods sections have been revised to better explain the experimental rationale and the scientific motivation for evaluating both chitosan incorporation into the sausage batter and chitosan surface coating. Additional discussion has also been included to clarify the biological and technological significance of the observed effects and their practical implications for fermented meat production.

Comment 2: The Introduction provides sufficient general background on chitosan and fermented sausages, but the novelty of the study should be stated more clearly. In particular, the authors should better explain why chitosan was used both as a powder added to the batter and as a surface coating, as this is not clearly presented.

This issue becomes particularly important when interpreting the results, as the addition of chitosan to the batter appears to provide more consistent benefits than coating.

Response 2: Thank you to the reviewer for this valuable comment. In accordance with the reviewer’s suggestion, the novelty of the present study has been more clearly emphasized in the revised Introduction. In particular, we have clarified the rationale for investigating two different modes of chitosan application: its incorporation into the sausage batter and its application as a surface coating.

Comment 3: The Materials and Methods section is generally detailed, but several points require clarification. The coating preparation procedure should be briefly described in the manuscript rather than referring to previous publications.

Response 3: Thank you to the reviewer for this valuable comment. In accordance with the reviewer’s suggestion, the revised Materials and Methods section now includes a brief description of the preparation and application of the chitosan coating, as well as the main characteristics of the chitosan used in the study, including its source, manufacturer, product specification, and relevant physicochemical properties. This additional information was included to provide a clearer and more complete description of the experimental materials and procedures.

Comment 4: Similarly, the use of the term “lean pork meat” for pork shoulder is questionable and should be reconsidered or supported with compositional data.

Response 4: Thank you to the reviewer for this comment. We have revised the terminology accordingly and replaced “lean pork meat” with “pork shoulder meat”

Comment 5: What is the allspice extract?

Response 5: Thank you for the comment. The terminology has been clarified in the revised manuscript, and “allspice extract (Pimenta dioica)” has been used.

Comment 6: In addition, the phrase “left to rest overnight” is too vague and should be replaced with precise conditions.

Response 6: Thank you for this comment. The precise conditions for the overnight conditioning step, including temperature and relative humidity, are provided in Table S1 of the Supplementary Material, as indicated at the end of the corresponding paragraph.

Comment 7: Although smoking, fermentation and ripening conditions are presented in the Supplementary Material, the most important process parameters should also be summarized in the main manuscript in my opinion.

Response 7: Thank you for this comment. We agree that the most important processing conditions should be readily accessible to the reader. The key parameters of the process are now provided in the main manuscript, while the detailed process conditions are presented in Table S1 of the Supplementary Material.

Comment 8: Another important issue concerns the experimental design and statistical analysis. The authors states that three independent production batches were prepared, but it is not entirely clear what constituted the experimental unit. It would be helpful if the authors explicitly stated whether statistical analyses were performed using production batches, individual sausages or analytical measurements as replicates.

Response 8: We thank the reviewer for this important comment. We agree that the definition of the experimental unit should be stated more clearly.

In the present study, three independent production batches were prepared and served as biological replicates of the experiment. Multiple sausages were produced within each batch and used for subsequent physicochemical, sensory, and microbiological analyses. However, individual analytical measurements were not treated as independent experimental replicates. Instead, the statistical analysis accounted for the replication structure by including batch (replication) as a random factor in the statistical model, thereby incorporating between-batch variability into the estimation of treatment effects.

Comment 9: The Results and Discussion section contains a large amount of information, but it is often difficult to identify the most important findings. A considerable part of the discussion is repetitive. For example, similar discussion patterns are repeated for pH, colour, texture and microbiological characteristics, where the authors first present their results and then provide long lists of studies reporting similar, contradictory or neutral effects. This approach considerably increases the length of the manuscript without always improving interpretation (the colour section is a good example. The manuscript discusses numerous colour parameters (L*, a*, b*, C*, h and λ), as well as multiple interaction effects between treatment factors. However, the practical significance of many of these differences remains unclear. For instance, several significant differences in colour traits are numerically very small and may not be perceptible to consumers or relevant from a technological perspective). The authors themselves note in the Supplementary Material that some significant differences were not of great magnitude. This issue deserves greater emphasis in the discussion.

Response 9: We thank the reviewer for this valuable comment. We are aware that the detailed presentation of the results contributes to the overall length of the manuscript. However, this approach was intentional, as we aimed to provide a detailed analysis and discussion of all investigated parameters. The present study has limitations regarding the relatively limited number of quality parameters evaluated, as clearly stated in the dedicated “Study Limitations and Future Perspectives” section. Therefore, we considered it important to present and discuss all statistically significant differences, including those that were relatively small in numerical magnitude.

We agree that some statistically significant differences were numerically small and may therefore have limited immediate practical or consumer relevance. Nevertheless, we believe that such findings may indicate certain trends and provide useful guidance for future research on the application of chitosan in fermented meat products. Therefore, these differences were also considered and discussed in the Discussion section.

As previously noted, the reviewers expressed different opinions regarding the appropriate length of the manuscript—some suggested that the manuscript should be expanded by incorporating additional results from the Supplementary Material, whereas others recommended substantial shortening. Considering these differing recommendations, as well as the limitations of the present study, we decided to retain the original structure of the manuscript, with a detailed presentation and discussion of the investigated parameters.

Comment 10: A similar situation occurs in the sensory analysis. The reported overall sensory scores ranged approximately from 4.81 to 4.87 depending on treatment. Although some of these differences reached significance, it is questionable whether such small differences would be meaningful from a consumer perspective.

Response 10: Thank you for this valuable comment. A similar concern was also raised by another reviewer. Accordingly, we have added a paragraph to the Sensory Characteristic of Tea Sausages section discussing the relatively small numerical differences observed among treatments and emphasizing that statistically significant differences do not necessarily imply meaningful differences from a consumer perspective.

