Fermentation-Induced Changes in Nutritional, Antinutritional, and Microbial Characteristics of Calabash Fruit (Crescentia cujete L.) Seeds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript describes the evaluation of a new type of condiment prepared by fermenting calabash seeds. This process is traditionally applied to other seeds, but this approach may not only expand the possible processing of calabash seeds but also yield a new type of product. Introduction clearly describes situation and opens the problem of composition. Material and methods are well described, just with a few typos – line 92 potable water not portable; line 114 30 °C. In the part 2.2 can be mentioned how the samples were stored and the temperature of fermentation is also missing.

Results and discussion are interesting text, only some technical details can be improved. Table 1 conclude changes in composition during fermentation. The label lacks the units of the main components content and also whether they are given for fresh sample or based on dry matter. This is a relatively significant difference and could also shed light on the increasing content of the main components. This must be cleared up and reflected in discussion. Presentation of condiments as good source of proteins looks strange, can authors present ow much protein will a typical dose of fermented spices added to a meal?

Acidity is presented as total titratable acidity and authors present it as the evidence of fermentation. But the amount is not so high and pH, as the important value is not mentioned. Because the decrease of pH is usually reported as a sign of fermentation. Evidence of the presence of organic acids such as lactic and acetic acid is required. This part needs to be supplemented and explained.

The breakdown of antinutrients is presented clearly, as are other results.

Isolation of fermenting bacteria with MRS and their biochemical characterization is the standard operation. Authors presented four main strains as the fermenters. Problem I see is, these strains are well known as the strains withs many pathogenic strains. Usually, food with Klebsiella sp. is released, but the authors present it as a safe? Bacillus strains differ also from safe to dangerous, make the authors some observation about it? This part must be improved with some information about safety of product.

Conclusions present all information clearly, but practical use is omitted. Especially safety and real nutritional effect calculated with typical dose of condiment.

Author Response

Dear Editor,

 

Thank you for the constructive comments on our manuscript. We have revised the manuscript to incorporate the suggested changes, with changes added using a red font, deletions are not shown. We hope that our revision is satisfactory as look forward to a favourable response.

Samson Oyeyinka

Reviewer 1

Comment: Manuscript describes the evaluation of a new type of condiment prepared by fermenting calabash seeds. This process is traditionally applied to other seeds, but this approach may not only expand the possible processing of calabash seeds but also yield a new type of product.

 

Response: We sincerely thank the reviewer for this positive and insightful assessment. We agree that applying traditional fermentation techniques to calabash (Crescentia cujete L.) seeds represents a novel approach that broadens their potential utilization and supports the development of a new fermented condiment with improved nutritional and microbial characteristics.

 

Comment: Introduction clearly describes situation and opens the problem of composition.

Response: We thank the reviewer for this positive feedback.

 

Comment: Material and methods are well described, just with a few typos – line 92 potable water not portable

Response: This has been changed in the revised manuscript.

 

Comment: line 114 30 °C.

Response: Space has been added and the symbol put in superscript.

 

Comment: In the part 2.2 can be mentioned how the samples were stored and the temperature of fermentation is also missing.

Response: We thank the reviewer for this comment. As the fermentation was conducted using traditional methods, the temperature was not monitored. To preserve the samples for subsequent analyses, fermented seeds were immediately frozen and then freeze-dried. For microbial analyses (Sections 2.8 and 2.9), samples were withdrawn immediately after fermentation and processed without delay. The manuscript has been updated to clarify these details in Section 2.2 (Lines 95-100).

 

Comment: Results and discussion are interesting text, only some technical details can be improved.

Response: We thank the reviewer for this positive evaluation of the Results and Discussion sections. We appreciate the suggestion regarding technical details and have carefully revised the manuscript to improve clarity, precision, and consistency of the presented data.

 

Comment: Table 1 conclude changes in composition during fermentation. The label lacks the units of the main components content and also whether they are given for fresh sample or based on dry matter. This is a relatively significant difference and could also shed light on the increasing content of the main components. This must be cleared up and reflected in discussion.

Response: We thank the reviewer for this important observation. Table 1 has been revised to include appropriate units (% dwb). The values were initially expressed on wet basis but based on the reviewer’s suggestion, we have converted to dry weight basis to account for moisture differences. Additionally, the discussion has been updated to clarify that the observed increases in protein and carbohydrate contents are influenced by moisture reduction during fermentation as well as microbial biochemical transformations.

 

Comment: Presentation of condiments as good source of proteins looks strange, can authors present how much protein will a typical dose of fermented spices added to a meal?

Response: We thank the reviewer for this insightful comment. While we did not explicitly claim that the fermented calabash seed condiment is a primary protein source, we would like to provide context: many traditional African fermented condiments, including locust bean (iru), ogiri, and fermented calabash seed, are indeed relatively high in protein on a dry weight basis. However, these condiments are typically used in small amounts for flavour and aroma rather than as a main dietary protein source. To provide practical perspective, a typical serving of 5–10 g would contribute approximately 1–2.5 g of protein per meal, serving as a supplementary protein source rather than a primary one.

 

Comment: Acidity is presented as total titratable acidity and authors present it as the evidence of fermentation. But the amount is not so high and pH, as the important value is not mentioned. Because the decrease of pH is usually reported as a sign of fermentation. Evidence of the presence of organic acids such as lactic and acetic acid is required. This part needs to be supplemented and explained.

