Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The introduction should be completed with the argumentation why the Spray drying technique was chosen. What are the advantages of this technique compared to others. Why were maltodextrin (MD) and gum Arabic (GA) chosen as encapsulating agents and not, for example, alginate?

1. Chapter. Materials and methods: Preparation of cocoa microcapsules- The description is complete, but it is not mentioned how the process parameters were selected. For example, was the method for extracting active compounds from cocoa optimized in another article by the research team or is it taken from a literature study? The same question is for the preparation of the emulsion and the production of microcapsules.

2. Chapter. Results and discussions: The discussion is too general.

    - 3.1. Characteristics of cocoa extract: The results are compared with a literature study where extract phenolic compounds from lemon wastes (peels and edible pulps) was studied and not cocoa almond as specified in the article. I would recommend that the comparison be made with the same cocoa species.

3.2 Microcapsule preparation parameters and 3.3. Effect of spray drying: The influence of parameters on particle size is not discussed. Also, no analytical technique is specified to determine the particle size obtained.

- 3.4.3. Release kinetics of bioactive compounds- to understand the release behavior, experimental data should be fitted to various mathematical models. It should be complemented with a kinetic study.

- The Conclusion should be completed with more experimental data.

- I would recommend completing the study with SEM or DLS analysis of the microcapsule size, as well as with pictures regarding the application of microcapsules in chicken muscle- iInhibition of microbial growth rates. Also, using TGA analysis, the thermal stability of microcapsules can be studied, which is very important when discussing the controlled release of active substances.

Author Response

Comments 1: The introduction should be completed with the argumentation why the Spray drying technique was chosen. What are the advantages of this technique compared to others. Why were maltodextrin (MD) and gum Arabic (GA) chosen as encapsulating agents and not, for example, alginate?

Response 1: The authors appreciate the reviewer's comments, as they improve the scientific quality of the manuscript. With regard to the introduction, an explanation was added as to why the spray-drying technique was chosen and its advantages over other techniques. In addition, the advantages of maltodextrin (MD) and gum arabic (GA) as encapsulating agents were explained, as well as why they were selected for this research (fourth paragraph of the introduction).

Comments 2: 1. Chapter. Materials and methods: Preparation of cocoa microcapsules- The description is complete, but it is not mentioned how the process parameters were selected. For example, was the method for extracting active compounds from cocoa optimized in another article by the research team or is it taken from a literature study? The same question is for the preparation of the emulsion and the production of microcapsules.

Response 2: The authors appreciate the reviewer's comment. In this regard, the bibliographic references that were considered in selecting the procedures and parameters for the cocoa microcapsule preparation process were cited. In 2.2.1. Preparation of cocoa and extraction of compounds, it is mentioned at the end of the paragraph; in 2.2.2. Emulsion preparation, it is reported at the beginning of the paragraph; and in 2.2.3. Spray drying of microcapsules, it is mentioned at the beginning of the paragraph.

Comments 3: 2. Chapter. Results and discussions: The discussion is too general.

Response 3: The authors appreciate the reviewer's comment. Consequently, several sections of the manuscript's discussion were modified, with specific comparative analyses of results reported from cocoa extracts and more in-depth discussions. Changes were made in sections: 3.1. Characteristics of cocoa extract; 3.2.1. Effect of encapsulating agent type; 3.2.2. Effect of spray drying; 3.3.3. Release kinetics of bioactive compounds; 3.6.1. Inhibition of microbial growth rates; 3.7. Novelty, impact on the area of interest, and limitations of the study.

Comments 4: - 3.1. Characteristics of cocoa extract: The results are compared with a literature study where extract phenolic compounds from lemon wastes (peels and edible pulps) was studied and not cocoa almond as specified in the article. I would recommend that the comparison be made with the same cocoa species.

Response 4: The authors agree with the reviewer's comment. Therefore, the respective modifications were made in section 3.1. Characteristics of cocoa extract. Comparative analyses were performed on the results obtained with studies that reported the properties of microcapsules extracted from cocoa. In addition, the cited bibliographic references were added.

