Physicochemical and Biochemical Changes in Cocoa during the Fermentation Step

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

line 99: cocoa powder? consider a different terminology.

line 147: Authors should please check the numbering 2.4.2 comes after 2.4.3?

line 236: Authors mention (T24) which is not part of the experimental design.

Please see below additional comments

1. What is the main question addressed by the research?

The Physicochemical changes in cocoa during the fermentation step for different clones.

2. What parts do you consider original or relevant for the field? What 
specific gap in the field does the paper address? 

Comparison of the native cocoa varieties and the clones is relevant. The gap as claimed by authors is the lack of enough studies in terms of evaluating the content of bioactive compounds, their variability during post-harvest operations.

3. What does it add to the subject area compared with other published 
material?

 Physical and chemical changes during fermentation is well known. Authors applied the knowledge to these new clones specific to their geographical area.

4. What specific improvements should the authors consider regarding the 
methodology? What further controls should be considered?

Line 102: Can authors provide more information about the hybrid cocoa clones used? Example C1 is a hybrid of x and y variety. This might help readers understand some of the findings.

Also, are Guayaquil (G) and Criollo (Cr) both controls? Or just Criollo? It is not currently well explained why these two were added and whether they are controls. Authors should clearly specify.

Line 112-115: The whole description of the method is not very clear. Authors mention grains and blended cocoa. Are authors referring to the same thing? The images in the supplementary sheet does not give a good representation as to which is which. Can authors re-write this section explaining the stepwise process clearly.

5. Please describe how the conclusions are or are not consistent with the 
evidence and arguments presented. Please also indicate if all main questions 
posed were addressed and by which specific experiments.

The conclusion can be improved. How do the clones compare with the native cocoa? Are they better? Similar? worse? Will authors suggest alternative fermentation methods? That should clearly be outlined in the conclusion.

6. Are the references appropriate?

Yes

7. Please include any additional comments on the tables and figures and 
quality of the data. 

Authors limited their discussions to T0 and T120 when the experimental design considers other key times of fermentation T48 and 46. There could be a decrease, then an increase at a point in any of the parameters at any of these time periods. Can authors justify why T48 and T96 were ignored in most cases in the discussions.

 

Comments on the Quality of English Language

The paper needs a total english editing. The spellings and grammatical constructions are below standard. Please get help with editing.

Author Response

line 99: cocoa powder? consider a different terminology.

Cocoa powder was changed to fat-free cocoa

 

line 147 (Now 149): Authors should please check the numbering 2.4.2 comes after 2.4.3?

Thank you for your correction. It was corrected

 

Line 236 (Now 238): Authors mention (T24) which is not part of the experimental design.

Thank you, the correct number is, T48

Please see below additional comments

1. What is the main question addressed by the research?

The Physicochemical changes in cocoa during the fermentation step for different clones.

The physicochemical and biochemical changes that occur in cocoa beans of new clones and native varieties during a fermentation process

2. What parts do you consider original or relevant for the field? What 
specific gap in the field does the paper address? 

Comparison of the native cocoa varieties and the clones is relevant. The gap as claimed by authors is the lack of enough studies in terms of evaluating the content of bioactive compounds, their variability during post-harvest operations. Knowledge of the new clones is important for the chocolate production ecosystem in this geographic area.

3. What does it add to the subject area compared with other published 
material?

Physical and chemical changes during fermentation is well known. Authors applied the knowledge to these new clones specific to their geographical area. But not only physical and chemical changes (supplementary material), biochemical changes are important for further organoleptic characteristics of chocolate. This study is part of most extensive study, including enzymatic reactions, drying and roasting processes.

4. What specific improvements should the authors consider regarding the 
methodology? What further controls should be considered?

Room temperature can be difficult to control, but it can be measured in situ with thermocouples and the internal temperature of fermentation. It is important to analyse the changes with difference of temperature within room and fermentation temperature close to 10°C. One possible control is known % of the different components of the blend grains. Blend material is very important product around the geographic production region.

Line 102: Can authors provide more information about the hybrid cocoa clones used? Example C1 is a hybrid of x and y variety. This might help readers understand some of the findings.

