GC-Olfactometric Analysis as a Tool for Comprehensive Characterization of Aromatic Profiles in Cocoa-Based Beverages with a Natural Chocolate Substitute

Round 1

Reviewer 1 Report

The article is interesting and is a consistent continuation of the research of F. P. Spada & all. on the precise chromatographic and olfactometric characterization of dried and fermented jackfruit seeds for their use as substitutes for natural cocoa in cocoa-flavored drinks.

The methodology of chromatographic research is modern and consistent with the current state of knowledge. SPME-GC-MS analyses are performed professionally and at a high standard, using modern equipment. The authors use the Head Space technique.  This technique is complemented by olfactometric tests performed by two experts. My doubts are raised by such a small number of experts. Often, several or even a dozen experts participate in such research because the opinions of individual experts are subjective.  The authors should justify that they used only two experts. What were their (experts) professional qualifications?

Other comments of a procedural nature.
The title of the article uses the word "coca" but it should be "cocoa"
Keywords should not start with capital letters??
The authors use many acronyms. They partly explain them and partly don't. I suggest that the authors add a list of acronyms with explanations.
By using the SPME technique, the authors can only analyze volatile organic compounds. What about other substances present in breadfruit seeds: for example, macromolecular, ionic, and other compounds that cannot be detected by the SPME technique.  Don't these compounds pose any threat to consumers of such modified drinks and food products?

Author Response

Reviewer 1: a) My doubts are raised by such a small number of experts. Often, several or even a dozen experts participate in such research because the opinions of individual experts are subjective.  The authors should justify that they used only two experts. What were their (experts) professional qualifications?

Authors answer: a) We appreciate the insightful observations and comments. In response to the GCO examiners' feedback, we have incorporated additional details into the methodology section of the manuscript, specifically highlighting the average experience of the examiners. We used two examiners for our study, a choice supported by similarities found in scientific literature. Notably, two articles employing a comparable methodology, both published in peer-reviewed scientific journals, involved studies conducted with two or three examiners[1] ^and [2]. This approach aligns with the established practice recognizing that the technique requires exhaustive training and experience.

Reviewer 1: b) The title of the article uses the word "coca" but it should be "cocoa". Keywords should not start with capital letters? The authors use many acronyms. They partly explain them and partly don't. I suggest that the authors add a list of acronyms with explanations.

Authors answer: b) We changed the word to COCOA, we removed the capital letters in KEYWORDS, and added a list of acronyms.

Reviewer 1: c) By using the SPME technique, the authors can only analyze volatile organic compounds. What about other substances present in breadfruit seeds: for example, macromolecular, ionic, and other compounds that cannot be detected by the SPME technique.  Don't these compounds pose any threat to consumers of such modified drinks and food products?

Authors answer: c) The concerns raised about the limitations of the Solid Phase Microextraction (SPME) technique in analyzing volatile organic compounds (VOCs) in breadfruit seeds are noted. It is acknowledged that the SPME technique may not capture macromolecular, ionic, and other compounds that could be present in the seeds. The potential threat to consumers of modified drinks and food products due to these undetected compounds is a valid consideration.

In response to these concerns, our investigation has considered various studies that have addressed sensorial and toxicological aspects. Notably, these studies have not revealed sensory or toxic indicators that prioritize the analysis of ionic and other compounds. This suggests that, from a sensory and toxicological perspective, there may not be immediate cause for alarm regarding these specific substances.

Furthermore, we recognize the need for ongoing research, and your suggestion to proceed with new objectives is duly considered. Future studies could explore additional facets and unveil potentially significant findings that may have been overlooked in the current analysis.

It is also crucial to acknowledge existing literature highlighting the presence of toxic macromolecular and ionic compounds in some agricultural commodities within the human diet. This underscores the importance of comprehensive research to understand the composition of food products thoroughly. In the case of breadfruit seeds, it is noteworthy that most of the production in Brazil, particularly of jackfruit, occurs in extractive and organic systems. This information provides context to the agricultural practices and may influence the composition of the seeds.