Comments 5: 3.2 Microcapsule preparation parameters and 3.3. Effect of spray drying: The influence of parameters on particle size is not discussed. Also, no analytical technique is specified to determine the particle size obtained.

Response 5: The authors appreciate the reviewer's comment. In this regard, scanning electron microscopy (SEM) was used to determine the morphology and particle size of the microcapsules, with the procedure and equipment used specified in section 2.3.2. Morphological characterization and particle size of microcapsules. In addition, a figure was added showing the SEM image of the microcapsules obtained from maltodextrin and gum Arabic (Figure 1), and the effect of the microcapsule preparation operating parameters on particle size was analyzed, discussing these results in sections 3.2.1. Microcapsule preparation parameters, and 3.2.2. Effect of spray drying.

Comments 6: - 3.4.3. Release kinetics of bioactive compounds- to understand the release behavior, experimental data should be fitted to various mathematical models. It should be complemented with a kinetic study.

Response 6: The authors appreciate the reviewer's comment. In this regard, the discussion of the kinetic results of bioactive compound release (3.3.3. Release kinetics of bioactive compounds) was improved by comparing them with other studies that have evaluated the kinetic behavior of release in microcapsules prepared from plant extracts. In addition, Figure 4 shows the kinetic study of the release of phenolic compounds through the prepared microcapsules. On the other hand, it should be noted that the data did not fit any mathematical model for the following reasons: i) the objective of the research focuses on the preparation of microcapsules from cocoa extracts using spray drying, their characterization and study of antioxidant and antimicrobial properties, as well as their novel application in high-consumption food matrices (chicken muscles); ii) similar studies that have been developed recently (with cocoa extracts and other plant matrices) and that have been taken as a reference for this manuscript have not considered making adjustments with kinetic models, since this topic should be addressed in specific research that analyzes not only the adjustment of kinetic data, but also the effect of operational, technical, and performance parameters on kinetic behavior and its respective mathematical modeling. Among the manuscripts reviewed are:

Microencapsulation of Theobroma cacao L polyphenols: A high-value approach with in vitro anti-Trypanosoma cruzi, immunomodulatory and antioxidant activities: https://doi.org/10.1016/j.biopha.2024.116307

Evaluation of the Efficiency of Encapsulation and Bioaccessibility of Polyphenol Microcapsules from Cocoa Pod Husks Using Different Techniques and Encapsulating Agents: https://doi.org/10.3390/pr13103094

Microencapsulation of phenolic-enriched extract from cocoa pod husk (Theobroma cacao L.): https://doi.org/10.1016/j.powtec.2021.03.033

Enhancing Stability of Boesenbergia rotunda Bioactive Compounds: Microencapsulation via Spray-Drying and Its Physicochemical Evaluation: https://doi.org/10.3390/foods14152699

Comments 7: - The Conclusion should be completed with more experimental data.

Response 7: The authors agree with this comment from the reviewer. In this regard, the Conclusions were rewritten, adding experimental data to strengthen the conclusions drawn.

Comments 8: - I would recommend completing the study with SEM or DLS analysis of the microcapsule size, as well as with pictures regarding the application of microcapsules in chicken muscle- iInhibition of microbial growth rates. Also, using TGA analysis, the thermal stability of microcapsules can be studied, which is very important when discussing the controlled release of active substances.

Response 8: The authors appreciate the reviewer's recommendations regarding the incorporation of analytical techniques and representative figures. Consequently, the following changes were made: a SEM analysis of the microcapsules prepared with maltodextrin and gum arabic (Figure 1) was incorporated to define the morphology and size of the microencapsulated particles, thus improving the analysis of the results (sections 3.2.1. Effect of encapsulating agent type, and 3.2.2. Effect of spray drying). In addition, Figure 6 shows the inhibition of the microbial growth rate of enterobacteria and mesophilic aerobic bacteria over time in chicken breast samples treated with microencapsulated cocoa extracts. Finally, all the figures in the manuscript were revised, improving their graphic quality and verifying the English in each one.