Genotypes characteristics of the clones were add in the manuscript (Table 1, Now line 113)

Also, are Guayaquil (G) and Criollo (Cr) both controls? Or just Criollo? It is not currently well explained why these two were added and whether they are controls. Authors should clearly specify.

Guayaquil (G) and Criollo (Cr) are both controls.

Line 112-115 (Now 116-126): The whole description of the method is not very clear. Authors mention grains and blended cocoa. Are authors referring to the same thing? The images in the supplementary sheet does not give a good representation as to which is which. Can authors re-write this section explaining the stepwise process clearly.

Section was rewrite as follow:

In this study, the method of micro-fermentation by small sample insertion was used, as suggested by (Patent: WO2013025621 A1), Seguine E., 2013. Fermentation was carried out in a wooden box 100x100x90 cm. First, 100 kg of blended cocoa beans were placed in the box (step 1, Fig. S2). Soon, 2 kg of cocoa beans of each selected material were putted in a mesh bags (20x30 cm) and correctly identified (step 2, Fig. S2). Then, all mesh bags with the selected material were placed on the blended cocoa beans properly separated (roughly 15 cm, step 3, Fig. S2). Promptly, another 100 kg of blended cocoa beans were placed on the mesh bags containing the selected material (step 4, Fig. S2). The fermentation begins by covering all the material in the wooden box with banana leaves (step 5, Fig. S2). Additionally, the box can be cover by slight plastic mesh to avoid contact with insects (step 6, Fig. S2: supplementary material).

 

5. Please describe how the conclusions are or are not consistent with the evidence and arguments presented. Please also indicate if all main questions posed were addressed and by which specific experiments.

The conclusion can be improved. How do the clones compare with the native cocoa? Are they better? Similar? worse? Will authors suggest alternative fermentation methods? That should clearly be outlined in the conclusion.

During the fermentation process, the cotyledon of cocoa bean undergoes physicochemical and biochemical changes. The pH of cotyledon decreases for two reasons, one is the acidification of the cotyledon due to of acetic acid came into the seeds and likely by the enzyme activity on lipids, proteins, and carbohydrates. The fermentation index (FI) goes from 0.47 ± 0.04 to 1.001 ± 0.12 in all the INIFAP clones and Guayaquil, while Criollo a FI values from 1.3 to 2.5 were recorded. Compounds absorbing at 460 nm increase and compounds absorbing at 530 nm decreased in clones and Guayaquil. Besides, in Criollo beans, compounds absorbing at 460 nm increase but in compounds absorbing at 530 not changes were observed. With this data a final FI of 2.5 for the Criollo variety can be proposed as successful fermentation. The increase in total phenolic compounds is might due to an increase in flavonoids content. According to DPPH and ABTS determination method, the antioxidant capability was not affected after 120 h of fermentation. However, for the criollo variety a significant difference was observed using FRAP method perhaps due to its redox reducing capacity. The amount of methylxanthines did not affect the antioxidant potential of fermented cocoa. The FTIR scan of fat-free cocoa showed significant differences between unfermented beans and fermented beans, several peaks assigned to carbohydrates and protein decreased. The changes in the proteins and carbohydrates are might due to enzymatic activity during the fermentation period.

 

6. Are the references appropriate?

Yes

7. Please include any additional comments on the tables and figures and quality of the data. 

Authors limited their discussions to T0 and T120 when the experimental design considers other key times of fermentation T48 and 46. There could be a decrease, then an increase at a point in any of the parameters at any of these time periods. Can authors justify why T48 and T96 were ignored in most cases in the discussions.

Thank you for your comments. This study was focused to define main changes at the beginning of the fermentation to the end of this step. We know now there are changes in bioactive compounds, proteins and carbohydrates.

Reviewer 2 Report

Comments and Suggestions for Authors

The research of the current manuscript concerns the comparison of different types (native and hybrid) of cocoa seeds after fermentation.

To evaluate the effect of the fermentation on the quality of the relative cocoa seeds, the authors studied some aspects of the chemical composition strictly influenced by the fermentation process such as pH, total acidity, total phenolic and flavonoid content, theobromine and caffeine.

