Effects of Fermentation Temperature on the Physicochemical Properties, Bioactive Compounds, and In Vitro Digestive Profile of Cacao (Theobroma cacao) Seeds

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the corresponding revisions in highlighted changes in the re-submitted files.

In Lines 2-4, I suggest changing "Impact" to "Effects."

The suggestion was accepted. See highlight mark in the revised version of the manuscript. Line 2

In Line 11, the text reads, "Three fermentation conditions were evaluated: F40 (40°C), Control (50°C), and F60 (60°C)." Could it be changed to "Low," "Normal," and "High" groups? The current grouping is confusing and not uniform.

The suggestion was accepted. Changes are highlighted in the revised document. Lines 11-12

In Lines 25-27, it states, "Higher fermentation temperatures improve flavor and digestibility, making them suitable for chocolate production, while lower temperatures retain bioactive compounds, favoring functional food applications." The conclusion drawn in this paper is meaningless because it is common sense, which undoubtedly greatly affects the paper's innovation. It is suggested that the author extract other innovative points to attract more readers.

The suggestion was accepted. Changes are highlighted in the revised document. Lines 25-31

Please check the scientific name of Cacao in Line 34.

The suggestion was accepted. Changes are highlighted in the revised document. Line 38

There is a lack of effective logical connectors between the introductions in Lines 34-111, making this part of the content very dull. It is suggested that the author reorganize it. Also, I am curious why the author placed Figure 1 here. Has the content conveyed in Figure 1 not been reported before?

The suggestion was accepted. The introduction section was redone and adjusted. Lines 38-110

About Figure 1, it is positioned within the text to provide visual context for the fermentation setup described in the manuscript. Since fermentation conditions, including temperature and aeration, play a critical role in microbial activity and biochemical transformations, it is important to illustrate the experimental conditions under which these processes take place.

In Line 124, under "2.2. Fermentation profiles," the author needs to explain in detail why 50°C was chosen as the control group and why 40 and 60 were chosen as treatment groups, rather than 45 and 55, or more temperature gradients.

The suggestion was accepted. The section was redone and adjusted. Lines 125-155

In Line 136, "sixth day T60: gradually," is there a symbol missing before T60?

Section was edited. Changes are highlighted in the revised document. Lines 158-161

In Line 230, "All procedures were performed in triplicate unless otherwise specified" - what does this mean? Does it mean that each experiment was conducted three times? For example, in vitro Protein digestibility - if so, how did the author coordinate the data processing of these three times? Was it merged or treated as a covariate for the number of repetitions?

Changes are highlighted in the revised document. See lines 263-266

In Line 236, under "3. Results and discussion," it is suggested that the author separate the results and discussion to more clearly convey the feasibility and innovation of the current results to the readers.

The decision to present the Results and Discussion as a single section was intentional to maintain a cohesive narrative, allowing for a more integrated interpretation of the findings. Separating them may disrupt the logical flow, as each result is directly linked to its corresponding analysis and contextual significance.

readability.

In Figure 3, there is a lack of icons in the picture, that is, what do the three Lines in the picture represent respectively?

We appreciate the reviewer’s observation regarding Figure 3 and acknowledge the need for additional clarity. Additionally, we will update the figure legend to explicitly describe the meaning of each line. The revised legend will now specify the corresponding fermentation treatments (Control, T40, T60) and their respective temperature trends. Lines 294-298

From Figure 2 to Figure 7, the text format in the pictures is not uniform, and in the reference section, it is difficult to find the serial numbers in the text, which makes it difficult for me to assess the accuracy of the references you cited. The format of the references is not uniform, for example, the 22nd reference in Lines 539-540 does not have a DOI.

We appreciate the comment, however upon investigation, it appears that this article does not have a DOI assigned. This is likely due to its publication date in 1992, a period when the DOI system was not yet widely adopted. Consequently, the absence of a DOI in this reference is consistent with the practices of that time.

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the corresponding revisions in highlighted changes in the re-submitted files.

Although no major flaws were identified in the study, there are still some details that can be improved. In this regard, I recommend a minor revision of the manuscript before its publication:

1.) The references are not formatted according to the instructions for authors of the Fermentation journal (reference numbers in the text should be placed in square brackets, references must be numbered in the order in which they appear in the text, etc.).

We appreciate the observation, references were modified accordingly thorough the text.

2.) CO₂ (carbon dioxide) should be written as CO₂ and not CO² [lines 77 and 85].

Correction was made thorough the document.

3.) It would be good to explain in more detail how the chemical analysis was performed [lines 146-149], as the reference is not readily available, while a considerable part of the results are related to this analysis. Furthermore, it is not clear whether these methods are AACC methods or perhaps AOAC methods.

We appreciate the observation; a correction was made on the chemical analysis section. See corrected text highlighted in the revised version. Lines 169-175.

4.) A more detailed description of how the samples were pulverised [lines 151 and 152] is advisable.

We appreciate the observation; a correction was made on the chemical analysis section. See corrected text highlighted in the revised version. Lines 177-184

5.) It would be good to show an example of the chromatogram obtained in the HPLC analysis of free sugars [line 154] – it could be included in Supplementary Materials.

We appreciate the reviewer’s suggestion to include an example of the HPLC chromatogram of free sugars in the Supplementary Materials. The HPLC analysis was performed by an external analytical service, which provided the results in a printed format. Unfortunately, we do not have access to the original chromatogram in a digital file.