[1] Yaozheng Liu, Dylan C. Cadwallader, MaryAnne Drake,

Identification of predominant aroma components of dried pea protein concentrates and isolates,

Food Chemistry, Volume 406, 2023, https://doi.org/10.1016/j.foodchem.2022.134998.

[2] Fernanda Papa Spada, Lais Masson Zerbeto, Gabriel Bernardes Cabreira Ragazi, Érika Maria Roel Gutierrez, Miriam Coelho Souza, Jane K. Parker, and Solange Guidolin Canniatti-Brazaca

Optimization of postharvest conditions to produce chocolate aroma from jackfruit seeds.

Journal of Agricultural and Food Chemistry 2017 65 (6), 1196-1208,  DOI: 10.1021/acs.jafc.6b04836.

Reviewer 2 Report

Their manuscript “GC-olfactometric analysis as a tool for comprehensive characterization of aromatic profiles in coca-based beverages with a natural chocolate substitute” provides interesting information on the composition and main odor-active compounds responsible for the sensory similarity between beverages formulated with genuine cocoa powder and an alternative proposal obtained from jackfruit seeds processed to develop chocolate-like aromas.

Beyond possible risks with adulterant purposes that this alternative Jackfruit powder could represent in the Food Industry to replace the use of cocoa products (chocolate), well focused, its use can represent in the future an alternative for valorization of the byproducts of this tropical crop.

In the manuscript review, some aspects have been highlighted and must be considered to improve the document. Suggestions and comments for revision are outlined below and the PDF file is also noted.

L3: In the title, check the word “coca-based.” Should it be cocoa-based?

L15-L37: Limit the abstract to the maximum number of words suggested in Instructions for Authors.

L92: Should be section 2. MATERIAL AND METHODS

L97-L100: Clarify how many times the process was repeated to obtain DJS and FJS? Were the FJSBev and DJSBev formulations made with a single batch obtained from DJS and FJS? The development of aromas during fermentation is critical, assuming that drying and roasting were more controlled.

In Section 2.3, near L-36 (or where the authors consider appropriate), indicate how many replicates were made of the analysis of each sample in both SPME-GCMS and GC-O analysis.

L144: Should be subsection 2.4 Statistical analysis.

L171-173: Correctly indicate on the two graphs in Figure 1 which is A) and which corresponds to B). Do the same in the figure caption. Additionally, as indicated in the description in the manuscript, edit Figure 1B, adding I and II to identify the groups.

Check the comment in L192.

Check the comment in L214.

In Figure 5. Proceed as indicated in Figure 1.

Address comments indicated in Table 2.

Check the comment in L262.

Suggestions

1. Improve the resolution of Figures 1 and 4.

2. Since in this study, the aroma profile of drinks formulated with jackfruit seed flour as a cocoa substitute is characterized for the first time, it is suggested that the authors indicate in an additional table (which can be sent to Supplementary Material), the names of the 71 identified compounds related to the odor-active aroma descriptors. The above would let us know which compounds correspond to the keys indicated in Figure S1.

3. Please indicate the title of the Figure (Figure S1) considered in the supplementary material.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Reviewer 2  a) L3: In the title, check the word “coca-based.” Should it be cocoa-based?

Authors answer: a) We have updated the word to 'COCOA’.

Reviewer 2  b) L15-L37: Limit the abstract to the maximum number of words suggested in Instructions for Authors.

Authors answer: b) We have adjusted the abstract to 200 words, adhering to the maximum word limit suggested in the Instructions for Authors.

Reviewer 2  c) L97-L100: Clarify how many times the process was repeated to obtain DJS and FJS? Were the FJSBev and DJSBev formulations made with a single batch obtained from DJS and FJS? The development of aromas during fermentation is critical, assuming that drying and roasting were more controlled.

Authors answer: c) In the manuscript (Lines 102-109), we have provided clarification regarding the times and formulations for the batches, specifically outlining 3 batches for roasting blend and 1 batch for the beverage.