Response: We thank the reviewer for this insightful comment. Although pH values are usually reported as a sign of fermentation, ammoniacal fermentation process such as those involving condiments do not follow that pattern. In most cases, the pH values do not change or sometimes increases. While we did not directly measure specific organic acids, literature on similar fermented condiments (e.g., soy dawadawa, 0.08–0.26 mg lactic acid/g; kidney bean-based condiments) shows that modest acid production accompanies microbial fermentation, supporting the interpretation of our TTA data as evidence of active fermentation Molecular analysis of bacterial community dynamics during the fermentation of soy-daddawa condiment | Food Science and Biotechnology

 

Comment: The breakdown of antinutrients is presented clearly, as are other results.

Response: We thank the reviewer for this positive comment.

 

Comment: Isolation of fermenting bacteria with MRS and their biochemical characterization is the standard operation. Authors presented four main strains as the fermenters. Problem I see is, these strains are well known as the strains withs many pathogenic strains. Usually, food with Klebsiella sp. is released, but the authors present it as a safe? Bacillus strains differ also from safe to dangerous, make the authors some observation about it? This part must be improved with some information about safety of product.

Response: We thank the reviewer for raising this important concern regarding microbial identification and safety. Phenotypic characterization suggested the presence of several genera, including Bacillus, Lysinibacillus, Klebsiella, and Providencia. Molecular identification using 16S rRNA gene sequencing was attempted for all isolates; however, only five isolates produced high-quality sequences suitable for reliable identification. These sequences corresponded to Bacillus and Lysinibacillus species. Although faint PCR bands were observed for some other isolates, including those presumptively identified as Klebsiella and Providencia, the resulting sequences were of insufficient quality and were therefore excluded from definitive molecular identification and reporting. We have revised the manuscript to clarify this limitation and to avoid overinterpretation of provisional identifications. In addition, we expanded the Discussion to acknowledge that genera such as Bacillus include both non-pathogenic and pathogenic species, and that strain-level identification, virulence assessment, and toxin profiling were beyond the scope of the present study. We now explicitly state that further molecular and safety evaluations are required before large-scale or commercial application of the fermented product. These clarifications have been incorporated into Section 3.6.

 

Comment:  Conclusions present all information clearly, but practical use is omitted. Especially safety and real nutritional effect calculated with typical dose of condiment.

Response: We thank the reviewer for this valuable suggestion. The Conclusion section has been revised to include the practical application of the fermented calabash seed condiment. Specifically, we added an estimate of nutritional contribution based on a typical serving size (5 g dry condiment), showing that a portion could provide approximately 1.1 g of protein and 1.5 g of fat per meal. In addition, we explicitly acknowledged that while the microbial community reflects patterns of traditional spontaneous fermentations, further molecular and toxicological studies are required to fully establish product safety prior to large-scale or commercial use. These additions provide a more practical perspective on the nutritional and functional relevance of the condiment without overstating safety or efficacy.

Reviewer 2 Report

Comments and Suggestions for Authors

In my opinion, the manuscript entitled Fermentation-Induced Changes in Nutritional, Antinutritional, and Microbial Characteristics of Calabash Fruit (Crescentia cujete L.) Seeds by Oladunjoye et al.,

Introduction is enough to provide a current state of the art for the present study and material and methods are enough described by authors and results are presented, discussed and justified.

 

I have some comments as follows:

1. When an abbreviation is used for the fort time, please mention it under brackets such as PEM.
2. Please mention the pedoclimatic condition from the farm, the period of harvesting. How the authors decided that the fruits could be collected and are enough maturated?
3. Please better emphasize the novelty of the present study and add more information about calabash fruits such as growth condition, soil requirements and healthy human importance based on its content in bioactive compounds.

Comments for author File: Comments.pdf

Author Response

Dear Editor,

 

Thank you for the constructive comments on our manuscript. We have revised the manuscript to incorporate the suggested changes, with changes added using a red font, deletions are not shown. We hope that our revision is satisfactory as look forward to a favourable response.

Samson Oyeyinka

Reviewer 2

Comment: In my opinion, the manuscript entitled Fermentation-Induced Changes in Nutritional, Antinutritional, and Microbial Characteristics of Calabash Fruit (Crescentia cujete L.) Seeds by Oladunjoye et al., Introduction is enough to provide a current state of the art for the present study and material and methods are enough described by authors and results are presented, discussed and justified.

Response: We sincerely thank the reviewer for the positive feedback and for recognizing the clarity and adequacy of the Introduction, Materials and Methods, and Results and Discussion sections. We appreciate the reviewer’s encouragement.

Comment: I have some comments as follows: When an abbreviation is used for the fort time, please mention it under brackets such as PEM.

Response: We thank the reviewer for this suggestion. All abbreviations in the manuscript have been revised so that the full term is provided at first mention, followed by the abbreviation in parentheses (e.g., Protein-Energy Malnutrition (PEM)).

 

Comment: Please mention the pedoclimatic condition from the farm, the period of harvesting. How the authors decided that the fruits could be collected and are enough maturated?

Response: We thank the reviewer for this important comment. The calabash fruits used in this study were obtained directly from local farmers in Nigeria, who have traditional knowledge of fruit ripening. The maturity of the fruits was further confirmed by a botanist at the Department of Botany, University of Ibadan, where the seeds were also formally identified. The manuscript has been updated to reflect this information, providing clarity on the source, identification, and maturity of the fruits used in the study.

 

Comment: Please better emphasize the novelty of the present study and add more information about calabash fruits such as growth condition, soil requirements and healthy human importance based on its content in bioactive compounds.

Response: We thank the reviewer for this valuable comment. The Introduction has been revised to better emphasize the novelty of the present study by highlighting the lack of prior research on fermentation-induced changes in calabash seeds. Additional information on the growth conditions, soil preferences, and nutritional and health significance of calabash fruits based on their bioactive compound content has been added. These revisions provide a clearer context for the study and its potential contribution to functional foods and local dietary applications.