Chitosan Films in Food Applications. Tuning Film Properties by Changing Acidic Dissolution Conditions

Round 1

Reviewer 1 Report

The research article “Tuning chitosan films properties by changing acidic dissolution conditions” provides interesting insights on how the interplay between acid type and chitosan influences different properties of the resulting films. The overall experimental set-up generally looks plausible. Nevertheless, taking a closer look at the individual sections, some weak points were identified.

General comments:

Significant differences are mentioned several times throughout the manuscript. Is this statement supported by statistical analyses? If so, where is the information on the corresponding statistical analyses?

Throughout manuscript, there are a number of spelling, grammar and syntax errors. Therefore, it would be advantageous to check the English of the paper thoroughly.

Specific comments:

Lines 99 et seqq.: For the microbiological experiments, it is not clear how many technical and biological replicates were performed. Without this information, it is difficult to assess the drawn conclusions. The tested strains are suitable standard representatives of (potentially) pathogenic Gram-positive and Gram-negative bacteria associated with food. However, taking into account the potential application in the food packaging sector, analyzing further known food-associated microorganisms, such as spore-forming bacteria, would help demonstrating the efficacy of chitosan. Furthermore, it is of great interest to analyze the antimicrobial activity of the produced films as these are more likely to be applied in the packaging sector compared to chitosan solution.

Line 109 et seq.: it is stated that growth and purity controls were included. However, it is not clear if the effect of acid without chitosan was also addressed. According to line 193 et seq., the differences in the MIC were observed for different acid-chitosan solutions. However, it is not clear if this difference is only due to chitosan and/or the chitosan-acid-“interaction” or whether there is free acid which itself may exhibit antimicrobial activity.

Line 185 & 258 et seq.: citation/referencing error occurs which leaves the reader wondering if any important citation is missing.

Section “3.Results”: the manuscript would benefit from more details and discussion within the results part especially regarding the results descripted from line 252 onwards.

Author Response

Reviewer 1#

The research article “Tuning chitosan films properties by changing acidic dissolution conditions” provides interesting insights on how the interplay between acid type and chitosan influences different properties of the resulting films. The overall experimental set-up generally looks plausible. Nevertheless, taking a closer look at the individual sections, some weak points were identified.

Reply: We are grateful to the reviewer for recognizing the whole importance of our manuscript. The manuscript was improved according the reviewers comments.

General comments:

Significant differences are mentioned several times throughout the manuscript. Is this statement supported by statistical analyses? If so, where is the information on the corresponding statistical analyses?

Reply: Statistical analysis was performed and the results added to the manuscript. Also the materials and methods section was updated to include the description of the statistical analysis performed.

Throughout manuscript, there are a number of spelling, grammar and syntax errors. Therefore, it would be advantageous to check the English of the paper thoroughly.

Reply: The entire manuscript text was revised to correct the errors.

Specific comments:

Lines 99 et seqq.: For the microbiological experiments, it is not clear how many technical and biological replicates were performed. Without this information, it is difficult to assess the drawn conclusions. The tested strains are suitable standard representatives of (potentially) pathogenic Gram-positive and Gram-negative bacteria associated with food. However, taking into account the potential application in the food packaging sector, analyzing further known food-associated microorganisms, such as spore-forming bacteria, would help demonstrating the efficacy of chitosan. Furthermore, it is of great interest to analyze the antimicrobial activity of the produced films as these are more likely to be applied in the packaging sector compared to chitosan solution.

Reply: Thank you for your comments. Assays were performed in triplicate in two independent experiments for each strain.

The main barrier for an efficient antibacterial activity is the bacteria cell wall composition. Therefore, we chose two standard representative strains of Gram-negative and Gram-positive that are susceptible to known antibiotics (no antibacterial resistance mechanisms and not because they are potential pathogenic bacteria). Spore forming bacteria are important in the food industry (Clostridium spp., Bacillus spp.). However, spores are incredible resistant forms of bacteria and very difficult to eradicate. For example, Clostridium spores can be viable in the environment for 30 years (resist to heat, desiccation and UV radiation). If there is contamination of the food, they will germinate in the normal bacteria form (a Gram-positive bacteria). Additionally,  these are the forms that will produce the toxins that lead to food poisoning, not the spores. In the present work we demonstrate the efficiency of chitosan in the vegetative cells.

The main objective of this study was not yet testing the films. We acknowledge the comment of the reviewer and it will be done in a future work.