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the work presented here is of high potential interest, but there are some shortcomings which should be addressed prior to publication:

• In the abstract, what is meant by "metabolites" here? Specifically in the second sentence "However, these metabolites are susceptible..." Were the extracts subjected to any type of digestion process prior to use? If not, then this is probably a typo.
• Please elaborate on the choice of wall materials, especially since a surfactant was also used to impart emulsion stability. Why GA and MD - what are the differences with respect to e.g. barrier properties and thermal protection? Why not compare the pure wall materials with a linear combination of MD and GA?
• What type of GA was used? In the materials and methods section, there is a parenthesis labelled "Brand" which I suspect was supposed to be replaced by the brand (preferably including what species of GA) that was used.
• Please provide the equations for loading capacity and encapsulation efficiency
• How were the release profiles measured? There is no description of the analysis method used in section 2.3.3. Based on the amounts used, this was definitely not done gravimetrically.
• What type of spray dryer was used, and what was the spray drying yield?
• There is no mention of the characteristics of the microcapsules such as size distribution, despite its mention in the schematic in Figure 1. Please provide results for the size distribution (and ideally also morphology e.g. from SEM), as differences in size and thus surface area could affect many of the parameters studied here.
• Please use the correct number of significant digits when reporting uncertainty. For example, in Table 1 the loading capacity in 10% GA is listed as 47.9 +/- 2.3%. If the uncertainty exceeds unity, there is no justification for the use of decimal points.

Author Response

Comments 1: In my opinion, the work presented here is of high potential interest, but there are some shortcomings which should be addressed prior to publication:

In the abstract, what is meant by "metabolites" here? Specifically in the second sentence "However, these metabolites are susceptible..." Were the extracts subjected to any type of digestion process prior to use? If not, then this is probably a typo.

Response 1: The authors appreciate the reviewer's comment. Indeed, this is a typographical error. To correct this error, the word “metabolites” was replaced with “bioactive compounds” in the abstract and other sections of the manuscript (marked in red).

Comments 2: Please elaborate on the choice of wall materials, especially since a surfactant was also used to impart emulsion stability. Why GA and MD - what are the differences with respect to e.g. barrier properties and thermal protection? Why not compare the pure wall materials with a linear combination of MD and GA?

Response 2: We thank the reviewer for their comment. This aspect was addressed both in the introduction (fourth paragraph) and in the materials and methods section (2.2.2. Emulsion preparation). Specifically, it was indicated that maltodextrin and gum arabic were selected as wall materials because they are widely used encapsulating agents but with clearly differentiated structural, emulsifying, and functional properties in spray-drying systems for food products. In addition, the food-grade surfactant Tween 80 was used to improve interfacial stability during homogenization and prevent coalescence prior to drying, without replacing the barrier and thermal protection functions of the wall materials. Regarding the absence of mixtures of both encapsulants, the objective of the study was to compare the individual performance of the encapsulants under the same conditions. The inclusion of mixed matrices would have introduced additional studies on the effect of interaction and competition (synergism/antagonism), which can perfectly well be addressed as a future line of research.

Comments 3: What type of GA was used? In the materials and methods section, there is a parenthesis labelled "Brand" which I suspect was supposed to be replaced by the brand (preferably including what species of GA) that was used.

Response 3: We appreciate the reviewer's comment. The brand and type of GA used were added, replacing it with the word “Brand” in section 2.1. Materials.

Comments 4: Please provide the equations for loading capacity and encapsulation efficiency.

Response 4: The authors appreciate the reviewer's comment. Therefore, in section 2.3.3. Loading capacity and encapsulation efficiency, the equations used to calculate the loading capacity and encapsulation efficiency were added, with their respective units and the meaning of the variables involved.

Comments 5: How were the release profiles measured? There is no description of the analysis method used in section 2.3.3. Based on the amounts used, this was definitely not done gravimetrically.

Response 5: The authors are grateful for this question and comment from the reviewer. Consequently, section 2.3.3. Loading capacity and encapsulation efficiency, has been corrected, adding a description of the methods used to analyze and quantify the parameters used to determine loading capacity and encapsulation efficiency. Furthermore, section 2.3.4. Release kinetics of bioactive compounds, indicates the procedure for analyzing and quantifying the release of polyphenolic compounds.