To ensure transparency, we have included a detailed description of the HPLC method. including column specifications, mobile phase conditions, and retention times, to facilitate reproducibility. Lines 189-191

6.) Gas chromatography was used to analyse the fatty acids. It would be good to mention this fact explicitly (e.g. by writing “gas chromatograph” and not just “chromatograph” [line 163]).

We appreciate the observation; a correction was made. See corrected text highlighted in the revised version. Lines 193-195

7.) The abbreviation “pGI” is used in the text [line 217], but it is not explained that it stands for “predicted glycemic index”.

We appreciate the observation; a correction was made. See corrected text highlighted in the revised version. Line 250

8.) I suggest a slightly more concise caption for Figure 4: “… imply the time of fermentation.” -> “… imply the time of fermentation in hours.” [line 337]

We appreciate the observation; a correction was made. See corrected text highlighted in the revised version. Line 366-368

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the corresponding revisions in highlighted changes in the re-submitted files.

The introduction part must be significantly revised. From lines 56-111 must be rewritten. It seems more to a materials and methods part (it describes processes to obtain fermented cacao seeds in a too much extensive way!).

The introduction must present some innovation aspects, what new contribution bring this study.

Thank for the observations, the introduction was updated in the revised version.

Materials and methods: line 114-115 – what

year were harevest? Error bars from Figure 3 and 3 are not visibile. Also no significant differences on graph can be seen.

We acknowledge the reviewer’s observation. The error bars were included but may appear unclear due to scaling or overlapping with data points.

3.7- sub-chapter statistical analysis name – line 443 must be changed since statistical analysis it used in all study.

We appreciate the reviewer’s suggestion. To reflect that statistical analysis was applied to the entire study, we revise the sub-chapter title, see changes in yellow, line 470

Figure 7- observations and variables must be changed with samples and physico-chemical characteristics for example.

We appreciate the reviewer’s suggestion to refine Figure 7 by replacing "observations and variables" with more specific terms related to the study. To improve clarity and relevance, we update the figure to explicitly refer to samples and their physico-chemical characteristics. Figure 7 and lines 492-494

The conclusion are too short with few physicochemical data discussed.

Thank for the observations, the conclusion was updated in the revised version. Lines 496-511

The authors conclude that the sensory attributes are modified but this was not determined in this study.

Thank for the observations, this sentence was removed from the updated conclusion.

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the revisions in the highlighted changes in the re-submitted files.

In the abstract, on lines 11–12, the abbreviations for the groups are F40 and F60, while in the main text, the group abbreviations are still T40 and T60. The author should make these consistent.

The suggestion was accepted. See highlighted changes in the corrected version of the text.

Although Figure 3 has provided explanations for A, B, C, and each type of line, as a figure, the most important thing is to be intuitive and clear. Has the author considered splitting the three indicators into three separate figures, with each figure containing data from the three treatment groups? In this way, the groups could be directly labeled in the figures.

The suggestion was accepted. Changes were made in figure 3 to avoid overcrowded data. See Figure 3.

Figure 7 is very blurry. Please provide a high-resolution image to improve the visualization of the data. I don't know why there is a one-line gap between the three paragraphs in the conclusion section on lines 495–511, but I still do not recommend splitting the conclusion into three parts.

We upload the highest resolution available in this corrected version. We have uploaded the highest resolution available in this corrected version. However, I suspect that the PDF generation system may cause the deficiencies in the manuscript proofs. Also, the conclusion was updated according to the referee's suggestions.

At the end of the text, the heading for References is missing. I look forward to the author's revisions. Good Luck!

The suggestion was accepted. Changes are highlighted in the revised document. Line 503

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the corresponding revisions in highlighted changes in the re-submitted files.

The authors reply well and the article may be published in it current form

Thank you for your valuable contributions.

Thank you very much for taking the time to review this manuscript.

Please find the detailed responses below and the revisions in the highlighted changes in the re-submitted files.

I strongly recommend that the authors carefully revise the presentation of the results, especially the figures and tables. For example, in the revised version, the labels below Figure 2 are still T40 and T60, even though the figure caption and other parts of the text have been changed to F40 and F60.

We appreciate the referee's suggestions. The manuscript has been thoroughly revised, with corrections highlighted in yellow, particularly addressing misspellings and modifications in labels F40 and F60.

Figure 2 has been updated, along with its description, which has been revised and corrected accordingly. The corresponding text in the manuscript has also been updated in the relevant section. Please refer to lines 157–161.

Additionally, the updated Figure 3 is in black and white and the grouping is very unclear. I don't understand why the authors don't simply place the group icons in the upper left or upper right corner of the figure.

Corrections have been made to Figure 3 to improve clarity, and labels have been added within the figure. Additionally, the figure footnote has been modified for better comprehension.

The font and title of the data in Table 1 and Table 2 are different. I checked the journal's requirements, and they currently do not meet the journal's standards.

All tables and figures have been revised to meet the journal’s formatting requirements, with particular attention to font types. The table footnote has also been adjusted for improved clarity.

If the authors believe that our suggestions are unreasonable or not conducive to improving the quality of the manuscript, they can directly refuse to make the changes.

We sincerely appreciate your observations. Thanks to your feedback, several other improvements have been made in this version of the manuscript.