Recovery of Bioactive Compounds from Calabrian Bergamot Citrus Waste: Selection of Best Green Extraction
Round 1
Reviewer 1 Report
This manuscript is interesting because it presents a thorough analytical characterization of extracts obtained from subproducts of the Bergamot pomace. The high value extract products have various applications in different industrial sectors. I have the following comments to the authors:
Novel solvents, such as supercritical fluids, ionic liquids, and deep eutectic solvents, are being increasingly demanded by the industry for the extraction and concentration of valuable components in the development of pharmaceutical products. Recent reviews have explored the applications of these technologies for the processing of citrus fruits. The authors can cite these emerging technologies.
Accelerating solvent high-pressure extraction (ASE) is also being used in this field.
It's worth considering why the extraction experiments were carried out using a 50/50 ethanol/water mixture instead of other concentrations like 30/70 ethanol/water mixture, for instance.
Additionally, it can be discussed why it is necessary to carry out a drying process of the raw material before extraction if the solvent is water or ethanol/water at 50% mass.
A rotary evaporator may be used to evaporate the solvent after extraction, but the operating conditions of this unit should be considered. It's also worth noting the final water content in the product.
Total phenolic content (TPC) and total flavonoid content (TFC) are important considerations. Microwave-assisted extraction tends to show lower TPC contents.
It's important to consider how TPC and antioxidant activity may be affected if a dried extract product is required for commercialization.
Author Response
The authors thank the Reviewer for his useful advice, which we provided to bring.
- Novel solvents, such as supercritical fluids, ionic liquids, and deep eutectic solvents, are being increasingly demanded by the industry for the extraction and concentration of valuable components in the development of pharmaceutical products. Recent reviews have explored the applications of these technologies for the processing of citrus fruits. The authors can cite these emerging technologies. Accelerating solvent high-pressure extraction (ASE) is also being used in this field.
- we added a new sentence in the text and two new references.
- It's worth considering why the extraction experiments were carried out using a 50/50 ethanol/water mixture instead of other concentrations like 30/70 ethanol/water mixture, for instance.
A: Thank for the observation, during our experimentation we considered different extraction conditions, such as ratio solvent/sample; solvents mixture; times, temperatures. Initially, we also evaluated the application of different ratio of solvent, including 30/70 ethanol/water mixture, but between 30/70 ethanol/water mixture and 50/50 ethanol/water, the best results were showed by 50/50, for this reason we choice this concentration of solvents.
- Additionally, it can be discussed why it is necessary to carry out a drying process of the raw material before extraction if the solvent is water or ethanol/water at 50% mass.
A: We choice to dry the raw material for two main reasons: 1) to extend the availability of product over time, the high moisture content of the pomace, would make the product highly unstable and perishable; 2) As showed in different previous works, the drying process and the reduction of moisture content determine the increase of extraction yield.
- A rotary evaporator may be used to evaporate the solvent after extraction, but the operating conditions of this unit should be considered. It's also worth noting the final water content in the product.
A: Yes, the application of rotary evaporator could be a valuable tool for concentrating the extract. Indeed, in other our works we applicated this technique. However, in this study to reduce variables that could have increased the error, the calculations considered the dry matter of the product characterizing the content actually extracted from the sample.
- Total phenolic content (TPC) and total flavonoid content (TFC) are important considerations. Microwave-assisted extraction tends to show lower TPC contents.
A: These results, push us to carry out new experiments to understand the lesser effect of microwaves on the extraction of antioxidants.
- It's important to consider how TPC and antioxidant activity may be affected if a dried extract product is required for commercialization.
A: With this work we selected the best extraction method, in order to extract the highest amount of phenolic compounds from bergamot by-products. Of course, in the case of extracts commercialization, different aspects should be evaluated. This is the basis for our other work currently being written, which will include the use of dried and microencapsulated extracts obtained from the considerations raised in this work.