Comments 6: What type of spray dryer was used, and what was the spray drying yield?

Response 6: The authors appreciate the reviewer's comments. In section 2.2.3. Spray drying of microcapsules, information related to the model, brand, and country of origin of the spray dryer used was added. In addition, the spray drying performance was added to the first paragraph of section 3.2.2. Effect of spray drying.

Comments 7: There is no mention of the characteristics of the microcapsules such as size distribution, despite its mention in the schematic in Figure 1. Please provide results for the size distribution (and ideally also morphology e.g. from SEM), as differences in size and thus surface area could affect many of the parameters studied here.

Response 7: The authors agree with the reviewer's comment. Therefore, an SEM analysis of the microcapsules prepared with maltodextrin and gum Arabic (Figure 1) was added to define the morphology and size of the microencapsulated particles and analyze their effect on the experimental parameters evaluated (sections 3.2.1. Effect of encapsulating agent type, and 3.2.2. Effect of spray drying).

Comments 8: Please use the correct number of significant digits when reporting uncertainty. For example, in Table 1 the loading capacity in 10% GA is listed as 47.9 +/- 2.3%. If the uncertainty exceeds unity, there is no justification for the use of decimal points.

Response 8: We appreciate the reviewer's comment. Accordingly, we reviewed the correct use of significant digits in data uncertainty throughout the manuscript. The text and tables containing this error were corrected.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled: “Development of cocoa (Theobroma cacao L.) CCN-51 microcapsules with antioxidant and antimicrobial properties” overall seems not properly assess the objective set in the title. In particular regarding the antimicrobial activity. This point should be better described and substantited. Moreover, the experiments on animals should need the etical committee approval for the protocol and for the experimental approach. This shuold be added to the text (see section 2.5.). the criteria for selecting “fresh chicken muscle” should be exploited for better clarity. Contaminants could impact on the overall procedures: please clear this point. The Figure 1 end the other Figures in the text seem not clear to read: please indicate the source. The novelty and limits of the proposed study should be discussed and the Conclusions better substantiated.

Finally, an article (not peer reviewd) online availavle as pre print at the address: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=5345343 can be located and has the same title and Authors (Authors however seem different compared to the submitted version to the Journal). This point should be explained. Overall the novelty and the impact on the area of interest should be better assessed.

Comments on the Quality of English Language

The English language overall seems fine, minor check would be useful for better readability.

Author Response

Comments 1: The manuscript entitled: “Development of cocoa (Theobroma cacao L.) CCN-51 microcapsules with antioxidant and antimicrobial properties” overall seems not properly assess the objective set in the title. In particular regarding the antimicrobial activity. This point should be better described and substantited. Moreover, the experiments on animals should need the etical committee approval for the protocol and for the experimental approach. This shuold be added to the text (see section 2.5.). the criteria for selecting “fresh chicken muscle” should be exploited for better clarity. Contaminants could impact on the overall procedures: please clear this point. The Figure 1 end the other Figures in the text seem not clear to read: please indicate the source. The novelty and limits of the proposed study should be discussed and the Conclusions better substantiated.

Response 1: The authors appreciate the reviewer's comment. Consequently, the information regarding the antimicrobial properties of microcapsules was better substantiated in several sections of the manuscript: abstract (rewritten to highlight the antimicrobial applications of microcapsules); materials and methods (2.4. Bacteriostatic activity evaluation, 2.5. Application in food matrices); results and discussion (3.5. Antimicrobial activity of microcapsules, 3.6. Application in food matrices); Conclusions.

