Metabolomic Insights into the Potential of Chestnut Biochar as a Functional Feed Ingredient

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reggi et al., investigate the functional properties of chestnut derived biochar with a potential application as animal feed. It is the reviewer's opinion that this manuscript does not merit publication in Applied Sciences. There are several issues with this manuscript:

(i) there are many errors in the manuscript related to typos, formatting, English language and grammar.

(ii) It seems that a very limited amount of experiments have been conducted not allowing proper conclusions to be made. 

(iii) several key analytical procedures related to biochar are not performed. The pyrolysis process is not a focus of this study (although the authors briefly explain the process). What is the carbon content, etc of the biochar? Elemental analysis is missing. The authors mention the chemical composition of the chestnut biochar, but only crude protein is mentioned. What about the other constituents (hemicellulose, cellulose, lignin, ...). 

(iv) the analysis of the derived chestnut biochar extracts is not sufficient. QTOF-HPLC is a rather qualitative way of determining the composition, not quantitative. Therefore, the mentioned areas do not mean much on their own.

(v) the conclusion is very general.

Comments on the Quality of English Language

See comments above.

Author Response

Dear reviewer, Please see the attachment file

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript submitted for review concerns the issue of in vitro evaluation of functional properties of Biochar from Chestnut Agro-Residues biomasses for Sustainable Animal Feed. This is an important issue and requires deepening knowledge on this subject. The authors correctly constructed the manuscript. The literature review was performed correctly. The justification of the research is satisfactory. A procedure scheme in the Material and Methods section would be useful, which would allow for easier recognition of the conducted research. The graphs included in the results are difficult to read and require improvement. Their quality is definitely poor, they are also too small. The discussion is conducted correctly. The conclusions are justified and applicable.

Author Response

Dear review, Please see the attachment file

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study focused on chestnut-derived biochar, highlighting its functional properties, such as antimicrobial, antioxidant, and probiotic activities.　The use of chestnut bark charcoal in animal feed has been demonstrated to confer a number of benefits. These include the enrichment of the feed with minerals and the prevention of the growth of bacteria that are both beneficial and harmful.　The majority of research into bio-charcoal has focused on its adsorption properties. This report makes a valuable contribution to the field by examining the potential of bio-charcoal as a feedstock and gathering basic data on its use in this context.　The experimental methods and results have been validated; however, an additional explanation of the basic concept is required.　In other words, what form is it used as feed? It seems reasonable to posit that the substance will be incorporated into the basic feed, but  would be grateful if you could provide us with details of the envisaged percentage.　It is acknowledged that this is now a fundamental consideration; however, it is unlikely that the limited quantity of charcoal will constitute the entirety of the feed, and it is assumed that it would then be incorporated into the primary feed. It seems reasonable to question the quantity that should be mixed with the main feed.　

Author Response

Dear reviewer, Please see the attached file

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Please find my comments in the attached file.

Regards

Comments for author File: Comments.pdf

Author Response

Dear reviewer, Please see the attached file

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The work of Reggi et alia is in regard to morphological, chemical, and functional as well as metabolomic aspects of biochar.

Introduction is well written, however a brief chemical explanation of the process of carbonisation of the organic matter in question should be provided.

Materials and methods

HPLC/MS analysis for bioactive compound is mentioned chromatograms are lacking.

HPLC collumn temperature is lacking.

The microbial evaluation is relevant, however I strongly suggest the authors NOT to perform an in vivo evaluation on mammals as well (i.e. mice), as it will be a shame to sacrifice animals for well known facts, just for the pursue of another in vivo study. Thus said, the present article is relevant and of good quality, I suggest only a minor revision.

Author Response

Dear reviewer, Please see the attached file

Author Response File: Author Response.pdf

Reviewer 6 Report

Comments and Suggestions for Authors

Authors have prepared an interesting and original work.

Only a few recent references may be added

Conclusions may contain suggestions on dosage for in vivo trials based on in vitro findings.

Author Response

Dear reviewer, please see the attached file

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed most of the comments raised by the reviewer and performed an English proofread by an external party. Therefore, the reviewer accepts this manuscript for publication in Applied Sciences.

Author Response

Thank you for your positive feedback

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

Yo write that The analysis was conducted in biological duplicate, with three technical replicates each. But how many measurements or samples were made per each replicate?

You can not use ANOVA if you have 6 samples in total. In this case use nonparametric Kruskall Wallis test.

 

Author Response

Dear reviewer,

Please see the attachment

Author Response File: Author Response.docx