Furthermore, the authors also evaluated the antioxidant activity by ABTS, DPPH and FRAP as well as the relative FTIR spectrum.

In my opinion this work is interesting and the experimental procedure well organized.

The results obtained are useful to characterize the new hybrid and to confirm the chemical profile of the native ones.

However, I think that the research is uncompleted because of the lack of the entire aspect relative to the fermentation effect on the cocoa sensory profile.

In this regard, I would like to recognize that cocoa is an ingredient which is used to produce chocolate products, the impact of which on humans are fundamentally linked to the sensory responses. Thus, an evaluation of the volatile composition as well as the sensory profile of the four fermented cocoa analyzed, should be given.

Authors should explain why parameters relative to the impact of the fermentation on the sensory profile of the cocoa were not considered at all.

It seems that this paper is mostly oriented to explain the healthy importance of the cocoa for its content in phenols in particular than the hedonistic aspect, which in my opinion is the predominant.

 

Regarding the phenols’ evaluation, the authors dis not give a precise profile of these substances but I think that the characterization of the cocoa, in particular when the experimentation include new not still described cocoa hybrids, should include a quali-quantitative evaluation of the single phenols contained.

 

Moreover, I would like to suggest a full English editing and I would like to underline that the term “significative”, should be used only when the data are supported by a proper statistical analysis which was not always applied.

Comments on the Quality of English Language

The English language should be revised 

Author Response

The research of the current manuscript concerns the comparison of different types (native and hybrid) of cocoa seeds after fermentation.

To evaluate the effect of the fermentation on the quality of the relative cocoa seeds, the authors studied some aspects of the chemical composition strictly influenced by the fermentation process such as pH, total acidity, total phenolic and flavonoid content, theobromine and caffeine.

Furthermore, the authors also evaluated the antioxidant activity by ABTS, DPPH and FRAP as well as the relative FTIR spectrum.

In my opinion this work is interesting and the experimental procedure well organized.

The results obtained are useful to characterize the new hybrid and to confirm the chemical profile of the native ones.

However, I think that the research is uncompleted because of the lack of the entire aspect relative to the fermentation effect on the cocoa sensory profile.

In this regard, I would like to recognize that cocoa is an ingredient which is used to produce chocolate products, the impact of which on humans are fundamentally linked to the sensory responses. Thus, an evaluation of the volatile composition as well as the sensory profile of the four fermented cocoa analyzed, should be given.

Thank you for your comments, we agree with you, however this study is just a part of a team project. Furthermore, one important aspect during fermentation is the enzymatic processing of important precursors of sensory properties for chocolate. In parallel other studies are carrying out.

Authors should explain why parameters relative to the impact of the fermentation on the sensory profile of the cocoa were not considered at all.

It seems that this paper is mostly oriented to explain the healthy importance of the cocoa for its content in phenols in particular than the hedonistic aspect, which in my opinion is the predominant.

 Is correct, but hedonistic aspect is no less. We try to understand the complete roll of

Regarding the phenols’ evaluation, the authors dis not give a precise profile of these substances but I think that the characterization of the cocoa, in particular when the experimentation include new not still described cocoa hybrids, should include a quali-quantitative evaluation of the single phenols contained.

 

Moreover, I would like to suggest a full English editing and I would like to underline that the term “significative”, should be used only when the data are supported by a proper statistical analysis which was not always applied.

English edition has done

 

Other comments

Indeed, fermentation is an essential process with significant impact on the sensory profile of cocoa and future chocolate, as indicated by various works, although it must be remembered that the final profile also depends on other subsequent operations such as drying and roasting. It is considering the evaluation of the sensory profile of the new clones and their comparison with the native varieties after complete study of physicochemical and biochemical changes stepwise. In the future it would be important to determine the sensory profile of each clone and native variety and complement it with the data obtained in this study. Likewise, determine the phenol profile since it is also an indicator of quality because a high content of phenols, especially flavanol’s, is likely to provide greater health benefits. Along with this, it is important to be able to determine the enzymatic profile in the future to determine the variety and quantity of enzymes that act during fermentation since these play a crucial role in the quality and sensory aspects of the future chocolate.