For its part, the first paragraph of section 2.5, Application in food matrices, explained compliance with ethical aspects in the research carried out. On this topic, it should be noted that the research did not involve the slaughter or treatment of live animals, but was carried out using samples of chicken muscle for human consumption refrigerated in supermarkets and certified stores in Ecuador, and that these meat products were obtained from brands and distribution companies that have health registration and the respective permits from the National Agency for Health Regulation, Control, and Surveillance (ARCSA, in Spanish) for the production, preservation, and distribution of meat products in Ecuador. This state agency is responsible for monitoring the quality and safety of food and other goods for human consumption. In addition, the research complies with current Ecuadorian regulations and the guidelines of the ethics committee of Ecuadorian universities regarding the use for research purposes of meat products that have already been processed and are available for sale in stores and supermarkets. For this reason, ethical aspects were not mentioned in the initial submission of the manuscript, especially since other similar studies that evaluated refrigerated meat products and were used as references did not consider reporting ethical issues (https://doi.org/10.1016/j.foodhyd.2023.109496).

The first paragraph of section 2.5 also clearly indicated the selection criteria for fresh chicken muscle samples. The samples were taken directly from refrigerated meat products for human consumption and recently produced, from brands and supermarkets with certifications endorsed by ARCSA for the production, preservation, and distribution of meat products in Ecuador. As these are meat products for human consumption, there is no risk of contamination that could affect the experimental procedures of the research. However, as a precaution, the initial bacterial load of the chicken muscle samples evaluated was quantified.

All figures in the manuscript were reviewed, improving their graphic quality and verifying the English in each one. Regarding the source of the figures, all were generated from data obtained in this research.

Additionally, section 3.7, Novelty, impact on the area of interest, and limitations of the study, was added, discussing the aspects suggested by the reviewer. The conclusions were also revised, providing experimental data, the most relevant findings, and future prospects for research in this field.

Comments 2: Finally, an article (not peer reviewd) online availavle as pre print at the address: https://papers.ssrn.com/sol3/papers.cfm?abstract_id=5345343 can be located and has the same title and Authors (Authors however seem different compared to the submitted version to the Journal). This point should be explained. Overall the novelty and the impact on the area of interest should be better assessed.

Response 2: We appreciate the reviewer's comment. Regarding this point, we would like to clarify that the document mentioned is a preliminary version (preprint) from an early stage of the research review, disseminated for the purposes of rapid communication before submitting the work to a formal peer review process. The apparent differences in authorship are due to adjustments in scientific contributions and authorship agreements made during the development of the current version of the manuscript, in accordance with institutional and editorial ethical guidelines. The current paper includes additional authors who performed activities focused on improving the scientific quality of the paper, such as the development and analysis of microcapsule characterization studies, the development of microbiological assays, statistical analysis, discussion of results, and financing of analytical techniques and acquisition of chemical reagents. All authors included in the version submitted to Chemistry have made substantial contributions to the conception of the study, experimental design, characterization of materials, polyphenol release studies, microbiological studies, application of microcapsules in chicken muscles, data analysis, discussion, and interpretation of results.

Novelty and impact on the area of interest were also incorporated into the manuscript, specifically in section 3.7. Novelty, impact on the area of interest, and limitations of the study.

In addition, the English of the entire manuscript was reviewed, including figures and tables. Corrections were made to the texts in the following sections: 1. Introduction; 2.2.2. Emulsion preparation; 2.2.3. Spray drying of microcapsules; 2.3.1. Determination of phenolic content and antioxidant capacity; 2.3.2. Morphological characterization and particle size of microcapsules; 2.3.3. Loading capacity and encapsulation efficiency; 2.4. Bacteriostatic activity evaluation; 3.1. Characteristics of cocoa extract; 3.2.1. Effect of encapsulating agent type; 3.2.2. Effect of spray drying; 3.3.3. Release kinetics of bioactive compounds; and 4. Conclusions.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in this form.

Author Response

Comments 1: Accept in this form.

Response 1: The authors appreciate the reviewer's comments.

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the authors have addressed the questions and concerns

Author Response

Comments 1: In my opinion, the authors have addressed the questions and concerns.

Response 1: The authors appreciate the reviewer's comments.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript has been modified, nonetheless the main point remains: the proposed study approval by etical committee is not mentioned. Please add this information which seems necessary for the study conducted. Formal approval from an Institutional Animal Care and Use Committee (IACUC) or a similar ethics committee (like an Animal Ethics Committee/AWERB) should ne mentioned in the text. The ethical review assessment process confirms that researchers meet the existing national/institutional standards, even for tissue samples.