Comment 11: The observed reduction in LAB counts after coating deserves further discussion. Since lactic acid bacteria play an important role in fermented sausage quality, it would be useful to discuss whether this effect should be regarded as beneficial, neutral or potentially undesirable.

Response 11: Thank you for this valuable comment. We agree that the reduction in LAB counts following chitosan coating requires careful interpretation. We have therefore expanded the Discussion to clarify that LAB are essential for fermentation, acidification, flavour development, and stability of dry-fermented sausages, and that excessive inhibition could potentially be undesirable. However, the reduction observed in the present study was interpreted cautiously, as LAB remained detectable in the coated sausages and the effect may reflect partial control of microbial growth rather than complete inhibition. We have also acknowledged that further studies monitoring LAB dynamics and identifying the predominant LAB species would be valuable for determining the technological significance of this effect.

Comment 12: The Supplementary Material contains useful information regarding processing conditions and experimental design. However, a substantial part of the Supplementary Material consists of detailed narrative descriptions of ANOVA outputs, which largely repeat information already presented in the Results section. Considerably shortening these descriptions would improve readability and reduce redundancy.

Response 12: Thank you for this valuable comment. We agree that some of the descriptions of the ANOVA results in the Supplementary Material overlap with information presented in the main manuscript. However, this approach was intentional, as we wanted the reader to have access to the complete statistical analysis in one place, rather than having the statistical information divided between the main manuscript and the Supplementary Material. Therefore, we decided to retain the complete statistical analysis in the Supplementary Material to provide a comprehensive overview of the statistical results and facilitate their interpretation.

Comment 13: Finally, the Conclusions section should be more focused. At present, it largely summarizes individual analytical results. Instead, the authors should concentrate on the broader message of the study. Moreover, please delete numbering in conclusions.

Response 13: Thank you for this valuable comment. In accordance with the reviewer’s suggestion, the bullet points have been removed from the Conclusions section, and the individual findings have been presented as a continuous text. A general conclusion has also been added to emphasize the broader message and practical significance of the study. Directions for future research and further perspectives are already provided in the dedicated “Study Limitations and Future Perspectives” section.

 

Best Regards,

Authors

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed the major concerns raised during the previous round of review, and the revised manuscript has substantially improved in terms of methodological transparency, statistical treatment, discussion of the results, and acknowledgment of the study limitations. In particular, the clarification of the experimental design and statistical approach and the expanded discussion of the limitations strengthen the manuscript considerably.I have only one minor point remaining. The Methods state that, in addition to TPC and LAB, Enterobacteriaceae, E. coli, Salmonella spp., Listeria monocytogenes, sulfite-reducing clostridia, yeasts and moulds were investigated, and the Results report that these microorganisms were not detected. However, the Study Limitations section states that the microbiological evaluation was limited to TPC and LAB and that yeasts and moulds were not investigated. This apparent inconsistency should be corrected for clarity.

Author Response

Dear Reviewer,

Thank you very much for your comments and professional advice. These opinions help to improve our article. Based on your suggestion and request, we have made corrected modifications on the manuscript.

All corrections in the revised version of the manuscript are marked in red.

Furthermore, we would like to show the details as follows:

Comment: I have only one minor point remaining. The Methods state that, in addition to TPC and LAB, Enterobacteriaceae, E. coli, Salmonella spp., Listeria monocytogenes, sulfite-reducing clostridia, yeasts and moulds were investigated, and the Results report that these microorganisms were not detected. However, the Study Limitations section states that the microbiological evaluation was limited to TPC and LAB and that yeasts and moulds were not investigated. This apparent inconsistency should be corrected for clarity.

Response: Thank you for pointing out this inconsistency. We have corrected the Study Limitations section accordingly by removing the statement that yeasts and moulds were not investigated. The corresponding text has been revised to ensure consistency with the microbiological analyses reported in the Methods and Results sections.

 

Best Regards,

Authors

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript overall, taking my comments into account.

Author Response

Dear Reviewer,

Thank you for your positive and constructive feedback. We greatly appreciate the time and effort you have devoted to reviewing our manuscript. We are pleased that the revisions adequately address your comments and have improved the overall quality and clarity of the manuscript.

Best Regards,

Authors

Reviewer 3 Report

Comments and Suggestions for Authors

The experimental unit and replication structure still require clarification. Please explicitly state whether three independent production batches were prepared for each chitosan concentration, or whether only one production batch was prepared for each concentration (0, 0.25, and 0.50%). This information is essential to understand what constituted the biological replicates and how the random effect of experimental repetition was defined in the statistical model.

Author Response

Dear Reviewer,

Thank you very much for your comments and professional advice. These opinions help to improve our article. Based on your suggestion and request, we have made corrected modifications on the manuscript.

All corrections in the revised version of the manuscript are marked in red.

Furthermore, we would like to show the details as follows:

Comment: The experimental unit and replication structure still require clarification. Please explicitly state whether three independent production batches were prepared for each chitosan concentration, or whether only one production batch was prepared for each concentration (0, 0.25, and 0.50%). This information is essential to understand what constituted the biological replicates and how the random effect of experimental repetition was defined in the statistical model.

Response: We sincerely thank the Editor for raising this important point regarding the experimental design and model structure. We confirm that one batch of each type of sausage (Ch0/B, Ch0.25/B, and Ch0.50/B), each weighing 50 kg, was manufactured, and repeated measurements were conducted within each individual batch. Therefore, the term "batch" in our context does not refer to multiple manufacturing replications under the same conditions, but rather to a single production instance per treatment group.

We hope this clarification resolves the concern and appreciate the opportunity to improve the clarity of the manuscript.

Best Regards,

Authors