Line 109 et seq.: it is stated that growth and purity controls were included. However, it is not clear if the effect of acid without chitosan was also addressed. According to line 193 et seq., the differences in the MIC were observed for different acid-chitosan solutions. However, it is not clear if this difference is only due to chitosan and/or the chitosan-acid-“interaction” or whether there is free acid which itself may exhibit antimicrobial activity.

Reply: Yes, the effect of the different acids without chitosan was addressed. The results were added to the manuscript (lines 208-214).

Line 185 & 258 et seq.: citation/referencing error occurs which leaves the reader wondering if any important citation is missing.

Reply: The references are related with the table 1 and 2 and were corrected. The other references were not affected by this error.

Section “3.Results”: the manuscript would benefit from more details and discussion within the results part especially regarding the results descripted from line 252 onwards.

Reply: The whole results section was improved and also the discussion regarding the effect of the different acids in the mechanical properties was improved.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript polymers1024638 present the effect of three different acids on  chitosan solubilization and characteristics of chitosan film. The  subject is of interest for chitosan bioactive film formation and application. However, manuscript need improvements before publication.

The main weakness of the manuscript is related to statistical analysis.  Figures 2, Figures 3, Figures 5 presents error bars. These bars are standard deviation -  this need to be mentioned also in figure caption, not only in material and method section.  How these were calculated? What is the statistical significance? My suggestion to the authors is to add to the Material and Methods section a subsection related to Statistical Analysis. To each figures where error bars are used,  the type of statistical errors, number of replicates and statistical significance should be presented. Also, in Table 2 are presented  means and errors. For each determination, brightness, opacity and contact angle, number of replicates and statistical significance must be presented also in Table caption.

Others small mistakes from manuscript should be corrected.

Suppliers of materials and equipment must be presented with their whole name, headquarter city/town and country. Please add all these info.

L185 and L258 - Error! Reference source not found. – please solve this issue related to the reference software.

Author Response

Reviewer 2#

Manuscript polymers1024638 present the effect of three different acids on  chitosan solubilization and characteristics of chitosan film. The  subject is of interest for chitosan bioactive film formation and application. However, manuscript need improvements before publication.

Reply: We thank the reviewer for the positive comments on our manuscript. The manuscript was improved according the reviewer comments.

The main weakness of the manuscript is related to statistical analysis.  Figures 2, Figures 3, Figures 5 presents error bars. These bars are standard deviation -  this need to be mentioned also in figure caption, not only in material and method section.  How these were calculated? What is the statistical significance? My suggestion to the authors is to add to the Material and Methods section a subsection related to Statistical Analysis. To each figures where error bars are used,  the type of statistical errors, number of replicates and statistical significance should be presented. Also, in Table 2 are presented  means and errors. For each determination, brightness, opacity and contact angle, number of replicates and statistical significance must be presented also in Table caption.

Reply: We agree with the reviewer and the statistical analysis was added to the manuscript. The captions of the figures and tables were updated.

Others small mistakes from manuscript should be corrected.

Reply: The manuscript text was carefully revised to avoid mistakes.

Suppliers of materials and equipment must be presented with their whole name, headquarter city/town and country. Please add all these info.

Reply: We agree with the reviewer. The information about materials and equipments was updated.

L185 and L258 - Error! Reference source not found. – please solve this issue related to the reference software.

Reply: The issue in the reference was solved.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for accepting and implementing the reviewer comments! The manuscript benefited greatly from the revision and the resulting changes/improvements.

Still, I have some remarks.

The authors informed the reviewers of the number of replications of the microbiological experiments, but unfortunately, missed to include this information in the material and method section as well. Therefore, I kindly ask the authors to update the corresponding paragraph.

Moreover, the results section was indeed improved but still misses some details and discussion from line 283 onwards.

Best regards,

Reviewer

Author Response

Reviewer 1#

Thank you for accepting and implementing the reviewer comments! The manuscript benefited greatly from the revision and the resulting changes/improvements.

Still, I have some remarks.

The authors informed the reviewers of the number of replications of the microbiological experiments, but unfortunately, missed to include this information in the material and method section as well. Therefore, I kindly ask the authors to update the corresponding paragraph.

Reply: We are grateful to the reviewer for recognizing the improvements made in our manuscript. We apologize for the information missing in the materials and methods section. The information was added and highlighted in yellow.

Moreover, the results section was indeed improved but still misses some details and discussion from line 283 onwards.

Best regards,

Reviewer.

Reply: We added more details in the results section and also the discussion was improved, according to the reviewer's comment.

Author Response File: Author Response.docx