Reviewer 2 Report
missing a discussion part
the article was well written with minor grammatical typography, yet its originality was average (plagiarism 32%). The data was presented in a good way, clearly, and easy to read. However, I couldn’t proceed to the main discussion as the article was not written completely.
minor grammatical typography
Author Response
The authors thank the reviewer to have read and commented our work. Moreover, we thank him for pointing out that it is missing the word discussion in the text. In this article we decided to write at the same time "Results and discussion" and to no separated the sections. In the first version of the work, we forgot to write it in the section 3, we have now corrected the error.
Reviewer 3 Report
This paper presents a detailed study on the application of various extraction methods. The topic of the study is worth investigating, and the experimental design was acceptable. Although some parts and impressions of the article seem to be preliminary, but the detailed study may have an impact and be useful to readers.
Notes are on file.
Comments for author File: 
Comments.pdf
Author Response
The authors thank the Reviewer for contributing to the correction of our work, we proceeded to respond and edit errors in the text.
R: How many repetitions were there in the laboratory tests?
A: we carried out three repetitions for each sample
R: What equipment was used for extraction?
A: A heating magnetic stirrer (Velp Scientifica, Usmate Velate (MB), Italy); a Sonoplus Ultrasonic homogenizers, Series 2000.2, HD 2200.2 (BANDELIN, Ultraschall seit 1955) and a Microwave Digestion System (ETHOS EASY, Millestone, Bergamo, Italy) were used to assisted the extraction process (as described in 2.2. section)
A: Figure 1, we modified with the capital letter
A: as suggested, we changed TF with TFC.
R: What test was used to test the normality of the distribution?
A: Initially, all the results were elaborated statistically through excel and were reported as means an standard deviation, after we performed a Multivariate and One-way analysis of variance (ANOVA) with SPSS Software and Tukey’s post hoc test was applied; and a Pearson’s correlation test, with the same software (p<0.05).
R: lack of discussion of results (DPPH and FRAP)
A: In this work we analyzed the total antioxidant activity with two different text DPPH and ABTS. In order to improve the explanation of ABTS and DPPH, we compared both results, explaining that the higher quantity detected in the ABTS assay compared to the DPPH assay considering the question on the focal point of the data obtained.: “It could be dependently by the fact that ABTS assay generally is more sensitive to hydrophilic antioxidants, while DPPH assay is more sensitive to lipophilic antioxidants.” In fact the highest amount of phenolic compounds detected was represented by Neoeriocitrin, Naringin and Neohesperidin wich are hydrophilic compounds.
A: We checked the decimal place in table 7
Author Response File: 
Author Response.docx
Round 2
Reviewer 2 Report
Overall, the authors have already fulfilled the major revision as suggested in the 1st round of review. In particular to add a discussion part in the article. However, there are still some revisions and suggestions required to improve the quality of the article.
1. There is no explanation about the reason why those TPC and TFC are more extractable by UA in particular than others. It was just a values comparison and statistical analysis.
2. To talk about extraction efficiency, the distribution coefficient should be discussed, including the temperature and solvent effect.
3. The bioactivity comparison is commonly presented in IC50 value
4. How do the TPC & TFC data correlate with the DPPH & ABTS?
5. More explanation supported by references is required to discuss the different trends between the results of DPPH vs ABTS; what is the effect of extraction method, solvent, temp, and time towards the bioactivity of the compound at the molecular level. In particular, what was the stability of the compounds at high temperatures and its effects on their activities?
6. Please validate the discussion on lines 311-313 with the related literature
7. Please summarize the main reason why the maceration with H2O/EtOH, 1/1 at 70 C was chosen as the best extraction method.
8. There is a repetition of the paragraph in results and discussion (Lines 282-295)
9. The label of X & Y axis of the Figure should be presented clearly and readable
In general, the quality of the English Language is good, but there are still some corrections are required;
1. There are some typographical and grammatical errors such as neohesperidin vs neoesperidin, dw vs DW, line 376 e.t.c, please check again thoroughly.
2. Rewrite the paragraph (lines 442-452). it is too long for 2 sentences.
Author Response
The Authors thank the reviewer for his comments and Suggestions.
In attached the file with the answers
Author Response File: Author Response.docx