The paragraph: “Preparation of cocoa and extraction of compounds” mentions “some modifications: please describe whuch modifications have been done. The paragraph 2.5 reports a Reference whule the experimental procedure should be described in full. In the same paragraph all the cerifications and authorizations should be mentioned for better assessing the context. The “guidelines of the ethics committee of Ecuadorian universities” are mentioned: please give more details on this point.

Safetu aspects as follow up of the experiments presnted shoud also be part of the text:please exlain this oint and clear better.

The pre-print can be considered acceptable to be put online, nonetheless the Authors should be the same of the manuscipt as submitted for the peer review, and the change of the names should be better justified. The justification given by the Authors seems not acceptable in its present form.

Comments on the Quality of English Language

The English language overall seems fine, minor check would be useful for better readability.

Author Response

Comments 1: The manuscript has been modified, nonetheless the main point remains: the proposed study approval by etical committee is not mentioned. Please add this information which seems necessary for the study conducted. Formal approval from an Institutional Animal Care and Use Committee (IACUC) or a similar ethics committee (like an Animal Ethics Committee/AWERB) should ne mentioned in the text. The ethical review assessment process confirms that researchers meet the existing national/institutional standards, even for tissue samples.

Response 1: The authors appreciate the reviewer's comment. The study has been approved by the university's ethics committee, and this information has been added to section 2.5. Application in food matrices.

Comments 2: The paragraph: “Preparation of cocoa and extraction of compounds” mentions “some modifications: please describe whuch modifications have been done. The paragraph 2.5 reports a Reference whule the experimental procedure should be described in full. In the same paragraph all the cerifications and authorizations should be mentioned for better assessing the context. The “guidelines of the ethics committee of Ecuadorian universities” are mentioned: please give more details on this point.

Response 2: The authors agree with the reviewer's comments. In this regard, the modifications made to the bibliographic citation in paragraph 2.2.1. Preparation of cocoa and extraction of compounds were explained. In paragraph 2.4. Bacteriostatic activity evaluation, the modifications made to the reference included were also indicated. In addition, in the paragraph of section 2.5. Application in food matrices, the experimental procedure for determining the antimicrobial activity of microcapsules in fresh chicken muscle samples was added. All the certifications and authorizations required to carry out the research were also mentioned, both from the University and from the brands of fresh chicken available in supermarkets, which guarantee their safety and food safety. With regard to the mention made of the “guidelines of the ethics committee of Ecuadorian universities” in section 2.5. Application in food matrices, information was added about these guidelines, specifically referring to the regulations of the research ethics committee of the Technical University of Manabí (the institution where the research was conducted), which was supplemented with a bibliographic citation of the regulations issued by the university.

Comments 3: Safetu aspects as follow up of the experiments presnted shoud also be part of the text:please exlain this oint and clear better.

Response 3: The authors appreciate the reviewer's comment. Consequently, in section 2.5. Application in food matrices, a paragraph was added stating that the research complies with safety requirements, since all materials and waste from the bacterial study were sterilized or autoclaved before disposal.

Comments 4: The pre-print can be considered acceptable to be put online, nonetheless the Authors should be the same of the manuscipt as submitted for the peer review, and the change of the names should be better justified. The justification given by the Authors seems not acceptable in its present form.

Response 4: The authors understand the reviewer's comment. In this regard, we would like to explain once again that the pre-print document was sent to SSRN long before the final version was prepared for peer review at MDPI. In our case, an overview and advice regarding the preprint led to major modifications to the paper and additional work that warranted the inclusion of the participants as authors prior to submitting the manuscript to this journal. Specifically, the authors who were added contributed in the following roles or activities: validation, formal analysis, data curation, writing—review and editing, visualization, resources, funding acquisition. Furthermore, it is confirmed that this manuscript has not been submitted to or is being considered by another journal.

Comments on the Quality of English Language: The English language overall seems fine, minor check would be useful for better readability. Response 5: The authors appreciate the reviewer's comment. In this regard, we reviewed the English throughout the manuscript, including figures and tables, and made the necessary corrections.