Reviewer 2  d) In Section 2.3, near L-36 (or where the authors consider appropriate), indicate how many replicates were made of the analysis of each sample in both SPME-GCMS and GC-O analysis.

Authors answer: d) We appreciate the insightful observations and comments. In response to the replicates: SPME-GCMS analysis: The volatile compounds extracted in triplicated by SPME were separated and identified on a Shimadzu QP2010 GC-MS under the following experimental conditions: RTX5MS column (30 m × 0.25 mm i.d. × 0.25 μm film thickness, Restek, USA) (Lines 129-131). GC-O analysis: Two experienced examiners (ach with>150 h of GCO experience) performed the detection and verbal description of the odor-active components of the extracts. The sniffing time was approximately 20 min. Examiners sniffed SPME extracts twice (Lines 140-143).

Reviewer 2  e) L171-173: Correctly indicate on the two graphs in Figure 1 which is A) and which corresponds to B). Do the same in the figure caption. Additionally, as indicated in the description in the manuscript, edit Figure 1B, adding I and II to identify the groups.

Authors answer e) We appreciate the insightful observations and comments. During the initial submission, we added indications on the graphs; however, some may have suggested removing the text. Currently, we have revised and maintained these indications, and we hope they are acceptable. Additionally, we have enhanced the description in the edited manuscript.

Reviewer 2  f) Check the comment in L192.

Authors answer f) We appreciate the insightful observation. We have made a change to the letter in the Mint attribute, while the Chocolate letter remains correct.

Reviewer 2  g) Check the comment in L214.

Authors answer g) We have revised the manuscript, specifically focusing on lines 216-220. “The fact that the dry beverages were clustered in a separate group could be explained by the presence of green (A6) citrus (A6), and earth (A16) odor-active descriptors (Figure 5B). Heptanal (A6) and 2-phenylethyl ester (B8) were also found in the dry beverage (Figure 5B), mischaracterizing a typical cocoa beverage.”

Reviewer 2  h) In Figure 5. Proceed as indicated in Figure 1.

Authors answer h) We appreciate the insightful observations and comments. During the initial submission, we added indications on the graphs; however, some may have suggested removing the text. Currently, we have revised and maintained these indications, and we hope they are acceptable. Additionally, we have enhanced the description in the edited manuscript.

Reviewer 2  i) Address comments indicated in Table 2.

Authors answer i) Yes, they are isomers, and we used 'a' or 'an' in compound names to indicate just a compound when the radical position cannot be determined. This is due to the unavailability of an identical standard. We have enhanced all references in Table 2

Reviewer 2  j) Check the comment in L262.

Authors answer j) We appreciate the insightful observations and comments. We have revised the manuscript, specifically focusing on lines 216-220 according to figure 5B. “Cocoa powder demonstrated a lower concentration of 2,3-butanediol compared to beverages formulated with jackfruit seed flours (Figure 5B).”

Suggestions Reviewer 2.

1. Improve the resolution of Figures 1 and 4.
2. Since in this study, the aroma profile of drinks formulated with jackfruit seed flour as a cocoa substitute is characterized for the first time, it is suggested that the authors indicate in an additional table (which can be sent to Supplementary Material), the names of the 71 identified compounds related to the odor-active aroma descriptors. The above would let us know which compounds correspond to the keys indicated in Figure S1.
3. Please indicate the title of the Figure (Figure S1) considered in the supplementary material.

Authors answer Suggestions

Thank you for your suggestions. We have made improvements to Figures 1 and 4. Additionally, we have included a supplementary table containing 71 odor-active aroma descriptors. A Linear Retention Index (LRI) was utilized for tentative identification; however, some compounds remain unidentified through this method. It is possible that in a future objective study employing authentic standards, all compounds could be identified. The title for Figure S1 has been revised to: 'Heatmaps for odor-active aroma descriptors in cocoa-beverages replaced with chocolate made from natural jackfruit seed flour.